Abstract

   Non-alcoholic fatty liver disease (NAFLD) is a progressive disease of
   the liver covering a range of conditions from hepatic steatosis to
   liver fibrosis. NAFLD could be induced by High-fat-diet(HFD). Ionizing
   radiation is widely used in medical diagnosis and therapy as well as is
   a common risk factor in occupational environment. Whether the exposure
   of various dose of radiation has effects on HFD-induced NAFLD remains
   unclear. Here, we reported that radiation exposure promoted HFD-induced
   NAFLD in a dose-response manner. Furthermore, the gut microbiota
   composition had significant difference among mice with or without
   radiation treatment. Specifically, the Bacteroidetes/Firmicutes ratio,
   the abundance of A. muciniphila, Butyricococcus, and Clostridiaceae
   decreased significantly in the mice with co-exposure of high dose of
   radiation and HFD treatment. A fecal transplantation trial (FMT)
   further verified the role of gut microbiota in the regulation of the
   liver response to co-exposure of high dose of radiation and HFD
   treatment. Notably, the gut microbiome analysis showed plasma
   lithocholic acid (LCA) level increased in the mice with co-exposure of
   high dose of radiation and HFD treatment. Following antibiotic and
   probiotic treatments there was a significantly decreased LCA bile acid
   concentration and subsequent promotion of INSR/PI3K/Akt insulin
   signaling in the liver tissues. Our results demonstrate that the
   co-exposure of radiation and HFD aggravates the HFD-induced NAFLD
   through gut microbiota-LCA-INSR axis. Probiotics supplementation is a
   potential way to protect against co-exposure of radiation and
   HFD-induced liver damage. Meanwhile, our study provide a new insight
   that population with potential HFD-induced damage should pay more
   attention on preventing from liver damage while exposing radiation.

   Keywords: Gut microbiota, High fat diet, Radiation

Graphical abstract

   Image 1
   [37]Open in a new tab

Highlights

     * •
       Gut microbiota-lithocholic acid-insulin receptor (LCA-INSR) axis
       involves the promotion effects of radiation on HFD-induced NAFLD.
     * •
       Probiotics improve the liver damage induced by co-exposure of
       radiation and HFD.

1. Introduction

   Non-alcoholic fatty liver disease (NAFLD) is a progressive disease of
   the liver covering a range of conditions from hepatic steatosis to
   liver fibrosis ([38]Moore et al., 2020). It is associated with a
   five-fold increased risk of incident diabetes in young adults
   ([39]Chung et al., 2021). Approximately 30% of the general US
   population are affected by NAFLD, of whom 25% may develop further liver
   disease ([40]Chalasani et al., 2018), while 25% of the European
   population suffer from NAFLD ([41]Fazel et al., 2016). In China, which
   is the world's most populous country, the prevalence of NAFLD has
   surpassed the incidence in USA and Europe ([42]Nan et al., 2021).
   Although many studies have revealed that lifestyle behavior can
   increase the NAFLD risk, the synergistic role of environmental and
   lifestyle factors in the development and progression of NAFLD should be
   considered, due to human exposure to multiple risk factors in the real
   world.

   Ionizing radiation is a common environmental exposure factor, although
   it is not visible and cannot be touched. Many epidemiological and
   animal studies have suggested that radiation exposure is associated
   with an excess additive risk of secondary cancer ([43]Thorby-Lister et
   al., 2018), inflammation disease ([44]Yahyapour et al., 2018), and
   diabetes ([45]Nylander et al., 2016; [46]Mortazavi et al., 2016). There
   is evidence that ionizing radiation exposure can have multiple impacts
   on human health, that the hormesis and adaptive response to low dose
   radiation exposures ([47]Vaiserman et al., 2021), DNA double-strand
   breaks ([48]Huang and Zhou, 2021), and carcinogenesis induced by high
   doses of radiation ([49]Huang and Zhou, 2020). Indeed, human expose
   various radiation dosage in environment or occupational exposure
   including Radiological therapy/examination, space flight
   ([50]Cucinotta, 2022), nuclear power plant, accident of nuclear
   weapons, outdoor ultraviolet radiation or natural high background area
   such as Yangjiang, China ([51]Cuiju et al., 2020). Over the past
   decades, although lots of studies of the effects of radiation have been
   published, most of them focused on the high dose or low dose,
   respectively. A study by W Yu et al. showed that mice pre-exposed to
   chronic low dose rate radiation are less susceptible to chromosome
   aberration induced by subsequent acute higher X-irradiation ([52]Yu et
   al., 1995) indicating the dose exposure is a critical factor to
   influence human health. A study showed a dose-response relation between
   radiation exposure of pancreas and subsequent risk of diabetes ([53]de
   Vathaire et al., 2012). Also, Therapeutic radiation exposure of the
   abdomen may increase the risk of adipose tissue dysfucntion ([54]Huang
   et al., 2021). Importantly, as the fast food popular and HFD diet habit
   has been more and more public across the world, the obesity and
   diabetes incidences have been increasing largely during the past
   decades. Thus there may have larger amount of population encountered
   the chances of radiation including medical examination or radiotherapy.
   Furthermore, in some particular occupational environment including
   space flight, the astronauts may encounter various doses of radiation.
   Exposure of radiation from low dose to high dose may be in the
   astronauts from earth to the space, whereas exposure of radiation from
   high dose to low dose of radiation may be in the cancer patients with
   radiation therapy. Whether the effects of variation of radiation dosage
   on health remains unclear. In addition, the effects of an exposure to
   radiation in combination with lifestyle behavior, in particular,
   variation of radiation dosage in radiation exposure accompanied by most
   high-food diet (HFD) behavior, requires in-depth study. We therefore
   hypothesized that the effects of radiation on HFD-induced damage may be
   associated with the variation of radiation dosage.

   There is emerging evidence that the bile acid-gut microbiota axis
   contributes to liver disease susceptibility ([55]Shao et al., 2021),
   with the process being considered to be the language of an intricate
   molecular cross-talk between humans and their gut microbiota
   ([56]Guzior and Quinn, 2021). Liver disease pathogenesis and
   progression could be changed by gut microbiome modification, which
   influences the bile acid pool ([57]Hild et al., 2021). However, whether
   the bile acid-gut microbiota axis is involved in the synergistic
   effects of variation of radiation dosage and HFD patterns, and the
   potential molecular mechanisms, remains unknown.

   Here, we found that the variation of radiation dosage of radiation
   alters the HFD-induced NAFLD progression. In particular, it was found
   that in HFD fed mice, exposure to radiation that gradually changed from
   a high to low dose (H-L) significantly promoted the progression of
   NAFLD through the regulation of bile acid via the gut
   microbiota-lithocholic acid-insulin receptor (IA-INSR) axis. Our
   findings explained how the co-exposure of variation of radiation dosage
   in radiation exposure and HFD altered the progression of NAFLD in mice,
   which improve our understanding of regulatory interactions between
   these two entities.

2. Results

2.1. The synergistic effect of the HFD and radiation exposure presented
significantly different biological characteristics in mice

   The western diet, which is a typical HFD, has been shown to induce
   NAFLD ([58]Wan et al., 2021). In the real world, dietary behavior is
   often accompanied by exposure to multiple physical factors, in
   particular a varying range of natural radiation exposures. We designed
   an experiment to investigate the synergistic effects of a HFD and
   radiation exposure on liver function in mice. We treated mice with a
   radiation exposure that gradually changed from a low to high dose (L-H)
   or from a high to low dose (H-L) with or without a HFD (L-H + HFD,
   H-L + HFD) at abdominal region for 15 weeks ([59]Figs. S1A–C, [60]Figs.
   S2A–B). We found that, while there was no significant change in
   intestine tissues ([61]Fig. S1D), there were significant changes in
   body weight ([62]Fig. S2C) and glucose levels ([63]Figs. S2D–F). There
   were no significant differences in glucose levels between the L-H and
   H-L groups, but mice in the H-L + HFD group had a higher glucose level
   compared to the L-H + HFD group and this trend increased over time. In
   addition, there were significant changes in liver function parameters,
   including the levels of liver aspartate alanine transaminase(ALT)
   ([64]Fig. S3A), transaminase (AST)([65]Fig. S3B), liver total
   cholesterol(TC)([66]Fig. S3C), liver triglyceride (TG)([67]Fig. S3D),
   high density lipoprotein(HDL)([68]Fig. S3E), and low density
   lipoprotein (LDL)([69]Fig. S3F) among the four groups. An analysis of
   the liver tissue histology revealed that the lipid accumulation in the
   H-L + HFD group was greater than in the L-H + HFD, L-H, and H-L groups
   ([70]Fig. S3G). Collectively, the characteristics of glucose levels and
   liver function were as follows: (i) no significant difference was
   observed between the L-H and H-L groups; (ii) the L-H + HFD group had a
   higher liver dysfunction than the L-H group; (iii) the H-L + HFD group
   had a higher liver dysfunction than the L-H group; (iv) the L-H + HFD
   group had a higher liver dysfunction than the H-L group; (v) the
   H-L + HFD group had a higher liver dysfunction than the H-L group; and
   (vi) the H-L + HFD group had a higher liver dysfunction than the
   L-H + HFD group. These results indicate that: (i) a HFD in synergy with
   radiation exposure triggered strong liver dysfunction; and (ii) while
   there were no differences in mice in relation to the variation of
   radiation dosage of radiation, mice fed with a HFD were more sensitive
   to the variation of radiation dosage of radiation.

2.2. The HFD enabled the liver to respond more sensitively to the spatial
changes of radiation

   Considering the marked increase in liver dysfunction in the mice given
   a HFD diet accompanied by radiation exposure compared to those without
   the HFD, we determined the liver fibrosis-related inflammation factors
   and protein levels in mice. We found that mice in the H-L + HFD group
   exhibited a more severe activation and progressive inflammation
   response than the mice in the L-H + HFD, L-H, and H-L groups ([71]Fig.
   1A). The combination of the H-L and HFD treatments increased the
   expression of mice liver fibrosis-related biomarkers ([72]Fig. 1B). The
   expression of both liver inflammation factors and fibrosis-related
   proteins increased in a time-dependent manner in response to the H-L
   variation of radiation dosage. A transmission electron microscopy (TEM)
   analysis further revealed that in the H-L + HFD group, the H-L and HFD
   treatment impaired the mitochondrial structure more severely than in
   the other three groups ([73]Fig. 1C), with the ridge disappearing and
   appearing to have become fused and swollen. These results further
   indicated that the HFD made the liver more sensitive, with more
   inflammation and fibrosis in response to the H-L radiation exposure
   than in response to the L-H exposure. Figure D–F and [74]Figs. S4A–C
   showed the EMT-related proteins expression at 5, 10 and 15 weeks.

Fig. 1.

   [75]Fig. 1
   [76]Open in a new tab

   High-fat-diet synergized with repeated exposure to radiation variation
   from high dose to low dose triggers stronger liver fibrosis in mice. A.
   Liver cytokines levels, IFN-γ, TNF-a, IL-1β, IL-10 and NFkB in 4 groups
   at the 5th week. B. Liver cytokines levels, IFN-γ, TNF-a, IL-1β, IL-10
   and NFkB in 4 groups at the 10th week. C. Liver cytokines levels,
   IFN-γ, TNF-a, IL-1β, IL-10 and NFkB in 4 groups at the 15th week. D.
   Western blot analysis of effects of co-exposure HFD and radiation
   variations on liver fibrosis-related proteins E-cadherin, N-cadherin,
   Vimentin, TGF-β1 expression at the 5th week post various intervention
   in mice. GAPDH subjected to the control reference. E. Western blot
   analysis of effects of co-exposure HFD and radiation variations on
   liver fibrosis-related proteins E-cadherin, N-cadherin, Vimentin,
   TGF-β1 expression at the 10th week post various intervention in mice.
   GAPDH subjected to the control reference. F. Western blot analysis of
   effects of co-exposure HFD and radiation variations on liver
   fibrosis-related proteins E-cadherin, N-cadherin, Vimentin, TGF-β1
   expression at the 15th week post various intervention in mice. GAPDH
   subjected to the control reference. G. Representative electron
   microscopy images of liver tissues at indicated the 5th, 10th and 15th
   week among 4 groups, scale bar: 0.5 μM. Data are means ± standard
   deviation (SD). Mann-Whitney test or two-tailed unpaired Student's
   t-test were used for statistical analyses. *p < 0.05 indicates
   significant difference.

   Previous studies have reported that a HFD promotes multiple diseases by
   altering the gut microbiota, leading to changes in intestinal
   physiology ([77]Mayneris-Perxachs et al., 2021). We hypothesized that
   increasing gut barrier damage due to the HFD was associated with the
   progression of H-L + HFD-induced liver inflammation activation and
   fibrosis. The gut intestinal barrier is vital for maintaining gut
   integrity and permeability to avoid endotoxemia, such as that caused by
   lipopolysaccharide (LPS) entry into the plasma and the subsequent
   low-grade inflammation, which has been considered to play a causal role
   in the development of non-communicable diseases ([78]Huda et al., 2021;
   [79]Huang et al., 2020a).The results showed that, compared to the other
   three groups, the H-L + HFD group exhibited more severe damage to the
   gut barrier, with broken membranes ([80]Fig. 2A). The TEM analysis
   further showed that the mitochondrial structure was more severely
   altered ([81]Fig. 2B) with the disappearance of the ridge, which
   appeared to be fused and swollen; lower expression of tight junction
   proteins, including Occludin, ZO-1, Claudi-1, and E-cadherin, and
   higher expression of fibrosis-related proteins, including N-cadherin,
   occurred ([82]Fig. 2C–F). These results indicated the synergistic
   effect of the H-L radiation pattern and HFD, exacerbating gut barrier
   damage and inducing inflammation and fibrosis in the liver.

Fig. 2.

   [83]Fig. 2
   [84]Open in a new tab

   High-fat-diet synergized with repeated exposure to radiation variation
   from high dose to low dose triggers stronger intestinal barrier
   dysfunction and EMT progress. A. H&E staining of intestine of mice
   treated with radiation variants or combined with HFD at indicated the
   5th, 10th and 15th week among 4 groups, scale bar: 100 μM. B.
   Representative electron microscopy images of liver tissues at indicated
   the 5th, 10th and 15th week among 4 groups, scale bar: 0.5 μM. C.
   Immunofluorescence detection for Occludin in mice treated with
   radiation variants or combined with HFD at indicated the 5th, 10th and
   15th week among 4 groups, scale bar: 50 μM. D. Immunofluorescence
   detection for E-cadherin in mice treated with radiation variants or
   combined with HFD at indicated the 5th, 10th and 15th week among 4
   groups, scale bar: 50 μM. E. Intestine EMT-related E-cadherin,
   N-cadherin relative expression and intestinal barrier tight
   junction-related Occludin, ZO-1 and Claudin-1 relative expression by
   qRT-PCR in mice treated with radiation variants with or without HFD fed
   at the 5th week. F. Intestine EMT-related E-cadherin, N-cadherin
   relative expression and intestinal barrier tight junction-related
   Occludin, ZO-1 and Claudin-1 relative expression by qRT-PCR in mice
   treated with radiation variants with or without HFD fed at the 15th
   week. Data are means ± standard deviation (SD). Mann-Whitney test or
   two-tailed unpaired Student's t-test were used for statistical
   analyses*p < 0.05 indicates significant difference.

2.3. The H-L radiation pattern restructured the gut microbiota

   The gut microbiota composition was further determined from fecal
   samples of the mice at 15 weeks using bacterial 16S rRNA gene v3-v4
   amplicon sequencing as previously reported ([85]Liu et al., 2019). The
   significant alterations in gut microbiota abundance at the phylum and
   species levels among the four groups are listed in [86]Supplementary
   Tables 1 and 2, respectively. We found that mice in the H-L + HFD group
   had a significant difference in the beta diversity of gut microbiota
   abundance compared to the other three groups ([87]Fig. 3A and B).
   Specifically, the Bacteroidetes/Firmicutes ratio, the abundance of A.
   muciniphila, Butyricococcus, and Clostridiaceae decreased significantly
   in the H-L + HFD group, while the abundance of Fusobacteria and
   Actinobacteria increased in the H-L + HFD group compared to the other
   three groups ([88]Fig. 3D–I). A clusters of orthologous groups (COG)
   analysis of proteins predicted that the changed gut microbiota was
   mainly concentrated around lipid metabolism, signal transduction, and
   amino acid transport and metabolism ([89]Fig. 3J). A Kyoto Encyclopedia
   of Genes and Genomes (KEGG) analysis showed that the altered gut
   microbiota groups were associated with infectious diseases, lipid
   metabolism, and amino acid metabolism ([90]Fig. 3K).

Fig. 3.

   [91]Fig. 3
   [92]Open in a new tab

   High-fat-diet synergized with repeated exposure to radiation variation
   from high dose to low dose induces significant impact on gut microbiome
   in mice. A. Analysis of Beta diversity among 4 groups used to compare
   the difference in gut microbiota composition among 4 groups by QIME
   software through unweighted unifrac method. B. Heatmap of gut
   microbiota abundance among 4 groups. Color represents abundance. Color
   from red to blue means relative abundance increased from high to low.
   C. Gut microbiota distribution columnar diagram among 4 groups. Various
   color represents various species. D. Bacteroidetes/Firmicutes ratio
   among 4 groups. E. Relative abundance of Fusobacteria among 4 groups.
   F. Relative abundance of Actlnobacterla among 4 groups. G. Relative
   abundance of Akkermansia mucinipila among 4 groups. H. Relative
   abundance of Butyrlcococcus among 4 groups. I. Relative abundance of
   Clostridiaceae among 4 groups.J. Picrust2 software is used to annotate
   the characteristic sequence to be predicted with the existing
   phylogenetic tree in the software, and img microbial genome data is
   used to output functional information, so as to speculate the
   functional gene composition in the sample, so as to analyse the
   functional differences between different groups ([93]Luo et al., 2021).
   COG(Clusters of Orthologous Groups of proteins) analysis to predict the
   difference changed gut microbiota function. K. Potential KEGG (Kyoto
   Encyclopedia of Genes and Genomes) pathway analysis was used to predict
   changes in metabolic pathways of functional genes of microbial
   communities among samples in different groups. Data are
   means ± standard deviation (SD), n = 5 per group. Mann-Whitney test or
   two-tailed unpaired Student's t-test were used for statistical
   analyses. *p < 0.05 indicates significant difference.

   To clarify further the role of gut microbiota in the regulation of the
   liver response to H-L radiation and HFD, we conducted a fecal
   transplantation trial (FMT) ([94]Fig. S5A). As shown in [95]Fig. S5B,
   mice receiving a fecal transplant from the H-L + HFD-treated group had
   higher glucose levels than the mice receiving a fecal transplant from
   the L-H + HFD and control groups. Mice receiving a fecal transplant
   from the H-L + HFD-treated group had more severe liver dysfunction,
   with significantly increased serum levels of ALT, AST, TC, TG, and LDL,
   but decreased levels of HDL ([96]Figs. S5C–H). Hematoxylin and eosin
   (H&E) staining of the liver showed that mice receiving a fecal
   transplant from the H-L + HFD-treated group had a higher lipid
   accumulation in their liver and more severe gut barrier damage than
   mice receiving a fecal transplant from the L-H + HFD and control groups
   ([97]Fig. S5I). We also found that the amount of liver inflammation
   cytokines increased, while the gut barrier tight junction-related
   proteins decreased in mice receiving a fecal transplant from
   H-L + HFD-treated mice compared to the other two groups ([98]Figs.
   S5J–K).

   To understand more fully the effects of gut microbiota-mediation on the
   gut barrier and liver function, we used an antibiotic cocktail to
   eliminate the gut microbiota of the mice, while probiotics were used as
   a beneficial positive control ([99]Fig. S6A). We found that the plasma
   LPS level, and the serum hepatic-related TG and TC levels significantly
   decreased after the antibiotic and probiotic treatments ([100]Figs.
   S6B–C). Furthermore, the relative expression of liver fibrosis-related
   genes and liver inflammation-related cytokines significantly decreased
   after the antibiotic and probiotic treatments ([101]Figs. S6D–E), which
   indicated the critical role of gut microbiota. We conducted H&E
   staining, which revealed that the liver lipid accumulation was
   ameliorated. The degree of gut barrier damage was alleviated in the
   H-L + HFD group mice after the antibiotic and probiotic treatments
   ([102]Fig. S6F), which indicated that the gut microbiota could mediate
   the progress of H-L + HFD-induced gut barrier damage and liver
   fibrosis. To explore whether the mitochondrial structure could be
   restored, we performed a TEM analysis. We found that mitochondrial
   numbers increased, with the fusion and swelling ameliorated after the
   antibiotic and probiotic treatments ([103]Fig. S6G). These results
   demonstrated that the gut barrier damage and liver fibrosis induced by
   the synergistic effect of HFD and the H-L variation of radiation dosage
   of radiation were mainly mediated by modification of gut microbiota and
   gut metabolites. These effects could be ameliorated by antibiotic or
   probiotic treatments, and the plasma LPS level increased.

2.4. The insulin IRS-1/Akt/mTOR pathway was disrupted by the co-exposure to
glucose and radiation

   Because mitochondrial function is a hallmark of the insulin signaling
   pathway, we speculated that H-L + HFD-induced liver lipid accumulation
   may be caused by disruption to the insulin pathway. We then detected
   insulin resistance signaling using HepG2 cells by focusing on the
   IRS1/Akt/mTOR signaling pathway. In agreement with our hypothesis, we
   found that: (i) radiation exposure increased PI3K, NFkB, mTOR
   expression, but had no influence on p53 and p-p53 ([104]Fig. 4A and B)
   while the effects of radiation on PI3K, NFkB, mTOR expression were
   dose-dependent; (ii) compared to the cells that received radiation,
   those that received glucose + radiation exhibited a higher expression
   of PI3K, NFkB, Akt, mTOR, and GSK3-β, but there were no alterations in
   the p53 expression ([105]Fig. 4C and D), while the effects of
   glucose + radiation on NFkB, Akt, mTOR, and GSK3-β expression were
   dose-dependent([106]Fig. 4E and F); (iv) compared to the cells that
   received radiation, those that received glucose + radiation exhibited a
   higher expression of p-IRS-1([107]Fig. 4G and H). Further study showed
   that NFkB and PI3K expression increased with the accumulation of
   various doses of radiation and glucose ([108]Fig. 4I).

Fig. 4.

   [109]Fig. 4
   [110]Open in a new tab

   High glucose synergized with radiation treatment triggers insulin
   resistance in HepG2 cells. A. Representative of Western blot analysis
   of PI3K, NFkB, mTOR, p53, p-Akt, p-mTOR and p-p53 in liver cells
   treated with or without radiation. B. Quantitative of liver cell
   insulin resistance-related genes PI3K, NFkB, mTOR, p53, p-Akt, p-mTOR
   and p-p53 expression. C. Representative of Western blot analysis of
   PI3K, NFkB, mTOR, p53, p-Akt, p-mTOR and p-p53 in liver cells treated
   with or without high glucose combined with 6Gy radiation. D.
   Quantitative of liver cell insulin resistance-related genesPI3K, NFkB,
   mTOR, p53, p-Akt, p-mTOR and p-p53 expression with or without high
   glucose combined with 6Gy radiation. E. Representative of Western blot
   analysis of GSK3-β, NFkB, mTOR, p-Akt, mTOR, p-mTOR and p-p53 in liver
   cells treated with or without high glucose combined with 8Gy radiation.
   F. Quantitative of liver cell insulin resistance-related genes GSK3-β,
   NFkB, mTOR, p-Akt, mTOR, p-mTOR and p-p53 expression with or without
   high glucose combined with 8Gy radiation. G. Representative of Western
   blot analysis of p-IRS-1 in liver cells treated with or without high
   glucose combined with 8Gy radiation. H. Quantitative of liver cell
   insulin resistance-related genesp-IRS-1 expression with or without high
   glucose combined with 8Gy radiation. I. NFkB and PI3K mRNA expression
   were detected by qRT-PCR at indicated accumulation radiation exposure
   of various doses. Cells in L-H group were exposed 0.5Gy first and then
   exposed 3Gy radiation at 24 h, 5Gy radiation at 48h. Cells in H-L group
   were exposed 5Gy first and then exposed 3Gy radiation at 24 h, 0.5Gy
   radiation at 48h. Cells in L-H + Glu group were treated with glucose
   and exposed 0.5Gy first and then exposed 3Gy radiation at 24 h, 5Gy
   radiation at 48h. Cells in H-L + Glu group were treated with glucose
   and exposed 5Gy first and then exposed 3Gy radiation at 24 h, 0.5Gy
   radiation at 48h.

   To understand more fully the effects of glucose + radiation on the
   insulin signaling pathway, we used an antibiotic cocktail to eliminate
   the gut microbiota of the mice, with a probiotic treatment used as a
   beneficial positive control. We found that gut TLR4 and MYD88
   expression decreased after the antibiotic or probiotic treatments in
   the H-L + HFD group ([111]Fig. S7A). Immunohistochemistry (IHC)
   detection showed that E-cadherin and ZO-1 expression increased after
   the antibiotic or probiotic treatments in the H-L + HFD group
   ([112]Fig. S7B). Furthermore, INSR, PI3K, Akt, and mTOR expression
   decreased after the antibiotic or probiotic treatments in the H-L + HFD
   group ([113]Fig. S7C). We further found that in the HepG2 cells the
   protein expression of PI3K, Akt, and mTOR increased post radiation
   exposure, but decreased at a high radiation rose (8 Gy) compared with
   the increased expression at 4Gy. Consistent with the results of
   previous animal studies, the investigation of the cell insulin
   signaling pathway suggested that the H-L + HFD-induced liver fibrosis
   was mediated by gut microbiota through disruption of the gut barrier
   TLR4/MYD88 and liver IRS1/Akt/mTOR signaling pathways.

2.5. The H-L + HFD treatment promoted intestinal inflammation and liver
dysfunction through a reduction in the levels of isocaproic acid (IA)

   To determine the mechanism underlying the alteration of the gut
   microbiota composition and its association with increased gut barrier
   damage and liver fibrosis risk, we compared the gut microbiome between
   the HFD vs. control and H-L + HFD vs. L-H + HFD using high-performance
   liquid chromatography (HPLC). [114]Fig. 5A and B shows the INSR and Akt
   expression were increased to a greater extent in the H-L + HFD group
   than in the L-H + HFD group. Compared with the HFD group, the levels of
   L-homoserine, 4-demethylpremithramycinone, and epiteaflavic acid
   3′-gallate increased significantly in the HFD group ([115]Fig. 5C and
   D). These changed metabolites were mainly concentrated in the
   beta-alanine metabolism, phenylalanine, tyrosine, and tryptophan
   biosynthesis pathways ([116]Fig. 5E). Compared with the L-H + HFD
   group, the IA level decreased significantly, but the levels of
   3-methyladipic acid, tetradecanedioic acid, and L-homoserine increased
   in the H-L + HFD group ([117]Fig. 5F and G). Consistent with previous
   animal and cell studies, these changed metabolites were mainly
   concentrated in the glucose metabolism-related pathways, e.g., glycine,
   serine, and threonine metabolism ([118]Fig. 5H). [119]Fig. 5I showed
   the alteration of various bacteria species among four groups. [120]Fig.
   5J showed the Isocaproic acid (IA) molecular structure.

Fig. 5.

   [121]Fig. 5
   [122]Open in a new tab

   Profiling of plasma metabolomics under HFD synergizes L-H or H-L
   radiation setting. Mice were divided into four groups, con, HFD,
   L-H + HFD, H-L + HFD. Liver tissues were collected at 15th week post
   HFD synergizes L-H or H-L radiation setting. A. H&E staining of liver
   tissues in 4 groups, scale bar, 50 μM. Immunohistochemistry detection
   for INSR and Akt in liver tissues. Scale bar: 50 μM. B. Quantitative of
   INSR and Akt expression in liver tissues in 4 groups. C. Top 10
   upregulated plasma metabolites in mice with HFD fed compared with
   normal control. D. Cluster Heatmap with metabolite properties between
   HFD and control groups, respectively in mice plasma metabolomics. E.
   Potential metabolism pathway analysis was conducted between HFD and
   control groups, respectively in mice plasma metabolomics. F. Top 10
   upregulated plasma metabolites in mice with H-L synergized with HFD
   setting compared with L-H synergized with HFD setting. G. Cluster
   Heatmap with metabolite properties between H-L + HFD and L-H-HFD
   groups, respectively in mice plasma metabolomics. H. Potential
   metabolism pathway analysis was conducted between H-L + HFD and L-H-HFD
   groups, respectively in mice plasma metabolomics. I. Gut microbiota
   abundance among 4 groups. J. Isocaproic acid (IA) molecular structure
   and the OD value. Data are means ± standard deviation (SD).
   Mann-Whitney test or two-tailed unpaired Student's t-test were used for
   statistical analyses*p < 0.05 indicates significant difference.

   To explore the role of IA in H-L + HFD-induced liver function damage
   and intestinal inflammation, we compared the differences between the
   H-L + HFD and H-L + HFD + IA groups ([123]Fig. 6A). Compared with the
   H-L + HFD group, the levels of AST, ALT, TC, TG, and LDL decreased
   after IA treatment, but the HDL level increased ([124]Fig. 6B–G). We
   found that IA levels in the cecum extract and plasma decreased in the
   H-L + HFD group compared with the H-L or HFD groups ([125]Fig. 6H and
   I). We found that gut TLR4, MYD88, and NFkB expression decreased after
   IA treatment in the H-L + HFD group following an IHC determination
   ([126]Fig. 6J). We further found that Fusobacterium nucleatum had a
   slower growth rate and lower maximum growth concentration in the cecal
   extract from mice after the IA treatment in the H-L + HFD group than in
   mice without the IA treatment ([127]Fig. 6K), indicating that the
   increase in F. nucleatum abundance in H-L + HFD mice may have been due
   to the alteration of complex gut microbiota-produced metabolites. We
   then investigated the alteration of bile acid in H-L + HFD mice with or
   without an IA treatment. We found that two components of bile acid,
   ileum taurocholic acid (TCA) and lithocholic acid (LCA), decreased
   after the IA treatment (Figure 6L). Plasma LCA also decreased, but the
   levels of CA, DCA, and TUDCA were not influenced by IA (Figure 6M).
   These results were consistent with a previous report that the levels of
   LCA increased in patients with inflammatory bowel disease associated
   with an increased abundance of gut F. nucleatum ([128]Weng et al.,
   2019), indicating that IA influenced H-L + HFD-induced liver function
   damage.

Fig. 6.

   [129]Fig. 6
   [130]Open in a new tab

   IA attenuated H-L + HFD-induced liver dysfunction in mice. Mice were
   divided into 2 groups, H-L + HFD, H-L + HFD + IA. Liver tissues were
   collected at 5th week post HFD synergizes H-L radiation or IA setting.
   A. Timeline of mice subjected to H-L + HFD, H-L + HFD + IA. B. AST
   level. C. ALT level. D. TC level. E. TG level. F. HDL level. G. LDL
   level. H. IA level in cecum content. I. IA level in plasma. J.
   Immunohistochemistry detection for TLR4, MYD88 and NFkB in intestinal
   tissues. Scale bar: 50 μm. K. Growth curve of F. nucleatum in cecal
   extracts (0.2 mg/ml) from 2 groups (3 technical replicate of 6
   biological replicates for each group). L. ileum bile acid, CA, DCA,
   TCA, TUDCA and LCA levels in 2 groups. M. Plasma bile acid, CA, DCA,
   TCA, TUDCA and LCA levels in 2 groups. Data are means ± standard
   deviation (SD). Mann-Whitney test or two-tailed unpaired Student's
   t-test were used for statistical analyses*p < 0.05 indicates
   significant difference.

2.6. Lithocholic acid promoted H-L + HFD-induced liver dysfunction through
the inhibition of INSR signaling

   Previous reports have indicated that LCA is a secondary bile acid and
   an increase in its concentration is associated with accelerated
   insulitis, resulting in an increased risk of diabetes ([131]McGlinchey
   et al., 2020). We therefore investigated the potential mechanism
   underlying LCA-mediated liver dysfunction ([132]Fig. 7A). Because
   insulin signaling dysregulation is a hallmark of HFD-induced liver
   lipid accumulation through the critical receptor INSR ([133]Dongiovanni
   et al., 2017), we used INSR inhibitor linsitinib ([134]Meijles et al.,
   2021) to inhibit INSR signaling. We found that serum levels of AST,
   ALT, TC, TG, and LDL increased, but the HDL level decreased after an
   LCA treatment in the H-L + HFD + INSR inhibitor group ([135]Fig. 7B–G).
   Liver PI3K and Akt expression decreased, but NFkB expression increased
   after an LCA treatment in the H-L + HFD + INSR inhibitor group
   ([136]Fig. 7H), indicating that LCA decreased the expression of INSR
   signaling downstream genes, resulting in the inhibition of INSR
   signaling and promoting the progression of liver dysfunction. This
   result was consistent with a previous report that inhibition of the
   INSR/PI3K/Akt signaling pathway leads to glucose/insulin-stimulated
   hepatic stellate cell activation and extracellular matrix (ECM)
   production ([137]Zhang et al., 2014). We further used Western blotting
   to test the expression of liver fibrosis-related biomarkers, and found
   that the inhibition of INSR expression decreased E-cadherin expression,
   but increased N-cadherin and a-SMA expression ([138]Fig. 7I and J). The
   relative expression of liver inflammation factors increased after LCA
   treatment in the H-L + HFD + INSR inhibitor group ([139]Fig. 7K). The
   relative expression of E-cadherin decreased after radiation but
   increased after IA treatment, whereas N-cadherin and a-SMA increased
   after radiation but decreased after IA treatment (Figure 7L). LCA
   treatment had the opposite effects (Figure 7M). These observations
   indicated that IA may against the radiation-induced fibrosis but LCA
   may promote the radiation damage. Meanwhile, LCA may exert an
   acceleration effect on H-L + HFD-induced liver fibrosis by targeting
   liver INSR signaling.

Fig. 7.

   [140]Fig. 7
   [141]Open in a new tab

   LCA inhibited H-L-HFD-induced liver fibrosis through regulating insulin
   resistance signaling pathway. Mice were divided into 4 groups,
   H-L + HFD, H-L + HFD + INSR inhibitor, H-L + HFD + INSR inhibitor + LCA
   and H-L + HFD + INSR inhibitor + IA. Liver tissues were collected at
   5th week post HFD synergizes H-L radiation or LCA or IA setting. A.
   Timeline of mice subjected to H-L + HFD, H-L + HFD + INSR inhibitor,
   H-L + HFD + INSR inhibitor + LCA and H-L + HFD + INSR inhibitor + IA.
   B. AST level. C. ALT level. D. TC level. E. TG level. F. LDL level. G.
   HDL level. H. Immunohistochemistry detection for PI3K, Akt and NFkB in
   liver tissues. Scale bar:50 μm. I. Western blot analysis (and J.
   quantification) of effect of LCA (50 mg/kg) on liver fibrosis-related
   proteins, E-cadherin, N-cadherin a-SMA expression in mice. K.
   Quantification of liver inflammation-related mRNA expression. L.
   E-cadherin, N-cadherin and a-SMA mRNA expression in mice liver tissues
   with or without radiation and IA treatment were detected by qRT-PCR. M.
   E-cadherin, N-cadherin and a-SMA mRNA expression in mice lung tissues
   with or without radiation and IA treatment were detected by qRT-PCR.
   Data are means ± standard deviation (SD). Mann-Whitney test or
   two-tailed unpaired Student's t-test were used for statistical
   analyses*p < 0.05 indicates significant difference.

3. Discussion

   Radiation exposure has a number of complex outcomes, including
   beneficial and non-beneficial roles in health, but the mechanisms
   involved remain unclear. A number of studies have shown that a HFD is
   an NAFLD risk due to the alteration of the gut microbiota composition
   ([142]Mu et al., 2021). Recent studies have shown that cancer patients
   with obesity have a higher radiotherapy resistance in clinical settings
   ([143]Sabol et al., 2020), indicating that a HFD-related life style is
   not only a risk factor associated with many diseases but may also play
   a role in the development of diseases combined with other environmental
   factors. Radiation exposure, a common environmental physical factor
   known for its double-edged ability to provide beneficial or
   non-beneficial outcomes, is used in medical diagnosis or therapy and
   can improve the liver immune function at low radiation doses
   ([144]Zhang et al., 2021a). However, there is also a risk of oxidative
   liver damage after frequent high doses of radiotherapy ([145]Sharma et
   al., 2021). The molecular pathogenic mechanisms of radiation-induced
   liver injury remain unclear. Previous reports have indicated it
   associates with vascular changes, increased collagen synthesis,
   sequential activation of key growth factors and cytokines including
   tumor necrosis factor alpha (TNF-α), transforming growth factor beta
   (TGF-β), as well as early effects of DNA damage, oxidative stress and
   reactive oxygen species production which resulting in the
   hepatocellular apoptosis and inflammatory reaction. In addition, the
   activation of compensatory proliferation of myofibroblastic is a
   critical cellular event for underlying hepatic fibrosis ([146]Kim and
   Jung, 2017).

   In this study, through a comparison of treatments with different
   variation of radiation dosage exposures, and combined with a HFD diet,
   we identified the specific mechanism by which a varying pattern of
   radiation exposure and a HFD alters the intestinal microbiota, damages
   the intestinal barrier, and increases LCA levels, thus promoting the
   development and progression of HFD-induced NAFLD via the activation of
   liver INSR/PI3K/Akt signaling. Depleting the gut microbiota by
   antibiotic treatment and the inhibition of INSR signaling eventually
   ameliorated HFD-induced liver damage via the LCA-INSR/PI3K/Akt axis.

   Fusobacterium nucleatum, which is generally found in human dental
   plaque and is one of the most abundant oral commensal flora, is
   associated with various human diseases, including lung disease and
   periodontal infection after entering the blood circulatory system
   ([147]Stokowa-Soltys et al., 2021). Studies have shown that this
   bacteria can migrate from its original oral site to other parts of body
   and cause diseases such as cancer. However, F. nucleatum also has an
   anti-inflammatory role through the production of butyric acid
   ([148]Stokowa-Soltys et al., 2021). Asal Milhen et al. found that mice
   fed a HFD diet had an increased F. nucleatum abundance, with increased
   body weight and glucose tolerance ([149]Milhem et al., 2021). Another
   study found a causal link between the activation of the LPS pathway by
   F. nucleatum and HFD-induced inflammation ([150]Blasco-Baque et al.,
   2012). Consistent with these findings, we found that an increase in F.
   nucleatum abundance led to an increase in the LCA level and further
   promoted NAFLD through the activation of liver INSR signaling.

   We identified IA as the specific metabolite that inhibited the growth
   of F. nucleatum, which explained the inhibition of F. nucleatum in
   antibiotic or probiotic-treated mice. There is emerging evidence that
   IA, which is one of the short-chain fatty acids and the product of the
   anaerobic intestinal bacterial fermentation of dietary fiber
   ([151]Zietek et al., 2021), is positively associated with the body mass
   index(BMI) ([152]Irving et al., 2016) and hepatic lipid metabolism
   ([153]Zietek et al., 2021). Obesity in pregnancy can reduce the amount
   of IA in the stool and a decrease in the IA level would also reduce
   liver function ([154]Honkoop et al., 1997). Consistent with previous
   reports, our results further indicated that the reduction of IA levels
   can be caused by the co-exposure of long term radiation exposure and a
   HFD.

   Bile acids, a class of amphipathic steroids produced in the liver and
   extensively modified by the gut microbiota, are critical actors in the
   onset and progression of NAFLD ([155]Li et al., 2021). Bile acids can
   act as signaling molecules, regulating liver cellular homeostasis
   through interaction with the Farnesoid X receptor(FXR), resulting in
   liver lipid metabolism ([156]Radun and Trauner, 2021). The reported
   effects of bile acids on NAFLD make it an attractive therapeutic target
   in HFD-induced liver diseases. Masahiro M et al. developed an ileal
   bile acid transporter inhibitor and found that it improved HFD-induced
   hepatic steatosis by ameliorating gut microbiota dysbiosis in NAFLD
   model mice ([157]Matsui et al., 2021). The liver is the central organ
   in lipid metabolism and studies have found that bile acids can activate
   a series of signaling pathways, such as the protein kinase B (AKT)
   signaling pathway that regulates lipid metabolism ([158]Kwong et al.,
   2015). Lithocholic acid is a secondary bile acid, with high
   hydrophobicity and cytotoxicity, that is transformed by gut bacteria
   from a primary bile acid, causing more cell wall damage than the
   primary bile acids ([159]Chiang, 2017). Lithocholic acid has been found
   to promote carcinogenesis ([160]Ma et al., 2018) in the liver in animal
   models. A population-based epidemiological study showed that high LCA
   levels were strongly associated with advanced liver fibrosis ([161]Kwan
   et al., 2020). Liver injuries induced by LCA treatment include the
   impacts on signaling pathways associated with inflammation and the
   proliferation of G protein-coupled receptor (TGR5)-dependent signaling
   ([162]Klindt et al., 2019). Genetic polymorphisms related to insulin
   resistance will increase NAFLD susceptibility ([163]Nobakht et al.,
   2020). Consistent with previous reports, we found that LCA promoted
   H-L + HFD-induced liver dysfunction through the activation of the INSR
   signaling pathway. These findings indicate that LCA might be the direct
   cause of HFD-induced NAFLD in the context of a radiation exposure
   varying from a high to low dose, with radiation likely to be a
   concomitant factor.

   INSR/IRS/PI3K/Akt is classical insulin pathway in the process of
   glucose metabolism. Many studies have found that inhibition of this
   pathway due to environmental toxicants, e.g., bisphenol A, can activate
   an inflammatory response and lead to NAFLD ([164]Lin et al., 2019).
   Yang P et al. found that the use of liraglutide could ameliorate NAFLD
   in diabetic mice through the activation of the IRS/PI3K/Akt signaling
   pathway ([165]Yang et al., 2019). Zhang F et al. found the use of
   tetramethylpyrazine could reduce glucose and the insulin-induced
   activation of hepatic stellate cells through inhibition of the
   INSR-mediated PI3K/Akt pathway ([166]Zhang et al., 2014). Consistent
   with these reports, our results showed that an increased LCA level in
   plasma could inhibit the INSR/PI3K/Akt pathway, leading to the
   development and progression of combined HFD and radiation-induced
   NAFLD. This further confirmed that the insulin pathway in NAFLD induced
   by environmental radiation exposure and a HFD can be disrupted.
   Considering that the INSR/PI3K/Akt pathway is a well-clarified pathway
   in insulin resistance, we suggest that future studies should
   investigate the underlying mechanism of how the INSR/PI3K/Akt pathway
   plays a role in which NAFLD is induced by a combination of
   environmental physical factors and lifestyle factors.

   Limitations about this study should be mentioned. First, the radiation
   exposure levels in this study are remaining high, caution is required
   while interpreting these findings. Second, collagen levels in the liver
   and liver sections stained with Sirius red to reflect the degree of
   fibrosis and the effect of no experimental radiation exposure should be
   considered in the further study. Third, population study including
   cohort study or case-control study should be considered in the future
   to evaluation the effects of various radiation doses on human health.

4. Conclusions

   In summary, we determined that variation of radiation dosage of
   radiation exposure altered the HFD-fed gut microbiota composition in
   mice through the regulation of gut barrier tight junction-related
   protein expression, leading to lower IA levels and increased LCA
   levels, and eventually the inhibition of the liver insulin
   INSR/PI3K/Akt pathway, thus promoting NAFLD development and
   progression. These findings have improved our understanding of the
   molecular mechanisms by which the gut microbiome can be regulated by a
   combination of environmental and lifestyle factors, and provides a
   novel strategy for the prevention or treatment of HFD-induced NAFLD.
   However, coupled with the over-physiologic doses of radiation used in
   this study, caution is required while interpreting these findings to
   human accidental/occupational/environmental radiation exposure.

5. Methods and materials

5.1. Mice, radiation, chemicals and cell lines

   Male C57BL/6 mice were purchased at 8 weeks of age from the Hunan SJA
   Laboratory Animal Co., LTD, China. All of the mice were housed at the
   Animal Laboratory Division, Xiangya School of Public Health, Central
   South University, China. All animal procedures and testing were
   conducted according to the National Legislation and local guidelines of
   the Laboratory Animals Center at the Central South University. The
   study and research protocols were approved by the Institutional Animal
   Review Board of Central South University (2020sydw0110). In addition,
   all animals in the study were treated humanely with regard to the
   alleviation of suffering. All animal procedure experiments were
   approved by the Committee on Animal Use and Care of Central South
   University, China. All the mice were maintained in a
   specific-pathogen-free (SPF) environment with controlled conditions of
   a 12 h light/dark cycle at 20–22 °C and 45 ± 5% humidity. After 1 week
   of acclimation, the mice were used for the study with the group design
   detailed below.

   To investigate the effects of spatial-temporal of radiation and
   synergization with HFD on liver metabolic impairments, mice were
   divided into four groups: L-H group, H-L group, L-H + HFD group and
   H-L + HFD group. The mice in the L-H group and H-L were fed ad libitum
   with a conventional diet. The mice in the L-H + HFD group and the
   H-L + HFD were exposed to different spectrum of radiation accompanied
   by HFD fed. The ingredients of high fat diet contained 24.15% fat,
   22.51% protein and 38.34% carbohydrate purchased from XIETONG Bio.
   Company, Nanjing, China. For L-H treatment, the dosage spectrum was
   0.1Gy of radiation administered to mice 5 times at the first 5 weeks,
   then 0.5Gy of radiation administered to mice 3 times within the 5th
   week and 7th week, 3Gy was administered at abdomen part at the 7th
   week, 5Gy was administered at abdomen part at the 10th week, 10Gy at
   abdomen part at the 15th week at room temperature, whereas for H-L
   treatment, the dosage spectrum was 10Gy of radiation administered to
   mice 1 time at the first week, then 5Gy of radiation administered to
   mice at abdomen part 1 time at the 5th week and 3 Gy of radiation
   administered to mice at abdomen part 1 time at the 7th week, 0.5Gy was
   administered at the 10th week 3 times, 0.1Gy was administered at the
   15th week 5 times at room temperature. Cells and animals were subjected
   to irradiation treatment for 24 h with a dose range 0–10 Gy delivered
   with the Varian linear accelerator (2100EX, San Francisco, California,
   USA). Body weight, food intake and water consumption were recorded. The
   mice were killed at the 5th, 10th and 15th weeks to collect serum and
   tissues for further detection of various parameters. The radiation
   source was provided by the Department of Oncology, Xiangya Hospital,
   Central South University (CSU), Changsha, 410008, People's Republic of
   China ([167]Zhang et al., 2021b).

   Lithocholic acid (LCA) was purchased from Sigma (CAS No. 434-13-9).
   Linsitinib (HY-10191) were ordered from MedChem Express, MCE and
   dissolved in 25 mM tartaric acid). Isocaproic acid (IA) was purchased
   from Sigma (CAS No. 646-07-1). D-glucose was purchased from Solarbio
   (G8150). The antibodies used for Western blot analysis targeted the
   following proteins: GAPDH (CST, 5174), E-cadherin (CST, 14472), PI3K
   (Abcam, ab73262), Akt (Proteintech, 10176-2-AP), p-Akt (Abcam,
   ab81283), IRS-1 (CST, 2382), p-IRS-1 (CST, 2381), mTOR (Bioworld,
   bs1844), p-mTOR (CST, 5536S), PYCR1 (Abcam, ab279385), p53 (CST,
   2527S), p-PI3K (CST, 4228T), Occludin (Abcam, ab216327), ZO-1 (Abcam,
   ab221547), Claudin-1 (Abcam, ab211737), N-cadherin (CST, 31593), INSR
   (CST, 23413).

   The HepG2 cells were purchased from the Cell Bank of Type Culture
   Collection of the Chinese Academy of Sciences (Shanghai, China). Due to
   the previous reports that HepG2 was used in many metabolic studies
   (35893906, 35931467), we thus selected HepG2 in our study. The HepG2
   cells were cultured in Dulbecco's modified Eagle's medium (DMEM;
   HyClone, Utah, USA) with 10% foetal bovine serum (FBS) and 1%
   antibiotic in a 5% CO2 cell incubator at 37 °C as described in our
   previous reports ([168]Dai et al., 2020).

5.2. Antibiotic/probiotic treatment and fecal microbiota transplantation
(FMT)

   An antibiotic cocktail including ampicillin sodium salt (CA2031-5G),
   polymyxin B sulfate salt (CP8711-100 MG) and streptomycin sulfate (CS
   10481-5G) was purchased from Coolaber Science & Technology. The
   probiotics included Lactobacillus rhamnosus (No. CICC, 20259) and
   Lactobacillus reuteri (No. CICC 6118), which were purchased from the
   China Center of Industrial Culture Collection
   ([169]http://www.china-cicc.org/). Insulin was purchased from Coolaber
   Science & Technology (CI6561), China.

   For investigation of the gut microbiota effects, mice were divided into
   four groups: L-H group, H-L group, L-H + HFD group and H-L + HFD group
   (in which mice were co-exposed to L-H or H-L radiation with HFD fed for
   15 weeks). The mice fecal samples were collected at the 15th week and
   shipped to Biotree, Co., LTD, Shanghai, China for gut microbiome
   detection using 16rS technical. For investigation of the gut
   microbiota-dependent effects, mice were divided into four groups: Con,
   H-L + HFD, H-L + HFD + Antibiotics and H-L + HFD + Probiotics. In the
   H-L + HFD + antibiotics group (in which H-L + HFD + HFD mice were
   treated with an antibiotic cocktail that included 50 g/L ampicillin
   sodium, 100 U/L streptomycin, and 50 mg/L polymyxin B sulfate, as
   previously described ([170]Rozos et al., 2018)) and the
   H-L + HFD + HFD + probiotics group (in which mice were treated with
   probiotics with dosages chosen based on our previous report ([171]Ju et
   al., 2020)). Briefly, probiotics, including Lactobacillus rhamnosus
   (No. CICC, 20259) and Lactobacillus reuteri (No. CICC 6118), were
   purchased from the China Center of Industrial Culture Collection
   ([172]http://www.china-cicc.org/). The probiotics were administered to
   the mice orally after being dissolved in PBS solution containing
   50 × 10^9 CFU of probiotics. The mice were administered antibiotics and
   probiotics prior to H-L + HFD co-exposure for 2 weeks, and then the
   experiment was performed for 10 weeks. The mice were killed at 10 weeks
   to collect serum and tissues for further detection of various
   parameters.

   For the FMT study, the microbiota donors were mice treated with
   L-H + HFD or H-L + HFD for 10 weeks. Faeces of the donors were
   collected and dispersed in sterile Ringer working buffer, and the
   supernatant was mixed with skimmed milk for transplantation.
   Four-week-old germ-free male C57BL/6J mice were randomly divided into
   three groups(with 10 mice in each group), housed in sterile plastic
   package isolators and fed a sterilized normal diet. After 2 weeks of
   acclimation, the germ-free mice were orally gavaged with faecal
   suspensions from mice fed a HFD or subjected to L-H + HFD or H-L + HFD
   feeding. The mice were killed to collect tissues for further detection
   of various parameters at the end of the experiment. After all these
   experiments, the mice were fasted overnight before being euthanized.
   Blood samples were collected and kept at room temperature for 1 h to
   ensure complete clotting before centrifugation at 4 °C and 5000 rpm for
   5 min to obtain serum samples. Faeces and tissues such as liver tissue,
   BAT, hippocampal tissue, and intestinal tissue were carefully
   collected, flash-frozen in liquid nitrogen and then stored at −80 °C
   until assessment.

   To investigate the effects of IA, mice were divided into two groups:
   the H-L + HFD group, the H-L + HFD + IA group (in which mice were
   co-exposed to H-L radiation, HFD fed and fed IA). The mice were treated
   for 10 weeks and then killed to collected tissues for detection of
   various parameters.

   To investigate the effects of LCA, mice were divided into four groups:
   the H-L + HFD group, the H-L + HFD + Linsitinib group (in which mice
   were co-exposed to H-L radiation, HFD fed and Linsitinib), the
   H-L + HFD + Linsitinib + LCA group (in which mice were co-exposed to
   H-L radiation, HFD fed and Linsitinib and LCA), the
   H-L + HFD + Linsitinib + IA group (in which mice were co-exposed to H-L
   radiation, HFD fed and Linsitinib and IA),. The mice were treated for
   10 weeks and then killed to collected tissues for detection of various
   parameters.

5.3. Analysis of liver function parameters, bile acid measurement and growth
curves of F. nucleatum

   The insulin concentration was analysed with an enzyme-linked
   immunosorbent assay (ELISA) kit (Xinle Bio Co., Ltd., Shanghai, China)
   according to the manufacturer's instructions. For physiological
   function-related parameter detection, blood samples were collected in
   heparin-coated tubevia eye punctures and centrifuged, and then the
   plasma samples were stored at −80 °C. Plasma cholesterol, HDL, LDL, AST
   and ALT levels were measured with a biochemistry platform using an
   Olympus AU400 Chemistry Analyser provided by Central South University.
   Blood glucose concentrations were measured with a blood glucose test
   meter, and blood samples were obtained from the mice tails at 0, 15,
   30, 60, and 90min after intraperitoneal injection of glucose (1.5 g/kg
   body weight).

   For isocaproic acid assay, a 0.5 g fecal sample was suspended in a tube
   containing 5 mL of water and mixed intensively for 5 min. Using 5 M HCl
   solution, the pH of suspension was adjusted to 2–3. The samples were
   then shaken for 10 min and centrifuged for 20 min at 5.000 rpm.
   Subsequently, the supernatant was filtered (Ø 400 μm) and assayed using
   used an HPLC–MS gas chromatograph system (Shimadzu Corporation, Japan)
   ([173]Skonieczna-Zydecka et al., 2018).

   Bile acid including CA (cholic acid), DCA (deoxycholic acid), TCA
   (taurine-conjugated cholic acid), TUDCA and LCA were measured as
   previously described ([174]Qi et al., 2019). After prepared the plasma
   and ileum samples, bile acid were measured in Ultra Liquid
   Chromatography 100 system coupled to an AB 5600 TripleTOF system (AB
   SCIEX), with an XBridge Peptide BEH C18 column (2.13100 mm, 1.7 mm;
   Waters Corp.). The column temperature was 40 _C, the flow rate was
   0.4 mL/min, and the injection volume was 5 mL. The mobile phase solvent
   is a mixture of 0.1% (v/v) formic acid, 10 mM acetic acid-amine in
   water (phase A) and 0.1% formic acid in 80% (v/v) methanol and 20%
   (v/v) acetonitrile (phase B).

   F. nucleatum ATCC25586 was grown anaerobically (80% N2, 10% CO2 and 10%
   H2) in agar plate added with 1 μg/mL menadione, 10 μg/mL hemin acquired
   from Sigma, and 5% sheep blood. The optical density at 600 nm of F.
   nucleatum, was adjusted to 0.06–0.07. The concentration of 20 μg/mL
   Nal-P-113 was then added to the respective bacterial cultures. Cultures
   without an antibacterial agent were considered as control group. The
   plates were incubated for 48 h continuously, a microplate reader was
   used to analyse the optical density at 600 nm. The tests were performed
   more than three times.

5.4. H&E immunohistochemistry (IHC) and IF staining

   Intestine, liver tissues were embedded in paraffin for staining with
   H&E according to previous reports ([175]Wu et al., 2021). For IHC, the
   paraffin sections were placed in citrate buffer for antigen retrieval
   and blocked in 3% H2O2. After incubation with the appropriate primary
   antibody and secondary antibody, the paraffin sections were blocked
   with DAB chromogen solution and HRP substrate solution. For IF
   staining, tissues were fixed with 4% paraformaldehyde at room
   temperature and permeabilized with 0.05% Triton X-100. Then, the
   tissues were stained with primary antibodies against the following
   proteins overnight at 4 °C: Occludin, ZO-1, E-cadherin, N-cadherin.
   After washing with PBS 3 times, the cells were stained with secondary
   antibodies. The nuclei were stained with DAPI (Sigma). Images were
   acquired using a fluorescence confocal microscope (Nikon TI2-E, Crest
   Optics, X-Light V3, Italy).

5.5. Electron microscopy for structural analysis of the gut, and liver

   TEM analysis was performed after collection of the gut, and liver
   tissues by Shiyanjia Lab ([176]www.shiyanjia.com). The tissues were
   split and treated in a cold fixative solution composed of 2.5%
   glutaraldehyde at 4 °C for 4 h. After washing with PBS, the specimens
   were post-fixed in 1% OsO4 at 4 °C for 1 h and washed again with PBS. A
   graded series of ethanol solutions was used for further dehydration,
   and the specimens were transferred to be incubated. TEM was performed
   with a JEM-2100F at 80 kV, and images were acquired using a
   side-inserted BioScan camera.

5.6. Western blot analysis and quantitative real-time PCR (qRT-PCR)

   Protein and mRNA expression was assessed using Western blotting and
   qRT-PCR methods. For Western blotting, proteins from gut tissue, liver
   tissue, hippocampal tissue and BAT were extracted using protein
   extraction reagent (Thermo Scientific, 78501). The total proteins were
   separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
   transferred to a polyvinylidene fluoride(PVDF) membrane using a wet
   transfer apparatus (Bio-Rad PowerPac^TMBasic, Bio-Rad Mini-Protean
   Tetra System 10025025 Rev A 12–06250312) as described in our previous
   reports ([177]Huang et al., 2020b, [178]2020c). ImageJ was used for
   band densitometry analysis. For RNA extraction from frozen tissues,
   TRIzol reagent (Jingcai Bio., Xi'an, Shanxi, China) was used. The
   relative expression of mRNA was quantified using SYBR Green dye (TB
   Green Premix Ex Taq II). Specific primers were designed by Green
   Pharma, Shanghai, China. qRT-PCR was performed with the following
   program: 95 °C for 10 min, 95 °C for 15s, and 60 °C for 1 min in 40
   cycles. The program was performed in a CFX96 Touch apparatus (Bio-Rad).
   The relative expression was calculated using the 2-ΔΔCT method.

5.7. 16S rRNA microbiome sequencing

   Faecal samples were collected based on the experimental design. The
   faecal samples were shipped to BioTREE Company, Shanghai, China, for
   detection using an Illumina NovaSeq. Total cellular DNA was extracted
   with an E.Z.N.A. Stool DNA Kit (Omega) based on the manufacturer's
   instructions. The bacterial hypervariable V3–V4 region of 16S rRNA was
   amplified using the primers 341_F:5′-CCTACGGGNGGCWGCAG-3′ and
   802_R:5′-TACNVGGGTATCTAATCC-3′ according to our previously published
   study ([179]Liu et al., 2019). The library, which was constructed using
   a TruSeq® kit, was quantified with a Qubit instrument and qRT-PCR.
   After the library was qualified, a NovaSeq 6000platform was used for
   sequencing. According to the barcode sequences and the PCR
   amplification primer sequences, the data for the different samples were
   separated. After the barcode and primer sequences were removed, FLASH
   (v1.2.7) was used ([180]http://ccb.jhu.edu/software/FLASH/). The reads
   in each sample were spliced, and the spliced sequence was considered
   the raw tag. The raw tags obtained by splicing were filtered strictly
   to obtain high-quality tags. The QIIMEpipeline (v1.9.1,
   [181]http://qiime.org/scripts/split_libraries_) was used for tag
   quality control with FASTQ files. Briefly, for tag trimming, raw tags
   with continuous low quality scores (default quality threshold: ≤19)
   were first cut from the first low-quality base site when the base
   number reached the set length (default length value: 3). Next, for tag
   length filtering, tags with continuous high-quality bases with lengths
   less than 75% of the tag length were further filtered out from the tags
   that remained after trimming. After the above processing steps,
   chimaeric sequences were removed from the tags. The tag sequences were
   compared to a species annotation database to detect chimerism
   ([182]https://github.com/torognes/vsearch/), and the chimaeric
   sequenceswere removed. Finally, the clean tags were obtained. The
   UniFrac distance was calculated with QIIME software (version 1.9.1),
   and an unweighted pair group method with arithmetic mean (UPGMA) sample
   clustering tree was constructed. Principal component analysis (PCA),
   principal coordinate analysis (PCoA) and non-metric multi-dimensional
   scaling (NMDS) analysis were performed with R software (version
   2.15.3). PCA was performed with R software's ADE4 package and ggplot2
   package; PCoA analysis was performed with R software's WGCNA, stats and
   ggplot2 packages; and NMDS analysis was performed with R software's
   vegan package. R software was used to analyse the differences between
   groups for beta diversity indices, and parametric tests and
   nonparametric tests were carried out. If there were only two groups,
   t-tests and Wilcoxon tests were used. If there were more than two
   groups, Tukey's test and Wilcoxon test of the agricolae package were
   used.

5.8. Untargeted metabolomics

   Faecal samples were collected after mice were killed and stored at
   −80 °C until analysis. Analysis was carried out using
   ultrahigh-performance liquid tandem chromatography quadrupole
   time-of-flight mass spectrometry (UHPLC-QTOFMS) by BioTREE Company,
   Shanghai, China. During detection, both positive ion (POS) mode and
   negative ion (NEG) mode were used. Then, the missing values were filled
   with half of the minimum value. Additionally, the internal standard
   normalization method was employed in this data analysis. The final
   dataset containing the peak number, sample name and normalized peak
   area information was imported into the SIMCA15.0.2 software package
   (Sartorius Stedim Data Analytics AB, Umea, Sweden) for multivariate
   analysis. The data were scaled and logarithmically transformed to
   minimize the impacts of both noise and high variance of the variables.
   After these transformations, PCA, an unsupervised analysis that reduces
   the dimensionality of the data, was carried out to visualize the
   distribution and grouping of the samples. The 95% confidence interval
   in the PCA score plot was used as the threshold to identify potential
   outliers in the dataset. To visualize group separation and find
   significantly changed metabolites, supervised orthogonal projections to
   latent structures-discriminant analysis (OPLS-DA) was applied. Then, a
   7-fold cross validation was performed to calculate the values of R2 and
   Q2. The R2 value indicates how well the variation of a variable is
   explained, and the Q2 value indicates how well a variable can be
   predicted. To assess the robustness and predictive ability of the
   OPLS-DA model, 200 permutations were further conducted. Afterward, the
   R2 and Q2 intercept values were obtained. Here, the intercept value of
   Q2 represents the robustness of the model, the risk of over fitting and
   the reliability of the model (smaller value is better). Furthermore,
   the variable importance in the projection (VIP) value of the first
   principal component in OPLS-DA was obtained. This value summarizes the
   contribution of each variable to the model. The metabolites with VIP>1
   and p < 0.05 (Student's t-test) were considered significantly changed
   metabolites. In addition, commercial databases, including the KEGG
   ([183]http://www.genome.jp/kegg/) and Metabo Analyst
   ([184]http://www.metaboanalyst.ca/) databases, were used for pathway
   enrichment analysis.

5.9. Statistical analysis

   Gut microbiota abundance, relative metabolite levels, relative protein
   expression and mRNA expression are presented as the means±SDs.
   Significant differences between mean results were analysed by Student's
   t-test and one-way ANOVA, but the results of the antibiotic, IA and
   probiotic intervention experiments were analysed by two-way ANOVA. The
   statistical figures were drawn and multiple comparisons via Tukey's
   test were carried out with GraphPad Prism 8.0 software. ImageJ was used
   for band densitometry analysis. A value of p < 0.05 was considered to
   indicate a significant difference between means.

Ethical approval and consent to participate

   The study was approved by the Animal Care and Use Committee at the
   Central South University.

Consent for publication

   N/A.

Availability of supporting data

   N/A.

Funding

   This study was supported by grants from the National Natural Science
   Foundation of China (Grant Nos. 82073486, U1803124), Key Research and
   Development (R&D) Plan of Hunan Province (Grant Nos.2021SK2026),
   Natural Science Foundation of Hunan Province (2019JJ40397), and
   Scientific research project of Hunan Health Committee(202112010058),
   and the Graduate Research Innovation Program of Central South
   University (No.2021zzts0325,2021zzts0966,2021zzts0970).

CRediT authorship contribution statement

   Huiji Pan: Investigation. Meiling Zhou: Investigation. Zhao Ju:
   Investigation. Jinhua Luo: Investigation. Jing Jin: Investigation.
   Liangfang Shen: Investigation. Pingkun Zhou: critically revised
   manuscript. Ruixue Huang: Conceptualization, Formal analysis, Writing –
   original draft, Funding acquisition.

Declaration of competing interest

   The authors declare that they have no known competing financial
   interests or personal relationships that could have appeared to
   influence the work reported in this paper.

Acknowledgments