抑制JAK/STAT信号通路可减轻激素诱导的MRL/lpr狼疮鼠股骨头坏死

The Role of the JAK/STAT Signaling Pathway in Steroid-induced Necrosis of Femoral Heads in MRL/lpr Iupus Mice

股骨头坏死(osteonecrosis of femoral head,ONFH)是系统性红斑狼疮(systematic lupus erythematosus,SLE)并发症之一,其发病机制复杂,治疗棘手,是SLE致残的主要原因。已有研究证实,JAK/STAT信号通路参与SLE的病理过程,而JAK/STAT激活也被发现与ONFH的发生有关。我们推测并证实,JAK/STAT信号通路在SLE-ONFH发生发展中发挥了重要作用。30只雌性MRL/lpr小鼠随机分为3组:模型组(脂多糖/24 h,2次+甲基强的松龙/24 h,3次)、对照组(加等量PBS)和治疗组(模型组+JAK1/2抑制剂巴瑞替尼/d,6周),每组各10只。比较各组小鼠抓力的结果表明,模型组小鼠在第4周与第6周时,抓力值较对照组明显减少(P<0.05);治疗组小鼠在第6周时,抓力值优于模型组(P<0.05)。造模第6周处死小鼠取双侧股骨头,观察股骨头形态及HE染色病理改变。结果表明,对照组小鼠股骨头呈球型,透亮,骨质坚硬,无软骨缺损;模型组小鼠股骨头呈不规则型,粗糙,色泽灰暗,股骨头有部分缺损;治疗组小鼠表现基本与模型组相似,总体股骨头外观形态较对照组不规则,色泽较对照组暗,股骨头有部分缺损,但其程度无模型组严重。模型组与治疗组小鼠空骨陷窝率较对照组明显升高(P<0.05);治疗组小鼠空骨陷窝率低于模型组(P<0.05)。通过Western印迹、ELISA和RT-qPCR检测,局部骨组织JAK/STAT通路(JAK1、JAK2、JAK3、STAT3)蛋白质表达、磷酸化水平、mRNA表达,血清及局部组织IL-6、TNF-α表达。结果表明,模型组小鼠骨组织IL-6、TNF-α和STAT3的mRNA表达显著高于对照组及治疗组(P<0.05),且模型组小鼠血清IL-6、TNF-α的含量较治疗组、对照组明显升高(P<0.05)。模型组的软骨分解代谢物ADAMTS-4、MMP-13及JAK/STAT通路相关蛋白质JAK1、p-JAK1、JAK2、p-JAK2、STAT3、p-STAT3均显著高于对照组及治疗组(P<0.05)。综上所述,JAK/STAT信号通路参与了MRL/lpr狼疮小鼠ONFH发病过程。选择性JAK1/2抑制剂可有效抑制ONFH炎症,改善骨结构及关节功能,并可能成为系统性红斑狼疮ONFH的有效治疗药物。

Osteonecrosis of femoral head(ONFH)is one of the complications of systemic lupus erythematosus(SLE),which is characterized by complex pathogenesis and difficult treatment,and is the main cause of SLE-induced disability.Previous studies have confirmed that the JAK/STAT signaling pathway is involved in the pathological process of SLE,and the activation of JAK/STAT has also been found to be related to the occurrence of ONFH.Therefore,we speculated that the JAK/STAT signaling pathway may play an important role in the occurrence and development of SLE-ONFH.30 female MRL/lpr mice were randomly divided into three groups:the model group(Lipopolysaccharide/24 hours,twice+Methylprednisolone/24 hours,3 times),the control group(equal amount of PBS)and the treatment group(the model group+JAK1/2 inhibitors Baricitinib/day,6 weeks),with 10 mice in each group.The results showed that the grip value of the model group was significantly lower than that of the control group at the 4 th and 6 th week(P<0.05),and that of the treatment group was better than that of the model group at the 6 th week(P<0.05).At the 6 th week,the mice were sacrificed to take the bilateral femoral head,and the morphology and HE staining pathological changes of the femoral head were observed.The results showed that the femoral head of the control group was spherical,transparent,hard and without cartilage defects,while that of the model group was irregular,rough,gray in color and partial defects of the femoral head.The performance of the mice in the treatment group was basically similar to that in the model group.And the overall appearance of the femoral head was more irregular than that in the control group.The color was darker than that in the control group,and there was a partial defect of the femoral head,but the degree was not as serious as that in the model group.The empty bone lacuna rate in the model group and treatment group were significantly higher than that in the control group(P<0.05),and the empty bone lacuna rate in the treatment group was lower than that in the model group(P<0.05).Western blotting,ELISA and RT-qPCR were used to detect the protein expression,phosphorylation levels,mRNA expression of the JAK/STAT pathway(JAK1,JAK2,JAK3,STAT3)in local bone tissues,and the expression of IL-6 and TNF-αin serum and local tissues.The mRNA expression of IL-6,TNF-αand STAT3 in bone tissues of the model group were significantly higher than that of the control group and treatment group,and the content of serum IL-6 and TNF-αof the model group were significantly higher than that of the treatment group and control group.The cartilage catabolic metabolites ADAMTS-4,MMP-13 and JAK/STAT pathway related proteins JAK1,p-JAK1,JAK2,p-JAK2,STAT3 and p-STAT3 in the model group were significantly higher than those in the control group and treatment group.In summary,the JAK/STAT signaling pathway is involved in the pathogenesis of ONFH in MRL/lpr lupus erythematosus mice.Selective JAK1/2 inhibitors can effectively inhibit ONFH inflammation,improve bone structures and joint functions,and may become an effective therapeutic drug for SLE ONFH.