| Background: Diabetes is due to insulin secretion or insulin resistance,leading to high blood sugar,easily caused by a series of complications,such as diabetic nephropathy,retinopathy,cardiovascular disease.Especially cardiovascular disease which caused more than half of the deaths of diabetes and the consumption of a large number of social medical resources,especially in China after entering the aging society,medical expenses rose sharply.Although there are many clinical treatment of diabetes and its complications of drugs and means,but the drug treatment is facing many problems,such as lack of drug source,too much cost,treatment is not ideal,easy to cause various side effects,so clinically looking for diabetes And its complications of drug treatment,is extremely urgent,has become one of the current research focus.Melatonin is an endogenous molecule,and the molecular weight is very small 232.28,mainly from the pineal gland.The secretion of melatonin is controlled by the suprachiasmatic nucleus,reduced secretion during the day and increased secretion at night.Strong secretion of light under the dark secretion increased.The current study found that melatonin not only regulate circadian rhythm,improve sleep,but also has a strong antioxidant stress,can remove free radicals,eliminate inflammation and so on.In the cardiovascular protection,obesity,metabolic syndrome and diabetes and its complications play a useful role.Notch signaling pathway as a highly conserved classic pathway,through cell-cell activation,involved in cell growth,survival,apoptosis,differentiation and other processes.This group has confirmed that melatonin can activate Notch1/Hes1 pathway in diabetic myocardial ischemia / reperfusion injury,play a protective effect,and the study of diabetic macrovascular injury has not been reported.JAKs signaling pathway is also involved in many cell responses,including cell survival,differentiation,apoptosis,etc.The study group has studied that melatonin can activate JAK2/STAT3 signaling pathway against myocardial ischemia/reperfusion injury.Research on the damage of diabetic macrovascular disease has been unreported.Objective: In order to clarify the protective mechanism of melatonin in diabetic macrovascular injury,this study was explored by animal experiments and cell experiments.1)To explore the melatonin in diabetic vascular smooth muscle injury can play a protective effect.2)To explore whether melatonin can activate Notch1/Hes1 and JAK2/STAT3 signaling pathway antidiabetic vascular injury.3)To explore the relationship between Notch1/Hes1 and JAK2/STAT3 signaling pathway in melatonin antidiabetic vascular injury.Methods: 1)STZ induced the establishment of diabetic animal model.2)High fat and high glucose treatment of smooth muscle cells to establish an isolated diabetes model.3)Regular blood glucose meter to detect blood glucose changes in rats.4)EIISA method to detect melatonin,glycosylated hemoglobin,blood lipid changes.5)Arterial tissue superoxide production,malondialdehyde(MDA)and superoxide dismutase(SOD)determination.6)The expression of caspase3 was detected by immunohistochemistry.7)Western blot was used to detect the expression of apoptotic marker protein and accessory protein.8)Detection of cell viability(MTT method).9)TUNEL method to detect apoptosis.10)Statistics using Graphpad Prism 5 software for statistics.Results 1)In diabetic animal experiments,melatonin significantly improved blood glucose in diabetic rats,control weight,reduce glycosylated hemoglobin,total cholesterol,triglycerides,low density lipoprotein,thereby reducing the risk of diabetes.2)In diabetic rats,the content of superoxide,malondialdehyde(MDA),gp91 phox protein and superoxide dismutase(SOD)in thoracic aorta were measured.The levels of superoxide dismutase(SOD)DM+Mel group was significantly improved compared with diabetes mellitus(DM),confirming that melatonin played a significant role in anti-oxidative stress in diabetic macrovascular injury.3)In diabetic mellitus,the expression of Notch1,Hes1,JAK2 and Stat3 protein in DM + Mel group was significantly higher than that in diabetic group(DM).In addition,compared with the(DM+Mel)melatonin treatment group,the expression of caspase3 and Bcl2 in the diabetic group was too much and the expression of anti-apoptotic protein Bax was decreased.The immunohistochemical results of thoracic aorta were positive for caspase3 protein in diabetic group(DM).Therefore,we suspect that melatonin can activate Notch1/Hes1 and JAK2/STAT3 signaling pathway,play anti-apoptotic effect.4)In order to further confirm the hypothesis in animal experiments,we used tissue adherence method to culture primary smooth muscle cells.Vascular tissue block adherent culture flask 7 days the tissue around the radial crawling smooth muscle cells.30 days after a large number of smooth muscle cells covered with culture bottles,overlapping arrangement or radiation arrangement,was "honeycomb" shape.Individual cells were long spindle-shaped,both ends slender.5)In the high fat and high glucose environment to 50mmol/L,100mmol/L and 200mmol/L melatonin treatment,cell viability showed a certain gradient.And with the increase of melatonin concentration,the apoptosis rate was significantly reduced.In addition,the expression of gp91 phox protein in melatonin group(HG/HF + Mel)was significantly lower than that in HG/HF group(P <0.01).In addition,the results of superoxide dismutase are the same.The above results show that melatonin protects smooth muscle from oxidative stress injury during high fat and high glucose injury.6)To detect the effects of melatonin on Notch1/Hes1 and JAK2/STAT3 signaling in the high level and high glucose environment,the expression of Notch1,Hes1,JAK2 and STAT3 were detected.Compared with HG/HF group,the expression of Notch1,Hes1,JAK2 and STAT3 protein in melatonin group(HG/HF + Mel)were significantly increased(P <0.01).To investigate the effect of melatonin on smooth muscle cell apoptosis under high fat and high glucose,we found that the HG/HF group was significantly overexpressed in the melatonin-treated group,and the expression of proapoptotic protein totalcaspase3 and Bax was low Anti-apoptotic protein Bcl2(P <0.01).We found that the effect of melatonin on Notch1 / Hes1 and JAK2/STAT3 signaling pathways and apoptotic pathway was consistent with that of animal experiments at the cellular level,high fat and high glucose environment.7)Melatonin protects against high fat and high glucose by activating Notch1/Hes1 and JAK2/STAT3 signaling.There was no significant difference in DAPT or AG490 group compared with HG/HF(P> 0.05).However,compared with melatonin treatment group(HG/HF + Mel),the apoptosis was significantly increased and reduced the superoxide,gp91 phox,Notch1/Hes1 and JAK2/STAT3 signaling pathway protein expression.We found that the melatonin treatment group(HG/HF + Mel)was significantly lower than that of HG/HF group,HG/HF + Mel + DAPT group and HG/HF + Mel + AG490 group.The expression of Notch1,Hes1,JAK2 and STAT3 protein was significantly decreased in DAPT group compared with melatonin group(P <0.01),but the expression of Notch1 and Hes1 protein was significantly decreased(P <0.01)compared with AG490 group,but JAK2 and STAT3 protein expression(P> 0.05).Compared with HG/HF + Mel + DAPT group and HG/HF + Mel + AG490 group,the expression of pro-apoptotic protein total caspase3 and Bax decreased,and the anti-apoptotic protein Bcl2 was up-regulated in melatonin group and HG/HF group.These results suggest that melatonin exerts anti-oxidative stress and anti-apoptotic effects through Notch1/Hes1 and JAK2/Stat3 signaling pathways in high-fat and high-glucose environment,and Notch1/Hes1 and JAK2/STAT3 signaling path for upstream and downstream relations.Conclusions 1)As the first time in this study,it has been shown that melatonin has a protective effect on diabetic macrovascular injury.2)We have reported for the first time that melatonin can reduce oxidative stress injury by resisting Notch1/Hes1 and JAK2/STAT3 signaling pathways,resistance to diabetic macrovascular injury.3)Our study found a new mechanism of melatonin in the cardiovascular protection of diabetes,providing a new strategy for cardiovascular protection in diabetes. |
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