COX-2 Inhibits Autophagy Of HSC-T6 Through PI3K/Akt/mTOR Signaling Pathway

Objective:This experiment was designed to study the changes of autophagy level and PI3K/Akt/mTOR signaling pathway related proteins after COX-2 deletion in HSC-T6.To clarify whether COX-2 regulates autophagy of HSC-T6 through regulating PI3K/Akt/mTOR signaling pathway,in order to provide new ideas for the prevention and treatment of liver fibrosis.Methods:1.HSC-T6 was cultured in vitro and divided into control group(HSC-T6 was not treated),NC group(LV-NC-sg RNA virus transfected HSC-T6)and KO group(LV-COX-2-sg RNA virus transfected HSC-T6).Fluorescence microscopy was used to observe the transfection efficiency and Western blot was used to detect COX-2 protein expression.2.Cells were treated with the autophagy inducer Rapamycin and overexpressed virus.They were divided into control group(HSC-T6 without any treatment),Rap group(HSC-T6 with Rapamycin),Rap+OENC group(HSC-T6 with Rapamycin and LV-NC-OE)and Rap+OECOX-2 group(HSC-T6 with Rapamycin and LV-COX-2-OE),NC group(HSC-T6 sg RNA-NC stable sieve cells did not receive any treatment),KO group(HSC-T6 sg RNA-COX-2 stable sieve cells did not receive any treatment).The expression of LC3 was observed by immunofluorescence,the expression of LC3Ⅱ/Ⅰ and P-mTOR was detected by Western blot.3.Treating cells with the autophagy inhibitor 3-methyladenine(3-MA),They were divided into control group(HSC-T6 without any treatment),KO group(HSC-T6 sg RNA-COX-2 stable sieve cells without any treatment),3-MA group(HSC-T6 with 3-MA),KO+3-MA group(HSC-T6 sg RNA-COX-2 stable sieve cells with 3-MA),MDC staining was used to detect the formation of autophagosomes,and Western blot was used to detect LC3Ⅱ/Ⅰ expression.4.Cells were treated with PI3 K inhibitor LY294002 and Akt inhibitor Perifosine.They were divided into control group(HSC-T6 without any treatment),KO group(HSC-T6 sg RNA-COX-2 without any treatment),LY294002 group(HSC-T6 with 10μM LY294002),KO+LY294002 group(HSC-T6 sg RNA-COX-2 stable sieve cells were added with 10μM LY294002),Perifosine group(25μM Perifosine was added to HSC-T6),KO+Perifosine group(25μM Perifosine was added to HSC-T6 sg RNACOX-2 stable sieve cells).The expressions of LC3Ⅱ/Ⅰ,P-mTOR/mTOR and P-Akt / Akt were detected by Western blot.Results:1.The expression of COX-2 was significantly decreased after transfection of LV-COX-2-sg RNA with HSC-T6.2.Compared with the control group,the expression of p-mTOR in Rap group and KO group decreased,while the expression of LC3increased;Compared with the Rap group,the expression of p-mTOR increased and the expression of LC3 decreased after adding COX-2overexpression.3.Compared with the control group,LC3 expression was decreased and autophagosomes were decreased in 3-MA group.LC3 expression was increased and autophagosomes were increased in KO group.Compared with KO group,LC3 expression was decreased and autophagosomes were decreased in KO+3-MA group.4.Compared with the control group,the expressions of p-mTOR and p-Akt in KO group,LY294002 group and Perifosine group decreased,while the expression level of LC3 increased;Compared with KO group,the expression of p-mTOR and p-Akt in KO+LY294002 group and KO+Perifosine group decreased,while the expression level of LC3 increased.Conclusion:After the deletion of COX-2 in HSC-T6,the autophagy level of HSC-T6 was increased,COX-2 inhibits autophagy of HSC-T6 through PI3K/Akt/mTOR signaling pathway.