| Objective:To clarify the competitive binding relationship of VASH1-AS1/let-7d-5p/SLC5A6 and its regulatory function on osteogenic and angiogenic expression,and then to explore whether Bushen Huoxue capsule uses the ce RNA competitive mechanism of VASH1-AS1/let-7d-5p/SLC5A6 to regulate osteogenic and angiogenic differentiation and play a role in the prevention and treatment of SONFH.At the same time,the VASH1-AS1/let-7d-5p/SLC5A6 regulatory axis and its function were verified in the femoral head samples of clinical ONFH patients after THA.Method:1.Double luciferase reporter gene detection was used to detect the luciferase activity after co-transformation of let-7d-5p with corresponding reporter gene plasmids of target genes VASH1-AS1 and SLC5A6,and to analyze the targeted binding relationship between molecules of VASH1-AS1/let-7d-5p/SLC5A6 regulatory axis.Furthermore,lentivirus was used to transfect overexpressed / interfered VASH1-AS1,and q PCR and Western blotting were used to detect the expression of ce RNA regulatory axis and its regulation on osteogenic angiogenesis of BMSCs after bi-directional intervention of VASH1-AS1.2.Dex was used to interfere with BMSCs to simulate the effect of hormone on cells in the progression of SONFH disease.At the same time,the drug-containing serum of Bushen Huoxue capsule of SD rats was used to interfere with BMSCs.The growth,proliferation and exercise ability of BMSCs were observed.The expression of m RNA and protein of osteogenic and angiogenic factors in cells was detected by q PCR and Westernblotting,and the expression of related molecules on the axis regulated by VASH1-AS1/let-7d-5p/SLC5A6.3.A SONFH rat model was constructed using LPS in combination with MPS,and low,medium and high doses of kidney-supplementing capsules were set up to intervene in the model rats.8 weeks later,the effects of kidney-supplementing capsules on the expression of VASH1-AS1/let-7d-5p/SLC5A6 regulatory axis,bone tissue repair and revascularization expression in SONFH model rats.4.Postoperative bone tissue specimens from ONFH patients who underwent THA surgery in our hospital from December 2021 to June 2022 were collected,and the samples were distinguished into hormone,alcohol,and trauma groups according to etiology and nadir criteria,while patients admitted for THA surgery for femoral neck fracture were included in the control group.q PCR was used to detect VASH1-AS1/ let-7d-5p/SLC5A6 regulatory axis and the expression of osteogenic and angiogenic related factors in the necrotic femoral head tissues of the corresponding groups were examined by q PCR.Results:1.The results of double luciferase reporter gene detection showed that the relative luciferase activity of let-7d-5p mimics cotransfected with SLC5A6 3’UTR WT vector and VASH1-AS13’UTR WT vector was significantly lower than that of their respective control groups,while the relative luciferase activity of let-7d-5p mimics cotransfected with SLC5A6 3’UTR MT vector and UTR NC vector had no significant difference compared with their respective control groups.2.After VASH1-AS1 overexpression / interference lentivirus transfection into BMSCs,the expression of mi RNAlet-7d-5p was negatively correlated with lnc RNAVASH1-AS1,while the expression of m RNASLC5A6 was positively correlated with VASH1-AS1,and the m RNA expression and protein expression abundance of osteogenic related factors were positively correlated with VASH1-AS1.The m RNA expression and protein expression abundance of angiogenic factors were positively correlated with VASH1-AS1 as a whole,but the m RNA expression of VEGFA decreased slightly in VASH1-AS1 overexpression group.3.CCK-8 results showed that Dex of 400ng/m L could significantly reduce the cell survival rate of BMSCs at 48 h.The drug-containing serum of different concentrations of Bushen Huoxue capsule had no cytotoxicity to BMSCs.High-and medium-dose Bushen Huoxue capsule drug-containing serum could promote the growth,proliferation and cell activity of normal BMSCs.High-dose Bushen Huoxue capsule drug-containing serum could reverse the adverse effect of Dex on the growth and proliferation of BMSCs.4.The results of cell scratch test showed that Dex could inhibit the movement ability of BMSCs,while different doses of Bushen Huoxue capsule containing serum could weaken or even eliminate this adverse effect and improve the movement and migration ability of BMSCs.5.The results of q PCR in vitro showed that under the influence of Dex,the expression of VASH1-AS1 and SLC5A6 in BMSCs showed a significant downward trend,and there was a positive correlation between them,while let-7d-5p significantly increased the corresponding expression.After the addition of Bushen Huoxue capsule containing serum,the above trend changed in the opposite direction.The m RNA expression trend of osteogenic and angiogenic factors was the same,which decreased significantly under the influence of Dex,but increased in varying degrees with the participation of drug-containing serum of Bushen Huoxue capsule.6.The results of Western blotting in vitro showed that after the intervention of Dex,the protein expression of OCN and v WF in BMSCs did not change significantly,while other osteogenic and angiogenic factors showed a significant downward trend.The expression of COL1A1 protein in low-dose group and low-middle dose group of Bushen Huoxue capsule was lower than that in control group and model group.In addition,different doses of Bushen Huoxue capsule drug-containing serum could increase the protein expression abundance of osteogenic and angiogenic factors in BMSCs under the intervention of Dex.7.Micro-CT results showed that after 8 weeks,the morphology of trabeculae in the subchondral zone of rats in the model group and different doses of Bushen Huoxue capsule group were destroyed in different degrees than that in the normal group,but the damage degree in the Bushen Huoxue capsule group was slighter than that in the model group,and the trabeculae were relatively intact and evenly distributed.BMD,BS/BV,BV/TV,Tb.Th and Tb.Sp in the model group showed significant reverse changes compared with the control group,while Bushen Huoxue capsule intragastric administration showed significant reverse changes in different degrees compared with the model group.There was no significant difference in BMD and BS/BV,BV/TV between the low dose group and the high dose group and the model group,but there was only statistical difference in Tb.Th between the high dose group and the model group.There was no statistical difference in Tb.N among all groups.8.The results of HE staining showed that there were no obvious pathological changes in the control group;the trabecular morphology of the femoral head in the model group was slightly disturbed,with a large number of empty bone sockets compared with the normal group;the pathological changes in the histological structure of the trabecular bone of the rats were significantly reduced and the rate of empty bone sockets was significantly lower than that of the model group.9.Immunohistochemical results showed that: hormones reduced the osteogenic and vascularization-related expression in the subchondral region of the femoral head of rats in the model group to different degrees;the Chinese herbal medicine of kidney tonifying and blood activation capsules could correct the negative response of osteogenic and vascularization-related factors to hormones,but the expression of RUNX2 in the gavage group of kidney tonifying and blood activation capsules was still lower than that in the model group,and there was no significant difference in v WF between the groups.10.In vivo q PCR results showed that the expression of VASH1-AS1 and SLC5A6 in the femoral head tissues of rats in the model group were both significantly decreased compared with the normal group,and the two showed positive correlation,but the expression of let-7d-5p was significantly elevated compared with the normal group,and the gavage group of Kidney Renal Activation Capsule was able to significantly reverse the changes of VASH1-AS1/let-7d-5p/ SLC5A6.The expression of m RNA of osteogenesis-related factors was reduced in the model group,but the expression of RUNX2 and OCN in the model group was improved by renal activation capsule;the expression of VEGFA among the angiogenesisrelated factors was significantly reduced in the model group,while the expression of v WF and CD31 were not significantly different between the model and control groups.The expression of VEGFA and CD31 were not significantly different between the model and control groups.11.The Western blotting results of in vivo experiment showed that different doses of Bushen Huoxue capsule could improve the decreasing trend of osteogenic and angiogenic factor protein expression in the femoral head of model rats.12.The observation results of clinical samples showed that the femoral head samples of patients with osteonecrosis of the femoral head were obviously deformed,the surface was rough,the weight-bearing area collapsed and the surface cartilage was seriously damaged,and the weight-bearing area collapsed significantly and the cartilage layer became thinner or even disappeared.or there are subchondral fracture lines,there are many necrotic areas and bone trabeculae are sparse,disordered arrangement,mostly wrapped by fibrous bands,it can be seen that a large area of yellow-white necrotic area is accompanied by many cystic necrosis foci.The results of this q PCR showed that osteonecrosis caused by different etiology could lead to low expression of VASH1-AS1 and SLC5A6 and high expression of let-7d-5p in femoral head tissue,and the expression of osteogenic and angiogenic factor m RNA in bone tissue of patients with osteonecrosis of femoral head decreased compared with the control group,and the pathogenicity of trauma was the most obvious.Conclusion:1.VASH1-AS1/let-7d-5p/SLC5A6 targeting interaction regulates the expression of osteogenic vessels in BMSCs.2.The medicated serum of Bushen Huoxue capsule regulates the expression of BMSCs osteogenesis and vascularization through VASH1-AS1/let-7d-5p/SLC5A6 axis.3.Bushen Huoxue capsule regulates the balance of osteogenic vascular differentiation in SONFH model rats through VASH1-AS1/let-7d-5p/SLC5A6 axis.4.The decreased expression of osteogenic and angiogenic factors in ONFH patients with osteonecrosis of the femoral head caused by hormone,alcohol,trauma and other factors is highly related to the expression of related molecules on the VASH1-AS1/let-7d-5p/SLC5A6 regulatory axis. |
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