铅和脂多糖暴露致小鼠神经炎症的损伤机制

Mechanism of neuroinflammatory injury induced by lead and lipopolysaccharide in mice

[背景]铅和脂多糖(LPS)暴露均可以引起神经炎症和小胶质细胞极化,但是铅和LPS联合暴露是否会导致神经炎症加剧以及小胶质细胞的极化情况还未见报道。[目的]探讨铅和LPS联合暴露对小鼠神经炎症的影响以及机制。[方法]SPF级雄性C57小鼠64只随机分为8个组:对照组、LPS组、低铅组、中铅组、高铅组、低铅+LPS组、中铅+LPS组和高铅+LPS组。铅染毒:分别给予0.25、0.50、1.00 g·L^(-1)的乙酸铅饮水9周;LPS处理:乙酸铅染毒结束前一周,每天腹腔注射250 g·kg^(-1)(以体重计),每天注射一次,共7次,末次注射24 h后处死动物。用ELISA法检测皮质中炎症因子肿瘤坏死因子(TNF)-α、白介素(IL)-1β和IL-6的变化情况。用荧光定量PCR方法检测小胶质细胞M1型标志物[诱导型一氧化氮合酶(iNOS)、NADPH氧化酶2(NOX2)、趋化因子受体7(CCR7)]、M2型标志物[精氨酸-1(Arg-1)、转化生长因子-β(TGF-β)、趋化因子受体2(CCR2)]、髓样细胞触发受体2(TREM2)m RNA和微小RNA-34a(miR-34a)的表达情况。用Western blotting法检测TREM2、iNOS和Arg-1蛋白的表达情况。[结果]与对照组相比,LPS组、中铅组和高铅组小鼠皮质中炎症因子TNF-α、IL-1β和IL-6表达量均增加;与LPS组或者同剂量的铅组相比,中铅+LPS组和高铅+LPS组皮质中TNF-α(7.82±0.60,8.79±1.62)、IL-1β(8.80±0.95,11.18±0.92)和IL-6(7.53±0.90,8.66±0.93)含量增加(P<0.05)。LPS组、中铅、高铅组皮质中M1标志物iNOS、NOX2、CCR7 mRNA和iNOS蛋白表达高于对照组,而M2标志物Arg-1、TGF-β、CCR2 mRNA和Arg-1蛋白表达明显低于对照组,尤其是CCR2 mRNA表达下降尤为显著(P<0.05)。与LPS组或者同剂量的铅组相比,中铅+LPS组和高铅+LPS组皮质中iNOS、CCR7 mRNA和iNOS蛋白表达升高较为明显,而Arg-1m RNA表达减少。此外,中铅+LPS和高铅+LPS组皮质中TREM2 mRNA(0.20±0.03,0.04±0.01)和蛋白(0.32±0.02,0.21±0.02)表达均低于LPS组或相同剂量的铅组,而miR-34a表达升高(P<0.05)。[结论]铅和LPS联合暴露后加剧了神经炎症损伤,小胶质细胞M1标志物增加显著,这可能与miR-34a表达升高导致TREM2表达有关。

[Background]Both lead and lipopolysaccharide(LPS)exposures can result in neuroinflammation and microglia polarization,but it is not clear whether the combined exposure to lead and LPS will aggravate neuroinflammation and the polarization of microglia.[Objective]The in vivo experiment investigates the effect and mechanism of combined exposure to lead and LPS on neuroinflammation in mice.[Methods]A total of 64 SPF male C57 mice were randomly divided into eight groups:control group,LPS group,low lead group,medium lead group,high lead group,low lead+LPS group,medium lead+LPS group,and high lead+LPS group.Mice in the lead exposure groups were given 0.25,0.50,and 1.00 g·L^(-1) lead acetate through drinking water for nine weeks respectively.The LPS treatment was administered at a dose of 250 g·kg^(-1)(by weight)for 7 d at the 9 th week of lead exposure.The animals were sacrified 24 h after the last injection.The changes of inflammatory factors tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6)in the cortex were detected by ELLSA.The expressions of M1 markers[induced nitric oxide synthase(i NOS),NADPH oxidase 2(NOX2),andchemokine receptor 7(CCR7)],M2 markers[arginase 1(Arg-1),transforming growth factor-β(TGF-β),and chemokine receptor 2(CCR2)],triggering receptor expressed on myeloid cells 2(TREM2)m RNA and micro RNA-34 a(mi R-34 a)were detected by quantitative PCR.The protein expressions of TREM2,i NOS,and Arg-1 were detected by Western blotting.[Results]Compared with the control group,the levels of inflammatory cytokines TNF-α,IL-1β,and IL-6 in the cortex of mice were increased in the LPS group,the medium lead group,and the high lead group.Compared with the LPS group or the corresponding lead group,the levels of TNF-α(7.82±0.60,8.79±1.62),IL-1β(8.80±0.95,11.18±0.92),and IL-6(7.53±0.90,8.66±0.93)were elevated in the medium lead+LPS group and the high lead+LPS group.The expression levels of M1 markers i NOS,NOX2,and CCR7 m RNA and i NOS protein in the cortex of the LPS group,the medium lead group,and the high lead group were higher than those of the control group,while the expression levels of M2 markers Arg-1,TGF-β,and CCR2 m RNA and Arg-1 protein were lower,especially the expression of CCR2 m RNA(P<0.05).Compared with the LPS group or the corresponding lead group,the m RNA and protein expression levels of i NOS and the CCR7 m RNA expression levels of the medium lead+LPS group and the high lead+LPS group were increased,while the expression levels of Arg-1 m RNA were decreased.In addition,the TREM2 m RNA(0.20±0.03,0.04±0.01)and protein(0.32±0.02,0.21±0.02)expression levels in the cortex of the medium lead+LPS and the high lead+LPS groups were lower than those of the LPS group or the corresponding lead group,while the expression level of mi R-34 a was increased(P<0.05).[Conclusion]Combined exposure to lead and LPS aggravates neuroinflammatory injury and increases M1 polarization of microglia,which may be related to the increased expression of TREM2 up-regulated by mi R-34 a.