Dihydromyricetin ameliorated MCD mice by regulating the composition of serous lipids, bile acids and ileal microflora

doi:10.21203/rs.3.rs-2667860/v1

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Dihydromyricetin ameliorated MCD mice by regulating the composition of serous lipids, bile acids and ileal microflora

https://doi.org/10.21203/rs.3.rs-2667860/v1

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published 02 Aug, 2023

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Dihydromyricetin (DMY) is a natural flavonoid with anti-nonalcoholic steatohepatitis (NASH) activity. However, the effects of DMY on the composition of lipids and bile acids (BAs) in serum, and gut microbiota (GM) in ileum of mice with NASH are not clear. Herein, after male C57BL/6 mice were fed with methionine and choline deficiency (MCD) diet and simultaneously administered with DMY (300 mg/kg/day) by gavage for 8 weeks, the pathological changes of liver tissue were observed by Oil Red O, hematoxylin eosin and Masson staining, the levels of serum AST, ALT and liver TG, MDA were detected by the detection kits, the composition and contents of serum lipids and BAs were detected by LC-MS, the mRNA levels of hepatic BAs homeostasis-related genes were detected by RT-qPCR, and microbiological diversity in ileum was analyzed by 16S rDNA sequencing. The results showed the significant changes including 28 lipids, 4 BAs (23-norDCA, UCA, 7-KDCA and CA), 2 BAs transporters (Mrp2 and Oatp1b2) and 3 GMs (Actinobacteria, Akkermansiaand Faecalibaculum) between MCD and DMY groups. Among them, DMY treatment significantly down-regulated 21 lipids, 4 BAs mentioned above, Faecalibaculum abundance and the ratio of Firmicutes/Bacteroidota, significantly up-regulated 7 lipids and 2 GM (Actinobacteria andAkkermansia). The results suggested that DMY alleviated NASH by up-regulating intestinal probiotics (Actinobacteria and Akkermansia, etc) and inhibiting intestinal harmful bacteria (Erysipelotrichaceaeand Faecalibaculum), thereby regulating the composition and contents of serum BAs and lipids in MCD mice.

Dihydromyricetin

Nonalcoholic steatohepatitis

Lipidomic

bile acids

Gut microbiota

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease, characterized by bullae steatosis, hepatocellular balloonlike degeneration, lobular inflammation and varying degrees of fibrosis[1–3]. Abnormal lipids accumulation especially triglyceride (TG) in hepatocytes is considered to be the basis of the formation and development of NASH [4, 5].

It has been reported that bile acids (BAs)-gut microbiota (GM) axis plays an important role in the pathogenesis of NAFLD/NASH [6]. BAs are the final products of cholesterol metabolism mainly by hepatic cells. According to its source, it can be divided into the primary BAs (i.e., cholic acid [CA] and chenodeoxy-CA [CDCA]) generated by liver cells and the secondary BAs (i.e., deoxy-CA [DCA] and lithocholic acid [LCA]) formed via de-conjugation and de-hydroxylation by the resident bacteria of the distal small intestine and colon. BAs mainly exist in the enterohepatic circulation system and play various physiological functions through recycling, such as regulating cholesterol clearance, GM composition and hepatic glucolipid metabolism [7, 8]. Under normal physiological condition, the level of BAs in the body remains stable. When BAs homeostasis is destroyed, oxidative stress and inflammation can be activated, leading to cholestasis, hepatic steatosis and fibrosis [9]. The serum BAs level in NASH patients were significantly increased [10], and the severity of the disease was positively correlated with BAs synthesis and its serum level [11]. Additionally, GM can regulate the metabolism of endogenous ethanol, choline and BAs by influencing farnesoid X receptor (FXR) signal transduction [12, 13]. Human GM mediates the occurrence and progression of NAFLD/NASH through its metabolites, such as BAs, amino acids and short-chain fatty acids [6, 14]. There is evidence of changes in the content and composition of GM in small intestine of NASH patients [15], and such changes can affect the body's energy homeostasis, leading to liver steatosis [16], as well as changes in intestinal permeability and metabolic endotoxemia associated with liver inflammation and fibrosis [17, 18]. Therefore, the enteric-liver axis is considered as a new target for prevention and treatment of NASH.

Dihydromyricetin (DMY) is a natural type of flavonoids existed in Ampelopsis grossedentata (Hand.-Mazz.) and traditionally used to treat fever or cough [19]. As far as we know, in addition to anti-inflammatory, antioxidant and hepatoprotective pharmacological activities [19], DMY also has significant anti-NASH effects. In brief,

DMY promoted AMPK by inhibiting the expression of PPAR and phosphorylation of serine/threonine kinase Akt, and improved lipids deposition induced by oleic acidin L02 and HepG2 cells [20]. DMY reduced the levels of serum ALT, AST, TC, TG, LDLc and nonestesterified fatty acid, enhanced the synthesis and transport of intrahepatic BAs, and inhibited the reabsorption of ileal BAs in ob/ob mice by regulating FXR signaling pathway [21]. DMY inhibited the de novo lipid synthesis of fat in the liver of obese mice through FXR-SREBP-1C pathway, improved mitochondrial respiration capacity and redox homeostasis by regulating SIRT3 signaling, reduced liver lipid deposition of high fat diet (HFD) fed mice and palmic acid-treated mouse primary hepatocytes [22]. Moreover, DMY improved glucolipid metabolism and inflammation of NAFLD patients [23]. However, the effects of DMY on ileum GM and serum lipids and BAs of NASH mice is unclear.

Considering the important role of the enteric-liver axis in BA and lipid metabolism and its close relationship with NASH, mice fed by methionine and choline deficiency (MCD) diet were used in this work to study DMY-induced effects on the composition of serum lipids and BAs detected by LC-MS, as well as the diversity of ileal GM assayed by 16S rDNA sequencing. The results have certain reference value for elucidating the mechanism of DMY in preventing NASH.

Chemicals and reagents

DMY and obeticholic acid (OCA) (purity ≥ 98%) were got from Shanghai Source Leaf Biological Technology Co., Ltd (Shanghai, China). MCD and MCS (choline and methionine sufficient) diet were from Nantong Trophy Feed Technology Co., Ltd. (Jiangsu, China). Lipid and 50 BAs standards were purchased from CNW/IsoReag (Germany). RNase A was purchased from Promega. Trizol was from Invitrogen (USA). FSQ-301 ReverTra Ace® qPCR RT Kit was from Toyobo Co., Ltd (Japan). SYBR Green I fluorescent RT PCR kit was from Bio-Rad Laboratories (American). Phusion® High-Fidelity PCR Master Mix was purchased from NEB (USA). TruSeq® DNA PCR-free sample preparation kit was purchased from Illumina (USA). Qiagen Gel Extraction Kit was purchased from Qiagen (Germany). The detection kits of ALT, AST, TG and MDA were from Nanjing Jiancheng Bioengineering Research Institute (Jiangsu, China).

Animals and treatment

6–8 weeks SPF male C57BL/6 mice were from Hubei center for disease control and prevention. Mice were maintained in an SPF animal house with 12-hour cycles of light and darkness. The room temperature is maintained at 22 ± 2°C and the humidity is 50–60%. Mice were randomly divided into four groups, and subjected to the following treatments: MCS group, MCD group, DMY group and positive control (OCA) group, 7 mice in each group. Mice in MCS group and other groups were given respectively with MCS and MCD for 8 weeks, and mice in DMY and OCA groups were given the corresponding drug by gavage once a day during the MCD feeding period. The dosage of DMY was 300 mg/kg/day [22], and OCA was 6.5 mg/kg/day converted according to the clinically recommended dose for adults [24]. Mice in MCS and MCD groups administrated with an equal amount of 0.5% CMC-Na solution. Mice were monitored daily for their general health and their body weight once a week. At the end of 8 weeks administration, blood samples collected from orbital venous plexus were centrifuged (3000 g, 4°C, 10 min) to obtained serum. Liver and ileum content of each mouse were collected after sacrifice by cervical dislocation for subsequent experiments.

Biochemical parameters analysis

The activity of serum ALT and AST, and hepatic TG and MDA content were determined following the instructions of test kits.

Liver histopathologic analysis

The liver tissue was quickly fixed in 4% paraformal-dehyde for 24 h and made into 4 µm paraffin sections for H&E and Masson’s stainning. Liver tissues were frozen and cut into 10 µm frozen section for Red Oil O staining.

Serum untargeted lipidomic Investigations

Serum samples were pre-treated according to the method described in the literature [25]. In short, 50 µL serum sample, 1 mL mixture containing methanol, MTBE and internal standard mixture and 200 µL water were mixed evenly. After centrifugation, supernatant was collected and dried. The extract was redissolved with mobile phase.

UPLC coupled with QTrap mass spectrometer 6500+ (SCIEX, USA) was uesed for lipidomic analysis. Each sample was separated by C₃₀ column (2.6 µm, 2.1 mm × 100 mm). The mobile phase A was 60% acetonitrile and 40% water (containing 0.1% HCOOH and 10 mmol/L CH5NO2). The mobile phase B was 10% acetonitrile and 90% isopropanol (containing 0.1% HCOOH and 10 mmol/L CH5NO2). The flow rate was 0.35 mL/min. The elution gradient was listed in Table 1. The mass spectrometry analysis was using a electrospray ionization (ESI)⁺/ESI⁻ mode.

After processing the raw data, it was imported to R software for multiple statistical analysis, including PCA and OPLS-DA. Value of variable importance (VIP) ≥ 1 and fold change (FC) of lipid content (≥ 2 or ≤ 0.5) were used as criteria to screen differential lipids. The metabolic pathway analysis was carried out by the KEGG database.

Table 1

The gradient elution of lipids and BAs detection
	gradient elution
Lipids detection	0–2 min, 20–30% B 2–4 min, 30–60% B 4–9 min, 60–85% B 9–14 min, 85–90% B 14-15.5 min, 90–95% B 15.5–17.3 min, 95% B 17.3 min, 20% B 17.3–20 min, 20% B
BAs detection	0-0.5 min, 5%-40% 0.5–4.5 min, 40–50% B 4.5–7.5 min, 50–75% B 7.5–10 min, 75%-95% B 10–12 min, 95% B 12–14 min, 5% B

Serum BAs analysis

The BAs contents in serum were measured by ExionLC AD UPLC coupled with QTRAP® 6500 + Triple Quadrupoletriple mass spectrometry from SCIEX Corp (SCIEX, USA). Chromatographic separations were performed with an C₁₈ column (1.8 µm, 2.1 mm x 100 mm; Waters, USA). The mobile phase A was ultra-pure water (containing 0.01% CH₃COOH and 5 mmol/L CH3COONH4). The mobile phase B was acetonitrile (containing 0.01% CH₃COOH ). The flow rate was 0.35 mL/min. The gradient elution was listed in Table 1. ESI⁻ and scheduled multiple reaction monitoring were adopted for mass spectrometry detection.

Gut microbiota analysis

Total genomic DNA was extracted from the ileal contents and and tested for quality. Then PCR amplification was performed. The specific primers with Barcode (515F and 806R) were selected according to the sequencing region to be used. PCR products were tested with 2% agarose gel and purified. The library was constructed with the kit and sequenced on Illumina MiSeq 2 × 300. Uparse software was used for clustering, and sequences with 97% similarity were clustered into OTUs. Qiime software (Version 1.9.1) was used for α and β diversity analysis.

RNA Extraction and Quantitative Real-Time PCR

Total RNA was extracted from liver tissue (50–100 mg) from each mouse, and the quality and concentration of RNA were detected. Then reverse transcription reaction and PCR were performed. The expression of target genes were calculated according to the instructions. The primer sequence was listed in Table S1.

Statistical analysis

Experimental results were expressed as mean ± standard deviation. The one-way ANOVA analysis of SPSS 25.0 software was used for statistical test analysis of data. P value less than 0.05 indicated a significant difference and had statistical significance. Spearman method was used to analyze the relevance between GM and serum lipids and bile acids.

Effects of DMY on body weight and liver index

Figure 1A showed the weight of the mice. Compared with the MCS group, the weight of mice in the MCD, DMY and OCA groups decreased significantly. Liver indexes were similar in all groups (Fig. 1B).

Effects of DMY on biochemistry of liver and serum

Compared with MCS group, the contents of serum ALT, AST and liver TG and MDA in mice fed MCD diet were significantly increased, indicating that the model was successfully established. After 8 weeks of DMY treatment, the contents of ALT, TG and MDA were significantly reduced. Meanwhile, OCA treatment also significantly reduced AST and TG contents (Fig. 2).

Effects of DMY on liver pathomorphology

The results of pathological sections were shown in the Fig. 3. Compared with MCS group, The liver of MCD mice has many fat vacuoles and fat droplets, and swelling and balloon-like degeneration occurred, accompanied by inflammatory cell infiltration and fibrosis. DMY or OCA treatment significantly reduced the hepatic fat vacuoles, lipid droplets and hepatocyte ballooning, along with improved hepatic steatosis and lobular inflammation, whereas no improvement on fibrosis was observed.

Effects of DMY on serum lipid profile

Lipidomics was used to analyze the differences of serum lipid composition among the mice in MCS, MCD and DMY groups. It can be seen from the PCA score map (Fig. 4A), MCS and MCD groups could be separated, DMY group was also separated from MCD group, and was more closer to MCS group, suggesting that DMY treatment improved MCD-induced disorder of lipid metabolism.

Furthermore, the supervised OPLS-DA model was established to further identify differential lipid species.The OPLS-DA score map indicated that the MCS group was significantly separated from the MCD group (Fig. 4B), although MCD group and DMY group could also be distinguished (Fig. 4C), but the difference was not as significant as that between MCD group and MCS group. In the S-plots (Fig. 4D,E), Red dots indicate the lipids with VIP > 1, the green dots indicate the lipids with VIP < 1. As shown in Fig. 4F, 451 lipid species in MCD group were significantly changed in contrast to MCS group, including 23 aliphatic acyls (FA), 282 glycerophospholipids (GP), 44 sphingolipids (SP), 14 sterol lipid (ST) and 52 glycerides (GL). Among them, 401 lipids species were down-regulated (shaded green), 14 lipid species were up-regulated (shaded red). Compared with MCD group, there were 21 down-regulated lipid species and 8 up-regulated lipid species in DMY group (Fig. 4G-K), and 15 of 29 lipid species were the mutual differential lipid species among the three groups, including 7 up-regulated lipids PE(O-20:0_22:4), (PE(O-20:0_16:0), PE(O-20:1_22:4), PE(P-18:0_20:1), PE(O-20:2_22:6), PE(P-15:0_20:4), Hex2Cer(d18:2/16:0)) and 8 down-regulated lipids (12,13-EpOME, FFA(18:3), 14(S)-HDHA, 9,10-EpOME, 9,10-DiHOME, FFA(18:1), LPC(14:1/0:0), FFA(16:2)) (Fig. 4L). Of note, the levels of the above 4 eicosanoids and 3 FAAs, as well as PE(O-20:1_22:4) and LPC(14:1/0:0) in DMY group, were almost consistent with those of MCS group.

Pathway enrichment analysis showed that 15 lipid species mentioned above was involved in 20 metabolic pathways (Fig. 5). Based on the P-value, the biosynthetic pathway of fatty acids is considered to be the main pathway involved in the regulation of lipid metabolism disorders in MCD mice by DMY.

Effects of DMY on the composition of GM in ileum

The composition of GM in ileum was analyzed by using 16S rDNA sequencing. The statistical analysis of the α diversity indexes of different samples under the 97% consistency threshold were presented in Table 2. The Chao1, ACE and Shannon indexes of MCD group were lower than those of MCS group, but the difference was not statistically significant. but without significant difference. DMY treatment significantly increased these indexes as compared to MCD group, which suggested that DMY changed the α diversity of GM in ileum of MCD mice.

Table 2

The Alpha diversity indexes of tested mice (n = 7). # indicates comparison with MCD group. ## indicates P value less than 0.05.
Group	Chao1	ACE	Shannon
MCS	218.06 ± 12.58	226.54 ± 13.14	2.83 ± 0.20
MCD	192.31 ± 16.53	198.02 ± 13.73	2.50 ± 0.11
DMY	350.17 ± 32.37^##	354.69 ± 34.12^##	3.45 ± 0.33^##

The PCoA diagram shows the β-diversity values. As shown in Fig. 6A, the three groups were clearly separated, indicating that the composition of GM in ileum of MCS, MCD and DMY groups differed significantly. Figure 6B-D showed the top 10 most abundant GM. At phylum level, Firmicutes, Proteobacteria and Bacteroidota are the three predominant phyla. Compared with MCS group, the content of Firmicutes increased, while that of Verrucomicrobiota and Actinobacteria reduced in MCD group. DMY treatment significantly reduced the ratio of F/B, and elevated the contents of Verrucomicrobiota and Actinobacteria (Fig. 6E-F). At the family level, Erysipelotrichaceae, Enterobacteriaceae and Akkermansiaceae are the main family. MCD mice showed lower contents of Bifidobacteriaceae and Akkermansiaceae than those of MCS group mice. DMY treatment increased the contents of Akkermansiaceae and reduced the contents Erysipelotrichaceae as compared to MCD group (Fig. 6G). Faecalibacuium, Klebsiella and Akkermansia are the main genera of genus level. There was no statistical difference of contents of Faecalibacuium and Akkermansia between MCS group and MCD group, while DMY treatment significantly reduced the contents of Faecalibaculum and increased the contents of Akkermansia in MCD mice (Fig. 6H).

Effects of DMY on serum BAs profile

50 serum BAs contents were displayed in Table S2. Compared to MCS group, the contents of nor-deoxycholic acid (23-norDCA), ursocholic acid (UCA), 7-ketodeoxycholic acid (7-KDCA), ω-muricholic acid (ω-MCA), 3β-DCA and lithocholic acid (LCA) in MCD group were significantly increased. After 8 weeks of treatment with DMY, the levels of 23-DCA, UCA, 7-KDCA and cholic acid (CA) significantly were reduced (Fig. 7).

Correlation between differential GM and differential lipids or BAs induced by DMY

At the phylum level, Firmicutes was positively related with 12,13-EpOME, 14(S)-HDHA, 9,10-DiHOME, 9,10-EpOME, FFA(18:1), LPC14:1/0:0, FFA(16:2), FFA(18:3) and negatively correlated with PE(O-20:0_22:4) and PE(O-20:1_22:4). Verrucomicrobiota was positively related with PE(P-15:0_20:4), PE(O-20:0_22:4), and PE(O-20:1_22:4), negatively correlated with 12,13-EpOME, 14(S)-HDHA, 9,10-EpOME, 9,10-DiHOME, FFA(18:1), FFA(16:2), LPC(14:1/0:0) and FFA(18:3). Actinobacteria was positively related with PE(O-20:2_22:6), PE(O-20:0_16:0), PE(P-18:0_20:1), PE(O-20:0_22:4), PE(O-20:1_22:4), negatively correlated with 14(S)-HDHA, 9,10-EpOME, FFA(18:1), 9,10-DiHOME, FFA(16:2), 12,13-EpOME, FFA(18:3) (Fig. 8A). At the family level, Erysipelotrichaceae was positively related with FFA(18:3), 9,10-DiHOME, LPC(14:1/0:0) and 14(S)-HDHA. Akkermansiaceae was negatively correlated with 14(S)-HDHA, 12,13-EpOME, FFA(18:1), 9,10-EpOME, FFA(16:2), 9,10-DiHOME, FFA(18:3) and LPC(14:1/0:0), positively related with PE(O-20:0_22:4), PE(P-15:0_20:4) and PE(O-20:1_22:4) (Fig. 8B). At the genus level, Faecalibaculum was positively related with LPC(14:1/0:0), Akkermansia was negatively correlated with FFA(18:3), 9,10-DiHOME, FFA(18:1), 14(S)-HDHA, 12,13-EpOME, FFA(16:2), 9,10-EpOME and LPC(14:1/0:0) positively related with PE(O-20:1_22:4) and PE(P-15:0_20:4) (Fig. 8C).

The correlation between the differential GM and the differential serum BAs induced by DMY treatment was shown in Fig. 8D-E. Actinobacteria and Firmicutes were positively related with CA, Verrucomicrobiota was negatively correlated with 7-KDCA, UCA and CA at the phylum level (Fig. 8D). At the family level, Bifidobacteriaceae was negatively correlated with 23-DCA, Erysipelotrichaceae was positively related with 7-KDCA, UCA and CA, Akkermansiaceae was negatively correlated with 7-KDCA, UCA and CA (Fig. 8E). At the genus level, Faecalibaculum was positively related with 7-KDCA, UCA and CA, while Akkermansia was just opposite to that of Faecalibaculum (Fig. 8F).

Effect of DMY on mRNA expression of bile acid homeostasis related genes

Next, we studied the effect of DMY on mRNA expression of BA homeostasis related genes in liver. As presented in Fig. 9, Compared to MCS group, the mRNA expression levels of Cyp7a1 (cholesterol 7α-hydroxylase), Ntcp (sodium taurocholate co-transporting polypeptide), Bsep (bile salt export pump), Mrp (multidrug resistance protein) 2, Oatp1b2 (organic anion transporting polypeptide) and Cyp27a1 (sterol 27-hydroxylase) in MCD group were significantly reduced. DMY treatment remarkably increased the mRNA expression of Mrp2 and Oatp1b2. However, the expression of other genes did not change significantly after DMY treatment.

NASH is closely associated with lipid metabolism disorders [26]. The accumulation of liver lipids can be attributed to the amount of fatty acids obtained by the liver exceeding its processing capacity, which is one of the pathogenesis of NASH [27]. Abnormal increase of FFAs level in blood will directly stimulate FFAs uptake in liver, which stimulated TNF-α expression and lead to accumulation of TG and liver steatosis [28, 29]. Oxidative lipids generated by the oxidation of polyunsaturated fat acids, such as 12-HETE, leukotriene B4 and leukotriene D4, have been proved to be associated with obesity, type 2 diabetes and insulin resistance (IR) [30–32]. PE is a precursor to synthesize phosphatidylcholine (PC), which is involved the synthesis of VLDL. Intrahepatic TG is mainly transported out of the liver in the form of VLDL. If the synthesis of PE is inhibited, the synthesis of PC is reduced, which leads to reduced TG output in the liver. DMY treatment reduced the levels of 3 FFAs (18:1, 16:2 and 18:3) and 4 oxidative lipids [9,10-EpOME, 14(S)-HDHA) 9,10-DiHOME and 12,13-EpOME], and increased the levels of 6 PEs(P-18:0_20:1, O-20:0_22:4, P-15:0_20:4, O-20:2_22:6, O-20:1_22:4 and O-20:0_16:0) in serum of MCD mice, suggesting that the improvement of DMY on MCD mice is closely related to its regulation of the serum FFAs, oxidative lipids and PEs stated above.

GM participates in the occurrence and development of NASH through mediating energy metabolism and IR [17, 33]. Dysregulation of GM can release a large amount of lipopolysaccharide and activate liver inflammation by damaging mucosal barrier [34]. Transplanting GM from HFD mice into the intestines of normal mice significantly increased IR and body fat content [35]. HFD up-regulated the gut ratio of F/B in obese mice, which promoted the body to obtain energy and lead to obesity [36]. DMY has been reported to significantly alter the richness and diversity of GM in some animal models [37], alleviated intestinal dysbiosis in colitis mice by increasing the contents of Akkermansia and Lactobacillaceae [38], improved the contents of Bacterioidetes and suppressed Firmicutes in the intestinal tract of HFD mice [39]. Similarly, the present work found that DMY treatment significantly increased the contents of Actinobacteria, Verrucomicrobiota and Akkermansiaceae, decreased the ratio of F/B and contents of Erysipelotrichaceae and Faecalibaculum in MCD mice. Studies have found that Actinobacteria can promote energy metabolism and reduce fat content [40, 41]. Verrucomicrobia is mainly distributed in the intestinal mucus layer, Akkermansia is its dominant bacterium [42]. The abundance of Akkermansia is positively related with body health status, such as relieving obesity and IR [43], preventing fatty liver and maintaining intestinal homeostasis by regulating liver lipid synthesis and inflammation [44]. Faecalibactaculum belongs to Erysipelotrichia in Firmicutes [45]. Erysipelotrichia is an important bacterial marker for the susceptibility to fatty liver disease caused by choline deficiency [46]. Thereby, the alleviation of DMY on MCD mice may be closely related to its inhibition of harmful bacteria and induction of beneficial bacteria in intestinal tract.

It is well known that BAs, as signaling molecules, can regulate their self-synthesis, glucolipid metabolism, GM composition and energy homeostasis through various receptors. The contents of serum GCA, TCA and GCDCA in NASH patients were higher than those in health individuals [47], and the liver damage of NASH patients was related to abnormal changes of serum BAs, for instance, increased GCA and CA in plasma were positively connected with liver inflammation [48]. CA is a hydrophobic BA that can damage mitochondrial electron transport chain, and lead to ROS and oxidative stress [49]. Increased CA level was associated with hepatocyte ballooning in NAFLD patients [50], and with increased ratio of Firmicutes to Bacteroides in rats and mice [51, 52]. Additionally, the elevated level of some secondary BAs (free and conjugated UDCA, 7-KDCA, etc) in gut promoted the synthesis and excretion of BAs with irritable bowel syndrome patientsby inhibiting intestinal FXR/ FGF19 signaling pathway [44, 53], which was beneficial to maintain in vivo glucose homeostasis [54]. DCA is a highly toxic secondary BA that produces cytotoxicity through activation of JNK1 pathway [55], and is associated with ballooning of hepatocytes [48]. DCA is also a natural hepatic FXR antagonist. High concentration of DCA not only inhibited hepatic glycogen synthesis and promoted gluconeogenesis [10], but also inhibited the growth and reproduction of Bacteroidete that can improve IR [56]. The results indicated that LCA, 23-DCA, UCA, 7-KDCA, ω-MCA, 7-KDCA and 3β-DCA in MCD mice were significantly increased as compared to MCS mice, while DMY treatment reduced the contents of 23-DCA, UCA, 7-KDCA and CA. To know the potential signaling mechanism of DMY alters serum BAs content, the effect of DMY on the mRNA expression of BAs homeostasis related genes in liver of MCD mice was also detected. Primary BAs are synthesized though both classical and alternative pathways of cholesterol metabolism. Cyp7a1 and Cyp27a1 are main rate-limiting enzymes of classical and alternative pathways, respectively [13]. After binding with taurine (rats and mice) or glycine (human), the primary BAs are transported through Bsep and Mrp2 to form micelles with substances such as cholesterol and phospholipids, and stored in the gallbladder in the form of bile [7]. As well known, 95% of BAs is reabsorbed into the portal vein at the end of the ileum and circulated to the liver, then absorbed into hepatocytes by Ntcp and Oatp1b2 [7]. The results indicated that the mRNA expression levels of hepatic Cyp7a1, Cyp27a1, Bsep, Mrp2, Ntcp and Oatp1b2 in MCD mice were significantly decreased as compared to MCS mice, which was is basically consistent with reported literatures [57, 58]. DMY treatment significantly increased the mRNA expression levels of Mrp2 and Oatp1b2 in MCD mice. Up-regulated Mrp2 can reduce the accumulation of hepatic BAs, and up-regulated Oatp1b2 can promote BAs uptake by hepatocytes from blood. Song et al found that DMY alleviated obesity by up-regulating the genes related to BA conjugation (Bacs and Bat) and secretion (Bsep, Mrp2, Abcg5 and Abcg8), and down-regulating the genes related to bile acid re-absorption (Asbt, Ostα and Ostβ) of liver in obese ob/ob mice [21]. The increased hepatic Mrp2 and Oatp1b2 and decreased serum 23-DCA, UCA, 7-KDCA and CA observed in MCD mice indicated that DMY regulated BAs homeostasis of MCD mice partly due to its effect on hepatic BAs transporter expression, partly due to its effect on the abundance of certain GM.

Indeed, GM is closely related with the metabolism of lipids and BAs. Primary BAs secreted into gut are treated by GM-expressed bile salt hydrolysase and 7α/β dehydroxylase to generate secondary BAs [59]. BAs affect FXR signaling and TGR5 signal transduction in liver and intestine through enterohepatic circulation, and then regulate BA synthesis, lipid metabolism and inflammation [58, 60]. Additionally, BAs in gut can directly affect the composition of GM due to its antibacterial properties [61], or as a signal molecule to affect the expression of genes encoding antimicrobial peptides and lectins by activating BA receptors such as FXR [62], indirectly affecting GM [63]. It's worth noting that DMY induced regulatory trends on beneficial bacteria in ileum and beneficial lipids or BAs in serum of MCD mice were basically the same. More specifically, DMY treatment up-regulated probiotics (Akkermansia and Bifidobacteriaceae) and beneficial lipids [PE(O-20:0:4), PE (O-20:1:22.4), PE(P-15:0:4)], decreased harmful lipids [12, 13-EPOME, 9, 10-DIHOME, FFA(18:1), 14(S)-HDHA, FFA(16:2), LPC(14:1/0:0), FFA(18:3)] and harmful BAs (23-DCA and CA), and so it is for the regulatory trends of harmful bacteria (Erysipelotrichaceae and Faecalibaculum) and harmful lipids and BAs (Fig. 8).

In the study, serum lipids and BAs in mice were detected for the first time, and the 15 lipids may be biomarkers for DMY to exert therapeutic effect. Moreover, the correlation between the changed differential lipid metabolites and bile acids and gut microbiota was analyzed. The results suggest that dihydromyricetin may affect lipid metabolism and BAs by regulating gut microbiota in NASH mice induced by MCD diet.

Our study had some limitations. We found that dihydromyricetin may influence bile acid metabolism through gut microbiota, but we did not detect fecal BAs and the expression levels of BAs metabolity-related genes in intestinal tissues.

In summary, DMY treatment increased the levels of probiotics (Actinobacteria, Verrucomicrobiota, Bacteroidota and Akkermansia) in ileum and beneficial lipids (PE O-20:1:22.4, PE O-20:0:4 and PE P-15:0:4) in serum, decreased the levels of harmful bacteria (Erysipelotrichaceae and Faecalibaculum) and harmful lipids (12, 13-EPOME, 14(S)-HDHA, 9, 10-DIHOME and LPC 14:1/0:0, etc) and harmful BAs (23-DCA and CA). These changes should be conducive to the ameliorative effect of DMY on MCD mice. Dihydromyricetin can be used as functional food for NASH prophylaxis in daily life and clinical practice.

Acknowledgements

Not applicable

Authors’ contributions

Junjun Wang designed and directed the project. Ping Luo, Jiao Liu and Xiaolei Miao completed the experiments. Ping Luo analyzed the data. Xiaolei Miao and Ping Luo performed the statistical analysis and wrote the original draft. Junjun Wang and Yong Chen revised the manuscript.

Funding

The work was supported by the project of Education Department of Hubei Province (No.Q20181004), China.

Availability of data and materials

All data related to this study are available upon request.

Ethics approval and consent to participate

All animal experiments were conducted in accordance with the experimental guidelines of the Animal Ethics and Welfare Committee of Hubei University and were approved.

Consent for publication

Not applicable

Competing interests

The authors declare no confict of interest.

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No competing interests reported.

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Dihydromyricetin ameliorated MCD mice by regulating the composition of serous lipids, bile acids and ileal microflora

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Abstract

Figures

Introduction

Materials And Methods

Chemicals and reagents

Animals and treatment

Biochemical parameters analysis

Liver histopathologic analysis

Serum untargeted lipidomic Investigations

Serum BAs analysis

Gut microbiota analysis

RNA Extraction and Quantitative Real-Time PCR

Statistical analysis

Result

Effects of DMY on body weight and liver index

Effects of DMY on biochemistry of liver and serum

Effects of DMY on liver pathomorphology

Effects of DMY on serum lipid profile

Effects of DMY on the composition of GM in ileum

Effects of DMY on serum BAs profile

Correlation between differential GM and differential lipids or BAs induced by DMY

Effect of DMY on mRNA expression of bile acid homeostasis related genes

Discussion

Strengths And Limitations

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1