The Experimental Study On The Inhibitive Effect Of Mimeng Flower Decoction On The Hypoxia Human Umbilical Vascular Endothelial Cells By TGF-β Signal Transdution

ObjectiveDiabetic retinopathy (DR) is one of the diabetic serious complications, and damages visual function seriously. But the pathogenesis is not exactly known and nowerdays Troditional Chinese Medicine has the superiority and positive effect to delay the speed of the occurrence and development of DR.The objective of the thesis is to sysmetically investigate the inhibitive effect of Mimeng Flower decoction on the proliferative Human Umbilical Vascular Endothelial Cell (HUVEC) induced by chronic hypoxia, and study the mechanism of Transforming Growth Factor-β(TGF-β) signal transduction, in order to provide experimental foundation for Mimeng Flower decoction used in clinic to prevent and cure proliferative HUVEC.MethodsHUVECs were cultured in vitro. 100μmol/L CoCl2 was used to set up the cellar chemistry hypoxia model, so that HUVEC proliferation were induced. At the same time,10mg/ml,20mg/ml,40mg/ml water extract of Mimeng Flower decoction was added to the HUVEC separately. The following studies were made after incubation for 24 hours.1. Through MTT method to detect the optimal density of CoCl2 to induce the proliferation of HUVEC.2. Through MTT method to detect the optimal density of Mimeng Flower decoction.3. Through MTT method to detect the inhibitive effect of Mimeng Flower decoction in the proliferation of HUVEC induced by chronic hypoxia.4. Through Real Time-PCR method to detect the change of TGF-βmRNA,TGF-βReceptorⅠ/ⅡmRNA expression in proliferative HUVEC regulated by Mimeng Flower decoction.5. Through Western blot method to detect the change of TGF-βmRNA,TGF-βReceptorⅠ/Ⅱprotein expression in proliferative HUVEC regulated by Mimeng Flower decoction.Results1. The results of MTT test:1.1 The optimal density of CoCl2 was 100μmol/L to induce the proliferation of HUVEC compared with higher or lower density and normal groups (p<0.05)1.2 The results of MTT test about the proliferation of HUVEC showed that the hypoxia group could increase the optical density (OD) values obviously compared with normal group (p<0.01); Mimeng Flower decoction groups could reduce the OD values obviously compared with hypoxia group (p<0.01) and the effect was dose dependent.2. The results of Real Time-PCR test showed that the expression of TGF-βTGFβRⅠ/ⅡmRNA increased in hypoxia group compared with normal group (p< 0.01); Mimeng Flower decoction groups could reduce the expression of TGF-βTGFβRⅠ/ⅡmRNA compared with hypoxia group in different extent and the effect was dose dependent.The expression of mRNA of the midial density of Mimeng Flower decoction is nearest the normal group.3. The results of Western blot test showed that the protein expression of TGF-β,TGFβRⅠ/Ⅱincreased in hypoxia group compared with normal group (p<0.01); Mimeng Flower decoction groups could reduce the expression of TGF-β,TGFβRⅠ/Ⅱprotein compared with hypoxia group (p<0.01) and the effect was dose dependent. Mimeng Flower decoction groups were obviously different with each other in statistics (p<0.01) Conclusions1. The proliferation of HUVEC induced by 100μmol/L CoCl2 may contribute to upregulating the expression of TGF-β,TGFβRⅠ/ⅡmRNA and protein.2. Mimeng Flower decoction could markly inhibite the proliferation of HUVEC in hypoxia state and the effect was dose dependent.3. Mimeng Flower decoction could downregulate the expression of TGF-β,TGFβRⅠ/ⅡmRNA and protein, and the effect was dose dependent.4. The mechanism of inhibiting proliferation by Mimeng Flower decoction in HUVEC may be related to downregulating the expression of TGF-β,TGFβRⅠ/ⅡmRNA and protein, so that the TGF-βsignal transduction was regulated indirectly.5. Combinding the past animial experiment, Mimeng Flower decoction regulates the expression of TGF-β,TGFβRⅠ/ⅡmRNA and protein all the time, and keeps the dymamic equilibrium, to inhibite the proliferation of HUVEC. It accords with "YIN and YANG in a relative equilibrium" of Troditional Chinese Medicine (TCM) and embodies the distinctness and superiority of the whole therory and determination of treatment through the differentiation of symptoms in TCM.