| Objective:To evaluate the role of ventricular hypertrophy induced by establishing right heart hypertrophy model,arterio-venous fistula operated volume-overload rats,we investigated the regulation of plasma rennin and angiotensinⅡ(AngⅡ),and the myocardium expression of cardiotrophin-1(CT-1).Methods:1.Arterio-venous fistula operating used for increasing volume-overload in right ventricle to establish the rat model.28 gauge needles were piercing the inferior vena cava then perforate at Arterio-venous interstitial matrix.When the interstitial matrix at arterio-venous fistula success, then arterial blood flowing could be confirmed from the 28 gauge needle.After that,rinse arterio-venous fistula by using heparin saline solution and sutured the perforation.When removed artery clamp,the dilated vena cava could be confirmed that shows the successful of overload model.2.The male SD rats(n= 64) were randomly divided into 8 groups:(1) Normal group(n= 8):no treatment,normal diet;(2) Operation sham group(control group,n= 8):28 gauge needles were piercing the inferior vena cava but not perforating the Arterio-venous fistula,the procedure was as same as above groups;(3) Operation group:after operation,at the 1st,2nd,4th and the 8th week,the hemodynamic parameters for rats from each group were detected,then sacrificed and the heart weight parameters A-V fistula were measured(n=8);(4) Olmesartan group(Olm,Shanghai Sankyo Co.,Ltd.,n=8) was oral administration of 5mg/kg/d after the 5th week,for 4 weeks;(5) Lotensin group(Lotensin,Lot,Beijing Nuohua Co,n=8),which was oral administration of 5mg/kg/d after the 5th week,for 4 weeks.3.Measurement methods:Heart wet weights(Ratio of heart weight to body weight,HB/WB;left ventricular mass index, LVMI;right ventricular mass index,RVMI);Hemodynamic parameters were detected by using corresponding equipment,blood pressure of SBP,DBP and MBP) were obtained;RIA were suing for testing plasma concentration of rennin and AngⅡ;Immunohistochemistry method was used for identification of CT-1 expression and the localization in myocardium;Western-blot was used for semi-quantification of CT-1 and gp130 protein.4.Statistical analysis:SPSS 13.0 for Windows statistical package t test and one-way ANOVA.Homogeneity of variance between groups was compared by using LSD testing method.Variances of samples which were tested by using Games-Howell test.Data were presented by mean±standard deviation((?)±S),the testing standard was significantly differentα=0.05,P＜0.05.Results:1,Volume-overload cardiac hypertrophy in rat model:(1) Myocardium hypertrophy index measurements:four different time periods(1,2,4and 8 week) the model group of HB/WB,LVMI,RVMI followed the trend tended to increase,one of eight weeks of the change in model group,the most significant,these indicators data were: 2.7±0.3,2.1±0.2,0.6±0.1,with the sham-operated group(2.3±0.1,1.7±0.1,0.4±0.1) compared to both a significant difference(P＜0.05,Tablel,Figurel);with the model group after 8 weeks compared with Lotensin group and Olmesartan group treatment groups can respectively reduce the HB/WB,LVMI,RVMI:0.4±0.1,0.5±0.0(P＜0.05,Table 1,Figure 1).(2) Invasive hemodynamic measurement:comparison with the sham-operation group,model group,the first weekend mean arterial pressure(MBP) increased 111%,to the 2nd weekend to reach the largest 141%,the 4th weekend to reduce,the 8th weekend returned to normal; compared with the model group of the 8th week,after Lotensin and Olmesartan two drugs after the intervention group,only Olmesartan reduced the MBP group 10%,there is statistical significance(P＜0.05,Table2,Figure2).(3) Right ventricle sample HE staining and immunohistochemical results:HE staining:sham-operated group,the right ventricle Organize neatly arranged,and normal myocardial cells,no significant stormed hyperplasia,non-vascular ecstasies and vascular wall thickening,such as performance(Figure3A,3B);the model group of the 8th week HE stained sections,showing with myocardial tissue disorders,myocardial cell size increased, but mainly change in length(Figure3C);compared with the model group of the 8th week after Olmesartan(Figure3E) of drugs after the intervention,the right ventricle Organize with return to normal,myocardial cells near normal,no significant stromal hyperplasia,one of Olmesartan group myocardial biopsy morphology closer to sham-operated group;Lotensin group after the intervention do not change the morphology(Figure3D).Immunohistochemistry:8 weeks in model group with right ventricular organizations disorders,myocardial cell size increased,but mainly change in length.2.Plasma renin and AngⅡDetermination:plasma renin activity with the model group to reduce the extension of time,the model group of the 8th week minimum 170%,compared with the sham-operated group,there is statistical significance(P＜0.05,Figure 4-1),but levels of plasma AngⅡsignificantly increased the model group,one of eight weeks up to 192%,compared with the sham-operated group,there is statistical significance(P＜0.05,Figure4-2);and 8 weeks in model group comparison,the two groups after drug intervention,plasma renin activity were recovered (P＜0.01,Figure4-1),Olmesartan group plasma AngⅡlevels increased(P＜0.01,Table3, Figure4-2),but decreasing in Lotensin group(P＜0.05,Table 3,Figure 4-2).3.Myocardium CT-1 and gp130 expression(1) CT-1 expression:Immunohistochemistry:model group,CT-1 expression in brown positive reaction products are mainly distributed in the membrane region and cytoplasmic domain,while the sham-operated group,while the expression is mainly cytoplasmic;and sham-operated group,the average optical density(MOD) of model group,8 weeks significantly increased(P＜0.05,Table4, Figure5D,Figure6);after the intervention,eight weeks with the model group,Olmesartan group myocardial tissue form near normal,myocardial cells are arranged in rows,to reduce the interstitial cells,CT-1 expression in brown positive reaction product mainly reduce the membrane area-based(P＜0.05,Table 4,Figure5E).Western-blot: Sham-operated group and the comparison of the MOD,8 week model group MOD increased 161%(P＜0.01,Table 5,Figure7-8).(1,2,4and 8 week) the model group of MOD and RVMI correlation analysis showed significant correlation(r=0.925,P＜0.01,Figure9). After two groups of drug intervention,eight weeks with model group,Olmesartan group MOD to reduce 146%(P＜0.05,Table6,Figure10-11),the other group was not significant (P＞0.05,Table6,Figure10-11).(2) gp130 expression:Western-blot test results showed that sham-operated group,model group 1,2,4 and 8 weeks on behalf of gp130 protein expression followed by increased MOD,and 8 weeks in model group increased 139%(P＜0.05,Table 7,Figure12-13).After two groups of drugs after the intervention compared with the model group,Olmesartan group MOD a significant reduction for 140%(P＜0.05,Table 7,Figure12-13),the other group was not significant (P＞0.05,Table 7,Figure 12-13).Conclusion:1.AngⅡwas involved in myocardium hypertrophy induced by volume overload,this process was AT1R-mediated;2.The increase of AngⅡmay be not from plasma rennin regulation,but by non-enzyme-activation pathway;3.CT-1 relative myocardium hypertrophy which induced by volume overload was its receptor subunit gp130 mediated;4.CT-1 relative myocardium hypertrophy which induced by volume overload may be related with the RAS.
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