The Regulation Role Of P2X7R To NLRP3Inflammasome In Atherosclerosis

Purpose： Observation of the roles of P2X7R in atherosclerosis. Investigate themechanism of NLRP3inflammasome activation in atherosclerosis. Research the rolesof P2X7R/NLRP3inflammasome activation by oxLDL.Method:We observed the expression levels of P2X7R and NLRP3mRNA and proteinin peripheral blood monocytes by Real-time PCR and western blot. The plasma levelsof oxLDL and IL-1β analyzed by ELISA, in Non-CAD and CAD patients. We alsoabserved the expression and distribution of P2X7R and NLRP3in the atherosclerosisplaque of coronary artery from autopsy by immunofluorescence. For understanding themechanism of NLRP3inflammasome in macrophages activated during atherosclerosisby P2X7R, the specificity siRNA and some inhibitors to caspase-1and cathepsin-Bwere used. The co-immunoprecipitation was used to assay the interaction between PKRphosphorylation and NLRP3. Finally, expressions of P2X7R and PKR phosphorylationwere observed in apoE-/-mice aortic sinus by immunohistochemistry andimmunofluorescence.Results:1. The expression and roles of P2X7R and NLRP3in human atherosclerosis.The expression of P2X7R and NLRP3in CAD were higher than in Non-CADpatients. The concentration of IL-1β in CAD patient’s plasma is significantly higherthan in Non-CAD subjects’（28.99±5.98pg/ml vs14.76±2.96pg/ml，P<0.05）. Thelevel of oxLDL in CAD patients’ plasma was also higher than the Non-CAD subjects’ （183.86±39.31pg/ml vs81.38±19.51pg/ml，P<0.05）. From the results ofcoronary artery sections of autopsy, compared to right coronary artery, the lesion of leftanterior descending presents eccentric stenosis. In the atherosclerosis lesion of leftanterior descending, there are plenty of foam cells where the intima-media thicknessincreases. Immunofluorescence measurement shows that expression of P2X7R in theCD68positive area in artery has high intensity. And expression of NLRP3is also veryhigh in these locations.2.The mechanism by which P2X7R regulates NLRP3in atherosclerosis.2.1THP-1macrophages were treating by oxLDL in different concentration with0μg/ml,25μg/ml,50μg/ml and100μg/ml for24hours and found increasing concentration ofIL-1β in culture medium (3.80±1.05pg/ml，53.51±12.28pg/ml，139.79±34pg/ml，321.93±77.7pg/ml respectively，P＜0.01）. The expression of proIL-1β mRNA andprotein in cytoplasm of THP-1cells were also elevated.2.2When100μg/ml oxLDL stimulate THP-1cells for24hours, increased amount ofproIL-1β and mature IL-1βwere produced and the concentration of IL-1β in culturemedium raised. At the same time, the mRNA and protein expression of P2X7R andNLRP3in THP-1were also elevated.2.3After NLRP3was knocked down by siRNA, the expression of proIL-1β waskeeping the high level, but the level of IL-1β was significantly lower than the negativecontrol.2.4In vitro, After the P2X7R mRNA was knocked down by siRNA, THP-1macrophage production of IL-1β mRNA and proIL-1protein by oxLDL were notdecreased. However, the proIL-1β activation was inhibited.2.5OxLDL could improve PKR phosphorylation, up-regulate NLRP3expression, andpromote caspase-1production. When P2X7R was suppressed, the ability of oxLDLregulation of PKR phosphorylationwas attenuated. Co-immunoprecipitation shows PKRphosphorylation by oxLDL is related with NLRP3. When P2X7R is inhibited by siRNA,PKR phosphorylationby oxLDL decreased significantly, and less NLRP3was interacting with PKR phosphorylation. The activated caspase-1was also reduced.2.6On incubation with oxLDL, lots of lipid droplet were formed with cholesterol esteraccumulation in THP-1macrophage. The macrophage developed to foam cells. But, thelipid droplet and cholesterol ester were very found in decreased amount in the THP-1cells transfected by P2X7R siRNA. P2X7R siRNA inhibited the foam cells formation.3. The roles of P2X7R in apoE-/-mice atherosclerosis.3.1To analyze if P2X7R is related to atherosclerosis, we observed P2X7R expression inapoE-/-mice aortic sinus atherosclerotic lesion by high cholesterol diet. With12weekshigh cholesterol diet, atherosclerotic plaque was found in apoE-/-mice aortic sinus. Thesize of atherosclerotic lesion is larger than the control. In the lesion, there were not onlyplenty of foam cells, but also lots of needle cholesterol crystals. In the plaque, P2X7Rhad intensive expression, especially in foam cells genesis. We assayed the plasma levelsof oxLDL and IL-1β in apoE-/-mice by ELISA. It was found that IL-1βin plasma ofapoE-/-mice fed by high cholesterol diet for4weeks,8weeks,12weeks showedincreasingly higher levels than control（51.56±13.28pg/ml，140.27±36.11pg/ml，171.34±45.21pg/ml，vs8.87±2.27pg/ml，P＜0.01）.OxLDL in plasma of apoE-/-mice fedby high cholesterol diet for4weeks,8weeks,12weeks was2.61±0.93nmol/l,6.06±1.53nmol/l and6.93±2.12nmol/l, respectively. But the oxLDL in plasma ofapoE-/-mice in control could not be assayed.3.2For observing the effect of P2X7R in apoE-/-mice atherosclerosis, we injectedlentivirus with P2X7R siRNA to mice through vein to inhibit the roles of P2X7R inmice. We found that compared to apoE-/-and apoE-/-NC, the atherosclerosis inapoE-/-P2X7R-/-was attenuated. The IL-1β level in plasma of apoE-/-P2X7R-/-mice wasmuch lower than control and negative contro（l81.57±25.79pg/ml vs157.33±40.01pg/ml，P＜0.05；81.57±25.79pg/ml vs160.32±35.59pg/ml P＜0.05）.3.3We observed the level of PKR phosphorylation and its relationship with P2X7R inthe aortic sinus atherosclerotic lesion from apoE-/-mice fed by high cholesterol diets for2weeks,4weeks,8weeks and12weeks. PKR phosphorylationlevels was elevated in theP2X7R positive locaiton of atherosclerosis developement. Conclusion1) The expression of P2X7R，NLRP3and proIL-1β in CAD patients and the levels ofoxLDL and IL-1β in plasma are significantly higher than the Non-CAD subjects.2) P2X7R and NLRP3expression in macrophages may participate in atherosclerosisonset and development.3) By oxLDL，P2X7R regulates the assembly and activation of NLRP3inflammasomeby controlling the level of PKR phosphorylation, but P2X7R could not regulate theexpression of NLRP3mRNA and protein directly.4) P2X7R participates in the progression of foam cells whitch were treated by oxLDL.5) With progression of atherosclerosis, the expression of P2X7R in arteries of apoE-/-mice and the levels of oxLDL and IL-1β in plasma increased gradually.6) P2X7R plays important roles in the apoE-/-mice atherosclerosis development.