| 1.Objective1.1 This study was performed to illustrate the potential relationship between reduced serum and local LncRNA MALAT1 expressions with disease severity in patients with non-traumatic osteonecrosis of femoral head(ONFH).1.2 To explore the effect of "Xuehai" and "Shenshu" activating blood and tonifying kidney method combined with Huoxue Shenggu Decoction on LncRNA MALAT1,miR-145 and HIF-1α in ONFH patients with "kidney deficiency" and "blood stasis" TCM differetiation.1.3 The current study investigated the role of MALAT1 on angiogenesis of bone microvascular endothelial cells(BMECs)isolated from steroid-induced ONFH.1.4 To investigate the effects of "Xuehai" and "Shenshu" moxibustion on promoting blood circulation and tonifying the kidney on the proliferation and migration of BMECs,MALAT1/miR-145,VEGFA and HIF-1α.2.Methods2.1 A total of 104 patients with non-traumatic ONFH and 100 healthy controls were consecutively recruited from our hospital.Serum and local LncRNA MALAT1 expressions were detected using RT-PCR.Radiographic progression was defined by FICAT classification.Clinical severity was evaluated by VAS and Harris hip score.Receiver operating characteristic(ROC)curve was carried out to determine the diagnostic value of MALAT1 in the radiographic progression.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.The patients were informed of the procedure method before treatment.The patients,they were divided into 45 cases in the observational group and 45 cases in the control group.The control group was given Huoxue Shenggu Decoction orally,1 dose a day.On the basis of taking traditional Chinese medicine,the observation group added "Shenshu" and "Xuehai" moxibustion once a day for 2 months.The pain and function of ONFH patients were evaluated by visual analog score(VAS)and Harris hip score(HHS)before and 2 months.In addition,the expressions of MALAT-1 and miR-145 in serum were detected by PT-PCR and VEGFA and HIF-1α in serum were detected by ELISA.2.3 40 corticosteroid-induced ONFH patients received THA were enrolled in our study.Expressions of MALAT1,miR-145 and HIF-1α were detected by qRT-PCR in affected necrosis tissue and non-affected normal tissue.Bone microvascular endothelial cells(BMEC)were isolated from 6 patients and treated with 0.1 mg/mL hydrocortisone to establish a GC-damaged model of BMECs.MALAT1 plasmid and miR-145 mimic were transfected into BMECs.BMEC proliferation was assessed using MTT assays.The migration ability of cells was detected by scratch-wound assays.Matrigel assay was performed to detect angiogenesis in vitro.Western blot assay was used to detect HIF-1α,VEGF-A and ANGPT2 expressions.FISH,RNA pull down,RIP and luciferase assay were carried out to determine the interaction of MALAT1,miR-145 and HIF-1α.2.4 We prepared "Shenshu" and "Xuehai" blood activating and kidney tonifying moxibustion serum.In the moxibustion serum group,the Xuehai and Shenshu of rats were treated with direct moxibustion.The small moxa cone(1.5mg/unit)was made of refined moxa velvet,2 units/time,once every other day(6 consecutive times),and the eyeball blood was taken to separate the serum.Except that the rats in the normal control group were not treated with moxibustion,the rest were treated with moxibustion serum group.MTT assay was used to detect the proliferation of BMECs,the migration of BMECs cells were detected by Transwell assat and wound scratch assay,and the angiogenesis in vitro was detected by matrix gel assay.MALAT1,miR-145,VEGF-A and HIF-1α were detected by RT-PCR.The expression of VEGFA and angpt2 proteins were also detected by Western Blot analysis.3.Results3.1 Serum LncRNA MALAT1 expressions were significantly lower in non-traumatic ONFH patients than in healthy controls(P<0.001).In addition,local MALAT1 expressions in non-traumatic ONFH tissue were significantly lower in the affected area than that in non-affected area(P<0.001).FICAT stage 4 has significantly lower serum and local LncRNA MALAT1 expressions in comparison with stage 3(P=0.007、0.02<0.001),FICAT stage 3 showed markedly decreased serum and local LncRNA MALAT1 expressions compared with stage 2(P=0.008、0.001<0.001).Serum and local LncRNA MALAT1 expressions were significantly and negatively associated with VAS(serum r=-0.381,P<0.001;local r=-0.449,P<0.001)and positively related to Harris hip score.Further ROC curve analysis indicated that serum MALAT1 may act as a decent indicator in the diagnosis of non-traumatic ONFH.3.2 Compared with baseline,the VAS score of the two groups decreased significantly at 2 months after treatment,and the Harris score improved significantly at 2 months after treatment（P<0.05).The VAS score decreased and Harris score increased in the observation group at the second month after treatment,which was significantly different from that in the control group(P=0.007、0.005<0.05).In addition,compared with baseline,the expression of MALAT1,VEGFA and HIF-1α increased significantly and the level of miR-145 decreased significantly(P<0.05).Serum MALAT,VEGFA and HIF-1α in the observation group were significantly increased in comparison with the control group(P=0.003,0.014,0.000<0.05),whereas the level of miR-145 decreased significantly(P=0.000<0.05).3.3 Compared with non-necrotic femoral head tissue,the expression of MALAT1 and VEGFA decreased significantly,while the expression of miR-145 increased significantly in necrotic tissue.The expression of MALAT1 and VEGFA in necrotic tissues was significantly negatively correlated with miR-145.Compared with the negative control group,malat1 overexpression increased BMECs activity,decreased apoptosis,increased total angiogenesis density,and significantly increased the number of migrated BMECs;On the contrary,when MALAT1 was silenced,BMECs activity decreased,apoptosis increased,total angiogenesis density decreased,and the number of migrated BMECs decreased significantly.In addition,MALAT1 overexpression promoted VEGFA,HIF-1 and ANGPT2 expressions in comparison with the negative control group.FISH,RNA pulldown,RIP and luciferase experiments determined that VEGFA was the target of miR-145 and could be regulated by MALAT1.3.4 Compared with the normal control serum group,the proliferation rate and migration number of BMECs in "Xuehai" and "Shenshu" moxibustion Huoxue Bushen serum group were significantly higher than those in the normal control serum group.In addition,the angiogenesis in moxibustion serum group was significantly higher than that in normal serum group(P=0.002<0.05).Finally,compared with the normal control serum group,the expression of MALAT1,VEGFA and HIF-1α in moxibustion serum group expression increased significantly(P=0.028,0.003,0.002<0.05),while miR-145 expression decreased significantly((P=0.002<0.05).4.Conclusion4.1 Decreased serum and local MALAT1 expressions may reflect disease severity in non-traumatic ONFH patients.4.2 "Tonifying kidney and activating blood" moxibustion method combined with Huoxue Shenggu decoction is better than simple Huoxue Shenggu Decoction in the treatment of ONFH patients with kidney deficiency and blood stasis.4.3 MALAT1 upregulates VEGFA/HIF-1α through competitive miR-145 and Pathway promotes the angiogenesis and proliferation of BMECs.4.4 Xuehai-Shenshu blood activating and kidney tonifying serum can promote the proliferation,migration and angiogenesis of BMECs. |
PDF Full Text Request