那如三味片对大鼠创伤性脊髓损伤后炎症作用的影响及相关机制研究

Anti-inflammatory effect of Mongolian medicine Naru-3 on traumatic spinal cord injury in rats and its mechanism

目的观察那如三味片(Naru-3)对创伤性脊髓损伤大鼠炎症作用的影响,并探讨相关机制。方法 SD大鼠随机分为假手术组、模型组、甲基泼尼松龙(MP,30 mg/kg)组和Naru-3低、中、高剂量(10、20、30 mg/kg)组,每组12只。假手术组仅进行手术暴露,不进行打击;其他组别采用Allen’s打击法建立大鼠脊髓损伤模型。术后30 min各组ig给药,假手术组和模型组ig等体积生理盐水,每天1次,连续21 d。采用实时荧光定量PCR(qRT-PCR)和western blotting法检测脊髓组织中白介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)mRNA和蛋白的相对表达量;ELISA法检测血清中IL-1β、IL-6和TNF-α蛋白含量;免疫组织化学法确定脊髓组织单核细胞趋化蛋白-1(MCP-1)定位及表达;流式细胞术分析脊髓组织M1及M2表型巨噬细胞比例。结果与模型组比较,Naru-3、MP显著减少血清中IL-1β、IL-6和TNF-α蛋白含量,显著减少脊髓组织IL-1β、IL-6和TNF-αmRNA、蛋白水平(P<0.05、0.001),Naru-3作用呈剂量、时间相关性;免疫组织化学结果显示,与模型组比较,Naru-3、MP可以明显减少脊髓中MCP-1表达;流式细胞术结果表明,与模型组比较,Naru-3、MP显著诱导M1表型巨噬细胞分化为M2表型(P<0.001)。结论 Naru-3可以有效降低脊髓损伤引起的炎症反应,机制与诱导M1表型巨噬细胞分化为M2表型相关。

Objective To observe the anti-inflammation effect of the Mongolian medicine narru-3(Naru-3) prescription in the treatment of spinal cord injury,and explore its related mechanism.Methods SD rats were randomly divided into sham group,model group,methylprednisolone(MP,30 mg/kg) group and Naru-3 low,medium and high dose(10,20,30 mg/kg) group,with 12 rats in each group.The sham group was exposed to surgery only,but not attacked.The other groups were used Allen's attacking method to establish rat spinal cord injury model.Rats were ig administered in each group 30 min after operation,and rats in sham and model group was administered with normal saline once a day for 21 d.The relative expression levels of interleukin(IL)-1β,IL-6 and tumor necrosis factor-alpha(TNF-α) in spinal cord tissues were detected by real-time fluorescence quantitative PCR(qRT-PCR)and Western blotting.The levels of IL-1β,IL-6 and TNF-α in serum were detected by ELISA.The localization and expression of monocyte chemoattractant protein-1(MCP-1) in spinal cord tissues were determined by immunohistochemistry.The proportion of M1 and M2 phenotypic macrophages in spinal cord tissue was analyzed.Results Compared with model group,Naru-3 and MP significantly reduced the levels of IL-1β,IL-6 and TNF-α protein in serum and the mRNA and protein levels of IL-1β,IL-6 and TNF-α in spinal cord tissue(P<0.05,0.001).The effect of Naru-3 was dose-dependent and time-dependent.Immunohistochemical results showed that Naru-3 and MP could significantly reduce the expression of MCP-1 in spinal cord compared with the model group.The results of flow cytometry showed that,compared with model group,Naru-3 and MP significantly induced M1 phenotype macrophages to differentiate into M2 phenotype(P<0.001).Conclusion Naru-3 can effectively reduce the inflammatory response induced by spinal cord injury,and the mechanism is related to inducing M1 phenotype macrophages to differentiate into M2 phenotype.