Protective Effect Of The Extraction From Anoectochilus Formosanus On High Glucose·induced In Cultured Human Umbilical Vein Endothelial Cell And The Mechanism

ObjectiveDiabetes diagnoses are increasing at an alarming rate worldwide. Vascular complications are currently the principal causes of disability and mortality in patients with diabetes. Oxidative stress has been postulated to be a major contributor to the pathogenesis of these events. Many biochemical pathways adversely affected by hyperglycemia that are found at elevated levels in diabetic patients are associated with the generation of reactive oxygen species, ultimately leading to increased oxidative stress in a variety of tissues. Besides damaging the functions of these molecules, the increased oxidative stress also triggers a series of cellular factors, activates stress-sensitive intracellular signaling pathways, and leads to the late complications of diabetes. More and more reserchers explore the effects of antioxidative agents on the process and progression of diabetes, especially traditional Chinese medicine.Anoectochilus roxburghii(Wall.)Lindl. an orchidaceous phanerophyte, is a perennial and precious herb. It is a popular folk medicine used in diabetes, tumor, hyperliposis, curing snake bite and so on. The present study the therapeutic effects of the aqueous extract (Anoectochilus formosams polysaccharide, AFP) and the Methanol extract (compound J-9, kinsenoside) from A. formosanus on diabetes mellitus and its mechanisms of actions in vitro experiments. MethodsThe antioxidant capacities of AFP were investigated in the scavenging effects of AFP on oxygen radicals and its inhibitory effect on lipid peroxidation by in vitro assay. In the primary culture medium of human umbilical vein endothelial cells (HUVEC), added AFP, J-9, kinsenoside respectively and incubate with high glucose(80mM) 72h, measured cellular CAT activity and T-AOC in the cell. MMP-2 and MMP-9 enzymatic activities were assayed by gelatin zymography. Applied Real time qRT-PCR to detect the mRNA expression of MMP-2, MMP-9, TIMP-1, TIMP-2, NF-κB.Results1 Study on Antioxidative Activity of AFP in vitroThe experimental results demonstrate that, AFP exhibited inhibitory effects on the hydroxyl free radical and superoxide anion free radical in a dose-dependent manner. It also showed substantial antioxidant activities in the mice liver tissue homogenate.2 The effect and mechanism of AFP on high glucose-induced HUVECsThe results indicate that, AFP has no toxicity or influence on HUVECs proliferation. Moreover, it has a notablely protective effect on HUVECs induced by high glucose. AFP also can enhance the CAT activity of the cells. Further more, AFP participate in the protection of endothelium function by inhibiting the activities of extracellular MMP-2/-9 in HUVECs induced by high glucose. Real time qRT-PCR revealed that high glucose-induced MMP-2/-9 mRNA expression levels were attenuated by pretreatment with AFP. In contrast, AFP increased TIMP-1/-2 expression significantly. AFP downregulated high glucose-induced NF-κB expression .3 The mechanism of compound J-9 and kinsenoside affect in HUVECs induced by High glucoseThe results show that, both compound J-9 and kinsenoside can also enhance the anti-oxidative ability of the cells by increasing CAT activity and T-AOC. Pretreatment with compound J-9 and kinsenoside respectively decreased high glucose-induced increase of MMP-2/-9 activities. Real time qRT-PCR revealed that both compound J-9 and kinsenoside inhibited high glucose-induced MMP-2/-9 expression and increased TIMP-1/-2 expression significantly, thus completely reversing the increases MMP-2/TIMP-2 mRNA expression associated with high glucose. Also, both compound J-9 and kinsenoside downregulated high glucose-induced NF-κB expression.ConclusionIn conclusion, we found that AFP shows favorable antioxidant activities. From previous reserch, we kown both compound J-9 and kinsenoside have effective antioxidation in the cell experiments. AFP, J-9, kinsenoside all blunted oxidant stress with diabetic vascular complications, prevented excessive degradation of ECM, and blocked the intracellular signaling pathways and reversed the endothelial dysfunction associated with diabetes vascular complications.