摘要
目的:探讨桃红四物汤对去势(OVX)雌性小鼠心肌缺血再灌注损伤(MIRI)的影响及机制研究。方法:雌性小鼠建立去势模型后,采用酶联免疫吸附测定法(ELISA)检测小鼠的雌二醇(E2)水平验证去势模型;对成功建立去势模型的小鼠抽取60只随机分为6组:假手术组、模型组、桃红四物汤低、中、高剂量组(9、18、36 g·kg^(-1))、核因子E2相关因子2(Nrf2)抑制剂组,每组10只;采用激光散斑血流仪验证MIRI模型;采用2,3,5-氯化三苯基四氮唑(TTC)染色法与苏木素-伊红(HE)染色法检测心肌组织病理变化;ELISA检测小鼠血清中肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白Ⅰ(CTnⅠ)、丙二醛(MDA)、超氧化物歧化酶(SOD)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)水平;采用免疫荧光染色法观察Nrf2、血红素加氧酶-1(HO-1)的表达情况;蛋白免疫印迹法(Western blot)检测Nrf2、HO-1与凋亡蛋白B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、炎症因子白细胞介素-18(IL-18)、白细胞介素-1β(IL-1β)的表达。结果:与假手术组比较,模型组小鼠血清中CK-MB、CTnⅠ、MDA、IL-6水平升高(P<0.01),SOD、IL-10水平降低(P<0.01),心肌组织TTC染色、HE染色损伤评分增加(P<0.01),心肌组织免疫荧光Nrf2、HO-1的表达减少(P<0.01),心肌组织Nrf2、HO-1、Bcl-2蛋白表达量减少,Bax、IL-18、IL-1β蛋白表达量增加(P<0.01);与模型组比较,桃红四物汤剂量组小鼠血清中CK-MB、CTnⅠ、MDA、IL-6明显下降(P<0.05,P<0.01),SOD、IL-10明显增加(P<0.05,P<0.01),心肌组织的TTC染色、HE染色损伤评分显著降低(P<0.01),心肌组织免疫荧光Nrf2、HO-1的表达增加(P<0.01),心肌组织Nrf2、HO-1、Bcl-2蛋白表达量明显增加(P<0.05,P<0.01),Bax、IL-18、IL-1β蛋白表达量明显减少(P<0.05,P<0.01),其中以桃红四物汤高剂量组效果最为显著;Nrf2抑制剂组小鼠血清中CK-MB、CTnⅠ、MDA、IL-6水平明显升高(P<0.05,P<0.01),SOD、IL-10水平明显降低(P<0.05,P<0.01),心肌组织TTC染色、HE染色损伤评分显著增加(P<0.01),心肌组织免疫荧光Nrf2、HO-1的表达显著减少(P<0.01),心肌组织Nrf2、HO-1、Bcl-2蛋白表达量明显减少,Bax、IL-18、IL-1β蛋白表达量显著增加(P<0.01)。结论:桃红四物汤能够减轻去势小鼠心肌缺血再灌注损伤,减轻氧化应激反应,减少炎症因子释放并抑制组织凋亡,对去势MIRI小鼠发挥保护作用,并且该保护作用可能与激活Nrf2/HO-1信号通路有关。
Objective:To investigate the effect and mechanism of Taohong Siwutang(TSD)on myocardial ischemia reperfusion injury(MIRI)in ovariectomized(OVX)female mice.Method:After the OVX model of female mice was established,the estradiol(E2)level was detected by enzyme-linked immunosorbent assay(ELISA)to verify the model.Sixty OVX mice were randomly divided into six groups:Sham operation group,model group,low,medium,and high dose groups(9,18,36 g·kg^(-1))of TSD,and nuclear factor E2-related factor 2(Nrf2)inhibitor group,with 10 mice in each group.The MIRI model was verified by a laser speckle flowmeter.The pathological changes in myocardial tissue were detected by 2,3,5-triphenyltetrazolium chloride(TTC)and hematoxylin-eosin(HE)staining.Serum creatine kinase isoenzyme(CK-MB),cardiac troponinⅠ(CTnⅠ),malondialdehyde(MDA),superoxide dismutase(SOD),interleukin-6(IL-6),and interleukin-10(IL-10)levels were detected by ELISA.The expression of Nrf2 and heme oxygenase-1(HO-1)were observed by immunofluorescence staining.The expressions of Nrf2,HO-1,apoptotic B-cell lymphomato-2(Bcl-2),Bcl-2 associated X protein(Bax),inflammatory cytokines interleukin-18(IL-18),and interleukin-1β(IL-1β)were detected by Western blot.Result:Compared with the sham operation group,the serum levels of CK-MB,CTnⅠ,MDA,and IL-6 in the model group were increased(P<0.01),and the levels of SOD and IL-10 were decreased(P<0.01).The damage scores of TTC and HE staining in myocardial tissue were increased(P<0.01).The expressions of Nrf2 and HO-1 in myocardial tissue by immunofluorescence were decreased(P<0.01),and the protein expressions of Nrf2,HO-1,and Bcl-2 in myocardial tissue were decreased.The protein expressions of Bax,IL-18,and IL-1βwere increased(P<0.01).Compared with the model group,serum levels of CK-MB,CTnⅠ,MDA,and IL-6 of TSD groups were significantly decreased(P<0.05,P<0.01),and SOD and IL-10 were significantly increased(P<0.05,P<0.01).TTC staining and HE staining damage scores of myocardial tissue were significantly decreased(P<0.01).The expressions of Nrf2 and HO-1 in myocardial tissue by immunofluorescence were increased(P<0.01).The protein expressions of Nrf2,HO-1,and Bcl-2 were significantly increased(P<0.05,P<0.01),and those of Bax,IL-18,and IL-1βwere significantly decreased(P<0.05,P<0.01).The effect of the high dose group of TSD was the most significant.The serum levels of CK-MB,CTnⅠ,MDA,and IL-6 in the Nrf2 inhibitor group were significantly increased(P<0.05,P<0.01),and the levels of SOD and IL-10 were significantly decreased(P<0.05,P<0.01).The damage scores of TTC and HE staining in myocardial tissue were significantly increased(P<0.01).The expressions of Nrf2 and HO-1 in myocardial tissue by immunofluorescence were significantly decreased(P<0.01).The protein expressions of Nrf2,HO-1,and Bcl-2 in myocardial tissue were significantly decreased,and those of Bax,IL-18,and IL-1βwere significantly increased(P<0.01).Conclusion:TSD can alleviate MIRI in OVX mice,reduce oxidative stress response and the release of inflammatory factors,and inhibit apoptosis,playing a protective role in OVX mice with MIRI,which may be related to the activation of Nrf2/HO-1 signaling pathway.
作者
杨钰铭
祝莹
刘常昳
程婧
何非
YANG Yuming;ZHU Ying;LIU Changyi;CHENG Jing;HE Fei(School of Nursing,Anhui University of Chinese Medicine,Hefei 230012,China;The Second Hospital of Anhui Medical University,Hefei 230601,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第13期11-19,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81903833)
安徽省高校科研重点项目(2023AH053177)
老年护理与健康重点实验室项目(RZ2300000592)
2023年度安徽省高校人文重点项目(2023AH050722)。
关键词
桃红四物汤
心肌缺血再灌注损伤
炎症因子
凋亡
去势
Taohong Siwutang
myocardial ischemia reperfusion injury
inflammatory factors
apoptosis
ovariectomy
