Study On The Mechanism Of Hepatocellular Carcinoma Pyroptosis Induced By Endoplasmic Reticulum Stress

Objective: 1,To observe the relationship between pyroptosis and endoplasmic reticulum stress-related protein expression in hepatocellular carcinoma,and understand the correlation between endoplasmic reticulum stress and pyroptosis in hepatocellular carcinoma.2,By regulating the changes of endoplasmic reticulum stress,detecting related protein expression,furtherly confirming the endoplasmic reticulum stress in the regulation of possible mechanisms of pyroptosis in hepatocellular carcinoma.Methods: With the approved of the Ethical Committee of Southwest Medical University and informed consent of all patients,47 patients with primary hepatocellular carcinoma were previously treated in our hospital and collect surgical resection of human hepatocellular carcinoma and its corresponding paracancerous liver tissues.At the same time,we also collected 15 cases of hepatic hemangioma patients(with surgical indications)in our hospital for surgical resection when the hepatic tissue around the normal liver tissue as experimental normal control group.HE staining was performed on each group of liver tissues,and then observe the histopathological changes in each group under light microscope to verify the accuracy of the materials.Immunohistochemistry(IHC)and Western blot(WB)were used to analyze the relationship of expression of protein between caspase-1 and pro-inflammatory cytokines IL-1?,and ERS related proteins GRP78,CHOP,ATF6,PERK,and IRE1 in human normal liver tissues,paracancerous tissues,and cancer tissues.The gray scale value of the band diagram measured by WB was analyzed with the Quantity One 5.4 software;In order to verify the expression of caspase-1,IL-1? and ERS-related proteins GRP78,CHOP,ATF6,PERK,IRE1 in hepatoma cells and normal cells,we cultured the hepatocellular carcinoma cell line 7402 and human normal cell line L02;We again cultured hepatocellular carcinoma cell line 7402,with a concentration of 3u M ERS activator-tunicamycin(tunicamycin,TM)and 100 u M ERS inhibitor tauroursodeoxycholic acid(TUDCA)alone stimulates hepatoma cells and samples are collected after 24 hours of co-culture.WB was used to analyze the expression of pyroptosis-associated protein caspase-1,pro-inflammatory cytokines IL-1?,and ERS-related proteins GRP78,CHOP,ATF6,PERK and IRE1 in human hepatocellular carcinoma cell line 7402,and to understand the trends of these proteins after drug intervention.To analyze the possible mechanism of endoplasmic reticulum stress and pyroptosis in hepatocellular carcinoma cell.RESULTS:(1)The expression levels of caspase-1 and IL-1? in human hepatocellular carcinoma and human hepatocellular carcinoma cell line 7402 were highly expressed: HE staining was performed under light microscope to analyze the histopathological characteristics,confirming that the collected tissue was the ideal site;The expression levels of caspase-1 and IL-1? in hepatocellular carcinoma were significantly higher than those in normal tissues and paracancerous tissues(P<0.001).The expression of paracancerous tissues was higher than that of normal tissues.The increase was statistically significant(P<0.001).We further applied IHC to analyzing the changes of caspase-1protein in hepatocellular carcinoma.The results showed that caspase-1 was highly expressed in hepatocellular carcinoma compared with normal tissues and paracancerous tissues.The caspase-1 were performed brown-yellow particles which localized in cytoplasm;In vitro experiment we confirmed that the expression levels of caspase-1 protein and pro-inflammatory cytokine IL-1? in hepatocellular carcinoma cell line 7402 were also significantly higher than that of normal cell line L02(P<0.001).(2)ERS-associated pathway proteins were activated in human hepatocellular carcinoma and human hepatocellular carcinoma cell line 7402:The results of WB showed that ERS-related proteins GRP78,CHOP,ATF6,PERK,and IRE1 were overactivated in hepatocellular carcinoma and hepatoma cells.Significantly higher than normal liver tissues and paracancerous tissues(P<0.001);IHC further tested ERS-related proteins GRP78,CHOP,ATF6,PERK,and IRE1.The results showed that ERS-related proteins were performed brownish-yellow particles and highly expressed in the cytoplasm of HCC tissues,a very small amount of protein was translocated to the nucleus.(3)TM can induce further activation of ERS in hepatocellular carcinoma cells and promote the expression of protein caspase-1 and pro-inflammatory cytokines IL-1?.In vitro experiment,3?M concentration of TM was co-cultured with hepatoma cells for 24 hours,and we passed WB analysis to collecting samples found that the expression of ERS marker protein GRP78 and CHOP protein in the TM group was significantly higher than that in the control group,the difference was statistically significant(P<0.001),the expression levels of the pyroptosis-associated protein caspase-1 and the pyroptosis-associated pro-inflammatory factor IL-1? was also significantly higher than that of the control group(P<0.001).(4)TUDCA can inhibit the activation of endoplasmic reticulum stress and the downregulation of the expression of pyroptosis-associated protein in hepatoma cell line 7402:Co-culture with hepatoma cells with a concentration of 100 u M TUDCA for 24 hours,WB detection found that the ERS marker protein GRP78 and CHOP protein was down-regulated.The expression of caspase-1 and pro-inflammatory factor IL-1? was also down-regulated,and the difference was statistically significant(P<0.001).Conclusions:(1)The pyroptosis-associated protein caspase-1 and the pyroptosis-associated pro-inflammatory factor IL-1? was highly expressed and the ERS-related protein was activated in HCC.(2)ERS activator TM can further stimulate the activation of ERS-related proteins in hepatocellular carcinoma,promote the expression of ERS-related proteins GRP78,CHOP,ATF6,PERK,and IRE1,initiate the unfolded protein response,and the expression of caspase-1 and IL-1? was further increased after treated with TM.(3)The ERS inhibitor TUDCA can inhibit the activation of ERS in hepatocellular carcinoma cells,down-regulate the expression of ERS-related proteins GRP78,CHOP,ATF6,PERK and IRE1,and down-regulate expression of pyroptosis-associated proteins caspase-1 and pro-inflammatory cytokines IL-1?.It shows that there is indeed a new type of programmed cell death,that is,pyroptosis,in hepatocellular carcinoma.At the same time,the changes of pyroptosis-associated protein and the ERS-related protein keep synchronized,the ERS may have a potential regulatory effect on pyroptosis in HCC.It further clarify the way of hepatocellular carcinomar cell death and explore the treatment of hepatocellular carcinoma provides a new strategy.