Effect And Mechanism Of High Fat Microenvironment On Hepatic Stellate Cells Activation

Background and objectiveNon-alcoholic fatty liver disease(NAFLD)can progress to liver fibrosis,which can eventually lead to liver cirrhosis and liver cancer.The mechanism of its progression is not clear,but there is evidence that hepatic stellate cell(HSCs)activation has a key role in liver fibrosis.In NAFLD,HSCs may be exposed to high concentrations of free fatty acid(FFA)from circulating lipids like normal liver cells.However,the role of the high-fat microenvironment in activating HSCs is still unclear.Studies have shown that the Rho/ROCK signaling pathway plays a crucial regulatory role in the activation,proliferation and migration of HSCs.The purpose of this research is to investigate the impact of high-fat microenvironment on HSCs and its regulation of Rho/ROCK signaling pathway.Method1.Primary HSCs were isolated from adult male SD rats(body weight 300.0g-400.0g)by Percoll density gradient centrifugation method,and cultured with complete culture medium.328 nm ultraviolet light and inverted microscope observation of primary HSCs autofluorescence and cell morphology.Trypan blue staining was used to determine cell viability.The perinuclear lipid droplet staining of HSCs was observed by oil red O staining.The expression of Desmin and α-SMA was detected by immunocytochemistry(ICC),and finally the primary HSCs were identified.2.Male SD rats(body weight 350.0g-400.0g),fasted for 48 hours(8:00am-8:00am),and then fed with high fat die(energy 5.58kcal/g).The portal vein blood was extracted at 0h,2h,3h,and 4h,and the portal vein high-fat serum of re-feeding rats was separated and obtained.Palmitic acid(PA)powder was added to 10% bovine serum albumin(BSA)solution,stir at 50°C for 24 hours to fully dissolve it,and prepare an8 m M PA solution.Use PA solution and portal vein high-fat serum to construct an in vitro high-fat microenvironment model,with different concentrations of PA solution(0,100,200,300,500,1000μmol/L)and different concentrations high-fat serum(low,medium,high concentration)culture rat primary HSCs and human hepatic stellate cell line(LX-2),and cell proliferation was detected by flow cytometry and CCK kit.Western Blotting and real time PCR were used to detect the protein and mRNA expression levels of PDGF,TGF-β,α-SMA,Collagen Ⅰ and Collagen Ⅲ in cells.The cell scratch test was used to detect changes in the migration ability of HSCs.3.Real time PCR detected the expression levels of Rho and ROCK mRNA,and studies the mechanism of the high-fat microenvironment on the proliferation,activation and migration of HSCs.Results1.Primary rat HSCs were successfully isolated by Percoll density gradient centrifugation,Trypan blue rejection rate is greater than 95%,autofluorescence can be observed under 328 nm ultraviolet light.The inverted phase-contrast microscope can observe the typical morphological changes of hepatic stellate cells: the transparent small round cells gradually enlarge into polygons and small stellate shapes.Finally,the cells fuse into sheets,the cell body is wide,the cytoplasmic axons are fully extended,and the cell skeleton is obvious.The staining of primary HSCs perinuclear lipid droplets was revealed by Oil red O staining.Immunocytochemical staining showed Desmin(+)and α-SMA(-)in the quiescent primary HSCs,and Desmin(+)and α-SMA(+)in the activated primary HSCs.2.Compared with the contents of TG(0.87±0.18mmol/L)and FFA(0.77±0.05mmol/L)in portal vein serum of normal control rats,the contents of TG in portal vein serum of starving rats at 2h,3h and 4h were 1.64±0.28,2.48±0.23 and 3.12±0.18mmol/L,respectively(P<0.05),and the contents of FFA were 0.91±0.03,1.50±0.11,1.79±0.28 mmol/L(P<0.05).Exposure of LX-2 cells to different concentrations of PA and high-fat serum in rat portal vein significantly promoted lipid accumulation in a dose-dependent manner(P<0.05),indicating that a high fat microenvironment cytological model was successfully established in vitro.3.PA and high-fat serum have different effects on HSCs.(1)LX-2 cells and primary HSCs were treated with different concentrations of PA and high-fat serum respectively.PA inhibited the proliferation of LX-2 cells,while high-fat serum promoted the proliferation of LX-2 cells,in a time-dose response relationship.(2)Higher concentration PA increased the expression of α-SMA in LX-2 cells,decreased type Ⅰ and Ⅲ collagen,and low concentration PA(100μM)promoted the expression of type Ⅲ collagen.The results of real time PCR and western blot showed high-fat serum promotes the expression of α-SMA,type Ⅰ and Ⅲ collagen.(3)After the primary HSCs of rats were treated with various concentrations of PA for 12 hours,the mRNA expression levels of α-SMA,type Ⅰ and Ⅲ collagen in the cells increased to varying degrees.However,after treatment with high-fat serum,there was only high concentration high-fat serum group promoted the increase in the expression of α-SMA,type Ⅰ and Ⅲ collagen.(4)The scratch test found that higher concentrations of PA and high-fat serum could promote the migration of LX-2 cells.4.1000μM PA promoted the expression of TGF-β and PDGF mRNA in LX-2 cells,and the high concentration high-fat serogroup promoted the expression of PDGF mRNA in LX-2 cells,but inhibited the expression of TGF-β mRNA.All concentrations of PA and both the high concentration high-fat serogroup promoted the expression of TGF-β and PDGF mRNA in primary HSCs.The 1000μM PA and high-concentration high-fat serogroup promoted the expression of Rho and ROCK mRNA in LX-2 cells,which was basically consistent with the expression of TGF-β and PDGF.Conclusions1.Primary HSCs of rats were successfully isolated by liver perfusion in situ,digestion in vitro and Percoll density gradient centrifugation,and the quiet and activated primary HSCs were identified.2.A high fat microenvironment model was successfully established to simulate cell culture under different high-lipid conditions.It was found that PA could lead to lipid accumulation in LX-2 cells,and the high fat microenvironment could promote the expression of α-SMA,Collagen Ⅰ and Collagen Ⅲ in HSCs,thus affecting the activation,proliferation and migration of HSCs.3.High fat microenvironment may up-regulate the expression of Rho/ROCK signaling pathway by promoting the secretion of TGF-β and PDGF in HSCs,thereby promoting the activation of HSCs and leading to the development of liver fibrosis.