Role And Mechanism Of INOS And NLRP3 Inflammasome In Myocardial Fibrosis Induced By Cigarette Smoke Exposure

PART I THE DEGREE OF MYOCARDIAL FIBROSIS AND EXPRESSION OF INOS AND NLRP3 WERE DIFFERENT BETWEEN SMOKERS AND NON-SMOKERSObjective: Smoking is an independent risk factor for cardiovascular disease.At present,there are no clinical data of myocardial fibrosis caused by smoking.This part mainly detects the degree of myocardial fibrosis between smoking and non-smoking patients and screens the differentially expressed proteins in order to find potential research targets related to myocardial fibrosis.Methods: 1.Right atrial appendage tissues were collected from patients undergoing cardiac surgery from June 2021 to September 2021 in the Department of Cardiothoracic Surgery,The First Affiliated Hospital of Chongqing Medical University.Right atrial appendage tissues from 10 rows of patients were collected,and patients were divided into smoking group（n=5）and non-smoking group（n=5）according to their smoking history.Masson staining showed collagen fiber deposition in myocardium of patients in smoking group and non-smoking group.2.Immunohistochemistry was used to detect iNOS and NLRP3 expression in myocardium of patients in smoking group and non-smoking group.3.The expression of iNOS and NLRP3 in myocardium of patients in smoking group and non-smoking group was detected by Western Blot.Results: 1.The positive staining rate of Masson of collagen fiber in myocardial tissue of smokers was（21.40 ± 3.43）% higher than that of nonsmokers（5.612 ± 2.15）%,and the difference was statistically significant.Immunohistochemical results showed that: The positive rates of iNOS（29.22 ± 5.28）% and NLRP3（44.95 ± 4.38）% in smokers were significantly higher than those in nonsmokers（5.21 ± 1.86）% and NLRP3（7.30 ± 1.90）%.Western Blot results showed that the expression of iNOS and NLRP3 in myocardial tissues of smokers was significantly higher than that of non-smokers.Conclusion: Collagen fiber deposition in myocardial tissue of smoking patients was significantly higher than that of non-smoking patients,and iNOS and NLRP3 were differentially expressed in myocardial tissue of smoking and non-smoking patients,suggesting that iNOS and NLRP3 may be potential targets for studying myocardial fibrosis induced by cigarette smoke.PART ⅠI CIGARETTE SMOKE EXPOSURE INDUCES INOS EXPRESSION AND PROMOTES CARDIAC FIBROSISObjective: Continuous exposure to tobacco smoke leads to aseptic inflammatory response,which is one of the important causes of myocardial fibrosis.Inducible nitric oxide synthase（iNOS）can be activated by a variety of inflammatory factors,and then participate in the regulation of myocardial fibrosis.This part mainly explores the role of iNOS in myocardial fibrosis induced by tobacco smoke exposure.Methods: 1.C57BL/6 mice and iNOS^-/-mice were divided into C57BL/6 wild-type（WT）group,C57BL/6 tobacco smoke exposure group（CS）,iNOS^-/-mouse group(iNOS^-/-),iNOS^-/-mouse tobacco smoke exposure group(CS + iNOS^-/-).Four in each group.Mice in the CS and CS+ iNOS^-/-groups were exposed to tobacco smoke,while mice in the WT and iNOS^-/-groups were exposed to air in the same environment.Small animal ultrasonic cardiac function detector was used to detect the cardiac function of mice.Masson staining was used to reveal the collagen fiber deposition in the myocardial tissue of mice,and Western Blot was used to detect the protein expression.2.Neonatal rat cardiac fibroblasts were cultured in vitro and co-cultured with cigarette smoke extract（CSE）.The expression of iNOS,a-SMA,Collagen I and III in myocardial fibroblasts of Suckling rats were detected after CSE treatment at different concentration and time.Myocardial fibroblasts of Suckling rats were pretreated with LNIL（iNOS inhibitor）for 1h,and then CSE was co-cultured for 24 h.Western blot was used to detect the expressions of iNOS,a-SMA,Collagen I and III.Results: 1.In CS group,the left ventricular ejection fraction（LVEF）and left ventricular short axis shortening fraction（LVFS）decreased gradually.LVFS（32.44 ± 1.31）% and LVEF（84.87 ± 1.22）% and LVFS（46.74 ± 0.89）% in CS group were significantly lower than those in Control group at week 4.2.The positive staining rate of Masson of collagen fiber in myocardial tissue of mice in CS group（12.37 ± 1.66）%was significantly higher than that in Control group（1.20 ± 0.25）%.3.Compared with Control group,the protein expression levels of iNOS,aSMA,Collagen I and III in CS group were significantly increased.4.Cardiac function of CS+iNOS^-/-group decreased gradually,and LVEF（72.94 ± 1.14）% and LVFS（42.41 ± 0.78）% were significantly better than those of CS group（61.10 ± 2.85）% and LVFS（32.44 ± 1.31）% at week 4.5.The positive staining rate of Masson fiber in myocardial tissue of CS+iNOS^-/-group（1.99 ± 0.39）% was significantly lower than that of CS group（12.37 ± 1.66）%.6.Compared with the control group（WT）,iNOS^-/-group and CS+iNOS^-/-group,the protein expression levels of iNOS,a-SMA,Collagen I and III in myocardial tissue of mice in CS group were significantly increased（P<0.05）.7.In vitro experiment,CSE promoted the expression levels of iNOS,a-SMA,Collagen I and III in concentrationtime dependent manner.L-NIL（iNOS inhibitor）could significantly reverse the increase of iNOS,a-SMA,Collagen I and III protein levels induced by CSE.Conclusion: In mice exposed to tobacco smoke,the left heart function decreased,myocardial fibrosis and the protein levels of iNOS,a-SMA,Collagen I and III increased.However,iNOS knockout could significantly improve the cardiac function decline and myocardial fibrosis induced by tobacco smoke.These results suggest that exposure to tobacco smoke can induce iNOS expression in mouse myocardial fibroblasts,promote myocardial fibrosis,and further cause cardiac function decline.PART ⅡI CIGARETTE SMOKE EXPOSURE INDUCES ACTIVATION OF NLRP3 INFLAMMASOME AND PROMOTES CARDIAC FIBROSISObjective: NLRP3 inflammasome,as an important component of innate immunity,plays a central role in inflammatory response.It has been reported that cigarette smoke exposure can induce NLRP3 inflammasome activation,but it is not clear whether it is involved in the process of cardiac fibrosis induced by tobacco smoke exposure.This part of the experiment mainly explored the role of NLRP3 inflammasome in cardiac fibrosis induced by cigarette smoke exposure.Methods: C57BL/6 mice and NLRP3 knockout(NLRP3^-/-)mice were divided into C57BL/6 wild-type（WT）group,C57BL/6 with cigarette smoke exposure group（CS）,NLRP3^-/-mouse group(NLRP3^-/-)and NLRP3^-/-mouse with cigarette smoke exposure group(CS+NLRP3^-/-),4mice in each group.Mice in CS group and CS+NLRP3^-/-group were exposed to cigarette smoke,while mice in WT group and NLRP3^-/-group were placed in the air under the same environment.The cardiac function of mice was detected by animal ultrasonic cardiac function detector;Masson staining was performed to show the deposition of collagen fibers in mouse myocardial tissue and Western blot was used to detected the protein expression.The neonatal rat cardiac fibroblasts were treated with CSE.To detect the expression of NLRP3,a-SMA,collagen I and collagen III in neonatal rat cardiac fibroblasts treated with CSE at different concentrations and times.Neonatal rat cardiac fibroblasts were pretreated with MCC950（NLRP3 inhibitor）for 1h,and then co-cultured with CSE for 24 h.The expressions of NLRP3,a-SMA,collagen I and collagen III were detected by Western blot.Results: 1.Cardiac function of mice in WT group and NLRP3^-/-group did not change significantly,while LVEF and LVFS of mice in CS group and CS+NLRP3^-/-group gradually decreased.At week 4,cardiac function indexes of mice in CS group were as follows: LVEF（61.10±2.85）% and LVFS（32.44±1.31）% were significantly lower than those in CS+NLRP3^-/-group: LVEF（71.96±1.26）% and LVFS（41.54±0.66）%.2.The positive staining rate of collagen fiber Masson in CS group（12.37±1.66）% was significantly higher than that in CS+NLRP3^-/-group（1.97±0.49）%,and the difference was statistically significant.3.Compared with WT group,NLRP3^-/-group and CS+NLRP3^-/-group,the protein expression levels of NLRP3,caspase-1p20 and IL-βp17 in CS group were significantly increased（P < 0.05）.4.In vitro experiments showed that CSE could induce the protein expression levels of NLRP3,a-SMA,Collagen I and Collagen III to increase in concentration-time dependent manner.After intervention with MCC950（NLRP3 inhibitor）,the CSE induced elevation of a-SMA,Collagen I and Collagen III protein levels was significantly reversed.Conclusion: Cigarette smoke exposure caused the decrease of left cardiac function,cardiac fibrosis and the increase of NLRP3,Caspase-1p20,IL-1βp17,a-SMA,collagen I and collagen III protein levels in mice,but NLRP3 knockout could significantly improve the decrease of cardiac function and cardiac fibrosis caused by cigarette smoke,suggesting that cigarette smoke exposure induced the activation of NLRP3 inflammasome in cardiac fibroblasts and promoted cardiac fibrosis in mice,and cause the dysfunction of cardiac function.PART IV EXPERIMENTAL STUDY ON EXPLORING THE REGULATORY MECHANISM BETWEEN INOS AND NLRP3Objective:Bioinformatics analysis showed that there was a potential regulatory mechanism between iNOS and NLRP3.This part of the experiment mainly reveals the internal regulation mechanism between iNOS and NLRP3.Methods: 1.The protein interaction between iNOS and NLRP3 was predicted on Genemania website by bioinformatics methods such as physical interaction,gene co-expression,gene co-location,gene enrichment analysis and website prediction.2.Western blot was performed to detect the expression of iNOS,NLRP3,Caspase-1p20,and IL-1βp17 in mouse myocardial tissue.Subsequently,neonatal rat myocardial fibroblasts were pretreated with L-NIL（iNOS inhibitor）and MCC950（NLRP3 inhibitor）for 1h and then co-cultured with 1.5% CSE for 48 h,then western blot was used to detect the expression of iNOS,NLRP3,Caspase-1p20 and IL-1βp17.3.Neonatal rat cardiac fibroblasts were pretreated with L-NIL（iNOS inhibitor）,TAPI-1（TACE inhibitor）,MCC950（NLRP3 inhibitor）,ODQ（s GC inhibitor）and KT5823（PKG inhibitor）for 1h,and then cocultured with CSE for 48 h.Western blot was used to detect the expression of iNOS,active-TACE and NLRP3,and real-time quantitative PCR（QRTPCR）was used to detect the expression of m RNA-TNF-a.Results: 1.The protein interaction between iNOS and NLRP3 was predicted by bioinformatics.It was found that there was no direct interaction between iNOS and NLRP3,however,the indirect interaction regulation mainly occurs through TNF（tumor necrosis factor）bridging.2.In vivo experiments have found that iNOS knockout can significantly reverse the elevated expression of NLRP3,Caspase-1 p20 and IL-1β p17 proteins caused by CS exposure,while NLRP3 knockout cannot change the effect of CS exposure on iNOS expression.In vitro experiments showed that the expression of protein iNOS,NLRP3,caspase-1p20 and IL-1βp17in neonatal rat cardiac fibroblasts treated with CSE was significantly upregulated.The increase of iNOS,NLRP3,caspase-1p20 and IL-1βp17induced by CSE could significantly reversed by L-NIL treatment;The increase of NLRP3,caspase-1p20 and IL-1βp17,except the iNOS,induced by CSE could be significantly reversed by MCC950 treatment.3.After CSE treatment,the expressions of iNOS,active TACE,NLRP3 protein and m RNA-TNF-a in neonatal rat cardiac fibroblasts were significantly upregulated.After L-NIL intervention,the increase expression of iNOS,active TACE,NLRP3 protein and m RNA-TNF-a induced by CSE could be significantly reversed;After intervention with ODQ,KT5823 and TAPI-1could significantly reverse the increase expression of active TACE,NLRP3 protein and m RNA-TNF-a induced by CSE,but it cannot reverse the effect of CSE on iNOS expression;After intervention with MCC950,it could significantly reverse the increase expression of NLRP3 expression induced by CSE,but could not reverse the effects of CSE on the expression of iNOS,active TACE and m RNA-TNF-a.Conclusion : Cigarette smoke exposure induced the expression of iNOS,then further activated the sGC/cGMP/PKG/TACE/TNF-a axis and regulated the activation of NLRP3 inflammasome,which provided a new pathological mechanism for elucidate the cardiac fibrosis induced by cigarette smoke exposure.