| Coronary heart disease(CHD)is a major disease in our country with high morbidity and mortality,which seriously threatens human health.Professor Wang Jie found in many years of clinical practice that CHD mostly occurs in middle-aged and elderly people over 40 years old.On the basis of blood stasis syndrome,patients often have symptoms such as weak waist and knees,dizziness,tinnitus and forgetfulness.And patients often have clinical manifestations of kidney deficiency and blood stasis(KD&BS).Using the method of Bushen Huoxue could significantly improve the symptoms and the clinical efficacy of patients.However,the mechanism of Traditional Chinese Medicine(TCM)for Bushen Huoxue in the treatment of CHD is still unclear,and the scientific mechanism is still unclear.Therefore,this study aims to reveal the mechanism of Bushen Huoxue Decoction(BSHXD)in the treatment of unstable angina pectoris of CHD with KD&BS syndrome through clinical research,transcriptome high-throughput sequencing and cell experiments.Objective:To evaluate the efficacy and safety of BSHXD in treating unstable angina pectoris of CHD with KD&BS syndrome.Transcriptome high-throughput sequencing and qRT-PCR technology were used to screen and verify the targets of BSHXD.To investigate the mechanism of BSHXD in inhibiting H2O2 induced apoptosis of human umbilical vein endothelial cells(HUVEC)and human kidney proximal tubular epithelial cells(HK-2)by lncRNANEAT1/miR-139-5p/Fos axis.Methods1.A randomized,double-blind,placebo-controlled clinical study of BSHXD on unstable angina pectoris of CHD with KD&BS syndrome.A total of 80 patients with unstable angina pectoris of CHD with KD&BS syndrome were included and randomly divided into the treatment group and the control group.The treatment group was given BSHXD plus conventional western medicine treatment,and the control group was given placebo plus conventional western medicine treatment.The course of treatment was 4 weeks and the follow-up was 6 months.The general clinical data of the two groups of patients,frequency and duration of angina pectoris,TCM syndrome scores,single symptom and sign scores,seattle angina pectoris(SAQ),blood lipid level,high-sensitivity c-reactive protein(hs-CRP),blood routine,liver and kidney function,coagulation function,urine routine,stool routine,electrocardiogram and other safety indicators were observed before and after treatment.And the occurrence of major adverse cardiovascular events(MACEs)in patients within 6 months was also observed.2.Transcriptome high-throughput sequencing screening and qRT-PCR verification of the targets of BSHXD for the treatment of CHD with KD&BS syndrome.Five patients with good clinical efficacy were selected from the treatment group of BSHXD,and the significant differences in lncRNA,miRNA and mRNA in patients of CHD with KD&BS syndrome before and after treatment with BSHXD were analyzed by using transcriptome next-generation high-throughput sequencing.Then the competitive endogenous RNA(ceRNA)regulatory network was constructed by using the method of bioinformatics analysis.At the same time,another 20 patients were selected from the treatment group of BSHXD,and qRT-PCR technology was used to verify the regulatory effect of BSHXD on the lncRNA-miRNA-mRNA axis.Besides,ELISA technology was used to detect the expression of Fos protein in plasma before and after treatment.3.The mechanism of BSHXD regulating lncRNA NEAT1/miR-139-5p/Fos axis against H2O2-induced apoptosis of HUVEC and HK-2.The target-binding relationship between lncRNA NEAT1 and miR-139-5p,and between miR-139-5p and Fos was detected by dual-luciferase reporter gene assay.H2O2-induced HUVEC and HK-2 cells were used to construct an oxidative stress injury model.The cells were divided into six groups,group A:HUVEC control group;group B:HUVEC H2O2 model group;group C:HUVEC H2O2+BSHXD treatment group;group D:HK-2 control group;group E:HK-2 H2O2 model group;group F:HK-2 H2O2+BSHXD treatment group.CCK8 method was used to detect the effect of BSHXD on the proliferation of cells in each group.Flow cytometry was used to detect the expression of ROS and apoptosis of cells in each group.Tunel staining was used to analyze the apoptosis of cells in each group.The expression levels of lncRNA NEAT1,miR-139-5p,Fos,Caspase 3 and Caspase 9 mRNA in each group of cells were detected by qRT-PCR.And the expression levels of Fos,AP-1,Caspase 3 and Caspase 9 proteins in each group of cells were detected by Western Blot.Results1.After 4 weeks of treatment,compared with the placebo group,BSHXD treatment group could significantly reduce the frequency and duration of angina pectoris(P<0.01),reduce TCM syndrome scores(P<0.01),improve the clinical symptoms of chest pain,chest tightness,fatigue,shortness of breath,soreness and weakness of waist and knees,dizziness,tinnitus,wheezing,forgetfulness and frequent nocturia(P<0.01 or P<0.05),and improve the scores of the physical activity limitation,the anginal stability,the anginal frequency and the disease perception in the SAQ(P<0.01).However,there were no statistical differences between BSHXD and placebo in improving blood lipid level,hs-CRP and the incidence of MACEs(P>0.05).In addition,BSHXD had no effect on the safety indicators such as blood routine,liver and kidney function,coagulation function,urine and stool routine,and electrocardiogram.Besides,BSHXD could reduce the level of patient’s blood urea(P<0.05),and increase thrombin time within the normal range(P<0.05).2.The results of transcriptome high-throughput sequencing showed that there were 696 differential lncRNAs(314 up-regulated and 382 down-regulated),86 differential miRNAs(74 up-regulated and 12 down-regulated)and 382 differential mRNAs(249 up-regulated and 133 down-regulated)before and after treatment with BSHXD.The ceRNA regulatory network of 13 lncRNA-2 miRNA-4 mRNA was constructed by using bioinformatics,and the lncRNA NEAT1/miR-139-5p/Fos regulatory axis was obtained by further analysis.In the qRT-PCR validation,it was found that BSHXD could significantly reduce the expression of lncRNA NEAT1(P<0.05)and Fos(P<0.05),increase the expression of miR-139-5p(P<0.05),and reduce the expression of apoptosis-related gene Caspase 3(P<0.05).And there was a trend to reduce the expression of Caspase 9,but with no statistical difference(P>0.05).In further ELISA verification,it was found that BSHXD could also significantly reduce the level of Fos protein in plasma(P<0.05).3.The luciferase activity was decreased significantly when lncRNA NEAT1 wild-type transfection with miR-139-5p mimic(P<0.05),while the luciferase activity of lncRNA NEAT1 mutant type transfected with miR-139-5p mimic had no significant change.Besides,the luciferase activity of Fos wild-type transfected with miR-139-5p mimic was significantly decreased(P<0.05),while the luciferase activity of Fos mutant type had no significant change after miR-13 9-5p mimic was transfected.It was confirmed that there was a regulatory relationship for targeted binding between lncRNA NEAT1 and miR-139-5p,and between miR-139-5p and Fos.H2O2-induced oxidative damage significantly inhibited the proliferation of HUVEC and HK-2 cells(P<0.01),increased the level of ROS in cells(P<0.01),and promoted the apoptosis of HUVEC and HK-2 cells.For HUVEC,the BSHXD could significantly increase the OD value of HUVEC(P<0.01),alleviate the inhibition of H2O2 on the proliferation of HUVEC,improve cell survival rate,reduce the ROS level in HUVEC(P<0.01),reduce the early apoptosis rate,late apoptosis rate,total apoptosis rate and apoptotic index(P<0.01),decrease the expression of lncRNA NEAT1,Fos,Caspase 3 and Caspase 9 mRNA(P<0.01 or P<0.05),increase the level of miR-139-5p(P<0.05),inhibite the expression of Fos,AP-1,Caspase 3 and Caspase 9 proteins(P<0.05)in HUVEC,and could play the role of anti-apoptotic effect on HUVEC.And for HK-2 cells,BSHXD could also significantly increase the OD value of HK-2 cells(P<0.05),alleviate the inhibition of H2O2 on the proliferation of HK-2 cells,improve the survival rate of HK-2 cells,reduce the level of intracellular ROS(P<0.01),decrease the late apoptosis rate,total apoptosis rate and apoptotic index(P<0.01 or P<0.05),decrease the expression of lncRNA NEAT1,Fos,Caspase 3 and Caspase 9 mRNA(P<0.05),increase the level of miR-139-5p(P<0.05),and inhibit the expression of AP-1,Caspase 3 and Caspase 9 proteins(P<0.01)in HK-2 cells.Furthermore,BSHXD could play an anti-apoptosis role in HK-2 cells.Conclusion1.BSHXD has a good clinical efficacy and safety in the treatment of unstable angina pectoris of CHD with KD&BS syndrome.2.The lncRNA NEAT1/miR-139-5p/Fos axis might be a potential target of BSHXD for the treatment of CHD with KD&BS syndrome.3.BSHXD could inhibit the apoptosis of HUVEC and HK-2 cells induced by H2O2 through regulating the lncRNA NEAT1/miR-139-5p/Fos axis,so as to play a role in delaying the progression of atherosclerosis and protecting the kidney. |
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