松萝酸调节cGAS-STING信号通路对大鼠心肌缺血再灌注损伤的保护作用研究

Protective effect of usnic acid on myocardial ischemia-reperfusion injury in rats by regulating the cGAS-STING signaling pathway

目的 探究松萝酸调节环磷酸鸟苷-磷酸腺苷合成酶(cGAS)-干扰素基因刺激因子(STING)信号通路对大鼠心肌缺血再灌注损伤的保护作用。方法 选取50只SD健康大鼠,分为假手术组、模型组、松萝酸低剂量组(25 mg/kg)、松萝酸高剂量组(100 mg/kg)和RU.521组(cGAS抑制剂,5 mg/kg),每组10只通过结扎冠状动脉左前降支再灌注方法建立大鼠心肌缺血再灌注模型。HE染色观察大鼠心肌组织病理学变化;TUNEL染色检测心肌细胞凋亡情况;NBT染色检测心肌梗死面积百分比;商品化试剂盒检测心肌组织超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和丙二醛(MDA)水平;ELISA法检测大鼠血清肌酸激酶同工酶(CKMB)、肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)水平;RT-PCR法检测心肌组织cGAS mRNA、STING mRNA表达;Western blot检测大鼠心肌组织中Bax、Bcl-2、Caspase-3、cGAS、STING蛋白表达。结果 假手术组大鼠心肌组织结构形态正常,凋亡率较低;与假手术组比较,模型组大鼠心肌细胞受损严重、凋亡率、心肌梗死面积百分比、血清CKMB、TNF-α、IL-6水平、MDA水平、cGAS mRNA、STING mRNA表达水平、Bax、Caspase-3、cGAS、STING蛋白表达水平显著升高,SOD、CAT、Bcl-2蛋白表达水平显著降低(P<0.05);与模型组相比,松萝酸低、高剂量组和RU.521组大鼠心肌细胞形态结构明显改善,炎性细胞浸润显著减少、细胞凋亡率、心肌梗死面积百分比、血清CKMB、TNF-α、IL-6水平、MDA、cGAS mRNA、STING mRNA表达水平、Bax、Caspase-3、cGAS、STING蛋白表达水平显著降低,大鼠心肌组织SOD、CAT、Bcl-2蛋白表达水平显著升高(P<0.05);RU.521组各项指标与松萝酸高剂量组几乎相当。结论 松萝酸对心肌缺血再灌注大鼠具有保护作用,可能是通过抑制cGAS-STING信号通路实现的。

Objective To investigate the protective effect of usnic acid on myocardial ischemia-reperfusion injury(MIRI) in rats by regulating the cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING) signaling pathway.Methods Fifty healthy Sprague-Dawley(SD) rats were selected and randomly grouped into sham operation group,model group,low-dose usnic acid group(25mg/kg),high-dose usnic acid group(100mg/kg),and RU.521 group(cGAS inhibitor,5mg/kg),with 10 rats per group.A rat MIRI model was established by ligating the left anterior descending coronary artery and reperfusion.The hematoxylin and eosin(H&E) staining was applied to observe the pathological changes in rat myocardial tissues.The terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) was applied to detect cardiomyocyte apoptosis.Nitro Blue Tetrazolium(NBT) staining was applied to detect the percentage of myocardial infarction area.Commercial kits were applied to detect the levels of superoxide dismutase(SOD),catalase(CAT),and malondialdehyde(MDA) in myocardial tissues.Serum levels of creatine kinase MB(CK-MB),tumor necrosis factor-alpha(TNF-α),and interleukin 6(IL-6) were detected by enzyme-linked immunosorbent assay(ELISA).The mRNA expressions of cGAS and STING in myocardial tissues were detected by real-time reverse transcription polymerase chain reaction(RT-PCR).Protein expressions of Bax,Bcl-2,Caspase-3,cGAS and STING were detected by Western blot.Results In the sham operation group,the myocardial tissue morphology was normal and the apoptosis rate was low.Compared with those of the sham operation group,rats in the model group presented severely damaged myocardial cells,significantly increased apoptosis rate,the percentage of myocardial infarction area,serum levels of CK-MB,TNF-α and IL-6,MDA,mRNA levels of cGAS and STING,and protein levels of Bax,Caspase-3,cGAS,and STING,and significantly decreased SOD and CAT levels,and the protein level of Bcl-2(P<0.05).Compared with those of the model group,rats in the low-dose and high-dose usnic acid group and RU.521 group presented improved morphology and structure of myocardial cells,significantly reduced infiltration of inflammatory cells,significantly decreased apoptosis rate,the percentage of myocardial infarction area,serum levels of CK-MB,TNF-α and IL-6,MDA,mRNA levels of cGAS and STING,and protein levels of Bax,Caspase-3,cGAS,and STING,and significantly increased SOD and CAT levels,and the protein level of Bcl-2(P<0.05).The above indexes were comparable between RU.521 group and high-dose usnic acid group.Conclusion Usnic acid has a protective effect on MIRI in rats by inhibiting the cGAS-STING signaling pathway.