The Improvement Effects And Mechanisms Of Baicalin Against High Fat Diet-induced Nonalcoholic Fatty Liver Disease In Mice

Non-alcholic fatty liver disease(NAFLD)is the most prevalent chronic liver disease in the world,affecting approximately 25 percent of adult popultion and imposing a significant burden on human health and global healthcare system.Currently,there are no effective clinical-approved drugs.Therefore,the research and development of drug candidates have become an urgent need.Traditional Chinese medicine monomers have attracted increasing attention from researchers due to due to their multi-target and broadspectrum action mechanism Although reports have revealed the lipid lowering,anti-oxidant,antiinflammatory and hepatoprotective effects of baicalin(BA),an active ingredient of traditional Chinese medicine,whether and how BA exerts protective effect against NAFLD is largely unclear.Here,BA was selected as the research object to explore its improvement effect against NAFLD by in vivo and in vitro models.Then,liver transcriptomics was used to find the key signaling pathway of BA ameliorative effect from a global perspective.Finally,the molecular mechanisms of BA against NAFLD was explored at cellular level.The main results are as follows:1 BA can effectively relieve high fat diet(HFD)-induced NAFLD in miceTo investigate the effects of BA on NAFLD mice,a total of 120 8-week-old male C57BL/6J mice were randomly divided into 4 groups after 2 weeks of adaptation: conventional diet control group,conventional diet +BA control group,HFD group and HFD+BA group.After 16 weeks of feeding to establish a mouse NAFLD model,except for the normal diet control group and HFD group which all were gavaged the same volume of 0.5% sodium carboxymethyl cellulose,the other two groups were gavaged with BA(100 mg/kg/d),and the intervention period lasted for 8 weeks.Then,the improvement effect of BA against HFD-induced NAFLD mice was detected.The results showed that compared with the normal diet control group,the parameters of the normal diet +BA group were not significantly different.Compared with HFD group,HFD+BA group significantly reduced feed intake,liver weight and liver weight/body weight ratio,improved liver lipid deposition,reduced blood or liver triglyceride(TG)and total cholesterol(TC)content,reduced liver function injury,and alleviated liver oxidative stress and inflammatory response.These results illustrated that BA can improve NAFLD in vivo.2 Effects of BA on palmitic acid(PA)-induced AML-12 cellsTo determine the safe dosage of BA at the cellular level,CCK-8 assay was performed on AML-12 cells,and then the concentration of BA administration in vitro model was explored using co-treated cells with PA.Then AML-12 mouse liver cells were induced by PA(400 μM,24 h)to establish NAFLD cell model.Finally,the efficacy of BA in alleviating NAFLD was discussed at the cellular level.The results showed that BA concentration less than or equal to 50 μM had little effect on the activity of AML-12 cells.Co-treatment with PA showed that BA less than or equal to 25 μM showed good cell protective activity.The NAFLD cell model was then treated with 25 μM BA,and the results showed that BA treatment could significantly reduce the abnormal lipid metabolism,oxidative stress and inflammation caused by PA.These results indicate that BA can effectively improve NAFLD in vitro,which lays a foundation for further discussion on the molecular mechanism.3 Liver transcriptomics was used to investigate the key signaling pathways of BA improving NAFLDTo systematically explain the improvement effect of BA against NAFLD,RNA was extracted from the liver tissues of mice of HFD group and HFD+BA group for RNA-sequencing analysis,and the core signaling pathway was determined according to the analysis results,and then RT-PCR and Western blot verification were performed on the liver tissue to determine the reliability of the core signaling pathway.Finally,inhibitor of the signaling pathway was used for validation in vitro.The results showed that,compared with HFD group,BA intervention caused significant changes in liver tissue gene expression.Functional analysis of 409 significantly differentially expressed genes(DEGs)showed that these DEGs were closely related to lipid metabolism.Further enrichment analysis of signal pathway showed that AMPK signal pathway exhibited the most significant changes.The results of liver tissue and cells RT-PCR and Western blot fully corroborated the transcriptomic analysis results.The addition of AMPK inhibitor Compound C(CC)in the in vitro model weakened the lipid accumulation reduction,anti-oxidation and anti-inflammatory effects of BA,and Western blot showed that CC inhibited the activation of AMPK signaling pathway by BA.These results indicate that AMPK signaling pathway is the key signaling pathway for BA improving NAFLD,and this result lays a foundation for further discussion on the molecular mechanism of BA improving NAFLD.4 Based on transcriptome results,the molecular mechanisms of BA improving NAFLD was exploredIn vitro and in vivo experiments have shown that the improvement of BA against NAFLD is closely related to its reduction of lipid deposition,oxidative stress and inflammatory response.Further transcriptome analysis shows that the core signaling pathway of BA’s improvement effect is AMPK signaling pathway.Therefore,three signaling pathways downstream of AMPK signaling pathway,which are closely related to lipid metabolism,oxidative stress and inflammatory response,were taken as research objects to further explore the molecular mechanisms of BA-mediated AMPK improving NAFLD.The results showed that BA inhibited lipid accumulation in hepatocytes by down-regulating SREBP1 signaling pathway.BA can also upregulate Nrf2 signaling pathway to reduce oxidative stress.BA also reduces inflammation by downregulating the NF-κB signaling pathway.These results suggest that BA improves NAFLD by regulating the SREBP1/Nrf2/NF-κB signaling pathway in an AMPK-dependent manner.In summary,the results of this study indicate that BA can effectively improve NAFLD both in vitro and in vivo.Transcriptome analysis shows that AMPK signaling pathway plays an important bridging role in this process.Further mechanism analysis reveals that AMPK mediated by BA regulates SREBP1/Nrf2/NF-κB signaling pathway to reduce lipid accumulation,oxidative stress and inflammatory response in the liver,thereby maintaining liver homeostasis and improving NAFLD.These findings will expand our understanding of the molecular mechanism of BA improving NAFLD and provide new drug targets for effective prevention and treatment of NAFLD.At the same time,the application of transcriptome provides a new paradigm for analyzing the mechanisms of traditional Chinese medicine components in prevention and treatment of diseases.