针刀干预对LDH模型大鼠TGF-β/BMP信号通路水平的影响

Effects of needle knife interventions on lumbar disc herniation rats by regulating the TGF-β/BMP signaling pathway

目的探讨针刀干预对腰椎间盘突出症(LDH)模型大鼠椎间盘退变及转化生长因子β/骨形态发生蛋白(TGF-β/BMP)信号通路水平的影响。方法建立LDH大鼠模型,大鼠分为空白组、假手术组、模型组、塞来昔布组(0.34g·kg^(-1)·d^(-1)塞来昔布溶剂)、针刀组、TGF-β/BMP抑制剂组(针刀+SB-431542,针刀+100μL/鼠/2d的SB431542)、TGF-β/BMP激动剂组(针刀+SRI-011381 hydrochloride,针刀+10 mg·kg^(-1)·2 d^(-1)的SRI-011381 hydrochloride),每组20只。观察7组大鼠一般行为状态;HE染色观察椎间盘软骨组织形态学变化;RT-qPCR检测TGF-β1 mRNA、BMP-7 mRNA、Smad2/3 mRNA、Smad1/5/8 mRNA、Smad4 mRNA及TIMP-3 mRNA的表达;ELISA法检测肿瘤坏死因子(TNF-α)、白介素-6(IL-6)、基质金属蛋白酶-3(MMP-3)水平;免疫组化检测椎间盘软骨组织TIMP-3表达;Western blot检测TGF-β1、BMP-7、Smad2/3、Smad1/5/8、BMPR-1A、ALK5、Sox9、COL2A1蛋白水平。结果与空白组及假手术组比较,模型组大鼠行为异常,椎间盘出现破裂,基质丢失,软骨细胞减少,软骨终板与髓核边界结构紊乱,Smad4 mRNA、TIMP-3 mRNA表达与TGF-β1、BMP-7、Smad2/3、Smad1/5/8 mRNA及蛋白、BMPR-1A、ALK5、Sox9、COL2A1蛋白表达、TIMP-3平均光密度值显著降低(P<0.05),TNF-α、IL-6、MMP-3水平显著升高(P<0.05)。与模型组比较,针刀组、塞来昔布组大鼠异常行为减少,椎间盘软骨组织损伤减轻,Smad4 mRNA、TIMP-3 mRNA表达与TGF-β1、BMP-7、Smad2/3、Smad1/5/8 mRNA及蛋白、BMPR-1A、ALK5、Sox9、COL2A1蛋白表达、TIMP-3平均光密度值显著升高(P<0.05),TNF-α、IL-6、MMP-3水平显著降低(P<0.05)。与针刀组比较,TGF-β/BMP抑制剂组大鼠异常行为及椎间软骨组织损伤加重;Smad4 mRNA、TIMP-3 mRNA表达与TGF-β1、BMP-7、Smad2/3、Smad1/5/8 mRNA及蛋白、BMPR-1A、ALK5、Sox9、COL2A1蛋白表达、TIMP-3平均光密度值显著降低(P<0.05),TNF-α、IL-6、MMP-3水平显著升高(P<0.05)。TGF-β/BMP激动剂组大鼠异常行为减少,椎间盘软骨组织损伤减轻;Smad4 mRNA、TIMP-3 mRNA表达与TGF-β1、BMP-7、Smad2/3、Smad1/5/8 mRNA及蛋白、BMPR-1A、ALK5、Sox9、COL2A1蛋白表达、TIMP-3平均光密度值显著升高(P<0.05),TNF-α、IL-6、MMP-3水平显著降低(P<0.05)。结论针刀干预可能通过激活TGF-β/BMP信号通路,抑制细胞外基质降解、抑制椎间盘组织内炎症,起到延缓椎间盘退变发展、达到治疗LDH的目的。

Objective To investigate the effects of needle knife interventions on intervertebral disc degeneration of rats with lumbar disc herniation(LDH)and the involvement of the Transforming growth factor-beta/bone morphogenic protein(TGF-β/BMP)signaling pathway.Methods The LDH rat model was established.Rats were randomly divided into blank group,sham operation group,model group,celecoxib group(0.34 g/kg/d celecoxib solvent),needle knife group,TGF-β/BMP inhibitor group(needle knife+100μL of SB431542 every 2 days),and TGF-β/BMP agonist group(needle knife+10 mg/kg SRI-011381 hydrochloride every 2 days),with 20 rats in each group.The general behavioral status of rats in each group was observed.Hematoxylin and eosin(H&E)staining was applied to observe the morphological changes in intervertebral disc cartilage tissue.Real-time reverse transcriptase-polymerase chain reaction(RT-qPCR)was applied to detect the mRNA expressions of TGF-β1,BMP-7,Smad2/3,Smad1/5/8,Smad4 and TIMP-3.Enzyme-linked immunosorbent assay(ELISA)was applied to detect relative levels of TNF-α,IL-6 and MMP-3.Immunohistochemistry was applied to detect the positive expression of TIMP-3 in intervertebral disc cartilage tissues.Western blot was applied to detect protein levels of TGF-β1,BMP-7,Smad2/3,Smad1/5/8,BMPR-1A,ALK5,Sox9 and COL2A1.Results Compared with those of blank group and sham operation group,rats in the model group presented abnormal behaviors,intervertebral disc rupture,matrix loss,decreased chondrocytes,disordered boundary between cartilage endplates and nucleus pulposus,significantly lower mRNA levels of Smad4 and TIMP-3,mRNA and protein levels of TGF-β1,BMP-7,Smad2/3 and Smad1/5/8,protein levels of BMPR-1A,ALK5,Sox9 and COL2A1 and mean optical density of TIMP-3(P<0.05),but significantly higher levels of TNF-α,IL-6 and MMP-3(P<0.05).Compared with those of model group,rats in needle knife group and celecoxib group presented reduced abnormal behaviors,alleviated intervertebral disc damages,significantly higher mRNA levels of Smad4 and TIMP-3,mRNA and protein levels of TGF-β1,BMP-7,Smad2/3 and Smad1/5/8,protein levels of BMPR-1A,ALK5,Sox9 and COL2A1 and mean optical density of TIMP-3(P<0.05),but significantly lower levels of TNF-α,IL-6 and MMP-3(P<0.05).Compared with those of needle knife group,rats in the TGF-β/BMP inhibitor group presented aggravated abnormal behaviors and intervertebral disc rupture,significantly lower mRNA levels of Smad4 and TIMP-3,mRNA and protein levels of TGF-β1,BMP-7,Smad2/3 and Smad1/5/8,protein levels of BMPR-1A,ALK5,Sox9 and COL2A1 and mean optical density of TIMP-3(P<0.05),but significantly higher levels of TNF-α,IL-6 and MMP-3(P<0.05).Rats in the TGF-β/BMP agonist group presented relieved abnormal behaviors and intervertebral disc rupture,significantly higher mRNA levels of Smad4 and TIMP-3,mRNA and protein levels of TGF-β1,BMP-7,Smad2/3 and Smad1/5/8,protein levels of BMPR-1A,ALK5,Sox9 and COL2A1 and mean optical density of TIMP-3(P<0.05),but significantly lower levels of TNF-α,IL-6 and MMP-3(P<0.05).Conclusion Needle knife interventions delay the development of intervertebral disc degeneration in LDH rats by activating the TGF-β/BMP signaling pathway,inhibiting extracellular matrix degradation,and relieving inflammation in intervertebral disc tissue.