二甲双胍重塑创伤性脑损伤后免疫环境抑制神经元死亡

Metformin suppresses neuronal death after traumatic brain injury by remodeling immune environment

目的观察二甲双胍对创伤性脑损伤免疫环境及神经元的影响。方法将SPF级健康成年雄性C57BL/6小鼠随机分为实验组和对照组,每组15只,实时定量聚合酶链反应(Real-time PCR)技术检测促炎性细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6和抗炎性细胞因子IL-4、IL-10、IL-13的表达;流式细胞术检测CD206和诱导型一氧化氮合酶(iNOS)的表达水平;通过Fluoro-Jade B染色的实验方法,计算神经元的死亡个数,组间采用t检验。结果通过Real-time PCR技术检测炎性细胞因子表达,实验组促炎因子IL-6(1.00±0.74)、IL-1β(2.08±0.86)、TNF-α(1.32±0.15)表达量低于对照组IL-6(1.67±1.31)、IL-1β(2.92±0.20)、TNF-α(2.47±0.14),差异有统计学意义IL-6(t=-17.212,P<0.05)、IL-1β(t=14.872,P<0.05)、TNF-α(t=-20.929,P<0.05);实验组抗炎因子IL-4(1.47±0.43)、IL-10(2.16±0.25)低于对照组IL-4(2.12±0.12)、IL-10(2.86±0.24),差异有统计学意义IL-4(t=-5.961,P<0.05)、IL-10(t=-7.942,P<0.05);通过流式细胞术检测免疫细胞标志物的表达水平,实验组iNOS(0.378±0.033)低于对照组(0.566±0.028),差异有统计学意义(t=-16.813,P<0.05);实验组CD206(0.628±0.035)表达高于对照组(0.313±0.18),差异有统计学意义(t=30.720,P<0.05);通过FJB染色统计FJB阳性细胞个数,实验组神经元个数(18.27±1.98)明显低于对照组神经元个数(53.60±2.16),差异有统计学意义(t=-46.638,P<0.05)。结论二甲双胍治疗TBI,不仅抑制神经元死亡而且减少神经炎症。

Objective To investigate the effect of metformin on the immune environment and neurons in traumatic brain injury(TBI).Methods The animal models of TBI in mice injected with metformin and normal saline were used as experimental group and control group,respectively.The real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect pro-inflammatory cytokines[tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6]and anti-inflammatory cytokines.The expression of IL-4,IL-10,IL-13,CD206 and inducible nitric oxide synthase(iNOS)was detected by flow cytometry.The number of dead neurons was calculated by the experimental method of FJB staining.The SPSS25.0 statistical software was used to analyze.Quantitative data were expressed as mean±standard deviation(±s),and t test was used.Results The expression of IL-6(1.00±0.74),IL-1β(2.08±0.86),TNF-α(1.32±0.15),IL-4(1.47±0.43)and IL-10(2.16±0.25)in experimental group was significantly lower than in the control group[IL-6(1.67±1.31),IL-1β(2.92±0.20),TNF-α(2.47±0.14),IL-4(2.12±0.12),IL-10(2.86±0.24)]with the difference being statistically significant[IL-6(t=-17.212,P<0.05);IL-1β(t=14.872,P<0.05);TNF-α(t=-20.929,P<0.05);IL-4(t=-5.961,P<0.05);IL-10(t=-7.942,P<0.05)].The expression level of iNOSin the experimental group(0.378±0.033)was significantly decreased as compared with that in the control group(0.566±0.028)(t=-16.813,P<0.05).The expression of CD206 in the experimental group(0.628±0.035)was significantly higher than that in the control group(0.313±0.18)(t=30.720,P<0.05).The number of FJB positive cells in the experimental group(18.27±1.98)was significantly less than that in the control group(53.60±2.16),and the difference was statistically significant(t=-46.638,P<0.05).Conclusion Metformin in the treatment of TBI not only inhibits neuronal death but also reduces neuroinflammation.