| Myocardial fibrosis?MF?is a common pathological change in various heart diseases that develop to a certain stage.In pathological conditions,myocardial injury is usually accompanied by normal cardiomyocyte necrosis and activation of cardiac fibroblasts?CFs?,which continuously proliferate and release fibrotic factors that cause MF.The occurrence of MF will reduce the compliance of the ventricular wall and restrict the diastolic filling which will further damage myocardial function.Therefore,it is particularly important to reduce and slow down MF and explore possible mechanisms,and it has become a major problem that needs to be solved urgently.When the heart is pathologically stimulated,RAAS is activated to produce angiotensin??Ang??,which induces the occurrence of MF and the accumulation of extracellular matrix collagen.Studies have shown that AT1-AA,angiotensin ? type 1 receptor autoantibody can exert an Ang ?-like agonistic effect by specifically binding to the extracellular second loop of the angiotensin ? type 1 receptor?AT1R-EC??.Whether AT1-AA can also cause myocardial fibrosis and how it affects remains to be further explored.MF is a chronic inflammatory response.When the heart is injured,it will promote the activation and release some factors such as the interleukin family.Among them,interleukin-1?IL-1??is one of the most studied factors which is related to inflammation,and it has an important effect on cardiovascular disease.The IL-1?precursor is cleaved and activated by the IL-1?converting enzyme?caspase1?when it was injured.Caspase-1 is activated by the NLRP3 inflammasome,which cause two adjacent caspase 1 to undergo autolysis and cleaved into P10 and P20 fragments which can exert biological activity.Studies have found that Ang?can induce inflammatory response and IL-1?secretion.So,can AT1-AA also induce IL-1?secretion,whether NLRP3 inflammasome are involved,and how does it affect MF?Mammalian rapamycin target protein?mTOR?is a homologue of yeast rapamycin target protein,which can promote the development of myocardial fibrosis mainly by regulating collagen secretion.In many diseases,mTOR can activate the expression of NLRP3 inflammasome,so does mTOR have effect on the activation of AT1-AA-induced MF and NLRP3 inflammasome?Therefore,this study explored whether the long-term presence of AT1-AA can induce MF and whether the mTOR-NLRP3 inflammsome-IL-1?pathway is involved in this process.Objective:1.Establish AT1-AA positive rat model and observe the changes of cardiac morphology and function,mTOR,NLRP3 inflammasome and IL-1?in rats.2.Confirm the function of mTOR-NLRP3-IL-1?pathway in AT1-AA-induced MF.Methods:1.Modeling of AT1-AA positive rats:Using adjuvant?complete adjuvant for the first time,incomplete adjuvant for the rest of time?,Na2CO3 solution and biosynthetic AT1R-EC? antigen peptides which are formulated according to the ratio to actively immunize SD rats,whereas the Vehicle group was treated with normal saline in adjuvant.The immunization method is:subcutaneous injection on the back?the first injection was a subcutaneous multi-point injection,and the subsequent strengthening of the immune was a subcutaneous single-point injection?.2.Separation and purification of AT1-AA.3.Small animal ultrasound to detect damage to heart structure and function.4.HE staining was used to observe changes in cardiac structure.5.Masson staining to detect collagen deposition in the heart.6.Immunohistochemical staining was used to observe the expression levels of collagen?,Collagen?and NLRP3 in cardiac collagen.7.Western blot to detect the expression levels of p-mTOR/mTOR,NLRP3,ASC,Caspase1 protein.8.ELISA to detect the titer of AT1-AA and IL-1?secretion.9.Immunofluorescence staining to detect collagen?and collagen?expression of myocardial fibroblast cells.Results:1.Successfully established AT1-AA positive rat modelSD rats were actively immunized with the artificially synthesized antigen peptides,twice per week,and rat serum was collected incidentally during the immunization.ELISA experiments showed that compared with the Vehicle group,the antibody titer in rat serum increased gradually with the immunization time.The titer increased significantly at the 2nd week?P<0.05?,and the AT1-AA antibody titer reached to the peak at the 8th week then maintained this peak value?P<0.01?.The above results suggested that the AT1-AA positive rat model has been successfully constructed?fig.1?.2.Myocardial fibrosis occurred in AT1-AA positive ratsUltrasound results showed that the heart structure of AT1-AA positive rats was damaged at 12 weeks compared with Vehicle group.The specific manifestations were decreased EF and FS which are indicators of cardiac function and IVSd and IVSs which are indicators of cardiac structure?P<0.05?.By 16 weeks,the damage of heart's strcture and function had reduced?Fig.2A-2E?.HE staining showed that the myocardial fibers in the Vehicle group did not show obvious rupture,and the cardiomyocytes were dense,arranged neatly,and the nucleus size was uniform.The cardiac structure of AT1-AA positive rats did not change significantly in the early stage of immunization.At 12 week of immunization,the myocardial fibers of the rats were ruptured,the cells were arranged disorderly,the rat myocardial cells were enlarged,the nucleus size was significantly different,and some myocardial cells were lysed.Heart damage was more significant at 16 week of immunization?Fig.3?.Masson staining showed that the cardiomyocytes of the rats in the Vehicle group were neatly arranged,and there was no obvious collagen fibrosis or only a small amount of collagen deposition in the myocardial matrix.AT1-AA positive rats started collagen deposition from the 4th week.With the extension of the immunization time,to the 12th and16th weeks,the myocardial cell space increased and the collagen fibers deposited in the myocardial space increased?P<0.05,Fig.4?.The results of immunohistochemical staining?IHC?showed that compared with Vehicle group,with increasing of immunization time,both type I collagen and type ?I collagen of AT1-AA positive rats increased.And the Col?/Col?ratio increased?P<0.01,Fig.5A,5B?.These results suggested that AT1-AA can promote myocardial fibrosis.AT1-AA positive rats have impaired heart structure,function and collagen deposition.3.AT1-AA promoted cardiac NLRP3 inflammasome expression and IL-1?secretionImmunohistochemical staining results showed that compared with Vehicle group,the expression of NLRP3 protein increased with increasing of immunization time?P<0.05,Fig.6A,6B?.Western blot results showed that compared with Vehicle group,the expressions of NLRP3,ASC,P10 and caspase1 which are composed NLRP3 inflammasome increased with increasing of immune time?P<0.05,Fig.6C-6G?.ELISA results showed that,with increase of immunization time,the secretion of IL-1?in the serum of immunized rats gradually increased?P<0.01,Fig.7?.The above results suggested that AT1-AA can promote the expression of cardiac NLRP3 inflammasome and increase the secretion of IL-1?in serum.4.NLRP3 inflammasome participated in AT1-AA-induced myocardial fibrosisImmunohistochemical staining results showed that,compared with the AT1-AA group,the expression of NLRP3 protein in the heart of the immunized rats which are injected intraperitoneally with the NLRP3 inflammasome inhibitor MCC950 decreased?P<0.05,Fig.8A,8B?.Western blot results showed that compared with the AT1-AA group,the expression of NLRP3 inflammasome in the hearts of rats that immunized intraperitoneally with NLRP3inflammasome inhibitor MCC950 decreased?P<0.05,Fig.8C-8G?.ELISA test results showed that compared with the AT1-AA group,the secretion of IL-1?in the AT1-AA+MCC950 group was significantly reduced?P<0.01,Fig.9?.The above results suggested that MCC950 can inhibit the expression of NLRP3inflammasomes and reduce the secretion of IL-1?.Ultrasound results showed that compared with the AT1-AA group,MCC950 improved the cardiac function and structural damage caused by AT1-AA.The specific manifestation was that EF,IVSs and IVSd were all recovered?P<0.05?.FS was improved but it was not statistically significant?Fig.10A-10E?.The HE staining results showed that compared with the AT1-AA group,the myocardial fiber breakage was significantly improved after injection of MCC950,the myocardial cells were relatively dense,the cells were arranged neatly,the nucleus size was uniform,and the intercellular substance was less?Fig.11?.Masson staining showed that compared with the AT1-AA group,the myocardial cells in the AT1-AA+MCC950 group were stained more neatly,and a smaller amount of collagen was deposited in the myocardial matrix?P<0.05,Fig.12?.IHC staining results showed that,compared with the AT1-AA group,both the type I collagen and the type ?I collagen in the heart tissue of rats in the AT1-AA+MCC950 group decreased,and the ratio of Col?/Col?decreased?P<0.05,Fig.13A,13B?.These results suggested that NLRP3 inflammasomes are involved in AT1-AA-induced myocardial fibrosis.5.Increased expression of mTOR protein in heart tissue of AT1-AA positive ratsWestern blot results showed that compared with the Vehicle group,the expression of p-mTOR/mTOR in the heart of AT1-AA group increased with increasing of immunization time?P<0.05,Fig.14?.The results suggested that AT1-AA can activate the expression of p-mTOR.6.mTOR participated in AT1-AA-induced cardiac NLRP3 inflammasome expression,IL-1?secretion,and collagen deposition.Rat myocardial fibroblasts were isolated and the expression of different groups was detected by Western blot.Compared with the AT1-AA group,the expression of p-mTOR/mTOR and NLRP3 inflammasome proteins was reduced after the addition of mTOR inhibitor RAPA?P<0.05,Fig.15A-15F?.ELISA showed that compared with the AT1-AA group,IL-1?secretion was significantly reduced after RAPA addition?P<0.01,Fig.16?.Immunofluorescence showed that AT1-AA induced collagen type I and type ?I collagen expression was reduced after RAPA addition?Fig.17A,17B?.These results suggested that mTOR was involved in AT1-AA-induced cardiac NLRP3inflammasome expression,the secretion of IL-1?,and collagen deposition.7.AT1-AA induced NLRP3 inflammasome expression,IL-1?secretion and myocardial fibrosis through AT1-RWestern blot results showed that compared with the AT1-AA group,the expression of NLRP3 inflammasomes in the heart of rats with ARB-type TM decreased,and the expression of p-mTOR/mTOR also decreased?P<0.05,Fig.18A-18F?.ELISA results showed that compared with the AT1-AA group,the secretion of IL-1?in the serum of rats in the AT1-AA+ARB group was reduced?P<0.01,Fig.19?.Ultrasound results showed that TM can improve cardiac function and structural damage caused by AT1-AA.Specifically,compared with the AT1-AA group,rats IVSs and IVSd value in the AT1-AA+ARB group were significantly increased,and FS and EF were improved,but there was no statistical difference?P<0.05,Fig.20A-20E?.The results of HE staining showed that TM can improve myocardial structural damage and myocardial fiber breakage induced by AT1-AA?Fig.21?.Masson staining showed that the myocardial cells of the AT1-AA+ARB group were arranged more neatly,and the collagen deposition in the myocardial interstitial tissue was less?P<0.05,Fig.22?.IHC results showed that both type?collagen and type?collagen were reduced in the heart tissue of rats in the AT1-AA+ARB group,and the ratio of Col?/Col?decreased?P<0.05,Fig.23A,23B?.The above results suggested that TM can reduce the expression of NLRP3inflammasomes,IL-1?secretion and myocardial fibrosis induced by AT1-AA.AT1-AA induced NLRP3 inflammasome expression,IL-1?secretion,and enhanced myocardial fibrosis through AT1-R.Conclusion:1.The long-term presence of AT1-AA can lead to myocardial fibrosis,impaired heart structure and function in rats,and can promote the increase of mTOR,NLRP3inflammasome and IL-1?in cardiac tissues.2.mTOR-NLRP3-IL-1?signaling pathway was involved in AT1-AA-induced myocardial fibrosis.3.AT1-AA worked through AT1-R to induce mTOR-NLRP3 inflammasome-IL-1?pathway to induce myocardial fibrosis. |
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