Effects Of Melatonin On Alleviating Vascular Endothelial Dysfunction In Type 1 Diabetic Rats

Objective To observe the effect of melatonin（MLT）on vascular endothelial dysfunction in type 1 diabetic rats,and to explore the feasible mechanism of melatonin in improving vascular endothelial dysfunction in diabetic rats.Methods（1）Establishment of animal model: 24 feeding and rearing clean male rats were randomly selected and randomly assigned to three experimental groups,with 8 rats in each group with similar growth status: control group,model group and melatonin treatment group.The model group and the treatment group were given streptozotocin55 mg /kg by intraperitoneal injection to establish T1DM（type 1 diabetes）model rats.After modeling,the treatment group was given melatonin 10mg/kg/d by intraperitoneal injection,and the remaining two groups were given equal volume of normal saline injection.After 16 weeks of continuous administration,the aortic arch tissue of rats was taken for the endothelial function test of the arterial ring.Observation of changes in aortic morphology by performing HE staining.Western blot was used to detect endoplasmic reticulum stress-related proteins（GRP78,CHOP）,and inflammasome related proteins（AIM2,IL-1β）in aortic tissues.（2）HUVEC were cultured and divided into three groups: control group,model group and treatment group.The control group（Ctrl group）was treated with DMEM medium at a normal low glucose level（5.5mmol/L glucose）,and the model group（HG group）was treated with DMEM medium at a high glucose level（30 mmol/L glucose）.Treatment group（HG+MLT group）was treated with DMEM medium at a high glucose level containing 100 μmol/L melatonin.The method of MTT was used to detect the effects of high glucose and melatonin on HUVEC proliferation.The production of reactive oxygen species（ROS）was detected by flow cytometry.The release of lactate dehydrogenase（LDH）in cell culture supernatant was determined.Autophagic flow was detected by mRFP-GFP-LC3 tandem fluorescent protein adenovirus transfection.Hoechst 33342/PI double-staining was used to detect cell apoptosis and necrosis.Western blot was used to detect the effects of high glucose and melatonin on the expression of endoplasmic reticulum stress-related proteins（GRP78,ATF4,CHOP,IRE1α,PERK,ATF6α）,autophagy related proteins（LC3B,P62,Beclin1）and inflammatory body related proteins（NLRP3、cleaved caspase-1、IL-1β、AIM2、ASC）in HUVEC.Result（1）Compared with the control group,the endothelial function of aorta in model group was impaired,HE staining showed that the endothelial cells in the aorta tissue were disordered and vacuolated,the protein expression levels of GRP78,CHOP,AIM2 and IL-1β increased.Compared with the model group,the endothelial function of aorta in treatment group was restored,HE staining showed that the endothelial cells in the aorta tissue were closely arranged,the protein expression of GRP78,CHOP,AIM2 and IL-1β decreased.（2）Compared with the control group,cell proliferation level of the model group decreased,ROS production increased,LDH releasing in cell culture supernatant increased,the green fluorescence increased compared with red fluorescence after transfection with autophagy double label adenovirus,the blue and red fluorescence increased in the double-staining,the expressions of GRP78,ATF4,CHOP,IRE1α,PERK,ATF6α,P62,NLRP3,cleaved caspase-1,IL-1β,AIM2,ASC increased,LC3 B,Beclin1 decreased.Compared with model group,cell proliferation level of the treatment group increased,ROS production decreased,LDH releasing in cell culture supernatant decreased,the green fluorescence decreased compared with red fluorescence after transfection with autophagy double label adenovirus,the blue and red fluorescence decreased in the double-staining,the expressions of GRP78,ATF4,CHOP,IRE1α,PERK,ATF6α,P62,NLRP3,cleaved caspase-1,IL-1β,AIM2,ASC decreased,LC3 B,Beclin1 increased.Conclusion Melatonin can ameliorate vascular endothelial dysfunction in type 1diabetic rats,and the mechanism may be related to endoplasmic reticulum stress,autophagy and inflammasome activation.