Protection Effect And Molecular Mechanism Of Xinjiang Coreopsis Tinctoria Nutt.on Diabetic Retinopathy

Objective: Diabetes mellitus(DM)is a kind of metabolic disease characterized by hyperglycemia,which has become the third global public health problem after cardiovascular disease and malignant tumor due to its complicated pathogeny.Diabetic retinopathy(DR)constitutes one of major diabetic microvasscular complications with complicated pathogenesis;however,currently there is still no therapy with remarkable efficacy.Therefore,it is imperative to seek effective drugs.Xinjiang Coreopsis Tinctoria Nutt.is a rare alpine wild plant with highly medicinal value and preliminary studies have demonstrated its functions such as blood glucose reducing,lipid-lowering,antioxidation and etc.The study is intended to explore the function of the extract from Coreopsis tinctoria Nutt.on Diabetic Rat and its influence on diabetic retinopathy,to explore the protection effect and molecular mechanism of the extract from Coreopsis Tinctoria Nutt.on endothelial cell damage,providing theoretical basis for tap into the medicinal effect of Coreopsis Tinctoria Nutt.and DR development of new drug.Method: 1)diabetic rat models were established by raised by high sugar and high fat feed and induced by small-dose STZ which were divided into normal group,model group,high&medium&low-dose groups of ethyl acetate extract from Coreopsis tinctoria Nutt.and melbine positive drug invention group,such indexes as weight,blood glucose,serum insulin,glycosylated hemoglobin,blood lipid 4 items,glutamic-pyruvic transaminase,glutamic oxalacetic transaminase,?-glutamyl transpeptidase,total serum protein,albumin,serum creatinine,blood uric acid and urea nitrogen of rats in all groups were tested after 4-week intervention,and changes of VEGF and ICAM1 in retinal tissue were observed using immunohistochemical method;2)db/db type 2 diabetic mice model were adopted,effective parts of Coreopsis Tinctoria Nutt.were obtained through alcohol extraction of Coreopsis Tinctoria Nutt plus macroporous resin enrichment amplification process,which were divided into normal group,model group,high & low-dose groups of Coreopsis Tinctoria Nutt,intervention group of melbine(DMBG),positive drug,and combination intervention group of melbine(DMBG)and Coreopsis Tinctoria Nutt.After 10-week intervention on db/db type-2 diabetic mice model,indexes including weight,blood glucose,glycosylated hemoglobin,MDA and SOD,serum of all groups were tested;pathology changes in retina of mice were observed using HE staining method;expression changes in factors such as VEGF,ICAM1,PEDF,PDGF,Bcl-2,IGF-1 of mice retina by immunohistochemical method;3)HUVEC damage models induced by high glucose were established which were intervened by high&medium&low doses from effective parts of Coreopsis Tinctoria Nutt.Changes in contents of MDA,SOD,CAT and GXH-PX in cell supernatant were tested;Changes of VEGF,ICAM1,LC3,p62,Bcl-2 proteins in cells of all groups were tested by Western-Blot method;cells were intervened by cell signal pathway inhibitors and protein expressions of VEGF and ICAM1 in cells before and after intervention were tested.Results: 1)the main chemical components in ethyl acetate extract from Coreopsis Tinctoria Nutt consist of marein,flavanomarein,chlorogenic acid,dicaffeoylquinic acid,wherein,marein content is the highest,181.61mg/g;2)Diabetic rat models were established by raised by high sugar and high fat feed and induced by small-dose STZ.Rats in these model groups appeared abnormal glucose,liver and kidney function indexes,showing that diabetes models were successfully established;3)ethyl acetate extract from Coreopsis Tinctoria Nutt can remarkably reduce glucose level of diabetic rats,improve relative indexes such as liver and kidney functions of diabetic rats,and down-regulate VEGF and ICAM1 expressions in retinopathy;4)The effective parts of Coreopsis Tinctoria Nutt obtained from alcohol extraction plus macroporous resin enrichment amplification process are mainly comprised by chlorogenic acid,flavonoid marein,marein,dicaffeoylquinic acid,lavanokanin with the total content of 532.82mg/g including total flavonoid content(by naringin,mg/g)of 330mg/g;5)The effective parts of Coreopsis Tinctoria Nutt significantly reduce glucose level of rats with type-2 db/db diabetics,by hemoglobin glycosylation and improvement of oxidative stress level;6)The effective parts of Coreopsis Tinctoria Nutt can decrease the expressions of VEGF?ICAM1?PDGF?IGF-1 in retinopathy,and increase the expressions of Bcl-2 and PEDF,and the combination invention group of Coreopsis Tinctoria Nutt and melbine achieves most remarkable effect;7)MDA content in HUVEC damage model induced by high glucose is increased while SOD,CAT,GXH-PX levels are reduced,establishing high-glucose damage model;8)The effective parts of Coreopsis Tinctoria Nutt can significantly enhance the ability of high-glucose damaged HUVEC cells,reduce MDA content,and raise the levels of SOD,CAT and GXH-PX,which are related to drug intervention concentration;9)The effective parts of Coreopsis Tinctoria Nutt can obviously down-regulate the expressions of VEGF,ICAM1;after antagonism of PI3 K pathway,the effective parts of Coreopsis Tinctoria Nutt has more remarkably down-regulating effect on VEGF;after antagonism of NF-?B pathway,the effective parts of Coreopsis Tinctoria Nutt has more remarkably down-regulating effect on ICAM;10)The effective parts of Coreopsis Tinctoria Nutt can obviously down-regulate the expressions of p62 and up-regulate the expressions of LC3 and Bcl-2 in HUVEC damaged by high glucose;Conclusion: 1)Coreopsis Tinctoria Nutt has certain therapeutic action on diabetics by significantly improve the blood glucose level and lipid disorder of diabetic rats;2)Coreopsis Tinctoria Nutt exerts preventive and therapeutic effect on retinopathy by affecting the expressions of factors related to retinopathy;3)Coreopsis Tinctoria Nutt exerts the protection effect on HUVEC damage induced by high-glucose by improving oxidative stress level by improving oxidative stress level,down-regulating the expressions of VEGF and ICAM1,and enhancing autophagy.