SFR reduced myocardial hypertrophy caused by pressure overload.
A pressure overload model of rats was constructed firstly by the AB constriction method. Then, rats in the experimental group were treated with different concentrations of SFR (1, 2 and 4 mg/kg/day); After six weeks, the hearts were obtained from rats, the heart weight and tibia length of rat were measured, heart mass/ tibin length (mg/ cm) was calculated, then the heart was stained via HE staining to observe cardiac morphology, the diameter of myocardial fibers was quantitatively measured to evaluate myocardial hypertrophy.
The results showed that, the average diameter of myocardial cells and heart mass/ tibin length (mg/ cm) increased in AB group, and the mRNA expression of cardiac hypertrophy markers ANP and BNP also were significantly increased at the same period compared with Sham group, the difference was statistically significant (n=6, #p<0.05) (Figure 1). That was, a pressure overload myocardial hypertrophy model was successfully constructed.
Compared with the AB group, the diameter of myocardial fibers and heart mass/ tibin length (mg/ cm) were decreased in SFR 1, SFR 2, and SFR 4 groups, and the mRNA expression of ANP and BNP also were reduced, the difference was statistically significant (n=6, *p<0.05) (Figure 1). It showed that treatment with different concentrations of SFR alleviated the degree of myocardial hypertrophy in the process of pressure overload.
SFR improved cardiac function during the process of pressure overload.
In order to understand the influence of SFR on the cardiac function during the process of pressure overload, Cardiac Echocardiography was used to detect the cardiac function indexes of left ventricular end-diastolic posterior wall thickness (LVPWd), left ventricular end-systolic posterior wall thickness (LVPWs), left ventricular ejection fraction (LVEF) in each group of rats. The results showed that LVEF decreased in AB group compared with the Sham group, while LVPWs and LVPWd increased, the difference was statistically significant (n=8, #p<0.05) (Figure 2), The change of heart rates (HR) was not statistically significant. This showed that the cardiac function was reduced in the process of pressure overload.
Compared with the AB group, the LVPWs and LVPWd decreased in SFR 1, SFR 2, and SFR 4 groups, LVEF increased, and the difference was statistically significant (n=8, *p<0.05) (Figure 2); The results showed that different concentrations of SFR could protect heart function during pressure overload and this protective effect increased with the increased of drug concentration.
SFR reduced apoptosis during pressure overload induced- myocardial hypertrophy.
In order to reveled the effect of SFR on myocardial apoptosis during pressure overload, the mRNA and protein expressions of Capase-3 and Capase-9 were detected by PCR and WB. Studies have shown that the protein and mRNA expression of Capase-3 and Capase-9 in the AB group increased compared with the Sham group, and the difference was statistically significant; (n=6, #p<0.05) (Figure 3). It showed that in the process of pressure overload of rats, the cardiac apoptotic was enhanced. Compared with the AB group, the protein and mRNA of Capase-3 and Capase-9 in the SFR 1, SFR 2, and SFR 4 groups decreased sequentially, and the difference was statistically significant; (n=6, *p<0.05) (Figure 3). It showed that SFR reduced the apoptosis during pressure overload as the concentration of SFR increased.
SFR inhibited inflammation during pressure overload- induced myocardial hypertrophy.
In order to reveled the influence of SFR on myocardial inflammation during pressure overload, The mRNA and protein expressions of inflammatory factors IL-1β and TNFα were detected by PCR and WB. Studies have shown that the protein and mRNA expression of IL-1β and TNFα in the AB group increased compared with the sham group, and the difference was statistically significant; (n=6, #p<0.05) (Figure 4). It showed that the inflammation of the heart was enhanced during the pressure overload of rats. Compared with the AB group, the protein and mRNA of IL-1β and TNFα in the SFR 1, SFR 2, and SFR 4 groups decreased sequentially, and the difference was statistically significant; (n=6, *p<0.05) (Figure 4). It showed that with the concentration of SFR increased, SFR reduced the inflammation of cardiac during pressure overload.
AMPK signaling pathway was activated during pressure overload- induced cardiac hypertrophy.
In order to studied the specific mechanism of SFR on myocardial hypertrophy during pressure overload, AMPK signaling pathway was detected via PCR and WB. Studies have shown that the protein and mRNA expression of p-AMPK, p-ACC in the AB group decreased compared with the Sham group, and the difference was statistically significant (n=6, #p<0.05) (Figure 5). It showed that in the process of pressure overload of rats, the AMPK signal pathway of heart was inhibited. Compared with the AB group, the phosphorylation and mRNA expression of AMPK, ACC in the SFR 1, SFR 2, and SFR 4 groups increased. The difference was statistically significant; (n=6, *p<0.05) (Figure 5). It showed that with the concentration increased of SFR, the phosphorylation of AMPK and ACC enhanced, that is the AMPK pathway was activated.
Inhibition of AMPK signaling pathway reversed the protective effect of SFR on myocardial hypertrophy during pressure overload.
In order to further understand whether SFR protected myocardial hypertrophy by activating AMPK signaling pathway during pressure overload. AMPK inhibitor (Dorsomorphin dihydrochloride, Compound C, CPD) was used for further research. The experiment was divided into four groups: Sham group, AB group, AB+ SFR group, AB+ SFR+ CPD group (2mg /kg SFR was injected internationally), After 6 weeks, the results showed that compared with the AB group, the average diameter of myocardial cells and heart mass/ tibin length (mg/ cm) were decreased, simultaneously, the mRNA expression of ANP and BNP were also reduced in AB+SFR group the difference was statistically significant; (n=6, #p<0.05, *p<0.05)(Figure 6). These results were consistent with previous findings that SFR reduced pressure overload myocardial hypertrophy.
Compared with AB+ SFR group, the average diameter of myocardial cells and and heart mass/ tibin length (mg/ cm) increased, the mRNA expression of ANP and BNP in AB+ SFR+ CPD group also increased, the difference was statistically significant; (n=6, #p<0.05, *p<0.05) (Figure 6). In summary, this showed that inhibition AMPK reversed the protective effect of SFR on myocardial hypertrophy during pressure overload.
Inhibition of AMPK signaling pathway reversed the protective effect of SFR on cardiac function during pressure overload.
The LVPWd, LVPWs, LVEF were detected by Echocardiography (Figure 7). The result showed that compared with the AB group, the LVPWs and LVPWd were decreased in AB+ SFR group, while the LVEF was increased, and the difference was statistically significant (n=8, *p<0.05) (Figure 7); In additions, compared with AB+SFR groups, the LVPWs and LVPWd were increased in the AB+ SFR+CPD group, while the LVEF was decreased, and the difference was statistically significant; (n=8, &p<0.05) (Figure 7). It showed that inhibition of AMPK signaling pathway reversed the protective effect of SFR on cardiac function during pressure overload. That was, SFR improved myocardial hypertrophy by activating AMPK signaling pathway, exerted a protective effect on heart function.