Protective Effect Of Paeonol On Diabetic Cardiomyopathy In Rats And The Mechanism Of Oxidative Stress Signal Transduction

Diabetes meilitus（DM）is a group of metabolic diseases characterized by chronic hyperglycemia caused by insulin secretion disorder or impaired biological function.The number of adults with diabetes worldwide is 285million by 2010,which is estimated to increase to 439 million by 2030,which poses a serious threat to human health.Continuous high glucose stimulation results in vascular damage and involvement of heart,brain,kidney,eyes and other organs.Diabetic cardiomyopathy（DCM）is one of the common complications of diabetes,which is independent of hypertension,myocardial ischemia and myocardial valve disease that leads to abnormal cardiac structure and function.The main pathological manifestations include myocardial inflammation,myocardial fibrosis,myocardial apoptosis and cardiac dysfunction.At present,the treatment of DCM mainly aims at relieving and inhibiting heart failure by effectively controlling blood sugar,lowering blood pressure and lipids.However,western medicine treatment has different degrees of side effects and the treatment effect is not targeted.Chinese herbal medicine has natural advantages in the prevention and treatment of cardiovascular diseases due to its low toxicity and multi-target characteristics.Peony skin is often used to treat cardiovascular and cerebrovascular diseases in traditional Chinese medicine.Paeonol（Pae）,derived from the root bark of peony,is one of the most important effective components of peony bark,and has many pharmacological effects such as anti-oxidation,anti-blood lipid,anti-blood glucose,anti-arrhythmia and anti-inflammation.Previous studies in our group have confirmed that paeonol can improve ventricular remodeling after myocardial infarction in rats.However,the protective effect of paeonol on DCM in rats has not been reported.In this study,the protective effect of paeonol on DCM and the mechanism of oxidative stress signal transduction were further investigated by constructing DCM rat model and observing the changes of myocardial structure and function in rats.Part Ⅰ Protective effect of paeonol on diabetic cardiomyopathy in rats.Objective:To observe the effect of paeonol on myocardial structure and function changes of DCM in rats,and further confirm the protective effect of paeonol on DCM.Methods:1 Preparation of DCM mode:SPF male SD rats weigh（150+10）g,and the normal diet is suitable for feeding for 1 week.The mode rats are fed with high fat and high sugar diet for 6 weeks,fasting for 18 hours,intraperitoneal injection of small dose STZ 25mg·kg^-1 for 2 days to construct type II diabetes rat model.After injection of STZ,tail venous blood of rats is taken and blood sugar is measured on the 3td and 7th days respectively.When fasting blood glucose≥16.7 mmol L,the rat model of type II diabetes was successfully prepared.After 12 weeks,one rat from each group was taken for histological examination,which showed myocardial pathological changes,hypertrophy of myocardial cells,disorder of myocardial collagen fiber arrangement,and myocardial fibrosis of different degrees,indicating the successful preparation of DCM mode.2 Experimental grouping:male SD rats were divided into normal group（NC group）、model group（MC group）、paeonol low dose group(Pae-L group,30 mg kg^-1)、paeonol medium dose group(Pae-M group,60 mg kg^-1)、paeonol high dose group(Pae-H group,120 mg kg^-1)、metformin(DMBG group,157mg kg^-1)positive drug group.At the 9th week,drug intervention was started.Paeonol and metformin were dissolved in sodium carboxymethyl cellulose（1%）and iused into stomach respectively.NC group and MC group were iused with sodium carboxymethyl cellulose of equal volume once/d for 12 weeks.3.Index detection:The fasting blood glucose（FBG）and body weight（BW）of rats in each group were measured,and the heart weight（HW）of rats in each group was weighed to measure the heart weight index（HWI,heart weight/body weight）;Total cholesterol（TC）and triglyceride（TG）were measured by automatic biochemical analyzer.Double antibody sandwich enzyme-linked immunosorbent assay（ELISA）was used to measure the contents of advanced glycation end products（AGEs）、glycosylated hemoglobin（GHb）、lactate dehydrogenase（LDH）、aspartate aminotransferase（AST）and creatine kinase isoenzyme MB（CK-MB）;Hematoxylin-eosin staining（HE staining）and Masson staining were used to observe myocardial pathological changes and myocardial fibrosis degree in each group of rats.;Western blot was used to detect the expression of type I collagen and type III collagen.Results:1 Effects of Pae on BW and HWI of Rats in Each Group:Compared with NC group,BW and HWI in MC group decreased significantly（P<0.05）and increased significantly（P<0.05）;Compared with MC group,the BW of Pae-L group、Pae-M group、Pae-H group and DMBG group increased to different degrees（P<0.05）and HWI decreased to different degrees（P<0.05）,among which the influence of Pae-H group on BW and HWI was more significant than that of Pae-L group and Pae-M group.2 Effects of Pae on blood sugar and blood lipid levels of rats:Compared with NC group,the effects of Pae on blood sugar and blood lipid levels of rats in MC group increased significantly in FBG、GHb、AGEs、TC and TG（P<0.05）;Compared with MC group,FBG、GHb、AGEs、TC、TG of Pae-L group、Pae-M group、Pae-H group and DMBG group are all reduced to different degrees（P<0.05）,and Pae-H group has more significant improvement effect on various indexes.3 Effect of Pae on CK-MB、AST and LDH in Serum of Rats in Each Group:The results of myocardial tissue markers CK-MB、AST and LDH showed that compared with NC group,CK-MB、AST and LDH in serum of MC group were significantly increased（P<0.05）.Compared with MC group,CK-MB、AST and LDH in Pae-L group、Pae-M group and Pae-H groupand DMBG group decreased to different degrees（P<0.05）,among which Pae-H group had more significant improvement effect on various indexes.4 HE staining resultsAfter HE staining and fixation,the myocardial tissue of rats was observed by optical microscope.The morphology of myocardial cells and myocardial fibers in NC group were normal;Compared with NC group,MC group has hypertrophy of myocardial cells,disordered arrangement of myocardial fibers,fiber breakage and dissolution,uneven staining between myocardial cells,and nucleus breakage or even disappearance;Compared with MC group,the pathological changes of myocardium in Pae-L group、Pae-M group、Pae-H group and DMBG group have been improved to different degrees.The arrangement of myocardial fibers is regular and the coloration is uniform.The improvement in Pae-H group is more obvious than that in Pae-M group and Pae-L group.5 The results of Masson staining showed that the degree of myocardial fibrosis in each group was observed by optical microscope.Red myocardial cells were dominant in the NC group,and there was no significant increase of myocardial collagen fibers in the myocardial interstitium.Compared with the NC group,myocardial cells and interstitial collagen fibers（blue）in the MC group were significantly increased.Compared with MC group,myocardial collagen fiber hyperplasia in Pae-H group,Pae-L group,Pae-M group and DMBG group showed different degrees of improvement,among which the Pae-H group had the least blue bands and the most obvious improvement of collagen fiber.6 Western blot method was used to detect the expression of typeⅠcollagen（Col-Ⅰ）and typeⅢcollagen（Col-Ⅲ）.The expression of col-I and col-Ⅲproteins in myocardial interstitium of NC group is relatively low;compared with NC group,the deposition of col-I and col-Ⅲin MC group will increase,which is positively correlated with the degree of myocardial fibrosis（p<0.05）;Compared with MC group,the expression of col-I and col-Ⅲproteins in Pae-H group,Pae-L group,Pae-M group and DMBG groupreduced to different degrees,and the improvement of col-I and col-Ⅲin Pae-H group was more obvious.Conclusion:1 Pae can reduce the contents of FBG,GHb,AGEs,TC and TG in DCM rats,improve the levels of blood glucose and lipid,and play a protective role in myocardial injury in DCM rats.2 Pae can reduce the contents of CK-MB,AST and LDH in serum of DCM rats,reduce the release of myocardial enzyme and reduce the degree of myocardial injury in DCM rats.3 Pae can reduce the level of HWI and improve the myocardial structure.4 Pae suppresses the DCM myocardial tissue of rat col-I and col-Ⅲexpression,improved the disorder of myocardial cell center muscle fibers arranged,reduces the myocardial tissue fibrosisdegree,to DCM rats myocardial protection.Part Ⅱ Effects of paeonol on oxidative stress signaling pathway in diabetic cardiomyopathy in rats.Objective:To observe whether paeonol can alleviate the occurrence and development of diabetic cardiomyopathy by activating Nrf2/ARE pathway and inhibiting AGEs/RAGE/NOX signaling pathway.Methods:The preparation of the model and experimental grouping are the same as those of 1 and 2 in the first part of the method.Superoxide dismutase（SOD）test box（WST-1 method）and malondialdehyde（MDA）test box（TBA method）were used to measure SOD activity and MDA content changes in myocardial tissue respectively.Real Time PCR（qRT-PCR）was used to detect cardial tissue of rats in each group.The expression levels of Nrf2、RAGE、NOX and NF-κB p65 pro mRNA expression levels of Nrf2、RAGE、NOX and NF-κB p65 in myoteins were detected by Western blot.Results:1 Compared with the NC group,the activity of SOD in myocardium of MC group was significantly decreased（P<0.05）,and the content of MDA was significantly increased（P<0.05）.Compared with MC group,SOD activity of Pae-L group,Pae-M group and Pae-H group increased to different degrees（P<0.05）,and MDA content decreased to different degrees（P<0.05）.Among them,Pae-H group has more obvious improvement effect on various indicators.2 Compared with the NC group,the mRNA expression level of Nrf2 in the myocardium of the MC group was significantly decreased（P<0.05）,and the mRNA expression levels of RAGE,NOX,and NF-κB p65 were increased（P<0.05）.Compared with MC group,the mRNA expression of Nrf2 in myocardial tissue of Pae-L group,Pae-M group and Pae-H group was increased to different degrees（P<0.05）,and the mRNA expression of RAGE,NOX and NF-κB p65 was decreased to different degrees（P<0.05）.3 Compared with the NC group,the protein expression level of Nrf2 in the myocardium of the MC group was significantly decreased（P<0.05）,and the protein expression levels of RAGE,NOX and NF-κB p65 were significantly increased（P<0.05）.Compared with MC group,Nrf2 protein expression in myocardial tissue of Pae-L group,Pae-M group and Pae-H group was increased to different degrees（P<0.05）,and the expressions of RAGE,NOX and NF-κB p65 protein were decreased to different degrees（P<0.05）.In addition,Nrf2 was significantly increased and RAGE,NOX and NF-κB p65 were significantly decreased in Pae-H group compared with Pae-L group and Pae-M groupConclusion:1 Pae can improve the gene expression of Nrf2 in the cardiac tissue of DCM rats,up-regulate the expression of SOD and other antioxidant enzymes,and reduce the content of MDA,suggesting that Pae’s protective effect on the cardiac muscle of DCM rats may be achieved by activating the Nrf2/ARE signaling pathway and improving the body’s ability to remove ROS.2 Pae can reduce the generation of AGEs in DCM rats,inhibit the expression of RAGE and NF-κB p65 genes,reduce the activity of NADPH oxidase,and reduce the production of ROS,suggesting that the protective effect of Pae on the myocardium of DCM rats may be related to the inhibition of AGEs/RAGE/NOX signaling pathway.