Effect Of Melatonin And Tongluo Recipe On Glucose And Fat Metabolism And The Activity Of Hypothalamo-pituitary-adrenal Axis In Experimental Diabetes Rats

Objective:Four metabolic pathways have been thought participate in genesis and development of diabetes mellitus chronical complications,including polyol pathway flux,advanced glycosylation end product formation,protein kinase C pathway and increased hexosamine pathway flux.Now oxidative stress is thought its colletive mechanism.Hyperglycemia induced increase of reactive oxygen species(ROS),oxidative stress and activation of the four pathways which caused increase of ROS ulteriorly,formed infemal circle and injured target organ.Simultaneously,diabetes mellitus as stressor,if hyperglycemia can not be controlled for long time,hypothalamo-pituitary-adrenal axis is in strate of excess activation,homeostasis is destroyed,which further aggravate hyperglycemia damage. Melatonin,as one of neuroendocrine hormones secreted by pineal gland,is one of strong antioxidants,could decrease production of free oxygen radicals and protect antioxidant enzyme.Recent research find that melatonin also can regulate the activity of hypothalamo-pituitary-adrenal axis.Taking its complex regulation on nerve,endocrine and immune network into consideration,antioxidation may not be unique mechanism of melatonin protect diabetes mellitus metabolic disorder.Whether melatonin stabilize internal environment and ameliorate metabolic disorder through regulating hypothalamo-pituitary-adrenal axis in diabetes mellitus has no final conclusion.TongLuo could promote blood flow and stabilize endothelial function,Ginseng saponin has antioxidative function.Our research study the effect of melatonin and TLR on glucose and fat metabolism and the activity of hypothallamo-pituitary-adrenal axis in experimental diabetes rats,approach possible mechanism of melatonin and TLR protecting diabetes mellitus.Methods:Diabetic rats model were induced by injection of streptozotocin(STZ) (65mg/kg,intraperitoneal).Rats were randomly divided into 5 groups,each 6-7:large dose of melatonin(MEL 1):10mg/kg/d;small dose of melatonin(MEL2):0.2mg/kg/d; Tongluo recipe treatment group(TLR):Tongluo recipe 1.0g/kg/d(all groups received gavage once daily);and diabetic control group(DM) and the normal control group(NC). The experiment lasted for 12 weeks.1.To measure the rats' weight,double adrenal weight,serum total cholesterol and triglycerides,serum malondialdehyde and blood glucose.2.To observe the ultrastructure of rat pituitary adrenotropic hormone cell and adrenal cortex fascicular zone cell with transmission electron microscope.3.To detect antepituitary adrenocorticotropic hormone with immunohistochemistry methods.4.To measure blood corticotrophin releasing hormone,adrenocorticotropic hormone and corticosterone with radioimmunoassay.Result:1.Blood glucose,MDA,TC,TG and AW/BW in DM group were significantly higher and body weight were significantly lower than those in NC group(P<0.01);compared with DM group,blood glucose,MDA,TC,TG and AW/BW were decreased markedly(P<0.01 or P<0.05) and body weight were increased markedly(P<0.01 or P<0.05) in DM+MEL1 group and DM+MEL2 group,MDA,TC,TG in TLR group were decreased markedly(P<0.05),blood glucose,AW/BW and body weight had no significant changes.2.TEM observation on pituitary adrenotropic hormone cell:Compared with NC group, DM group rat adrenotropic hormone cell mark significant ultrastructural changes:chondrosome quantity increase,part of cristae amotio,rough endoplasmic reticulum dilate obviously,ambinucelus diastem appear,secretory granule with different particle size and electron density distribute along cytomembrane,its quantity increase obviously.Compared with DM group,in melatonin group,chondrosome quantity decrease,cristae approximate normal,rough endoplasmic reticulum has no viorus dilation,secretory granule decrease,MEL1 group approach NC group.TLR group has no significant changes.3.TEM observation on adrenal cortex fascicular zone cell:Compared with NC group, DM group rat adrenal cortex fascicular zone cell mark significant ultrastructural changes:powerful tracheid-cristae chondrosome,with larger volume and increased quantity,partial liposome enclosed with intensive disposed chondrosome,smooth endoplasmic reticulum proliferate and dilate.Compared with DM group,melatonin group has manifest improvement,MEL1 group approach NC group.TLR group is similar to DM group.4.Immunohistochemistry for antepituitary adrenocorticotropic hormone express: Compared with NC group,pituitary corticotrophin gray scale ratio was significantly higher(P<0.01) in DM group.Compared with DM group,pituitary corticotrophin gray scale ratio was significantly lower(P<0.01) in DM+MEL1 group,pituitary corticotrophin gray scale ratio was lower in DM+MEL2 group and TLR group,but its difference has no statistical significance.5.Compared with NC group,blood CRH,ACTH,CORT in DM group was significantly higher(P<0.01);Compared with DM group,blood CRH,ACTH,CORT in DM+MEL1 group was significantly lower(P<0.01),blood CORT in DM+MEL2 group was significantly lower(P<0.05),blood CRH and ACTH in DM+MEL2 group has no significant changes,blood CRH,ACTH,CORT in TLR group has no significant changes.Conclusion:1.After intraperitoneal injection of STZ-induced diabetic rats with 12-weeks' average forage feeding,blood MDA,TC,TG and blood glucose increased virously,treating with melatonin and TLR,these biochemistry index decreased,suggest that oxidative stress is their colletive mechanism,antioxidants can obviously improve glucose and fat metabolism disorder in DM,TLR may act as antioxidant through its composition ginseng saponin,specific mechanism need further study.2.AW/BW and blood CRH,ACTH,CORT increased markedly in DM group,suggest that hypothalamo-pituitary-adrenal axis is in the excessively activated state,further aggravate internal enviorment disorder.Melatonin inhibit blood CRH,ACTH and CORT,large dose is better than small dose,protect acute metabolic disorder,specific mechanism need further study.3.TEM observation displayed that pituitary adrenotropic hormone cell in DM group has more secretory granule,pituitary immunohistochemistry also suggest that the ACTH express of pituitary cell enhanced,which was coincidence with the result of blood ACTH measured by RIA.Adrenal cortex fascicular zone cell ultrastructure has manifest changes,imply cortex hyperfunction.Melatoin can inhibit these changes,but TLR has no significant inhibition.