Research On The Mechanism Of Corydalis Extract In Lowering Blood Sugar Based On Insulin Secretion Effect

Diabetes mellitus is a kind of chronic metabolic disease resulting from insulin secretion deficiency and/or biological function disorders of islet beta cell characteristic of high blood glucose.People suffered type 2 diabetes mellitus(T2DM)account for 90%of all diabetic patients approximately.The complex pathogenesis of diabetes has not been fully understood as yet.It has been acknowledged that beta cell secretory dysfunction and insulin resistance are two main factors during the course.Thus,searching for new regulatory targets of pancreatic βcell to improve its secretion function and elucidating its related molecular mechanisms is one of the hotspot problems of the prevention and treatment of T2DM to be solved.Corydalis edulis Maxim,is a plant of Papaveraceae Corydalis of which the whole plant is used as a Chinese herb.It is a grey-green,annual plant wildly grown in many provinces of china,usually at an altitude of 400 to 1200 meters.Corydalis edulis Maxim.is traditionally used to clear heat and remove toxicity,moisten lung for relieving cough,stop itching and induce astringency.However,there was no modern research about the plant.Recently this natural Chinese herb was found to be prepared as beverages for improving T2DM in the folk.Thus,experiments were carried out to identifiy the possible effect and explore the molecular targets of Corydalis edulis Maxim.extract(CE)on improving diabetes,aiming at providing new ideas for its possible role to be a new complementary drug for treating diabetes.This experiment can be divided into the following two parts:(1)To evaluate the hypoglycemic effect of CE on high-fat and high-glucose diet induced T2DM diabetic ApoE-/-mice.All mice were randomly divided into six groups(n= 8-9 for each group):the control group(CG),the model group(HG),the low,middle,high doses of CE groups,and gliclazide group(G),and were administered orally with 100,200,400 mg/kg CE,and 100 mg/kg gliclazide once a day,respectively.After 8 weeks,in accordance with gliclazide,CE-treated groups decreased the glucose levels dose dependently(P<0.05).Serum TC and TG levels in CE treated mice were also significantly lower than those of the HG treated mice(P<0.05).Intraperitoneal glucose tolerance test(IPGTT)indicated that the CE treated mice showed ameliorated glucose clearance.Likewise,insulin tolerance test(ITT)showed that the CE-treated mice had increased insulin sensitivity.As for blood insulin levels,ELISA assays showed that CE treatment groups were significantly elevated compared to vehicle group(P<0.05).Similarly,immunohistochemical staining of pancreas sections of CEM-treated mice showed an increasing expression of insulin.Moreover,middle and high doses of CE groups showed significant body weight increment during the 4th,6th and 8th week under the condition of no influence on food intake.Collectively,it was considered that CE could decrease the blood glucose and improve their metabolic disorders of high-fat and high-glucose diet induced T2DM diabetic ApoE-/-mice.(2)Further investigations were conducted to explore the mechanism of CE’s hypoglycemic effect in vitro.Firstly an a-glucosidase inhibitor screening model was used,however,CE failed to inhibit the activity of a-glucosidase.Later effect of CE was studied on an insulin secretagogue screening model using HIT-T15 cells.Results of HTRF assays showed a remarkable enhancement in insulin secretion by CE treatment in a dose-dependent manner and a glucose-independent manner,i.e.,CE could promote insulin secretion in glucose-free,low-glucose and high glucose conditions.Then further study to explore the underlying mechanism of CE’s insulinotropic effect.Under physiological conditions,glucose stimulates insulin secretion by generating triggering and amplifying signals in β cells.Multiple kinases are involved in insulin secretion amplifying pathways including protein kinase A and protein kinase C.PKA related signaling pathway and PKC related signaling pathway play important roles in the secretory process.Experiments about the former were done to determine the intracellular cAMP content using an EIA assay after CE treatment.While no significant difference was seen among the groups.Subsequently,experiments related the latter were performed.PKC agonists and/or inhibitors were chosen to function alone or in combination with CE.Results showed that the insulinotropic action of CE on HIT-T 15 cells was suppressed to different extent.Then to study the performances of different iso forms of PKC influenced by CE,western blotting assays were carried out to test the expression of them.It was demonstrated that the translocations of PKC 8,ζ and μ were promoted while the one of PKC ε was on the reverse.Besides,CE didn’t affect the expressions of nPKCs(PKC α,βⅡ).Later experiments of immunofluorescence staining of PKC α,δ,μ and siRNA assays of PKC δ,μ,ζ were well consistent with the western blotting results.Therefore,in vitro investigations indicated that CE promoted insulin secretion through activating nPKCs and aPKCs in HIT-T15 cells.In conclusion,CE decreased the blood glucose levels and alleviated dietary-induced diabetes in ApoE-/-mice by increasing insulin secretion.Further results revealed that the insulinotropic effect of CE was mediated by the activation of nPKCs and aPKCs.Proofs provided in the thesis indicated that CE may serve as a new antidiabetic agent with a potential insulinotropic effect.