Study On The Efficacy And Mechanism Of Puerariae Lobatae Radix,and Puerariae Thomsonii Radix Against Arrhythmia Caused By MIRI

Objective:To observe the difference in antiarrhythmic effects and possible mechanisms of Puerariae Lobatae Radix（PLR）and Puerariae Thomsonii Radix（PTR）on arrhythmia of rats with ischemia/reperfusion（I/R）injury,and the effects of PLR and PTR medicated intestinal ab-sorption fluid（IAF）and medicated serum（MS）on H9c2 cells injured by oxygen-glucose depri-vation/reoxygenation（OGD/R）and discussed through systemic pharmacological methods and molecular docking techniques.Methods:（1）SD was adaptively reared for one week and then divided into groups,each with 15 animals,and continuous intragastric administration of corresponding drugs for 15 days:propranolol group（Propranolol,Pro,15mg/kg/d）,PLR low,medium and high groups（PLRL=1.6,PLRM=3.2,PLRH=6.4g/kg/d）,PTR low,medium and high group（PTRL=1.6,PTRM=3.2,PTRH=6.4g/kg/d）,sham operation group（Sham）,The model group（I/R）was given the same volume of normal saline every day.One hour after the last administration,theⅡlead electrocardiogram was monitored.The left anterior descending coronary artery（Left anterior descending,LAD）method was used to establish an ischemia/reperfusion model for 15 minutes and 30 minutes of reperfusion.ECG was recorded throughout the process.After the reperfusion,six animals in each group were randomly selected to confirm the area of the cardiac ischemic site with the Evans blue staining method.After the ECG,eight animals were randomly selected from each group to take blood from the abdominal aorta to detect SOD,MDA,CK-MB,GSH-Px,TNF-α,and IL-6.Furthermore,immediately remove the heart.After cleaning,eight were randomly selected from each group to calculate the myocardial hypertrophy index.Then,the myocardial tissue was stored at-80°C for the left ventricular myocardial tissue c Tn T content,Na⁺-K⁺-ATPase,and Ca²⁺-ATPase activity detection.Randomly select 4 animals in each group to take part of the left ventricular myocardium tissue of the same position and fix it with 4%paraformaldehyde for HE,Masson staining,TUNEL detection and immunohistochemical detec-tion;each group randomly select 6 animals to take the same part of the left ventricle Myocardial tissue is used for Western Blot to detect the protein expression of MAPK p38 and NFκB p65;（2）Use Q-TOF to detect the active ingredients in IAF and MS of PLR and PTR,use Pro,2.5%,5%,7.5%IAF or MS and Puerarin（Pue）intervened in H9c2 cells,established OGD/R injury model to detect H9c2 cell viability,intracellular SOD activity and LDH activity,and Western Blot was used to detect the protein expression of cardiomyocytes MAPK p38 and NFκB p65;（3）Through systemic pharmacology and molecular docking technology,the active ingredients and targets of Radix Pueraria obtained in TCMSP and chemical databases are crossed with targets related to arrhythmia in Gene Cards and OMIM databases,and the common targets are analyzed by GO and KEGG Obtain their biological processes in the organism and the pathways involved in them.Finally,the active ingredients with higher priority and correlation were selected for molecular docking with MAPK p38 and NFκB p65 proteins,and the binding methods and char-acteristics between these ingredients and proteins were analyzed.The potential mechanism of Radix Pueraria root against arrhythmia was explored.Results:（1）ECG parameters:Compared with Sham,normal rats have abnormal ECG pa-rameters after I/R treatment,which manifests as frequent ventricular premature contractions,joint rhythms,ventricular tachycardia,ventricular fibrillation,etc.（P<0.05）,the overall ar-rhythmia score was significantly higher than that of Sham（P<0.05）.Compared with the I/R group,PLRL,PLRM,and PTRL can improve the abnormal ECG parameters of I/R rats and re-duce the arrhythmia score（P<0.05）.All treatment groups can shorten the duration of arrhythmia（P<0.01）,reduce the incidence of VT and VF in rats;Compared with PTR at the same dose,the frequency of paired VPC in the PLRL group was less than that in the PTRL group（P<0.05）,and the frequency of bigeminy and trigeminy and arrhythmia score in PLRM group were lower than those in PTRM group（P<0.01）;Myocardial hypertrophy index and area of ischemic area:Compared with Sham,I/R treat-ment had no significant effect on rat myocardial hypertrophy index（P>0.05）,and there was no statistical difference between all administration groups and I/R group（P>0.05）;Compared with Sham,the area of the myocardial ischemic site in I/R rats was significantly increased（P<0.01）,and all drug pretreatment groups reduced the size of the ischemic area in rats（P<0.01）;Elisa kit detection:Compared with Sham,I/R can reduce rat serum SOD,GSH-Px activity,increase MDA,TNF-αcontent,and CK-MB activity（P<0.01）,PLR and PTR can regulate these to varying degrees of the activity of myocardial enzymes and the content of inflammatory fac-tors（P<0.05）;I/R can reduce the activity of Na⁺-K⁺-ATPase and Ca²⁺-ATPase in the heart of normal rats（P<0.01）,and the content of c Tn T increases（P<0.01）,Compared with the I/R group,the drug pretreatment group Pro,PLRL,PLRM,PTRL,PTRM increased Na⁺-K⁺-ATPase activ-ity（P<0.01）,Pro and PLRM increased Ca²⁺-ATPase activity（P<0.01）,Pro,PLRL PLRM can reduce the content of c Tn T in the heart of rats treated with I/R（P<0.01）.The Ca²⁺-ATPase ac-tivity and c Tn T content of the PTR group were not statistically different from those of the I/R group（P>0.05）;In addition,TNF-αin the PLRL group was higher than that in PLRL group TNF-αcontent of PTRL was lower than that of PTRL（P<0.05）;GSH-Px activity in PLRH group was higher than that in PTRH group（P<0.05）;The activity of CK-MB in PLRM and PLRH groups was lower than that in PTRM and PTRH groups（P<0.05）.The content of c Tn T in PLRL and PLRM group was lower than that in PTRL and PTRM group（P<0.05）;Pathological changes:Compared with Sham,I/R rats showed cardiomyocyte fiber swelling,inflammatory cell infiltration,disordered arrangement,and intercellular collagen deposition.The drug pretreatment group improved these changes to varying degrees.Pro,PLRM,PTRH（P<0.01）and PLRL,PLRH,PTRM（P<0.05）reduced the volume of rat myocardial interstitial collagen;Pro,PLRM,PLRH,PLRL（P<0.01）,and PTRL（P<0.05）reduced the apoptosis index of cardiomyocytes;Apoptotic cells in PLRL and PLRM groups were less than PTRL and PTRM,respectively（P<0.05）;Immunohistochemistry:The positive expression of connexin Cx43 in rat cardiomyocytes in the I/R group was significantly increased（P<0.01）,and the expression of Cx43 in the drug pre-treatment group was expanded to varying degrees.Among them,the positive expression of Cx43in the Pro and PLRM groups（P<0.01）and PLRL（P<0.05）was higher than that in the I/R group,and the remaining groups had no statistical difference compared with I/R;The expression of Cx43 in PLRL group and PLRM group was higher than PTRL and PTRM respectively（P<0.05）;Western Blot:Compared with Sham,the relative expression of MAPK p38 and NFκB p65protein in the heart of I/R rats increased significantly（P<0.01）;compared with I/R,Pro,PLRL,PLRM,PLRH,PTRH（P<0.01）,and PTRM（P<0.05）can inhibit the expression of MAPK p38protein,and the relative expression of NFκB p65 protein in all treatment groups was signifi-cantly reduced（P<0.01 vs I/R）;The relative expression of NFκB p65 protein in PLRL and PLRH groups was lower than that of PTRL and PTRH respectively（P<0.05）.（2）Q-TOF detection:IAF and MS of PLR and PTR identified by Q-TOF test contain Pue,daidzin,3’-methoxypuerarin,daidzein,formononetin,scoparone,formononetin,and genistein.Cell viability:After OGD/R treatment,the viability of H9c2 cells was significantly lower than that in the Normal group（P<0.01）,and the viability of H9c2 cells in the Pro,IAF,MS,and Pue groups was increased to varying degrees compared with the OGD/R group（P<0.01）;The cell survival rate of the IAF and MS groups at the three concentrations of PLR was higher than that of the same concentration of PTR（P<0.05）;SOD and LDH activity:Compared with the Normal group,the SOD activity of the OGD/R group was significantly reduced（P<0.01）,and the LDH activity was significantly increased（P<0.01）;Compared with OGD/R,Pro,Pue,IAF,and MS of PLR and PTR can all increase SOD activity of H9c2 cells（P<0.01）,SOD activity of PLR group is higher than that of PTR group;Pro,Pue,and Both of IAF and MS of PLR and PTR can reduce the LDH activity of H9c2 cells（P<0.01）;The SOD activity of the IAF group at the three concentrations of PLR was higher than that of the same concentration of PTR（P<0.05）,and the SOD activity of the MS group with 2.5%PLR and 5%PLR was higher than that of the same concentration of PTR（P<0.05）;After IAF treatment in the 2.5%PLR and 5%PLR groups,the LDH activity was lower than the same concentration of PTR（P<0.05）;Western Blot:the protein expressions of MAPK p38 and NFκB p65 in H9c2 cells in the OGD/R group were significantly higher than those in the Normal group（P<0.05）.The IAF and MS of different concentrations of PLR and PTR can be different degrees of inhibition of MAPK p38 and NFκB p65 protein expression（P<0.05）,the PTR-MS group NFκB p65 protein expres-sion was not statistically diverse from the OGD/R group（P>0.05）.Comparison of IAF and MS:The survival rate of H9c2 cells treated by IAF was higher than that of MS treatment group（P<0.01）,SOD activity of IAF group was higher than that of MS group（P<0.01）,and LDH activity of IAF treatment group was lower than that of MS treat-ment group（P<0.05）.There was no significant difference in the relative expression of MAPK p38 protein in H9c2 cells treated with IAF and MS（P>0.05）.The relative expression of NFκB p65 protein in the IAF group was lower than that in the MS group（P<0.01）.（3）According to the analysis,12 active ingredients in Radix Pueraria interfere with 135 ar-rhythmia-related genes to play an antiarrhythmic effect.These are mapped to 268 KEGG path-ways,mainly PI3K-Akt,MAPK,Fox O,TNF,IL-17,HIF-1,c AMP,C-type lectin receptor,Toll-like receptor,Ras,p53,NFκB,VEGF.The five components with the highest correlation were scored by molecular docking with MAPK14 and NFκB3 proteins,and it was found that they were combined by hydrogen bonding orπ-πconjugated form.Conclusion:（1）PLR and PTR have varying degrees of protection against MIRI and reperfusion arrhythmia.The effect of PLR against reperfusion injury and arrhythmia is better than that of the same dose of PTR;（2）The IAF and MS of PLR and PTR can protect the dam-age of OGD/R to H9c2 cells to different degrees.The IAF and MS of PLR protect the damage of H9c2 cells by OGD/R better than the IAF and MS of the same concentration of PTR;（3）IAF avoids the interference of a variety of exogenous and endogenous components,and is more suitable for in vitro pharmacological research of traditional Chinese medicine than MS;（4）PLR and PTR may act on the MAPK p38/NFκB p65 signaling pathway through daidzein,puerarin,genistein,formononetin,and scoparone to exert anti-ischemic/reperfusion antiarrhythmic effects to protect the ischemic myocardium from reperfusion injury.