脊髓康对脊髓损伤模型大鼠血清外泌体中miRNA表达的影响

Effect of Jisui Kang(脊髓康)on the Expression of Serum Exosomal mRNAs in Spinal Cord Injury Model Rats

目的探讨脊髓康治疗脊髓损伤的可能作用机制。方法将80只SD大鼠随机分为假手术组、模型组、强的松组和脊髓康组,每组20只。除假手术组外其余各组大鼠采用改进Allen′s方法建立脊髓损伤模型,假手术组大鼠脊髓暴露但未受伤。造模后假手术组和模型组大鼠给予生理盐水20ml/(kg·d)灌胃,强的松组大鼠给予强的松溶液60mg/(kg·d)灌胃,脊髓康组大鼠给予脊髓康溶液25g/(kg·d)灌胃。各组大鼠分别于术后7、14、21和28天进行指标观察,每个时间点5只。大鼠的运动功能采用双后肢运动功能评分(BBB评分)和斜板试验评价,采集血清后提取外泌体并用外泌体标志蛋白(Tsg101、CD63、CD9)进行鉴定,检测损伤区RhoA、ROCK蛋白及mRNA表达,并检测外泌体中miR-21、miR-126、miR-133b表达。结果与假手术组同时间比较,模型组大鼠斜板试验角度和BBB评分均降低,模型组在术后7、28天时RhoA蛋白及mRNA表达升高,术后28天时ROCK蛋白及mRNA表达升高,术后7、28天时外泌体中miR-21、miR-126、miR-133b表达降低(P<0.05);与模型组同时间比较,脊髓康组和强的松组大鼠在术后21、28天时斜板试验角度和BBB评分增加,术后28天时RhoA、ROCK蛋白及mRNA表达均降低,外泌体中miR-21、miR-126、miR-133b表达均升高(P<0.05或P<0.01)。结论脊髓康可能通过促进急性脊髓损伤模型大鼠血清外泌体中miR-21、miR-126、miR-133b表达,进而抑制RhoA/ROCK通路,从而修复脊髓损伤,改善大鼠的运动功能,在28天时作用最明显。

Objective To explore the possible mechanism of Jisui Kang(JSK,脊髓康)in the treatment of spinal cord injury(SCI).Methods Eighty SD rats were randomly divided into sham operation group,model group,prednisone group,and Jisui Kang group,with 20 rats in each group.Except for the sham operation group,the rats in the other groups used the modified Allen′s method to establish the acute SCI model.The spinal cord of the rats in the sham operation group were exposed but not injured.After modeling,rats in the sham operation group and model group were administered with 20 ml/(kg·d)normal saline by gavage,while rats in the prednisone group and JSK group received 60 mg/(kg·d)prednisone solution and 25 g/(kg·d)JSK solution by gavage,respectively.The outcomes were assessed at the 7 th,14 th,21 st and 28 th day after operation,with five rats at each time point.The motor function of hindlimbs determined by BBB score and oblique plate test were evaluated.After collecting the serum,the exosomes were extracted and identified by the exosome′s marker proteins(Tsg101,CD63,CD9).The expression of RhoA,ROCK protein and mRNA in the injured area,and the expression of miR-21,miR-126,miR-133 b mRNA in exosomes were detected.Results Compared with the sham operation group at the same time point,the model group had significantly reduced angle of the oblique plate test and the BBB score at all time points,higher expression of RhoA protein and mRNA at the 7 th and 28 th day,higher expression of ROCK protein and mRNA at the 28 th day,and lower expression of miR-21,miR-126,miR-133 b mRNA in exosomes at the 7 th and 28 th day(P<0.05).Compared with the outcomes in the model group at the same time point,the angle of the oblique plate test and the BBB score significantly increased at the 7 th and 21 st day in the JSK group and prednisone group;the expression of RhoA,ROCK protein and mRNA decreased at the 28 th day;and the expression of miR-21,miR-126,and miR-133 b mRNA increased in exosomes at all time points(P<0.05 or P<0.01).Conclusion JSK may promote the expression of miR-21,miR-126,miR-133 b mRNA in serum exosomes of acute SCI model rats,thereby inhibiting the RhoA/ROCK pathway,repairing spinal cord injury and improving the motor function of rats.The effect works best at 28 days.