硒-甲基硒代半胱氨酸促进周围神经再生的实验研究

Experimental study on promotion of peripheral nerve regeneration by selenium-methylselenocysteine

目的探讨硒-甲基硒代半胱氨酸(selenium-methylselenocysteine,SMC)促进周围神经再生的可行性及其作用机制。方法将大鼠雪旺细胞RSC96细胞随机分为5组,分别为A组(无任何处理对照组)、B组(加入100μmol/L H_(2)O_(2))、C组(加入100μmol/L H_(2)O_(2)+100μmol/L SMC)、D组(加入100μmol/L H_(2)O_(2)+200μmol/L SMC)、E组(加入100μmol/L H_(2)O_(2)+400μmol/L SMC);通过MTT法检测SMC对细胞增殖的影响,确定合适剂量组别后,通过自由基(reactive oxygen species,ROS)免疫荧光检测细胞氧化应激水平。将36只4周龄雄性SD大鼠随机分为3组,分别为假手术组(Sham组)、坐骨神经损伤组(PNI组)及SMC治疗组(SMC组),每组12只;PNI组大鼠造模术后正常喂食、水,SMC组大鼠于每日饮用水中加入0.75 mg/kg SMC。术后4周各组大鼠坐骨神经取材行神经电生理检测复合肌肉动作电位(compound muscle action potential,CMAP)最高电位,ELISA法检测炎症因子IL-17、IL-6、IL-10和氧化应激因子过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)水平,劳克坚劳蓝(luxol fast blue,LFB)染色观测髓鞘密度,免疫荧光染色观察胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和髓鞘碱性蛋白(myelinbasicprotein,MBP)荧光强度,透射电镜观察髓鞘形态并测量轴突直径,Western blot检测p38丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)、磷酸化p38MAPK(phosphorylation p38MAPK,p-p38MAPK)、血红素氧合酶1(heme oxygenase 1,HO-1)、核因子红细胞系2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)蛋白相对表达量。结果MTT检测示加入SMC后显著促进RSC96细胞增殖,且低浓度即可达有效效果,故选择C组进入后续实验;ROS免疫荧光检测示B组较A组ROS荧光强度明显上升,C组较B组ROS荧光强度明显下降(P<0.05)。动物实验神经电生理检测示,SMC组CMAP最高电位显著高于PNI组和Sham组(P<0.05)。ELISA检测示,PNI组较Sham组IL-6、IL-17、MDA水平明显上升,IL-10、SOD、CAT水平明显下降;SMC组较PNI组IL-6、IL-17、MDA水平明显下降,IL-10、SOD、CAT水平明显上升(P<0.05)。LFB染色和透射电镜检测示SMC组的髓鞘密度和轴突直径明显优于PNI组和Sham组(P<0.05)。免疫荧光染色示SMC组GFAP和MBP荧光强度显著强于PNI组和Sham组(P<0.05)。Western blot检测示SMC组Nrf2、HO-1蛋白相对表达量显著高于PNI组和Sham组,PNI组p-p38MAPK/p38MAPK蛋白表达量比值显著高于SMC组和Sham组(P<0.05)。结论SMC可能通过上调Nrf2/HO-1通路抑制神经损伤后的氧化应激和炎症反应,进而抑制p38MAPK通路磷酸化促进雪旺细胞增殖,最终促进髓鞘形成和加速周围神经再生。

Objective To investigate the feasibility of selenium-methylselenocysteine(SMC)to promote peripheral nerve regeneration and its mechanism of action.Methods Rat Schwann cells RSC96 cells were randomly divided into 5 groups,which were group A(without any treatment,control group),group B(adding 100μmol/L H_(2)O_(2)),group C(adding 100μmol/L H_(2)O_(2)+100μmol/L SMC),group D(adding 100μmol/L H_(2)O_(2)+200μmol/L SMC),group E(adding 100μmol/L H_(2)O_(2)+400μmol/L SMC);the effect of SMC on cell proliferation was detected by MTT method,and the level of oxidative stress was detected by immunofluorescence for free radicals[reactive oxygen species(ROS)]after determining the appropriate dose group.Thirty-six 4-week-old male Sprague Dawley rats were randomly divided into 3 groups,namely,the sham operation group(Sham group),the sciatic nerve injury group(PNI group),and the SMC treatment group(SMC group),with 12 rats in each group;the rats in the PNI group were fed with food and water normally after modelling operation,and the rats in the SMC group were added 0.75 mg/kg SMC to the drinking water every day.At 4 weeks after operation,the sciatic nerves of rats in each group were sampled for neuroelectrophysiological detection of highest potential of compound muscle action potential(CMAP).The levels of inflammatory factors[interleukin 17(IL-17),IL-6,IL-10 and oxidative stress factors catalase(CAT),superoxide dismutase(SOD),and malondialdehyde(MDA)]were detected by ELISA assay.The luxol fast blue(LFB)staining was used to observe the myelin density,fluorescence intensity of glial fibrillary acidic protein(GFAP)and myelin basic protein(MBP)was observed by immunofluorescence staining,and myelin morphology was observed by transmission electron microscopy with measurement of axon diameter.Western blot was used to detect the protein expressions of p38 mitogen-activated protein kinases(p38MAPK),phosphorylated p38MAPK(p-p38MAPK),heme oxygenase 1(HO-1),and nuclear factor erythroid 2-related factor 2(Nrf2).Results MTT assay showed that the addition of SMC significantly promoted the proliferation of RSC96 cells,and the low concentration could achieve an effective effect,so the treatment method of group C was selected for the subsequent experiments;ROS immunofluorescence test showed that group B showed a significant increase in the intensity of ROS fluorescence compared with that of group A,and group C showed a significant decrease in the intensity of ROS fluorescence compared with that of group B(P<0.05).Neuroelectrophysiological tests showed that the highest potential of CMAP in SMC group was significantly higher than that in PNI and Sham groups(P<0.05).ELISA assay showed that the levels of IL-6,IL-17,and MDA in PNI group were significantly higher than those in Sham group,and the levels of IL-10,SOD,and CAT were significantly lower;the levels of IL-6,IL-17,and MDA in SMC group were significantly lower than those in PNI group,and the levels of IL-10,SOD,and CAT were significantly higher(P<0.05).LFB staining and transmission electron microscopy showed that the myelin density and the diameter of axons in the SMC group were significantly higher than those of the PNI group and the Sham group(P<0.05).Immunofluorescence staining showed that the fluorescence intensity of GFAP and MBP in the SMC group were significantly stronger than those in the PNI group and Sham group(P<0.05).Western blot showed that the relative expressions of Nrf2 and HO-1 proteins in the SMC group were significantly higher than those in the PNI group and Sham group,and the ratio of p-p38MAPK/p38MAPK proteins was significantly higher in the PNI group than that in the SMC group and Sham group(P<0.05).Conclusion SMC may inhibit oxidative stress and inflammation after nerve injury by up-regulating the Nrf2/HO-1 pathway,and then inhibit the phosphorylation of p38MAPK pathway to promote the proliferation of Schwann cells,which ultimately promotes the formation of myelin sheaths and accelerates the regeneration of peripheral nerves.