| Background and objective Diabetes mellitus(DM)is a chronic metabolic disease characterized by hyperglycemia and metabolic disorders.Diabetic cardiomyopathy(DCM)is one of the common cardiovascular complications of diabetes,which can lead to heart failure and seriously threaten the life safety of patients.Melatonin(MLT)is a neurohormone mainly produced in the pineal gland of humans and mammals,and secreted into various cells through the blood circulation to play a regulatory role.Melatonin can play a key role in alleviating many chronic diseases by inducing cellular antioxidant defenses and alleviating cellular inflammation and apoptosis.Non-codingRNA(ncRNA)is transcribed from the genome,not translated into protein,and plays a regulatory role at theRNA level.Long non-codingRNA(lncRNA)and microRNA(miRNA)are two common non-codingRNAs.LncRNAs can act as competing endogenousRNAs or sponges,reducing the functional availability of targeted miRNAs and participating in the regulation of gene expression,and their lengths are generally more than 200 nucleotides.MiRNAs usually bind to mRNAs to regulate gene expression at the post-transcriptional level and are generally about 18-25 nucleotides in length.Recent studies have shown that ncRNAs play a crucial role in the pathophysiological process leading to diabetic cardiomyopathy.This study aimed to further explore the anti-apoptotic effect of melatonin on cardiomyocytes from diabetic patients and to elucidate the underlying mechanisms associated with ncRNAs.Methods In vivo: Sprague Dawley(SD)rats were randomly divided into normal group(N),diabetes group(DM),insulin treatment group(DM+insulin),melatonin treatment group(DM+MLT),12 per group.Animals was adaptively reared for one week,and then the type 1 diabetes model was established by intraperitoneal injection of streptozotocin(Streptozotocin,STZ,55 mg/kg).After a week,blood glucose was measured,and the model was considered successful when the blood glucose was ≥ 11.1 mmol/L.And then insulin(IN,1 u/kg/d,once a day for the first 10 weeks and twice a day for the next 6weeks)and melanin(10 mg/kg/d)were administered respectively.During the treatment,SD rats were fed with ordinary food and water.And their body weight and blood glucose were measured once a week.After 16 weeks,they were euthanized by anesthesia,and the hearts were separated rapidly.Langendorff perfusion experiments were performed on the hearts.Serum and cardiac tissue were collected and stored for subsequent experiments.The melatonin concentration of SD rats in serum were detected by ELISA kit.The changes of heart morphology of diabetic rats were observed by H&E staining and Masson staining.The effect of melatonin on cardiac function of diabetic rats was observed by Langendorff perfusion experiments.Western blot was used to observe melatonin receptorrelated proteins(Mel-1A/B-R and NQO2),apoptosis-related proteins(Bax,Bcl-2,caspase-3 and caspase-9),MAPK pathway-related proteins(p-JNK/JNK,p-ERK/ERK and p-p38/p38),and endoplasmic reticulum stress-related protein(GRP-78,PERK,IRE1α,ATF-6α,ATF4 and CHOP).The expressions of lncRNA H19 and miRNA-29 c were observed by TR-qPCR.In vitro: The H9c2 cells were stimulated with a medium containing high concentration of glucose(33 mM)to simulate the diabetic state.The cells were divided into normal glucose group(NG),high glucose group(HG),solvent group(HG+DMSO),insulin group(HG+insulin,1 μM),and melatonin group(HG+MLT,10μM).Western blot was used to observe melatonin receptor-related proteins(Mel-1A/B-R and NQO2),apoptosis-related proteins(Bax,Bcl-2,caspase-3 and caspase-9),MAPK pathway-related proteins(p-JNK/JNK,p-ERK/ERK and p-p38/p38),endoplasmic reticulum stress-related protein(GRP-78,PERK,IRE1α,ATF-6α,ATF4 and CHOP).The apoptosis of H9c2 cells under high glucose stimulation was observed by Hoechst staining.In addition,lncRNA H19 shRNA,miRNA-29 c inhibitor and miRNA-29 c mimic were also transfected.RT-qPCR was used to observe the expression of lncRNA H19,miRNA-29 c and MAPK13 after transfection.Western blot was used to detect the expression of MAPK13 protein.The possible target genes of lncRNA H19 and miR-29 c were predicted based on bioinformatics analysis,and then the targeting relationships between lncRNA H19 and miRNA-29 c,miRNA-29 c and MAPK13 were detected by dual luciferase reporter assay.Meanwhile,RNA immunoprecipitation(RIP)experiment further determined the targeting relationship between lncRNA H19 and miRNA-29 c.Cardiomyocytes apoptosis was detected by caspase-3 activity kit and DNA fragment kit after transfection.Results In vivo: During the experiment,we found that the weight gain of the diabetic rats was much slower than that of the normal group,and the blood glucose was always at a high level.Insulin treatment could promote weight gain and blood glucose reduction in diabetic rats.However,the treatment melatonin group did not have these effects.The results of melatonin concentration showed that diabetes inhibited the expression of melatonin in rats,and the melatonin concentration in the melatonin treatment group was significantly higher than that in the diabetes group.Histological staining revealed that long-term diabetes significantly damaged myocardial fibers and exacerbated collagen accumulation.Treatment with melatonin alleviated this pathological damage caused by diabetes.Heart rate,LVDP,coronary flow,+dp/dt max,and-dp/dt max were significantly lower in diabetic rats compared with the normal group,indicating that chronic hyperglycemia lead to severe myocardial dysfunction,and melatonin treatment significantly improved cardiac function in diabetic rats.We found that the expression of melatonin receptors MT1 and MT2(Mel-1A/B-R)and NQO2 was significantly reduced in the myocardial tissue of diabetic rats,which was significantly improved after melatonin treated.Compared with normal rats,the expressions of Bax/Bcl-2,cleaved-caspase9 and cleaved-caspase3 proteins in myocardial tissue of diabetic rats were significantly upregulated.And they tended to normalize under melatonin treatment.Melatonin could also significantly reduce the increase of JNK/ERK/p38 phosphorylation in myocardial tissue of diabetic rats.At the same time,melatonin could significantly down-regulate the protein expressions of GRP-78,PERK,IRE1α,ATF-6α,ATF4 and CHOP in myocardial tissue of diabetic rats.In the pathological state of diabetes,the expression of lncRNA H19 was increased and the expression of miRNA-29 c was decreased,while melatonin significantly down-regulated lncRNA H19 and up-regulated the level of miR-29 c.In vitro: In cell experiments,melatonin treatment could significantly improve the decreased expression of MT1 and MT2(Mel-1A/B-R)caused by high concentration of glucose,but high glucose and melatonin treatments had no significant effect on NQO2.Compared with the normal group,high-concentration glucose treatment could significantly increase the expression of Bax/Bcl-2,cleaved-caspase9 and cleaved-caspase3 in cardiomyocytes,which were significantly improved after melatonin treated.Melatonin treatment could significantly attenuate high glucose-induced cardiomyocyte apoptosis.The phosphorylation level of JNK/ERK/p38 in cardiomyocytes was significantly increased with high glucose stimulated,and significantly decreased after melatonin treated.In addition,melatonin treatment also significantly ameliorated the elevated protein expressions of GRP-78,PERK,IRE1α,ATF-6α,ATF4 and CHOP caused by high concentrations of glucose.When lncRNA H19 shRNA was transfected,the expression of lncRNA H19 was significantly inhibited,and the addition of melatonin treatment further inhibited its expression.While the expression of miRNA-29 c was further up-regulated,and the expression of MAPK13 protein was further decreased.After treatment with miRNA-29 c inhibitor,the expression of miRNA-29 c was inhibited,and the expression rebounded after simultaneous treatment with melatonin.At the same time,melatonin treatment could also improve the increase of MAPK13 protein expression caused by miRNA-29 c inhibitor,but there was also non-significant influence at transcriptional level.The dual-luciferase report showed that miRNA-29 c mimic induced decrease in the luciferase activity of wild-type lncRNA H19.However,when a site in lncRNA H19 was mutated,this effect disappeared.And melatonin treatment obtained similar result.RIP experiments showed that the expressions of lncRNA H19 and miRNA-29 c were obviously aggregated in the immunoprecipitates,indicating that lncRNA H19 directly bound miRNA-29 c.In addition,miRNA-29 c mimic also induced decrease in the luciferase activity of wild-type MAPK13,which was abolished when a site in MAPK13 was mutated.And similar results were obtained with melatonin treatment.High glucose treatment resulted in marked increase in caspase-3 activity and DNA fragment in cardiomyocytes.When lncRNA H19 shRNA transfected,the activity of caspase-3 and DNA fragment in cardiomyocytes were significantly reduced in high glucose conditions.Treatment with melatonin and miRNA-29 c mimic also reduced the increase in caspase-3activity and DNA fragment in cardiomyocytes caused by high concentration glucose.Significant increase in caspase-3 activity and DNA fragment was detected in the high glucose group transfected with miRNA-29 c inhibitor alone,which was significantly inhibited when melatonin and miRNA-29 c inhibitor,lncRNA H19 shRNA and miRNA-29 c inhibitor were simultaneously treated in the high glucose group.Conclusion Melatonin can alleviate high glucose-induced cardiomyocyte apoptosis.And the mechanism is related to the effect of melatonin on the expression of lncRNA H19/miR-29 c,MAPK signaling pathway and endoplasmic reticulum stress. |
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