| Background:Osteonecrosis of the femoral head（ONFH）is a common bone and joint disease.The etiology of ONFH can be divided into two categories:traumatic and non-traumatic ONFH.Various systemic diseases are usually related to non-traumatic ONFH.These diseases include systemic lupus erythematosus（SLE）,vasculitis,Raynaud’s phenomenon,thrombophlebitis and so on[1-6].Studies have reported that the incidence of ONFH in patients with SLE is between 4%-15%[7].Patients with SLE often use glucocorticoids,so the incidence of ONFH is higher.ONFH is one of the important causes of disability in SLE patients,which seriously affects the quality of life of patients,and there is no effective treatment drug at present.The pathogenesis of ONFH is complex.At present,some studies believe that ONFH is due to the imbalance between osteoblast osteogenesis and osteoclast bone resorption[8].Janus kinase（JAKs）is a non-receptor tyrosine protein kinase family,which plays a pivotal role in the signaling process of more than 50 cytokines or hormone receptors.The inflammatory cytokines transmitted by JAK/STAT signaling pathway play a very important role in the formation of osteoclasts[9].At present,it has been proved that the activation of JAK/STAT signaling pathway is involved in the occurrence of ONFH under the stimulation of various inflammatory cytokines[10].However,there is no study on the role of JAK/STAT signaling pathway in SLE-ONFH,so this study intends to establish ONFH model in MRL/lpr mice with lupus background to explore the role of JAK/STAT signaling pathway in SLE-ONFH.Purpose:（1）To establish SLE-ONFH model with MRL/lpr lupus mice;（2）To explore whether JAK/STAT signaling pathway is involved in the occurrence of SLE-ONFH,so as to provide a therapeutic target for clinical treatment of SLE-ONFH.Method:（1）Thirty MRL/lpr female lupus mice aged about 15 weeks were randomly divided into groups A,B and C with 10 mice in each group according to random number table method.Group A was the control group,group B was the model group,and group C was the treatment group.（2）Mice in group B were injected with lipopolysaccharide（LPS）intraperitoneally at a dose of 10μg/kg twice,with an interval of 24 hours each time.24 hours after the second injection of LPS,methylprednisolone（MP）was injected intramuscularly at a dose of 20mg/kg for three consecutive injections with an interval of 24 hours each time[11].Mice in group A were injected with the same dose of normal saline at the same site.Mice in group C were given intragastric administration of baricitinib（10mg/kg）on the basis of the modeling method of group B,and baricitinib was dissolved in DMSO,once a day,for 6 weeks,and a total of 6 weeks were observed.（3）Six weeks later,the mice were sacrificed by neck removal.and the degree of ONFH in MRL/lpr lupus mice was judged by observing the appearance of bilateral femoral heads and HE staining of bilateral femoral heads.The expression levels of STAT3m RNA in the JAK/STAT signaling pathway and the m RNA levels of inflammatory cytokines IL-6 and TNF-αwere detected by RT-q PCR.The protein expression levels of JAK1,p-JAK1,JAK2,p-JAK2,STAT3,p-STAT3,ADAMTS-4 and MMP-13 were detected by WB,and the contents of IL-6 and TNF-αin the serum of mice were detected by ELISA.Result:（1）The results of grasping force showed that compared with group A,the grasping strength of mice in group B was significantly decreased at the 4th and 6th week（P<0.05）.At the 6th week,there was significant difference in grasping force between group B and group C（P<0.05）.（2）the general appearance of femur showed that the femoral head of mice in group A was spherical,transparent,with hard bone and no cartilage defect;In group B,the femoral head of mice was irregular,rough,dark in color and partly defective.The performance of mice in group C is basically similar to that in group B,but the overall appearance of femoral head is irregular and darker than that in group A,and the femoral head has some defects,but the degree is not as serious as that in group B.（3）the histomorphology results of HE staining showed that the bone trabecular structure of mice in group A was complete,bone cells were clearly visible,and A few empty bone lacunae were occasionally seen.The structure in the bone marrow was normal,and the cartilage tissues were arranged neatly without fibrosis and hyperplasia.In group B,the distribution of bone trabecula was irregular,thin,broken and disordered.Bone cells and chondrocytes were rare,empty bone lacunae were significantly increased,and a small amount of fibrous tissue hyperplasia was observed on the surface of cartilage and around bone trabecula.The trabecular structure of mice in group C was relatively complete,few fractures were found,and the structure arrangement was orderly.The number of empty bone lacunae in group C was significantly reduced compared with that in group B.After HE staining,the rate of empty bone lacunae in each group was calculated under A microscope and statistically analyzed.It was found that the rate of empty bone lacunae in groups B and C was significantly higher than that in group A,and the difference was statistically significant（P<0.05）.The difference between groups B and C was statistically significant（P<0.05）.（4）ELISA results showed that the contents of IL-6 and TNF-αin serum of mice in groups B and C were significantly higher than those in group A,and the difference was statistically significant（P<0.05）.There were significant differences in the contents of IL-6 and TNF-αbetween group B and C（P<0.05）.（5）The results of WB experiment showed that the expression of ADAMTS-4 and MMP-13 were increased in group B,and the difference was statistically significant compared with group A（P<0.05）,the expression of ADAMTS-4 and MMP-13 was decreased after intragastric treatment with baricitinib,an inhibitor of JAK/STAT signaling pathway,which was statistically different from group B（P<0.05）;JAK/STAT pathway related proteins JAK1,p-JAK1,JAK2,p-JAK2,STAT3 and p-STAT3 were significantly different between group A and group B（P<0.05）.The protein expressions of JAK1,P-JAK1,JAK2,P-JAK2 and STAT3 in group B and group C were significantly different（P<0.05）.（6）The results of RT-q PCR showed that the m RNA expressions of IL-6,TNF-αand STAT3 in group B were significantly increased compared with group A,and the difference was statistically significant（P<0.05）.The m RNA expressions of IL-6,TNF-αand STAT3in B and C groups were significantly different（P<0.05）.Conclusion:（1）the ONFH model of MRL/lpr lupus mice was successfully established in this experiment;（2）It is confirmed that JAK/STAT signaling pathway is involved in the pathogenesis of ONFH in MRL/lpr lupus mice.JAK inhibitor can block JAK-STAT signaling pathway,down-regulate the levels of IL-6 and TNF-αinflammatory cytokines,and reduce the inflammatory response,which may be a new treatment for SLE-ONFH. |
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