| PART ONE Expression of endoplasmic reticulum stress and pyroptosis related protein in rat liver ischemia-reperfusion injuryObjective: to study the expression of endoplasmic reticulum stress and pyroptosis in rat liver during heat ischemia reperfusion injury.Methods:(1)rat model of hepatic ischemia-reperfusion injury and its grouping: 36 SD male healthy rats weighing 280-300 g were selected and the first hepatic hilum was blocked by vascular occlusion clip to establish rat model of hepatic ischemia-reperfusion occlusion.The rats were divided into control group(sham group),ischemia 30 minutes reperfusion 0 hours group(IR0h group),reperfusion 1 hour group(IR1h group),reperfusion 6 hours group(IR6h group),reperfusion 12 hours group(IR12h group),reperfusion24 hours group(IR24h group),6 rats in each group,blood and liver tissue samples were collected at each time point.(2)the liver tissues of each group were stained by he,and the pathological changes were observed under light microscope to evaluate the inflammation and tissue necrosis.(3)the serum levels of alanine transaminase(alt),aspartate transaminase(AST)and(LDH)in rats were detected by automatic biochemical analyzer.The expressions of caspase-1 and caspase-11 weredetectedby immunohistochemistry(IHC)and western blot.Western blot(WB)was used to detect the expression of endoplasmic reticulum stress related proteins GRP7878 and at ATF6.The data collected by the above methods were described statistically by mean SD,andanalyzed statistically by SPSS 19.0 statistical software,p<0.05,the difference was statistically significant.Results:(1)the establishment of the model of heat ischemia reperfusion injury: the rat liver heat ischemia reperfusion injury model was successfully established.the pathological changes in each group of heat ischemia reperfusion injury: he staining under light microscope showed normal liver cell morphology,nuclear blue staining,uniform cytoplasm,clear hepatic lobule structure,tight arrangement of hepatic sinuses in a cord shape,normal central venous structure.the other IR groups showed different degrees of hepatic sinuses congestion,inflammatory infiltration and tissue necrosis.the liver tissue of IR6 h group and IR24 h group showed large flake necrosis,of which the liver tissue inflammation injury in IR24 h group was the most serious.Suzuki score showed:(2)liver function: AST 、 ALT and LDH increased gradually with the time of ischemia-reperfusion in rats,reached its peak at the time of IR6,and then gradually began to decline with the time.the difference between IR group and sham group was statistically significant(p<0.05).(3)the activity of caspase-12 in liver tissue: caspase-12 begans to increase in IR0 h group and reached the peak at IR6 h,with the gradual decline.(4)the expression of endoplasmic reticulum stress protein GRP78,ATF6,CHOP:GRP8 、ATF6 and CHOP began to increase in IR1 h,while at IR6 h began to increase significantly,IR12 h reached the peak,IR24 h later decreased,but still in a higher expression level,compared with the sham group has.(5)changes of expression level of pyroptosis: caspase-1 and case pase-11 wereexpressed at basic level in liver tissue of the control group,but not significantly in IR1 h,IR6h began to express significantly increased,IR2 h reached the peak,IR24 h later decreased,but still in a higher expression level.(6)the expression of proinflammatory factor IL-1β: IL-1β was expressed at the basic level in the liver tissue of the sham group.IR1 h begans to increase,and IR6 h begans to increase significantly.IR12 h reached the peak,and after IR24 h decreased..Conclusion:(1)In the rat liver thermal ischemia-reperfusion injury,the tissue damage may be related to apoptosis.(2)In the rat liver ischemia-reperfusion injury,there is another kind of programmed cell death way besides death ways such as necrosis and apoptosis,that is,pyroptosis,and the liver tissue damage may be related to pyroptosis to some extent.PART TWO Regulation mechanism of endoplasmic reticulum stress on pyroptosis in liver cold preservationObjective: to study the expression of endoplasmic reticulum stress and pyroptosis related protein in rat liver during cold storage,And to explore the regulation and signal pathway of endoplasmic reticulum stress on pyroptosis.Methods:(1)rat liver tissue and normal rat liver cell cold preservation model and grouping:(1)rat liver tissue cold preservation: 18 SD male healthy rats weighing about 280-300 g were selected to establish rat liver cold preservation model.Divided into control group(Normal group),cold storage 12 hours group(CS12h group),cold storage 24 hours group(CS24h group),6 rats in each group,at the corresponding time point to collect liver tissue;(2)cold preservation model of normal rat liver cells: BRL cells of normal rat liver cell line were cultured,and the cold preservation model of liver cells was established,which was divided into control group(sham group),cold preservation group(cs12h group)and cold preservation group(cs24h group).liver cells were collected at corresponding time points.(2)rat liver tissue preservation solution was sent to clinical laboratory of affiliated hospital of southwest medical university,and LDH level was detected by automatic biochemical analyzer.(3)the rat liver BRL cells were stimulated with 2.5 ug / ml endoplasmic reticulum stress agonist tunicamycin(TM)and100 u M endoplasmic reticulum stress inhibitor tudca(TUDCA),respectively.the control group was added with dimethyl sulfoxide(DMSO)of equal volume,and the samples were collected after 24 hours of culture.Western blot(WB)was used to detect the expression of apoptosis related proteins,endoplasmic reticulum stress and focal necrosis related proteins.(4)the liver tissues of each group were stained by he,the pathological changes were observed under light microscope,and the inflammation and tissue necrosis were evaluated.The expressions of caspase-1 and caspase-11 were detected by immunohistochemistry(IHC)and western blot.Western blot(WB)was used to detect the expression of caspase-12,endoplasmic reticulum stress related protein and proinflammatory factor IL-1β.The data collected by the above methods were described statistically by mean SD,and analyzed statistically by SPSS 19.0 statistical software,p < 0.05,the difference was statistically significant.Results:(1)the rat model of liver tissue cryopreservation was successfully established,and the pathological changes of each group were as follows: cell edema and cytoplasmic loosening were observed in each group,inflammatory cell infiltration was observed,no tissue necrosis was found,and the liver tissue injury was the most serious in the 24-hour cryopreservation group.Expression of apoptosis-related protein in liver tissue during cold storage: caspase-12 was almost not expressed in the control group,gradually increased after 12 hours of cold storage in liver tissue,reached the peak at 24 hours.(2)expression of endoplasmic reticulum stress protein GRP78、ATF6 and CHOP : the expression of GRP78,ATF6 and CHOP in normal liver tissue increased after 12 hours of cold storage and reached the peak after 24 hours(3)expression of caspase-1,case pase-11 and proinflammatory factor IL-1β in fpyroptosis related protein: caspase-1,case pase-11 and IL-1β were expressed at basic level in liver tissue of the control group,and began to increase after 12 hours of cold storage,and reached the peak after 24 hours.(2)after cold storage,the expression of apoptosis related protein caspase-12,pyroptosis related protein caspase-1,caspase-11,proinflammatory factor IL-1β and endoplasmic reticulum stress related protein in BRL cells of rat liver were significantly higher than those in control group.with the extension of cold storage time,the expression level of apoptosis related protein caspase-12,pyroptosis related protein caspase-1,caspase-11 and endoplasmic reticulum stress related protein increased gradually,which was consistent with the change rule of cold storage results of liver tissue.(2)the expression of caspase-1,caspase-11,IL-1β and endoplasmic reticulum stress-related protein were significantly higher than those of the control group,suggesting that tunicamycin induced endoplasmic reticulum stress and promoted the expression of pyroptosis-related protein.(3)the expressions of caspase-1,caspase-11,IL-1β,GRP78,ATF6 and CHOP were significantly lower than those of the control group,suggesting that Niuhuang ursodeoxycholic acid inhibited the occurrence of endoplasmic reticulum stress and the expression ofapoptosis and pyroptosis-related proteins during liver cold storage.Conclusion:(1)Apoptosis was induced in rat liver during cold storage,that is,apoptosis may be related to liver cell damage.(2)during cold storage of rat liver,the damage of liver tissue cells may be related to pyroptosis.(3)endoplasmic reticulum stress protein at ATF6-CHOP may have potential regulatory effect on pyroptosis of cells. |
PDF Full Text Request