Protective Effect Of β-blockers On Heart Valve Damage In Spontaneously Hypertensive Rats

Background Senile degenerative valvular heart disease is the most common structural heart disease in the elderly.Hypertension is an important risk factor for senile degenerative heart valvular disease^[1,2],and blood pressure management can effectively reduce the incidence of senile degenerative heart valvular disease.Currently,five first-line antihypertensive drugs are recommended in the hypertension prevention and treatment guidelines.While effectively lowering blood pressure,do different antihypertensive drugs have a protective effect on heart valves?Are there differences between them?Objective By observing the protective effect ofβ-blocker on heart valve damage in spontaneously hypertensive rats,the protective effect and mechanism ofβ-blocker on heart valve in spontaneously hypertensive rats were preliminarily discussed.Methods Twenty-four male spontaneously hypertensive rats were randomly divided into4 groups according to feeding methods.Group A was fed with ordinary diet+edible starch（placebo）.Group B was fed with common diet+CCB（nifedipine 60mg bid）;Group C was fed with common diet+CCB+βB（nifedipine 40mg bid,metoprolol 75mg bid）;Group D was fed ordinary diet+βB（metoprolol 150mg bid）.Blood pressure,heart rate and general condition of rats were observed during feeding.9 months later,the rats were killed and the heart samples were taken under sterile conditions for examination.The pathological features of rat aortic valve were observed by HE staining.The expressions of MMP-2,MMP-9,collagenⅠandⅢwere detected by immunohistochemical staining.The expression of Runx2,BMP-2,Osteocalcin,Osteopontin and MGP were detected by immunofluorescence staining.Aortic valve calcification was detected by Von Kossa staining.Quantitative data such as blood pressure and heart rate were processed by SPSS 25.0 software,expressed as mean±standard deviation（x±s）,and normality test was conducted,and then Kruskal-Wallis test was used to compare whether the differences between groups were statistically significant.Quantitative data such as MMP-2,MMP-9,collagenⅠ,collagenⅢ,BMP2,Runx2,Osteocalcin,Osteopontin and MGP were firstly scanned by Image J software to detect the expression of the above indexes and obtain statistical data.Then SPSS 25.0 software was used to process the experimental data,expressed as mean±standard deviation（x±s）.After normal test and homogeneity test of variance,Man-Whitney U test or one-way analysis of variance（ANOVA）were conducted to compare whether the differences between groups were statistically significant.Results（1）The mean values of the initial blood pressure of rats in group A,B,C and D were 190/138mm Hg,187/140mm Hg,191/139mm Hg and 180/135mm Hg,respectively.Compared with the initial systolic blood pressure,the changes of systolic blood pressure of rats after 9 months were as follows:Systolic blood pressure of group A had no significant change,systolic blood pressure of group B decreased by 41%,group C decreased by 35%and group D decreased by 38%.Compared with group A,systolic blood pressure of group B,C and D decreased significantly（p<0.01）,had statistical difference;Compared with initial diastolic blood pressure,diastolic blood pressure of rats after 9 months:diastolic blood pressure of rats in group A did not change significantly,diastolic blood pressure of rats in group B decreased by 22%,that of rats in group C decreased by 34%and that of rats in group D decreased by 33%.Compared with diastolic blood pressure of rats in group A,diastolic blood pressure of rats in group B,C and D decreased significantly（p<0.01）,had statistical difference;The initial heart rate of rats in group A,B,C and D was 464bpm,461bpm,465bpm and 466 bpm,respectively.Compared with the initial heart rate of rats in group A,B,C and D treated with different drugs,the changes of heart rate of rats after 9months were as follows:The heart rate of group A had no significant change,group B increased by 2%,group C decreased by 35%,and group D decreased by 41%.Compared with group A,the heart rate of group B had no significant change,and the heart rate of group C and D decreased significantly（p<0.01）with statistical difference.The initial body weight of rats in groups A,B,C and D ranged from 250g to 290g.After 9 months of feeding,the body weight of rats in the four groups increased by about 50g,and there was no difference between the four groups in body weight.（2）HE staining results of aortic valves in groups A,B,C and D showed that the valve lobes in group A were dark red in HE staining;compared with group A,HE staining of the valve in group B was similar to that in group A,while HE staining of the valve in groups C and D was light pink,and the color of the valve in group D was the lightest.The valve of rats in group A had extensive collagen fiber hyperplasia,and the degree of fibrosis was serious.There was no significant change between group B and group A,the degree of fibrosis in group C and D was reduced,and the degree of fibrosis in group D was the lightest.There was no obvious calcium salt deposition in the valve of rats in the 4 groups.（3）Immunohistochemical staining was used to detect the expression levels of MMP-2,MMP-9,collagenⅠandⅢin aortic valvae of rats in groups A,B,C and D.The results showed that,The expressions of MMP-2,MMP-9,collagenⅠandⅢin the aortic valve of group A were dark brown,while the expressions of MMP-2,collagenⅠandⅢin group D were light brown.The expressions of MMP-2,MMP-9,collagenⅠandⅢin the aortic valve of spontaneously hypertensive rats were analyzed by Image J scanning.The expressions of MMP-2,collagenⅠand collagenⅢin the aortic valve of rats in group A were the highest.The expressions of MMP-2,MMP-9,collagenⅠandⅢin aortic valve of rats in group B were not significantly decreased（P>0.05）,while the expressions of MMP-2,collagenⅠandⅢin aortic valve of rats in group D were significantly decreased（P<0.01）.There was a statistical difference.（4）The expression levels of BMP2,Runx2,Osteocalcin,Osteopontin and MGP in aortic valves of rats in groups A,B,C and D were detected by immunofluorescence staining.The results showed that,The expression of BMP2,Runx2,Osteocalcin and Osteopontin in the aortic valve of group A was dark orange-red,while the expression of BMP2,Runx2,Osteocalcin and Osteopontin in group D was light orange-red.The expression of MGP in the aortic valve of group A was light orange-red and that of group D was dark orange-red.The expressions of BMP2,Runx2,Osteocalcin,Osteopontin and MGP in the aortic valve of spontaneously hypertensive rats were analyzed by Image J scanning.The expressions of BMP2,Runx2,Osteocalcin and Osteopontin in aortic valves of rats in group B were not significantly decreased（P>0.05）.The expressions of BMP2,Runx2,Osteocalcin and Osteopontin in aortic valves of rats in group D were significantly decreased（P<0.05）,with statistical difference.Compared with group A,the expression level of MGP in aortic valve of rats in group B was not significantly increased（P>0.05）,while the expression level of MGP in aortic valve of rats in group D was significantly increased（P<0.05）,with statistical difference.（5）Vonkossa staining was used to detect calcium salt deposition in aortic valves of rats in groups A,B,C and D.The results showed that no obvious brown-black granular calcium salt deposition was found in aortic valves of rats in groups A,B,C and D under ordinary microscope.Conclusion（1）The expressions of MMP-2,MMP-9,collagenⅠ,collagenⅢ,BMP2,Runx2,Osteocalcin and Osteopontin were significantly increased in the heart valves of spontaneously hypertensive rats,while the expressions of MGP were significantly decreased.The expressions of MMP-2 and MMP-9 were positively correlated with valve thickening.The expressions of collagenⅠandⅢwere positively correlated with valve rigidity.The expression of BMP2,Runx2,Osteocalcin and Osteopontin was positively correlated with valve osteoblast-like differentiation,while the expression of MGP was negatively correlated with valve osteoblast-like differentiation.The above findings suggest that there is indeed valve damage in spontaneously hypertensive rats in this study.（2）The blood pressure of spontaneously hypertensive rats decreased significantly after the use ofβ-blockers,and the antihypertensive effect was not significantly different from that of CCB.At the same time,β-blockers also significantly reduced the heart rate of spontaneously hypertensive rats,but CCB could not reduce the heart rate of rats.The above indexes changed after antihypertensive treatment.There was no significant change in the above indexes and no significant reduction in valve damage when blood pressure dropped.When blood pressure and heart rate decreased,the expressions of MMP-2,MMP-9,collagenⅠ,collagenⅢ,BMP2,Runx2,Osteocalcin and Osteopontin decreased significantly,and the expressions of MGP increased.（3）The valve damage of spontaneously hypertensive rats was not necessarily alleviated under the condition of pure antihypertensive treatment,but the valve damage was significantly alleviated after the use ofβ-blockers.It was considered that the mechanism of valve damage reduction was related to heart rate.