Study On The Myocardial Protective Effect Of Zhenwu Decoction On CTnT^R141W Transgenic Dilated Cardiomyopathy Mice And Its Mechanism

PurposeIn this experiment,we observed the effect of Chinese herbal compound ZhenwuDecoction on mice with dilated cardiomyopathy and explored its mechanism of action on transgenic mice with dilated cardiomyopathy from multiple levels to provide new ideas and theoretical basis for the treatment of dilated cardiomyopathy..Materials and Methods:60 cTnT^R141W141W transgenic dilated cardiomyopathy mice and 15 normal C57BL/6J mice were purchased from the Institute of Laboratory Animals,Chinese Academy of Medical Sciences and bred at the Animal Experimental Center of Liaoning University of Traditional Chinese Medicine.Fifteen 4-month-old C57BL/6J mice were used as the normal group,and60 mice with cTnT^R141W141W transgenic dilated cardiomyopathy at 4 months of age were randomly divided into 4 groups according to body weight.Model group,captopril group,Zhenwu decoction High-dose group and Zhenwu Decoction group,15 in each group.The normal group and the model group were given the same amount of saline to gavage.According to the equivalent dose conversion method between human and mice,the mice were given the following gavage doses:the mice in the traditional Chinese medicine group were treated with Zhenwu Decoction and the dosage of Zhenwu Decoction in the middle-dose group was 18.2g/kg/d?crude drug?;the dose of Zhenwu Decoction in the high-dose group was 36.4g/kg/d?crude drug?;The benefit group to captopril gavage,captop consumption of10mg/kg/d.Twice daily,0.3 ml each time,continuous administration for 1 month.In the first experiment,the pathological changes of the heart were observed by section scanning and light microscopy;the ultrastructure of cardiomyocytes was observed under transmission electron microscope;the expression level of cTnT^R141W141W transgene in myocardial tissue of each group was identified by HpaII digestion and RT-PCR;Pulser ultrasound detects cardiac function in mice,including ejection fraction and shortening of the short axis;enzyme-linked immunosorbent assay?ELISA?detects serum BNP levels.In the second experiment,collagen fibers were detected by masson staining in mouse myocardium.The mRNA and protein expressions of Col1a1 and Col3a1 in myocardium were detected by RT-PCR and western blotting.In the third experiment,the expression of Acta1,Atp2a2 and Nppb mRNA and protein in myocardium were detected by RT-PCR and western blotting.Result:1?Slice scanning and light microscopy to observe the pathological changes of the heart:The heart of the model group cTnT^R141W141W transgenic mouse was significantly enlarged compared with that of the normal group,and the left ventricle was significantly enlarged and the wall of the chamber became thinner.After the administration of Zhenwu Decoction,the heart of the mouse became smaller,and the left ventricular cavity was smaller than that of the model group.Small,the relative thickness of the chamber wall becomes larger;HE staining of the left ventricular myocardium under the light microscope shows that:compared with the myocardium of mice in the model group,the myocardium of the mouse in the model group is disordered,and the arrangement of the myocardial cells after the administration of Zhenwu Tang is relatively neat.2?Transmission electron microscopy:Observing the heart of 5-month-old mice showed that the myocardial fibers of the normal mice were regular,well-structured,abundant in mitochondria,and the discs were tidy and smooth;whereas in the model group,the myocardial fibers became thinner,dissolved,or even disappeared.Z band disappeared,mitochondria swollen,vacuolized,disc curled, partially dissolved;Western medicine group most complete mitochondria,some sacral breaks,and the electron density is not uniform,muscle tow breaks,dissolves,disappear,rub disk The edge is relatively clear,but there is a partial rupture phenomenon,and there are still ambiguous boundaries;most of the mitochondria in the Zhenwu Decoction administration group are relatively complete,but some mitochondria show local cavitation,septal breakage,local breakage and dissolution of the myofilament.The boundary of the disk was relatively clear,but there was a partial blur,and the arrangement of myocardial fibers was more regular than that of the model group.The above results show that after the intervention of Zhenwu Decoction,the damage of myocardial ultrastructure of cTnT^R141W141W transgenic dilated cardiomyopathy mice was alleviated.3?The mice with transgenic dilated cardiomyopathy were identified:By HpaII digestion and RT-PCR identification,the normal group did not detect the transgenic cTnT^R141W141W after enzyme digestion,and the transgenic group had 1.13 times more cTnT^R141W141W than endogenous cTnT.4?Left ventricular end-diastolic diameter?LVEDd?,left ventricular ejection fraction?EF%?,and short-axis shortening?FS%?were measured by echocardiography:The wall of the ventricle was thinner and the heart chamber was enlarged in the model group compared with the normal group.The exercise decreased,the left ventricular end-diastolic diameter increased,the left ventricular ejection fraction and the short axis shortening rate decreased,and the myocardial systolic function decreased.After administration of Zhenwu Decoction,both the high-dose group and the middle-dose group decreased the left ventricular end-diastolic diameter,increased the left ventricular ejection fraction and the short axis shortening rate.5?BNP levels in serum were detected by ELISA:The levels of BNP in each group were compared:Compared with the normal group,the level of BNP in the model group was significantly higher?P<0.01?,which was statistically significant.The heart function of the model group mice was decreased,indicating that the transgenic gene was expanded.The cardiomyopathy mice had reduced cardiac function,and the levels of BNP in the captopril group and the high dose group of Zhenwu Tang increased,but there was no statistical significance.Compared with the model group,the captopril group and the Zhenwu Tang middle high dose group the level of BNP was significantly lower ?P<0.01?.Compared with the positive group?captopril group?,there was no significant difference in the level of BNP in the high-dose group of Zhenwu Tang,indicating that Zhenwu Decoction can improve the dilated cardiomyopathy to some extent Rat heart function was non-inferior to the captopril group.6?Masson staining of myocardial tissue:Masson staining by light microscope showed normal myocardial tissue in mice,collagen fibers content is less;model group transgenic mice disorganized myocardial interstitial collagen fibers increased significantly;captopril group Compared with Zhenwu Decoction group,myocardial cells were arranged more neatly,collagen was less,and interstitial fibrosis was reduced.7?The expression of Col1a1 and Col3a1 mRNA and protein in each group was compared:Compared with the normal group,the expression of Col1a1 mRNA in the myocardial tissue of the model group was significantly increased?P<0.01?.The mice in the captopril group and the Zhenwu Tang high-dose group were given the expression of Col1a1 mRNA was increased in different degrees?P<0.01?.Compared with the model group,the expression of Col1a1 mRNA in the myocardial tissue of captopril group and Zhenwu Tang high-dose group was decreased to varying degrees?P<0.01?.Comparing with captopril group,the expression of Col1a1 mRNA in the myocardial tissue of Zhenwu Decoction high dose group was higher,but there was no significant difference.The expression of Col1a1 mRNA in the myocardial tissue of Zhenwu Decoction group was higher than that of Captopril group.Difference?P<0.01?;Compared with the high-dose Zhenwu Decoction group,the expression of Col1a1 mRNA in the myocardial tissue of the middle-dose group was increased?P<0.05?.Compared with the normal group,the expression of Col3a1 mRNA in the myocardial tissue of the model group was significantly increased?P<0.01?,and the expression of Col3a1mRNA in the myocardial tissue of the captopril group and the Zhenwu Tang high-dose group was increased to varying degrees?P<0.01?.<0.01);Compared with the model group,the expression of Col3a1 mRNA in the myocardial tissue of captopril group and Zhenwu Decoction high-dose group was decreased to varying degrees?P<0.01?,among which the high-dose group of Zhenwu Decoction had the most significant decrease;Compared with captopril group,the mRNA expression of Col3a1 in the high-dose group of Zhenwu Decoction was lower,but there was no statistically significant difference.The expression of Col3a1 mRNA in the myocardial tissue of Zhenwu Decoction group was higher than that of the control group?P<0.05?.<0.01);Compared with the high-dose Zhenwu Decoction group,the expression of Col1a1 mRNA in the myocardial tissue of the middle dose group was increased?P<0.01?.Compared with the normal group,the expression of Col1a1 protein in the myocardial tissue of the model group was significantly increased?P<0.01?.The expression of Col1a1protein in the myocardial tissue of the captopril group was reduced,but there was no statistically significant difference,Zhenwu Decoction high dose The expression of Col1a1protein in myocardial tissue of mice was similar to that in the normal group.There was no significant difference between the two groups.The expression of Col1a1 protein was higher in Zhenwu Decoction group than in the normal group?P<0.01?.Compared with the model group,the captopril group The expression of Col1a1 protein in the myocardial tissue of the high-dose group of Zhenwu Decoction was decreased?P<0.01?,but the decline was most obvious in the captopril group,followed by the high-dose group of Zhenwu soup,and the middle-dose group of Zhenwu decoction was the least.Compared with the normal group,the expression of Col3a1 protein in the myocardial tissue of mice in the model group and captopril group was significantly increased?P<0.01?,but the increase was most significant in the model group,and there was no significant difference in the Zhenwu Tang high-dose group?P>0.05?;compared with the model group,captopril group,Zhenwu Tang high-dose group mice myocardial tissue Col3a1 protein expression was significantly reduced?P<0.01?;each drug group compared to captopril group down Col3a1 protein expression The best effect?P<0.01?,Zhenwu Tang high school dose group did not see significant differences?P>0.05?.8?Expression of Acta1,Atp2a2,and Nppb mRNA and Protein in Cardiac Tissues of Different Groups:Compared with the normal group,the expression of Acta1 mRNA in the myocardial tissue of the model group,the captopril group,the Zhenwu Decoction high-and middle-dose group had significant differences?P<0.01,vs captopril group P<0.05?;In group comparison,the expression of Acta1 mRNA in the myocardial tissue of captopril group,Zhenwu decoction high-and middle-dose group was significantly decreased?P<0.01,v.Zhenwu Decoction middle dose group P<0.05?;Captopril group,Zhenwu Tanggao There was no significant difference in the expression of Acta1 mRNA in the myocardium of the mice between the moderate and medium dose groups?P>0.05?.Compared with the normal group,Atp2a2 mRNA expression was not significantly different in the captopril group?P>0.05?,and there was a significant difference in other groups?P<0.01?.Atp2a2 mRNA expression was compared with the model control group.Significantly up-regulated?P<0.01,vs.Zhenwu Decoction,P<0.05?,among groups,it was found that Captopril group and Zhenwu Decoction high-dose group had the best effect on improving myocardial Atp2a2 mRNA expression in mice?Captop Lee group vs Zhenwu soup high dose P>0.05?.Compared with the normal group,the expression of Nppb mRNA in the myocardial tissue of the model group was significantly increased?P<0.01?,and the expression of Nppb mRNA in the myocardial tissue of each drug group was increased to varying degrees?P<0.01?;each drug was compared with the model group.The expression of Nppb mRNA was significantly decreased in the group?P<0.01?,and it was significantly decreased in the high-dose group of Zhenwu Decoction;the expression of Nppb mRNA was significantly decreased in the high-dose group of Zhenwu Decoction compared with the captopril group and the middle-dose group of Zhenwu Decoction?P<0.01?.There was no significant difference between Captopril group and Zhenwu Decoction group?P>0.05?.Compared with the normal group,the expression of Acta1 protein in the control group was significantly increased?P<0.01?,and there was no significant difference in each group?P>0.05?.Compared with the model group,the expression of Acta1 protein in the myocardial tissue was significantly decreased in each group?P<0.01?;There was no significant difference between groups in each drug group?P>0.05?.Compared with the normal group,Atp2a2 protein expression in the captopril group showed no significant difference?P>0.05?,the rest of the groups were significantly different?P<0.01?;Compared with the model group,the expression of Atp2a2 protein in the cardiomyocytes of each group was significantly higher?vs,Zhenwu Decoction,P<0.05, P<0.01?;Comparison of each drug group found that Captopril group up-regulated expression of Atp2a2 protein was the best?P<0.01?,Zhenwu Decoction high-dose group up-regulated expression of Atp2a2 protein Better than Zhenwu Decoction group?P>0.05?.Compared with the normal group,the expression of Nppb protein in the model group as significantly increased?P<0.01?;compared with the model group,the expression of Nppb protein in the myocardial tissue of each drug group was significantly reduced?P<0.01?;compared with the captopril group,Zhenwu The expression of Nppb protein was significantly decreased in the Tanggao and middle-dose groups?P<0.01?.There was no significant ifference between the middle and high-dose groups of Zhenwu Decoction?P>0.05?.Conclusion:1?Zhenwu Decoction can improve the cardiac function of cTnT^R141Wtransgenic mice with dilated cardiomyopathy,including EF and FS,and reduce the expression of BNP in erum.2?Zhenwu Decoction can improve the cardiac structure and ultrastructure of cTnT^R141W141W transgenic mice with dilated cardiomyopathy and down-regulate the expression of cTnT^R141W.3?Zhenwu Decoction can inhibit the fibrosis of cardiomyocytes and improve ventricular remodeling by down-regulating the expression of Col1a1 and Col3a1 mRNA and protein in myocardial tissue.4?Zhenwu Decoction can prevent and treat DCM by down-regulating the expression of Acta1 and Nppb mRNA and protein in hypertrophic myocardium,upregulating the expression of Atp2a2 mRNA and protein,inhibiting the hypertrophy of cardiomyocytes and improving ventricular remodeling.