| Objective:Western blotting was used to detect the general pathological morphology and the expression of osteogenic and angiogenic related proteins in the femoral head samples of patients with ONFH caused by different etiologies,which provided a theoretical basis and reference for the follow-up basic SONFH related experiments and clinical treatment;then observed the effect of Bushen Huoxue Capsule on GC related ONFH The effects of BMSCs treated by GC on osteogenesis and angiogenesis in SD rats were studied.The mechanism of Bushen Huoxue Capsule on osteogenesis,angiogenesis and hedgehog signal pathway related factors in animal and cell experiments was further studiedMethod:1.Collect the femoral head samples of patients with ONFH and femoral neck fracture who underwent THA operation in our hospital from December 2017 to December 2019,and select the femoral head that meets the standard to be classified into the control group,the idiopathic group,the alcohol group,the hormone group and the posttraumatic group according to the etiology,using western The protein expression of osteogenic and angiogenic indexes in necrotic bone tissues of different etiologies was detected by using blotting.2.Establish GC related ONFH rat model by injecting MPs into gluteus muscle of SD rats,and then give different doses of Bushen Huoxue Capsule to intervene.After 8weeks,evaluate the effect of Bushen Huoxue Capsule on bone tissue reconstruction and angiogenesis of GC related ONFH rats by histopathology observation,fluorescence quantitative PCR detection and Western blotting analysis.3.The BMSCs of SD rats were purified and amplified by adherent method,and the serum and DEX of different dosages of Bushen Huoxue Capsule were used for intervention culture.The growth status,proliferation ability and differentiation potential of BMSCs were observed,and cell scratch test,q PCR test and Western were used Meanwhile,the co culture system of RAOECs and BMSCs in SD rats was established.The angiogenesis and Transwell migration experiments of RAOECs after co culture were carried out to observe the changes of angiogenesis ability and cell activity under the intervention of serum containing Bushen Huoxue Capsule and DEX.Results:1.By observing the approximate morphology and coronal plane of each group’s femoral head samples,we found that the femoral head samples of ONFH patients with different etiologies had similar pathological manifestations in the approximate morphology and profile,and had similar histological characteristics of necrosis and repair Results: in osteogenesis,the expression of ALP protein in the trauma group was significantly lower than that in the control group,but there was no significant difference among the other groups(P > 0.05)There was no significant difference(P > 0.05)in the expression of I protein between the two groups,and the expression of Runx2 in the other groups was significantly lower than that in the control group(P < 0.05);compared with the control group,the expression of OPN protein in the hormone group and the trauma group was significantly lower(P < 0.05),but there was no significant difference between the two groups(P > 0.05);the expression of Runx2 in the control group was significantly higher than that in the other groups In terms of angiogenesis,the expression of CD31,VEGFA and KDR related marker protein in ONFH femoral head samples caused by different etiologies was significantly lower than that in the control group(P < 0.05);while the expression of v WF was not significantly different among the groups(P > 0.05);in terms of hedgehog signaling pathway,the expression of Shh protein in ONFH patients with different etiologies was significantly lower than that in the control group(P < 0.05)There was no significant difference between the two groups(P > 0.05);compared with the control group,there was a significant decrease in the protein expression of Gli1,a signal factor downstream of the signal pathway,in the alcohol group and the posttraumatic group(P < 0.05),but there was no significant difference between the idiopathic group and the hormone group(P > 0.05);the protein expression of Gli2,a signal factor,appeared in each group compared with the control group Significantly reduced(P < 0.05).2.After 8 weeks of intervention in SD rats,there was no significant difference in the general view of femoral head samples in each group.The results of HE staining showed that the ratio of bone trabecular area in CG group was significantly higher than that in mg group(P < 0.05),and the data of LDG,MDG and HDG were significantly higher than that in mg group(P < 0.05).The ratio of empty bone pit in rats was consistent with that in CG group,which was significantly higher than that in mg group(P < 0.05)Compared with CG group,ALP and colgen in mg group were significantly higher than those in mg group(P < 0.05)1.The expression of OPN and Runx2 decreased in different degrees(P < 0.05);the three groups of different dosages of Bushen Huoxue Capsule by gavage were significantly higher than that of mg group(P < 0.05);the expression intensity of CD31,VEGFA,KDR and v WF in femoral head of mg group was significantly lower than that of CG group(P < 0.05),while that of different dosages of Bushen Huoxue Capsule by gavage was significantly higher than that of mg group(P < 0.05)< The results of q PCR showed that: in osteogenesis,the m RNA expression of ALP,colgen I and Runx2 in CG group was significantly higher than that in mg group(P < 0.05);the expression of colgen I and Runx2 in LDG group was significantly higher than that in mg group(P < 0.05);the expression of ALP and colgen I in MDG group was significantly higher than that in mg group(P < 0.05);while the expression of colgen I in HDG group was significantly higher than that in mg group(P < 0.05)The m RNA expression of CD31,VEGFA and KDR in CG group was significantly higher than that in mg group(P < 0.05);the m RNA expression of VEGFA in LDG group was significantly higher than that in mg group(P < 0.05);the m RNA expression of CD31 and VEGFA in MDG group was significantly higher than that in mg group(P < 0.05);the m RNA expression of VEGFA in HDG group was significantly lower than that in mg group(P < 0.05);the m RNA expression of VEGFA in Hedgehog group was significantly higher than that in mg group(P < 0.05)In terms of signal pathway,the m RNA expression of Shh,Gli1 and Gli2 in CG group was significantly higher than that in mg group(P < 0.05);the m RNA expression of Gli1 and Gli2 in LDG group was significantly higher than that in mg group(P < 0.05);the m RNA expression of Shh,Gli1 and Gli2 in MDG group and HDG group was significantly higher than that in mg group(P < 0.05);Western blotting analysis showed that ALP and collegen related to osteogenesis were significantly higher in CG group 1.The expression of OPN and Runx2 in LDG group was significantly higher than that in mg group(P < 0.05);the expression of ALP,colgen I and OPN in MDG group was significantly higher than that in mg group(P < 0.05);the expression of ALP and colgen in HDG group was significantly higher than that in mg group(P < 0.05)The expression of CD31,VEGFA,KDR and v WF in CG samples was significantly higher than that in mg group,while the expression of VEGFA and KDR in LDG group was significantly higher than that in mg group(P < 0.05);the expression of VEGFA and KDR in MDG group was significantly higher than that in mg group(P < 0.05),while the expression of CD31 and v WF in HDG group was significantly lower than that in mg group(P < 0.05)KDR and v WF were significantly higher than mg group(P < 0.05);hedgehog signal pathway related Shh,Gli1 and Gli2 protein expression in CG group were significantly higher than mg group,but only Gli2 protein expression in LDG group was significantly higher than mg group(P < 0.05);Shh and Gli2 protein expression in MDG group were significantly higher than mg group(P < 0.05);HDG group was also significantly higher than mg group(P < 0.05)Only the expression of Gli2 protein was higher in mg group(P < 0.05).3.In this study,BMSCs of primary SD rats with high purity can be purified by adherent method;primary BMSCs can be successfully induced to differentiate into osteogenesis,lipogenesis and chondrogenesis;different concentrations of serum containing Bushen Huoxue Capsule can promote the proliferation of primary BMSCs,and can also significantly improve the proliferation activity of primary BMSCs treated by DEX;3weeks after osteogenesis induction,alizarin red staining shows that The results of staining in the low,middle and high dose groups of serum containing medicine of different concentrations of Bushen Huoxue Capsule were similar to those in the control group,which was significantly higher than that in the model group;the average width of scoring in the CG group and LDG,MDG and HDG groups was significantly lower than that in the DG group(P < 0.05)at 24 hours,and the trend at 48 hours was consistent with that at 24 hours;the content of serum containing medicine of Bushen Huoxue Capsule in different concentrations of BMSCs and Dex in SD rats were increased The number of vascular branches in CG,LDG,MDG and HDG groups was significantly higher than that in mg group(P < 0.05),while the total length of vascular branches in CG group was significantly higher than that in DG group(P < 0.05),while the total length of vascular branches in MDG and HDG group was significantly higher than that in DG group(P < 0.05)The number of migrating cells in the MDG group was significantly lower than that in the CG group(P < 0.05);the number of migrating cells in the MDG group was significantly higher than that in the DG group(P < 0.05);however,the number of migrating cells in the LDG and HDG groups was not significantly different from that in the model group(P > 0.05);the results of q PCR showed that the m RNA expression of Shh,Gli1 and Gli2 in the DG group was significantly lower than that in the CG group(P < 0.05);the expression of Shh,Gli1 and Gli2 in the drug containing serum was significantly lower than that in the CG group(P < 0.05)On the other hand,the m RNA expression of Shh,Gli1 and Gli2 in LG and Hg group was significantly higher than that in CG group(P < 0.05),while the m RNA expression of Shh and Gli2 in mg group was significantly higher than that in CG group(P < 0.05);on the intervention,compared with DG group,the m RNA expression of Shh,Gli1 and Gli2 in MDG group was significantly higher than that in HDG group(P <0.05),while that of Shh and Gli2 in LDG group was significantly higher(P < 0.05)In terms of osteogenesis,the m RNA expression of ALP,collegen I and Runx2 in DG group was significantly lower than that in CG group(P < 0.05);in terms of drug containing serum,the m RNA expression of ALP,collegen I and Runx2 in LG Group was significantly higher than that in CG group(P < 0.05),while the m RNA expression of ALP and Runx2 in Mg and Hg group was significantly higher than that in CG group(P< 0.05);in terms of intervention,the m RNA expression of ALP,collegen in MDG group was significantly higher than that in CG group(P < 0.05)The m RNA expression of I and Runx2 in LDG group was significantly higher than that in DG group(P < 0.05),while that of ALP and collagen in HDG group was significantly higher than that in DG group(P < 0.05)The m RNA expression of CD31,VEGFA and KDR in CG group was significantly higher than that in DG group(P < 0.05);the m RNA expression of CD31,VEGFA and KDR in LG,Mg and Hg group was significantly higher than that in CG group(P < 0.05);the m RNA expression of CD31,VEGFA and KDR in MDG and HDG group was significantly higher than that in DG group(P < 0.05),and the m RNA expression of VEGFA and KDR in LDG group was significantly higher than that in DG group(P < 0.05)The expression was significantly higher in DG group(P < 0.05)The results of blotting showed that: in the hedgehog signaling pathway,the expression of Shh,Gli1 and Gli2 in the DG group was significantly lower than that in the CG group(P < 0.05);the expression of Shh,Gli1 and Gli2 in the LG Group was significantly higher than that in the CG group(P < 0.05);the expression of Gli1 in the Mg and Hg groups was significantly higher than that in the CG group(P < 0.05);the intervention was also significant Compared with the DG group,the expression of Shh,Gli1 and Gli2 in LDG and MDG group increased significantly(P < 0.05);while in HDG group,only Gli2 protein expression was significantly higher than that in DG group(P < 0.05);in osteogenesis,the expression of ALP,collagen I,OPN and Runx2 protein in BMS in DG group decreased significantly(P < 0.05)compared with CG group;ALP,collagen in Hg group 1.The protein expression of OPN and Runx2 in mg group was significantly higher than that in CG group(P < 0.05);the protein expression of colgen I,OPN and Runx2 in LG Group was significantly higher than that in CG group(P < 0.05);the protein expression of colgen I and OPN in LG Group was significantly higher than that in CG group(P < 0.05);in intervention,MDG and HDG group were higher than that in DG group(P < 0.05)1.OPN and Runx2 protein expression levels were significantly increased in LDG group(P < 0.05),while ALP and collegen were significantly increased in LDG group(P < 0.05)The expression level of CD31,VEGFA,KDR and v WF in BMSCs of CG group was significantly higher than that of DG group(P < 0.05);in CD31 and VEGFA,the expression level of LG,Mg and Hg was significantly higher than that of CG group(P < 0.05),but there was no significant difference between KDR and v WF(P > 0.05)In terms of intervention,compared with DG group,LDG and MDG group significantly increased the protein expression level of CD31,VEGFA,KDR and v WF(P < 0.05),while HDG group significantly increased the protein expression level of CD31,KDR and v WF(P < 0.05).Conclusion:1.Through the analysis of clinical necrosis of femoral head samples,this study preliminarily explored the primary mechanism of ONFH caused by different causes.2.Bushen Huoxue Capsule can promote bone remodeling and angiogenesis of GC related ONFH SD rats through hedgehog signaling pathway.3.Different concentrations of serum containing Bushen Huoxue Capsule can regulate and promote the differentiation of BMSCs to osteogenic and angiogenic direction in SD rats through hedgehog signal pathway. |
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