Effect Of Irbesartan On MMPs Pathway In Diabetes Induced Myocardial Fibrosis In Rat Model

Objective: To explore the role of MMPs pathway and AGEs-RAGE system in the process of myocardial fibrosis in diabetic rat model and analyze the possible correlation between MMP-14, MMP-2, TIMP-1 and TIMP-2. Further to explore the effect of irbesartan intervention on the happening of myocardial fibrosis and the changes of MMPs pathway and AGEs-RAGE system.Methods:1. Diabetic rat model preparation and grouping. All male SD rats were randomly divided into three groups: normal control group(CON), high glucose and high caloric diet group(HC) and diabetic group. The rats in normal control group were treated with normal diet, the rats in other groups were treated with high sugar and high fat diet. After 4 weeks, diabetes rat model was mimicked by intraperitoneal injection with STZ at 30 mg/kg and the diabetic rats were randomized into 2 groups: diabetic group(DM) and irbesartan + DM group(Ir+DM). The rats in Ir+DM group were treated with irbesartan at 50 mg/kg/d for 4weeks, 8weeks and 12 weeks.2. The rat’ general conditions including mental state, diet, reaction and hair colour were observed.3. The dosage of irbesartan was adjusted according to the rat’s weight weekly.4. Fasting blood glucose(FBG) level was measured at 4 w, 8 w and 12 w.5. Rats were killed at 4w, 8w and 12 w, respectively. The ratio of heart weight/ body weight(H/B) and left ventricular weight index(LVWI) were measured. The plasma triglyceride(TG) and total cholesterol(TC) levels were measured.6. The myocardial morphological changes were observed by HE staining and Masson staining.7. The contents of col I and col III in heart tissue and the levels of plasma TIMP-1 andTIMP-2 were evaluated using ELISA method.8. The protein expressions of AGE, RAGE, col I, MMP-2, TIMP-2 and MMP-14 in heart tissue were analyzed by Western blotting.Results: 1. The changes of general conditionThe rats in model group were appeared obvious diabetic symptoms including polyphagia, polydipsia and weight loss, hair messy and dull, slow reaction, and some rats had some symptoms such as cataract and gangrene. The general condition was improved in Ir+DM group.2. The changes of fasting blood glucose(FBG), body weight(BW), heart / body ratio(H/B), left ventricular mass index(LVWI), triglyceride(TG) and total cholesterol(TC)Compared with CON 4w group, BW level in HC 4w group was increased significantly(P < 0.01), while the levels of FBG, H/B, LVWI had no obviously change. Compared with CON 4w and HC 4w groups,, FBG levels in DM 4w group was increased while BW, H/B and LVWI had no change(P < 0.01). Compared with DM 4w group, there had no obvious change of BW, H/B, LVWI, FBG in Ir+DM 4w group.Compared with CON 8w group, in HC 8w group, BW level was increased significantly(P < 0.01), there had no obviously changes of the levels of FBG, H/B and LVWI. Compared with CON 8w and HC 8w groups, BW level was decreased(P < 0.01), while FBG level, H/B and LVWI were increased significantly(P < 0.01). Compared with DM 8w group, BW level was increased significantly in Ir+DM 8w group(P < 0.01), FBG, H/B and LVWI were decreased significantly(P < 0.01), FBG had no obviously change. The trend of 12 w was similar to 8w.Compared with CON group, in HC, DM and Ir+DM groups, plasma TG and TC levels were increased significantly(P < 0.01). Compared with DM group, in Ir+DM group, there had no obviously changes of TG and TC levels.3.The changes of cardiac pathologyHE staining was shown that: in DM group, myocardial fibers were arranged irregularly, fibers were ruptured, the gaps between cells were widen. In Ir+DM group, the pathological conditions had improved. Masson staining was shown: rats in DM group had a large amount of interstitial collagen fiber hyperplasia, collagen accumulation, or uneven distribution, while pathological conditions had improved in Ir+DM group.4. The contents of col I and col III in heart tissue and TIMP-1, TIMP-2 contents in plasma by ELISA methodThe contents of col I, col III and the ratio of col I/col III had no significant change at 4 weeks. Compared with CON 8w group, the contents of col I, col III, the ratio of col I/col III and plasma TIMP-1, TIMP-2 levels were no change in HC group. Compared with CON 8w and HC 8w groups, the contents of col I, col III, the ratio of col I/col III, the plasma TIMP-1 and TIMP-2 levels were increased significantly in DM 8w group(P<0.01). After irbesartan treatment, all parameters were lower than in DM 8w group. With the course extension, the change trend were more obviously.5. The protein expressions of coⅠ, AGE, RAGE, MMP-2, TIMP-2 and MMP-14 by Western blotThe protein expression of col I was no statistical difference between CON group and HC group. Compared with CON and HC groups, the expression of col I protein had no change in DM 4w group, but increased in DM 8w group, and increased more obviously in DM 12 w group. Compared with DM 12 w group, the expression of col I protein was significantly decreased in Ir+DM group.Compared with CON group, the protein expression levels of AGE, RAGE MMP-2, TIMP-2 and MMP-14 were no significant change in myocardial tissue in HC 12 w group. Compared with CON and HC 12 w groups, the protein expression levels of AGE, RAGE, TIMP-2 and MMP-14 in DM 12 w group were increased, but the expression level of MMP-2 and the ratio of MMP-2/TIMP-2 were decreased significantly. After irbesartan treatment, the protein expression levels of AGE, RAGE, TIMP-2, MMP-14 were decreased and the expression level of MMP-2, the ratio of MMP-2/TIMP-2 were increased significantly.Conclusions: 1. The type 2 diabetes induced myocardial fibrosis in rat model was succeeded.2. In myocardium of diabetic rat model, with the progress of diabetes,(1) The expressions of AGE, RAGE and col I protein were upregulated, suggesting fibrosis synthesis was increased.(2) Downregulation of MMP-2 protein expression and the ratio of MMP-2/TIMP-2, upregulation of TIMP-2 protein expression implied that fibrosis breakdown was decreased.(3) The expression of MMP-14 protein was increased, which suggested that MMP-14 was involved in the differentiation of fibroblasts and induced ventricular remodeling. The increases of TIMP-1, TIMP-2 levels suggested the dysfunction of left ventricle.3. Irbesartan can improve myocardial fibrosis in diabetic rats, the mechanisms may be:(1) Downregulating the expressions of AGEs, RAGE protein, then reducing collagen synthesis;(2) Upregulating the expression of MMP-2 protein, downregulating the expression of TIMP-2, increasing the ratio of MMP-2/TIMP-2 contributed to the collagen metabolism and promoted collagen degradation.