Adropin Ameliorates Cardiac Fibrosis In Type 2 Diabetic Cardiomyopathy Mice

Background and objective: Diabetic Cardiomyopathy(DCM)is a specific form of cardiomyopathy caused by insulin resistance,type 2 diabetes and hyperinsulinemia,independent of coronary artery disease and hypertension.Imbalance of glucose and lipid metabolism and myocardial fibrosis are its common pathophysiological manifestation.The mechanisms of diabetic cardiomyopathy have not yet been fully elucidated yet.Adropin is a secreted protein encoded by energy homeostasis associated gene(Enho),which plays an important role in maintaining energy balance,regulating glucose and lipid metabolism,improving endothelial function,promoting angiogenesis,and delaying atherosclerotic thrombosis.Studies have shown that Adenosine Monophosphate-Activated Protein Kinase(AMPK),a key molecule in the regulation of bioenergy metabolism,is one of the key signals involved in diabetes and its metabolic-related diseases,and an important component in the regulation of autophagy-related signal pathways.This study aims to explore the role and mechanism of Adropin in diabetic cardiomyopathy,and to explore whether its pathway is related to oxidative stress and AMPK signaling pathways regulating autophagy levels,so as to propose novel mechanism of Adropin and provide new treatment strategies for the treatment of diabetic cardiomyopathy.Methods: Thirty male Kunming rats aged 4-5 weeks were selected,and after adaptive feeding for 1 week,they were randomly divided into 3 groups,each with 10 rats.Respectively: the normal control group(Control,CON),diabetic cardiomyopathy group(DCM),and diabetic cardiomyopathy + Adropin group(DCM + ADR).The CON group was fed with standard feed for 16 weeks,fasted for 12 hours at the end of the 7th week,and injected an equal volume of citric acid-citrate buffer into the intraperitoneal cavity at one time.The same volume of saline was injected intraperitoneally at the end of the 10 th week for 7 days.The DCM group was fed with high-fat diet for 16 weeks,fasted for 12 hours at the end of the 7th week,and intraperitoneally injected 120mg/kg of streptozotocin(STZ)at one time.At the end of the 10 th week,the same volume of normal saline was injected intraperitoneally for a total of 7 day.The DCM + ADR group was fed with high-fat feed for 16 weeks,fasted for 12 hours at the end of the7 th week,and received a one-time intraperitoneal injection of STZ 120mg/kg.At the end of the10 th week,intraperitoneal injection of Adropin(2mg/kg.day)was started for 7 days.At the same time,the weekly food,drinking,body weight and fasting blood glucose levels of each group of mice were recorded.The fasting blood glucose of mice in DCM group and DCM + ADR group which were intraperitoneally injected with STZ for 3 days and 7 days was detected.Mice with fasting blood glucose ≤ 10mmol/L were excluded,and then fed with high-fat diet until the 16 th week.Glucose tolerance test and insulin tolerance test were used to compare the changes in glucose tolerance and insulin sensitivity of mice in each group;mice in each group underwent cardiac ultrasound at the end of the 16 th week to assess mouse heart function;the kit detected four serum lipids(TC,TG,LDL-C,HDL-C),oxidative stress-related indicators(SOD,MDA,GSH);HE and Sirius red staining were used to observe the morphology and structure of myocardial tissue and collagen fiber deposition;The protein levels of p-AMPK,mTOR,UII and UT in myocardial tissue were detected by immunohistochemical staining;Real-time fluorescent quantitative PCR was used to detect the gene expression of TGF-β1,Collagen1,ATG5,ATG 8,Beclin1,AMPK and mTOR.Result: 1.Basic physiological situation: At the end of the 16 th week,compared with the CON group,the average food intake,water intake,and fasting blood glucose of the DCM and DCM +ADR group mice were significantly increased(P<0.01),and the weight of the DCM group mice significant lower than CON group(P<0.05);Compared with the DCM group,the average food intake,water intake,body weight and fasting blood glucose of mice in the DCM + ADR group were no statistical difference.2.Glucose tolerance and insulin tolerance test: The fasting blood glucose of the mice in the DCM + ADR group began to decrease at the 15 th minute and gradually dropped to the baseline value.The DCM group began to decline slowly at the 30 th minute,indicating that Adropin intervention can enhance the glucose tolerance of the mice.In the insulin tolerance test,compared with the DCM group,the fasting blood glucose of the DCM + ADR group decreased significantly from 15 min to 60 min,and then slowly recovered,further indicating that Adropin intervention can enhance the insulin sensitivity of mice and improve insulin resistance.3.Cardiac ultrasound: Compared with the CON group,the left ventricular end-diastolic volume and end-systolic volume of the DCM group increased significantly(P <0.01),and the left ventricular ejection fraction and short axis shortening rate decreased(P <0.05);and Compared with the DCM group,the left ventricular end-diastolic volume and end-systolic volume of the DCM + ADR group decreased significantly(P <0.01),the left ventricular ejection fraction increased(P <0.05),and there was no significant difference in the short-axis shortening rate.The results showed that the left ventricular ejection function of diabetic cardiomyopathy mice was weakened,and the diastolic and contractile functions of the heart were impaired.After Adropin intervention,the left ventricular ejection fraction of the mice increased and the cardiac function improved.4.Serological indicators: Four tests of serum lipids in mice showed that the serum TC,TG and LDL-C concentrations of the DCM group increased significantly than the CON group(P <0.05),and the serum TC concentration of the DCM + ADR group was higher than the CON group(P<0.01).Compared with the DCM group,the serum LDL-C concentration of mice in the DCM +ADR group decreased(P <0.05);the differences in HDL-C concentration among mice in each group were not statistically significant.The detection of serum oxidative stress related indicators showed that compared with the CON group,the serum SOD activity in the DCM group was down-regulated(P <0.05),and the GSH concentration was significantly reduced(P <0.001),which further revealed that the anti-oxidation effect of diabetic cardiomyopathy mice was weakened,The level of oxidative stress increased.Compared with the CON group,the serum GSH concentration of the DCM + ADR group decreased(P <0.05).Compared with the DCM group,the serum SOD activity and the GSH concentration of the DCM + ADR group increased(P <0.05),indicating that Adropin might improve the antioxidant ability of mice.However,there was no significant difference in serum MDA levels of mice in each group.5.HE and Sirius red Staining: Compared with the CON group,the HE staining of mouse myocardial tissue found that in the DCM group,the structure of myocardial cells was disordered,some of the myocardial cells were hypertrophy and uneven distribution,and the connections between cells were untight,and fibers ruptured significantly;Compared with the DCM group,the structure of myocardial cells in the DCM + ADR group is relatively normal,the cells were arranged tightly,and the rupture of muscle fibers was improved.Sirius red staining of myocardial tissue in mice found that the collagen fibers around the blood vessels and between the myocardial tissues in the DCM group increased significantly than the CON group,the collagen fibers were thicker,and the degree of myocardial fibrosis was severe.Compared with the DCM group,the collagen fibers around the blood vessels and between the myocardial tissues in the DCM + ADR group were reduced,the collagen fibers were thinner,and the degree of myocardial fibrosis was reduced.6.Immunohistochemical staining: Compared with the CON group,the expression of p-AMPK in the myocardial tissue of the DCM group decreased,and the expression of mTOR,UII and UT increased.The expression of UII and UT in the DCM + ADR group increased slightly than the CON group,while the expression of p-AMPK and mTOR in the DCM + ADR group was not significantly different.Compared with the DCM group,the expression of p-AMPK in myocardial tissue of mice in the DCM + ADR group increased significantly,and the positive expressions of mTOR,UII and UT were significantly reduced.7.Real-time PCR: Compared with the CON group,the expressions of AMPK,ATG5,ATG8 and Beclin1 m RNA in the myocardial tissues of the DCM group were all down-regulated(P<0.01),and the expressions of mTOR,TGF-β1,and COL1 m RNA were significantly up-regulated(P<0.001).There were no significant differences in the expression of AMPK m RNA in the myocardial tissue of the DCM + ADR group and the CON group.The expressions of ATG5,ATG8 and Beclin1 were all down-regulated than(P<0.05),and the expressions of mTOR,TGF-β1,and COL1 m RNA were up-regulated than the CON group(P<0.05).Compared with the DCM group,the expression of AMPK,ATG5,ATG8 and Beclin1 in the myocardial tissue of mice in the DCM + ADR group were all up-regulated(P<0.05),and the expression of mTOR,TGF-β1 and COL1 m RNA was down-regulated(P<0.001).Conclusion: This study showed that Adropin might increase glucose tolerance in diabetic cardiomyopathy mice,improve insulin resistance,regulate lipid metabolism by reducing serum low-density lipoprotein cholesterol,and enhance the body’s antioxidant capacity,and further indicated that exogenous Adropin intervention might decrease collagen deposition and the expression of fibrogenic cytokines TGF-β1,COL1,delay the process of myocardial fibrosis in diabetic cardiomyopathy,and improve cardiac function in mice with diabetic cardiomyopathy.Its mechanism may be related to activation of AMPK and inhibition of mTOR signaling pathway and promotion of autophagy.The interaction and mechanism of Adropin and UII/UT system in myocardial fibrosis of diabetic cardiomyopathy need to be further explored.