淫羊藿苷通过核转录因子KappaB通路减轻大鼠椎间盘退变

Icariin Alleviates Intervertebral Disc Degeneration in Rats via the NF-κB Signaling Pathway

【目的】探讨淫羊藿苷对椎间盘退变大鼠的治疗作用及机制。【方法】将50只大鼠采用纤维环穿刺法建立椎间盘退变模型。将存活的48只大鼠随机分为模型组、淫羊藿苷组、激动剂组、激动剂+淫羊藿苷组,每组12只,另取10只作为假手术组。激动剂+淫羊藿苷组给予淫羊藿苷6.0 g/kg灌胃、尾静脉注射脂多糖5 mg/kg,淫羊藿苷组给予淫羊藿苷6.0 g/kg灌胃,激动剂组给予尾静脉注射脂多糖5 mg/kg,每日1次,连续2周。给药过程中,激动剂组和激动剂+淫羊藿苷组各死亡1只大鼠。给药结束后,酶联免疫吸附分析(ELISA)检测血清白细胞介素(IL)-6、IL-1β、肿瘤坏死因子α(TNF-α)含量,苏木素-伊红(HE)染色法观察椎间盘组织病理学改变,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测髓核细胞凋亡率,蛋白免疫印迹(Western Blot)法检测椎间盘组织核转录因子kappaB(NF-κB)p65、p-p65、B淋巴细胞瘤2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)的蛋白表达水平,免疫组织化学法检测椎间盘组织p-p65蛋白阳性表达。【结果】模型组大鼠椎间盘髓核与纤维环之间界限模糊、纤维环排列紊乱且存在缝隙,髓核细胞聚集、皱缩,基质减少;与模型组比较,淫羊藿苷组椎间盘纤维环结构和髓核形态及排列趋于正常;激动剂组大鼠椎间盘病变程度较模型组严重;激动剂+淫羊藿苷组大鼠椎间盘病变较激动剂组减轻。与假手术组比较,模型组血清IL-6、IL-1β、TNF-α含量,髓核细胞凋亡率,p-p65、Bax蛋白表达水平升高,Bcl-2蛋白表达水平降低(均P<0.05);与模型组比较,淫羊藿苷组IL-6、IL-1β、TNF-α含量,髓核细胞凋亡率,p-p65、Bax蛋白表达水平降低,Bcl-2蛋白表达水平升高(均P<0.05),而激动剂组IL-6、IL-1β、TNF-α含量,髓核细胞凋亡率,p-p65、Bax蛋白表达水平升高,Bcl-2蛋白表达水平降低(均P<0.05);与淫羊藿苷组比较,激动剂+淫羊藿苷组IL-6、IL-1β、TNF-α含量,髓核细胞凋亡率,p-p65、Bax蛋白表达水平升高,Bcl-2蛋白表达水平降低(均P<0.05);与激动剂组比较,激动剂+淫羊藿苷组IL-6、IL-1β、TNF-α含量,髓核细胞凋亡率,p-p65、Bax蛋白表达水平降低,Bcl-2蛋白表达水平升高(均P<0.05)。【结论】淫羊藿苷可改善椎间盘退变大鼠椎间盘病理损伤,减轻炎症反应,减少髓核细胞凋亡,其机制可能与抑制NF-κB信号通路有关。

Objective To investigate the therapeutic effects and mechanisms of icariin in rats with intervertebral disc degeneration(IDD).Methods An IDD model was established in 50 rats using the fibrous ring puncture method.The surviving 48 rats were randomly divided into the model group,the icariin group,the agonist group and the agonist+icariin group,12 rats in each group,and other 10 rats were used as sham-operation group.The agonist+icariin group was given icariin 6.0 g/kg by gavage and lipopolysaccharide 5 mg/kg by tail vein injection,while the icariin group was given icariin 6.0 g/kg by gavage and the agonist group was given lipopolysaccharide 5 mg/kg by tail vein injection.All the treatments placed once a day and lasted for 2 weeks.During administration,one rat died in the agonist group and the agonist+icariin group,respectively.After ending the medication,serum interleukin(IL)-6,IL-1βand tumor necrosis factorα(TNF-α)levels were measured by enzyme-linked immunosorbent assay(ELISA),intervertebral disc pathological changes were observed by hematoxylin-eosin(HE)staining,apoptosis rate of myeloid cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling(TUNEL),the protein expression levels of nuclear factor-kappaB(NF-κB)p65,p-p65,Blymphocytoma 2 gene(Bcl-2)and Bcl-2 associated X protein(Bax)in intervertebral disc tissues were detected by Western Blot method,the protein expression of p-p65 in intervertebral disc tissue was detected by immunohistochemistry.Results In the model group,the intervertebral disc nucleus pulposus and the fibrous ring were blurred,the arrangement of the fibrous ring was disorganized and there were gaps,the nucleus pulposus cells were aggregated and wrinkled,and the stroma was reduced;compared with the model group,the intervertebral disc fibrous ring structure and nucleus pulposus morphology and arrangement in the icariin group tended to be normal;the intervertebral disc lesion of rats in the agonist group was serious compared with that in the model group;the intervertebral disc lesions of rats in the agonist+icariin group were reduced compared with those in the agonist group.Compared with the sham-operation group,the expression levels of serum IL-6,IL-1βand TNF-αlevels,apoptosis rate of nucleus pulposus cells,p-p65 and Bax protein expression levels were increased,and the protein expression level of Bcl-2 was decreased in the model group(all P<0.05);compared with the model group,IL-6,IL-1βand TNF-αlevels,apoptosis rate of nucleus pulposus cells,protein expression levels of p-p65 and Bax were decreased and the protein expression level of Bcl-2 was increased in the icariin group(all P<0.05),and in the agonist group,IL-6,IL-1βand TNF-αlevels,myeloid apoptosis rate,protein expression levels of p-p65 and Bax were increased and protein expression level of Bcl-2 was decreased(all P<0.05);compared with the icariin group,IL-6,IL-1βand TNF-αlevels,myeloid apoptosis rate,the protein expression levels of p-p65 and Bax were increased and the protein expression level of Bcl-2 was decreased in the agonist+icariin group(all P<0.05);compared with the agonist group,the levels of IL-6,IL-1βand TNF-α,apoptosis rate of myeloid cells,protein expression levels of p-p65 and Bax were decreased and protein expression level of Bcl-2 was increased in the agonist+icariin group(all P<0.05).Conclusion Icariin could improve intervertebral disc pathological damage,reduce inflammatory response and decrease apoptosis of nucleus pulposus cells in rats with IDD,and the mechanism may be related to the inhibition of NF-κB signaling pathway.