Effects And Mechanism Research Of Dangguiliuhuang Decoction On Nonalcoholic Fatty Liver

Aim:To establish a steatosis cell model of nonalcoholic fatty liver(NAFL)by free fatty acid(FFA),study the effect and mechanism of Dangguiliuhuang Decoction(DGLHD)on this cell model,and evaluate the underlying effects and mechanisms of DGLHD on NAFL mice.Methods:Firstly,HepG2 cells were cultured with different concentrations of free fatty acids(oleic acid:palmitic acid=2:1).Cell viability was determined by MTT assay.The intracellular lipid droplets were observed by Oil red O staining.Lipid content was measured by Oil Red colorimetric assay.The level of triglyceride(TG)in the model group was detected by enzymatic method.Secondly,NAFL cell model was induced by FFA,and then treated with different doses(2.5 or 5 or 10 mg/mL)of DGLHD.The intracellular lipid accumulation was observed by Oil red O staining,and intracellular TG contents were measured by enzymatic method.The mRNA expression of PI3K,Akt,mTOR,GSK3β,GLUT4,ACC-1 and CD36 were detected by real-time PCR.Western blot method was used to determine the protein expression levels of p-PI3K,t-PI3K,p-Akt and t-Akt.Finally,the NAFL model was induced in mice by high fat-diet(HFD).Four weeks after HFD administration,mice were administered with different doses of DGLHD(10 or 5 or 2.5 g/kg)for 8 weeks.(1)To study the effect of DGLHD on lipid metabolism and liver function,the pathological sections of liver and adipose tissue were analyzed,Oil red O staining was used to detect lipid droplets in the liver,and the levels of TG,free fatty acid(NEFA),asparta te aminotransferase(GOT)and alanine aminotransferase(GPT)in serum and tissues were measured.(2)The effect of DGLHD on insulin sensitivity were studied by oral glucose tolerance and insulin tolerance test as well as serum insulin and adiponectin levels.(3)To investigate the effect of DGLHD on inflammation,the levels of TNF-α,IFN-γ,TGF-βand IL-10 in serum were measured by ELISA,and the mRNA levels of TNF-α,IFN-γ,TGF-βand IL-10 in liver were measured by Real-time PCR.(4)In order to study the immunological activity of DGLHD on T cells,in addition to lymphocyte proliferation test,the expression of CD3~+CD4~+T cells,regulatory T cells(Tregs),PD-1 and TCR expression on T cell surface in spleen and liver,as well as the expression of CD11c~+CD11b~+and CD11c~+CD8a~+cells in spleen were detected by flow cytometry.Additionally,we also detected the percentage of MHC-II,PD-L1 and costimulatory molecules CD86 on bone marrow-derived dendritic cells(BMDCs),and anlyzed the function of DCs by mixed lymphocyte reaction(MLR)and assessing the content of IL-12p70 secreted by DCs.Results:Firstly,HepG2 cells were cultured in medium containing 1mM concentration of free fatty acid for 24 h.The activity of HepG2 cells was not significantly affected.Under light microscope,there were a lot of lipid droplets in HepG2 cells,and the content of TG was significantly increased.The TG content in the model group was significantly higher than that in the control group.Secondly,compared with the model group,DGLHD could decrease the lipid droplet formation and lipid co ntent as well as the TG content of intracellular in HepG2 steatosis cells.Among them,while the dose of DGLHD is 2.5mg/mL,show the best role.Compared with the model group,DGLHD(2.5mg/mL)could increase the mRNA expression of PI3K,Akt,mTOR,GSK3βand GLUT4,and decrease the mRNA contents of ACC-1 and CD36.The expression of PI3K,Akt,GSK3βand CD36 was statistically different from that of model group.Meanwhile,DGLHD significantly increased the phosphorylation level of Akt protein.Finally,fed C57BL/6J mice with HFD could establish NAFL model.There a lot of vacuoles and lipid droplets in the liver.Meanwhile,TG,NEFA content in the serum and liver tissue was significantly increased.oral tolerance to glucose tolerance,insulin tolerance(TNF-α,IFN-γ)and Th2 anti-inflammatory cytokines(TGF-β,IL-10)in serum were decreased.Oral impaired glucose tolerance and insulin tolerance were occurred.The levels of Th1-type pro-inflammatory cytokines(TNF-α,IFN-γ)in serum were increased,while Th2 anti-inflammatory cytokines(TGF-β,IL-10)were decreased.(1)DGLHD reduced the number of vacuoles and red lipid droplets in the liver of NAFL mice as well as the size of fat cells in adipose tissue.DGLHD decreased the content of TG,NEFA,GOT and GPT in the serum and liver of NAFL mice.Besides,the mRNA levels of SREBP-1C,ACC-1,FAS and CD36 in NAFL mice were significantly down-regulated.(2)DGLHD improved oral glucose tolerance and insulin tolerance in NAFL mice,reduced insulin levels in serum,and increased the adiponectin levels.(3)Consistent with the gene expression in the liver,DGLHD reduced the levels of TNF-α,IFN-γcytokines,but increased TGF-βand IL-10 in the serum of NAFL mice.(4)DGLHD inhibited the proliferation of splenic T lymphocytes from NAFL mice and induced the differentiation of Tregs while had no effect on the percentage of CD3~+CD4~+T cells in spleen and liver.In addition,there was no significant effect of DGLHD on the ratio of CD11c~+CD11b~+and CD11c~+CD8a~+in the spleen.DGLHD increased the expression of PD-1 on the surface of CD4~+T cells in the spleen and liver of NAFL mice,and decreased the ratio of TCR in the liver.DGLHD reduced the expression level of CD86 on DCs surface and increased the expression of PD-L1,but had no significant effect on the expression of MHC-II.In addition,DGLHD significantly inhibited the ability of DCs stimulating T cell proliferation and attenuated the secretion of IL-12p70 from DCs.Conclusion:HepG2 cells were induced by free fatty acids(oleic acid:palmitic acid=2:1)to establish NAFL cell model,the best time was 24h,and the optimal concentration was 1mM.DGLHD can effectively reduce the levels of lipid accumulation and triglyceride in HepG2 steatosis cells.DGLHD improves insulin resistance by activating insulin-related pathways and up-regulating the expression of related genes.Simultaneously,DGLHD suppresses lipid metabolism-related gene expression levels,improves lipid metabolic disorders in vitro.In vivo experiments,DGLHD has the effect of improving lipid metabolism and liver function in NAFL mice,which the mechanism is related to the inhibition of fatty acid synthesis and transport-related genes.DGLHD improves NAFL by improving insulin resistance in NAFL mice.DGLHD exerts anti-inflammatory and hepatoprotective effects by promoting the transformation of Th1 type cytokines into Th2 cells in NAFL mice.DGLHD plays an anti-inflammatory role by inhibiting the proliferation of T cells in NAFL mice,inhibiting the T cell immune response,maintaining the immature state of bone marrow-derived DCs,and inducing Tregs differentiation.In summary,DGLHD improves the cellular and animal models of NAFL,the mechanism of which may be related to the ameliorated lipid metabolism,insulin resitance,inflammation and immune response.This study provides a theoretical basis for the prevention and treatment of nonalcoholic fatty liver by Dangguiliuhuang Decoction.Innovative points:1.We firstly investigated the effect and mechanism of Dangguiliuhuang Decoction on nonalcoholic fatty liver.2.The effect of Dangguiliuhuang Decoction on the percentage and function immune cel s of nonalcoholic fatty liver mice was studied for the first time.