Jiang Chen-Chen, Shi Lei, Zhao Xin-Ya, Zhang Hui, Li Zi-Xu, Lu Jia-Jun, He Yu-Xiang, Cao Di, Hu Hao-Ran, Han Jun
Third-Grade Pharmacology Laboratory of National,Administration of Traditional Chinese Medicine Wuhu 241002,China School of Pharmacy,Drug Research and Development Center,Wannan Medical College Wuhu 241002,China.
Third-Grade Pharmacology Laboratory of National,Administration of Traditional Chinese Medicine Wuhu 241002,China Anhui Provincial Engineering Laboratory for Screening and Re-evaluation of Active Compounds of Herbal Medicines in Southern Anhui Wuhu 241002,China.
Zhongguo Zhong Yao Za Zhi. 2023 Jan;48(2):455-464. doi: 10.19540/j.cnki.cjcmm.20221010.402.
This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL(-1)) group, and TFR(30 μg·mL(-1))+gene overexpression plasmid group. Intracellular Ca(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
本研究探讨了杜鹃总黄酮（TFR）对大鼠大脑中动脉闭塞（MCAO）诱导的脑损伤以及PC12细胞氧糖剥夺/复氧（OGD/R）损伤的影响及其潜在机制。采用MCAO法诱导大鼠局灶性缺血性脑损伤。雄性SD大鼠随机分为假手术组、模型组和TFR组。MCAO术后，给予TFR（60 mg·kg⁻¹），连续给药3天。采用酶联免疫吸附测定（ELISA）法检测血清中肿瘤坏死因子-α（TNF-α）、白细胞介素-1（IL-1）和白细胞介素-6（IL-6）的含量。基于苏木精-伊红（HE）染色和2,3,5-三苯基氯化四氮唑（TTC）染色观察脑组织的病理变化和脑梗死情况。采用RT-qPCR和蛋白质免疫印迹法检测脑组织中钙释放激活钙通道调节剂1（ORAI1）、基质相互作用分子-1（STIM1）、基质相互作用分子-2（STIM2）、蛋白激酶B（PKB）和半胱氨酸天冬氨酸特异性蛋白酶3（caspase-3）的mRNA和蛋白水平。采用OGD/R法诱导PC12细胞损伤。细胞随机分为正常组、模型组、基因沉默组、TFR（30 μg·mL⁻¹）组和TFR（30 μg·mL⁻¹）+基因过表达质粒组。通过激光扫描共聚焦显微镜和流式细胞术检测PC12细胞内Ca²⁺浓度和凋亡率。基于基因沉默和基因过表达技术探讨STIM-ORAI调节的钙库操纵性钙内流（SOCE）途径对TFR的影响。结果显示，给药3天后，TFR显著减轻了MCAO大鼠脑的组织病理学损伤，降低了血清中TNF-α、IL-1和IL-6的含量，下调了MCAO大鼠脑组织中ORAI1、STIM1、STIM2和caspase-3基因的表达，并上调了PKB基因的表达。TFR显著降低了OGD/R诱导的PC12细胞内Ca²⁺超载和凋亡。然而，通过沉默ORAI1、STIM1和STIM2基因可诱导产生类似TFR的效应。然而，这些基因的过表达显著阻断了TFR降低PC12细胞内Ca²⁺超载和凋亡的作用。综上所述，在局灶性脑缺血再灌注损伤早期以及PC12细胞OGD/R诱导的损伤中，TFR通过抑制STIM-ORAI调节的SOCE途径，减少Ca²⁺超载、炎症因子表达及凋亡，从而减轻缺血性脑损伤。
