摘要
探究杜鹃花总黄酮(total flavonoids of Rhododendron simsii,TFR)减轻大鼠局灶性脑缺血再灌注损伤(cerebral ischemia-reperfusion injury,CIRI)的保护作用及其与钙离子传感器基质相互作用分子(stromal interaction molecule,STIM)和钙释放激活钙通道调节分子(calcium release-activated calcium modulator,Orai)调控的钙库操作性钙离子内流(store-operated calcium entry,SOCE)通路之间的关系。大鼠随机分为假手术(sham)组、模型(MCAO)组、TFR(60 mg·kg^(-1))组、TFR(60 mg·kg^(-1))+SOCE通路阻断剂2-氨基乙基联苯基硼酸酯(2-aminoethoxydiphenyl borate,2-APB)(2.5 mg·kg^(-1))组、2-APB(2.5 mg·kg^(-1))组。sham组、MCAO组大鼠给予生理盐水,用药组给予TFR(60 mg·kg^(-1)),连续灌胃14 d直至取材,SOCE通道阻断剂2-APB(2.5 mg·kg^(-1))术后给予腹腔注射。ELISA法检测大鼠血清中白细胞介素-1(interleukin-1,IL-1)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的活性;TTC染色检测大鼠的脑组织梗死情况;HE染色检测大鼠缺血脑组织的病理状况;Western blot和RT-qPCR技术分别检测脑组织STIM1、STIM2、Orai1、半胱氨酸天冬氨酸蛋白酶-3(cysteinyl aspartate specific proteinase 3,caspase-3)、蛋白激酶B(protein kinase B,PKB)的蛋白及mRNA表达水平;免疫荧光检测大鼠脑神经元的生长情况。结果显示,相较于MCAO组TFR组大鼠神经功能评分明显降低,脑组织病理损伤显著改善,梗死面积明显减少,血清中IL-1、IL-6、TNF-α的活性显著降低,脑组织中STIM1、STIM2、Orai1、PKB蛋白及mRNA表达显著上调,caspase-3表达显著下调,在荧光显微镜下可观察到双标阳性细胞增多;而加入2-APB后,以上作用明显减弱。综上结果表明,TFR可能通过上调SOCE通道相关信号分子的基因表达,诱导缺血区周边的神经发生,改善缺血区周边的神经元生存状态,抑制炎症因子活性,从而发挥对抗局灶性脑缺血再灌注损伤的作用。
This paper explored the protective effect of total flavonoids of Rhododendron simsii(TFR) on focal cerebral ischemia-reperfusion injury(CIRI) in rats and its relationship with the store-operated calcium entry(SOCE) pathway regulated by stromal intera-ction molecule(STIM) and calcium release-activated calcium modulator(Orai).Rats were randomly assigned into the sham group, model(middle cerebral artery occlusion, MCAO) group, TFR(60 mg·kg) group, TFR(60 mg·kg)+SOCE pathway inhibitor 2-aminoethoxydiphenyl borate(2-APB, 2.5 mg·kg) group, and 2-APB(2.5 mg·kg) group.The rats in the sham group and MCAO group were administrated with normal saline, and those in the TFR group and TFR+2-APB group were administrated with TFR(60 mg·kg) by gavage for 14 days until sampling.The rats in the 2-APB group and TFR+2-APB group were intraperitoneally injected with 2-APB(2.5 mg·kg) after operation.The levels of interleukin-1(IL-1), interleukin-6(IL-6), and tumor necrosis factor-alpha(TNF-α) in serum were measured by ELISA.The cerebral infarction and the pathological status of ischemic brain tissue were detected via TTC staining and HE staining, respectively.The protein and mRNA levels of STIM1, STIM2, Orai1, cysteinyl aspartate specific proteinase 3(caspase-3), and protein kinase B(PKB) in brain tissue were respectively determined by Western blot and RT-qPCR.The growth of brain neurons in each group was observed via immunofluorescence method.The results showed that compared with the MCAO group, TFR lowered the levels of IL-1, IL-6 and TNF-α in serum and the score of neurological function, ameliorated the pathological injury of brain tissue, and decreased the infarct size.Moreover, TFR up-regulated the mRNA and protein levels of STIM1, STIM2, Orai1, and PKB, down-regulated those of caspase-3 in brain tissue, and increased the double-labeled positive cells under fluorescence microscope.However, the above effects were significantly weakened by the addition of 2-APB, a SOCE inhibitor.The results suggested that TFR may play a protective role against focal cerebral ischemia-reperfusion injury by up-regulating the expression of SOCE-related signal molecules, promoting neurogenesis around the ischemic area, improving the survival state of neurons, and redu-cing the activity of inflammatory mediators.
作者
陆嘉珺
姜晨晨
施磊
赫玉香
王书凡
尹秀云
陈卓
曹迪
胡浩然
韩军
LU Jia-jun;JIANG Chen-chen;SHI Lei;HE Yu-xiang;WANG Shu-fan;YIN Xiu-yun;CHEN Zhuo;CAO Di;HU Hao-an;HAN Jun(Third-Grade Pharmacology Laboratory of State,National Administration of Traditional Chinese Medicine,Wuhu 241002,China;Provincial Engineering Laboratory for Screening and Re-evalualion of Active Compounds of Herbal Medicines in SouthernAnhui,Wuhu 241002,China;Anhui Provincial Engineering Research Center for Polysaccharide Drugs,Wuhu 241002,China;School of Pharmacy,Drug Research and Development Center,Wannan Medical College,Wuhu 241002,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2022年第14期3828-3836,共9页
China Journal of Chinese Materia Medica
基金
国家自然科学基金面上项目(81973510)
安徽省高校自然科学研究重大项目(KJ2019ZD32)。
