雷公藤甲素对脂多糖诱导脊髓损伤小鼠及BV2小胶质细胞的调节作用及机制研究

Regulation effect and mechanism of triptolide on lipopolysaccharide-induced spinal cord injury in mice and BV2 microglia

目的:探讨雷公藤甲素(TPL)对脂多糖(LPS)诱导的脊髓损伤(SCI)小鼠及BV2小胶质细胞的调节作用,并探究TPL对抗炎症的可能机制。方法:将45只C57小鼠作为研究对象。15只作为对照组。30只采用改良Allen’s法制备小鼠SCI模型,其中15只不加干预作为SCI组,另15只行TPL治疗[0.2 mg/(kg·d)连续注射7 d]作为TPL组。采用HE染色评估各组脊髓组织损伤情况。采用运动功能评分(BBB)评估各组运动能力。再以BV2细胞为研究对象,分为对照组、LPS诱导组(100 ng/ml)以及TPL处理组(LPS 100 ng/ml+TPL 50 nmol/L)。采用ELISA法检测BV2细胞各组及小鼠各组炎症因子[白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)]含量。采用生化方法检测一氧化氮(NO)含量。采用Western blot检测诱导型一氧化氮合酶(iNOS)表达。采用Western blot及免疫荧光法检测BV2细胞及脊髓损伤组织NOD样受体蛋白3(NLRP3)通路相关蛋白的表达。结果:TPL可抑制LPS诱导的BV2小胶质细胞及SCI小鼠IL-1β、IL-6、TNF-α的表达及iNOS介导的NO产生,促进SCI小鼠脊髓修复。同时,TPL可通过抑制NLRP3通路调节LPS诱导的BV2小胶质细胞炎症及SCI小鼠模型的炎症。结论:TPL可通过抑制NLRP3通路及iNOS-NO通路在BV2小胶质细胞及SCI小鼠模型中发挥抗炎作用,这为脊髓损伤的治疗用药提供了理论支持。

Objective:To investigate the regulation effect of triptolide(TPL)on lipopolysaccharide(LPS)-induced spinal cord injury(SCI)mice and BV2 microglia,and explore the possible mechanism of TPL against inflammation.Methods:45 C57 mice were used as research objects.Fifteen animals were used as control group.Thirty mouse models of SCI were prepared by modified Allen’s method,15 of them without intervention were taken as SCI group,and the other 15 were treated with TPL[0.2 mg/(kg·d)continuous injection for 7 days]as TPL group.HE staining was used to evaluate the spinal cord tissue injury in each group.The BBB score was used to evaluate the sports ability of each group.Taking BV2 microglia as the research object,and they were divided into control group,LPS induction group(100 ng/ml)and TPL treatment group(LPS 100 ng/ml+TPL 50 nmol/L).The ELISA method was used to detect the levels of inflammatory factors(IL-1β,IL-6,TNF-α)in each group of BV2 cells and each group of mice.Biochemical methods was used to detect NO content.Western blot was used to detect the expression of iNOS.Western blot and immunofluorescence were used to detect the expression of NLRP3 pathway related proteins in BV2 cells and tissues of SCI mice.Results:TPL could inhibit the expression of IL-1β,IL-6,TNF-αand NO mediated by iNOS in BV2,and promote spinal cord repair in SCI mice.At the same time,TPL can regulate LPS-induced inflammation of BV2 microglia and SCI mice models by inhibiting the NLRP3 pathway.Conclusion:TPL can exert anti-inflammatory effects in BV2 microglia and SCI mice model by inhibiting the NLRP3 and iNOS-NO pathway,which provides theoretical support for the treatment of SCI.