| Guided by TCM Vessel-collateral Theory,based on the saying in?Ling Shu · Diseases before Health?: “xu xie invades to person,which starts in the skin,…… stays attached to the venation,gradually gathered without dissipating,masses obstruction of collaterals,or stays attached to the sun-venation,or stays attached to the collateral-venation”,this study proposed that TCM pathogenesis of myocardial fibrosis after AMI is “qi deficiency,blood stasis,collateral blockage and stagnation formation” and established the basic treatment method of “benefiting qi,activating blood circulation,dredging collaterals and removing stagnation”.Starting with End MT,the AMI model was established in SD rats in vivo,and End MT was induced by hypoxia in HCMECs in vitro.Representative tongxinluo drugs were given as intervention,not only helpful to reveal the pathological mechanism of myocardial fibrosis after AMI,but also of great significance to elucidation the mechanism of TXL based on “microvascular” protection and regulation of NRG-1-mediated End MT in myocardial fibrosis after AMI.Part ? Effect of Endothelial-to-Mesenchymal Transition in Myocardial Fibrosis after AMI and Intervention of TongxinluoObjective: To investigate the role of End MT in myocardial fibrosis after AMI in vivo and bring to light the effect of TXL intervention.Methods: The AMI model was established in SD rats by ligating left anterior descending coronary artery.The success of AMI model establishment was evaluated according to the changes of ST segment of Electrocardiogram(ECG)before and after ligation.SD rats with AMI successfully established were randomly divided into Sham group,Model group,low dose Tongxinluo group(TXL-L),medium dose tongxinluo group(TXL-M),high dose tongxinluo group(TXL-H)and benazepril group(10 rats in each group).Drug intervention was initiated from the day after completion of the model successfully established.Before the experiment,0.5% Sodium carboxymethyl cellulose aqueous solution(CMC-Na)was used to prepare the drug into the required concentration.The rats were administered with low(0.2g/kg/d)-,mid(0.4 g/kg/d)-,high(0.8g/kg/d)-dose Tongxinluo and benazepril(10mg/kg/d)for 4 weeks,respectively.Oral dosing volume of 3ml/kg.Cardiac function was measured by small animal ultrasonic cardiogram,the weight of heart and body were weighed to calculate the heart weight index,the degree of myocardial fibrosis was detected by Masson staining,and protein expression of myocardial fibrosis related indicators such as Collagen I,Collagen ?,MMP-2 and MMP-9 were detected by Western Blot.The protein and m RNA expression of End MT-related markers such as CD31,VE-cadherin,?-SMA and FSP-1 were determined by Western blot analysis and RT-q PCR to investigate the role of End MT in myocardial fibrosis after AMI.Results: 1 Electrocardiogram changes before and after coronary artery ligation Before ligation of left anterior descending coronary artery,the P wave and PR intervals were obvious,and R wave descends branch and T wave ascends branch intersect,without apparent ST segment;After ligation of the anterior descending branch of the left coronary artery,the R wave and T wave fused into a tall tented wave,and the “damaged” ST segment changes appeared,indicating the successful preparation of the rat AMI model.2 Tongxinluo(TXL)improved cardiac function in rats with myocardial fibrosis after AMI Echocardiography was used to examine cardiac function 4 weeks after AMI.Model group significantly increased LVEDd and LVESd and decreased EF and FS compared with Sham group(P<0.01);Compared with Model group,although the TXL-L has not significant effect on cardiac function(P>0.05),it significantly decreased LVEDd,LVESd and increased EF,FS in TXL-M,TXL-H and benazepril group(P<0.01);Compared with benazepril group,there was no significant difference in the protective effect of TXL-H on cardiac function(P>0.05).3 TXL decreased the HW/BW index of myocardial fibrosis rats after AMI The heart weight index was obtained by weighing the heart and calculating the heart weight/body weight.The results showed that compared with Sham group,the heart weight index of Model group rats was significantly increased(P<0.01);After 4 weeks of drug intervention,compared with the Model group,the heart weight index of TXL for each dose groups and benazepril group was significantly reduced(P<0.01);Compared with benazepril group,TXL for each dose groups had no significant difference in reducing HW/BW index(P>0.05).4 TXL attenuated myocardial fibrosis after AMI in rats Myocardial fibrosis was detected by Masson trichrome staining,blue represents collagen fibers and red represents myocardium.Myocardial fibers of Sham group were closely arranged and orderly without obvious collagen fiber hyperplasia.The myocardial fibers of Model group were disordered and hyperplasia of collagen fibers was observed.TXL and benazepril variously improved myocardial fibrosis.Protein expressions of type I and III Collagen,MMP-2 and MMP-9 in the myocardial fibrosis were determined by Western blot analysis.Compared with Sham group,protein expression of type I and III Collagen,MMP-2 and MMP-9 showed a distinct increase in Model group(P<0.01);Compared with Model group,protein expression levels of type I and III Collagen,MMP-2 and MMP-9 were downregulated in TXL and benazepril group(P<0.05,P<0.01).Moreover,there was no difference in preventing MF between the TXL-H and benazepril group(P>0.05).TXL reduced myocardial fibrosis after AMI in a dose-dependent manner.5 TXL significantly inhibited End MT in myocardial fibrosis rats after AMI As showed,compared with Sham group,Model group progressively decreased the level of CD31,VE-cadherin whereas increased that of ?-SMA,FSP-1 at both protein and m RNA levels(P<0.01),which indicates that End MT is take part in myocardial fibrosis.However,compared with Model group,expression level of endothelial marker genes such as CD31,VE-cadherin showed a distinct increase whereas mesenchymal marker genes such as ?-SMA,FSP-1 were significantly decreased at both protein and m RNA levels in TXL for each dose group and benazepril group(P<0.05,P<0.01).Compared with benazepril group,there was no significant difference in the inhibitory effect of TXL-H on End MT after AMI(P>0.05),indicating that TXL could inhibit End MT in rats with myocardial fibrosis after AMI.Part ? Effect of hypoxia-induced HCMECs on End MT and Intervention of TongxinluoObjective: To reveale the role of hypoxia in inducing End MT in human cardiac microvascular endothelial cells(HCMECs)in vitro and bring to light the effect of TXL intervention.Methods: HCMECs were incubated in hypoxia conditions(1% O2,37?)for 3 days to induce End MT model.HCMECs were divided into Control group,Hypoxia group and TXL group.MTS was used to test the survival activity of HCMECs;The protein expression of End MT-related markers such as CD31,VE-cadherin,?-SMA and FSP-1 were determined by Western blot analysis;FITC phalloidin-labeled Phalloidin was used to detect the changes of F-actin protein in HCMECs cells;Immunofluorescence was used to detecte the expression of HCMECs End MT-related markers such as CD31,v WF,?-SMA and Vimentin.Results: 1 MTS was used to detect the effect of tongxinluo on the survival activity of HCMECs TXL has a protective effect on HCMECs.Compared with the Control group,the survival activity of TXL 200 ?g/m L group had no significant difference,but it showed an upward trend(P>0.05).TXL 400 ?g/m L and 600 ?g/m L had proliferative effects on HCMECs(P<0.01),with the most significant effect at 600 ?g/mL.2 TXL could inhibit the hypoxia-induced HCMECs to occur End MT Effect of TXL on End MT induced by hypoxia-induced HCMECs were detected by Western Blot analysis.Intervention was conducted with TXL 600 ?g/m L,400?g /m L and 200 ?g/m L,respectively.Results showed that the proteins of mesenchymal markers such as ?-SMA,FSP-1 in hypoxia-induced HCMECs were up-regulated,while the expressions of HCMECs markers such as CD31,VE-cadherin in HCMECs were decreased(P<0.01);However,End MT induced by hypoxia in HCMECs could be inhibited to varying degrees by each dose group of TXL,including up-regulation the protein expression of CD31,VE-cadherin,down-regulation the protein expression of ?-SMA and FSP-1(P<0.05,P<0.01).TXL 600 ?g/m L showed the best inhibitory effect in a dose-dependent manner.FITC phalloidin-labeled Phalloidin was used to detect F-actin changes in HCMECs.TXL 600 ?g/m L was used for further study.In control group,HCMECs presented cobblestone morphology,and F-actin presented fine filamentous uniform distribution.Hypoxia could induce HCMECs to transform from cobblestone morphology to fusiform.F-actin clustered into bundles and expressed a large number of stress fibers.Tongxinluo could improve the changes of cell morphology and F-actin induced by hypoxia.Immunofluorescence staining method were used to detected the effect of TXL on hypoxia-induced End MT,the results showed that HCMECs in control group presented a typical cobblestone morphology.When exposed to hypoxia conditions,HCMECs lost its pebblestone characteristics and presented a dispersed spindle appearance.Protein expression of HCMECs markers CD31 and v WF were down-regulated,and the interstitial cell markers ?-SMA and Vimentin were were up-regulated.After TXL intervention,protein expressions of CD31 and v WF were up-regulated,and ?-SMA and Vimentin were down-regulated,indicating that TXL could inhibit the hypoxia-induced End MT of HCMECs,which was consistent with the results of in vivo experiments that TXL could inhibit End MT in AMI rats.Part ? Research of Mechanism of NRG-1/Erb B/PI3K/AKT signaling pathway on Endothelial-to-mesenchymal Transition and Intervention of TongxinluoObjective: To investigate whether the NRG-1/Erb B/PI3K/AKT signaling pathway mediates End MT in myocardial fibrosis after AMI.Further to reveale the role of NRG-1/Erb B/PI3K/AKT signaling pathway in hypoxia-induced End MT in HCMECs in vitro and bring to light the effect of TXL intervention.Methods: In vivo,methods of model establishment and administration are same as part one.After 4 weeks of administration,the protein expression of the upstream and downstream factors of signaling pathway such as NRG-1,p-Erb B2,p-Erb B4,PI3 K,p-AKT and snail were determined by Western blot analysis,to investigate whether the NRG-1/Erb B/PI3K/AKT signaling pathway mediates End MT in myocardial fibrosis after AMI.In vitro,HCMECs were incubated in hypoxia conditions(1% O2,37?)for 3 days to induce End MT model.(1)Cells were divided into control group and hypoxia group,effect of hypoxia on protein level of NRG-1 in HCMECs was detected by Western Blot;(2)Flow cytometry and Western blot were used to determine the optimal silencing condition of si RNA NRG-1 on NRG-1 gene in HCMECs,respectively;(3)Cells were divided into control group and si RNA NRG-1 group.Western Blot was used to detect the protein expressions of End MT marker and NRG-1/Erb B/PI3K/AKT pathway upstream and downstream factor,to investigate the effect of si RNA NRG-1 on HCMECs;(4)To further explore the specific molecular mechanism of the inhibition of End MT by TXL.HCMECs were divided into 5 groups: control group,hypoxia group,hypoxia+si RNA NRG-1 group,hypoxia+TXL group and hypoxia+si RNA NRG-1+TXL group.HCMECs End MT-related markers v WF,Vimentin were detected by immunofluorescence;The protein expression of End MT-related markers CD31,VE-cadherin,?-SMA,FSP-1 and upstream and downstream factors of NRG-1/Erb B/PI3K/AKT pathway were determined by Western blot.Results: 1 TXL activates the NRG-1/Erb B/PI3K/AKT pathway in myocardial fibrosis rats after AMI Compared with sham group,the protein expressions of NRG-1,p-Erb B 2,p-Erb B 4,PI3 K and p-AKT were down-regulated and protein expression of End MT transcription factor snail were up-regulated in model group(P<0.01);Compared with model group,TXL for each dose and benazepril were able to up-regulate the protein expression of NRG-1,p-Erb B 2,p-Erb B 4,PI3 K and p-AKT in myocardial fibrosis tissues to varying degrees,and down-regulate protein expression of snail(P<0.05,P<0.01);and there was no statistically significant difference between TXL-H group and benazepril group(P>0.05).In conclusion,NRG-1/Erb B/PI3K/AKT is involved in myocardial fibrosis after AMI,and TXL may inhibit End MT in rats with myocardial fibrosis after AMI by activating NRG-1/Erb B/PI3K/AKT pathway.2 Expression of NRG-1 were down-regulated in hypoxia-induced End MT The protein expression of NRG-1 was significantly down-regulated in hypoxia group compared with the control group(P<0.01),which is consistent with the down-regulation of NRG-1 protein expression after AMI in vivo.3 Optimum silence condition of si RNA NRG-1 on NRG-1 gene in HCMECs To determine whether NRG-1 participated in hypoxia-induced End MT,si RNAs NRG-1 were applicated to silence NRG-1 gene in HCMEC.The transfection rate of three kinds of si RNA NRG-1 to HCMECs was detected by Flow Cytometry,results showed that the transfection rate of si RNA-1626 was high;The silencing effect of si RNA NRG-1 on NRG-1 protein in HCMECs were detected by Western Blot,results showed that si RNA-1626 had a significant silencing effect on NRG-1 expression in HCMECs(P<0.01).4 Silencing NRG-1 gene promote End MT in HCMECs cells Western Blot was used to detect the protein expression of End MT marker.Compared with control group,the protein levels of HCMECs markers such as CD31,VE-cadherin were down-regulated in si RNA NRG-1 group,while the protein expressions of interstitial markers such as ?-SMA,FSP-1 were up-regulated(P<0.01),demonstrating that silencing the gene expression of NRG-1 by si RNA NRG-1 in vitro could promote the End MT of HCMECs.5 Silencing NRG-1 gene inhibit the NRG-1/Erb B/PI3K/AKT pathway HCMECs were pretreated with si RNA NRG-1 in vitro to silence NRG-1 gene expression.Western Blot was used to detect the protein expression of NRG-1/Erb B/PI3K/AKT pathway upstream and downstream key factors.Compared with the control group,si RNA NRG-1 pretreated with HCMECs significantly reduced NRG-1,p-Erb B 2,p-Erb B 4 and p-AKT protein levels(P<0.01),indicating that si RNA NRG-1 inhibited the activation of NRG-1/ Erb B/PI3K/AKT pathway.6 TXL inhibits the hypoxia-induced End MT in HCMECs by activating NRG-1/Erb B/PI3K/AKT pathway 6.1 TXL activates the NRG-1/Erb B/PI3K/AKT pathway in HCMECs Compared with the control group,the protein level of NRG-1,p-Erb B2,p-Erb B4 and p-AKT significantly decreased,while the protein level of HIF-1? and snail,a zinc?finger binding transcription factors of End MT,was increased in hypoxia treatment(P<0.01);Compared with the hypoxia group,TXL pronouncedly elevated the levels of NRG-1,p-Erb B2,p-Erb B4 and p-AKT and reduced the level of HIF-1? and snail in HCMECs under hypoxia(P<0.05,P<0.01),indicating that TXL could activate NRG-1/Erb B/PI3K/AKT signaling pathway of HCMECs.The protein level of HIF-1? and snail were increased,while the protein level of NRG-1,p-Erb B2,p-Erb B4 and p-AKT were significantly decreased in hypoxia+TXL+si RNA NRG-1 compared with the hypoxia+TXL group(P<0.05,P<0.01).Compared with hypoxia+si RNA NRG-1 group,hypoxia+TXL+si RNA NRG-1 reduced the level of HIF-1?,snail and recreased the levels of p-AKT(P<0.05,P<0.01).It showed that the application of si RNA NRG-1 could partially reverse the activation of NRG-1/Erb B/PI3K/AKT signaling cascade and reverse the inhibition of TXL on snail.6.2 TXL inhibits EndMT by activating NRG-1/Erb B/PI3K/AKT pathway Immunofluorescence staining results clearly demonstrated that expression of v WF was decreased and the expression of ?-SMA was increased in the hypoxia group.Hypoxia+TXL treatment increased the expression of v WF and decreased the expression of ?-SMA.Pretreatment with si RNA NRG-1 significantly suppressed the upregulation of v WF and the downregulation of ?-SMA at protein level in the presence of TXL treatment.Western Blot analysis were used to detected End MT marker protein expression.Results showed that,compared with control group,the expression of CD31,VE-cadherin were down-regulated,the expression of ?-SMA,FSP-1 were up-regulated in hypoxia group(P<0.01);Hypoxia+TXL increased the expression of CD31,VE-cadherin,and decreased the expression of ?-SMA,FSP-1 compared with hypoxia group(P<0.01);Compared with the hypoxia+ TXL group,protein expression of CD31 and VE-cadherin were down-regulated,and ?-SMA and FSP-1 were up-regulated in hypoxia+TXL+ si RNA NRG-1 group(P<0.05,P<0.01);Compared with hypoxia+ si RNA NRG-1 group,hypoxia+TXL+si RNA NRG-1 partially inhibit the inhibitory effect of TXL on End MT(P<0.05,P<0.01),suggesting that NRG-1/Erb B/ PI3K/AKT signal cascade is involved in the protective effect of TXL on HCMEC.Conclusions: 1 Guided by TCM Vessel-collateral Theory,this study proposed that TCM pathogenesis of myocardial fibrosis after AMI is “qi deficiency,blood stasis,collateral blockage and stagnation formation”,which is interrelated with the process of myocardial fibrosis mediated by “microvessels” after AMI.Based on the pathogenesis of myocardial fibrosis after AMI,the basic treatment method of “benefiting qi,activating blood circulation,dredging collaterals and removing stagnation” was established,which opens up an effective treatment approach for myocardial fibrosis after myocardial infarction.2 The study focused on the pathological mechanism of NRG-1-mediated End MT in myocardial fibrosis after AMI.SD rat AMI model was established in vivo and End MT model induced by hypoxia-induced HCMECs was successfully constructed in vitro,respectively.The results revealing that End MT is a key pathological link of myocardial fibrosis after AMI,and NRG-1/Erb B/PI3K/AKT signaling pathway participated End MT to mediate the myocardial fibrosis course after AMI.3 Tongxinluo,a representative agent of collateral drug intervention,could improve cardiac function,inhibit myocardial fibrosis after AMI and End MT course at the level of overall animal.At the level of isolated cells,TXL could protect the structural integrity of microvascular endothelium,inhibit the hypoxia-induced End MT of HCMECs by activating NRG-1/Erb B/PI3K/AKT signaling pathway.This study combined experimental data to verify the pathogenesis hypothesis of “qi deficiency,blood stasis,collateral blockage and stagnation formation”,and to provide scientific data and evidence for the treatment method of “benefiting qi,activating blood circulation,dredging collaterals and removing stagnation” guided by Vessel-collateral Theory. |
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