目的 研究注射用丹参多酚酸(salvianolic acids for injection,SAFI)对氧糖剥夺后恢复(oxygen and glucose deprivation reperfusion,OGD/R)损伤PC12细胞的保护作用及机制。方法 采用网络药理学方法预测SAFI抗脑缺血再灌注损伤的作用靶点。建立OGD/R损伤PC12细胞模型,采用噻唑蓝(MTT)比色法和检测乳酸脱氢酶(LDH)的释放考察SAFI对PC12细胞活力的影响,吖啶橙/溴化乙锭(acridine orange/ethidium bromide,AO/EB)染色检测细胞凋亡,蛋白质印迹法(Western blot)检测B淋巴细胞瘤-2关联X蛋白(Bax)和B淋巴细胞瘤-2(Bcl-2)凋亡蛋白水平,进一步通过免疫荧光法研究SAFI保护OGD/R损伤PC12细胞的作用通路。结果 OGD/R损伤PC12细胞,导致细胞存活率明显降低,1和10 mg·mL-1的SAFI可以显著提高OGD/R后PC12细胞存活率,降低LDH释放。并且SAFI能够显著增加OGD/R损伤后抗凋亡蛋白Bcl-2的表达,抑制促凋亡蛋白Bax过表达。进一步研究表明,SAFI可以显著上调蛋白激酶B(protein kinase B,Akt)和信号转导及转录激活蛋白3(signal transducer and activator of transcription 3,STAT3)的磷酸化水平。结论 SAFI能够抑制OGD/R对PC12细胞的损伤,是通过促进Bax和Bcl-2平衡的恢复,激活磷脂酰肌醇激酶(PI3K)/Akt和蛋白酪氨酸激酶2(janus kinase 2,JAK2)/STAT3信号通路实现的。
Abstract
OBJECTIVE To research the protective effect and mechanism of salvianolic acids for injection(SAFI) on PC12 cells injured by oxygen and glucose deprivation reperfusion(OGD/R). METHODS The method of network pharmacology was used to predict the action pathway of SAFI against cerebral ischemia-reperfusion injury. The model of PC12 cells injured by OGD/R was established. The effect of SAFI on the viability of PC12 cells was investigated by MTT assay and LDH release assay. Apoptosis was detected by acridine orange/ethidium bromide, BCL-2-associated X proteinx and B-cell lymphoma-2 protein levels were detected by Western blot. The protective pathway of SAFI against OGD/R-injured PC12 cells was studied by immunofluorescence. RESULTS OGD/R damaged PC12 cells, resulting in a significant decrease in cell survival. SAFI of 1 and 10 mg·mL-1 could significantly reduce the damage and lactate dehydrogenase release of PC12 cells caused by OGD/R. SAFI could increase the expression of anti-apoptotic protein Bcl-2 and inhibit the overexpression of Bax after OGD/R injury to some extent. SAFI could significantly up-regulate the phosphorylation levels of protein kinase B and signal transducer and activator of transcription 3. CONCLUSION SAFI can inhibit the injury of PC12 cells induced by OGD/R by promoting the recovery of Bax and Bcl-2 balance and activating PI3K/Akt and JAK2/STAT3 signaling pathways.
关键词
注射用丹参多酚酸 /
氧糖剥夺后恢复 /
细胞凋亡 /
PC12细胞 /
作用机制
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Key words
salvianolic acids for injection /
OGD/R /
apoptosis /
PC12 cells /
action mechanism
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中图分类号：
R966
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脚注
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基金
国家自然科学基金项目资助(82004071);山东省重点研发计划资助(2019JZZY020909);中国医学科学院医学与健康科技创新工程重大协同创新项目资助(2021-I2M-1-005);中国科协青年人才托举工程项目资助(YESS20200108)
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