Honokiol affects the composition of gut microbiota and the metabolism of lipid and bile acid in methionine-choline deficiency diet-induced NASH mice

Honokiol (HNK), one of the main active components of Magnolia officinalis, has a positive effect on non-alcoholic steatohepatitis (NASH). However, the effects of HNK on the composition of serum lipids and bile acids (BAs) and gut microbiota (GM) of NASH mice are still unknown.C57BL/6 mice were fed with methionine-choline deficiency (MCD) diet and gavaged with HNK (20 mg/kg/d) for 8 weeks, then the serum lipids and BAs were detected by LC–MS, the composition of ileum microflora and the mRNA expression of hepatic BAs homeostasis related genes were analyzed by 16S rDNA sequencing and RT-qPCR, respectively. HNK treatment decreased the degree of hepatic lipid drops, inflammatory cell infiltration and fibrosis. Meantime, the serum levels of 34 lipids and 4 BAs in MCD mice were significantly altered by HNK treatment, as well as the increased abundance of Ruminococcaceae, Caulobacteraceae and Brevundimonas, and the decreased abundance of Firmicutes and Dubosiella. Besides, HNK treatment increased the hepatic mRNA expression of Oatp1b2 in MCD mice. The ameliorating effect of HNK on NASH may be partly related to its correction on the disorders of GM, serum lipids and BAs of MCD mice.

detected by commercial test kits.The results showed that HNK had no effect on weight loss and hepatic index of mice fed by MCD diet ((Fig.1a and b).Compared with MCS group, serum ALT, AST and liver TG, MDA levels in MCD group were significantly increased.After HNK treatment, serum ALT activity, liver TG and MDA content decreased remarkably, AST level was reduced similarly, but there was no significant difference compared with MCD group.In addition, the levels of liver TG, MDA and serum AST in OCA group were obviously lower than those in MCD group (Fig. 1c-f).
HNK attenuated hepatic steatosis, inflammation and fibrosis of NASH mice.Hepatic lipid accumulation, steatosis, inflammation and fibrosis of the mice were analyzed by Oil red O staining, HE staining and Masson staining (Fig. 2).Compared with MCS group, the liver cells of MCD mice were disordered and showed ballooning changes, with a large number of lipid droplets and fat vacuoles, accompanied by inflammatory cell infiltration and fibrosis.After treatment with HNK and OCA, the number of lipid droplets and fat vacuoles in liver cells were reduced significantly, the degree of inflammatory cell infiltration and fibrosis decreased, and the liver tissue morphology was closer to MCS group.

Effects of HNK on serum lipids of NASH mice.
The principal component analysis (PCA) showed the separation trend of serum lipid composition in each group.As shown in Fig. 3a, there was an obvious separation trend between the MCS group and the MCD group, indicating that MCD diet caused significant changes of serum lipid composition in mice.Compared with MCD group, the sample distribution of HNK group showed no change.
To further clarify the differential lipids among each group, we analyzed the serum lipid composition of MCS, MCD and HNK groups using orthogonal partial least squares discriminant analysis (OPLS-DA).As shown in Fig. 3b, MCS and MCD group were significantly separated.R 2 Y = 0.997, Q 2 = 0.967, and both R 2 Y and Q 2 were greater than 0.4, indicating that the OPLS-DA model was effective.Figure 3c showed that HNK group and MCD group were significantly differentiated, with R 2 Y = 0.998 and Q 2 = 0.719, indicating that the OPLS-DA model was effective.
The mRNA expression of BAs metabolism-related genes in mice liver were shown in Fig. 4. Compared with MCS group, the mRNA expression of CYP7A1, CYP27A1, Bsep, Mrp2, Ntcp and Oatp1b2 in MCD group were significantly decreased.HNK treatment markedly increased Oatp1b2 mRNA expression of MCD mice (Fig. 4f), whereas there were no significant effect on the mRNA expression of CYP7A1, CYP27A1, Bsep, Mrp2 and Ntcp (Fig. 4a-e).
Effects of HNK on the composition of GM in MCD mice.The Exponential dilution curve showed that the amount of 16Sr DNA sequencing data in this experiment was progressive and reasonable, and the sequencing depth was reliable (Fig. 5a).The statistical analysis of the Alpha diversity index of different samples under the 97% consistency threshold were shown in Table S3.There were no significant differences in Chao1, ACE, Simpson and Shannon indexes of GM among the three groups, indicating that HNK had no significant effect on the diversity of GM.Beta diversity analysis was applied to further study the composition of GM in each group, and non-metric multi-dimensional scaling (NMDS) showed significant differences in the composition of GM among the three groups (Fig. 5b).
The composition of GM of the tested mice at phylum, family and genus levels were shown in Fig. 5c-e.Compared with MCS group, the abundance of Firmicutes and Turicibacter were markedly increased, and the abundance of Eggerthellaceae was significantly decreased in MCD group.MCD mice treated by HNK significantly increased the relative abundance of Ruminococcaceae, Caulobacteraceae and Brevundimonas, and the abundance of Firmicutes and Dubosiella significantly decreased.Based on LEFSe analysis, species with LDA > 4 www.nature.com/scientificreports/were considered as the set value, and the microbiota with statistical differences among three groups were screened out and showed in Fig. 5f.

Discussion
One of the sources of hepatic FAs is the input of FFAs in the blood.Excessive FFAs entering the liver will break the stable balance of hepatic TG and promote the occurrence of NAFLD 14 .If the body is continuously hit by high concentration of FFAs, it will not only interfere with multiple metabolic pathways and induce IR of multiple organs 15 , but also lead to fibrosis of liver tissues 16 .Phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) are the important lipid components of GP.Once the synthesis of PC in the body is blocked, the secretion and release of very low density lipoprotein cholesterol will be inhibited, so that TG in liver tissue cannot combine with it and transport out of liver, resulting in TG accumulation in liver tissue 17 .PE is the precursor of PC synthesis, and its level directly affects the degree of liver lipid accumulation.The levels of PC and PE in liver of NAFLD mice were down-regulated and further decreased with the progression of fatty liver disease 18 .Eicosanoids are the endogenous lipid signaling molecules produced by polyunsaturated fatty acids such as arachidonic acid under the catalysis of enzymes, which play important role in inflammation and tumor.12,13-EPOME can increase the expression of pro-inflammatory cytokines in mouse macrophage RAW 264.7 and human colon cancer cell HCT-116 19 .9, 10-DIHOME can not only destroy mitochondrial function by changing the integrity of cell intima and increasing the release of cytochromic C 20 , but also inhibit the respiratory burst of neutrophils, enhance cellular oxidative stress, dilate blood vessels, induce apoptosis and other reactions 21,22 .Additionally, thromboxane B3 (TXB3) is an eicosapentaenoic acid with anti-inflammatory effects 23 .The present work found that HNK reduced the levels of hepatic TG and MDA and alleviated liver inflammation of MCD mice.This process may be related to HNK-induced reduction of serum FFAs (14:0, 16:1, 17:1, 18:1, 19:1,16:2, 18:3, 22:6, etc.) and eicosanoids (12, 13-EPOME, 9, 10-DIHOME), as well as elevation of serum PEs (O-18:0:0, O-20:0:0, O-16:16:0, O-20:0:0:6, O-15:1:22:6, P-18:0:0:6, P-16:0:0), TXB3 and TLCA-3-S.In our study, KEGG pathway enrichment analysis showed that fatty acid and unsaturated fatty acid biosynthesis pathways were related to differential lipids induced by HNK.Previous studies have shown that HNK can alleviate lipid metabolism disorders of NASH by suppressing the expression of SREBP-1c, FAS, SCD-1 and promoting the phosphorylation of ACC, which are the key genes involved in fatty acid and unsaturated fatty acid biosynthesis pathways [11][12][13] .These results are consistent with the results of this study.
The abnormal changes of serum BAs are closely related to many chronic liver diseases.The levels of primary and secondary BAs in serum of NASH patients were significantly increased, and the severity of NASH was positively related with serum BAs levels 24 .The risk of NAFLD progressing to fibrosis is related to the ratio of secondary BAs to primary BAs and the concentration of conjugated BAs.The higher the ratio of secondary BAs to primary BAs and the higher the concentration of conjugated BAs, the higher the risk of fibrosis 25 .Hydrophobic BAs (such as DCA, 7-KDCA, 23-DCA and TDCA) are often regarded as toxic BAs due to their ability to activate death receptors and stimulate the production of pro-inflammatory mediators 26 , or due to their ability to weaken the enteric FXR/FGF19 signaling, thereby resulting in increased bile acid synthesis 27 .TDCA can damage mitochondria and induce apoptosis of HepG2 cells 28 .Additionally, the serum concentration of hydrophilic bile acid GCA in patients with NAFLD is significantly higher than that in healthy people 29 .In this study, HNK treatment reduced the levels of 10 BAs (HCA, UCA, DCA, 7-KDCA, 3β-UDCA, 3-oxo-DCA, 23-DCA, HDCA, TDCA and GCA) and ALT in serum of MCD mice, suggesting that HNK may reduce the risk of fibrosis and hepatotoxicity of MCD mice by regulating the metabolism of secondary BAs.The synthesis of primary BAs depends on the rate-limiting enzymes CYP7A1 and CYP27A1 in the liver.Bsep and Mrp2 are responsible for the transport of BAs out of the liver, NTCP and OATPs are responsible for uptake of BAs into the liver 30 .The present study showed that the mRNA expressions of CYP7A1 and CYP27A1 in MCD mice were significantly down-regulated, which is similar to human liver with NASH 31 .Notably, the mRNA expression of Bsep, Mrp2, Ntcp and Oatp1b2 in MCD mice liver were also significantly down-regulated, suggesting that the hepatotoxicity induced by MCD diet was probably due to the reduced BAs output from the liver, and the increased levels of serum BAs was mainly due to the decreased BAs input from the liver.After HNK treatment, only the mRNA expression of Oatp1b2 was up-regulated in MCD mice.Combined with the effect of HNK on serum BAs of MCD mice, we speculated that the improvement of HNK on BA metabolism disorder in MCD mice resulted from, on one hand, its effect on liver Oatp1b2 expression, and on other hand, its effect on the composition and abundance of GM.
The GM in NAFLD patients, obese people, ob/ob and db/db mice is characterized by a higher proportion of Firmicutes and Bacteroidetes, an increased Proteobacteria producing endotoxins, and a decreased immunohomeostasis-related bacteria 32,33 .Firmicutes is harmful bacteria, and its main metabolites acetic acid and propionic acid are substrates for liver gluconeogenesis and adipose formation 34 .Ruminococcaceae is a probiotics that can degrade a variety of polysaccharides and fibers to produce SCFAs such as butyrate, its abundance is significantly negatively correlated with alcoholic cirrhosis, non-alcoholic liver fibrosis, and increased intestinal permeability [35][36][37] .Effective regulation of GM has a positive impact on decreasing liver fat accumulation and alleviating NAFLD 38,39 .Our results showed that HNK markedly decreased the abundance of Firmicutes and Dubosiella, and increased the abundance of Ruminococcaceae, Caulobacteraceae and Brevundimonas, suggesting that HNK may improve MCD mice by regulating the abundance of GM mentioned above.Notably, the oral bioavailability of HNK in rats was reported to be only 5.3% due to extensive first-pass metabolism and low absorption 40 , suggesting that the regulatory effect of HNK on lipids and bile acids may be realized through the regulation of gut microbiota.
In the occurrence and development of NASH, lipids, BAs and GM are closely related.Secondary BAs are mainly metabolized and generated by BA hydrolase encoded by intestinal microorganisms.Changes in intestinal microbial community will change the expression level of BA hydrolase, thus affecting the composition of the host BA pool, and thereby affecting the signal transmission of BAs 41 .GM regulates the synthesis and metabolism of liver BAs through FXR negative feedback, and the composition of BA pools also affects the composition of GM 42 .GM is also closely related to lipid metabolism.Obese people have a higher ratio of Firmicutes/Bacteroidetes in the intestinal tract, which has a higher ability to obtain energy from food 43 .Our results showed that the abundance of Ruminococcaceae was negatively correlated with the serum levels of 14 FFAs and 23-DCA, Firmicutes was negatively correlated with TLCA-3-S and TxB3, and positively related with 23-DCA, 7-KDCA, DCA, UCA, 3-oxo-DCA, HCA, 3β-UDCA.The correlation analysis results showed that the up-regulation effect of HNK on the beneficial bacteria Ruminococcaceae of MCD mice was well coordinated with its regulating effect on the levels of serum beneficial lipids, harmful lipids and bile acids.This coordination of regulation is also shown in its effects on the abundance of intestinal harmful bacteria Firmicutes and serum lipid and bile acid levels.
Our results suggested that HNK ameliorated the disorder of lipid and bile acid metabolism, up-regulated the abundance of intestinal probiotics and down-regulated the abundance of harmful bacteria in MCD mice.These may be closely related to its improvement effect of NASH.BAs metabolism runs through the enterohepatic circulation, and many regulatory factors in the gut, such as FXR and a variety of transporters, are directly involved.In this paper, only the effects of HNK on BAs homeostasis related enzymes and transporters in MCD mice were studied, and there was a lack of intestinal research data.In subsequent study, the effect of HNK on BAs metabolism related genes in the gut of mice should be further explored, and it should be linked with the effect of HNK on the alteration of GM, to determine whether the regulatory effect of HNK on BAs is related to the alteration of the abundance and composition of GM, and to provide a more accurate reference for the preclinical study of HNK against NASH.
All animal experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and followed the recommendations in the ARRIVE guidelines.The protocol was approved by the Experimental Animal Ethics Committee of Hubei University (Protocol Number: 20220039).All efforts were made to minimize suffering.

Histological examination and biochemical analysis.
Fresh mouse livers of about 1 cm 3 in size were dissected and immediately fixed in 4% paraformaldehyde for 24 h.Fixed tissues were embedded in paraffin and 4-µm sections were prepared and respectively stained with hematoxylin-eosin (HE), masson trichromatic staining or oil red O staining for morphological observations.The levels of ALT and AST in serum, as well as TG, MDA in liver homogenate (liver-saline, 1:9, w/v) were determined by using commercial test kits according to the manufacturer's instructions.
Sample preparation.50 μL serum of each mouse was mixed with 1 mL internal standard lipid extract (Methyl tert-butyl ether: Methanol = 3:1, V/V), vortex for 15 min, then add 200μL pure water, vortex for 1 min, centrifuge at 4 °C (12,000 rpm/min) for 10 min.Add 200μL acetonitrile/isopropyl alcohol (10/90, V/V) to the 200μL supernatant concentrate, vortex for 3 min, centrifugation at 12,000 r/min for 3 min.The supernatant was used for sample analysis.Identification of lipids and bile acids was based on self-built database MWDB (Metware database).The quality control sample (QC) is prepared by mixing the extracts of the samples in equal quantities.During the analysis, one QC was inserted for every 10 samples.The stability of the instrument during the project inspection can be judged by the overlapping display analysis of the total ion flow chromatogram (TIC) of the same quality control sample.
The LC-MS data was preprocessed by Analyst 1.6.3software, and the processed data was analyzed by R software (www.r-project.org/) for multivariate statistical analysis, including Principal Component Analysis (PCA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA).According to Variable Importance (VIP) value and fold change value, abnormal lipid components were screened out.The abnormal lipid components were imported into KEGG (Kyoto Encyclopedia of Genes and Genomes) database for metabolic pathway enrichment analysis.

Figure 5 .
Figure 5. Effects of HNK on the composition of gut microbiota in the tested mice (n = 7).(a) Exponential dilution curve.(b) Non-metric multidimensional scaling (NMDS) on the OTU level.(c) Microbiota composition at the phylum level.(d) Microbiota composition at the family level.(e) Changes in gut microbiota of mice at the genus level.(f) LDA bar plot of LEFSe analysis on gut microbiota.MCD, methionine-and choline-deficient diet; MCS, methionine-and choline-sufficient diet; HNK, honokiol.

Figure 6 .
Figure 6.Spearman correlation analysis between gut microbiota and serum lipids or BAs in MCD mice.(a) Correlation between gut microbiota and serum lipids.(b) Correlation between gut microbiota and serum BAs.*Indicates that the p value of the correlation coefficient significance test is < 0.05, and **Indicates that the p value is < 0.01.MCD, methionine-and choline-deficient diet; MCS, methionine-and choline-sufficient diet; HNK, honokiol.