基于NLRP3泛素化-苏素化调控通路研究针刺防治心肌缺血再灌注损伤的抗细胞焦亡作用及机制

Pyroptosis is a newfound mode of programmed cell death after apoptosis and autoghagy. Researches show that pyroptosis can occur in the process of myocardial ischemia reperfusion, and aggravate myocardial ischemia reperfusion injury (MIRI). Thus, inhibition of myocardial pyroptosis can improve MIRI. Pyroptosis depends on activation of NLRP3 and its effect of activating caspase1. The mechanism of pyroptosis is closely related to the ubiquitination and SUMOylation of NLRP3. Our previous researches show that acupuncture can decrease the expression level of caspase1, then remind that acupuncture may has ability to inhibit myocardial pyroptosis. Thus, we propose a hypothesis that acupuncture can regulate and control the ubiquitination and SUMOylation of NLRP3, then inhibition myocardial pyroptosis to improve MIRI. We adopt MIRI mice as carrier, and co-immunoprecipitation (co-IP), chromatin Immunoprecipitation (CHIP), polymerase chain reaction (PCR) et al. to conduct following researches: ① efficacy of acupuncture in inhibiting myocardial pyroptosis; ② efficacy and mechanism of acupuncture in activating ubiquitination of NLRP3 and regulating STUB1; ③ efficacy and mechanism of acupuncture in inhibiting SUMOylation of NLRP3 and regulating SENP3; ④ after restrictedly specific genes knock-out, efficacy of acupuncture for myocardial pyroptosis and regulation of acupuncture to the ubiquitination and SUMOylation of NLRP3 will be verified.

细胞焦亡是继细胞凋亡、细胞自噬之后新发现的一种细胞程序性死亡方式。已有研究表明细胞焦亡可发生于心肌缺血再灌注过程中，并会加重心肌缺血再灌注损伤（MIRI），因此抑制心肌细胞焦亡有助于减轻MIRI。细胞焦亡依赖于炎症小体NLRP3的激活及其对caspase1的活化，其机制与NLRP3泛素化-苏素化调控密切相关。前期研究发现针刺可显著下调心肌caspase1水平，提示针灸或可抑制心肌细胞焦亡，故本研究提出“针刺可调控NLRP3泛素化-苏素化通路抑制心肌细胞焦亡改善MIRI”的机制假说。本项目以MIRI小鼠为载体，运用 co-IP,ChIP, PCR等技术，研究：①针刺抑制MIRI小鼠心肌细胞焦亡的效应；②针刺活化NLRP3泛素化及对STUB1基因通路的调控；③针刺抑制NLRP3苏素化及对SENP3基因通路的调控；④限制性基因敲除后，验证上述针刺抗细胞焦亡作用及NLRP3泛-苏素化调控机制。

本项目以经脉学说理论、细胞程序性死亡为立项基础，以人类重大疾病“急性心肌梗死”的病理改变“心肌缺血再灌注损伤”为研究载体，以NLRP3介导的心肌细胞焦亡为研究靶点，以NLRP3泛素化-苏素化为切入点，采用co-IP、CHIP、PCR、ELISA、western blot等技术，观察针刺后处理改善心肌缺血再灌注损伤的心肌细胞焦亡机制。研究结果表明针刺后处理可有效上调STUB1及SENP3的表达水平，促进NLRP3的泛素化并抑制NLRP3的苏素化，以降低NLRP3表达，从而抑制GSDMD蛋白介导的心肌细胞焦亡；且针刺后处理调控NLRP3改善心肌细胞焦亡的作用机制可能依赖于STUB1及SENP3的参与。研究验证了“针刺可调控NLRP3泛素化-苏素化通路抑制心肌细胞焦亡改善MIRI”的机制假说。