The Effect Of Weikangning On SOD2、GSTP1、VDAC1 和 Enolase Through Nrf2/ARE Signal Way In The Human GES-1 Cell Line

Background:Functional dyspepsia (FD), according to the Rome Ⅲ’Consensus Report, is defined by 12 wk in the past 12 mo, of persistent or recurrent pain or discomfort in the upper abdomen, in the absence of organic disease and no association with bowel habit. The pathogenesis of Functional Dyspepsia (FD) remains unclear, motor dysfunction of upper gastrointestinal, drink and food, psychological and society would be possible relevant. Especially, research has demonstrated an association between this common diagnosis and psychosocial factors. Now the challenge is how to elucidate the pharmaceutic mechanisms of "Wei Kangning", which will greatly benefit the development of traditional Chinese medicines for prevention and therapy of FD. Preliminary work has already identified the target spots from functional dyspepsia by two-dimensional electrophoresis (2DE), mass spectrometry, such as VDAC-1, Enolase, SOD2, GSTP1. We will use fluorescence quantitative PCR technology and western blotting to test them.Purpose:To study the expression of WeiKangning on VDACl, Enolase, SOD2, GSTP1, and evaluate the effect of the Nrf2/ARE signal way in the induction of GSTP1 and SOD2 on human GES-1 cell line by WeiKangning,the human GES-1 cell lines was used. This study is to investigate the protection effect of WeiKangning against oxidation stress of GES-1 cells and the mechanisms involved.Method:Using qRT-PCR and Western blot, to evaluate the effect of Weikangning on the mRNA and protein expression level of GSTP1,VDAC1, Enolase and SOD2 in GES-1 cells; Nrf2 translocation was detected by Western blotting. Real time Q-PCR was used to detect expressions of NQO1,H0-l,GCLM and GCLC mRNA level;to further explore the role of Nrf2 in the effect of lycopene on GSTP1 and SOD2 expression, transfected either siRNA-Nrf2 or Nrf2 plasmid, the level of mRNA and protein expression of GSTP1,VDACl, Enolase and SOD2 in GES-1 cells were detected once more. SOD activities, GSH and MDA content wre were detected to assess the level of oxidative stress in GES-1 cells.Result:It was shown that, after treated with WeiKangning 24h, induced a significantly increase in the level of mRNA and protein of GSTP1. VDAC1, Enolase and SOD2(P<0.05). The results showed that WeiKangning could cause nuclear accumulation of Nrf2 in association with downstream activation of Nrf2 mediated oxidative response genes such as GCLC, NQO1,GCLM and HO-1(P<0.05). As expected, an siRNA-Nrf2 transfected was unable to support WeiKangning induction of GSTP1 and SOD2 expression(P<0.05). Futhermore, in GES-1 cells transfected with a Nrf2 plasmid, an significant increase in the induction of GSTP1 and SOD2 mRNA and protein expression level(P<0.05). SOD activities and GSH content of GES-1 cell were higher in Weikangning group than the normal control group (P<0.05). Compared with the normal control group, the MDA content in Weikangning group were decreased obviously (P<0.05). While an siRNA-Nrf2 transfected, compared with the shMock 0.05 group and shMock 0.3 group, SOD activities and GSH content were obviously lower (P<0.05), the MDA content were increased obviously (P<0.05).Conclusion:The results indicated the effect of lycopene induction of GSTP1 mRNA and protein level depends on a functional Nrf2/ARE signal way in GES-1 cell line. Weikangning could protect against the oxidative damage of GES-1 cells via the activation of Nrf2/ARE signal pathway. The information provided here, therefore, in order to further study, we will deepen our knowledge on how "WeiKangning" is against the Functional dyspepsia. Oxidative stress in gastric prove to be one of reason in the development of Functional dyspepsia.Herbs of-Weikangning play an active role in improving gastric motility by decreasing oxidative stress, which afford new theoretical bases for treating the Functional dyspepsia.