The Role Of Sumoylation Of PML In Human HSCs Fibrosis Induced By Arsenic And The Intervention Effect Of Procyanidins

ObjectiveArsenic trioxide(ATO)can activate the TGF?/Smad signaling pathway and cause HSCs activation,and ATO also has a regulatory effect on PML Sumoylation.Grape seed proanthocyanidin extract(GSPE)can regulate the TGF?/Smad signaling pathway to antagonize liver fibrosis caused by arsenic.However,it has not been reported whether PML Sumoylation plays a role in ATO-induced HSCs activation and whether procyanidins regulate PML Sumoylation to activate the TGF?/Smad signaling pathway.Therefore,this study used ATO and GSPE to intervene in HSCs to verify whether PML Sumoylation plays a role in ATO-induced HSCs activation,and to provide strategies for the prevention and treatment of ATO induced liver fibrosis;To explore whether proanthocyanidins can act on PML Sumoylation to antagonize ATO induced HSCs activation,and provide theoretical basis for improving the mechanism of action of GSPEMethodsThis experiment uses HSCs as the experimental objects,we use ATO(2 ?mol/L),GSPE(50 mg/L),PML siRNA,UBC9 siRNA and RNF4 siRNA to interpose HSCs.Cells activity was performed by CCK-8 kit;Western Blot was used to detect the levels of PML Sumoylation related proteins(S-PML,PML,SUMO-1,SUMO-2/3),TGF?/Smad signaling pathway,HSCs activation indicators(?-SMA,Collagen I)and inflammatory indicators(IL-1?,TNF-?);Immunofluorescence staining to detect the formation of PML-NBs;qRT-PCR to detect the expression levels of TGF-?1,?-SMA and Collagen I.One-way ANOVA method was used to compare the differences between multiple groups,and Bonferroni method was used for multiple comparisons between groups.Analysis of the interaction between ATO and GSPE by factorial design ANOVA was performed.P<0.05 was considered as statistically significant.Results1.Effects of ATO and GSPE on HSCs activity In the CCK-8 experiments,2?mol/L,3?mol/L and 5?mol/L ATO interfered with HSCs,the cell viability increased(P<0.05);Compared with the control group,after 24 h of 25 mg/L and 50 mg/L GSPE intervention,there was no significant change in cell viability(P>0.05);50 mg/L GSPE can reduce ATO-induced cell activity(P<0.05)2.Effects of ATO and GSPE on inflammation indexes in HSCs The levels of IL-1?(0.45±0.01)and TNF-?(0.35±0.02)in the ATO group were significantly increased;25mg/L and 50mg/L GSPE can reduce the expression of IL-1? and TNF-? induced by ATO(P<0.05)3.Effects of ATO and GSPE on HSCs activation index The levels of ?-SMA and Collagen I in the ATO intervention group were up-regulated(P<0.05);GSPE down-regulated the expression level of a-SMA induced by ATO(P<0.05)4.Effects of ATO and GSPE on TGF?/Smad signaling pathway Treatment of HSCs with 2?mol/L ATO increased the expression of TGF-?1(0.51±0.04)and phosphorylated Smad2/3(0.28±0.03)(P<0.05);GSPE down-regulated the expression of TGF-?1(0.24±0.01)and p-Smad2/3(0.18±0.02)induced by ATO(P<0.05).5.Effects of ATO and GSPE on PML Sumoylation 2 ?mol/L ATO treatment for 4 h and 3 ?mol/L ATO treatment for 2/4 h increased the expression levels of PML Sumoylation-related proteins(P<0.05);ATO treatment of HSCs did not stimulate PML Sumoylation after 24h(P>0.05);There was no significant change in the expression of PML Sumoylation-related proteins in the GSPE treatment group(P>0.05);50 mg/L GSPE down-regulated the expression levels of S-PML(0.27±0.03),PML(0.18±0.01),SUMO-1(0.37±0.02)and SUMO-2/3(0.40+0.02)induced by ATO(P<0.05).6.Effects of ATO and GSPE on PML Sumoylation after silencing PML,UBC9 and RNF4,respectively Compared with the non-transfected group,the expression levels of PML Sumoylation-related proteins in the PML-transfected group was significantly reduced(P<0.05);Based on PML transfection,GSPE reduced the expression levels of S-PML(0.25±0.02),PML(0.36±0.02),SUMO-1(0.46±0.02),and SUMO-2/3(0.48±0.03)induced by ATO(P<0.05).After UBC9 was silenced,the expression of PML Sumoylation related proteins were all down-regulated(P<0.05);while the expression of S-PML,PML,SUMO-1 and SUMO-2/3 proteins were significantly increased after RNF4 was silenced(P<0.05);After transfection with UBC9 or RNF4,the application of GSPE intervention significantly down-regulated the expression levels of S-PML,PML,SUMO-1 and SUMO-2/3 induced by ATO(P<0.05).7.Effects of ATO and GSPE on HSCs activation indexes after silencing PML,UBC9 and RNF4,respectively Compared with the non-transfected group,the PML-transfected group reduced the expression levels of TGF-?1,?-SMA,and Collagen I(P<0.05);After silencing PML,GSPE down-regulated ATO-induced expression of TGF-?1(0.69±0.03),?-SMA(0.73±0.03)and Collagen I(0.48±0.02)proteins(P<0.05).Silencing UBC9 down-regulated ATO-induced mRNA and protein expression levels of TGF-(31,p-Smad2/3,a-SMA,and Collagen content induced by ATO(P<0.05),while silencing RNF4 increased expression levels of HSCs activation indicators(P<0.05);On the basis of transfection with UBC9 or RNF4,GSPE down-regulated the expression levels of ATO-induced TGF-?1 and ?-SMA(P<0.05).ConclusionATO can induce HSCs activation by stimulating PML Sumoylation,causing liver fibrosis.GSPE may antagonize ATO-induced liver inflammatory injury by reducing the expression of IL-1 ± and TNF-?;GSPE may also antagonize ATO-induced HSCs activation by inhibiting PML Sumoylation and down-regulating TGF?/Smad signaling pathway.