| It is urgent to treat complications induced by old age and diabetes mellitus because of rapidly increase of the elder people and morbidities of diabetes. Dysfunctions of the cardiovascular system that mainly cause the death of aged people and declining life quality of diabetic patients is the most prominent complication caused by aging and diabetes mellitus. Accordingly, the development of targeted treatment is of great significance.Glucose and other reducing sugars react with proteins by a series of reactions to form a class of heterogeneous, nonenzymatic sugar-amino adducts that are called advanced glycation endproducts (AGEs). Formations and accumulations of AGEs crosslinks in longevity proteins of cardiovascular were closely related to cardiovascular stiffness and the decrease of cardiovascular tissues'sensitivity to bioactive molecules of aging people and diabetic patients. Breaking AGEs crosslinks is a novel therapeutic strategy based on reversing cardiovascular stiffness. Preclinical and clinical studies both indicated that AGEs breakers could explicitly improve cardiovascular system dysfunction associated with ageing and diabetes mellitus. However, AGEs breakers are developing drug with innovative ideas and mechanisms. Novel compounds with higher AGEs breaking activity still need to be further studied.The aims of this study include two parts: In partâ… , to research the effectiveness and mechanisms of C36D2, the main hydrolysate of the novel AGEs breaker C36, on reversing cardiovascular system stiffness in experimental diabetic rats, and to confirm whether it is the active form of C36 in vivo, and to provide experimental evidences for in vivo efficacy of C36. In partâ…¡, to explore whether AGEs breakers can improve sensitivity of diabetic-hypertensive rats to antihypertensive agents and its possible mechanism.In the first part, with C36 as the control, C36D2 was studied by classical method which was used to evaluate AGEs breakers. First of all, the specific enzyme labeled immunosorbent assay (ELISA) method was used to evaluate the ability of C36D2 to break formed crosslinks of glycated bovine serum albumin (AGEs-BSA) to the collagen in vitro, and the experiment of breaking immunoglobulin G crosslinked to diabetic rats'red blood cell surface (RBC-IgG) was used to evaluate the ability of C36D2 to break established AGEs crosslinks in vivo. And then, STZ-induced DM rats (course of DM for 3 monthes) treated with C36D2 (9 mg/kg, 18mg/kg, and 36mg/kg, respectively) for 4 weeks were studied by hemodynamic study with Doppler technique. In the measurement of cardiovascular system, systolic and diastolic blood pressure, cardiac output, and heart rate were measured. Total peripheral resistance (TPR) was determined as the quotient of mean arterial blood pressure and cardiac output. Stroke volume was calculated as the quotient of cardiac output and heart rate. Systemic arterial compliance (SAC) was calculated as the quotient of stroke volume and pulse pressure. In the measurement of left ventricle, the left ventricular systolic pressure and the left ventricular diastolic end pressure were measured and the maximal rate of left ventricular pressure rise (pos dp/dtmax) and pressure fall (neg dp/dtmax) were calculated from the digitized left ventricular pressure recording. Meanwhile, AGE contents in aorta, left ventricular myocardium, and kidney were determined by fluorescence detection. And solubility of tail-tendon collagen and ventricular myocardium were investigated by limited pepsin digestion under acidic conditions. Experimental results indicated that C36D2 could break AGEs crosslinks in vitro, and could significantly increase systemic arterial compliance, reduce total peripheral resistance and increase cardiac output in experimental diabetic rats. Moreover, the compound had the abilities to increase the left ventricular systolic pressure, the maximal rate of left ventricular pressure change and to reduce the left ventricular diastolic end pressure, and finally restored left ventricular functions in experimental diabetic rats. In addition, treatment with C36D2 resulted in an increase of solubility of tail tendon collagen and left ventricular myocardial collagen and a reduction of AGEs fluorescence in aorta, left ventricular and kidney. In conclusion, the effects of C36D2 almost resembled all those of ALT-711 and C36, and the mechanism was also similar to that of ALT-711 and C36. For the reason that C36D2 can restore cardiovascular system disorder in DM rats through the mechanism relating to breaking AGEs crosslinks over-accumulated in vivo, C36D2 should be the active form of C36, and a novel AGEs breaker.In the second part, at the very beginning, A novel experimental diabetic-hypertensive model wad established by treating STZ-diabetic rats with 1% NaCl-drinking chronically. And then, after AGEs breakers treatment for 4 weeks, diabetic-hypertensive rats were anesthetized with urethane- chloralosane-combination. A fluid-filled catheter was introduced through the right carotid artery for blood pressure monitoring. Another catheter was introduced into femoral vein for injecting Nifedipine of gradient concentration. The influence of AGEs breakers on effect of lowering blood pressure of antihypertensive which acts on vascular smooth muscle were observed. Meanwhile, contents and gene expressions of some bioactive factors in circulation system and kidney were measured as well. Immunohistochemical method was used to assay ET-1 expression in the kidney. In addition, aorta rings experiment was applied to evaluate effects of the compound on vascular systems. The results indicated that Nifedipine had significantly better anti-hypertensive effects on diabetic-hypertensive rats with AGEs breakers treatment than those vehicle treated control . Moreover, in AGEs breakers treatment diabetic-hypertensive rats, plasma NO contents were significantly increased while expressions of preproendothelin gene in aorta and Angiotensinogen gene in renal were significantly decreased. But there was no remarkable change on plasma Angâ…¡or ET-1. In addition, the relaxation of thoracic aorta rings from AGEs breakers treated diabetic-hypertensive rats induced by ACh significantly exceeded those from non-treated rats. Those studies demonstrated that STZ-diabetic rats that underwent chronic 1% salt loading showed a stable elevation in blood pressure, accompanying with increases in vasoconstriction substance ET-1 and renal Angâ…¡. AGEs breakers could significantly augment anti-hypertensive effects of Nifedipine in diabetic-hypertensive rats. Its mechanisms may relate to improving functions of vascular endothelial cells which had been disordered by diabetes. Besides, effects of AGEs on local renal RAS may participate in these mechanisms as well.In conclusion, as candidates of treating diabetic and aging cardiovascular complications, C36, an noevl AGEs breaker, and its hydrolysate C36D2 have great values of further development.
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