Experimental Study On NAFL Fibrosis Induced By Activated Hepatic Stellate Cells Under Mechanical Pressure

Objective:Establish a pressure microenvironmental cytology model to explore the effect of increased intracarotid sinus pressure on the activation of hepatic stellate cells and liver fibrosis in the NAFL stage.Methods:1.Grouping:(1)Control group:pressure was 0 mmHg(control,transwell)(2)Experimental group:pressures respectively were 4 mmHg,6 mmHg,8 mmHg,10 mmHg.2.Establish a pressure microenvironment cytology model:cut the transwell chamber and place it in a 6-well plate;place a weighing bottle on the top of the chamber;add ceramic grinding beads of appropriate quality into the weighing bottle.3.LX-2 cells were cultured in groups for 12 h,24 h,36 h,48 h,72 h,96 h,and the pressure conditions were 4 mmHg,6 mmHg,8 mmHg,and 10 mmHg,respectively.Observe the effect of mechanical pressure on the morphology of LX-2 cells with an inverted phase contrast microscope.LX-2 cells were cultured in groups for 48 h,72 h,96 h,and the pressure conditions were 4 mmHg,6 mmHg,8 mmHg,and 10 mmHg,respectively.LX-2 cells were stained with FITC-labeled phalloidin,and the cytoskeleton changes were observed by laser confocal microscope.The Young’s modulus of LX-2 cells was detected by biological atomic force microscope at 48 h and 72 h.4.LX-2 cells were cultured in groups for 24 h,48 h,72 h,96 h,and pressures were 4 mmHg,6 mmHg,8 mmHg,and 10 mmHg,respectively.The proliferation of LX-2 cells was detected by flow cytometry for 72 h.Scratch test detects 24 h and 48 h LX-2 cell migration.Quantitative Real-time PCR detects the expression of integrin αvβ3 and ITGB3 mRNA in LX-2 cells at 48 h,72 h and 96 h.5.LX-2 cells were cultured in groups for 48 h,72 h,96 h,and the pressure conditions were 4 mmHg,6 mmHg,8 mmHg,and 10 mmHg,respectively.Western-blotting was used to detect the expression of α-SMA protein in LX-2 cells.Quantitative Real-time PCR was used to detect the expression of ACTA2,COL1A1,COL3A1,COL4A1,TGFB1 and HGF mRNA.6.The rat primary hepatic stellate cells were cultured in groups for 24 hours under pressure conditions of 4 mmHg,6 mmHg,8 mmHg,and 10 mmHg.Quantitative Real-time PCR was used to detect the expression of Acta2 and Col1a1 mRNA.Results:1.The effect of mechanical pressure on the morphology and cytoskeleton of LX-2 cells:(1)When the pressure conditions were 4 mmHg,6 mmHg,8 mmHg,and 10 mmHg,the culture time was 36 h,48 h,72 h,and 96 h,respectively,the LX-2 cells appeared protruding,resembling"fluffy",and their morphology changed from polygonal to fusiform.(2)When the pressure conditions were 4 mmHg and 6 mmHg,the culture time was 48 h and 72 h,the F-actin expression of LX-2 cells increased.When the pressure conditions were 8 mmHg and 10 mmHg,the expression of F-actin in LX-2 cells decreased at 48 h,and the expression of F-actin in LX-2 cells increased at 72 h.(3)When the pressure conditions were 4 mmHg and 8 mmHg,the Young’s modulus of LX-2 cells increased at 48 h,and the Young’s modulus of LX-2 cells decreased at 72 h.2.The effect of mechanical pressure on the growth behavior of LX-2 cells:(1)Mechanical pressure promoted the proliferation of LX-2 cells(4 mmHg,6 mmHg,8 mmHg,72 h).(2)When the pressure conditions were 4 mmHg,6 mmHg,8 mmHg,and 10 mmHg,the culture time was 24 h and 48 h,respectively.Compared with the control group,the migration of LX-2 cells in the experimental group did not change significantly.(3)When the pressure conditions were 4 mmHg,6 mmHg,8 mmHg,and 10 mmHg,the culture time was 48 h,72 h,and 96 h,respectively.Compared with the control group,the expression of integrin αvβ3 mRNA in the LX-2 cells of the experimental group was decreased,but the expression of ITGB3 mRNA did not change.3.The effect of mechanical pressure on the activation and fibrosis of LX-2 cells and rat primary hepatic stellate cells:(1)Mechanical pressure promoted the expression of α-SMA protein in LX-2 cells(8 mmHg,10 mmHg,36 h).(2)Mechanical pressure promoted the expression of COL1A1 mRNA in LX-2 cells(6 mmHg,72 h).(3)Mechanical pressure inhibited the expression of TGFB1 mRNA in LX-2 cells(4 mmHg,6 mmHg,8 mmHg,10 mmHg,48 h,72 h and 96 h).(4)Mechanical pressure promoted Col1al mRNA expression in rat primary hepatic stellate cells(4 mmHg,24 h).Conclusions:1.Mechanical pressure may promote the expression of α-SMA and collagen I by activating hepatic stellate cells,which is involved in the process of NAFL fibrosis2.TGF-β may not be the main factor of NAFL stage stress-induced liver fibrosis,and the specific mechanism still needs to be further explored.