Effect Of Caveolim-1on The Er Stress-related Apoptosis Induced By Thapsigargin In RAW264.7and Mechanism Research

Background and purposeIn the study the regulation of caveolin-1(cav-1) on RAW264.7apoptosisinduced by thapsogargin and the expression of apoptosis-related protein Bcl-2and Bcl-xl was observed to explore the interaction between caveolin-1andEndoplamic reticulum(ER) stress and the molecule mechanism of cell apoptosiscaused by adjustment of downstream factor. It provides the new interventiontarget to the mechanism study of cav-1versus atherosclersis.Materials and Methods1. Establishment of the macrophage-derived ER stress model: RAW264.7cells were cultivated with TG in concentration of0.3μmol/L,1μmol/L and3μmol/L respectively. Then the cell viability was measured at12h,24h,36h and48h. The less than50%survival rate regarded as the mark of ER stress-derived,the optimum condition can be chosen to esta blish the mode of ER stress-relatedapoptosis induced by TG in RAW264.7.2. Gene Silence of cav-1by si-RNA technology: cav-1siRNA wasintroducted into RAW264.7and the expression of cav-1was detected by westernblot after48-96h cultivation to screen succeeded transfection cells (calledcav-1-siRNA） the and determine the best transfection condition.3. Detection of the effects of cav-1on ER stress-related apoptosis ofRAW264.7: RAW264.7was pretreated by siRNA technology for96h and thenadded1umol/l TG for24h cultivation. The apoptosis rates of cells were detectedby Annexin V-FITC/PI on FCM.4. To detection the effect of Cav-1on mRNA content of RAW264.7cell endoplasmic reticulum stress related genes Bcl-2and Bcl-xl: After cav-1genesilenced,the mRNA level changes of Bcl-2and Bcl-xl were detected bysemi-quantitative PCR in the different groups.Analysis of Cav-1effect of Bcl-2,Bcl-xl gene in RAW264.7cells endoplasmic reticulum stress response inmRNA.5. To detection the effect of Cav-1on expression of RAW264.7cellendoplasmic reticulum stress related protein Bcl-2and Bcl-xl：After cav-1genesilenced,the exprssion level of Bcl-2and Bcl-xl were detected by westernblotting so as to determine whether the adjustment of cav-1on RAW264.7ERstress goes the Bcl-2signal channel.Results1. Establishment of macrophage-derived apoptosis cells: RAW264.7cellswere cultu-red with1μmol/L TG for24h and the survival rate is41.07±3.35%.Accord to the sign of less than50%survival rate, this time the cells havetranslated into the ER stress related cell apoptosis. Thus, choose1μmol/L TG toreact on24h to establish the ER stress apoptotic cells model to conduct thefollow up experiment.2. Result of Gene Silence of cav-1by siRNA technology: cav-1siRNA wasintrod-ucted into RAW264.7and the expression of cav-1was detected bywestern blot after48h cultivation. Compared with the normal group andnagative control group, the result both decrease by80%. The succeeded transfe-ction cells were used for follow up experiment.3. Effect of cav-1on RAW264.7ER stress apoptosis: After the inhibitionof the cav-1expression in RAW264.7, cav-1-siRNA can promote the ER stressdamage in RAW264.7and compared with the TG group, the cell apoptosisrises(P<0.05).4. Effect of cav-1on mRNA content of RAW264.7cell endoplasmicreticulum stress related genes Bcl-2and Bcl-xl: After the Gene Silence of cav-1in RAW264.7, mRNA quantity of Bcl-2and Bcl-xl decrease apparentlycampared with the TG group(P<0.05).5. Effect of cav-1on expression of RAW264.7cell endoplasmic reticulumstress related protein Bcl-2and Bcl-xl: After the Gene Silence of cav-1inRAW264.7, the result of protein expression shows that Bcl-2and Bcl-xl decrease apparently compared with the TG group(P<0.05).Conclusion1. After the inhibition of the cav-1expression in RAW264.7, cav-1-siRNAcan promote the ER stress damage in RAW264.7and compared with the TG,cav-1can suppress the ER stress induced by TG in RAW264.7cells.2. Cav-1can promote the expression of Bcl-2and Bcl-xl in the process ofER stress induced by TG in RAW264.7. And in the meantime, cav-1can promotethe surpression effect of RAW264.7ER stress.