Study On The Mechanism Of Bushen Huoxue In Regulating MAPK Signaling Pathway In Rabbits With Femoral Head Necrosis Based On Four Routes Of Administration

Objective:Observe the effects of four different administration routes of Huogu perfusion solution on rabbits with steroid-induced femoral head necrosis,and explore its regulatory mechanism for MAPK signaling pathway,which provides a theoretical basis for the further development and application of the Huogu perfusion in the treatment of steroid necrosis of the femoral head.Method:Male New Zealand white rabbits were randomly divided into two groups:blank group(15),model group(75).The animal model of steroid-induced femoral head necrosis in the model group was randomly divided into 5 groups:auricular vein group(E),hip injection group(G),gavage group(W),external application group(F),model group(M)and each with 15 animals.Matsui model was used to replicate the steroid-induced osteonecrosis of the femoral head.The last abdominal cavity Methylprednisolone Sodium Succinate for Injection began treatment after 4 weeks.The blank group(K)was fed normally without any medication.The auricular vein was given to the Huogu perfusion 10mg·kg-1,twice a week.Hip injection of Huogu perfusion solution 0.70ml/times/hip.Gavage group was given 7.8mg.kg-1 of Huogu perfusion,once daily.The external application group was given a transdermal patch on the inner side of the upper part of both hindlimbs,7.5g/stick each time,Once every 3 days.After 12 weeks of treatment,the pathological and imaging changes of the femoral head were observed by HE,electron microscopy and MRI.Serum levels of TG and TC were tested by automatic biochemical analyzer.Serum levels of IL-1β,IL-6,IL-8,TNF-α,AP-1 and TRAP-5b were tested by ELISA.Immunohistochemical method was used to test the expression of p38,ERK and JNK in femoral head.Western blot and Real-time PCR were used to test p38,ERK,JNK,TRAF6,MyD88 protein and gene expression in the femoral head.Result:1.Expression of TG and TCThe contents of TG and TC in the model group were higher than those in the normal group,and there was a significant difference between the normal group and the normal group(P<0.01).In the other four ways of administration,the content of TG and TC in group G was significantly lower than that in group F,W and E,and there were significant differences compared with group F,W group and E group(P<0.05 or P<0.01).The content of TG and TC in the model group had an increasing trend compared with the normal group,and there was a significant difference compared with the normal group(P<0.01).In the other four routes of administration,the TG and TC levels in the G group were significantly lower than those in the F group.Group,W group and E group were significantly different from F group,W group and E group(P<0.05 or P<0.01).2.HEThe surface of the femoral head is smooth in the normal group,subchondral vascular plentiful,arrangement of bone cells at all levels,normal bone cell morphology,a small amount of bone lacunae can be seen,bone trabecula in large arrangement,uniform-distribution and abundant hematopoietic cells in bone marrow.Surface defect of femoral head in model group,disarranged osteoblasts in subchondral bone,bone cell atrophy,nuclear pyknosis,a significant increase in bone lacunae,bone trabecula thinning,disorder of arrangement,obvious enlargement of the bone marrow cavity,a significant increase in intraluminal adipocytes,significant reduction in hematopoietic cells.The above pathological changes in various administration groups were improved in varying degrees.The arrangement of bone cells at all levels of the femoral head is regular,bone lacunae have a degree of reduction,trabecular bone becomes thicker,more regular arrangement,decrease of fat cells in bone marrow,hematopoietic cell increase.Among them,the improvement of group G was the most obvious,followed by F,W and E.3.ELISA test resultsThe levels of IL-1β,IL-6,IL-8,TNF-α,AP-1 and TRAP-5b in the model group were significantly higher than those in the normal group,which compared with the normal group,P<0.01.Compared with model group,four different routes of administration,the levels of IL-1β,IL-6,IL-8,TNF-α,AP-1,and TRAP-5b proteins decreased to varying degrees.Among them,the improvement of group G was the most obvious,followed by F,W and E(P<0.01).4.The expression of p38,ERK and JNK proteinsImmunohistochemical results showed:The content of p38,ERK and JNK protein in the model group increased significantly,there is a statistical significance compared with the normal group(P<0.01).Compared with model group,four different routes of administration,the protein content of p38,ERK,and JNK decreased to varying degrees.Among them,the improvement of group G was the most obvious,followed by F,W and E(P<0.01).5.p38,JNK,ERK,TRAF6,MYD88 proteinsWestern blot results show:The contents of ERK,p38,JNK,TRAF6 and MyD88 in model group increased significantly.Compared with normal group,it has statistical significance(P<0.01).There are significant differences from the normal group(P<0.01).Compared with model group,four different routes of administration,the levels of ERK,p38,JNK,TRAF6 and MyD88 proteins were decreased to varying degrees.Among them,the improvement of group G was the most obvious,followed by F,W and E(P<0.01).6.p38,JNK,ERK,TRAF6,MYD88 genesReal-time PCR results show:The ERK,p38,JNK,TRAF6 and MyD88 gene levels in the model group increased significantly.Compared with normal group,it has statistical significance(P<0.01).There are significant differences from the normal group(P<0.01).Compared with model group,Four different routes of administration.The levels of ERK,p38,JNK,TRAF6 and MyD88 genes were decreased to varying degrees.Among them,the improvement of group G was the most obvious,followed by F,W and E(P<0.01).In conclusion:1.Four different routes of administration,treatment of rabbit SONFH with injection of Huogu perfusion into hip joint cavity is the best.2.Four different routes of administration have different degrees of inhibition on IL-1β,IL-6,IL-8,TNF-α,AP-1 and TRAP-5b expression in SONFH.The method of hip joint injection is the most obvious improvement of inflammation.3.Four different routes of administration have different inhibitory effects on ERK,p38,JNK,TRAF6,MyD88 protein and gene expression in SONFH.In summary,we hypothesized that Huogu perfusion solution may regulate the expression of inflammatory cytokines through MAPK and its downstream ERK/p38/JNK signaling pathway,it improve hormone-induced pathological changes of the femoral head,so as to treat SONFH.Four different routes of administration,the best treatment effect by injection of the hip joint cavity.