摘要
目的：观察P2X7R在动脉粥样硬化中的作用。探索NLRP3炎症小体在动脉粥样硬化中的激活机制。研究oxLDL对P2X7R/NLRP3炎症小体的激活作用。
方法：我们先通过Real-time PCR和Western blot的方法观察了非冠状动脉疾病患者和冠状动脉疾病患者外周血单核细胞上P2X7R，NLRP3的mRNA和蛋白的表达水平，并且用ELISA检测了这两组人群血浆oxLDL和IL-1β的水平。然后我们用免疫荧光的方法观察了P2X7R和NLRP3在尸体解剖冠状动脉粥样硬化斑块中的表达和分布。为了了解巨噬细胞NLRP3炎症小体在动脉粥样硬化中由P2X7R激活的机制，我们使用了特异性siRNA以及针对caspase-1和cathepsin B的抑制剂来进行研究。免疫共沉淀用来检测磷酸化PKR与NLRP3的相互作用。最后，我们还用免疫组化和免疫荧光的方法观察了P2X7R和磷酸化PKR在apoE-/-小鼠主动脉窦中的表达。
结果：
1. P2X7R与NLRP3在人动脉粥样硬化发展中的表达及作用。
冠状动脉疾病患者（CAD）P2X7R和NLRP3的表达都显著高于非冠状动脉人群（Non-CAD）。CAD患者血浆中IL-1β的浓度也显著高于非CAD人群（28.99±5.98pg/ml vs14.76±2.96pg/ml）。CAD患者血浆中oxLDL浓度也高于非CAD患者（183.86±39.31pg/ml vs81.38±19.51pg/ml，P<0.05）。从尸检标本切片中，我们可以看到，与右冠状动脉比较，左前降支病变处呈明显呈偏心性狭窄。在左前降支动脉粥样硬化病变中亦可见大量的泡沫细胞堆积。左前降支病变处的动脉内-中膜厚度（intima-media thickness）也明显增加。免疫荧光显示，在血管壁中CD68阳性的区域，P2X7R也有很高的表达。并且与无动脉粥样硬化斑块的右冠状动脉相比，前降支的动脉粥样硬化病变中P2X7R的表达更为强烈。而在P2X7R强烈表达的区域，NLRP3的表达也非常高。
2. P2X7R在动脉粥样硬化中对NLRP3炎症小体的调节机制
2.1我们用0μg/ml，25μg/ml，50μg/ml，100μg/ml不同浓度的oxLDL去刺激THP-1巨噬细胞24小时，发现THP-1巨噬细胞释放到培养基中的IL-1β浓度逐渐增高（分别为3.80±1.05pg/ml，53.51±12.28pg/ml，139.79±34pg/ml，321.93±77.7pg/ml，P＜0.01）。THP-1巨噬细胞胞浆内表达的proIL-1β的mRNA水平和蛋白水平也逐渐被上调，并且胞浆内IL-1β蛋白水平也相应提高了。
2.2100μg/ml oxLDL刺激巨噬细胞24小时以后，细胞产生了大量的proIL-1β和IL-1β。并且，培养基中IL-1β的浓度也明显增加了。在同样100μg/ml oxLDL刺激24小时后，THP-1巨噬细胞上P2X7R和NLRP3的mRNA和蛋白表达水平也明显提高了。
2.3NLRP3被siRNA敲减以后，在oxLDL刺激下，巨噬细胞上proIL-1β的表达水平仍然很高，但是细胞产生的IL-1β水平均明显低于阴性对照组。
2.4在体外实验中，我们用siRNA敲减了P2X7R的mRNA的表达后，THP-1巨噬细胞在oxLDL的刺激下产生的IL-1β mRNA水平和proIL-1β的蛋白表达水平并没有降低，不过，proIL-1β的成熟却明显被抑制了。
2.5OxLDL可以促进PKR的磷酸化，同时上调了NLRP3的表达水平，并且此时产生了大量有活性的caspase-1。当P2X7R水平受到抑制以后，明显减缓了oxLDL对PKR磷酸化的调节作用。免疫共沉淀实验显示：oxLDL促使PKR磷酸化以后，大量的NLRP3与PKR结合到一起，发生了相互作用。当P2X7R被siRNA抑制以后，oxLDL诱发的PKR磷酸化水平明显降低，与PKR结合的NLRP3也明显减少，有活性的caspase-1也明显减少了。
2.6在oxLDL持续作用下，THP-1巨噬细胞内由于堆积了大量的以胆固醇酯为主要成分的脂滴，转变成了泡沫细胞。但是，转染了P2X7R siRNA的巨噬细胞内，胆固醇酯和脂滴明显较少。P2X7R siRNA抑制了泡沫细胞的形成。
3. P2X7R在apoE-/-小鼠动脉粥样硬化病变中的作用
3.1为了明确P2X7R是否与动脉粥样硬化的发展有关，我们观察了P2X7R在高胆固醇饮食饲养的apoE基因敲除小鼠动脉窦粥样硬化病变中的表达。高胆固醇饮食12周时，在apoE-/-小鼠主动脉窦中，动脉粥样硬化斑块已经形成。动脉粥样硬化病变面积明显大于对照组。在病变区，不但有大量的泡沫细胞形成，还出现了针状的胆固醇结晶。在斑块中，P2X7R有较强的表达。特别是在新生成的泡沫细胞上，P2X7R的表达呈现出强阳性。用ELISA的方法检测了apoE-/-小鼠血浆oxLDL的水平和IL-1β的水平。结果表明，高胆固醇饮食饲养4周、8周和12周的apoE-/-小鼠血浆中的IL-1β水平显著高于对照组（51.56±13.28pg/ml，140.27±36.11pg/ml，171.34±45.21pg/ml，vs8.87±2.27pg/ml，P＜0.01）。高胆固醇饮食饲养4周、8周和12周后apoE-/-小鼠血浆中的oxLDL水平分别为2.61±0.93nmol/l，6.06±1.53nmol/l，6.93±2.12nmol/l，而正常饮食组apoE-/-小鼠血浆中的oxLDL几乎检测不到。
3.2为了明确P2X7R影响apoE-/-小鼠动脉粥样硬化病变形成的作用机制，我们给apoE-/-小鼠从尾静脉注射携带有P2X7R siRNA的慢病毒，来抑制小鼠体内P2X7R的作用。我们发现，与apoE-/-和apoE-/-NC组相比，apoE-/-P2X7R-/-小鼠动脉粥样硬化的发展受到了明显的抑制。apoE-/-P2X7R-/-小鼠血浆IL-1β的水平明显低于对照组和阴性对照组（apoE-/-+NC组）（81.57±25.79pg/ml vs157.33±40.01pg/ml，P＜0.05；81.57±25.79pg/ml vs160.32±35.59pg/ml P＜0.05）。
3.3我们用免疫荧光的方法观察了高胆固醇饮食分别饲养2，4，8，12周的apoE-/-小鼠主动脉窦动脉粥样硬化病变中的PKR的磷酸化水平以及与P2X7R的关系。在P2X7R表达阳性的部位，PKR的磷酸化水平也随着动脉粥样硬化病变的发展，逐渐增高。
结论：
1) CAD患者外周血单核细胞上P2X7R，NLRP3和proIL-1β的表达以及血浆中oxLDL和IL-1β的水平显著高于非CAD人群。
2)人巨噬细胞上高表达的P2X7R和NLRP3可能共同参与了动脉粥样硬化的发生和发展。
3)在oxLDL刺激下，P2X7R通过调控PKR的磷酸化水平，影响NLRP3炎症小体的组装和激活，但P2X7R不能直接调控NLRP3mRNA和蛋白的表达水平。
4)在oxLDL的作用下，P2X7R参与了THP-1巨噬细胞的泡沫化进程。
5)随着apoE-/-小鼠动脉粥样硬化病变的发展，P2X7R在动脉壁中的表达逐渐增高，血浆中oxLDL和IL-1β水平也逐渐增高；
6) P2X7R参与了apoE-/-小鼠动脉粥样硬化的发生发展。
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.
Conclusion
1) 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.
引文
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