Protective Effect Of Trehalose On Diabetic Cardiomyopathy

Objective:In this study,we established mouse model of type 1 diabetes and cultured H9c2 cardiomyocytes in vitro with high glucose,to explore the protective effects of trehalose of cardiomyocytes injured by high glucose.We further explored the role of PK2/PKR signaling pathway in the treatment of cardiomyocytes injured by high glucose with trehalose.Methods:1.C57BL/6J mice were intraperitoneally injected with 50mg/kg/d streptozotocin(STZ)for 5 days to establish diabetic mice model.After 7 days,the random blood glucose value was measured,random blood glucose was ? 16.7 mmol/L,the mice were considered as the diabetes mellitus mice.The mice were divided into normal control group(Con group,n = 15),diabetes group(DM group,n = 15),trehalose group(Con-TLS group,n = 15)and trehalose treatment group(DM-TLS group,n =15).Con-TLS group and DM-TLS group mice were injected with TLS solution(1mg/g/d)intraperitoneally for two consecutive days,and fed with 2 %(w/v)trehalose solution for 24 weeks;The Con group and DM group mice were fed with drinking water for 24 weeks.The body weight and random blood glucose of mice were measured every 4 weeks.At the end of the experiment,the glucose tolerance and echocardiogram were measured;The weight of heart and lung were measured;serum myocardial enzyme and lipid expression were measured by automatic biochemical analyzer;myocardial microstructure,myocardial morphology,degree of fibrosis and number of autophagic vesicles were observed by HE staining and Masson trichrome staining;myocardial apoptosis were observed by TUNEL staining;Western blot detected apoptosis related proteins,pyroptosis related proteins and autophagy related proteins and PK2/PKR signaling pathway protein expression levels.2.H9c2 cardiomyocytes were cultured in vitro and randomly divided into groups:(1)normal group(NG),high glucose group(HG),high glucose plus 50 mmol/L TLS group(HG-TLS(50)),high glucose plus 100 mmol/L TLS group(HG-TLS(100)),high glucose plus 150 mmol/L TLS group(HG-TLS(150)),normal group plus 150mmol/L TLS group,(NG-TLS(150));(2)normal group(NG),normal group plus 100mmol/L TLS group(NG-TLS),normal group plus 10?mol/L PKRA7 group(NGPKRA7),high glucose group(HG),high glucose plus 100 mmol/L TLS group(HG-TLS),high glucose plus 100 mmol/L TLS group and 10?mol/L PKRA7group(HG-TLS-PKRA7).Cells were collected after 72 hours of drug intervention.Western blot detected apoptosis related proteins,pyroptosis related proteins,autophagy related proteins and PK2/PKR signailing pathway protein expression levels.Results:Part 1 Protective effect and mechanism of trehalose on myocardial injury in type I diabetic mice1.Blood glucose and body weight at different time points: With the prolongation of the course of the disease,the blood glucose of the mice in the Con group and the Con-TLS group was stable,and the blood glucose of the mice in the DM group and the DM-TLS group was also at a stable high level;With the prolongation of the course of disease,the weight of mice in Con group and Con-TLS group increased gradually,while that in DM group decreased slowly.At the end of 24 weeks,compared with con group,the weight of DM group mice was significantly reduced,the body weight of DM-TLS group was significantly higher than that of DM group(P<0.05),but it did not reach the normal level.TLS had no effect on the body weight of Con group mice(P>0.05).2.Organ weight coefficient: the HW,LW of DM group were significantly lower than that of Con group,and the HW/BW,LW/BW were slightly increased.TLS decreased HW/BW and LW/BW in DM mice.TLS had no effect on organ weight of normal mice(P>0.05).3.Glucose tolerance results: At 120 minutes,the blood glucose value of DM group was still at a high level,the blood glucose of mice in Con,Con-TLS and DM-TLS groups fell back to the initial concentration;AUC of DM group was significantly higher than that of Con group(P<0.05).Compared with DM group,AUC of DM-TLS group decreased slightly,and was still at a high level(P>0.05).TLS had no effect on Con group.4.Metabolic characteristics: compared with Con group,the values of TC,TG and myocardial enzymes activities in DM group were significantly increased(P <0.05).TLS partially or significantly reduced the abnormal increase.TLS had no effect on serum lipid level and myocardial enzyme activity in normal mice.5.Echocardiography: Compared with control group,HR?EF?FS?E/A ratio decreased.After TLS treatment,HR,EF,FS,E/A ratio increased,not reaching the normal level.6.HE staining results: in Con group,the fibers were uniform pink and were closely arranged in rows,the thickness of fibers was uniform.In DM group,myofibrillar cells appeared dissolution,rupture and vacuole,the arrangement was not close,myofilament thickness was not uniform.Compared with DM group,DM-TLS group had more regular myocardial morphology,more orderly arrangement,less muscle fiber breakage and cavitation damage.The myocardial morphology of mice in Con-TLS group was similar to that in Con group.7.Masson' trichrome staining results: Compared with Con group,the collagen deposition increased significantly in DM group.Compared with DM group,the collagen deposition of DM-TLS group was significantly reduced,and the myocardial morphology and fibrosis degree of Con-TLS group were similar to those of Con group.8.Transmission electron microscope: Compared with Con group,the number of autophagic vesicles in DM group decreased significantly,while TLS increased the number of autophagic vesicles in DM group,and the number of autophagic vesicles in Con TLS group increased slightly.9.TUNEL: The number of myocardial cell apoptosis was in DM group was more than that in Con group,and the number of myocardial cell apoptosis was decreased after TLS treatment.The number of myocardial cell apoptosis was in Con-TLS group had no change compared with Con group.10.Western blot: the expression levels of Cleaved-Caspase3,NALP3,Caspase-1,IL-1?,IL-18,p62 protein and Bax/Bcl-2 ratio in DM group were significantly higher than those in Con group,and the expression levels of Beclin-1,Atg5,PK2,PKR1,PKR2 protein and LC3II/I ratio in DM group were significantly decreased(P<0.05).TLS significantly reversed the protein expression in the myocardium of DM mice.TLS had no effect on the expression of these proteins in normal mice(P>0.05).Part 2 Protective effect and mechanism of trehalose on H9c2 cardiomyocytes injured by high glucose1.Western blot:(1)Compared with NG group,Bax/Bcl-2 ratio and Cleaved-Caspase3 protein expression increased significantly(P<0.05),but decreased significantly after TLS treatment.TLS had no effect on the expression of apoptosis protein in NG group.(2)The expression of pyroptosis related proteins in HG group was significantly increased than that in NG group(P < 0.05).Compared with HG group,the expression level of pyroptosis related protein in TLS treatment groups partially or overtly decreased,the expression of NALP3,Caspase-1 and IL-1?decreased in a dose dependent manner.TLS had no effect on the expression of pyroptosis protein in NG group.(3)HG stimulation significantly reduced the expression of Beclin-1,Atg5 and LC3II/I ratio(P<0.05),and the expression level of p62 protein was significantly increased(P < 0.05).HG-TLS(100)significantly reversed the abnormal level of protein expression(P<0.05).TLS(150)had no effect on the expression level of autophagy protein in NG group(P>0.05).(4)Compared with NG group,the expression of PK2,PKR1 and PKR2 in HG group significantly decreased(P<0.05),and TLS(100)significantly increased PK2,PKR1 and PKR2 expression to the normal levels(P<0.05).TLS had no effect on PK2/PKR signal pathway expression.(5)PKRA7 significantly inhibited PK2 expression(P<0.05),PKRA7 significantly reversed TLS induced PK2/PKR protein expression increased in HG group.In addition,TLS had no effect on PK2/PKR signal pathway protein expression in NG group.(6)PKRA7 partially attenuated the TLS induced decrease of Cleaved-Caspase 3,NALP3,Caspase-1,IL-18 and IL-1? protein expression and Bax/Bcl-2 ratio(P>0.05).In addition,PKRA7 had no effect on the expression level of Beclin-1,p62,Atg5 protein and LC3II/I ratio in HG-TLS group.Conclusion:The salient findings from our current study suggest that trehalose has a protective effect on myocardial injury induced by high glucose,and its mechanism may be related inhibition of myocardial apoptosis,pyrosis and enhancement of autophagy.In addition,PK2/PKR signaling pathway may also be activated to protect cardiomyocytes from high glucose injury.