Effects Of Adiponectin On Myocardial Fibrosis Of Heart Failure Mice

IntroduceMyocardial Fibrosis (MF) is defined as the excess collagen accumulation ofextracellular matrix and high collagen concentration of heart tissue because of Suchpathological changes happen on many kinds of cardiovascular diseases, and it’s a keyfactor in turning heart function from compensation to uncompensated period. Past researchindicated that serum adiponectin was negatively and significantly related to MF, butwhether adiponectin is an endogenous protective mechanism against MF is still uncertain.In order to exclude the interference factor of the clinic, the myocardial fibrosis model isused to illuminate the causal relationship between adiponectin and MF.Objective(1) To create a myocardial fibrosis model intraperitoneal injection of isoproterenol;(2) To explore the effects of adiponectin on myocardial fibrosis in heart failure mice.Method1. Create MF model of mice by intraperitoneal injection of isoproterenol (ISO)a) SD male mouse,30-45g of weight. The experimental animals were divided intofour groups on the basis of different injection of ISO: the normal control group,3mg/kggroup,5mg/kg group and7.5mg/kg group. Each group had their mice intraperitonealinjected of ISO for3continual days.b) After5weeks, FS%value was measured using Doppler echocardiography.c) In order to evaluate the model rate, tissue paraffin sections were dyed by Sirius red,the morpha of collagen Ⅰ and Ⅲ and the CVF wasobserved and semi-quantified under apolar microscope.2. Influence of adiponectin on the expression of mRNA and protein ofcollagen Ⅰ andⅢa) SD male mouse,30-45g of weight. The experimental animals were divided intothree groups: ISO group, APN+ISO group, and normal control group.The animal model established by intraperitoneal injected by ISO (7.5mg/kg) was used in group ISO andgroup APN+ISO. In group APN+ISO, adiponectin was intraperitoneally injected by10μg/(kg.d) from the forth day. Otherwise, normal saline was intraperitoneally injectedgroup ISO and the normal control group instead.b)5weeks later, the rats were killed to detect the myocardial fibrosis degree. Thetissue paraffin sections of left ventricular myocardial were dyed by Sirius red, the morphaof collagen Ⅰ and Ⅲand the CVF was observed under a polar microscope.c) The expression of collagen Ⅰ and Ⅲ mRNA of myocardium were detected byRealtime-PCR method.And the expression of collagenⅠ and Ⅲprotein of myocardium weresemi-quantified using Western Blot method.3. All quantitative data were expressed as mean±standard deviation. Statisticalcomparisons between groups were analyzed with a one-way analysis of variance(SPSS13.0). A value of P<0.05was considered statistically significant.Result1. Establish MF model of mice by intraperitoneal injection of isoproterenol (ISO)a) FS%value of left ventricle was significantly lower in7.5mg/kg group than that incontrol group (26.92±2.90%vs32.36±2.81%. P<0.05), which means left ventricularsystolic function was damaged by intraperitoneal injection of ISO.b) Tissue paraffin sections dyed by Sirius red displayed that collagen Ⅰa nd Ⅲincardiac interstitium increased obviously in7.5mg/kg group by a polarizing microscope.Fibrosis of collagen Ⅰ were thick with bright red,and had a messy arrangement andrepeated overlap. Fibrosis of collagen Ⅲwere yellow green and thrust deep into collagenⅠ. Thecollagen volume figure (CVF) value was significantly higher in7.5mg/kg groupthan that in control group (6.22±0.43%vs4.58±0.41%. P<0.01). Polarizing lightmicroscope examination showed that fibrosis in group3mg/kg and5mg/kg wasred&yellow and light green. There was no significant difference between these two groupsand control group (5.02±0.33%,5.20±0.55%vs4.58±0.41%. P>0.05).These indicated thatintraperitoneal injection of isoproterenol of7.5mg/kg could establish rat myocardialfibrosis model.2. Influence of adiponectin on the expression of mRNA and protein of collagenⅠand Ⅲ.On the base of the study above, the MF model mice were established byintraperitoneal injection of ISO with doses of7.5mg/kg.a) Tissue paraffin sections dyed by Sirius red displayed that collagen Ⅰ andⅢ in control group were less and lightly colored. Collagen Ⅰa nd Ⅲ in heartinterstitiumincreased obviously in ISO group, which means interstitial fibrosis. Fibrosis of collagen Ⅰwere thick with bright red, and messy arrangement and repeated overlap were observed.Fibrosis of collagen Ⅲ wereyellow-green and intersected with collagen Ⅰ. CVF valueinISO group was significantly higher than that in control group (6.41±0.37%vs4.68±0.30%.P<0.01). Comparing to ISO group, CVF value in ISO+APN group declined significantly(5.94±0.35%vs6.41±0.37%. P<0.01).b) The mRNA expression of collagen Ⅰ and Ⅲ inISO group was significantly higherthan that in control group (collagen Ⅰ3.01±0.78vs1.00, collagen Ⅲ2.39±0.70vs1.00,P<0.01for both),. The mRNA expression of collagen Ⅰ and Ⅲin ISO+APN group wasmuch lower, than that in ISO group (collagen Ⅰ2.32±0.82vs3.01±0.78, collagenⅢ1.62±0.57vs2.39±0.70.P<0.01for both).c) The protein expression of collagen Ⅰ and Ⅲ in the ISO group was significantlyhigher than that in control group (collagen Ⅰ0.73±0.19vs0.28±0.15, collagen Ⅲ0.68±0.18vs0.25±0.17. P<0.01for both, respectively).Similarly, The protein expressionof collagen Ⅰ andⅢ in ISO+APN group was significantly higher than that in controlgroup (collagen Ⅰ0.53±0.17vs0.28±0.15, collagen Ⅲ0.45±0.19vs0.25±0.17, P<0.01forboth). Comparing to ISO group, there was a much lower expression of of collagen ⅠandⅢ in ISO+APN group(collagen Ⅰ0.53±0.17vs0.73±0.19, collagen Ⅲ0.45±0.19vs0.68±0.18. P<0.05for both, respectively).Conclusion1. The mice MF model can be created by intraperitoneal injection of ISO using doseof7.5mg/(kg. d).2. Exogenous adiponectin could inhibit the expression of mRNA and protein ofcollagen Ⅰ and Ⅲ.