现代中药研究与实践

目的 探讨三化汤抗大鼠脑缺血再灌注损伤(cerebral ischemia reperfusion injury,CIRI)作用机制。方法 采用网络药理学筛选三化汤抗CIRI的活性成分、作用靶点及信号通路。通过Longa线栓法建立大鼠大脑中动脉阻塞(MCAO)模型，诱导CIRI，给予尼莫地平/不同剂量组三化汤治疗之后，进行神经功能缺损评分，2,3,5-三苯四氮唑(TTC)法检测脑梗死体积，苏木素-伊红(HE)染色观察脑组织病理损伤，TUNEL染色评估神经元凋亡程度，化学比色法评价氧化应激相关标志物水平，ELISA法测定炎症因子水平，Western Blot法测定脑组织中细胞凋亡和磷脂酰肌醇-3-激酶(PI3K/AKT)信号通路相关蛋白的表达水平。结果 网络药理学结果显示，共筛选出三化汤有效成分66个及相关靶点705个，CIRI相关靶点1 267个，交集靶点171个。其中，核心成分包括橘皮素、芹菜素和香叶木素等；核心靶点主要有蛋白激酶B-1(AKT1)、胱天蛋白酶3 (CASP3)与B细胞淋巴瘤-2(BCL2)等；关键通路有PI3K/AKT信号通路等。动物实验结果显示，三化汤能够减轻CIRI大鼠神经功能缺损，减小脑梗死体积及病理损伤并降低神经元凋亡程度。此外，三化汤可以改善CIRI大鼠氧化应激反应，显著降低丙二醛(MDA)水平，同时升高超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)与过氧化氢酶(CAT)水平，还可以显著降低肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平并升高IL-10水平。同时，三化汤能上调p-PI3K、p-AKT及Bcl-2的表达，并下调Bax和Caspase-3蛋白的表达。结论 三化汤通过发挥抗炎、抗氧化应激、抗细胞凋亡作用以及调控PI3K/AKT信号通路来改善脑缺血再灌注大鼠的脑组织损伤。

Objective To explore the mechanism of Sanhua Decoction (SHD) in treating cerebral ischemia reperfusion injury (CIRI) in rats based on network pharmacology and animal experiments.Methods The active components,targets and signaling pathways of SHD against CIRI were screened by network pharmacology.The MCAO model was established using the Longa suture method to induce cerebral ischemia reperfusion injury.After treatment with nimodipine/different dose groups of SHD,neurological scores were evaluated.The cerebral infarct volume was measured using TTC method,the pathological damage of brain tissue was observed using HE staining,the degree of neuronal apoptosis was evaluated using TUNEL staining,the levels of oxidative stress-related markers were evaluated using chemical colorimetry,the levels of inflammatory factors were measured using ELISA,and the expression levels of cell apoptosis and PI3K/AKT signaling pathway related proteins in brain tissue were measured using Western Blot.Results The results of network pharmacology showed that a total of 66 active ingredients and 705 related targets were screened from SHD,and 1 267 targets related to CIRI were predicted.The compounds and CIRI shared 171 common targets.Among them,the core components included tangeretin,apigenin and diosmetin;the core targets mainly included AKT1,CASP3 and BCL2;the key pathways included PI3K/AKT signaling pathway.The results of animal experiments showed that SHD could alleviated the neurological scores in CIRI rats,reduced the volume of cerebral infarction and pathological damage,and reduced the degree of neuronal apoptosis.In addition,SHD could inhibited the oxidative stress reaction in CIRI rats,significantly reduced the level of MDA,increased the levels of SOD,GSH-Px and CAT,significantly reduced the levels of TNF-α and IL-6,and increased the levels of IL-10.Meanwhile,SHD could up-regulated the expression of p-PI3K,p-AKT and Bcl-2,and down-regulated the expression of Bax and Caspase-3 proteins.Conclusion SHD can improved brain tissue injury in rats with cerebral ischemia reperfusion by exerting anti-inflammatory,anti-oxidative stress,anti-apoptosis and regulating PI3K/AKT signaling pathway.