Protective Effect And Mechanism Of Coreopsis Tinctoria Nutt.on Diabetic Nephropathy Rat

Objective: Diabetic nephropathy(DN)is one of the common microvascular complications of diabetes mellitus.It is a kind of secondary kidney disease and the leading cause of end stage renal disease which the main characteristic is glomerulosclerosis.The mechanism of DN is complex and not clarified.Research found that oxidative stress and inflammation which actived by metabolism disorder might be the main cause and renal fibrosis and the pathologic change of DN.Glomeruli mesaginal cell is one of the functional cells in renal.GMC release amount of oxidative stress,inflammation and fibrosis cytokines during hyperglycemia which directly and indirectly lead mesangial expansion,ECM accumulation and ultimately the renal fibrosis.Coreopsis tinctoria Nutt.is a kind of annual herb which belongs to the composite family.Its capit?lum is used as medicine or health care tea.Coreopsis tinctoria Nutt.has the effects of anti-hypolipidemic,anti-hypertension,hyperglycemic,anti-oxidative and so on.Flavonoids are its main and effective constituent.Whether Coreopsis tinctoria Nutt.has the preventive effect on DN is still unknown.For the purpose of development of Coreopsis tinctoria Nutt,our research will confirm the anti-inflammation and anti-fibrotic effect of Coreopsis tinctoria Nutt.and the possible mechanism by vitro and vivo DN model.Method: 1)HPLC/MS analysis is to confirm the main constitutes of ethyl acetate of Coreopsis tinctoria Nutt.2)High-fat-diet feeding 8 weeks and low-dose of STZ intraperitoneal injection SD rats are made for 72 h to establish diabetic rats model.Renal index and pathological character will be analyzed and evaluated after 4 weeks of high-fat-diet feeding.3)SD rats are made into DN model based on the modeling method of DN and preventively gavaged administration of ethyl acetate of Coreopsis tinctoria Nutt for 4 weeks.Rats were sacrificed to test biochemical,renal indexes and pathological character in order to confirm the renal protective effect of Coreopsis tinctoria Nutt on DN rats.4)Inflammatory cytokines of MCP-1 and ICAM-1 are detected both on vitro and vivo DN models by Immunohistochemistry,Real-time PCR and western blot in order to confirm the anti-inflammation effects of ethyl acetate of Coreopsis tinctoria Nutt and marein.HBZY-1 cell is pretreated with high glucose of ethyl acetate of Coreopsis tinctoria Nutt,marein and PDTC to find the anti-inflammatory mechanism of Coreopsis tinctoria Nutt and marein by NF-?B signaling pathway.5)MTT and CCK8 kits are used to test the cell multiplication effect of Coreopsis tinctoria Nutt and marein;Fibrotic constitutes of TGF-?1,Collagen?and FN and related NF-?B,AMPK,TGF-?1/Smads signaling proteins were detected by Real-time PCR and western blot.HBZY-1 cell is pretreated with high glucose,AICAR,Dorsomorphin and PDTC to find the anti-fibrotic mechanism of Coreopsis tinctoria Nutt and marein by NF-?B,AMPK,TGF-?1/Smads signaling pathway.Results: HPLC/MS analysis confirm that there are four main constitutes of ethyl acetate extract of Coreopsis tinctoria Nutt which are marein,Chlorogenic acid,Flavanomarein,4,5-dicaffeoyl-quinic acid.Marein is the highest constitutes in ethyl acetate of Coreopsis tinctoria Nutt.Compared with normal rats,DN rats show the significant elevation of albuminuria,?2-MG and ?1-MG in urine after high-fat-diet feeding 8 weeks and low-dose of STZ intraperitoneal injection SD rats for 4 weeks.Renal pathological characters show basement membrane thickening and mesangial expansion,deposition of glycogen and fibrinogen in both glomerulus and tubule.This modeling method could mock the early physiological and pathological characteristics of DN.Pretreatment of ethyl acetate extract of Coreopsis tinctoria Nutt on DN rats for 4 weeks later,blood glucose,lipid disorder as well as renal injury and disfunction are significantly improved.In vivo experiment,ethyl acetate extract of Coreopsis tinctoria Nutt suppresses renal inflammatory cytokines of MCP-1,ICAM-1 mRNA and protein expression in DN rats.In vitro experiment,ethyl acetate extract of Coreopsis tinctoria Nutt and marein significantly decrease high glucose induced mRNA and protein expression of MCP-1,ICAM-1 through NF-?B signaling pathway in HBZY-1cell.The anti-inflammation activitiy of marein was better than Ethyl acetate extract of Coreopsis tinctoria Nutt.Fibrotic constitutes of TGF-?1,Collagen?and FN expression are also remarkably ameliorated by both ethyl acetate of Coreopsis tinctoria Nutt in DN rats.In high glucose induced of HBZY-1 cell,Ethyl acetate extrat of Coreopsis tinctoria Nutt and marein significantly decreases fibrotic constitutes of TGF-?1,Collagen?and FN mRNA and protein expression through NF-?B?AMPK/Smad?TGF-?1/Smads signaling pathway.The anti-fibrotic activity of ethyl acetate extract of Coreopsis tinctoria Nutt.is better than marein.Conclusion: Ethyl acetate extract of Coreopsis tinctoria Nutt has the preventive effect of early DN renal injury of SD rats.The possible anti-inflammation mechanism is by NF-?B signaling pathway and marein is the main active constitute;The possible anti-fibrotic mechanisms are by NF-?B?AMPK/Smad?TGF-?1/Smads signaling pathway and marein is one of the active constitutes...