Experimental Study Of Gubitongxiao Formula Regulating OPG/RANK/RANKL System In The Treatment Of Steroid-induced Necrosis Of The Femoral Head In Mice

Objective To observe the regulatory effect of Gubitongxiao recipe on OPG/RANK/RANKL system in mice with steroid-induced osteonecrosis of femoral head,in order to explore the efficacy of Gubitongxiao prescription in the treatment of steroid-induced osteonecrosis of femoral head.Method 1.To establish the animal model of steroid-induced osteonecrosis of the femoral head: 90 two-week-old Kunming male mice of SPF grade were randomly divided into two groups: model group(n = 78)and normal control group(n = 12).The model group was injected intraperitoneally with lipopolysaccharide LPS(1 mg/kg),followed by gluteal muscle injection of prednisolone acetate((PND)(100mg/kg)every 3 days until the 24 th day.The normal control group was injected with normal saline of the same dose.After the establishment of the model,two model mice were randomly selected for magnetic resonance detection to observe the osteonecrosis of the femoral head,and the blood of 24 mice in the two groups were taken to detect the levels of BALP and PINP by ELISA method,in order to observe whether the model of steroid-induced osteonecrosis of the femoral head was established successfully.2.Sixty mice who successfully established the model of steroid-induced osteonecrosis of the femoral head were randomly divided into treatment group(n = 20),positive control group(n = 20)and model group(n = 12).The positive control group was given Tongluo Shenggu capsule,the model group and the blank control group were treated with the same amount of normal saline for 12 weeks.After 12 weeks,all the mice were killed and the expression levels of OPG,RANK,RANKL,PLC? 2,CTSK and TRAP were determined by fluorescence quantitative analysis and Western blotting.Result 1.The body weight of mice in all the model groups was lower than that in the control group,and there were symptoms such as reduced food intake,curling up and less movement,messy and unsmooth body hair,and a few mice developed claudication.2.Magnetic resonance showed that the left hip of the model group showed high signal intensity;the contents of BALP and PINP in the two groups were measured by ELISA method,and the BALP and PINP in the model group were less than those in the normal group,and the difference was statistically significant.3.The results of RT-q PCR detection showed that the m RNA expression contents of OPG,RANKL,RANK,PLC?2,CTSK and TRAP in the model group were significantly different from those in the blank control group(p < 0.0l).The melting curve conformed to the unimodal characteristic,and the amplification curve conformed to the characteristic of proliferation curve,which was S-shaped,and the four periods were more obvious than those in the blank control group.Compared with the treatment group,the m RNA expression content of OPG and PLC?2 was significantly different(p < 0.05),the m RNA expression content of),RANKL,RANK,CTSK and TRAP was significantly different,and the m RNA expression content of OPG and m RNA was significantly different from that of the positive control group(p < 0.05),RANKL,RANK,PLC?2,CTSK,TRAP).Compared with the positive control group,there was no significant difference in the m RNA expression of OPG,RANKL,RANK,CTSK and TRAP between the treatment group and the positive control group,except for the content of PLC?2 m RNA.The content of OPG m RNA expression in the model group was the lowest,and that in the treatment group was lower than that in the other two groups,while the m RNA expression content of RANKL,RANK,PLC?2,CTSK and TRAP was the highest in the model group,higher in the treatment group than that in the positive control group,and the lowest in the blank control group.The results of WB detection showed that the protein expression contents of OPG,RANKL,RANK,PLC?2,CTSK and TRAP in the model group were significantly different from those in the blank control group(p < 0 01),and the protein expression levels of OPG,RANKL,PLC?2 and CTSK in the model group were significantly different from those in the treatment group(p < 0 05),RANK and TRAP protein expression).Compared with the positive control group,the expression of OPG,RANKL,RANK,PLC?2 and CTSK protein in the treatment group was significantly different from that in the positive control group(p < 0.05),while there was no significant difference in the protein expression content of OPG,RANKL,RANK,PLC?2,CTSK and TRAP between the treatment group and the positive control group.The expression of OPG protein in the model group was the lowest,and that in the treatment group and the positive control group was lower than that in the blank control group,while the expression of RANKL,RANK,PLC?2,CTSK and TRAP was the highest in the model group,and the RANK protein content in the treatment group was lower than that in the positive control group,while the protein contents of RANKL,PLC?2,CTSK and TRAP in the treatment group were higher than those in the positive control group,and the protein contents of RANKL,RANK,PLC?2,CTSK and TRAP in the blank control group were the lowest.Conclusion 1.The pathological model of SANFH can be successfully established by the method of lipopolysaccharide combined with hormone,and the pathological model is similar to that of patients with clinical overuse of corticosteroids,which can more directly observe the development of the course of SANFH and explore more efficient treatment methods.2.Gubitongxiao recipe can up-regulate the expression of OPG and inhibit the expression of RANKL/RANK,PLC?2,CTSK and TRAP.It can increase the number of OB,activate its function,decrease the number of OC or inhibit its function,and promote its apoptosis,increase bone formation and bone resorption in the body,so as to delay or even prevent the collapse of the femoral head,and there is no statistical difference between Gubitongxiao recipe and Tongluo Shenggu capsule.