| BackgroundIschemic arrhythmias are one of the most common complications of heart disease and cause serious health loss worldwide.The main antiarrhythmic drugs used in clinical practice are ion channel blockers,which have some side efects and are not very satisfactory in reducing the risk of death and improving prognosis.The concept of anti-arrhythmia therapy has been constantly innovated and developed,and more and more scholars are committed to improving the substrates of arrhythmia.In the new treatment strategies,the characteristics of traditional Chinese medicine have attracted wide attention,among which,Wenxin Granules have shown good efficacy and safety in long-term clinical practice.The anti-arrhythmia mechanism of Wenxin Granules has not reached a broad consensus.Although there are many basic studies,the research methods and evaluation criteria vary,so it is necessary to conduct systematic analysis and combing to search for valuable clues and evidence.It is noteworthy that the role of Wenxin Granules seems to be related to ER stress and gap junctions.Intervention in myocardial gap junction remodeling is an important research direction to improve the substrate of arrhythmias.Cx43 is mainly expressed in myocardium,and ER stress signaling pathway is involved in the regulation of Cx43 expression.Therefore,this research firstly summarizes the anti-arrhythmic mechanism characteristics and quality of evidence of studies on Wenxin Granules.Then the Wenxin Granules effects on cardiac electrophysiological remodeling after myocardial ischemia has carried on the systematic review,and the evidence relating to the Cx43 was used to guide subsequent in cells experiment.what's more,It was verified from the level of isolated cells that whether Wenxin Granules could interfere the expression of Cx43 by regulating ER stress pathway,so as to provide new evidence-based and experimental evidence for the in-depth understanding of the mechanism of Wenxin Granules in preventing and treating arrhythmia.Objective1.Through evidence-based studies,the effect characteristics and evidence quality of basic studies on Wenxin Granules were comprehensively analyzed,and the effect of Wenxin Granules on electrophysiological remodeling of rat heart after myocardial ischemia was systematically evaluated,so as to provide evidence-based basis for in-depth understanding of the mechanism of Wenxin Granules in preventing and treating arrhythmias.2.Experimental studies were designed under the guidance of evidence-based evidence to investigate the mechanism of interference of Wenxin Granules on Cx43 expression in H9C2 cells through ER stress pathway at the level of isolated cells.Methods1.Sy stematic review of basic research on Wenxin Granules1.1 Retrieval of journal academic papers in CNKI,CBM,Wanfang,VIP,EBbase,Pubmed,The Cochrane Library and Web of Science databases in the form of " Wenxin" in Chinese and English.The literatures were screened according to inclusion and exclusion criteria,the feature information of the included studies was extracted,and the effect characteristics and evidence quality of the basic studies on Wenxin Granules were analyzed.1.2 The effects of Wenxin Granules on myocardial electrophysiological remodeling after myocardial ischemia were systematically evaluated,and the quality of the literature was assessed using the SYRCLE animal experiment bias risk assessment tool and outcome indicators were analyzed.2.The ER stress model of H9C2 cells was constructed and the changes of Cx43 expression were observed2.1 H9C2 cells were cultured in vitro,and the growth curve was simulated to establish a stable research carrier.2.2 The cck-8 method was used to find out the effective stimulation concentration range of TG and the optimal modeling conditions(concentration-time).2.3 The ER stress marker molecules and apoptosis rate were detected by WB and flow cytometry to verify whether the ER stress model of H9C2 cells was successfully constructed.2.4 Morphological changes of H9C2 cells after modeling were observed under light microscope,and the expression of Cx43 was preliminarily observed by immunofluorescence staining.3.Effect of Wenxin Granules on Cx43 expression of H9C2 cells under ER stress3.1 The cck-8 method was used to screen out the non-toxic concentration range and the optimal effect concentration of Wenxin Granules leaching solution.3.2 The cck-8 method was used to explore the non-toxic concentration range of specific ER stress related protein degradation pathway blockers MG-132,Chloroquine and Salubrinal,as well as their effects on TG toxicity.3.3 According to the result of 3.2 to choose blockers and its blocked signaling pathways,experiment were randomly divided into the control group,the model group,the ERAD group,the WXKL group.Using flow cytometry,WB method and immunofluorescence method to detect the change of the expression of maker molecules and Cx43 in each group,analysis the mechanism of Wenxin Granules.Result1.Systematic review of basic research on Wenxin Granules1.1 Effect characteristics and evidence quality of Wenxin Granules:A total of 49 basic studies on Wenxin Granules were included,of which 18 mainly used patch clamp technology to directly detect myocardial electrophysiology,and the other 31 were related to myocardial ischemia,heart failure,and cardiac toxicity,using randomized control vivo/vitro experiments verified the interventional effect of Wenxin Granules.In general,Wenxin Granules mainly interfere with ischemic myocardial injury.However,the route of administration,the concentration of administration and the period of action are different,and the outcome indicators of each study also vary greatly.1.2 Evaluation results of the effects of Wenxin Granules on myocardial electrophysiological remodeling after myocardial ischemia:A total of 7 effective animal experimental studies were included,and the quality was considered low after evaluation.The main outcome indicators are electrocardiogram,gap junction structure,connexins,related miRNAs,and cardiomyocyte action potential parameters.Since the outcome indicators included in the study are lack of specific values and cannot be quantified,a descriptive system review was used to evaluate the outcome indicators.In summary evaluation,on the whole,all indicators can reflect the intervention effect of Wenxin Granules to a certain extent.Among them,the intervention mechanism of Wenxin Granules on the gap junction and connexins is worthy of further in-depth study.2.The ER stress model of H9C2 cells was constructed and the changes of Cx43 expression were observed2.1 The influence of TG on the activity of H9C2 cells was time-concentration dependent,and the effective stimulation concentration range was greater than 0.1?mol/L.When the TG solution was treated at 0.1 and 0.5?mol/L for 24 hours,the growth activity of H9C2 cells reached the inhibition rate of half.2.2 The 0.1?mol/L TG solution was used to stimulate H9C2 cells for 24 hours as the modeling condition.WB showed that compared with the control group,the expression of ER stress marker molecules GRP78 and CHOP were significantly increased in the model group.Flow cytometry suggested that a large number of cells in the model group showed early apoptosis,which proved that the cell model was successfully constructed,2.3 Under the light microscope,the cells in the model group showed obvious morphological changes,and the expression of Cx43 in the model group was decreased by immunofluorescence staining.3.Effect of Wenxin Granules on Cx43 expression of H9C2 cells under ER stress3.1 The cck-8 colorimetric analysis results showed that the non-toxic concentration range of Wenxin Granules leaching solution was less than 20g/L,and the optimal effect concentration was 10g/L.3.2 The cck-8 colorimetric analysis results showed that the non-toxic concentration range of MG-132 was less than or equal to 30?mol/L,and the non-toxic concentration range of Chloroquine and Salubrinal was less than or equal to 40?mnol/L.In the above concentration range,MG-132 failed to reduce the cytotoxicity of TG,and there was no statistically significant difference in the effects of Chloroquine on TG toxicity,while Salubrinal had a significant inhibitory effect on TG toxicity.Therefore,the ER stress apoptosis pathway blocking agent Salubrinal was selected as the positive control in the subsequent experiments of this study.3.3 According to the results of 3.2,the experimental scheme was determined and randomly divided into the control group,the model group,the Salubrinal apoptosis inhibition group and the Wenxin Granules intervention group.Under the light microscope,the cell morphology of the model group was significantly changed,and Salubrinal and Wenxin Granules both improved the cell morphology significantly.3.4 The results of flow cytometry showed that compared with the control group,the apoptosis rate of the remaining three groups increased significantly.Compared with the model group,the apoptosis rate of the Salubrinal apoptosis inhibition group and the Wenxin Granules intervention group were both significant decline.3.5 The WB results showed that compared with the control group,the model group's ER stress receptor protein GRP78,apoptosis signal molecule CHOP and caspase hydrolysis pathway marker molecule Caspase12 were significantly increased,while the expression of Cx43 protein was significantly reduced Compared with the model group,the expressions of CHOP and Caspase 12 in the Salubrinal apoptosis inhibition group and the Wenxin Granules intervention group were significantly decreased,while Cx43 was significantly increased.There was no significant difference in the expression of GRP78 in the Salubrinal apoptosis inhibition group.However,GRP78 in the Wenxin Granules intervention group was significantly upgraded.3.6 The results of immunofluorescence staining indicated that the expression of Cx43 around the apoptosis cells in the model group was significantly reduced and tended to accumulate toward the nuclear membrane,while the expression of Cx43 was significantly increased in the Salubrinal apoptosis inhibition group and the Wenxin Granules interference group,which were more evenly distributed in the cytoplasm and closer to the edge of the cell membrane.Conclusion1.Wenxin Granules play a protective role in myocardial ischemia,heart failure,cardiac toxicity and other disease models.The specific effects are reflected in the improvement of cardiac function indexes such as myocardial pathology,cardiac electrophysiology,and hemodynamics,as well as the regulation of genes and proteins related to ventricular remodeling.Although the basic studies on Wenxin Granules vary greatly,the quality of the studies is low,and the methodology design needs to be improved,the effect of Wenxin Granules on improving the electrophysiological remodeling of ischemic myocardium by intervening gap junction is worthy of attention and further study..2.At the level of isolated cells,the TG-induced ER stress apoptosis pathway can affect the expression of Cx43,Wenxin Granules can improve the expression of Cx43 in H9C2 cells by inhibiting the ER stress apoptosis pathway. |
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