Study On The Effect Of Inorganic Arsenic On The Autophagy Level Of Human Hepatic Stellate Cells

Objective:To study the effect of different doses of inorganic arsenic on the autophagy level of human hepatic stellate cells,and to explore the blocking effect of trimethyl adenine on inorganic arsenic-induced autophagosome-lysosomal fusion.Methods:1.RFP-GF P-LC3 lentivirus was used to infect LX-2cells,and cell lines stably expressing RFP-GFP-LC3 gene were screened by puromycin.The infection efficiency was analyzed by flow cyt ometry and laser confocal microscopy,respectively.Stably infected cells were treated with starvation,and the expression of RFP and GFP fluorescence and the formation of autoph agolysosomes were observed under alaser confocal microscope.2.CCK8 was used to det ect cell viability;the IC50 was determined and the arsenic concentration was determined a ccording to the specific conditions,including sodium arsenite(A_S³⁺)high,medium and low dose groups and sodium arsenate(A_S⁵⁺)high,medium and low dose groups.3.Flowcy tometry（FCM）was used to detect the apoptosis and cell cycle of LX-2cells after lentivirus infection with different doses of inorganic arsenic.4.Real-Time Quantitative PCR was used to detect the expression levels of LC3,Beclin-1,ATG4,ATG5,ATG8,BCL-2,BCL-X L mRNA in LX-2cells after lentiviral intervention with inorganic arsenic.5.Western blot detection of inorganic arsenic intervention on LX-2cells after microtubule-associated prote in light chain 3（LC3）,recombinant human autophagy effector protein（Beclin-1）,ubiquiti n-binding protein P62（SQSTM-1/P62）,transmembrane protein 49（TMEM-49）,mammalian rapamycin target protein（mTOR）,B lymphoma 2（BCL-2）,recombinant human B-cell lymphoma factor 2XL（BCL-XL）,αsmooth muscle actin（α-SMA）protein expression in cells.6.CCK8 method was used to detect the metabolic activity of trimethyl adenine+inor ganic arsenic on LX-2cells after lentivirus infection.7.Laser confocal microscopy was used to observe the expression of RFP,GFP and the formation of autophagolysosomes in LX-2cells infected with trimethyl adenine+inorganic arsenic.8.The expression levels of LC3,Beclin-1,SQSTM-1/P62 mRNA and protein in LX-2cells infected with trimethyl adenine+inorganic arsenic were detected by PCR and Western blot.Results:1.After the RFP-GF P-LC3 lentivirus stably infected LX-2 cells,the lentivirus infection rate was determined to be about 70%by flow cytometry,and its cell morphology was not significantly different from that of the normal group under confocal laser observation.There was also no significa nt difference between the red fluorescence intensity and the green fluorescence intensity.After starvation,an unobstructed fusion pathway between autophagosome and lysosome w as observed.2.Inorganic arsenic-exposed LX-2cells,grouped according to IC50,the high,medium,and low doses of the sodium arsenite group were 5μmol/L,0.5μmol/L,and 0.05μmol/L,respectively.The high,medium and low doses of the sodium arsenate group were60μmol/L,6μmol/L,and 0.6μmol/L,respectively.The control group and transfection g roup were（0μmol/L）.Cell metabolic activity gradually decreased with increasing dose.3.Analysis of the detection results by flow cytometry.Compared with the blank group,with the increase of the concentration of inorganic arsenic,the apoptosis rate gradually increas ed（all P<0.05）.Cell cycle test results showed that compared with the blank group,except for the infection group,infection+trivalent arsenic low dose group,infection+pentavale nt arsenic low dose group,the proportion of cells in the G0-G1 phase decreased,and the proportion of cells in the other dose groups increased significantly（All P<0.05）;compared with the blank group,the proportion of S-phase cells in the infection group,infection+tri valent arsenic low-dose group,infection+pentavalent arsenic low-dose group increased,a nd the proportions of the remaining dose groups decreased（P<0.05）).4.Compared with t he blank group,inorganic arseniccan up-regulate the expression of LC3,SQSTM-1/P62,ATG4,ATG5,and ATG8 mRNA.BCL-2 mRNA increases with the increase of inorganic arsenic dose,and BCL-XL mRNA in trivalent arsenic dose It decreased with increasing dose in the group（P<0.05）.5.Compared with the blank group,inorganic arsenic up-regulated the protein expressions of LC3,SQSTM-1/P62,TMEM-49,and BCL-XL,and down-re gulated the expression of mTOR protein.Beclin-1 decreased with the increase of inorgani carsenic dose In the trend,BCL-2 showed an upward trend with the increase of the inorganic arsenic dose.The inorganic arsenic significantly increased the expression ofα-SMA pr otein,and the trivalent arsenic medium-dose group increased significantly（P<0.05）.6.Th ere was no statistically significant difference in cell viability in the infection group compared with the blank group at 24 and 48 hours（P>0.05）,and the cell viability in the other inte rvention groups decreased significantly（P<0.05）.7.LX-2cells treated with inorganic arsenic alone,trimethyl adenine（3-MA）alone,and trimethyl adenine+inorganic arsenic treat ed lentivirus infection.Observe RFP,GFP and double fluorescence under a confocal micr oscope Fusion,double fluorescence fusion has more yellow fluorescence spots and red fluorescence spots.The trimethyl adenine combined with inorganic arsenic treatment grouph as stronger green fluorescence than red fluorescence.The yellow fluorescent spots after double fluorescence fusion increased compared with the inorganic arsenic alone treatment group.8.Compared with the blank group,both the inorganic arsenic group and the 3-MA+inorganic arsenic group up-regulated the expression of LC3,Beclin-1,SQSTM-1/P62 genes and proteins.Conclusion:Inorganic arsenic can induce apoptosis and autophagy in human hepatic stellate cells,which may have antagonistic effects.Trimethyl adenine inhibits LX-2cell activity and reduces the level of autophagy induced by inorganic arsenic,which may inhibit the progression of liver fibrosis caused by inorganic arsenic.The mechanism of arsenic-induced liver fibrosis remains to be further studied.