Effect Of Ginsenoside Rd On Hepatic Stellate Cells Activated By Oxidized Low-density Lipoprotein And Its Mechanism

Hepatic fibrosis is a dynamic process of wound healing to a variety of acute and/or chronic stimuli.The pathogenesis involves the activation and transformation of quiescent hepatic stellate cells(HSCs)into myofibroblast-like cells and the excessive accumulation with proteins such as ?-smooth muscle actin,collagens.Transforming growth factor-?1(TGF-?1)is one of the most potent cytokines to induce the activation of HSC s,it also initiates TGF-?1/Smad signals to promote the production and accumulation of extracellular matrix(ECM),and subsequently made the wound healing more difficult.CD36 is a scavenger receptor that is widely expressed in many cells and functions the uptake of long chain fatty acids(FA).In the liver CD36 is lowly expressed on the endothelial,parenchymal,and Kupffer cells,but it can be induced by fasting or activation of some transcription factors and it enhances hepatic uptake of FA and triglyceride(TG)accumulation.Autophagy involves in the activation of HSCs,which can provide energy by degrading the damaged organelles and promote the progression of hepatic fibrosis.Autophagy also participates in lipid metabolism in which it decomposes the lipid droplets and attenuates inflammation of hepatocytes.Ginsenoside Rd(Rd),a rare monomer compound,is one of the most active ingredients extracted from ginseng.Owing to its promising biological activities,Rd is widely used for protecting brain and cardiovascular disease as well as for regulating immune response and wound healing.But the effect of Rd on hepatic fibrosis involved by lipid metabolism keeps unknown.Therefore,this study aimed to explore the underlying effect of Rd on anti-fibrosis using HSCs activated by oxidized low-density lipoprotein(ox-LDL)in vitro,in order to provide a new perspective for its pharmacological activities.Methods1.MTT assay for viability and proliferation of HSCsHSCs were subcultured in 96 well plate and Rd was added with different concentrations for 24 hours and 48 hours respectively;add MTT working solution for 4 hours,then add DMSO for assay at 492 nm.2.Activation of HSC-T6 treated with ox-LDL in vitroHSCs were subcultured in 24 well plate when reaching 80%.Twelve hours later,ten microgram/ml ox-LDL was used to activate HSCs for 48 hours.3.Group dividing and Rd treatmentExperiments were divided into 4 groups:Control group,model group,low dose group,high dose group.HSCs were incubated with PBS,10 ?g/ml ox-LDL,10 ?g/ml ox-LDL+20 ?mol/L Rd and 10 ?g/ml ox-LDL+40 ?mol/L Rd for 48 hours.4.Cholesterol assay kit for total cholesterol assayHSCs lysate was divided into two parts,one for total protein assay and another for cholesterol assay.HSCs' total cholesterol was measured using cholesterol assay kit;the total protein concentration was measured using BCA assay kit,using total cholesterol/total protein to compare the difference of each group.5.Oil red staining for lipid uptakeHSCs were passaged on coverslips.4% paraformaldehyde was used to fix cells.Oil red working solution was added and 60% isopropyl alcohol was used to rinse coverslips.Observe directly under inverted microscopy.6.Immunofluorescence assay for COL1A1HSCs were passaged on coverslips.4% paraformaldehyde was used to fix cells,blocking buffer was used to block antibody and anti-COL1A1 antibody was added on the coverslips.After incubation overnight,FITC-labeled goat anti-rabbit IgG was added.DAPI staining solution was added to stain nucleus.Observe directly under inverted fluorescence microscopy.7.Determination of m RNA expression level by real-time PCRTotal RNA Kit was used to purified RNA,total concentration of RNA was measured using spectrophotometer,PrimeScript? RT reagent Kit was used to reverse RNA into cDNA,m RNA expression level was determined by real-time PCR,using Rat GAPDH as a reference,2-??Ct method was used to calculate the relative fold of target gene.8.Determination of the protein expression by Western blotRIPA lysate was used to extract total protein,BCA assay was used to determine the concentration of total protein.Western blot was used to assay the expression of protein:CD36,collagen type I(COL1A1),Smad2,Smad4,LC3?.Gray analysis was measured by Quantity One.Results1.Rd has no cytotoxicity under the lower concentration of 40?M;model group slightly proliferated after 24 hours,low dose group and high dose group significantly inhibited the proliferation of HSCs(p<0.01),compared with control group.Model group markedly proliferated after incubation for 48 hours(p<0.05),compared with control group;low dose group and high dose group significantly inhibited the proliferation of HSCs(low dose group: P<0.05;high dose group: P<0.01),compared with model group.2.Compared with control group,total cholesterol content of model group increased significantly(P<0.01);high dose group markedly inhibited the cholesterol accumulation in HSCs compared with model group(P<0.05).3.The number of lipid droplets and lipid uptake of model group increased no tably compared with control group,while high dose group significantly decreased the number of lipid droplets in HSCs compared with model group.4.Compared with control group,fluorescence intensity of model group was significantly increased,while the high dose group notably decreased the fluorescence intensity of HSCs compared with model group.5.TGF-?1 and CD36 mRNA increased significantly in HSCs stimulated with ox-LDL by comparison of those in control group(P<0.01).CD36 m RNA increased significantly after treatment of Rd by comparison of model group(P<0.01).6.Compared with control group,protein expression of COL1A1,CD36 and LC3? of model group increased significantly(COL1A1: P<0.05;CD36: P<0.01;LC3?: P<0.05,respectively);Smad2 and Smad4 protein expression had no obvious change.Compared with model group,low dose group and high dose group both inhibited the COL1A1 expression significantly(low dose group: P<0.05;high dose group: P<0.01,respectively);high dose group markedly inhibited the protein expression of CD36 and Smad4(P<0.01);low dose group and high dose group both significantly inhibited the expression of LC3?(low dose group: P<0.05;high does group: P<0.01,respectively).Conclusion1.Low concentration of ox-LDL could activate the HSCs in vitro and notably increase TGF-?1 and CD36 m RNA expression and COL1A1 protein expression.2.Effect of Rd on anti-fibrosis may have relationship with the inhibition of proliferation of activated HSCs and COL1A1 protein expression by reducing CD36 protein expression and lipid uptake.3.Rd inhibited the activation of HSCs probably by inhibiting TGF-?/Smad pathway and reducing the level of autophagy.