基于RAP1信号通路探讨右归丸延缓膝骨关节炎的软骨退变

Investigating Effect of Yougui Pill(右归丸)on Chondrocyte Apoptosis in Knee Osteoarthritis Model Rats Based on RAP1 Signaling

目的通过右归丸治疗膝骨关节炎(KOA)模型大鼠,观察大鼠关节软骨形态及检测右归丸对RAP1信号通路表达和大鼠血清中的炎性因子表达,探讨右归丸对RAP1信号通路的表达及防治KOA的作用机制,为治疗提供理论基础。方法72只SPF级SD大鼠(雌雄各半)随机数字表法分为假手术组12只和造模组60只。造模组采用改良Hulth法建立膝关节OA模型,假手术组接受假手术。将造模成功的大鼠分为模型组(n=11)、右归丸低剂量组(n=12)、右归丸中剂量组(n=12)、右归丸高剂量组(n=12)及塞来昔布组(n=12)。给药阶段模型组、假手术组给予0.9%氯化钠溶液;右归丸低剂量组为等效浓度的1/2为0.5 g/d、右归丸中剂量组等效浓度为1 g/d、右归丸高剂量组为等效浓度2倍为2 g/d,塞来昔布组配置成悬浮(等效浓度为0.0072 g/d)。驱赶7周后检测并比较各组大鼠软骨病理,血清TNF-α、IL-1β和IL-18含量,以及大鼠软骨组织中RAP1、RAF、MEK1/2、ERK1/2、CREB、MMP3、MMP13、ADAMTS、C-fos、C-myc和C-Jun的表达水平。结果各组大鼠软骨病理检测结果显示:假手术组大鼠软骨组织未见异常,模型组大鼠关节软骨部分区域出现明显缺损,右归丸低剂量组大鼠关节软骨表面磨损较模型组有所减轻,而中、高剂量组及塞来昔布组大鼠软骨表面可见侵蚀现象明显改善。右归丸对KOA大鼠血清中的IL-1β、TNF-α、IL-18含量与模型组相比,右归丸高、中、低剂量组和塞来昔布组在大鼠血清中表达显著降低,差异具有统计学意义。与软骨组织中RAP1-RAF-MEKl/2-ERKl/2-CREB信号通路活化水平检测结果显示:与假手术组比较,模型组大鼠软骨组织中RAP1、RAF、MEK1/2、ERK1/2、CREB、MMP3、MMP13、ADAMTS、C-fos、C-myc和C-Jun的表达水平均显著升高,差异有显著统计学意义(P<0.01)。结果提示,在KOA发生时,RAP1-RAF-MEKl/2-ERKl/2-CREB信号转导通路激活,介导软骨组织中炎症反应及细胞凋亡的发生,这与以往研究结果一致。右归丸干预后,软骨组织中RAP1、RAF、MEK1/2、ERK1/2、CREB、MMP3、MMP13、ADAMTS、C-fos、C-myc和C-Jun蛋白表达水平均显著降低,差异有显著统计学意义(P<0.01)。结论右归丸能够抑制KOA大鼠软骨组织中RAP1-RAF-MEKl/2-ERKl/2-CREB信号通路的活化,降低炎性因子表达,从而延缓软骨退变。

Objective To explore the treatment of knee osteoarthritis(KOA)model rats with Yougui Pill(右归丸),observe the morphology of rat articular cartilage,detect the effect of Yougui Pill on the expression of RAP1 signaling pathway and the expressions of inflammatory factors in the serum and explore the mechanism of Yougui Pill on the expression of RAP1 signaling pathway and the prevention and treatment of KOA so as to provide a theoretical basis for treatment.Methods Seventy-two SPF SD rats(half male and half female)were randomly divided into sham operation group(n=12)and modeling group(n=60).The KOA model was established by modified Hulth method in the model group,and sham operation group received sham operation.The rats were divided into model group(n=11),Yougui Pill low-dose group(n=12),Yougui Pill medium-dose group(n=12),Yougui Pill high-dose group(n=12)and celecoxib group(n=12).The model group and sham operation group were given 0.9%sodium chloride solution at the administration stage.The low-dose group of Yougui Pill was 1/2 of the equivalent concentration(0.5 g/d).The equivalent concentration of the medium-dose group of Yougui Pill was 1 g/d and the high-dose group of Yougui Pill was twice the equivalent concentration(2 g/d).Celecoxib group was configured as suspension(the equivalent concentration was 0.0072 g/d).After 7 weeks of driving,cartilage pathology,serum contents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-18(IL-18),and the expression levels of RAP1,RAF,MEK1/2,ERK1/2,CREB,MMP3,MMP13,ADAMTS,C-fos,C-myc and C-Jun in cartilage tissue were detected and compared in each group.Results Pathological examination results of cartilage in each group of rats showed that abnormalities were found in the cartilage tissue of rats in the sham operation group.There were significant defects in some areas of the joint cartilage of the model group rats.The surface wear in the Yougui Pill low-dose group was reduced compared to that of the model group.However,the erosion phenomenon on the cartilage surface of rats in the Yougui Pill medium-dose group,Yougui Pill high-dose group and the Western medicine group was significantly improved.Compared with those of the model group,the contents of IL-1β,TNF-αand IL-18 in Yougui Pill low-dose group,Yougui Pill medium-dose group and Yougui Pill high-dose group were obviously decreased with statistical significance.The detection results of RAP1 RAF MEKL/2-ERKL/2-CREB signaling pathway activation level showed that compared with those of the sham operation group,the expressions of RAP1,RAF,MEK1/2,ERK1/2,CREB,MMP3,MMP13,ADAMTS,C-fos,C-myc and C-Jun were significantly increased(P<0.01)with statistical significance.The results indicated that during the occurrence of KOA,the RAP1-RAF-MEKl/2-ERKL/2-CREB signaling pathway was activated,inflammation and cell apoptosis in cartilage tissue were mediated,which was consistent with the previous research results.After intervention with Yougui Pill,the expression levels of RAP1,RAF,MEK1/2,ERK1/2,CREB,MMP3,MMP13,ADAMTS,C-fos,C-myc and C-Jun proteins in cartilage tissue were significantly reduced(P<0.01)with statistical significance.Conclusions Yougui Pill can inhibit the activation of RAP1-RAF-MEK1/2-ERK1/2-CREB signaling Pathway in cartilage tissue of KOA rats,reduce the expression of inflammatory factors,and thus reduce chondrocyte apoptosis.