| Despite recent advances in the treatment of cardiovascular diseases(CVD),this devastating disease remains a leading cause of morbidity and mortality and imposes a significant economic burden worldwide.Myocardial fibrosis(MF)is the common pathology of many cardiovascular diseases at the end stage,hence,effectively preventing or reversing myocardial fibrosis is an important strategy for adverse cardiovascular events,including heart failure,arrhythmia and sudden cardiac death.While the mechanism of myocardial fibrosis remains unclear,it is urgent to explore it from a new perspective,thereby to reduce the occurrence of cardiovascular events.we developed a rat model of maternal non-bacterial inflammatory response induced by lipopolysaccharide(LPS)during early and mid-pregnancy where the offspring developed elevated blood pressure as early as 6 weeks of age,which progressively increased with age.Furthermore,cardiac hypertrophy and cardiac insufficiency was detected in 16 and 32-week-old offspring,respectively.Given the persistent increase of ventricular afterload due to high blood pressure,which can induce a series of pathophysiological changes,such as myocardial fibrosis,ventricular remodeling and heart failure,we speculated that maternal LPS exposure during pregnancy may cause myocardial fibrosis in offspring rats.As the membrane channel for substance exchange,gap junction plays a pivotal role in cardiac function and connexin43(Cx43)is the main connexin in heart.In the present study,the rat model of maternal LPS exposure during pregnancy was employed to address the impact of prenatal inflammation on myocardial fibrosis in offspring and to further assess the gap junction,Cx43,autophagy and DNA methylation involved in this pathology.Methods1.The animal modelthe pregnant rats were randomly divided into 2 groups,i.e.,control group and LPS group.The rats in these two groups were administered intraperitoneally with normal saline 0.5 ml and 0.79 mg/kg LPS respectively,on gestation days 8,10 and 12.Neonatal rats were cared for by their mothers until 4 weeks of age,after which they were fed standard rat chow.2.Index detection in offspring ratThe body weight and body length were measured weekly,and the food intakewas measured every other day,respectively,from 4 weeks of age in offspring rats.Histopathological alteration of the left ventricle was observed by Masson staining and transmission electron microscopy,?-SMA and Cx43 immunofluorescent patterns were detected by confocal laser-scanning microscope,Cx43,LC3,DNMT 1,DNMT 3A and DNMT 3B in the left ventricle,whose m RNA expression were analyzed by Real Time-PCR and proteins expression were performed by western blotting in 8-week-old and 16-week-old offspring.The primary cardiac fibroblasts were cultured and stimulated with LPS(10?g/ml),Ang II(20?M),CBX(400?M),LPS(10?g/ml)+Ang II(20?M),LPS(10?g/ml)+CBX(400?M)and Ang II(20?M)+CBX(400?M),respectively,for 24 h.Cell morphology was observed by microscopy and Cx43,LC3,DNMT 1,DNMT 3A and DNMT 3B,whose mRNA expression were analyzed by Real Time-PCR and proteins expression were performed by western blotting.Results1.Compared to Con,the food intake(P<0.001),Lee's index(P<0.05)and body weight(P<0.01)in LPS group were all increased significantly from 4 to 8-week-old.In addition,prenatal LPS exposure induced obviously long body length compared with Con.2.The myocardial fibers were thickened and arranged in disorder,and there was a lot of collagen deposition in the interstitium and around vessels,which can be observed in LPS exposure group at 8 weeks of age under light microscope while compared with control group in the Masson staining.As well,the collagen volume fraction(CVF)index increased significantly.Furthermore,the injuries became distinctive in LPS offspring at 16 weeks of age.3.Left ventricle ?-SMA immunofluorescence staining showed that compared with Con group,?-SMA expression in LPS group was obviously higher and Cx43 expression in LPS group was obviously reducer in the quantity and intensity of fluorescence both at 8 and 16 weeks of age.4.Compared with control group,the left ventricle of LPS group exhibited lesions,the number of gap junction was decreased as well as the number of intracellular autophagosomes was increased.5.In 8-week-old offspring,the m RNA expression of Cx43(P<0.05),DNMT 1(P<0.05)and LC3(P<0.001)was decreased obviously.In 16-week-old rats,the m RNA expression of Cx43(P<0.01)and DNMT 1(P<0.05)was significantly increased than that of Con group.However,the m RNA expression of LC3 was significantly decreased(P<0.01).There was no statistically significant difference in m RNA expression of DNMT 3A and DNMT 3B between the groups.6.The protein expression of Cx43(P<0.05),LC3(P<0.05)and DNMT 1(P<0.01)in 8-week-old rats was decreased significantly than that in Con group.The protein expression level of Cx43 at 16 weeks of age was lower than that of Con group(P<0.05).The protein expression of LC3 and DNMT 1 was obviously increased than that of Con group(P<0.05).The protein expression of DNMT 3A and DNMT 3B was not statistically different between groups.7.Fibroblasts stimulated by LPS,AngII,CBX,LPS+Ang II,LPS+CBX,Ang II+CBX appeared to differentiate and increased in sheet-like protrusions,and complex network structures were formed between the cells.The cell gap in CBX group,LPS+CBX group and Ang II+CBX group was wider than that of other groups.8.The mRNA expression of Cx43 in primary cardiac fibroblasts was significantly decreased in LPS group(P<0.001),AngII group(P<0.01)and CBX group(P<0.01)tha n that in Con group.Compared with LPS group,the m RNA expression of Cx43 was significantly reduced in LPS+CBX group(P<0.05).The group of Ang II+CBX's Cx43 mRNA expression was significantly lower than in Ang II group(P<0.05).The mRNA expression of Cx43 have no significant difference between LPS group and LPS+Ang II group,Ang II group and LPS+AngII group,CBX group and LPS+CBX group,CBX group and Ang II+CBX group.Compared with Con group,the mRNA expression of LC3 was significantly lower in LPS group(P<0.01),AngII group(P<0.05)and CBX group(P<0.01).The expression of LC3 mRNA was significantly decreased in the LPS+CBX group than that in the LPS group(P<0.05).In Ang II+CBX group,the m RNA expression of LC3 was significantly reducer(P<0.01)than that in AngII group.Compared with CBX group,the mRNA expression of LC3 was significantly lower in LPS+CBX group(P<0.05).The mRNA expression of LC3 have no significant difference between LPS group and LPS+Ang II group,AngII group and LPS+Ang II group,CBX group and AngII+CBX group.The expression of DNMT 1 m RNA was significantly increased in the LPS group(P<0.01)than that in the Ang II group(P<0.05),but decreased DNMT 1 m RNA expression in the CBX group(P<0.05).Compared with Con group,the mRNA expression of DNMT 1 was significantly lower in CBX group(P<0.05).In LPS+CBX group,the mRNA expression of DNMT 1 was significantly reducer(P<0.01)than that in LPS group.Compared with Ang II+CBX group,the mRNA expression of DNMT 1 was significantly lower in Ang II group(P<0.05).The m RNA expression of DNMT 1 have no significant difference between LPS group and LPS+Ang II group,AngII group and LPS+Ang II group,CBX group and LPS+CBX group,CBX group and AngII+CBX group.Compared with Con group,the mRNA expression of DNMT 3A in CBX group was significantly decreased(P<0.01).Compared with LPS group,the m RNA expression of DNMT 3A was significantly lower in LPS+CBX group(P<0.01).In Ang II+CBX group,the m RNA expression of DNMT 3A was significantly reducer(P<0.01)than that in AngII group.The m RNA expression of DNMT 3A have no significant difference among Con group vs LPS group and Con group vs Ang II group,LPS group vs LPS+AngII group,Ang II group vs LPS+Ang II group,CBX group vs LPS+CBX group and CBX group vs Ang II+CBX group.The DNMT 3B m RNA expression was decreased in AngII group(P<0.05)and CBX group(P<0.001)compared with Con group.In LPS+CBX group,the mRNA expression of DNMT 3B was significantly reducer(P<0.001)than that in LPS group.Compared with Ang II group,the mRNA expression of DNMT 3B in LPS+Ang II group was significantly increased(P<0.05).The m RNA expression of DNMT 3B have no significant difference among Con group vs LPS group and LPS group vs LPS+AngII group,Ang II group vs Ang II+CBX group,CBX group vs LPS+CBX group and CBX group vs AngII+CBX group.9.Compared with Con group,Cx43 protein expression was significantly decreased in LPS group(P<0.01),Ang II group(P<0.05)and CBX group(P<0.01).In the Ang II+CBX group,the Cx43 protein expression was significantly lower than in Ang II group(P<0.05).The Cx43 protein expression was no significant difference between LPS group and LPS+AngII group,LPS group and LPS+CBX group,Ang II group and LPS+AngII group,CBX group and LPS+CBX group,CBX group and Ang II+CBX group.The LC3 II/I protein expression was significantly decreased in LPS group(P<0.05),AngII group(P<0.05)and CBX group(P<0.01)compared with Con group.Compared with LPS group,the protein expression of LC3II/I was significantly higher in LPS+CBX group(P<0.05).The LC3II/I protein expression was no significant difference between LPS group and LPS+Ang II group,Ang II group and LPS+Ang II group,Ang II group and AngII+CBX group,CBX group and LPS+CBX group,CBX group and Ang II+CBX group.The expression of DNMT 1 protein in LPS group and AngII group was increased than that in Con group(P<0.05).In LPS+CBX group,the protein expression of DNMT 1 was significantly reducer(P<0.05)than that in LPS group.Compared with AngII group,the protein expression of DNMT 1 was significantly lower in Ang II+CBX group(P<0.01).The DNMT 1 protein expression have no significant difference between Con group and CBX group,LPS group and LPS+Ang II group,Ang II group and LPS+Ang II group,CBX group and LPS+CBX group,CBX group and Ang II+CBX group.Compared with CBX group(P<0.05),the protein expression of DNMT 3A was decreased significantly.In Ang II+CBX group,the protein expression of DNMT 3A was significantly reducer(P<0.05)than that in AngII group.The DNMT 3A protein expressionhave no significant difference among Con group vs LPS group and Con group vs Ang II group,LPS group vs LPS+Ang II group,LPS group vs LPS+CBX group,Ang II group vs LPS+Ang II group,CBX group vs LPS+CBX group and CBX group vs AngII+CBX group.The DNMT 3B protein expression was significantly decreased in the CBX group(P<0.01)than that in the Con group.In Ang II+CBX group,the protein expression of DNMT 3B was significantly reducer(P<0.05)than that in Ang II group.Compared with CBX group,the protein expression of DNMT 3B in LPS+CBX group was significantly increased(P<0.05).Theprotein expression of DNMT 3B have no significant difference among Con group vs LPS group,Con group vs Ang II groupand LPS group vs LPS+Ang II group,LPS group vs LPS+CBX group,Ang II group vs LPS+AngII group,CBX group vs Ang II+CBX group.Conclusion1.Maternal LPS exposure during pregnancy leads to myocardial fibrosis in offspring rats.2.Alteration of gap junction and abnormal expression of Cx43 in left ventricle may be related to myocardial fibrosis in offspring rats.Additionally,autophagy may mediate the down-regulated expression of Cx43.3.DNA methylation induced by maternal LPS exposure during pregnancy should participate in myocardial fibrosis through direct or indirect pathway in offspring rats. |
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