补肾活血汤通过调控Runt相关转录因子2基因的表达改善大鼠椎间盘退变的实验研究

Experimental study on Bushen Huoxue Decoction in improving intervertebral disc degeneration by regulating the expression of Runx2 Gene in rats

目的观察补肾活血汤对椎间盘退变模型大鼠椎间盘内Runt相关转录因子2(RUNX2)基因及其相关蛋白髓核基质大分子Ⅱ型胶原引链(COL2α1)、蛋白多糖(aggrecan)表达的影响。方法60只8周龄健康SPF级雄性SD大鼠,造模成功后随机分成空白组8只,假手术组8只,模型组18只和实验组18只,各组进行相应的干预措施,实验分为2周和4周两个周期,各周期结束后处死大鼠,取腰(L)3/4~L5/6椎间盘行HE染色观察椎间盘组织形态、免疫组化检测观察COL2α1和aggrecan的表达情况、蛋白免疫印迹法(Western blot)检测Runx2、COL2α1和aggrecan的蛋白表达情况、实时荧光定量聚合酶链反应(RT-qPCR)法检测Runx2、COL2α1和aggrecan的mRNA表达情况。结果与模型组比较,中药组Runx2基因表达水平显著低于模型组(P<0.05),COL2α1和aggrecan蛋白含量显著高于模型组(P<0.05)。结论补肾活血汤可有效抑制椎间盘退变模型大鼠Runx2基因的表达,起到治疗或延缓椎间盘退变的作用。

Objective To observe the effect of Bushen Huoxue Decoction on the expression of Runt-related transcription factor 2(Runx2)and its related proteins collagen typeⅡalpha-1 gene(COL2A1)and aggrecan(ACAN)in intervertebral disc.Methods Sixty 8-week-old healthy SPF male SD rats were enrolled.Except forrats in the blank group(n=8),the rest were randomly grouped after successful modeling:sham operation group(n=8),model group(n=18)and traditional Chinese medicine(TCM,n=18)group.Each group received corresponding intervention measures.The experiment was divided into two weeks and four weeks,and the rats were killed following each cycle end.The hematoxylin-eosin(HE)staining was conducted to observe the tissue morphology of intervertebral disc degeneration from the facet joints at L3/4 to L5/6,the immunohistochemistry was used to detect expression status of COL2a1 and ACAN,Western blot analysis was performed to assess the protein expression of Runx2,COL2a1 and aggrecan,and mRNA expression of Runx2,COL2a1 and ACAN was detected by reverse transcription quantitative PCR(RT-qPCR).Results The expression level of Runx2 gene in the TCM group was significantly lower than that in the model group(P<0.05),while the protein contents of COL2a1 and ACAN were significantly higher(P<0.05).Conclusion Bushen Huoxue Decoction can effectively inhibit the expression of Runx2 gene and play a role in treating or delaying intervertebral disc degeneration.