| Objective:We established mice models by ambient PM2.5 real-time exposure system to explore the adverse effects of PM2.5 on cardiac function in mice and its related mechanism.Methods:Forty-eight healthy male C57BL/6J mice were divided randomly into three groups and exposed to filtered air?FA?,unfiltered air?UA?and concentrated air PM2.5?CA?for 8 or 16 weeks,6 h per day,7 d per week,respectively.All mice were inhaled clean air for the rest of the time.The FA and UA mice were exposed to air in the chamber with or without three-layers of high-efficient particulate air?HEPA?filters,respectively.CA mice were exposed to concentrated PM2.5 by the PM2.5 concentration enrichment system.We monitored continuously the distribution of the particulate matters by ultraviolet aerodynamic particle sizer and the real-time concentrations of PM2.5 in exposure chambers by aerosol monitor.Simultaneously,the temperature and humidity in chambers were monitored continuously.The cardiac structure and function of mice were evaluated by echocardiography and electrocardiogram.The pathological changes,morphological structure and fibrosis of the heart were observed by HE staining,WGA staining and Masson staining.Immunofluorescence was used to detect the expression of macrophage marker?F4/80?and fibrosis specific protein??-SMA?.qRT-PCR was used to detect the expression of cardiac hypertrophy marker?Anf m RNA,Bnp m RNA,?-mhc m RNA?,fibrosis marker?Col1?m RNA,Col3?m RNA?,macrophage marker?F4/80 m RNA?and pro-inflammatory cytokines?Mcp-1m RNA and Ifn-?m RNA?.We also detected the expression of PI3K/Akt/Fox O signal pathway and inflammation-related proteins?TNF-?,IL-1?,IL-6,IL-8?in heart tissue by Western blot.Results:1. Changes of temperature and humidity,particle size distribution and PM2.5 concentration in the exposed chamberDuring the exposure period,the average temperature in different exposed cavities were 22.89±1.11??21.65±1.03??22.24±1.98?,respectively.And the average humidity were 45.96±9.71%RH,46.02±8.82%RH and47.24±8.52%RH,respectively.The size distribution of particles was measured.As a result,no particulate matter was found in the FA.90.46%and 99.48%particles in the UA and CA chamber were less than 2.5?m?aerodynamic diameter?,respectively.For 8 weeks of exposure,the average PM2.5 concentrations were 0,94.84and 900.21?g/m3 in the FA,UA and CA exposure chambers,respectively.For16 weeks of exposure,the average PM2.5 concentrations were 0,86.78 and671.87?g/m3 in the FA,UA and CA exposure chambers,respectively.2. Cardiac dysfunction in mice after PM2.5 exposureThe results of echocardiography showed that for 16 weeks exposure,ejection fraction?EF?and short axis fractional shortening?FS?significant decreased in a dose-dependent manner?P<0.05?,respectively.For 8 and 16weeks PM2.5 exposure,stroke volume?SV?of mice decreased significantly in a dose-dependent manner?P<0.05?,the thickness of diastolic left ventricular anterior wall?LVAW,d?and systolic left ventricular anterior wall?LVAW,s?of CA mice was significantly thicker than that of FA mice?P<0.05?.The SV of CA mice exposed for 16 weeks was significantly lower than that of 8 weeks exposure?P<0.05?.For ECG recordings,the QRS interval of CA mice after PM2.5 exposure for 16 weeks was significantly prolonged compared with the FA mice?P<0.05?.3. Histopathological changes of heart in mice after PM2.5 exposureFor 8 and 16 weeks PM2.5 exposure,the myocardial fibers in mice were thicker and disordered,infiltrated inflammatory cells were obvious by HE staining.The cross-sectional area?CSA?of cardiomyocyte in CA mice were significantly increased in a dose-dependent manner?P<0.05?by WGA staining.The myocardial collagen fibers in CA and UA mice were significantly increased in a dose-dependent manner?P<0.05?,and there was a time-effect relationship?P<0.05?by Masson staining.4. Cardiomyocyte hypertrophy in mice after PM2.5 exposureThe results of qRT-PCR showed that the cardiac hypertrophy markers?Anf,Bnp and?-mhc?increased in a dose-dependent manner after PM2.5exposure for 8 and 16 weeks?P<0.05?,and there was a time-effect relationship?P<0.05?.Western blot showed that after PM2.5 exposure for 16weeks,the PI3K expression of the heart decreased in a dose-dependent manner?P<0.05?,the ratio of p-Akt/Akt in CA group was significantly decreased compared with FA mice?P<0.05?,the ratio of p-Fox O1/Fox O1 was markedly increased in heart of CA mice in a dose-dependent manner?P<0.05?.5. Cardiac fibrosis in mice after PM2.5 exposureThe results of immunofluorescence determination showed the specific macrophage marker F4/80 expression in heart of CA mice was significant increased in a dose-dependent manner?P<0.05?.Compared with FA mice,the expression of?-SMA in CA mice was significantly increased?P<0.05?.qRT-PCR showed that Col1?and Col3?m RNAs levels were remarkably increased in a dose-dependent manner after PM2.5 exposure?P<0.05?,and there was a time-effect relationship?P<0.05?.Furthermore,the expression of F4/80 and inflammation cytokines?Mcp-1,Ifn-??were significantly increased after PM2.5 exposure for 16 weeks?P<0.05?.For 16 weeks exposure,the TNF-?,IL-1?,IL-6 and IL-8 expression in heart of CA mice were markedly increased compared with FA and UA mice,respectively?P<0.05?,and the TNF-?,IL-1?and IL-6 expression in UA mice showed significant increases compared to FA?P<0.05?.Conclusions:1.PM2.5 exposure induced cardiac dysfunction,which might relate with myocardial hypertrophy and myocardial fibrosis in a time-and dose-dependent manner.2.Cardiac hypertrophy induced by PM2.5 exposure might be related to the increased level of Fox O1 phosphorylation by PI3K/Akt-mediated.3.Cardiac fibrosis induced by PM2.5 exposure might be due to inflammation infiltration by macrophage activation. |
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