| Objectives: 1.To explore the mechanism of Vernonia anthelmintica(L.)Willd.in the treatment of vitiligo.2.Based on the pathogenesis of oxidative stress in vitiligo,an in vitro oxidative stress model of vitiligo was to be established by using 2,2-Azobis(2-methylpropyl)dihydrochloride(AAPH)and immortalized keratinocyte line Ha Ca T cells.3.To explore the antioxidant effect and its possible mechanism of kaempferol(KP)on Ha Ca T cells under oxidative stress.Methods: 1.Using selective reaction / multi-reaction monitoring technique(SRM/MRM),ultra high performance liquid chromatography(HPLC)and MRM mass spectrometry analysis,the flavonoids in the seeds of Vernonia anthelmintica(L.)Willd.were quantitatively analyzed;2.The effects of different concentrations of AAPH(0,10,15,20,25,30,40,50,60 mmol/L)on the proliferation of Ha Ca T cells were detected by cell counting kit-8(CCK-8),and the appropriate concentration of drug modeling was determined.The effects of different concentrations of kaempferol(0,10,20,30,40,60,80,100 μmol/L)on the proliferation of Ha Ca T cells were detected by CCK-8 method,and the optimal intervention concentration of kaempferol was determined.The experiment was divided into five groups: control group,model group,low,medium and high dose kaempferol groups.3.inverted phase contrast microscope to observe the effects of different concentrations of kaempferol on the morphology of Ha Ca T cells;4.real-time fluorescence quantitative PCR(real-time quantitative PCR,RT-q PCR)was used to detect the effects of different concentrations of kaempferol on the m RNA expression of Nrf2,HO1,NQO1,GCLC and GCLM in cells;5.western blot(WB)was used to detect the expression of Nrf2 at protein level,and the effect of kaempferol on Nrf2-related signal pathway was analyzed.Results:1.The content of total flavonoids in the seeds of Vernonia anthelmintica(L.)Willd.was 0.97g/100 g.A total of 36 flavonoids were obtained by quantitative analysis,of which the content of kaempferol was 62.96ng/100 g.2.CCK-8 showed that AAPH could inhibit cell proliferation,and the higher the concentration,the stronger the inhibitory effect(P <0.001).When the concentration of AAPH was 25mmol/L,the proliferative activity of Ha Ca T cells was(62.33 ±4.98)%.At this time,the cells were in the state of oxidative stress and there were no too many dead cells.Kaempferol had no significant inhibitory effect on the proliferation of Ha Ca T cells in the range of 0 ～ 40 μmol/L,but when the concentration of kaempferol was greater than or equal to 60 μmol/L,the viability of Ha Ca T cells decreased with the increase of kaempferol concentration(P < 0.001).3.Kaempferol could reduce the cell injury induced by AAPH,and the viability of Ha Ca T cells recovered gradually with the increase of kaempferol concentration.4.PCR results showed that AAPH could increase the m RNA expression of Nrf2,HO-1,GCLC and GCLM in cells.Low dose kaempferol could significantly increase the m RNA expression of Nrf2,HO-1 and GCLC in Ha Ca T cells under oxidative stress,and the effect of kaempferol on HO-1 was the most significant,which was close to 48 times of that of the control group and 4 times of that of the model group.There was no significant difference in the expression of NQO1 m RNA among the four groups.5.WB results showed that AAPH could increase the protein expression of Nrf2 in Ha Ca T cells,and kaempferol increased the protein expression of Nrf2 in oxidative stress Ha Ca T cells in a dosedependent manner.Conclusion:1.Kaempferol,a component of Vernonia anthelmintica(L.)Willd.,can reduce the damage of Ha Ca T cells induced by AAPH.2.Kaempferol can up-regulate the expression of key antioxidant transcription factor Nrf2 and its downstream antioxidant genes,indicating that Vernonia anthelmintica(L.)Willd.has a certain application prospect in the field of antioxidant stress. |
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