Molecular Mechanism Of Berberine Intervention In Nonalcoholic Steatohepatitis

ObjectivesUp to now, the operational therapeutic strategy and pathogenesis for nonalcoholic steatohepatitis (NASH) are uncertainty yet. In the present studies, we plan to investigate:1) The TCM patterns (syndromes) and pathogenesis in NASH;2) To evaluate the efficacy of berberine on two kinds of NASH models, i.e. Methionine and Choline deficiency (MCD) diet induced mice NASH, and high fat diet (HFD) induced rats NASH;3) To explore the molecular mechanisms of berberine for intervention NASH via inducing macrophage phenotypic switch in hepatic tissue, against insulin resistance, and modulating metabolic disorder.Material and Methods1Based on SinoMed and PubMed database, an own research and development kit of text mining was adopted to analyze the text information according to the process of data retrieving, data pre-treating, data analyzing, and data visualization. Furthermore, the TCM theory of’formula-pattern-disease’correlation was addressed to understand the material basis and potential drug targets for NASH.2On the pharmacodynamical experiments of berberine, C57BL/6mice and SD rats were divided into5groups:1) normal control group fed with ordinary diet;2) NASH model group fed with MCD or HFD;3) positive drug group coupled with rosiglitazone treatment;4) high dose berberine treatment group; and5) low dose berberine treatment group. The animal models were prepared through conventional approach. In brief, except normal control group, all mice will burden MCD diet for two weeks, and all rats will be fed HFD for eight weeks. Berberine was preventive medication by intragastric administration. After the experiment finished, blood sample was collected by abdominal aorta puncture in rats and picking eyeball in mice and then serum obtained. A part of liver tissue was excised and formalin-fixed immediately, and some liver tissue was frozen into liquid nitrogen. And then, the detecting techniques of serum biochemistry, histopathology, and enzyme linked immunosorbent assay (ELISA) were used to evaluate the efficacy of berberine.3Based on MCD induced mouse NASH model, physiological saline was injected into left ventricle for liver perfusion, and the nonparenchymal cells of liver tissue were extracted. The sub-phenotypes of macrophages in hepatic tissue were detected by flow cytometry (FCM). Regarding this, total macrophages were labeled by F4/80antibody, M1type macrophages were marked by F4/80+CD11c+antibody, and M2type macrophages were marked by F4/80+CD206+. And then, the M1/M2ratio was calculated to indicate macrophage phenotype transformation in hepatic tissue. 4Based on HFD induced rat NASH model, mRNA and protein expression of peroxisome proliferator-activated receptor-y (PPAR-y) on hepatic tissue was respectively detected by real-time fluorescent quantitation PCR and immunohistochemistry (IHC) methods. The global, unbiased metabolic profiling platform was based on a Waters AcquityTM Ultra Performance Liquid Chromatography system coupled with a XevoTM G2-Q-TOF-MS (UPLC-Q-TOF-MS). The endogenous metabolic profile coupled with principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA) techniques were carried to screen the different metabolites and related metabolic pathways.5The statistical analysis of the quantitative data was performed by SPSS17.0. Numerical value was signified with X±S. Statistic analysis of One-way ANOVA was also performed for difference in means between groups. Differences were considered significant at a value of P<0.05.Results1The text mining results showed that the main types of pattern in NAFLD involved liver stagnation and spleen defieiency (Gan Yu Pi Xu), dampness heat syndrome (Shi Re Nei Yun), and phlegm and stasis (Tan Yu Hu Jie). The core herbs for treating NAFLD include Dan Shen, Chai Hu, He Shou Wu, Ze Xie, Shan Zha, Bai Zhu, and Yu Jin. The key TCM pathogenesis corresponded to insulin resistance, lipid metabolic disorder, and chronic inflammatory response.2The result of pathological score shown two kinds of NASH animal model were successful prepared. Base on the animal models, both high dose and low dose berberine all showed better efficacy, such as markedly improving the degree of hepatic tissue pathology; decreasing serum ALT, CHO levels (p<0.05, vs. NASH model group); down-regulating TNF-a but up-regulating IL-10level in serum (p<0.05, vs. NASH model group). The data also suggested that there is not significantly difference among rosiglitazone (positive drug), low dose and high dose berberine on the treatment of NASH.3The results of flow cytometry showed that compared to normal control mice, the number of Ml type macrophages significantly increased and the number of M2type macrophages significantly decreased in hepatic tissue of MCD induced NASH mice. The ratio of M1/M2markedly increased (P<0.01). On the contrary, the number of M1decreased, but M2significantly increased on berberine and rosiglitazone treatment mice. Therefore, the ratio of M1/M2also significantly decreased (p<0.01). These results suggested that both berberine and rosiglitazone could improve the pathological process in MCD diet induced NASH model possibly through modulating macrophage phenotype transformation, i.e. the ratio of M2type was increased and M1type was decreased in hepatic tissue. 4The results of PPAR-y protein expression shown that compared to normal control group, HFD could reduce PPAR-y protein expression on hepatic tissue (p<0.01, vs. normal control). Berberine and rosiglitazone could up regulate PPAR-y protein expression (p<0.01, vs. NASH model control). On the contrary, the results of PPAR-ymRNA expression on hepatic tissue was up-regulated in NASH model(p<0.01, vs. normal control). Berberine and rosiglitazone could down-regulate PPAR-ymRNA expression (p<0.01, vs. NASH model control). These results suggested that berberine improve IR and alleviate the pathological process in HFD induced NASH model possibly through modulating the course of transcription and translation of PPAR-y.5A total of1494ions peaks were obtained from UPLC-Q-TOF/MS spectra (870in positive mode and624in negative mode). The score plots of PCA showed well delineated clusters and separations trends of normal control group, NASH model group, rosiglitazone treated group, and berberine treated group in both positive ion mode and negative ion mode, highlighting the disease diagnostic potential and drug intervention effect. Univariate statistical method was performed on all serum features derived from calculated between HFD induced NASH group and normal control group, rosiglitazone treated group and model group, berberine treated group and model group, respectively. A total of57metabolites were identified. In brief, the level of creatine,3-indoxyl-sulfuric acid, arachidonic acid,3-Hydroxychola-7,22-dien-24-oic acid, phosphatidylcholine (PC32:0,34:0,36:3,37:4,38:4,38:6,40:4,40:5,40:6,40:8), lysophosphatidylcholine (LPC16:0,17:0,18:0,20:4), sphingomyelin(SM34:1,34:2,36:1,40:1,42:1,42:2,42:3), and diacylglycerol (DG38:6) were significantly decreased (p<0.01; p<0.05); while the level of13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE), Eicosatrienoic acid, Phytomonic acid, Docosatrienoic acid, Eicosenoic acid, and phosphatidylcholine (PC34:1,34:3,34:4,35:2,36:1,36:2,36:4,36:5,38:2,38:3,38:4), lysophosphatidylcholine (LPC14:0,16:1,17:1,18:1,18:2,20:2), and diacylglycerol (DG34:2,36:3) were significantly increased in HFD rats than that in normal control rats (p<0.01; p<0.05). On the contrary, berberine and rosiglitazone could cause a systemic recovery from the HFD induced metabolic perturbation in rats. In the context of changed metabolic profiles, we sketched the perturbed metabolic network associated with NASH and recovered with drugs, which involved the metabolic pathways of β oxidation of fatty acids, metabolism of phosphatide-arachidonic acid, metabolism of phenylalanine-tyrosine-thyroid hormones, and cholesterol-bile acid metabolism.ConclusionOur results initially illustrate the profiles of patterns and the key TCM pathogenesis of NAFLD, which involved insulin resistance, inflammation, and metabolic disorder. Berberine could alleviate the pathological process in MCD and HFD induced NASH model possibly through certain mentioned biological processes:1Berberine could modulate macrophage phenotype transformation, e.g. suppressing the ratio of M1type and promoting M2type macrophages in hepatic tissue. Thus, pro-inflammatory cytokines were down-regulated. Meanwhile, anti-inflammatory cytokines were up-regulated to against pathological development and progression.2Berberine could partly alleviate the pathological process in HFD induced NASH model possibly through PPAR-y pathway.3Metabolomics results suggested that HFD could perturb kinds of metabolic networks involved metabolism of lipids and protein. According to the results, berberine could possible lead to a switch on the metabolic disorder of NASH.Comparing with other conventional methods, we reported a better approach for studying the pathogenesis of NASH and pharmacological mechanisms of berberine using UPLC-Q-TOF/MS techniques coupled with histopathology and biochemical analysis. These insights triggered a major change in the strategies adopted in the botanical drug research:the shift from’single compound drug to single target to single signal pathway’ to ’single compound drug to multiple targets’. Berberine can prevent NASH through multiple pathways, such as improving IR, regulating inflammatory factors and metabolism disorder. The research provides experimental data and new strategy on treating NASH for berberine.