[关键词]
[摘要]
目的 以NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)炎症小体为切入点,研究黄芩苷-栀子苷(7∶3)抗脑缺血损伤及并发心肌损伤的作用机制。方法 将雄性SD大鼠随机分为对照组、模型组、尼莫地平(10 mg/kg)组和黄芩苷-栀子苷低、中、高剂量(30、45、60 mg/kg)组,每组10只。ig药物7 d后,采用线栓法构建大鼠局灶性脑缺血模型,采用Zea-Longa评分标准进行神经功能评分;干湿质量法检测脑组织水肿情况;激光多普勒检测脑血流量;苏木素-伊红(HE)染色观察脑和心肌组织病理变化;TUNEL染色观察心肌组织凋亡情况;采用ELISA法测定心肌组织中心肌酶谱活性。利用网络药理学方法预测黄芩苷-栀子苷抗脑缺血损伤及并发心肌损伤的作用机制;采用ELISA法检测血清中白细胞介素-1β(interleukin-1β,IL-1β)、IL-6、IL-18、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;采用Western blotting法检测脑组织、心肌组织中NLRP3炎症小体相关蛋白表达。结果 与对照组比较,模型组大鼠脑、心组织损伤严重,炎性因子水平升高(P<0.01),心肌酶谱活性显著升高(P<0.01);与模型组比较,各给药组脑、心组织病理损伤不同程度减轻,脑含水量、神经功能评分、心肌细胞凋亡率、血清中心肌酶谱显著降低(P<0.05、0.01),脑血流量显著增加(P<0.05、0.01)。网络药理学结果显示,NOD样信号通路在黄芩苷-栀子苷防治脑缺血及其并发心损伤中发挥重要作用,选取NOD样受体家族NLRP3炎性小体继续深入研究,ELISA和qRT-PCR结果表明,模型组大鼠血清中IL-1β、IL-6、IL-18、TNF-α水平及脑组织皮层、心肌组织中NLRP3炎症小体蛋白表达水平显著升高(P<0.01),而各给药组血清中炎症因子水平和脑组织皮层、心肌组织中NLRP3炎症小体蛋白表达水平均显著降低(P<0.01)。结论 黄芩苷-栀子苷能抑制炎性介质的表达,通过下调NLRP3炎症小体发挥抗脑缺血损伤及卒中后心肌损伤作用。
[Key word]
[Abstract]
Objective To study the mechanism of baicalin-geniposide (7∶3) in preventing cerebral ischemia injury and myocardial injury by taking NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome as the breakthrough point. Methods Male SD rats were randomly divided into control group, model group, nimodipine (10 mg/kg) group and baicalin-geniposide low-, medium- and high-dose (30, 45, 60 mg/kg) groups, with 10 rats in each group. After 7 d of administration, the rat model of focal cerebral ischemia was established by thread embolism method, and neurological function was scored by Zea-Longa scoring standard. The edema of brain tissue was detected by dry-wet mass method; Cerebral blood flow was detected by laser Doppler; HE staining was used to observe the pathological changes of brain and myocardium; TUNEL staining was used to observe myocardial apoptosis; The activities of myocardial zymogram in myocardial tissue was determined by ELISA. Network pharmacology method was used to predict the mechanism of baicalin-geniposide against cerebral ischemia injury and myocardial injury; Levels of interleukin-1β (IL-1β), IL-6, IL-18 and tumor necrosis factor-α (TNF-α) in serum were detected by ELISA; Western blotting was used to detect the expressions of NLRP3 inflammasome related protein in brain tissue and myocardial tissue. Results Compared with control group, brain and heart tissue of model group were seriously damaged, levels of inflammatory factors were increased (P < 0.01), and activities of myocardial enzymes were significantly increased (P < 0.01). Compared with model group, pathological damage of brain and heart tissue were alleviated in different degrees, brain water content, neurological function score, myocardial cell apoptosis rate and myocardial enzyme spectrum in serum were significantly decreased (P < 0.05, 0.01), and cerebral blood flow was significantly increased (P < 0.05, 0.01) in each administration group. The results of network pharmacology showed that NOD-like signaling pathway played an important role in prevention and treatment of cerebral ischemia and its complicated myocardial injury by baicalin-geniposide. NOD-like receptor family NLRP3 inflammasome were selected for further study. The results of ELISA and qRT-PCR showed that, levels of IL-1β, IL-6, IL-18, TNF-α in serum and expressions of NLRP3 inflammasome protein in brain cortex and myocardial tissue of rats in model group were significantly increased (P < 0.01), while levels of inflammatory factors in serum and expressions of NLRP3 inflammasome protein in brain cortex and myocardial tissue were significantly decreased (P < 0.01). Conclusion Baicalin-geniposide could inhibit the expression of inflammatory mediators, and play an anti-cerebral ischemia injury and myocardial injury after stroke by down-regulating NLRP3 inflammasome.
[中图分类号]
R285.5
[基金项目]
国家自然科学基金资助项目(81473385);中医药“双链融合”中青年科研创新团队(2022-SLRH-YQ-006);陕西省中医药科研项目(2021-ZZ-JC023);咸阳市重点研发科技项目(JBGS-001)