| OBJECTIVE: To observe the therapeutic effect of Warming Yang to Resolving Fluid(WYRF)on mice with chronic heart failure induced by intraperitoneal injection of doxorubicin.To explore the mechanism of WYFR method in preventing and treating chronic heart failure(CHF)based on LXR-RAAS/NF-κB signaling pathway.METHOD: The fifteen male C57 BL/6 mice were divided into group A(Control)and fed on a regular diet.The remaining 91 mice were divided into group B and intraperitoneal injected with doxorubicin to prepare a model of CHF.After the model was established,the mice in group B were randomly divided into the high dose of WYRF group(WYRF-HD),the middle dose of WYRF group(WYRF-MD),the captopril group(Captopril)and the group of WYRF combined with captopril(WYRF+ Captopril).The Control and the Model group were gavaged with normal saline at a dose of 0.2ml/20 g body weight at 10 am for 4 weeks,and the remaining groups were administered intragastrically with corresponding drugs once a day for 4weeks.Mice in WYRF-MD group were gavaged with WYRF decoction at a dose of0.2ml/20 g body weight,the WYRF-HD group were gavaged with WYRF decoction at a dose of 0.4ml/20 g,the Captopril group were oral administrated with captopril suspension at a dose of 0.1ml/20 g,and mice in the WYRF+ Captopril group were fed with both WYRF decoction(0.2ml/20g)and captopril suspension(0.1ml/20g).The pharmacodynamics indexes of this experiment include mice body weight and mortality.The echocardiogram of the mice was conducted respectively when the CHF model established and the oral administration were complete.Moreover,the concentration of brain natriuretic peptide(BNP)and arginine vasopressin(AVP)detected by ELISA and the change of mouse ventricular histopathology detected by HE and Masson staining were used to evaluate the efficacy of WYRF.Moreover,the mechanism study of WYRF were based on the LXR-RAAS/NF-κ B pathway.Western-blot and PCR experiments were performed to detect the expression of LXRα,NF-κ B,TNF-α and INOS in the level of protein and mRNA.And ELISAmethod was conducted to detect the serum concentration of Renin,ANG-II and ALD.Apoptosis of myocardial cells in mice was detected by Tunel staining.RESULTS: 1.There wasn’t significant difference in body weight between group A and group B in the first three weeks of modeling(P<0.05).From the fourth week,mice in group A were gradually weight gain,remaining significant higher than mice in group B until the last week of modeling(P < 0.05).The results of heart Doppler ultrasound showed higher LVIDs,LVESV and lower EF,FS and HR in group B than group A(P<0.05),which reminded the CHF model have been established in group B.2.After 4 weeks of treatment,the model group showed significant higher LVIDs,LVESV,and lower EF,FS,HR compared to the Control group(P<0.05).Compared with the Model group,the color Doppler ultrasound of mice in the WYRF-MD,WYRF-HD,Captopril and WYRF+ Captopril group were relatively improved(P<0.05).ES and FS of WYRF-HD group were significant higher than the WYRF-MD group(P<0.05).3.The results showed significantly increased of serum BNP and AVP in the Model group than the Control group(P < 0.05).And decrease of BNP and AVP were observed in the WYRF-MD,WYRF-HD,Captopril and WYRF+ Captopril group compared to the Model group(P<0.05).4.Compared to the control group,disordered myocardial muscle fibers and myocardial myocyte nuclear vacuole changes were found in mice hearts of the Model group.The Masson staining showed increased mouse myocardial collagen fiber area in the Model group than the Control group(P<0.05).The WYRF-MD group and WYRF + Captopril group showed a small number of disordered myocardial muscle fibers and nuclear deflation.In the Captopril group and the WYRF-HD group,the nucleus of myocardial muscle cells was partially swollen,vacuoles changed,myocardial muscle fibers were curled,wavy,and some myocardial muscle fibers were dissolved.When compared to the Model group,mouse myocardial collagen fiber area in the WYRF-HD group,the Captopril group and the WYRF + Captopril group were significant decreased(P<0.05).5.The expression of LXRα mRNA and protein of mice myocardium in the Model group were significant lower compared to the Control group(P<0.05).The mRNA and protein expression of LXRα in the WYRF-HD group was significantly higher than the Model group(P<0.05)while there was no statistical difference from the Control group(P>0.05).Compared with the Captopril group,the expression of LXRαmRNA and protein were significantly upregulated in the WYRF-HD group and the WYRF + Captopril group(P<0.05).6.According to the ELISA experiment,the serum concentration of Renin was significant higher in the Model group than the Control group(P<0.05).In contrast to the Model group,the concentration of Renin in WYRF-HD group,the Captopril group and the WYRF + Captopril group were significantly decreased(P<0.05).The serum concentration of ANG-II were obviously increased in the Model group than the Control group(P<0.05).The concentration of ANG-II in the WYRF-HD group was significantly decreased than that in the Model group(P<0.05),and there was no statistical difference compared with the Control group(P>0.05).In contrast to the Control group,the expression of ALD in the Model group was obviously increased(P<0.05).The concentration of ALD in the WYRF-HD group was significant lower than the Model group(P<0.05).7.The NF-κB mRNA and protein expression were higher in the Model group than the Control group(P<0.05).The NF-κB mRNA expression in the WYRF-MD group,the WYRF-HD group,the Captopril group and the WYRF+ Captopril group were significant decrease than the Model group(P<0.05).Compared to the Model group,the expression of NF-κB protein in the WYRF-HD group was significantly decreased(P<0.05),and there was no significant difference between the other treatment groups and the model group.Compared to the Control group,the expression of TNF-α mRNA and protein in the Model group was obviously higher(P<0.05).The expression of TNF-α mRNA in the WYRF-HD group and the WYRF+ Captopril group were significantly decreased than the Model group(P<0.05).The expression of TNF-α in theWYRF-HD group and the Captopril group were displayed a downward trend,but there was no statistical difference compared to the Model group(P>0.05).The INOS mRNA and protein expression was upregulated in the Model group than the Control group(P<0.05).In contrast to the Model group,the INOS mRNA expression in the WYRF+ Captopril group was significantly decreased(P<0.05).Although downward trend were shown in the WYRF-HD group,WYRF-MD group and the Captopril group,there was no statistical difference compared to the Model group(P>0.05).Compared to the Model group,the expression of INOS in the WYRF-HD group and the WYRF+ Captopril group were obviously decreased(P<0.05).8.The results of myocardial Tunel cell apoptosis staining showed that the apoptosis index in the Model group increased significantly than the Control group(P<0.05).Compared with the Model group,the myocardial apoptosis index of the WYRF-HD group,the WYRF-MD group,the WYRF+ Captopril group and the Captopril group were significantly reduced(P <0.05),of which the WYRF-HD group and the Captopril group decreased more than the WYRF-MD group(P <0.05).CONCLUSION: 1.The CHF model of mice can be established by intraperitoneal injection of doxorubicin,causing abnormal cardiac function and ventricular hypertrophy in the model mice.And the modeling time lasts 8 weeks.2.The survival rate and life quality of CHF mice can be significant improved by the WYRF method.By using the WYRF method,mice heart function,EF and FS were obviously improved,and LVESV,LVIDs,serum BNP and AVP were remarkably decreased.3.The WYRF method can reduce the deposition of myocardial collagen fibers and myocardial cell apoptosis in CHF mice,and improve ventricular remodeling.4.The mechanism of WYRF method in improving heart function and ventricular remodeling of CHF mice may be through the regulation of LXR-RAAS signaling pathway.That is,by regulating the expression of LXRα,regulating RAAS,reducing water and sodium retention,reducing left ventricular load.5.The WYRF method can down-regulate NF-κB,decrease the expression of TNF-αand INOS,reduce myocardial inflammatory response,and reduce myocardial cell apoptosis by regulating the expression of LXRα.6.The high dose group of WYRF is more efficacy and safe than the medium dose. |
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