| ObjectTo study the regulatory effect and mechanism of a new type of melatonin receptor agonist Neu-p11on glucose metabolic through the impacting the mitochondria balance in different tissues in type 2 diabetic rats,to seek new ideas idea for the treatment of T2DM and opne up new wayofthe treatment for T2DM.Method1.Molding and grouping:After 10 days'adjusting in the experimental environment,10 of 70 female Wistar rats were randomly selected as normal control group and the remaining rats were used as the model group.The rats of normal control group was given regular feed,and the other rats were fed with high fat feed and free drinking water during the period.Two months later,small dose of intraperitoneal injection of STZ?30 mg·kg 1?were administered to other rats to induce the diabetic model,and the rats with blood glucose value over 13.0 mmol/L were used in the later expeiments.According to the blood glucose and body weight,the rats were divided into diabetic model group,low,medium and high doses of melatonin receptor agonist Neu-p11 groups,with has nine rats of each group.2.Drug administration:The experimental drugs were given at 8:30 am every day.The normal control group and the model group were given the same amount of water,and the positive drug group was given20mg·kg-1 melatonin,and the low-,medium-and high-dose groups were given 5mg·kg-1,10mg·kg-1,and20mg·kg-1 of agonist Neu-p11,respectively,.The volume of gavage was 1 ml·200g-1,and the drug was continuously administered to 4w.3.The determination of indicators:The rats'weight,feeding and drinking water were recorded daily,and the fasting blood glucose?FBG?level was measured every week.The fasting blood glucose?FBG?,insulin?INS?content,the amount of glucose tolerance?IPGTT?were determined at the 21th day of the experiment;At the 28th day one rat in each group were used as the executed ratto obtainthe brain,liver and pancreas and soaked in 4%paraformaldehyde for pathological observation;The rest of the rats were killed by amputation,blood serumwas separated,and the liver,muscles and hippocampuswere extracted and reloaded.The anthrone method was used to determine the content of glycogen by taking appropriate amount of liver and muscle tissue.The ATP content and ATPase activity of different tissues in rats were determined.4.Gene and protein determination:RT-PCR method was used for the determinations of mitochondria fusion protein?Mfn2?,glycogen metabolism enzyme glucose kinase?GK?,enol phosphate typepyruvate carboxylase?PEPCK?,glucose-6-sugar phosphatase?G-6-P?,glycogen synthesis kinase-3?GSK-3?mRNA expression in the livers,the hippocampus,muscle of rats.The mRNA expression levels of the activated protein kinase?AMPK?,silent regulating protein 1?Sirt1?,nuclear respiration factor?Nrf-1/2?and estrogen receptor alpha?ERR alpha?were measured in the tissues of rats.Western Blot method was used to determine the relative expression of cytokines?pgc-1?,mitochondrial fusion protein?Mfn2?and mitochondrial split protein?drp-1?.5.Pathological observation:HE staining was used to observe the morphological changes of different tissues in different groups.Results1.Effects of Neu-p11 on glucose metabolism in T2DM rats Compared with normal group,fasting blood glucose?FBG?,index of insulin resistance?IR?and glucose tolerance weresignificantly increased in T2DM rats;ATP content and ATP enzyme activity were reduced in different tissues,especiallyin model group rats;Compared with the model group,the hyperglycemia in drug administration group was reduced to some degrees,and Also,IR and glucose tolerance wereimproved,and ATP content and ATPase activity were increasedin T2DM rats.especially in low dose group of Neu-p11,the FBG and INSwere significantly decreased,;in melatonin group and low-dose group,IPGTT and IR were?P<0.05?improved?P<0.05?,and ATP content and enzyme activity were increased in T2DM rats?P<0.05?.2.Effects of Neu-p11 on tissue gene and protein in T2DM rats:Compared with normal group,glycogen metabolism enzyme gene expression were abnormal in liver tissue of T2DM rats PEPCK,G-6-P mRNA expression was significantly increased?P<0.01?;GK,GSK-3?mRNA expression were significantly decreased?P<0.01?;liver,Mfn2,AMPK,Sirt1,GCN5 and Nrf1 mRNA expression were significantly reduced in hippocampus and muscle tissue?P<0.01?,while ERR?mRNA expression was increased?P<0.01?,espeially the muscle tissue Nrf2 mRNA expression was significantly increased?P<0.01?and Nrf2mRNA were decreased in liver and hippocampal tissues?P<0.01?,ATPase mRNA expression was significantly reduced?P<0.05?.Mfn2 and PGC-1?protein expression were decreased,Drp-1 protein expression in liver was increased,Drp-1 protein expression in hippocampus and muscle;Compared with model group,tissue factor and abnormal protein expression of gene were improved in Neu-p11 groups,melatonin and Mfn2 and GK mRNA expression were increased in low-dose group of Neu-p11?P<0.05?,PEPCK and G-6-P mRNA expression were decreased?P<0.05?;Increase AMPK,Sirt1,GCN5,Nrf1 and Nrf2 mRNA expression,abate ERR?in tissues and Nrf2mRNA express in muscle have a trend of increasing?P<0.05?,Mfn2 and PGC-1?protein expression were upregulated in low-dose Neu-p11group,Drp-1protein expression was down-regulated–and the mRNA expression of ATP enzyme was increased in different tissue of T2DM rats in?P<0.05?.3.Effects of Neu-p11 on physiological morphology of different tissues in T2DM rats:Compared with normal group,the liver,brain and pancreas tissue were in damaged state in T2DM rats;while most of them were improved in the tissues of Neu-p11 treated groups,especially in low dose group of Neu-p11.ConclusionThe hypoglucemic effect of Neu-p11 may be mediatedby improving IR,impaired glucose metabolism and abnormal mRNA expression of hepatic glucose metabolism enzyme in T2DM rats..Melatonin receptor agonist,neu-p11,showed the regulatory effects on the mitochondrial balance-related factors in various tissues of T2DM ratsto maintain normal mitochondrial function and improve insulin resistance. |
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