Abstract

   Chronic kidney disease (CKD) is a leading public health problem with
   high morbidity and mortality, but the therapies remain limited. Bupi
   Yishen Formula (BYF) - a patent traditional Chinese medicine (TCM)
   formula - has been proved to be effective for CKD treatment in a
   high-quality clinical trial. However, BYF’s underlying mechanism is
   unclear. Thus, we aimed to reveal BYF pharmacological mechanism against
   CKD by network pharmacology and experimental studies. Network
   pharmacology-based analysis of the drug-compound-target interaction was
   used to predict the potential pharmacological mechanism and biological
   basis of BYF. We performed a comprehensive study by detecting the
   expression levels of fibrotic and inflammatory markers and main
   molecules of candidate signal pathway in adenine-induced CKD rats and
   TGF-β1-induced HK-2 cells with the treatment of BYF by western blotting
   and RT-qPCR analyses. Using small interfering RNA, we assessed the
   effect of BYF on the TLR4-mediated NF-κB mechanism for CKD renal
   fibrosis and inflammation. Network pharmacology analysis results
   identified 369 common targets from BYF and CKD. Based on these common
   targets, the BYF intervention pathway was analyzed by Gene Ontology
   (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
   analysis. We found that Toll-like receptor (TLR) and NF-κB signaling
   pathways were enriched. Then, we demonstrated that BYF significantly
   improved the adenine-induced CKD rat model condition by kidney
   dysfunction improvement and reversing renal fibrosis and inflammation.
   Subsequently, we investigated BYF’s effect on the TLR4/NF-κB signaling
   pathway. We found that TLR4 and phospho-NF-κB (p-p65 and p-IKβα)
   expression was significantly upregulated in adenine-induced CKD rats,
   then partially downregulated by BYF. Furthermore, BYF inhibited
   fibrotic and inflammatory responses, as well as TLR4, p-p65, and p-IKβα
   in TGF-β1-induced HK-2 cells. Additionally, the BYF inhibitory effect
   on fibrosis and inflammation, and NF-κB pathway activation were
   significantly reduced in TGF-β1-induced HK-2 cells transfected with
   TLR4 siRNA. Altogether, these findings demonstrated that the
   suppression of TLR4-mediated NF-κB signaling was an important
   anti-fibrotic and anti-inflammatory mechanism for BYF against CKD. It
   also provided a molecular basis for new CKD treatment drug candidates.

   Keywords: traditional Chinese medicine, bupi yishen formula, chronic
   kidney disease, network pharmacology, experimental study

Introduction

   Chronic kidney disease (CKD) is a worldwide public health problem with
   an estimated global prevalence of 8–16% ([38]Jha, et al., 2013). CKD
   affects approximately 8–10% of the Western countries population
   ([39]Lameire, et al., 2005), and >15% of U.S. adults may have CKD. In
   China, the total CKD prevalence is around 10.8% and affects more than
   120 million individuals ([40]Zhang, et al., 2012). Also, CKD is
   associated with adverse cardiovascular events and high mortality risk
   ([41]Bello, et al., 2011). However, there are limited treatment options
   available for CKD patients. The main approach to delay CKD progression
   is renin-angiotensin system (RAS) blockade, as well as blood pressure
   and glycemic control ([42]Palmer, et al., 2015; [43]Breyer and Susztak,
   2016). These therapeutic strategies are insufficient to impair CKD
   progression to end-stage renal disease (ESRD). Therefore, it is urgent
   to develop effective medications to prevent CKD progression.

   Traditional Chinese medicine (TCM) is commonly used in conjunction with
   Western medications for CKD treatment in China and other Asian
   countries ([44]Li and Wang, 2005; [45]Wojcikowski, et al., 2006;
   [46]Zhong, et al., 2013). However, the use of TCM in CKD treatment
   remains controversial, especially because of renal toxicities present
   in some TCM ([47]Feng, et al., 2018; [48]Yang B et al., 2018; [49]Omer
   Mohamed, et al., 2020). There is also emerging solid evidence of the
   beneficial effects of TCM prescribed for CKD patients in mainland China
   ([50]Zhang, et al., 2014; [51]Li, et al., 2020; [52]Wu, et al., 2021)
   and Taiwan ([53]Lin, et al., 2015), supporting that TCM can be
   promising for the development of new therapeutic drugs for CKD
   treatment.

   The Bupi Yishen Formula (BYF), a patent TCM, is composed of eight
   herbs, which are modified from one traditional Si-Jun-Zi Decoction
   (SJZD). SJZD was recorded in Tai Ping Hui Min He Ji Ju Fang
   (A.D.1078-1,110) and is used to replenish “Qi” and reinforce “Spleen”.
   Based on TCM theory, BYF can “reinforce the Spleen and invigorate the
   Kidney” and “dispel dampness and resolve turbidity”, suggesting that it
   could be used for CKD treatment. Over the past decade, BYF has been
   clinically applied as a basic treatment for CKD patients. Our recently
   published multicenter randomized controlled trial demonstrated that BYF
   significantly improved kidney function in non-diabetic CKD4 patients,
   as evidenced by a slower decline slope of the estimated glomerular
   filtration rate (eGFR) and a lower composite outcome risk ([54]Mao, et
   al., 2020). However, BYF’s underlying reno-protective mechanism remains
   unknown and requires investigation.

   In this study, we first identified the BYF and CKD common targets.
   Then, we analyzed the intervention pathways based on these common
   targets using network pharmacology. Second, we examined BYF’s
   therapeutic effect on CKD in vivo. We used an adenine-induced CKD rat
   model and found that BYF reduced renal fibrosis and inflammation, and
   simultaneously inhibited the TLR4/NF-κb pathway. Finally, the BYF
   regulatory mechanism on renal fibrosis and inflammation was validated
   in vitro with TGF-β1-induced HK2 cells and TLR4 siRNA. Altogether, our
   study demonstrated that BYF reduced renal fibrosis and inflammation by
   TLR4-mediated NF-κb signaling pathway suppression, which may be a key
   mechanism of its therapeutic effect on CKD.

Materials and Methods

Active Compounds and Corresponding Drug Targets Collection

   The BYF chemical compounds were screened using TCMSP
   ([55]http://tcmspw.com/tcmsp.php) ([56]Ru, et al., 2014), TCMID
   ([57]http://www.megabionet.org/tcmia/), and BATMAN-TCM
   ([58]http://bionet.ncpsb.org/batman-tcm/index.php.Home/Index/index).
   Compounds that showed DL ≥ 0.18 and OB ≥ 30% were retrieved as active
   by ADME analysis ([59]Wang, et al., 2015). We collected active
   compounds targets with the TCMSP and SYMMAP ([60]http://www.symmap.org)
   databases ([61]Wu, et al., 2019), then retrieved the target’s gene name
   and ID using the Uniprot ([62]https://www.uniprot.org/) database.

Disease and Drug-Disease Common Targets Collection

   The genes of targets related to “Chronic kidney disease” were screened
   via GeneCards ([63]https://www.genecards.org/) ([64]Stelzer, et al.,
   2011), OMIM ([65]https://www.omim.org/) ([66]Amberger, et al., 2015),
   TTD ([67]http://db.idrblab.net/ttd/), MALACARDS
   ([68]https://www.malacards.org/) ([69]Rappaport, et al., 2017), and
   DisGeNET database ([70]http://disgenet.org/) ([71]Piñero, et al.,
   2017). After removing duplicated genes, the common targets associated
   with BYF and CKD were collected as the candidates.

Drug-Compound-Target Interaction Network Construction

   To analyze the relationship between drugs, active compounds, and
   candidate targets, a drug-compound-target interaction network was
   constructed using Cytoscape 3.6.1 software. In the network, different
   targets, active compounds, and drugs were represented as different
   colors and shapes nodes.

Protein-Protein Interaction Network Construction

   The String database contains a large number of PPI relationships
   ([72]von Mering, et al., 2005). The candidate targets were unloaded
   onto the String database ([73]https://string-db.org/) to obtain related
   information about protein interactions. Then, a PPI network was
   Constructed using Cytoscape 3.6.1 software. Besides, the median of
   three topological indexes was calculated (BC, CC, and DC), and the core
   PPI network targets were screened.

Enrichment Analysis

   Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathway enrichment analysis were performed based on the
   candidate targets using DAVID 6.8. The downloaded results were sorted
   using p and count values. The workflow used for this study was shown in
   [74]Supplementary Figure S1.

BYF Water Extract Preparation

   BYF contains eight Chinese herbs. The related BYF herbal information
   and chemical composition analysis were performed as previously
   described ([75]Zhang, et al., 2018; [76]Mao, et al., 2020). Raw herbs
   were purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd.
   (Jiangsu, China). The eight BYF components, including Astragali radix
   (30 g), Codonopsitis radix (15 g), Atractylodis macrocephalae rhizome
   (12 g), Poria (15 g), Diosscoreae rhizome (15 g), Coicis semen (20 g),
   Cuscutae semen (12 g), and Salviae miltiorrhizae radix et rhizome
   (15 g), were boiled twice (1 h each) in ddH2O (w/v). The extract was
   condensed and stored at −20 °C. Before treatment, the BYF extract was
   dissolved in distilled water.

Animals and Experimental Treatment

   This animal experiment was performed according to protocols approved by
   the Institutional Ethics Review Boards of the Second Clinical College
   of Guangzhou University of Chinese Medicine, Guangzhou, China (approval
   No. 2020021). Twenty-four male Spraque-Dawley (SD) rats (∼200 g of
   weight) were purchased from the Guangdong Experimental Animal Center
   (Guangzhou, China). They were housed in the SPF animal breeding room
   with a 12-h light/12-h dark cycle and the temperature was maintained at
   22–25°C. Rats were randomly divided into four groups (n = 6 for each
   group): 1) control (CTL); 2) untreated CKD (CKD); 3) CKD treated with
   BYF-Low dose (BYF-L); and 4) CKD treated with BYF-High dose (BYF-H).
   The CKD in rats was induced by intra-gastric gavage with adenine
   (Sigma-Aldrich, St Louis, MO, USA) at 200 mg/kg for 4 weeks ([77]Chen,
   et al., 2017; [78]Thakur, et al., 2018). Rats in the CKD treatment
   groups received BYF extract doses of 15 g/kg/d (BYF-L) and 30 g/kg/d
   (BYF-H) for 4 weeks with simultaneous adenine administering. Rats in
   CTL received normal adenine-free saline solution for 4 weeks.

Biochemical Analysis of Serum and Urine Samples

   Serum creatinine and urea, 24 h urinary protein, urinary albumin to
   creatinine ratio, aspartate transaminase (AST), and alanine
   transaminase (ALT) were measured using a Roche automatic biochemistry
   analyzer (Hitachi, 7180, Tokyo, Japan) following the manufacturer’s
   instructions. Analyses were performed in the Laboratory Department of
   the University City Hospital of Guangdong Hospital of Traditional
   Chinese Medicine.

Histological Examination

   Rat kidney samples were fixed with 4% buffered paraformaldehyde (pH
   7.4) at 4°C overnight, dehydrated in graded alcohols, and embedded in
   paraffin. The paraffin-embedded kidneys were cut into 2 μm sections and
   stained with hematoxylin and eosin (HE), periodic acid-Schiff (PAS),
   and Masson’s trichrome for pathological changes evaluation. The tubular
   atrophy score was performed in PAS staining. The interstitial fibrosis
   was assessed by collagen deposition area in Trichrome staining using
   the ImageJ software (NIH, Bethesda, MD, United States).

Immunohistochemical Analysis

   For immunohistochemistry staining, 2 μm paraffin-embedded sections were
   deparaffinized and rehydrated. The antigens were repaired with 1% (w/v)
   Tris-EDTA solution by high temperature and pressure for 10 min.
   Sections were blocked with BSA, then incubated with primary antibodies.
   The following antibodies were used: TGF-β1 (3711; Cell Signaling
   Technology), α-SMA (19,245; Cell Signaling Technology), fibronectin
   (NBP1-91258, Novus), and TLR4 (sc-293072, Santa Cruz). The secondary
   antibody was horseradish-peroxidase (HRP) goat anti-rabbit IgG (J31126;
   Transgen Biotech).

Cell Culture and Treatment

   The HK-2 cells were cultured in DMEM/F12 supplemented with 10% FBS,
   25 mM glucose, and 1% penicillin and streptomycin. They were incubated
   in a 37°C humidified incubator supplied with 5% CO[2]. The HK-2 cells
   were passaged using 0.25% Trypsin at a 1:6–8 ratio every 3–4 days. At
   80% confluence, the cells were starved in 0.5% FBS overnight. Then,
   cells were divided into negative control (CTL), TGF-β1, and BYF groups.
   The TGF-β1 group was treated with 10 ng/ml TGF-β1 for 48 h. The BYF
   group was treated with 10 ng/ml TGF-β1 for 24 h, then treated with
   32 mg/ml BYF for 24 h.

Cell Viability

   The CCK-8 assay was applied to assess the BYF effect on cell viability,
   following the manufacturer’s instructions. First, serum-starved HK-2
   cells were treated with different BYF concentrations (0, 32, 64, 128,
   188, 256, 375, 512, 750, 1,500 mg/ml) with or without TGF-β1 for 48 h.
   Second, 10 μl of CCK-8 reagent reacted with HK-2 cells at 37°C for 2 h.
   Finally, the supernatant was removed, and the optical density was
   measured at 490 nm using a microplate reader (TECAN, Mannedorf,
   Switzerland).

TRL4 Downregulation by Small Interfering RNA

   Transfection of siRNA was used to downregulate TRL4 in the HK-2 cells.
   The HK-2 cells were transfected with 10 μM siRNA targeting TRL4
   (si-TRL4) or control siRNA (si-CTL) using Lipofectamine 2000 reagent
   (Invitrogen, Carlsbad, CA, United States), according to the
   manufacturer’s protocols. After siRNA transfection, cells were
   incubated with or without BYF for 48 h, with or without TGF-β1.

RNA Extraction and qPCR Analysis

   Total RNA was extracted from the kidney cortex and HK2 cells using
   TRIzol Reagent (Invitrogen, Carlsbad, CA). About 500 ng of extracted
   RNA was reversely transcribed to cDNA with the QuantiTect Reverse
   Transcription Kit. Then, cDNA samples were used to conducted real-time
   PCR analysis with an SYBR Green I Kit. Gene expression quantification
   was normalized to Glyceraldehyde-3-Phosphate dehydrogenase (GAPDH). The
   expression level fold change relative to the control group was
   calculated using the 2^-△△Ct method. The primers used for this study
   are described in [79]Supplementary Table S1.

Western Blotting Analysis

   The kidney cortex and cultured cells were lysed in
   radioimmunoprecipitation assay (RIPA) buffer containing a protease
   inhibitor cocktail (Roche) and phosphatase inhibitor. Protein
   concentration was measured by a BCA detection kit (Thermo Fisher
   Scientific). The same protein amount (50 μg) was electrophoresed using
   10% SDS-PAGE gels, then transferred to polyvinylidene difluoride
   membranes (Millipore, Billerica, MA, United States). The membranes were
   blocked with 5% nonfat milk for 1 h, then incubated with primary
   antibodies overnight at 4°C. Membranes were incubated with horseradish
   peroxidase (HRP)-conjugated anti-mouse IgG (Boster, BA1050) or
   HRP-conjugated anti-rabbit IgG (Boster, BA1054) for 1 h at room
   temperature. Their HRP activity was visualized using an enhanced
   chemiluminescence reagent (Bio-RAD, Bio-Rad universal Hood II,
   California, United States) and Image Lab System (Bio-RAD 5.2.1) was
   used for densitometric analysis. The primary antibodies used for this
   study included TGF-β1 (3711; Cell Signaling Technology); α-SMA (19,245;
   Cell Signaling Technology); fibronectin (NBP1-91258, Novus); Collagen I
   (NB600-408, Novus); Collagen III (NBP1-05119, Novus); Smad3 (9523S;
   Cell Signaling Technology); p-Smad3 (9520S; Cell Signaling Technology);
   TLR4 (sc-293072, Santa Cruz); NF-κb (p65, ab16502, Abcam); p-NF-κb
   (p-p65, 3033S, Cell Signaling Technology); IKβα (4814S, Cell Signaling
   Technology); p-IKβα (2859s, Cell Signaling Technology); and MyD88
   (ab219413, Abcam).

Statistical Analyses

   Each analysis is representative of at least three independent repeats
   of experiment. GraphPad Prism five software (GraphPad Software Inc, La
   Jolla, CA) was used for statistical analysis. The data are represented
   as mean ± standard deviations (SD). Differences between two groups were
   analyzed using a 2-tailed Student’s t-test and two-way analysis of
   variance (ANOVA) was used for comparison between three or more groups.
   A p-value < 0.05 was considered statistically significant.

Results

BYF Active Compounds and Candidate Targets in CKD

   Using TCMSP, TCMID, and Batman-TCM databases, 603 chemical compounds
   were screened in BYF’s eight components. Based on the DL ≥ 0.18 and OB
   ≥ 30%, 294 active compounds were selected acting on 2134 potential
   targets ([80]Supplementary Tables S2, S3). According to OMIM, TTD,
   MALACARDS, and DisGeNET databases, 1,157 predicted CKD-associated
   targets were obtained ([81]Supplementary Table S4). After merging
   active compounds and CKD targets, 369 common targets were recognized as
   candidates, which could be the biological basis of BYF’s effect on CKD
   ([82]Figure 1A and [83]Supplementary Table S5). Through network
   analysis, a drug-compound-target interaction network was established to
   generate the BYF active compounds for 369 candidate targets based on
   the 294 active compounds identified in the eight herbs ([84]Figure 1B).
   From this network, we found that different compounds act on multiple
   targets, and vice versa. Additionally, a PPI network was established
   based on the 369 candidate targets by importing the candidate targets
   gene IDs to the STRING database ([85]Figure 1C). The PPI network showed
   that there is a close interaction between those targets. The 30 core
   targets obtained from this PPI network showed that PI3K, AKT, IL6, TNF,
   NF-κb, and TLR4 were the most relevant ([86]Supplementary Table S6).

FIGURE 1.

   [87]FIGURE 1
   [88]Open in a new tab

   BYF network pharmacology analysis against CKD. (A) Venn diagram
   revealing BYF and CKD common target genes; (B) Drug-compound–target
   interaction network diagram; (C) PPI network and the proteins with the
   top 30° among common BYF and CKD targets.

Functional Enrichment Analysis of BYF Candidate Targets in CKD

   GO functional enrichment analysis was annotated from three aspects:
   biological process, molecular function, and cellular component. To
   discover the BYF pharmacological mechanisms in CKD treatment, 369
   candidate targets were inputted to the DAVID 6.8 database for GO
   enrichment analysis ([89]Figures 2A–C). The results indicated that the
   following mechanisms were related to BYF against CKD: inflammatory
   response, NF-κB transcription factor activation, apoptotic process,
   plasma membranes, and protein binding. To further explore the
   relationship between these candidate targets and their corresponding
   pathways, KEGG pathway enrichment analysis was performed via the DAVID
   6.8 database ([90]Figure 2D). The results indicated the Toll-like
   receptor (TLR) signaling, TNF signaling, PI3K-AKT signaling, apoptosis,
   and HIF-1 signaling pathways are related to BYF mechanisms against CKD.

FIGURE 2.

   [91]FIGURE 2
   [92]Open in a new tab

   Enrichment analysis of BYF candidate targets against CKD. GO enrichment
   analysis revealed the related biological process (A), molecular
   function (B), and cellular component (C); KEGG pathway analysis
   revealed the related pathways (D).

BYF Improved Renal Function in an Adenine-Induced CKD Rat Model

   Treatment with BYF significantly increased the body weight and
   decreased the urine volume in adenine-induced CKD rats ([93]Figures
   1A,B). Compared with CTL, the levels of 24 h urinary protein excretion
   and urine albumin-to-creatinine ratio in CKD were increased, and in BYF
   groups they were reduced ([94]Figures 1C,D). The CKD group showed
   higher serum creatinine and urea compared to CTL, which could be
   restored by BYF treatment ([95]Figures 1E,F). Moreover, the ALT and AST
   serum levels were not significantly different between the four groups
   ([96]Figures 1G–I). This indicates that the two BYF treatment dosages
   that we utilized are safe. In HE staining, CKD rats showed obvious
   renal tubular dilation and massive inflammatory cells infiltration, and
   BYF treatment inhibited these changes. In PAS staining, CKD rats
   indicated severe loss of tubular epithelial cells, chronic tubular
   atrophy, and glomerulosclerosis, recovered by BYF ([97]Figures 3J,K).
   Masson staining showed interstitial fibrosis in the CKD, and that BYF
   treatment significantly decreased collagen deposition ([98]Figures
   3J–L). Altogether, these results suggested that the CKD model was
   successfully established and that BYF improved kidney function and
   reduced structural damage in CKD rats.

FIGURE 3.

   [99]FIGURE 3
   [100]Open in a new tab

   BYF improves kidney function and reduces renal pathological injury in
   adenine-induced CKD rats. BYF administration effectively increased body
   weight (A) and reduced urine volume (B). In CKD rats, BYF reduced 24 h
   urinary protein excretion (C), urinary albumin-to-creatinine ratio (D),
   serum creatinine (E) and urea (F). (G) Serum ALT, (H) AST, (I) and
   ALT/AST levels. (J) HE, PAS, and Masson’s trichrome staining
   representative micrographs in indicated groups (scale bar = 100 μm).
   (K) Chronic tubular atrophy score. (L) Quantitative analysis of Masson
   trichromatic positive rate. All values were presented as means ± SD. n
   = 6 rats per group. *p < 0.05 and **p < 0.01 vs. the CTL group; ^# p <
   0.05 and ^## p < 0.01 vs. the CKD group.

BYF Attenuated Renal Fibrosis and Inflammation in Adenine-Induced CKD Rats

   Renal fibrosis has been long considered as the common and final
   manifestation of nearly all CKD progressive forms. Thus, we further
   examined the BYF effect on renal fibrosis by fibrotic markers.
   Immunohistochemical staining showed that the α-SMA, TGF-β1, and
   Fibronectin protein levels increased in CKD, which was decreased by BYF
   treatment ([101]Figures 4A,B). Western blotting (WB) indicated that the
   expression levels of TGF-β1, Fibronectin, α-SMA, Collagen I and III,
   and p-Smad3 also increased in CKD. In contrast, BYF administration
   significantly reduced these proteins level ([102]Figures 4C,D).
   Similarly, fibrotic markers mRNA expression increased in CKD and was
   reversed by BYF treatment ([103]Figure 4E). Interestingly, BYF
   treatment also remarkedly downregulated proinflammatory factors mRNA
   level, including interleukin-1β (IL-1β), IL-6, MCP-1, and tumor
   necrosis factor-alpha (TNF-α) in the kidneys of CKD rats ([104]Figure
   4F). Altogether, these results suggested that BYF could inhibit the
   increase in TGF-β1/Smad3-mediated fibrotic markers production and
   reduced inflammatory cytokines release. This provided further evidence
   of the BYF’s beneficial effect on CKD.

FIGURE 4.

   [105]FIGURE 4
   [106]Open in a new tab

   BYF reduces fibrotic and inflammatory markers expression levels in the
   kidney of adenine-induced CKD rats. α-SMA, TGF-β1, and Fibronectin
   immunohistochemical staining representative micrographs (A) and
   quantitative analysis (B). TGF-β1, Fibronectin, α-SMA, Collagen I and
   III, and p-Smad3 WB vaαlidation (C) and quantitative analysis (D). qPCR
   validation of fibrotic markers (E) and proinflammatory factors (F) in
   indicated groups. Scale bar = 100 μm. All values are presented as means
   ± SD. n = 3 rats per group. *p < 0.05 and **p < 0.01 vs. the CTL group;
   ^# p < 0.05 and ^## p < 0.01 vs. the CKD group.

BYF Inhibited TLR4/NF-KB Signaling Pathway in The Kidneys of Adenine-Induced
CKD Rats

   Recent studies revealed that the TLR4/NF-κB signaling pathways are
   closely associated with kidney fibrosis and CKD progression ([107]Wang,
   et al., 2008; [108]Liu, et al., 2015; [109]Pérez-Ferro, et al., 2016)
   by augmenting TGF-β/Smads responses ([110]Bhattacharyya, et al., 2013)
   and activating inflammatory cytokines ([111]Chen, et al., 2018). Since
   BYF could target TLR and NF-κB pathways based on the network
   pharmacology analysis, the anti-fibrotic and anti-inflammatory effects
   of BYF on CKD were explored. The TLR4 mRNA expression and protein
   expression by immunohistochemical staining were significantly
   upregulated in CKD and downregulated by BYF ([112]Figures 5A,B).
   Western blot analysis showed that the protein levels of TLR4, p-NF-κB
   (p-p65 and p-IKβα), and MyD88 in kidney tissues were significantly
   increased in CKD compared to CTL, while BYF treatment restored these
   proteins with the high doses effect being generally more evident
   ([113]Figures 5C,D). These results indicated that BYF treatment might
   attenuate CKD by improving renal fibrosis and inflammation via
   TLR4/NF-κB mechanism.

FIGURE 5.

   [114]FIGURE 5
   [115]Open in a new tab

   BYF suppresses TLR4/NF-κB signaling pathway in adenine-induced CKD
   rats. qPCR validation of TLR4 (A) and immunohistochemical staining of
   TLR4 (B) in indicated groups. TLR4, p65, p-p65, IKβα, p-IKβα, and MyD88
   WB validation (C) and quantitative analysis (D) in indicated groups.
   All values are presented as means ± SD. n = 3 rats per group. *p < 0.05
   and **p < 0.01 vs. the CTL group; ^# p < 0.05 and ^## p < 0.01 vs. the
   CKD group.

BYF Improved Fibrogenesis and Inflammation by Inhibiting TLR4-Mediated NFκB
Signaling Pathway in TGF-β1-Induced HK2 Cells

   Renal proximal tubular cells are the major sites of kidney injury and
   are critical in fibrosis development ([116]Liu, et al., 2018). Based on
   the inhibitory effect on renal fibrosis and inflammation in
   adenine-induced CKD rats, TGF-β1-induced HK-2 cells were used to study
   the BYF protection in vitro. To explore an optimal BYF concentration,
   different concentrations (0, 32, 64, 128, 188, 256, 375, 512, 750,
   1,500 mg/ml) were added to HK-2 cells for 24 h. CCK-8 assay showed that
   the cell viability at 32 mg/ml was optimum ([117]Supplementary Figure
   S2). Based on these results, 32 mg/ml of BYF was used in the subsequent
   experiments.

   To evaluate the anti-fibrotic and anti-inflammatory effects of BYF in
   vitro, the fibrotic and inflammatory markers expression level was
   detected in TGF-β1-induced HK-2 cells. Results showed that BYF markedly
   reduced the elevated α-SMA, Fibronectin, and TGF-β1, as well as TNF-α,
   IL-1β, and IL-6 mRNA levels in TGF-β1-induced HK-2 cells ([118]Figure
   6A,B). Likewise, TGF-β1, α-SMA, Fibronectin, Collagen III, and p-Smad3
   protein expression levels reduced after BYF treatment ([119]Figures
   6C,D). Next, we investigated the mechanisms whereby BYF inhibits renal
   fibrosis and inflammation in TGF-β1-induced HK-2 cells. Cells treated
   with TGF-β1 increased the TLR4, p-p65, and p-IKβα protein levels, while
   BYF treatment reduced it ([120]Figures 6E,F). These results suggested
   that BYF likely inhibited fibrogenesis and inflammation in vitro by
   TLR4/NF-KB pathway suppression.

FIGURE 6.

   [121]FIGURE 6
   [122]Open in a new tab

   BYF treatment decreases fibrotic and inflammatory markers expression
   levels and inhibits TLR4/NF-κB signaling pathway in the TGF-β1-induced
   HK2 cells. qPCR validation of fibrotic markers (A) and inflammatory
   factors (B) between groups. Fibrotic markers WB validation (C) and
   quantitative analysis (D) in indicated groups. TLR4/NF-KB signaling
   main proteins WB validation (E) and quantitative analysis (F) in
   indicated groups. All values are presented as means ± SD. n = 3
   independent experiments for each indicator. *p < 0.05 and **p < 0.01
   vs. the CTL group; ^# p < 0.05 and ^## p < 0.01 vs. the CKD group.

   To better understand TRL4/NF-κb signaling functional role in
   TRL4-mediated renal fibrosis and inflammation, we knocked down TRL4 in
   HK2 cells by siRNA. TLR4 mRNA expression was significantly
   downregulated ([123]Figure 7A). The expression levels of fibrotic and
   inflammatory markers showed that BYF treatment could not markedly
   decrease the fibronectin, TGF-β1, and α-SMA mRNA levels, as well as
   TNF-α, IL-1β, and IL-6 in TGF-β1-induced HK-2 cells silenced with TLR4
   siRNA ([124]Figures 7B,C). Next, WB showed that silencing TRL4 markedly
   suppressed the TGF-β1 activation effect on the NF-κb pathway in HK2
   cells with reduced p-p65, and p-IKβα expression levels, compared with
   the TGF-β1 group. Furthermore, BYF did not significantly reduce the
   levels when TLR4 was silenced ([125]Figures 7D,E). Altogether, these
   results demonstrated that the BYF might have a protective effect via
   the TLR4-mediated NF-κB mechanism to reduce CKD renal fibrosis and
   inflammation.

FIGURE 7.

   [126]FIGURE 7
   [127]Open in a new tab

   Silencing TLR4 significantly suppresses BYF inhibitory effect on
   TGF-β1-induced fibrogenesis and inflammation and NF-κB pathway
   activation in HK2 cells. (A) TLR4 qPCR validation in HK-2 cells
   transfected with TLR4 siRNA. (B) Fibrotic markers qPCR validation
   between groups. (C) Inflammatory factors qPCR validation between
   groups. TLR4/NF-κB signaling rescue assay WB validation (D) and
   quantitative analysis (E) in indicated groups. All values are presented
   as means ± SD. n = 3 independent experiments for each indicator. *p <
   0.05 and **p < 0.01 vs. the CTL group; ^# p < 0.05 and ^## p < 0.01 vs.
   the CKD group.

Discussion

   Although our previous clinical study revealed that BYF have protective
   effects on delaying kidney function progression among advanced CKD
   patients ([128]Mao, et al., 2020), its pharmacological mechanisms
   remain ambiguous. In this study, we investigated the BYF effect on CKD
   in vivo and in vitro. We explored its potential mechanisms combining
   network pharmacology, histopathology, and molecular biology. Network
   pharmacology-based analysis predicted that BYF protected against CKD
   through the TLR/NF-κB signaling pathway and the inflammatory and
   fibrotic response triggered by this pathway. Experimental validation
   indicated that BYF effectively inhibited the fibrosis and inflammatory
   response in adenine-induced CKD rats and TGF-β1-induced HK-2 cells.
   Also, these results showed that BYF alleviated renal fibrosis and
   inflammation via TLR4/NF-κB signaling pathway modulation. The putative
   anti-fibrotic and anti-inflammatory BYF mechanism in CKD is shown in
   [129]Figure 8.

FIGURE 8.

   [130]FIGURE 8
   [131]Open in a new tab

   BYF regulation on the TLR4/NF-κb signaling pathway in CKD.

   BYF is composed of eight Chinese herbs and has complex bioactive
   compounds. Therefore, it is difficult to clearfy its molecular
   mechanism through traditional pharmacological techniques. Network
   pharmacology-based analysis integrates a series of disciplines and
   techniques, including genomics, proteomics, and systems biology
   ([132]Kim, et al., 2019). Thus, network pharmacology provides an
   effective method to clarify the multifaceted biological phenomenon
   mechanism of such complex compounds in Chinese herbal formulations. To
   explore the BYF molecular mechanism, we selected 369 common BYF and CKD
   targets, constructed a drug-compound-target network, and executed GO
   and KEGG enrichment analysis. Results suggested that TLR and NF-κB
   signaling pathway plays an important role in BYF pharmacological
   mechanism during CKD treatment. Compounds associated with BYF may
   directly act on TLR4/NF-κB signaling pathway and interfere with the
   downstream TGF-β1/Smad3 signaling pathway and inflammatory response,
   resulting in the inhibition of profibrotic factors (α-SMA, Fibronectin,
   Collagen I and III) as well as the release of proinflammation cytokines
   (TNF-α, IL-1β, IL-8). Interestingly, the network pharmacology results
   showed that BYF may also interfere with the downstream targets, like
   PI3K, AKT, and TNF (except for the main molecules in the TLR4/NF-κB
   pathway), with or without TLR2/3 interference. These findings indicated
   that BYF may interfere with CKD by multiple pathways through multiple
   compounds from different Chinese herbs.

   Many animal models have been developed to study CKD pathogenesis and
   treatment in humans. However, most models do not mimic CKD complexity
   in humans, and the adenine diet or gavage model in rodents is an
   exception ([133]Yokozawa, et al., 1986; [134]Diwan, et al., 2018).
   Intra-gastric gavage of 200 mg/kg adenine in rats for 4 weeks has been
   well-accepted as a model to study kidney damage since this intervention
   mimick most of the functional and structural changes observed in human
   CKD ([135]Chen, et al., 2017; [136]Yang H, et al., 2018). We observed
   high levels of proteinuria, serum creatinine and urea, as well as
   tubular atrophy, inflammatory cells infiltration, and collagen
   synthesis during the 4 weeks in the adenine-induced CKD rats.
   Furthermore, BYF treatment partially recovered the kidney dysfunction
   and histopathological injury compared to the adenine-induced CKD group.
   These results suggested that a successful CKD rat model induced by
   adenine gavage was established.

   Inflammation and fibrosis are two CKD pathological features. It has
   been proven that TGF-β1, α-SMA, and extracellular matrix (ECM) proteins
   such as fibronectin, and collagen I and III are master markers in
   kidney fibrosis development ([137]Zeisberg and Neilson, 2010; [138]Liu,
   2011). Our results indicated that BYF markedly inhibited these fibrotic
   markers expression levels in adenine-induced CKD rat and TGF-β1-induced
   HK-2 cells, suggesting its anti-fibrotic effect in the kidney. Also, we
   found that BYF significantly decreased the proinflammatory factors mRNA
   levels, including IL-1β, IL-6, MCP-1, and TNF-α, in vivo and in vitro,
   indicating its anti-inflammatory effect in the kidney. Besides,
   TGF-β/Smads signaling prominent activation was observed (with increased
   TGF-β1 and p-Smad3 levels), which was reversed by BYF treatment. These
   results demonstrated that BYF protected against CKD through
   anti-fibrotic and anti-inflammatory effects.

   It is well known that the TLR signaling pathway is one of the most
   crucial pathways in the host immune response in an infected
   environment, responding to different microorganisms and endogenous
   ligands ([139]Mollen, et al., 2006). TLR4, an important member of the
   Toll-like family, is located in the cell membrane and cytoplasm and is
   crucial in the kidney fibrosis process ([140]Bhattacharyya, et al.,
   2013; [141]Pérez-Ferro, et al., 2016). NF-κB, an important
   transcription activator, modulates and regulates inflammatory
   mediators, and induces cytokines production ([142]Mitchell, et al.,
   2016). It was reported that TLR4 enhances the downstream activation of
   the NF-κB pathway, which ultimately results in the inflammation
   reaction ([143]Ciesielska, et al., 2021). In this study, we found that
   the TLR4, p-p65, p-IKβα, and MyD88 proteins levels were significantly
   increased in adenine-induced CKD rats, which markedly decreased after
   BYF treatment. These results suggested that BYF treatment may partially
   heal CKD by renal inflammation and fibrosis reduction via TLR4/NF-κB
   suppression mechanism.

   Many kidney cell types (e.g., tubular, myofibroblasts, endothelial, and
   inflammatory) are involved in the development and progression of renal
   fibrogenesis and inflammation under pathological conditions (Liu,
   2011). However, emerging evidence indicated that proximal tubular
   epithelial cells are the major injury sites and are critical in injury
   repair and fibrosis development ([144]Yang, et al., 2010; [145]Kang, et
   al., 2015; [146]Liu, et al., 2018). TGF-β1 is one of the most powerful
   profibrotic cytokines and is vital in renal inflammation and fibrosis
   by downstream Smad3 signaling activation ([147]Sutariya, et al., 2016;
   [148]Gu, et al., 2020). It was also demonstrated that TGF-β signaling
   could be activated by TLR4 in a hepatic fibrogenesis mice model
   ([149]Seki, et al., 2007). Therefore, we examined the BYF protective
   effects and TLR4/NF-κ b mechanism on TGF-β1-induced HK2 cells. We found
   that BYF markedly decreased fibrotic and inflammatory markers
   expression, and inhibited the protein expression of main molecules in
   the TLR4/NF-KB signaling pathway in TGF-β1-induced HK-2 cells. These
   results were consistent with previous research results obtained from
   adenine-induced CKD rat models. Then, we showed that the BYF
   anti-fibrotic and anti-inflammatory inhibition effects on the NF-κB
   pathway were diminished when TLR4 was silenced with siRNA in
   TGF-β1-induced HK-2 cells. Collectively, these findings demonstrated
   that the TLR4/NF-κB signaling suppression was an important
   anti-fibrotic and anti-inflammatory mechanism by which BYF partially
   healed CKD.

Conclusion

   Our results indicated that BYF could significantly reduce renal
   fibrosis and inflammation by TLR4/NF-κB signaling pathway inhibition.
   Although these preliminary findings could not fully explain the
   underlying mechanism of the BYF protective effect, they provided
   further support for clinical trials aiming to assess the BYF effects
   against CKD progression. However, whether BYF has a beneficial role in
   any non-adenine-induced CKD rat model needs to be further elucidated.
   Moreover, due to the TCM complex composition, new technologies are
   required to investigate the material basis and underlying mechanisms of
   BYF on CKD.

Acknowledgments