Radix Polygoni Multiflori Praeparata (No. 1901026) was purchased from Sichuan Neautus Traditional Chinese Medicine Co., Ltd., (Chengdu, China), and authenticated by professor Pei Jin, department of pharmacognosy, Chengdu University of Traditional Chinese Medicine, and the samples were deposited at the Herbal Medicine Museum. A high-fat diet (No. 2019022301) was provided by Biotech-HD Co., Ltd., (Beijing, China), and the diet composition was reflected in Table 1. HPLC grade acetonitrile, methanol, and formic acid were obtained from Merck Chemicals (Shanghai, China), Wokai Chemical Technology Co., Ltd. (Shanghai, China), and TCI Chemical Industry Development Co., Ltd. (Shanghai, China), respectively. The kit for detecting the levels of total cholesterol (TC) (No. 20200114), triglyceride (TG) (No. 20200114), low-density lipoprotein cholesterol (LDL-C) (No. 20200114), high-density lipoprotein cholesterol (HDL-C) (No. 20200114), Alanine transaminase (ALT) (No. 20200114), and Aspartate transaminase (AST) (No. 20200114) in serum and liver tissue were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). BCA Protein Quantification Kit (No. A00445) was obtained from Multi Sciences Biotech Co., Ltd. TRIzol reagents (No. 175702) used to extract total RNA from the liver and ileum tissue was purchased from Ambion Life Technologies (Carlsbad, CA, United States). 5X All-In-one MasterMix (No. 65170013) and Eva Green 2X RT-qPCR MasterMix-Low RoX (No. 65170013) were purchased from Applied Biological Materials Inc. (Richomnd, BC, Canada). PCR primer sequences were synthesized by TSINGKE Biological Technology (Chengdu, China). Other reagents used in this experiment were provided by Kelong Chemical Reagent Factory (Chengdu, China).

After precise weighing and sieving, 100 g of RPMP crude powder was placed in a round bottom flask with ten times the amount of 50% ethanol and soaked for 30 min, heat reflux for 60 min, filter. Then add eight times the amount of 50% ethanol, heat reflux for 60 min, filter, and combine the filtrate and the filtrate was concentrated by rotary evaporation at 45°C to contain 1 g of raw drug per ml and stored at 4°C.

Quantitative analysis of RPMP ethanol extract by HPLC based on a ZORBAX C18 analytical column (4.6 mm × 250 mm, 5 μm) with a pre-column ZORBAX C18 (4.6 mm × 12.5 mm, 5 μm). Refer to our previously established method (Yu et al., 2020). The gradient elution was carried out with 0.1% formic acid (A) and acetonitrile (B) as mobile phases at a column temperature of 30°C, a detection wavelength of 275 nm, and a flow rate of 1 ml/min according to the following mobile phase composition: (0–5 min) 5–10% B, (5–30 min) 10–22% B, (30–38 min) 22–25% B, (38–48 min) 25–32% B, (48–55 min) 32–45% B, (55–65 min) 45–85% B, (65–70 min) 85–95% B, (70–72 min) 95–95% B.

All experimental procedures were conducted in accordance with animal experimental protocols and guidelines approved by the Animal Ethics Committee of the Chengdu University of Traditional Chinese Medicine (Approval No. AEC-201611). 70 male Kunming mice (km) from Chengdu Dossy Experimental Animal Co., LTD. [Chengdu, China, No. SCXK (Chuan) 014–028] were housed for 1 week under standard environment (temperature of 25 ± 2°C, the humidity of 50 ± 5%, and a 12 h light/12 h dark cycle) with free access to sterile water and normal chow. All efforts were made to minimize animal suffering. And at the end of the adaptation, the following experiments were performed:

1) To determine the threshold for the therapeutic dose of RPMP ethanol extract, a dose conversion for mice was performed based on the guideline dosage of 9 g of RPMP for humans provided in the Chinese Pharmacopoeia (Mouse dose = Human dose/60 kg × 9.01). The km mice were randomly divided into five groups (n = 6/group) and treated with normal diet (ND) (CON), HFD (MOD), HFD and different doses of RPMP ethanol extract [0.68 g/kg (0.68 g/kg RPMP), 1.35 g/kg (1.35 g/kg RPMP), 2.70 g/kg (2.70 g/kg RPMP)] for 3 weeks. At the end of week 3, blood samples were collected from each group of mice.

2) To further investigate the role played by RPMP ethanol extract in the development of NAFLD, km mice were randomly divided into two groups for 3 weeks of ND (CON) (n = 8) and HFD administration (n = 32). And then the HFD-fed mice were randomly divided into HFD (MOD), HFD, and different doses of RPMP ethanol extract [0.34 g/kg (0.34 g/kg RPMP), 0.68 g/kg (0.68 g/kg RPMP), 1.35/kg (1.35 g/kg RPMP)] (n = 8/group) groups for 5 weeks of treatment, and continued to be supplemented with ND and HFD treatment during this process. Fecal samples were collected at week 8. And at the end of the experiment, blood, liver tissue, epididymal adipose tissue, and ileal tissue were collected.

Mice were treated with RPMP ethanol extract at 9:00 am and 18:00 pm daily. The body weight of mice was measured weekly and treatment volumes were adjusted. The volume of the extract administered was calculated according to the following formula. In order to reduce the interference of treatment volume, double distilled water was used to dilute RPMP ethanol extract with the initial concentration of 1 g/ml to 0.0625 g/ml (0.34 g/kg RPMP), 0.125 g/ml (0.68 g/kg RPMP), and 0.25 g/ml (1.35/ kg RPMP) for different dose groups. Also, during the RPMP ethanol extract intervention, the CON and MOD groups were given the same volume of double distilled water according to body weight. The     administration   volume   of   each   mouse ( ml ) = Body   weight   of   mice ( kg ) × Administration   dose ( g / kg ) The   concentration   of   the   extract ( g / ml )

After overnight fasting, fasting blood glucose values were measured in mice using a blood glucose meter (ACCU-CHEK Performa, Performa).

Blood samples. The blood of mice was obtained through the orbital vein plexus. The collected blood samples were allowed to stand at room temperature for 1 h, then centrifuged at 2,500 r/min for 15 min at 4°C. The supernatants were aspirated and stored in separate packs.

Tissue sample. The sacrifice of mice was accomplished by cervical dislocation. Abdominal dissection was performed to extract the liver, epididymal fat, and ileal tissue. The blood was removed by rinsing in ice-cold saline, and filter paper was swabbed dry before weighing and recording the weight of liver and epididymal adipose tissue. And the tissue samples were stored in 4% paraformaldehyde or liquid nitrogen.

Fecal samples. The mice were fixed in an ultra-clean worktable, their tails were lifted, and their abdomens were gently stroked with fingers to stimulate defecation. Fresh feces were collected in sterile lyophilized tubes, quickly placed in liquid nitrogen, and transferred to a refrigerator (−80°C) for storage.

Serum TC, TG, LDL-C, HDL-C, ALT, and AST levels were measured using the above-preserved blood samples and according to the kit instructions. Take 0.1–1 g liver tissue was homogenized by adding 0.86% cold saline into pipette in the proportion of homogenate medium: tissue weight (1:9). The prepared 10% homogenate was centrifuged at 2,000 r/min for 10 min, and the supernatant was reserved. Then, the liver levels of TC, TG, LDL-C, HDL-C, ALT, and AST were also measured according to the kit instructions.

The liver and colon samples were fixed with 4% paraformaldehyde, embedded in paraffin and sectioned (5 μm), stained with hematoxylin-eosin (HE). And lipid accumulation in the liver was observed by staining frozen sections of the liver with an oil red O stain. The frozen sections were fixed in fixative for 15 min, washed in PBS, dried and immersed in oil red O staining solution for 10 min, and then background fractionated with 60% isopropyl alcohol, and finally sealed with glycerol gelatin sealer after hematoxylin re-staining. And then observed under a light microscope (CX21FS1, OLMPUS, Japan) and the pathological changes of the liver and colon were photographed under a microscopic imaging system (DM1000, Leica, Germany).

The genomic DNA of fecal samples was extracted by using QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany), then detected by electrophoresis with 0.8% agarose gel. The V4 region of the bacterial 16S rDNA gene was amplified by polymerase chain reaction (PCR) with 515F–806R primer set (515F:5′-GTGYCAGCMGCCGCGGTAA-3′,806R:5′-GGACTACHVGGGTWTCTAAT-3′), three replicates per sample, each PCR reaction was terminated in the linear amplification period. PCR product of the same sample was mixed and detected by electrophoresis with 2% agarose gel, then purified by Qiagen Gel Extraction Kit (Qiagen, Germany). PCR products quality were evaluated on Qubit@ 2.0 Fluorometer (Thermo Scientific, United States), and then mixed in equidensity ratios. Sequencing libraries were prepared using the TruSeq DNA PCR-Free Sample Prep Kit (Illumina, No. FC-121-3001/3003). The library was sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated.

QIIME pipeline (qiime2) was used to process and analyze the raw data. Final Clean Reads were produced after the removal of chimera sequences in the tags, which were detected by the gold database (microbiomeutil-r20110519). Operational taxonomic units were clustered at 97% similarity, and sequences were taxonomically assigned against the SILVA database (Release_132). The raw data of libraries generated during this study is publicly available at the Sequence Read Archive (SRA) portal of NCBI (https://www.ncbi.nlm.nih.gov/sra/) under accession number PRJNA769303.

The quantitative analysis of mouse fecal BAs was done by liquid chromatography-mass spectrometry (LC-MS). Briefly, 10 mg of mouse feces was accurately weighed and suspended in 1 ml of methanol solution (−20°C). Ultrasonication at room temperature for 30 min, centrifugation at 12,000 rpm/min for 10 min at 4°C using a frozen centrifuge (H1850R, Cence). Then, 300 μl of the supernatant was filtered into the assay bottle with the assistance of a 0.22 μm membrane. Based on an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters), the elution was performed at a column temperature of 40°C and a flow rate of 0.25 ml/min using 0.01% formic acid (A) and acetonitrile (B) as mobile phases according to the following gradient of mobile phase composition: (0–4 min)25% B, (4–9 min)25–30% B, (9–14 min)30–36% B, (14–18 min)36–38% B, (18–24 min) 38–50% B, (24–32 min) 50–75% B, (32–35 min) 75–100% B, (35–38 min) 100–25% B. And scanned using multiple reaction monitoring (MRM).

Total RNA was extracted from liver and ileal tissues by using TRIZOL reagent and resuspended in 50 μl of enzyme-free water. RNA purity and integrity verified by optical density 260/280 values measured by Nucleic Acid/Protein Analyzer and Type 1 nucleic acid dye on 1% agarose gel. Reverse transcription for cDNA synthesis with 5X All-In-One MasterMix. Set reaction conditions according to the manufacturer’s instructions to complete the RT-qPCR reaction. Then, the relative trends of the genes were analyzed according to the amplification curves and the calculation method based on 2^−∆∆CT. Primer sequences were reflected in Table 2.

SPSS (Version 25.0) was used for statistical analysis. All experimental data were expressed as mean ± SEM. Comparisons between two groups and more than two groups in the experiment were done by Student’s t-test and one-way analysis of variance (ANOVA), respectively. And p < 0.05 was considered statistically significant.

According to Chinese Pharmacopoeia, the contents of TSG and free anthraquinone (calculated by the total amount of emodin and physcion) in the ethanol extract of RPMP were measured to determine whether they meet the quality standards (Commission, 2020). The quantitative results showed that the contents of TSG and free anthraquinone in the ethanol extract of RPMP were 4.01 and 0.21%, respectively, (Figure 1).

Prophylactic administration was taken to investigate the effective therapeutic dose of RPMP ethanol extract on serum biochemical parameters in mice under HFD administration. During the 3-weeks administration, there were no significant differences were observed between the dose groups in terms of the trend of body weight changes in the mice (Figure 2A). However, in terms of serum biochemical indicators, although each dose group did not show significant modulating effects compared with HFD mice, the results of each biochemical indexes showed that the 2.70 g/kg dose group was inferior to the 1.35 g/kg dose group in regulating serum lipid metabolism and liver function indexes induced by HFD (Figures 2B–E). The reason why RPMP ethanol extract did not show dose-dependence may be due to the fact that the dose of RPMP exceeded the threshold of treatment, and which disturbed the normal metabolic function of the liver, thus causing abnormal liver biochemical indexes. And during a 42-days study on the lipid regulation of RPMP in SD rats induced by high-fat diet (HFD), compared with middle dose (1.620 g/kg), low dose (0.810 g/kg), and high dose (3.240 g/kg) RPMP showed better effects in regulating liver lipid metabolism (TC, TG, LDL-C, HDL-C); However, in serum biochemical parameters, the medium dose of RPMP showed better results compared to the low and high doses (Li et al., 2012). And the middle dose (140 mg/kg) of liver TC was better than the low dose (70 mg/kg) and the high dose (280 mg/kg) of liver TC indices in 8 weeks Wistar rats treated with RPMP(Lin et al., 2020). And studies have shown that this phenomenon can return to normal after discontinuation of the drug. Therefore, a dose of 1.35 g/kg was chosen as the maximum dose of RPMP ethanol extract intervention in the follow-up experiments.

Obesity is closely related to the occurrence and development of NAFLD. Mice induced by HFD gained 113.93% body weight at the end of the experiment compared to the beginning, and this trend was significantly suppressed by the intervention of RPMP ethanol extract (Figure 3A). However, there was no significant difference in the food intake of mice between the groups (Figure 3B). HFD induced not only an increase in body weight, but also a significant increase in serum TC, TG, and LDL-C levels. However, significantly decreased HDL-C levels in lipid metabolism in mice. When the mice were treated with RPMP ethanol extract, the increase of serum TC, TG, and LDL-C levels was inhibited, while HDL-C levels were restored. In addition, serum levels of ALT and AST, markers of liver injury, which were significantly elevated in HFD administered mice, were also significantly improved by the intervention of RPMP ethanol extract. And this temporarily provided the ability of RPMP to improve the abnormalities of serum biochemical parameters caused by HFD (Figures 3C–H).

Pathological changes in the liver can directly reflect intrahepatic lipid droplet aggregation and inflammatory infiltration. HE staining observed that ND-fed mice had intact liver lobules, clear hepatic cord structure, rich hepatocyte cytoplasm, normal morphology and structure, no obvious dilatation or extrusion of liver sinusoids, and no obvious inflammation. In contrast, in HFD-fed mice, the tissues exhibited a large amount of hepatocyte steatosis, with round vacuoles of varying sizes visible in the cytoplasm, and focal infiltration of a small number of lymphocytes was seen around the local central vein. With the administration of different doses of RPMP ethanol extract, the hepatocellular steatosis present in the tissues was gradually suppressed, with clear hepatic cord structure, no significant dilatation or extrusion of the hepatic sinusoids, and no significant inflammation (Figure 4A).

Additionally, lipid deposition in the liver was assessed by oil red O staining. The results showed that, compared with ND, a large area of lipid tissue was stained red in the HFD-fed mice, demonstrating the presence of a large amount of lipid accumulation. In contrast, the degree of redness in the tissues decreased in a dose-dependent manner after the administration of RPMP ethanol extract. And these results showed that RPMP could reduce liver lipid deposition to a certain extent (Figure 4B).

The liver and epididymal fat weights of mice were examined, and it was found that the liver and epididymal fat weights of HFD-induced mice increased by 59.51 and 107.15%, respectively, compared with those of ND-fed mice. In contrast, the liver and epididymal fat weight of mice treated with RPMP ethanol extract showed a significant reduction relative to that of mice administered with HFD (Figures 5A,B). Meanwhile, an 8-weeks HFD administration resulted in a 16.54% increase in fasting blood glucose levels (GTT) in mice. In contrast, after RPMP ethanol extract treatment, fasting blood glucose levels in mice decreased, with a 13.31% decrease in the 1.35 g/kg RPMP-dose group (Figure 5C).

The liver is the main metabolic organ responsible for lipid and lipoprotein. Compared with ND-fed mice, we found that liver levels of TC, TG, and LDL-C showed an increase in the same way as in serum, while HDL-C also showed a tendency to decrease under the effect of HFD. Undoubtedly, these trends seem to recover in a dose-dependent manner after the intervention of RPMP ethanol extract (Figures 5D–G). Similarly, in HFD-fed mice, the levels of liver function indicators (ALT and AST) were significantly elevated, and which was significantly inhibited by the ethanol extract of RPMP (Figures 5H,I). Accordingly, it was concluded that RPMP ethanol extract could improve the abnormal lipid metabolism parameters and elevated liver function indexes caused by HFD.

The unique anatomical position of the intestine in relation to the liver makes the liver the first organ exposed to intestinal-derived factors, and thus the composition of the microbiota was examined next. A healthy microbial community was characterized by rich microbial diversity, which can reflect the stability and reduction ability of the ecosystem. The Chao 1 and Shannon indices, which were used to reflect microbial community diversity, showed a tendency to decrease under the effect of HFD compared to ND-fed mice, where a 10% loss of Shannon index was observed, which was suppressed when RPMP ethanol extract was given (Figure 6A). Meanwhile, Principal Co-ordinates Analysis (PCoA) results based on weighted Unifrac distances showed that mouse samples administrated with HFD were divided into different clusters with ND mice, and the administration of RPMP ethanol extract appeared to be reshaping the intestinal microbial community disrupted by HFD (Figure 6B).

Bacteroides abundance was reduced and Firmicutes abundance was increased in the microbial community of mice subjected to HFD. In contrast, the increased Firmicutes/Bacteroides (F/B) ratio was reduced under RPMP ethanol extract intervention (Figures 6C,D). The genus-level clustering heat map showed that separate clusters were formed between each group, but the samples from the RPMP ethanol extract were not completely separated from the ND-fed mice compared to the HFD-fed mice (Figure 7A). Further analysis was conducted and found that Bacteroides and short-chain fatty acids (SCFAs) producing bacteria Phascolarctobacterium exhibited a reduction in HFD intervention, where butyrate in SCFAs was able to reduce fat deposition and insulin resistance through lipoprotein-activated β-oxidation and cholesterol transport (Elvira-Torales et al., 2019). However, the reduced abundance of Bacteroides, Phascolarctobacterium were recovered after the administration of RPMP ethanol extract (Figures 6C, 7B). Desulfovibrio is a gram-negative bacterium that produces endotoxins and has been shown to increase intestinal permeability and production of enterogenic factors (mainly LPS) (Zhao et al., 2019). The increase in Desulfovibrio abundance caused by HFD was effectively reduced after RPMP ethanol extract treatment (Figure 6C).

Since the intervention of RPMP ethanol extract can affect the biochemical parameters and the structural composition of IM in mice administered with HFD, Pearson’s correlation was used to find the relationship between biochemical indicators and IM species in mice. As observed for changes in the abundance of IM species, there was a negative correlation between the decrease in the abundance of Bacteroides and Phascolarctobacterium and the changes in serum and liver biochemical parameters caused by HFD (Figures 8A,B). Moreover, in this process, Ruminococcaceae UCG-002, Ruminococcaceae UCG-013, Ruminiclostridium 9, Ruminococcaceae UCG-003, Ruminococcaceae UCG-014, and other Species of the Ruminococcaceae show inconsistent relationships, which was thought to be due to the fact that the Ruminococcaceae was a very heterogeneous family -containing both harmful and beneficial bacteria (Kong et al., 2019). Therefore, it was concluded that RPMP ethanol extract has a remodeling effect on the disorder of IM structure caused by HFD.

A major consequence of HFD induction was an increase in intestinal permeability. Disruption of the intestinal epithelial barrier increases the risk of liver exposure to enteric-derived factors. HE staining of the intestine showed that the intestinal wall of mice under the effect of HFD was obviously damaged, and the villi were shed and highly reduced. The morphological structure of the villi was restored to varying degrees after the administration of RPMP ethanol extract (Figure 9A).

The mRNA expression levels of tight junction (TJ) molecules, such as zonula occluden-1 (ZO-1), Junction adhesion molecule (JAM), and Occludin, which serve as markers of intestinal epithelial barrier integrity, were significantly downregulated under the management of HFD. And, the reduced gene expression levels were in line with the changes in IM structure. And as expected, administration of RPMP ethanol extracts prevented the reduction of mRNA expression levels of ZO-1, JAM, and Occludin (Figure 9B).

The ability of adjacent intestinal epithelial cells to form TJ was crucial for the formation and maintenance of the intestinal epithelial barrier. As a dynamic regulatory structure, it plays a double-sided role, preventing potentially harmful intestinal-derived factors from entering the body’s circulation, while at the same time providing the body with the absorption of essential nutrients and ions, etc. However, several results have shown that the function of TJ was influenced by the level of BAs in the organism (Münch et al., 2007; Raimondi et al., 2008). Increased levels of ZO-1, Occludin protein expression were found in the intestine of HFD-fed F11r ^−/− mice after sequestration of BAs (Gupta et al., 2020). And, in studies of animal models of NAFLD, supplementation with BAs and treatment of BAs-related receptors have also been used (McMahan et al., 2013; Wang et al., 2018). Thus, the composition structure of BAs in mouse feces was next examined.

The HFD-induced pool of mouse BAs was more significantly altered in terms of CA, CDCA, TCA, TCDCA, TLCA, GCA, GDCA, T-α-MCA, T-β-MCA, and other species. Among them, an increase in CA concentration could lead to an increase in the abundance of Firmicutes and a decrease in the abundance of Bacteroidetes (Staley et al., 2017). Also in patients with cirrhosis, an increase in the potentially pathogenic bacteria Enterobacteriaceae showed a positive correlation with the concentration of CDCA present (Kakiyama et al., 2013). Not only that, the study showed that the IM structure of liver tumor mice was significantly altered, in which DCA, TCA, TCDCA, and TLCA together contributed to the development of liver tumors. And this confirms the mutual crosstalk between BAs and IM.

T-β-MCA downregulated fibroblast growth factor 15 (FGF15) expression through inhibition of intestinal farnesoid X receptor (FXR) and, induced fibroblast growth factor receptor 4 (FGFR4)-dependent cytochrome P450 family seven subfamily A member 1 (CYP7A1) activation through inhibition of JUN transcriptional activity, thereby regulating the increased biosynthesis of BAs. In contrast, in mice, low levels of T-β-MCA activated intestinal FXR signaling, which explained the abnormally elevated intestinal FXR expression in our results (Sayin et al., 2013). Meanwhile, T-α-MCA and T-β-MCA were formed as α-MCA and β-MCA by the deconjugation of bile salt hydrolases (BSH). Bacteroides, Clostridium, Firmicutes, Bifidobacterium, Lactobacillus, Listeria, and many other microorganisms are able to activate the production of BSH. Thus, it was suggested that the abnormal elevation of α-MCA and β-MCA expression in HFD-induced mice might be due to the disordered IM structure (Figure 10A). And this may be responsible for the low level of T-β-MCA as well as the abnormal expression of intestinal FXR genes. These BAs species, which were altered by HFD administration, have recovered after treatment with RPMP ethanol extract (Figures 10A,B).

Since the administration of HFD caused a change in the composition of the IM and BAs pools in mice, correlation analysis was used to examine whether there was a dynamic relationship between the altered IM and BAs. We noticed a positive correlation between hydrophobic BAs –LCA, DCA, CDCA, CA, and UDCA, which are cytotoxic, and the abundance of most IM species (Figures 11A,B). Further, TDCA, alpha-MCA, beta-MCA, and CDCA were found to have more nodes in the correlation network compared to other species (Figure 10C). TDCA is a conjugate of deoxycholic acid and taurine in the form of sodium salt. In NASH patients, a more than 5-fold increase in TDCA metabolite levels compared to normal organism levels was observed (Lake et al., 2013). Meanwhile, abnormally elevated serum TDCA levels were found in high-fat diet-induced NASH mice (He et al., 2021). In our results, we also observed a significant increase in TDCA levels in mice induced by a high-fat diet. In addition, the cytotoxic hydrophobic bile acid CDCA also has multiple nodes (Figure 10C). A recent study also showed that increased transmission of CDCA to the colon, induced by a high-fat diet, disrupts the intestinal epithelial barrier (Gupta et al., 2020). We also observed a significant increase in the level of CDCA in the feces of mice induced by a high-fat diet, and the potency of FXR activation by endogenous bile acids is CDCA > LCA = DCA (Gonzalez et al., 2016). And this may explain the abnormal elevation of intestinal FXR gene expression in mice induced by a high-fat diet.

Cholesterol is a substrate for the synthesis of BAs, and the mRNA expression levels of ATP binding cassette subfamily A member 1 (ABCA1), scavenger receptor class B member 1 (SR-B1), and low-density lipoprotein receptor (LDLR), key genes responsible for transporting excess cholesterol from the diet and surrounding tissues to the liver, were significantly reduced by the intervention of HFD, while the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), responsible for the synthesis of cholesterol in the liver, was significantly increased. And, the mRNA expression of CYP7A1, the rate-limiting enzyme in the classical pathway of BAs synthesis, and mitochondrial sterol 27-hydroxylase (CYP27A1), which initiates the first step of the alternative synthesis pathway, was indeed significantly reduced (Figure 12A). That can be an important cause of cholesterol buildup in the liver.

In addition, bile salt export pump (BSEP), multidrug resistance protein 2 (MRP2), sodium taurocholate cotransporting polypeptide (NTCP), and organic anion transporting polypeptide (OATP-1) were significantly reduced in mice treated with HFD, except for the increased expression of the multidrug resistance protein 4 (MPR4) gene (Figure 12B). These genes were involved in the transport, release, and reabsorption of BAs in vivo.

The synthesis of BAs is regulated by a negative feedback mechanism. Furthermore, the expression of FXR, small heterodimer partner (SHP), liver receptor homolog 1 (LRH-1), and FGFR4 was significantly decreased, while sterol regulatory element-binding protein 1c (SREBP-1c) was significantly increased under the effect of HFD. On the other hand, the expression of FXR in the intestine did show an increasing trend (Figure 12C).

In addition, Niemann-Pick C1-Like 1 (NPC1L1) plays an important role in intestinal cholesterol absorption. However, in our results, the mRNA expression levels of NPC1L1 did not differ significantly between groups and even showed a slight tendency to decrease in the HFD-induced mice (Figure 12C). And the content of NCP1L1 protein in the intestinal epithelium of mice, also induced by a high-fat diet, was significantly reduced compared with that of a low-fat diet (Losacco et al., 2018). In a study examining the effects of a high cholesterol diet on the expression of NCP1L1 and intestinal transporters in rats and mice, the level of NCP1L1 mRNA in the ileum tissue of the mice was significantly reduced compared with that of the control group (Kawase et al., 2016). And in patients with hyperlipidemia treated with cholesterol synthesis by HMGCR inhibitor Atorvastatin, the level of NPC1L1 gene expression was significantly increased. At the same time, mice fed a cholesterol-free high-fat diet were found to inhibit both the cholesterol efflux transporter and the cholesterol absorption transporter NPC1L1, as well as the ratio of cholesterol absorption (Tremblay et al., 2011). Therefore, it was speculated that the decrease of NPC1L1 after ingestion of HFD may be due to the response mechanism of the body to supersaturated cholesterol. And decreased NPC1L1 disrupts the hepatic-intestinal circulation of cholesterol, which may account for the HFD-induced increase in the expression of hepatic cholesterol synthesis gene-HMGCR in mice.

The correlation results showed that there was an association between these changed gene expression levels and IM species. Bacteroides and Phascolarctobacterium were positively correlated with the expression of hepatic-FXR, JAM, ZO-1, LRH-1, peroxisome proliferator-activated receptor alpha (PPAR-α), NTCP, and Occludin genes which were statistically significant; and negatively correlated with ileum-FXR, SREBP-1c which were statistically significant (Figure 12D).

In contrast, after supplementation with RPMP ethanol extract, the tendency of genes related to BAs synthesis, transport, and regulatory functions was differentially suppressed in HFD-managed mice.