| Background:Atrial fibrillation(AF)is the most common tachyarrhythmia in clinic,which has high disability and mortality.It is one of the most important fatal diseases and brings great harm to human health.Current studies generally believe that atrial structural remodeling plays an important role in the occurrence and development of atrial fibrillation,and structural remodeling is marked by myocardial fibrosis.Many studies have found that interleukin-17A(IL-17A)can stimulate the release of pro-inflammatory cytokines.The effector protein of IL-17A/IL-17RA pathway is matrix metalloproteinases,which is also an important part of atrial structural remodeling.Inflammatory factors can participate not only in the inflammatory mechanism of atrial fibrillation,but also in the pathogenesis of atrial fibrillation fibrosis.Our previous studies found that IL-17 pathway is highly enriched in animal models of atrial fibrillation,indicating that IL-17 pathway plays an important role in the pathogenesis of atrial fibrillation and may be a potential target for the treatment of atrial fibrillation.The specific role and mechanism of IL-17A/IL-17RA pathway in atrial fibrillation myocardial fibrosis is worthy of further study.Objective:In order to explore the expression of IL-17A/IL-17RA signaling pathway in atrial fibrosis in patients with atrial fibrillation,in the first chapter of this study,the degree of inflammatory infiltration and fibrosis in left atrial appendage tissue of patients with AF and SR were observed by staining,and the expression levels of key genes and proteins in this pathway were compared.In order to further explore the role of IL-17A/IL-17RA signal pathway in the process of atrial fibrillation fibrosis,in the second chapter,we established a rat model of aseptic pericarditis,constructed adenovirus vector transfection target gene to regulate the expression of IL-17RA,and used esophageal pacing electrode to induce atrial fibrillation in an attempt to observe the changes of various indexes under different expression levels of IL-17RA.In the third chapter,in order to further explore the effect of IL-17A/IL-17RA signal pathway on rat cardiac fibroblasts,the target gene was transfected into rat cardiac fibroblasts by constructing adenovirus vector to regulate the expression of IL-17RA,and then the proliferation and migration ability of cardiac fibroblasts at different levels of IL-17RA expression was observed.The expression of corresponding genes and proteins in IL-17A/IL-17RA signal pathway was determined.To further explore the mechanism of IL-17A/IL-17RA signal pathway in atrial fibrillation fibrosis at cellular level.Materials and Methods:Ⅰ Expression of IL-17A/IL-17RA signal pathway in atrial fibrosis in patients with atrial fibrillation1.The specimens of left atrial appendage of patients with valvular disease in West China Hospital of Sichuan University were collected and the baseline data were collected.16 patients with atrial fibrillation were in the experimental group(AF group)and 7 patients with sinus rhythm were in the control group(SR group).The peripheral venous blood and left atrial appendage tissue of each patient were collected and preserved before and during operation;2.Left atrial appendage specimens were stained with HE to evaluate the degree of inflammatory infiltration,Masson staining to evaluate the degree of fibrosis,immunohistochemical staining to evaluate the expression of IL-17RA,and immunofluorescence to evaluate the expression of IL17-RA,MMP-2 and MMP-9.The ratio of Th17 cells to CD4+T cells in peripheral blood of the two groups was detected by flow cytometry,and the concentrations of serum IL-17A,IL-1β,IL-6,MMP-2 and MMP-9 were measured by ELISA method.RT-PCR and Westernblot were used to detect the expression of mRNA and protein related to IL-17A/IL-17RA pathway in left atrial appendage.Ⅱ The regulatory role of IL-17A/IL-17RA signal pathway in the rat model of aseptic pericarditis1.70 SD rats were randomly divided into 7 groups:Blank group(blank control group,n=10);Sham group(sham operation group,n=10);AF group(aseptic pericarditis model group,n=10);PC group(overexpression empty vector group,n=10);UP group(IL-17RA overexpression group,n=10);NC group(silent empty vector group,n=10);S group(IL-17RA silence group,n=10);2.The rat model of aseptic pericarditis was established,and adenovirus was injected into the left atrial appendage in situ to transfect the corresponding target gene.on the 4th day after operation,atrial fibrillation was induced by programmed stimulation of esophageal pacing electrode and the data of ECG baseline indexes were collected.the induction times of atrial fibrillation,the induction rate of atrial fibrillation and the maintenance time of atrial fibrillation were recorded;3.The rats in each group were killed immediately,and the samples of peripheral blood and left atrial appendage were taken;4.The concentrations of IL-17A,IL-1β,IL-6,MMP-2 and MMP-9 in serum were measured by ELISA method,the degree of inflammatory infiltration was evaluated by HE staining,the degree of fibrosis was evaluated by Masson staining,and the expression of IL17-RA,MMP-2 and MMP-9 was evaluated by immunohistochemical staining and immunofluorescence technique.RT-PCR and Westernblot were used to detect the expression of mRNA and protein related to IL-17A/IL-17RA pathway in left atrial appendage.Ⅲ The mechanism of IL-17A/IL-17RA signaling pathway in myocardial fibrosis in rat cardiac fibroblasts1.Cardiac fibroblasts from neonatal SD rats were isolated and subcultured;2.The phenotype of cardiac fibroblasts was identified;3.Adenovirus infected cells,up-regulated or silenced the expression of IL-17RA;4.MOI gradient was set up to observe the expression of green fluorescent protein under different MOI.Combined with CCK8 kit to detect cell proliferation-toxicity,the best MOI was selected;5.The growth and migration ability of cardiac fibroblasts infected by adenovirus was determined by scratch test and Transwell test;6.The phenotypic transformation of cardiac fibroblasts was detected by immunofluorescence staining of a-SMA,the expression of mRNA and protein related to IL-17A/IL-17RA pathway in left atrial appendage was detected by RT-PCR and Westernblot,and the concentration of MMP-2 and MMP-9 in the supernatant was measured by ELISA method.Results:I Expression of IL-17A/IL-17RA signal pathway in atrial fibrosis in patients with atrial fibrillation1.A total of 23 cases were included,including AF group(n=16)and SR group(n=7).Compared with SR group,LAD was significantly increased and EF decreased significantly in AF group;2.The ratio of Th17 cells to CD4+T cells in AF group was higher than that in SR group.Masson staining results showed that the degree of fibrosis in left atrial appendage tissue in AF group was significantly higher than that in SR group(P<0.05).The results of Masson staining showed that the degree of fibrosis in left atrial appendage tissue in AF group was significantly higher than that in SR group(P<0.05).The results of HE staining showed that the degree of inflammatory infiltration in left atrial appendage tissue in AF group was significantly higher than that in SR group;3.The results of immunofluorescence tissue staining showed that the levels of MMP-2 and MMP-9 in left atrial appendage tissue in AF group were higher than those in SR group.The results of immunohistochemistry showed that the level of IL-17A in left atrial appendage in AF group was higher than that in SR group;4.ELISA results showed that the contents of peripheral IL-17A,IL-1β and IL-6 in AF group were higher than those in SR group.QRT-PCR results showed that the mRNA levels of IL-1β and IL-6 in left atrial appendage tissue in AF group were higher than those in SR group.Western blot results showed that the expression of IL-17A,IL-17RA,p-P38,p-P42,p-P65,MMP-2,MMP-9,Collagen Ⅰ and collagen Ⅲ in IL-17A/IL-17RA signal pathway in AF group was higher than that in SR group.Ⅱ The regulatory role of IL-17A/IL-17RA signal pathway in the rat model of aseptic pericarditis1.On the 4th day after operation,all the surviving rats were normal in activity and diet,and the rats in the blank group were not in good spirits except the blank group.There was no death in Blank group,no death in Sham group,2 deaths in AF group,no death in PC group,3 deaths in UP group,1 death in NC group and 2 deaths in S group;2.After programmed stimulation of transesophageal pacing electrode,AVERP and AERP were significantly shortened(P<0.05).AVERP and AERP in UP group were significantly shorter than those in AF group(P<0.05).Compared with AF group,AVERP and AERP in);S group recovered significantly(P<0.05);3.Compared with sham group,the number of atrial fibrillation,the induction rate of atrial fibrillation and the total duration of atrial fibrillation in AF group were significantly increased after rapid stimulation of transesophageal pacing electrode(P<0.05).Compared with AF group,the number of atrial fibrillation,induction rate of atrial fibrillation and total duration of atrial fibrillation in UP group were significantly increased(P<0.05).Compared with AF group,the number of atrial fibrillation,the induction rate of atrial fibrillation and the total duration of atrial fibrillation in S group were significantly lower than those in S group(P<0.05);4.Masson staining results showed that the degree of fibrosis of left atrial appendage in AF group was significantly higher than that in Sham group,and that in UP group was significantly higher than that in AF group,and the degree of fibrosis in S group was significantly lower than that in AF group(P<0.05).The results of HE staining showed that the local inflammatory reaction in AF and UP groups was severe,a large number of inflammatory cells infiltrated and myocardial tissue was disordered,while in S group,the inflammatory reaction was slighter and inflammatory cell infiltration decreased;5.The results of immunofluorescence tissue staining and immunohistochemical staining showed that the expression of IL-17RA,MMP-2 and MMP-9 in AF group was significantly higher than that in Sham group,the expression in UP group was significantly higher than that in AF group,and the expression in S group was significantly lower than that in AF group;6.The results of qRT-PCR showed that the expression level of IL-17RA mRNA in AF group was significantly higher than that in Sham group,that in UP group was significantly higher than that in AF group,and that in S group was significantly lower than that in AF group,that in AF group was significantly higher than that in Sham group,that in UP group was significantly higher than that in AF group,and that in S group was significantly lower than that in AF group(P<0.05).The expression level of IL-1β in AF group was significantly higher than that in Sham group,UP group was significantly higher than that in AF group,and that in S group was significantly lower than that in AF group.Westernblot showed that the expression of IL-17A,IL-17RA,p-P38,p-P42,p-P65,MMP-2,MMP-9,Collagen Ⅰ and collagen Ⅲ in AF group was significantly higher than that in Sham group,that in UP group was significantly higher than that in AF group,and that in S group was significantly lower than that in AF group.Ⅲ The regulatory role of IL-17A/IL-17RA signal pathway in the proliferation and differentiation of cardiac fibroblasts1.The specific expression of FSP-1 and Vimentin proteins was observed by immunofluorescence staining,which confirmed that the isolated and cultured cells were cardiac fibroblasts;2.24 hours after adenovirus infection,green fluorescent protein and cell growth were observed.It was found that the proportion of cells expressing green fluorescent protein was the highest in MOI=100.The proliferation-toxicity of cardiac fibroblasts was detected by CCK8 kit,but the growth of cardiac fibroblasts was significantly inhibited in MOI=150,and the optimum MOI was 100.3.Both scratch test and Transwell test showed that the mobility of IL-17A group was higher than that of Blank group,that of UP group was higher than that of IL-17A group,and that of S group was lower than that of IL-17A group.4.After immunofluorescence staining,the green fluorescence intensity in AF group was significantly stronger than that in IL-17A group,and that in UP group was significantly stronger than that in AF group,and that in S group was significantly weaker than that in AF group(P<0.05).5.Verified by qRT-PCR,the expression level of IL-17RA mRNA in IL-17A group was significantly higher than that in Blank group,that in UP group was significantly higher than that in IL-17A group,that in S group was significantly lower than that in IL-17A group,that in IL-17A group was significantly higher than that in Blank group,that in UP group was higher than that in AF group,and that in S group was significantly lower than that in IL-17A group.The expression of IL-1β mRNA in IL-17A group was significantly higher than that in Blank group,that in UP group was significantly higher than that in IL-17A group,and that in S group was significantly lower than that in IL-17A group,and that the expression of IL-17RA,p-P38,p-P42,p-P65,MMP-2,MMP-9,Collagen Ⅰ and Collagen Ⅲ in IL-17A group was significantly higher than that in Blank group,UP group was significantly higher than IL-17A group,and S group was significantly lower than IL-17A group.Conclusion:1.The proportion of Th17 cells increased in patients with atrial fibrillation,the level of IL-17A increased in patients with atrial fibrillation,and the expression of IL-17A/IL-17RA signal pathway increased in patients with atrial fibrillation;2.The rat model of aseptic pericarditis was established successfully;the adenovirus vector was successfully transfected with the target gene;IL-17A/IL-17RA signal pathway can directly participate in the process of atrial fibrillation fibrosis through inflammatory mechanism and regulating the activities of MMP-2 and MMP-9;3.IL-17A can promote the transformation of fibroblasts to myofibroblasts and promote atrial fibrillation,IL-17A activates IL-17A/IL-17RA signal pathway by acting on the IL-17RA of cardiac fibroblasts,and directly participates in the process of atrial fibrillation by inflammatory mechanism and regulating the activities of MMP-2 and MMP-9. |
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