| Purpose:To explore the improvement effect of Shenxian-Shengmai oral liquid on sinoatrial node fibrosis in Sick sinus syndrome（SSS）mice,and further explore its mechanism,so as to provide further evidence support for Shenxian-Shengmai oral liquid in the treatment of SSS.Material and method:In the in vivo experiments,15 mice were randomly divided into three groups:sham operation group（SHAM,n=5）,SSS model group constructed by AngⅡ（SSS,n=5）and Shenxian-Shengmai oral liquid treatment group（SXSM,n=5）.The SSS model was constructed by slowly pumping AngⅡwith a microosmotic pump.The drug was administered by gavage on the second day after operation.The SXSM group was given Shenxian-Shengmai oral liquid,and the other groups were given the same amount of purified water.After 28 days of continuous administration,the comprehensive lead electrocardiogram of mice was recorded and analyzed to evaluate the heart rate changes by biological signal acquisition and processing system;The degree of fibrosis was evaluated by Masson’s trichrome staining and immunofluorescence staining of fibrosis marker protein.In vitro,mouse embryonic fibroblast NIH-3T3 cell line was used to simulate sinoatrial fibroblasts.The cells were divided into four groups:CON,AngⅡ,SXSMs and SC79 groups.CON was the blank control group,and no intervention measures were given.The cells in other groups were treated with AngⅡfor 48 hours after 12 hours of culture.At the same time,SXSMs group was given Shenxian-Shengmai oral liquid,SC79 group was given Shenxian-Shengmai oral liquid and Akt phosphorylation agonist SC79.CCK-8 was used to determine the optimal concentration of Shenxian-Shengmai oral liquid;The degree of fibrosis was evaluated by immunofluorescence staining of fibrosis marker protein;The level of autophagy was evaluated by Monodansylcadaverine（MDC）staining and transmission electron microscopy;The expression levels of Akt/m TOR signaling pathway related proteins Akt,p-Akt,m TOR,p-m TOR and autophagy related proteins beclin-1,p62,LC3-Ⅰand LC3-Ⅱwere detected by Western blotting.Results:1.In vivo experiments（1）Changes of mouse heart rate:the comprehensive lead electrocardiogram of mice showed that:compared with SHAM group,the heart rate of SSS group decreased significantly and the R-R interval was prolonged(##P<0.01);The heart rate and R-R interval in SXSM group were significantly improved(**P<0.01)（2）Changes of sinoatrial node fibrosis in mice:Masson’s trichrome staining and immunofluorescence staining showed that the sinoatrial node fibrosis in SSS group was significantly increased under the action of AngⅡ,accompanied by a large number of fibrosis marker proteins such as Fibronectin,Collagen I in sinoatrial node region(#P<0.05,##P<0.01);Shenxian-Shengmai oral liquid treatment can significantly improve the fibrosis of sinoatrial node in mice and decreased the expression of fibrosis marker proteins such as Fibronectin,Collagen I(**P<0.01).2.In vitro experiments（1）Screening of the optimal concentration of Shenxian Shengmai:When Shenxian-Shengmai oral liquid concentration was 0 ml/L,the cell viability was set at 100%.No cytotoxicity was observed in NIH-3T3 cell lines treated with different concentrations of Shenxian-Shengmai oral liquid for 12h,24h and 48h,and the cell viability reached its maximum at 1 ml/L.（2）Shenxian-shengmai oral liquid improved the fibrosis of NIH-3T3 cells by inhibiting the phosphorylation of AKT.Immunofluorescence staining showed that the fluorescence intensity ofα-SMA,Fibronectin and Collagen I in NIH-3T3 cells treated with AngⅡwas significantly higher than that in CON group(△P<0.05,△△P<0.01).At the same time,compared with AngⅡgroup,Shenxian-Shengmai oral liquid could improve the overexpression ofα-SMA,Fibronectin and Collagen I in NIH-3T3 cells induced by AngⅡ(##P<0.01)and this improvement was blocked by AKT phosphorylation agonist SC79(**P<0.01).（3）Shenxian-Shengmai oral liquid inhibits AKT phosphorylation to activate autophagy of NIH-3T3 cells:the results of Monodansylcadaverine（MDC）staining showed that the autophagy level of NIH-3T3 cells treated with AngⅡwas significantly decreased(△△P<0.01),while the autophagy rate of NIH-3T3 cells in AngⅡgroup was significantly increased by Shenxian-Shengmai oral liquid(##P<0.01),and the activation of autophagy by Shenxian-Shengmai oral liquid was blocked by AKT phosphorylating agonist SC79(**P<0.01).The results of transmission electron microscopy showed that:The number of autophagosomes in NIH-3T3 cells treated by AngⅡwas significantly decreased(△△P<0.01),while the number of autophagosomes in NIH-3T3 cells in AngⅡgroup was significantly increased after Shenxian-Shengmai oral liquid treatment（#P<0.05）,and the trend was reversed after adding SC79(**P<0.01),that is,the number of autophagosomes was reduced again.The results were consistent with those of Monodansylcadaverine（MDC）staining.（4）Shenxian-Shengmai oral liquid inhibits phosphorylation of AKT/m TOR signaling pathway to activate autophagy in NIH-3T3 cells:Compared with CON group,the expression level of p-AKT,p-m TOR in AngⅡgroup was significantly increased(△P<0.05,△△P<0.01),while the expression level of Beclin-1,LC3-Ⅱ/LC3-Ⅰwas significantly decreased and the consumption of autophagy substrate p62 was decreased(△P<0.05,△△P<0.01),suggesting that AKT/m TOR phosphorylation was activated and autophagy activity was inhibited in NIH-3T3 cell line.However,Shenxian-Shengmai oral liquid significantly reduced the overexpression of p-AKT,p-m TOR induced by AngⅡ（#P<0.05）,accompanied by increased expression of Beclin-1,LC3-Ⅱ/LC3-Ⅰand increased consumption of autophagy substrate p62(##P<0.01,#P<0.05).It is suggested that Shenxian-Shengmai oral liquid can enhance autophagy activity by inhibiting phosphorylation of AKT/m TOR signaling pathway,and AKT phosphorylation agonist SC79 can antagonize this effect(**P<0.01,*P<0.05).The quantitative analysis of CON,AngⅡ,SXSMs,SC79 has statistical significance among all groups.Conclusion:1.The SSS model of mice was successfully established by slowly pumping AngⅡinto the neck and back of mice,and the heart rate of mice in this model could be significantly improved by Shenxian-Shengmai oral liquid.2.Shenxian-Shengmai Oral Liquid can improve sinoatrial node fibrosis in SSS mice and reduce the expression of fibrosis marker proteins such as Fibronectin,Collagen I in sinoatrial node region,so as to improve sinoatrial node function.3.Shenxian-Shengmai Oral Liquid can regulate the expression of fibrotic proteins such asα-SMA,extracellular matrix（including Fibronectin and Collagen I）in NIH-3T3 cells by activating autophagy at the cellular level,thus inhibiting the transformation of fibroblasts into their activated phenotype myofibroblasts.4.Shenxian-Shengmai Oral Liquid can inhibit the activation of fibroblasts and the excessive secretion of extracellular matrix induced by AngⅡby inhibiting the phosphorylation of AKT/m TOR signaling pathway to activate autophagy,thus improving sinoatrial node fibrosis and producing sinoatrial node protection. |
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