The Mechanism Of PACAP Regulating MiR-132/VEGF Axis In The Prevention And Treatment Of Steroid Induced Osteonecrosis Of The Femoral Head With Huoxue Shenggu Decoction

1.Objective1.1 To explore whether there is a correlation between the expression of serum PACAP and the severity of disease in patients with non-traumatic ONFH1.2 To investigate the effect of Huoxue Shenggu Decoction on serum PACAP level,VEGF level and miR-132 in steroid-induced ONFH patients of kidney deficiency and blood stasis type1.3 To explore whether PACAP can promote angiogenesis differentiation of bone microvascular endothelial cells by upregulating miR-132/VEGF axis in vitro1.4 To explore whether the serum containing Huoxue Shenggu decoction can promote the angiogenesis differentiation of microvascular endothelial cells by regulating the PACAP/miR-132/VEGF axis in vitro2.Methods2.1 A total of 102 non-traumatic ONFH patients and 95 healthy controls were enrolled in this study.Serum PACAP levels were examined using enzyme-linked immunosorbent assay(ELISA).Serum levels of the ONFH marker IL-33 and β-CTX were also detected.Radiographic progression of ONFH was detected using the Association Research Circulation Osseous(ARCO)classification system.Harris hip score(HSS)and visual analogue scale(VAS)were analyzed to understand clinical severity.ROC curve analysis was performed to investigate the potential diagnostic value of serum PACAP in the radiographic progression of ONFH.2.2 A total of 90 patients with femoral head necrosis diagnosed as“kidney deficiency and blood stasis”by TCM differentiation were collected in our hospital.According to the principle of random control,they were divided into 45 cases treatment group and 45 cases control group.The control group was treated with traditional Chinese medicine for promoting blood circulation and removing blood stasis.The observation group took Huoxue Shenggu Decoction orally on the basis of external application of traditional Chinese medicine.VAS score and HHS score were used to evaluate the pain and function of ONFH patients before and 6 months after treatment;Serum miR-132 expression was detected by PT-PCR and serum VEGF and PACAP protein expression was detected by ELIS A.2.3 30 patients with steriod-induced ONFH who underwent total hip replacement were selected.The expressions of PACAP mRNA,miR-132 and VEGF mRNA in necrotic tissue of femoral head and adjacent non necrotic normal tissue were detected by qRT-PCR.BMECs were isolated from the femoral heads of 6 ONFH patients and treated with 0.1mg/ml hydrocortisone to establish a hormone injury model.PACAP dosing and miR-132 inhibitor were used for cell transfection respectively.MTT assay was used to detect the proliferation of BMECs,Transwell assay and wound scratch test were used to detect the migration ability of cells.Matrix gel assay was used to detect the angiogenesis in vitro,and Western blot assay was conducted to detect the expression of angiogenesis markers VEGF and HIF-1α expressions.2.4 The low dose and high dose serum containing Huoxue Shenggu decoction was prepared and added to the BMECs injury model group.The proliferation of BMECs was detected by MTT assay,the migration of BMECs was detected by Transwell cell and scratch test,and the angiogenesis in vitro was detected by matrix gel method.RT-PCR was used to detect the expressions of miR-132 and PACAP mRNA,and WB was used to detect the expression of VEGF and HIF-1α protein.3.Results3.1 Serum PACAP levels were significantly decreased in non-traumatic ONFH patients when compared to healthy controls.ARCO stage 4 showed markedly lower serum PACAP levels than stage 3.ARCO stage 3 had significantly decreased serum PACAP levels compared to stage 1/2.Serum PACAP levels were negatively correlated with the ARCO classification.In addition,serum PACAP levels were positively associated with HSS score and negatively related to VAS score.Last,PACAP levels were negatively correlated with serum IL-33 and positively associated with β-CTX.ROC curve analysis indicated that PACAP may serve as an early indicator for the radiographic progression of ONFH.3.2 Compared with baseline,the VAS score of the two groups decreased significantly at 6 months after treatment,and the Harris score improved significantly at 6 months after treatment.The VAS score decreased and Harris score increased in the observation group at the six months after treatment,which was significantly different from that in the control group.In addition,compared with baseline,the expression of PACAP,VEGF and miR-132increased significantly.Serum PACAP,VEGF and miR-132 expressions in the observation group were significantly increased in comparison with the control group.3.3 miR-132,PACAP mRNA,and VEGF mRNA expression were significantly decreased in necrotic femoral head tissues compared with adjacent non-necrotic femoral head tissues.The expressions of miR-132,PACAP mRNA and VEGF mRNA were positively correlated with each other in femoral head necrosis.Compared with the control group,the expressions of miR-132,PACAP mRNA and VEGF mRNA,the activity of BMECs,the density of total angiogenesis,and the number of migrating BMECs were significantly reduced in the GC damaged model.When PACAP was added,the viability of BMECs,the density of total angiogenesis,and the number of migrated BMECs and the expression of miR-132 also increased significantly.When miR-132 inhibitor was added,BMECs activity,total vessel formation density,and the number of migrated BMECs also significantly decreased.In addition,PACAP promoted vascular markers VEGF and HIF-la protein expressions.On the other hand,VEGF and HIF-1α expressions were significantly decreased when adding miR-132 inhibitor.3.4 Compared BMECs injury group,the proliferation rate and migration number of bmecs in the Huoxue Shenggu decoction containing low dose and high dose serum group increased significantly.In addition,the blood vessel formation in the Huoxue Shenggu decoction containing serum group was significantly higher than that in the BMECs injury group.Compared with the BMECs injury group,the expression of miR-132,PACAP mRNA and VEGF mRNA in the Huoxue Shenggu decoction containing serum group were significantly increased.Western blot experiments showed that VEGF and HIF-1α protein expressions in the Huoxue Shenggu decoction containing serum group were significantly higher than that in the BMECs injury.4.Conclusion4.1 Reduction of serum PACAP expression in patients with nontraumatic ONFH may reflect the severity of the disease.4.2 The effect of external application of traditional Chinese medicine for promoting blood circulation and removing blood stasis combined with Huoxue Shenggu Decoction on ONFH patients with kidney deficiency and blood stasis is better than that of external application of traditional Chinese medicine only.4.3 PACAP promotes the proliferation,migration and angiogenesis of BMECs in vitro through miR-132/VEGF axis.4.4 The serum containing Huoxue Shenggu decoction can promote the proliferation,migration and angiogenesis of BMECs in vitro possibly through PACAP/miR-132/VEGF axis.