Autophagy Activated By HBx In HepG2 Cell Elicits Inhibition Of NLRP3 Inflammasome Function

Background and Aim:Hepatitis B Virus X protein（HBx）is closely related to the development of Hepatocellular Carcinoma（HCC）.Previous studies have found that HBx can promote autophagy in liver cancer cells through a variety of signaling pathways,while our previous studies also found that HBx can induce mitophagy in HCC.Moreover,the relationship between autophagy and inflammasome is closely,and in most cases,if exposed to oxidative stress,cell will upregulate autophagy to inhibit NLRP3 inflammasome for self-protection.At the same time,other studies have found that the expression of NLRP3 in liver tumor tissues seem to be down-regulated,but not involve Hepatitis B Virus（HBV）.Therefore,the purpose of our research is to preliminarily study the effect of HBV on autophagy and NLRP3inflammasome in HBV-associated HCC,as well as their relationship,and to explore which antigen plays a major role in HBV.Methods:Firstly,we collected tumor tissues and adjacent tissues of 44 patients with HCC.Then immunohistochemical staining about LC3B,Beclin-1,NLRP3 and ASC were performed to calculate the average optical density value（IOD/area）by Image Pro Plus software.Secondly,T test was used to analyze the difference of target protein expression between tumor tissues and adjacent tissues.According to the preoperative HBV levels,patients were divided into four groups to analyze whether it affected the expression of target proteins in cancer tissues by one-way anova,and further verified in cell experiments by comparing Hep G2.2.15 cells with Hep G2 cells.Pearson correlation analysis was used to estimate the correlation between LC3B and NLRP3,and determine whether the two are still correlated after grouping according to HBV content.Next,after knocking down HBx expression in HBV plasmids or overexpressing HBx plasmids,the expression of LC3B and NLRP3 inflammasome were detected to explore the effects of HBx on autophagy and NLRP3 inflammasomes,respectively.Finally,When HBx was overexpressed in Hep G2,Bafilomycin A1,3-MA and Rapamycin were used to block or enhance autophagy to detect the expression of NLRP3 inflammasome.In addition,overexpressing NLRP3 or activating NLRP3 inflammasome by H₂O₂ to observe autophagy on the basis of HBx transfection.Furthermore,the specific NLRP3inhibitor MCC950 was added after H₂O₂ treatment to observe the changes in autophagy.Results:The expression of LC3B in tumor tissues was higher than that in paratumor tissues,and the expression of NLRP3 was opposite.Then in tumor tissues,The higher the HBV content,the higher the expression of LC3B and the lower the expression of NLRP3.What’s more,the expression of LC3B and NLRP3were negatively correlated on the whole,as same as in HBV-positive group after grouping,while the HBV-negative group was not statistically significant.Comparing Hep G2.2.15 with Hep G2,HBV up-regulated basic autophagy level and down-regulated NLRP3 inflammasome expression in Hep G2.2.15 cells.In the next part,LC3B expression was down-regulated and NLRP3 inflammasome expression was up-regulated after HBx was knocked down.Overexpression of HBx had the opposite effect and increase of autophagosome.Then,blocking autophagy when HBx was highly expressed enhanced the expression of NLRP3 inflammaosome,while enhancing autophagy inhibited the expression of NLRP3 inflammaosome.Finally,Overexpression of the NLRP3 molecule did not affect the expression of LC3B,whereas LC3B was up-regulated after activation of NLRP3 inflammasome with H₂O₂,and LC3B decreased after further treatment with MCC950.Conclusions:In the case of HBV infection,HBx promoted autophagy and further down-regulated the expression of NLRP3 inflammasome in Hep G2.Meanwhile,activated NLRP3 inflammasome can enhanced autophagy.It suggests that autophagy expression can be enhanced by HBx in Hep G2 to inhibit inflammation and form a protective effect of negative feedback.