Abstract

Background

   The compromised performance of laying hens in the late phase of
   production relative to the peak production was thought to be associated
   with the impairment of intestinal functionality, which plays essential
   roles in contributing to their overall health and production
   performance. In the present study, RNA sequencing was used to
   investigate differences in the expression profile of intestinal
   functionality-related genes and associated pathways between laying hens
   in the late phase and peak phase of production.

Results

   A total of 104 upregulated genes with 190 downregulated genes were
   identified in the ileum (the distal small intestine) of laying hens in
   the late phase of production compared to those at peak production.
   These upregulated genes were found to be enriched in little KEGG
   pathway, however, the downregulated genes were enriched in the pathways
   of PPAR signaling pathway, oxidative phosphorylation and glutathione
   metabolism. Besides, these downregulated genes were mapped to several
   GO clusters in relation to lipid metabolism, electron transport of
   respiratory chain, and oxidation resistance. Similarly, there were
   lower activities of total superoxide dismutase, glutathione
   S-transferase and Na^+/K^+-ATPase, and reductions of total antioxidant
   capacity and ATP level, along with an elevation in malondialdehyde
   content in the ileum of laying hens in the late phase of production as
   compared with those at peak production.

Conclusions

   The intestine of laying hens in the late phase of production were
   predominantly characterized by a disorder of lipid metabolism,
   concurrent with impairments of energy production and antioxidant
   property. This study uncovers the mechanism underlying differences
   between the intestinal functionality of laying hens in the late phase
   and peak phase of production, thereby providing potential targets for
   the genetic control or dietary modulation of intestinal hypofunction of
   laying hens in the late phase of production.

   Keywords: Laying hen, Late phase of production, Intestinal
   functionality, Transcriptome, Lipid metabolism, Energy generation,
   Oxidation resistance

Background

   Layer industry is one of the key components contributing to sustainable
   food sources in the world. The late phase of production (defined as a
   period in which the egg production is less than 90%), accounts for a
   large part of the whole cycle of layer production, during which laying
   hens are known to be characterized by the declined production
   performance and poor egg quality as compared with those at peak
   production, resulting in a restricted economic benefit of layer
   production [[33]1, [34]2]. One crucial reason for the compromises of
   production performance and egg quality of laying hens in the late phase
   of production could be the corresponding impairment of intestinal
   functional state [[35]3, [36]4]. The important roles of intestinal
   functional state have been increasingly recognized in contributing to
   the overall health and production performance of poultry [[37]5,
   [38]6], probably because the intestine possesses a wide variety of
   different physiological functions such as barrier function, immune
   defense, lipid metabolism, detoxification and neuroendocrine function
   [[39]6–[40]9], in addition to serving as the principal site for
   nutrient absorption. Since there was a deterioration of intestinal
   functioning such as absorption and barrier dysfunction, immune and
   defense defects in older animals as compared with young animals
   [[41]10, [42]11], the laying hens in the late and peak phase of
   production were speculated to display distinct differences in terms of
   intestinal functioning. This could be supported by the findings that
   aged laying hens had a destructed intestinal structure and an increased
   susceptibility of gut mucosal system to lose its integrity, as well as
   being more vulnerable to intestinal inflammatory responses relative to
   the young counterparts [[43]12, [44]13].

   It seems that the intestinal hypofunction of laying hens in the late
   phase of production after having undergone the intensive metabolism at
   peak production is associated with the aging-related down-regulations
   of the expression of certain functional molecules in the intestine
   [[45]14, [46]15], as supported by the finding that the age-related
   decline in the absorption of nutrients (carbohydrates, lipids and amino
   acids) was linked to the reduced abundances of their transporters in
   the intestine of rats [[47]16, [48]17], besides, aging-induced disorder
   of energy generation in the intestine was responsible by the
   mitochondrial respiratory chain deficiency, being mediated by the
   reduced expression of cytochrome c oxidase and succinate dehydrogenase
   [[49]18]. To date, comprehensive knowledge on the age-related
   discrepancies of intestinal functions between laying hens at different
   production stages is poorly understood. And far less is known regarding
   the differences between the intestinal functions of laying hens in the
   late phase and peak phase of production at the molecular level.

   Digital expression profiling using next-generation sequencing promises
   to reduce or eliminate some weakness of microarrays. As one of the
   powerful next-generation sequencing techniques, RNA sequencing has
   expanded knowledge on the extent and complexity of transcriptomes
   [[50]19]. Application of transcriptomic has been considered as an
   available method for nutrigenomics and physiological genomics studies
   in chickens, in order to obtain valuable information about the
   molecular mechanisms associated with the identification of key genes
   and pathways for the physiological changes following various treatments
   [[51]20, [52]21]. In this study, the RNA next-generation sequencing was
   employed to reveal intestinal differences in transcriptome profiles of
   laying hens at different laying periods, aiming to identify the
   important genes and critical pathways associated with the underlying
   mechanism for differences between the complex intestinal functionality
   of laying hens in the late phase and peak phase of production, thereby
   providing potential targets for improving the performance of laying
   hens in the late phase of production.

Results

Biochemical indices of the layer intestine

   The layer intestine from LP group had a reduced (P < 0.05) T-AOC and
   lower (P < 0.05) activities of T-SOD and GST, along with a higher
   (P < 0.05) content of MDA as compared with those from PP group
   (Table [53]1). With regard to the indices associated with energy
   metabolism, there were reductions (P < 0.05) in Na^+/K^+-ATPase
   activity and ATP level, concomitant with a decreasing trend (P < 0.10)
   of the activities of ALP and Ca^2+/Mg^2+-ATPase in the layer intestine
   of LP group relative to PP group (Table [54]2).

Table 1.

   Comparison of intestinal antioxidant status^1 of laying hens between
   groups^2 (n = 8)
   T-SOD
   (U/mg prot.) GST
   (U/mg prot.) T-AOC
   (U/mg prot.) GSH
   (nmol/mg prot.) MDA
   (nmol/mg prot.)
   PP 65.84 ± 10.29^a 106.78 ± 30.97^a 11.80 ± 1.15^a 24.91 ± 8.19
   3.33 ± 0.58^b
   LP 52.99 ± 8.08^b 77.95 ± 20.51^b 8.49 ± 1.18^b 20.69 ± 7.60
   4.32 ± 0.74^a
   P-value 0.015 0.046 < 0.001 0.304 0.010
   [55]Open in a new tab

   ^a,b Values with different superscripts within the same column differ
   significantly (P < 0.05)

   ^1 T-SOD total superoxide dismutase, GST glutathione S-transferase,
   T-AOC total antioxidant capacity, GSH reduced glutathione, MDA
   malondialdehyde

   ^2 PP laying hens in the peak phase of production, LP laying hens in
   the late phase of production

Table 2.

   Comparison of intestinal enzyme^1 activities of laying hens between
   groups^2 (n = 8)
   ALP
   (U/mg prot.) Na^+/K^+-
   ATPase
   (U/mg prot.) Ca^2+/Mg^2+-
   ATPase
   (U/mg prot.) SDH
   (U/mg prot.) ATP (μmol/mg prot.)
   PP 3.45 ± 0.53 1.24 ± 0.32^a 1.19 ± 0.34 12.36 ± 4.82 0.81 ± 0.18^a
   LP 2.98 ± 0.34 0.89 ± 0.30^b 0.92 ± 0.26 9.99 ± 3.62 0.60 ± 0.18^b
   P-value 0.074 0.043 0.092 0.285 0.036
   [56]Open in a new tab

   ^a,b Values with different superscripts within the same column differ
   significantly (P < 0.05)

   ^1 ALP alkaline phosphatase, SDH succinate dehydrogenase, ATP adenosine
   triphosphate

   ^2 PP laying hens in the peak phase of production, LP laying hens in
   the late phase of production

Summary of RNA sequencing data

   As shown in Table [57]3, RNA-Seq generated more than 40,910,976 raw
   reads for each library, with an average of 52,873,687 and 49,344,174
   paired-end reads for the PP and LP groups, respectively. The GC
   contents of the libraries were ranged from 49.28 to 50.87%, which were
   very close to 50%. All the samples had at least 92.04% reads equal to
   or exceeding Q30. The majority of reads in each library were mapped to
   the Gallus gallus 5.0 assembly of the chicken genome, and the average
   mapping rates were 87.79 and 90.87% for PP and LP groups, respectively,
   which had an average of 84.32 and 87.53%, respectively, of the reads
   mapped to the chicken genome in an unique manner.

Table 3.

   Characteristics^1 of RNA sequencing reads of the layer intestine
   (n = 4)
   Samples^2 GC contents (%) Q30
   (%) Total reads Mapped reads Mapping
   ratio Unique mapping ratio
   PP1 50.67 92.88 58,014,476 52,888,432 91.16% 87.47%
   PP2 50.06 92.49 50,793,752 46,281,638 91.12% 87.63%
   PP 3 50.37 92.89 56,232,772 50,630,631 90.04% 86..52%
   PP4 50.87 93.19 46,453,748 36,633,802 78.86% 75.66%
   LP1 49.85 92.35 49,218,916 44,799,521 91.02% 87.79%
   LP2 49.94 93.40 63,324,840 58,066,099 91.70% 88.36%
   LP3 49.28 92.04 40,910,976 36,615,172 89.50% 86.26%
   LP4 50.16 93.35 43,921,962 40,084,927 91.26% 87.70%
   [58]Open in a new tab

   ^1GC guanine-cytosine, Q30 the proportion of bases with a Phred quality
   score greater than 30

   ^2PP laying hens in the peak phase of production, LP laying hens in the
   late phase of production

Identification of DGEs between groups

   There was an obvious difference in gene expression profile of the layer
   intestine between groups, as revealed by the principal component
   analysis plot (Additional file [59]1). A total of 294 DGEs were
   identified in the intestine between groups, including 104 upregulated
   and 190 downregulated genes in LP group relative to PP group (Fig.
   [60]1a). Volcano plot visualized the difference in the expression
   profile of intestinal genes in these two groups (Fig. [61]1b). To
   confirm the accuracy of RNA sequencing data, we randomly selected 12
   genes including 3 upregulated genes (GYS2, INSR and Claudin-2) and 9
   downregulated genes (SOD3, FABP1, FABP2, LPL, APOA1, TXN, NDUFS6, GSTM2
   and GSTA3). The expression levels of these genes were quantified using
   RT-PCR, and the results were consistent with the findings obtained by
   RNA-Seq (Fig. [62]2), suggesting that the RNA sequencing reliably
   identified differentially expressed mRNAs in the ileal transcriptome.

Fig. 1.

   [63]Fig. 1
   [64]Open in a new tab

   The differentially expressed genes (a) and their visualization by
   volcano plot (b) of the layer intestine in LP group relative to PP
   group (n = 4). LP, laying hens in the late phase of production; PP,
   laying hens in the peak phase of production

Fig. 2.

   [65]Fig. 2
   [66]Open in a new tab

   Validation of the differentially expressed genes (DEGs) by RT-PCR
   (n = 8). a Comparison (fold change) of the RNA-Seq data of LP group
   relative to PP group. b Individual variability of validated DGEs in
   RT-PCR between the PP and LP groups. LP, laying hens in the late phase
   of production; PP, laying hens in the peak phase of production. Values
   are means and standard deviations represented by vertical bars.
   Significance of RT-PCR data was set at P < 0.05, while significance of
   RNA-seq data was set at false discovery rate (FDR) < 0.05

Functional annotation of DGEs between groups

   To obtain valuable information for functional prediction of DEGs,
   searches were made on standard unigenes in the COG and GO databases.
   The DEGs between groups were functionally distributed into 21 COG
   categories (Additional file [67]2). Thereinto, the greatest number of
   DEGs were assigned to the category of general function prediction only
   (25.6%), followed by the category of lipid transport and metabolism
   (9.6%), posttranslational modification, protein turnover, chaperones
   (8.8%), inorganic ion transport and metabolism (7.2%). When mapped to
   the GO database, the DEGs were distributed into three major functional
   categories including biological progress, cellular component and
   molecular function (Fig. [68]3). The most abundant terms annotated to
   the DEGs in the category of biological progress were cellular process,
   single-organism process, and metabolic process. While the most abundant
   terms among the category of cellular component were cell, cell part,
   and organelle. Within the category of molecular function, the majority
   of DEGs were assigned to the subcategories of binding and catalytic
   activity.

Fig. 3.

   [69]Fig. 3
   [70]Open in a new tab

   Gene oncology (GO) classification of differentially expressed genes in
   the layer intestine between groups (n = 4)

Pathway enrichment analysis of DEGs between groups

   The upregulated genes in LP group relative to PP group were found to
   confer little association (Q > 0.05) with any KEGG pathway except for
   tending to be enriched (Q < 0.10) in the pathway of SNARE interactions
   in vesicular transport (Table [71]4). Comparatively, the downregulated
   genes in LP group relative to PP group were enriched (Q < 0.05) in the
   pathways of peroxisome proliferator-activated receptor (PPAR) signaling
   pathway (rich factor (RF) = 11.7), oxidative phosphorylation
   (RF = 8.3), and glutathione metabolism (RF = 13.2) (Table [72]5). In
   addition, these downregulated genes were tended to be enriched
   (Q < 0.10) in the pathways of drug metabolism-cytochrome P450
   (RF = 13.1), metabolism of xenobiotics by cytochrome P450 (RF = 12.4),
   and glycine, serine and threonine metabolism (RF = 11.8).

Table 4.

   Pathway analysis (top ten) of upregulated genes of the intestine of
   laying hens in LP group relative to PP group^1 (n = 4)
   Pathway name Ko_ID Richment
   _factor P-value Q-value
   SNARE interactions in vesicular transport ko04130 19.0 0.005 0.090
   Starch and sucrose metabolism ko00500 13.6 0.009 0.175
   Cardiac muscle contraction ko04260 9.1 0.002 0.374
   Focal adhesion ko04510 4.2 0.032 0.617
   ECM-receptor interaction ko04512 6.8 0.034 0.648
   Mismatch repair ko03430 15.1 0.064 1
   Cell adhesion molecules ko04514 4.6 0.068 1
   Adrenergic signaling in cardiomyocytes ko04261 4.5 0.071 1
   Hedgehog signaling pathway ko04340 7.4 0.127 1
   Gap junction ko04540 3.3 0.267 1
   [73]Open in a new tab

   ^1PP laying hens in the peak phase of production, LP laying hens in the
   late phase of production

Table 5.

   Pathway analysis (top ten) of downregulated genes of the intestine of
   laying hens in LP group relative to PP group^1 (n = 4)
   Pathway name Ko_ID Richment
   _factor P-value Q-value
   PPAR signaling pathway ko03320 11.7 < 0.001 0.002
   Oxidative phosphorylation ko00190 8.3 < 0.001 0.003
   Glutathione metabolism ko00480 13.2 < 0.001 0.009
   Drug metabolism - cytochrome P450 ko00982 13.1 0.001 0.059
   Metabolism of xenobiotics by cytochrome P450 ko00980 12.4 0.002 0.068
   Glycine, serine and threonine metabolism ko00260 11.8 0.002 0.079
   Carbon metabolism ko01200 5.5 0.006 0.222
   Glyoxylate and dicarboxylate metabolism ko00630 10.7 0.015 0.583
   Renal cell carcinoma ko05211 53.7 0.018 0.740
   Circadian rhythm ko04710 40.3 0.025 0.984
   [74]Open in a new tab

   ^1PP laying hens in the peak phase of production, LP laying hens in the
   late phase of production

   In the PPAR signaling pathway, fatty acid-binding protein 1
   (FABP1|FC = 0.38), FABP2 (FC = 0.49), FABP3 (FC = 0.41), FABP5
   (FC = 0.69), FABP6 (FC = 0.58), lipoprotein lipase (LPL|FC = 0.56),
   apolipoprotein A1 (APOA1|FC = 0.56), sterol carrier protein 2
   (SCP2|FC = 0.75) and perilipin-1 (PLIN1|FC = 0.59) were lower expressed
   in LP group relative to PP group (Table [75]6). While the downregulated
   genes in LP group that mapped to the pathway of oxidative
   phosphorylation were identified as following: NADH dehydrogenase
   (ubiquinone) Fe-S protein 6 (NDUFS6|FC = 0.76), NADH dehydrogenase
   (ubiquinone) 1 alpha subcomplex subunit 1 (NDUFA1|FC = 0.66), NDUFA8
   (FC = 0.74), NDUFB2 (FC = 0.69), NDUFB9 (FC = 0.76),
   ubiquinol-cytochrome c reductase subunit 9 (UQCR9|FC = 0.65), ATP
   synthase subunit d (ATP5H|FC = 0.72), ATP synthase subunit e
   (ATP5I|FC = 0.68), ATP synthase subunit f (ATP5J|FC = 0.69), ATP
   synthase subunit g (ATP5L|FC = 0.66), and V-type proton ATPase subunit
   G 1 (ATP6V1G1|FC = 0.76). The downregulated genes in LP group that
   implicated in the pathway of glutathione metabolism were glutathione
   S-transferase (GST) omega-1 (GSTO1|FC = 0.73), GST mu 2
   (GSTM2|FC = 0.59), GST alpha 3 (GSTA3|FC = 0.69) and ornithine
   decarboxylase 1 (ODC1|FC = 0.68). Remarkably, the downregulated
   expression of GSTO1, GSTM2 and GSTA3 in LP group also mediated the
   decreasing trend of the pathways of drug metabolism-cytochrome P450 and
   metabolism of xenobiotics by cytochrome P450.

Table 6.

   The differentially expressed genes^1 (|fold change| > 1.3 at a false
   discovery rate < 0.05) that mapped to the enriched pathways (n = 4)
   KEGG pathways Pathway_ID Differentially expressed genes (Fold change)
   PPAR signaling pathway ko03320 FABP1 (0.38), FABP2 (0.49), FABP3
   (0.41), FABP5 (0.69), FABP6 (0.58), LPL (0.56), APOA1 (0.56), SCP2
   (0.75), PLIN1 (0.59)
   Oxidative phosphorylation ko00190 NDUFS6 (0.76), NDUFA1 (0.66), NDUFA8
   (0.74), NDUFB2 (0.69), NDUFB9 (0.76), UQCR9 (0.65), ATP5H (0.72), ATP5I
   (0.68), ATP5J (0.69), ATP5L (0.66), ATP6V1G1 (0.76)
   Glutathione metabolism ko00480 GSTA3 (0.69), GSTM2 (0.59), GSTO1
   (0.73), ODC1 (0.68)
   Drug metabolism-cytochrome P450 ko00982 GSTA3 (0.69), GSTM2 (0.59),
   GSTO1 (0.73)
   Metabolism of xenobiotics by cytochrome P450 ko00980 GSTA3 (0.69),
   GSTM2 (0.59), GSTO1 (0.73)
   Glycine, serine and threonine metabolism ko00260 LOC418544 (0.55), GLDC
   (0.51), LOC107051323 (0.51)
   [76]Open in a new tab

   ^1FABP fatty acid-binding protein, LPL lipoprotein lipase, APOA
   apolipoprotein A, SCP sterol carrier protein, PLIN perilipin, NDUFS
   NADH dehydrogenase (ubiquinone) Fe-S protein, NDUFA NADH dehydrogenase
   (ubiquinone) 1 alpha subcomplex subunit, NDUFB NADH dehydrogenase
   (ubiquinone) 1 beta subcomplex subunit, UQCR ubiquinol-cytochrome c
   reductase subunit, ATP5H ATP synthase subunit d, ATP5I ATP synthase
   subunit e, ATP5J ATP synthase subunit f, ATP5L ATP synthase subunit g,
   ATP6V1G V-type proton ATPase subunit G, GSTA3 glutathione S-transferase
   alpha 3, GSTM2 glutathione S-transferase mu 2, GSTO1 glutathione
   S-transferase omega-1, ODC1 ornithine decarboxylase 1, LOC418544
   cystathionine beta-synthase-like isoform, GLDC glycine dehydrogenase,
   LOC107051323 glycine hydroxymethyltransferase

GO clustering analysis of DEGs related to lipid metabolism, energy production
and oxidation resistance

   Since pathway analysis revealed that DEGs were predominantly enriched
   in the pathways of PPAR signaling pathway, oxidative phosphorylation
   and glutathione metabolism, the DEGs were subjected to deep-level GO
   clustering analysis in relation to lipid metabolism, energy generation
   and oxidation resistance, in order to better understand the network
   that responsible for the difference between groups. As shown in
   Table [77]7, there were reductions (Q < 0.05) of the clusters of
   transport, regulation of intestinal cholesterol absorption,
   phospholipid efflux, positive regulation of cholesterol esterification,
   reverse cholesterol transport, ATP synthesis coupled proton transport,
   hydrogen peroxide catabolic process, and removal of superoxide radicals
   within the category of biological process in LP group as compared to PP
   group. In terms of the category of cellular component, the layer
   intestines from LP group had less (Q < 0.05) clusters of very-low
   density lipoprotein particle and mitochondrial proton-transporting ATP
   synthase complex than those from PP group. Within the category of
   molecular function, we detected downregulated (Q < 0.05) clusters of
   lipid binding, transporter activity, phosphatidylcholine-sterol
   O-acyltransferase activator activity, cholesterol transporter activity,
   hydrogen ion transmembrane transporter activity, glutathione
   transferase activity, and antioxidant activity in LP group as compared
   with PP group.

Table 7.

   Gene oncology (GO) clustering analysis of differentially expressed
   genes^1 (|fold change| > 1.3 at a false discovery rate < 0.05) in
   relation to lipid metabolism, energy production and oxidation
   resistance (n = 4)
   GO terms GO_ID Differentially expressed genes (fold change) P-value
   Q-value
   Biological Process
    Transport GO:0006810 FABP6 (0.58) 0.010 0.010
    Regulation of intestinal cholesterol absorption GO:0030300 APOA1
   (0.56), APOA4 (0.52) < 0.001 0.004
    ATP synthesis coupled proton transport GO:0015986 ATP5H (0.72), ATP5I
   (0.68), ATP5L (0.66) < 0.001 0.006
    Phospholipid efflux GO:0033700 APOA1 (0.56), APOA4 (0.52) < 0.001
   0.011
    Positive regulation of cholesterol esterification GO:0010873 APOA1
   (0.56), APOA4 (0.52) < 0.001 0.021
    Hydrogen peroxide catabolic process GO:0042744 PRDX1 (0.74), APOA4
   (0.52) < 0.001 0.035
    Reverse cholesterol transport GO:0043691 APOA1 (0.56), APOA4 (0.52)
   < 0.001 0.035
    Removal of superoxide radicals GO:0019430 PRDX1 (0.74), APOA4 (0.52)
   < 0.001 0.047
   Cellular Component
    Mitochondrial proton-transporting ATP synthase complex GO:0000276
   ATP5H (0.72), ATP5I (0.68), ATP5L (0.66) < 0.001 < 0.001
    Very-low density lipoprotein particle GO:0034361 APOA1 (0.56), APOA4
   (0.52) 0.001 0.035
   Molecular Function
    Lipid binding GO:0008289 FABP1 (0.38), FABP2 (0.49), FABP3 (0.41)
   0.004 0.009
    Transporter activity GO:0005215 FABP6 (0.58) 0.008 0.008
    Antioxidant activity GO:0016209 APOA4 (0.52), FABP1 (0.38) 0.001 0.008
    Phosphatidylcholine-sterol O-acyltransferase activator activity
   GO:0060228 APOA1 (0.56), APOA4 (0.52) < 0.001 0.002
    Glutathione transferase activity GO:0004364 GSTA3 (0.69), GSTM2
   (0.59), GSTO1 (0.73) < 0.001 0.005
    Hydrogen ion transmembrane transporter activity GO:0015078 ATP5H
   (0.72), ATP5I (0.68), ATP5L (0.66) < 0.001 0.007
    Cholesterol transporter activity GO:0017127 APOA1 (0.56), APOA4 (0.52)
   < 0.001 0.027
   [78]Open in a new tab

   ^1FABP fatty acid-binding protein, APOA apolipoprotein A, ATP5H ATP
   synthase subunit d, ATP5I ATP synthase subunit e, ATP5J ATP synthase
   subunit f, ATP5L ATP synthase subunit g, PRDX1 peroxiredoxin-1, GSTA3
   glutathione S-transferase alpha 3, GSTM2 glutathione S-transferase mu
   2, GSTO1 glutathione S-transferase omega-1

Discussion

   PPAR signaling pathway is a key regulator of metabolism of the
   intestine [[79]22], which together with the liver are considered as
   important sites for lipid metabolism [[80]7]. In the present study, the
   lipid metabolism-related genes such as FABP1, FABP2, FABP3, FABP5,
   FABP6, LPL and APOA1 that mapped to PPAR signaling pathway were
   downregulated in LP group relative to PP group. FABP multigene can code
   for diversified kinds of FABPs such as liver-type FABP (encoded by
   FABP1), intestinal-type FABP (encoded by FABP2), heart-type FABP
   (encoded by FABP3), epidermal-type FABP (encoded by FABP5), and
   ileal-type FABP (encoded by FABP6) [[81]23]. These proteins display
   high-affinity binding for fatty acids and other hydrophobic ligands,
   facilitating the transport of lipids to the specific compartments of
   cells for storage or oxidation [[82]24]. Although FABPs share a highly
   conserved structure, each of them has its own sequence and exhibits
   distinct affinity for ligand preferences [[83]25]. Specifically,