Influence Of Suberoylanilide Hydroxamic Acid On Human Hepatic Stellate Cells Activation And Potential Mechanism

Aims The purpose of this study was to demonstrate the effects of Histone deacetylases inhibitor(HDACi)-SAHA(suberoylanilide hydroxamic acid) on biological characteristics of actived human hepatic stellate cell line LX2 and the possible mechanisms.Methods In this study, we investigated effects of SAHA on LX2 cell line, biologic behaviors by transwell and EdU experiments, And HSCs activation markers like α smooth muscle actin(α SMA) and collagen I mRNA and protein level were detected by real time PCR(q PCR) and Western Blots(WB). To learn the possible mechanisms involved in effects of SAHA, we test the differently expressed genes by microarray assay, and then analyzed bioinformation of 2-folds changes genes by performing IPA analysis, in our study, we focus on intracellular function of HMGB1 on pro-inflammatory nuclear transcription factor NFκB. We performed small interference RNA(siRNA) experiment to silence HMGB1 gene and then test the role of HMGB1 in SAHA’s effects on LX2.Results EdU and transwell experiments showed that after treated with SAHA, the proliferation and migration ability decreased by 76.25%( p=0.021) and 77.89 %( p=0.003), respectively. Gene microarrayanalysis suggested there were 504 genes showed 2-folds changes, in which, the HSCs activation related gene NFκB1(P50) was decreased by2.68 folds. IPA analysis showed that HMGB1/NFκB signaling pathway was remarkably inhibited. To verify the depression effect of SAHA on NFκB, we detected the total cell lysate of NFκB(P50,P65)and the activity of NFκB, WB showed P50 and P65 protein expression were decreased by 55%(p=0.02) and 67.0%(p=0.01), respectively. Dual Luciferase Reporter(DLR)system showed that after treated with SAHA,the transcriptional activity of NFκB was decreased by 70.1%(P=0.001).Next, we performed siRNA experiments to find out whether intracellular HMGB1 play a role in SAHA’s effects on NFκB inhibition:LX2 were treated with SAHA after blocked the HMGB1 gene,q PCR showed that P50 mRNA was back to untreated state(p>0.05),indicated that HMGB1 was a key factor of downregulation of P50 mRNA by SAHA treatment; Treated LX2 in the same conditions,WB showed that the downregulation of protein expression of P50 was restored to 73.7%(p=0.0013)of untreated state, and DLR system showed the activity of NFκB was restored to 53% of untreated state, these may indicated that intracellular HMGB1 may play a role in SAHA induced inhibition of NFκB. Next, WB showed that there were no significant change of HMGB1 protein level in total cell and nucleus lysate, and the immunofluorescent staining showed that HMGB1 accumulated in nucleus and perinuclear became scattered after SAHA treatment, immunoprecipitation experiment indicated that SAHA leads to a remarkable increase of Lysine residues acetylation of HMGB1 protein.Conclusions Our study demonstrated for the first time that SAHA inhibits human HSCs activation, we hypothesize that the SAHA effect results probably from HMGB1 depended downregulation NFκB1 and inhibition of NFκB activity.