Base On MicroRNA-130a Targeting Gax To Explore The Effect Of Compound Xiebai Capsule On Vascular Remodeling In Pulmonary Arterial Hypertension

Objective:To investigate the effect of Compound Xiebai Capsule on vascular remodeling of pulmonary arterial hypertension based on MicroRNA-130a targeting Gax.Methods:1.Healthy male SD rats were randomly divided into 6 groups:normal control group,PAH model group,positive drug control group,compound Xiebai capsule low(CXCL),medium(CHCM)and high(CXCH)dose groups.Rats in the normal control group were intraperitoneally injected with normal saline,and rats in the other groups were intraperitoneally injected with Monocrotaline(MCT)(60 mg/kg)to establish PAH model.On the second day of modelling,intragastric administration was performed for 21 consecutive days.Normal control group and PAH model group rats received normal saline,while positive control group rats received nifedipine 0.02g/kg daily.Rats in CHCL group were given CXC 0.67g/kg daily,in CHCM group were given CXC 1.0g/kg daily,and in CHCH group were given CXC 1.32g/kg daily.PAH model was evaluated by observing hemodynamic parameters and right ventricular hypertrophy index.Hematoxylin and eosin staining and transmission electron microscopy were used to observe the pathological features and ultrastructure of lung tissue.The expression of PCNA protein was detected by immunohistochemistry.Western blot was used to detect the expression of PCNA,cytochrome C,Caspase-3 and Caspase-9.Real-time quantitative polymerase chain reaction analysis(RT-qPCR)was used to detect miR-130a and Gax mRNA expression in rat lung tissue.2.Culture primary rat pulmonary artery smooth muscle cells(PASMCs).PASMCs were cultured under hypoxia.Different concentrations of CXC extract was to intervene cells.Cell proliferation was detected at 6h,12h,and 24h respectively.Flow cytometry was used to detect the cell cycle and apoptosis rate of PASMCs after the intervention of CXC.Western blot was used to detect the expression of PCNA,bcl-2,caspase-3,caspase-8,caspase-9 and Gax,RT-qPCR was used to detect miR-130a and Gax mRNA expression.3.Using RNA interference technology,to interfere miR-130a.After transfection,PASMCs intervented with CXC extract,RT-qPCR was used to detect miR-130a and Gax mRNA expression,MTT method was to detect PASMCs cell proliferation.The double luciferase reporter gene detection test was used to verify whether Gax was a target gene of miR-130a.The RNA interference technology was used to silence the expression of Gax,and at the same time,the CXC extract was used to interfere with PASMCs.The expression of Gax mRNA was quantitatively detected by RT-qPCR,and the cell proliferation of PASMCs was detected by MTT method.Western blot was to detect expression of Gax and PCNA.Results:1.The mPAP and RVSP values in the PAH model rats were significantly higher than those in the control group(P<0.01),indicating that the PAH model has been successfully established.After treatment with CXC,the mPAP,RVSP and RVHI in each treatment group decreased significantly(P<0.01 or P<0.05).The results of H&E staining showed that after treatment with each group of CXC,the thickening of the pulmonary vessel wall was significantly reduced,the lumen was enlarged,and the lesions such as pulmonary inflammation and fibroplasty were also reduced accordingly.Transmission electron microscopy observation of the ultrastructure of lung tissue showed that,after treatment with CXC,the ultrastructure of lung tissue showed decreased proliferation of smooth muscle cells,decreased endothelial cells,improved mitochondrial structure,and decreased collagen fibers.The percentage of PCNA-positive cells in the PAH model group was significantly higher(P<0.01),the percentage of PCNA-positive cells in each treatment group was reduced to varying degrees.Western blot results showed that,after treatment,PCNA expression levels decreased to varying degrees,while Cytochrome C,Caspase3 and Caspase9 expressions increased to varying degrees(P<0.01 or P<0.05).The expression of miR-130a in rat lung tissues in the PAH model group increased significantly(P<0.01),and the expression of Gax mRNA decreased significantly(P<0.01).After CXC intervention,the expression of miR-130a in each group decreased significantly,and the expression of Gax mRNA increased significantly(P<0.01 or P<0.05).2.Rat primary pulmonary artery smooth muscle cell culture was identified.After 24 hours of hypoxia,the proliferation of PASMCs in the hypoxia group was most significant(P<0.01),the inhibitory effect of CXC appeared concentration dependent.Flow cytometry analysis showed that in hypoxia group the percentage of cells in the G1 phase of the hypoxia group was lower and the percentage of cells in the S phase was higher,the percentage of cells in the G1 phase of the CXC intervention groups increased and the percentage of S phase decreased.Compared with the normal group,the percentage of apoptotic cells in the hypoxia group was significantly reduced(P<0.01),after the intervention of CXC,the rate of apoptotic cells increased significantly.The expression of PCNA and bcl-2 protein was significantly higher in the hypoxia group(P<0.01).After drug intervention,the PCNA and bcl-2protein expression level in the drug intervention groups decreased significantly(P<0.01).The expression of caspase3,caspase8,caspase9 and Gax in the hypoxia group was significantly lower(P<0.01).After drug intervention,the level of caspase3,caspase8,caspase9 and Gax protein increased significantly(P<0.05 or P<0.01).The results of RT-qPCR showed that the expression of miR-130a in PASMCs in the hypoxia group increased significantly(P<0.01),and the expression of Gax mRNA decreased significantly(P<0.01).After intervention with CXC extract,the expression of miR-130a decreased significantly,and the expression of Gax mRNA increased significantly(P<0.05 or P<0.01).3.After transfection with miR-130a mimic,it promoted the proliferation of PASMCs,after transfection with miR-130a inhibitor,it inhibited the proliferation of PASMCs.RT-qPCR results showed that the expression levels of Gax mRNA and miR-130a showed opposite trends after transfection.MTT results showed that the proliferation level of PASMCs in the hypoxia group was significantly increased(P<0.01),After the CXC intervention,the proliferation level of hypoxia+med group,hypoxia+med+miR-130a mimic NC group,hypoxia+med+miR-130a inhibitor NC group,hypoxia+drug+miR-130a inhibitor group significantly decreased,and of the hypoxia+med+mimic inhibitor group was the most significant,but of the hypoxia+med+miR-130a mimic group significantly increased.The results of the double luciferase reporter gene experiment verified that Gax is a target gene of miR-130a,and verified at the protein level that miR-130a can target the inhibition of GAX protein expression in PASMCs,miR-130a has a direct targeting relationship with the Gax gene.Further,siRNA transfection was used to silence the expression of Gax.After intervention with CXC,the expression level of Gax mRNA of PASMCs in the hypoxia group was significantly reduced(P<0.01),the expression level of Gax mRNA in hypoxia+drug group,hypoxia+drug+siRNA NC group increased significantly,the level in hypoxia+med+Gax-siRNA group was lower than that in the hypoxia group.After transfected with si-Gax,the results of MTT cell proliferation experiments showed that the proliferation level of PASMCs in the hypoxia group was significantly increased(P<0.01).After CXC intervened in PASMCs,the proliferation level of the hypoxia+med group,hypoxia+med+si-NC group was significantly reduced.The proliferation level of the hypoxia+med+si-Gax group was significantly increased(P<0.01).Compared with the control group,PCNA protein levels in hypoxia group were significantly increased(P<0.01),Gax protein levels in hypoxia group were significantly decreased(P<0.01).After CXC intervened in PASMCs,compared with the hypoxia group,in the hypoxia+med group,hypoxia+med+si-NC group,the PCNA protein expression level was significantly reduced,the Gax protein expression level was significantly increased(P<0.05 or P<0.01).In hypoxia+med+si-Gax group,,Gax protein level was significantly decreased(P<0.01),PCNA protein level was significantly increased(P<0.05).Conclusion:1.CXC is effective for the treatment of PAH rats,which can improve its clinical symptoms,reduce pulmonary artery pressure and right ventricular pressure,and right heart hypertrophy index.CXC can improve pulmonary vascular remodeling,improve the ultrastructure of pulmonary tissues related to vascular remodeling,inhibit the expression of the PCNA,and promotes the expression of apoptosis-related proteins cytochrome C,Caspase-3,and Caspase-9,reduces the expression of miR-130a and increases the expression of Gax mRNA in the lung tissue.2.The rat primary pulmonary artery smooth muscle cells were successfully isolated and cultured.Compound Xiebai Capsule has the effects of improving proliferation,cell cycle and promoting apoptosis in hypoxia-cultivated PASMCs cells.It can reduce the expression of PCNA,bcl-2,and increase the expression of caspase-3,caspase-8,caspase-9,Gax.CXC can reduce the expression of miR-130a and increases the expression of Gax mRNA.3.Gax was the target gene of miR-130a.miR-130a can inhibit the expression of Gax mRNA,it can promote the proliferation of PASMCs cultured in hypoxia,and Gax can inhibit the proliferation of PASMCs cultured in hypoxia.CXC interfered with miR-130a to regulate Gax,and exerted inhibitory effect on proliferation of PASMCs.4.Compound Xiebai Capsule interferes with miR-130a to regulate Gax,inhibits pulmonary artery smooth muscle cell proliferation,and achieves the effects of improving pulmonary vascular remodeling and reducing pulmonary arterial hypertension.