Influence Of Qing Re Li Shi Yin On The Proliferation Of HaCaT Cell Line Induced By TNF-α

Objective: To reveal the mechanism of the inhibition of the proliferation, theinductive apoptosis and the expression of IL-6、IL-8and IL-10,which is of traditionalChinese medicinal Qing Re Li Shi Yin on HaCat cells, and provide scientific basis for thetreatment of Qing Re Li Shi Yin on psoriasis vulgaris.Methods: To take the KeratinocytesHaCaT cell line as the research object to subculture, to observe the proliferation inhibitioneffect of Qing Re Li Shi Yin extracts on HaCaT cell cultured in vitro by MTT method; toobserve of the inductive apoptosis of Qing Re Li Shi Yin on HaCaT cells by flowcytometry; and to examine the effect of expression of IL-6mRNA、IL-8mRNA andIL-10mRNA by HaCaT cell lines through ELISA and real-time fluorescence quantitativePCR method.Result:1.Effect of TNF-α on proliferation of HaCaT cells TNF-αaffected on HaCaT cells for24h,48h and72h,and had the proliferation effect on HaCaTcells. Compared the effect of TNF-α on48h concentration which is2.5×10~5u/L with noTNF-α cells control group, its proliferation rate was14.11%. the research chose TNF-α2.5×10~5u/L as the induction dose of HaCaT cells.2. Comparison of inhibitory effect onthe proliferation of HaCaT cells in the liquid extracted from three kinds of traditionalChinese medicine Qing Re Li Shi Yin’s whole party①extract、split party②extract andsplit party③extract have a certain inhibitory effect on the proliferation of HaCaT cellsinduced by TNF-α at different concentrations (3.1g/L-100g/L) and different time (24h,48h and72h). compared with split party②extract and split party③extract, inhibitoryeffect of different concentration on proliferation of HaCaT cells in whole party①extractwas significantly higher than that of the latter two, and the difference was statisticallysignificant (P <0.05). This study researched that Qing Re Li Shi Yin extract had proliferation inhibitory effect on HaCaT cells induced by TNF-α further.3. Influenceof Qing Re Li Shi Yin with different concentration on the proliferation inhibitoryeffect of HaCaT cell induced by TNF-α. Compared drug effects for48h with MTXpositive controls, when Qing Re Li Shi Yin was on12.5g/L, the inhibition rate to HaCaTcell was40%, which was higher than that of MTX (38.76%). In the three concentration25g/L-100g/L, its inhibitory effect significantly increased and had evident difference (P <0.05or P <0.01) compared with MTX.4.Effect of Qing Re Li Shi Yin on the apoptosisof HaCaT cells After Qing Re Li Shi Yin extract with concentration of12.5g/L,10g/L,7.5g/L and5g/L effected HaCaT cells for48h, total number of cell apoptosis was45.75%,44.94%,41.26%and34.43%respectively, and TNF-α activation no drug control group (P<0.05or P <0.01).Obviously higher than no drug control group activated by TNF-α, itwas only24.65%, and the former is higher than the latter, difference was evident as well (P<0.05and P <0.01).5. Effect of Qing Re Li Shi Yin on cycle of HaCaT cells WhenMTX affected HaCaT cells for48h, cell in G1phase was65.94%, normal control groupand TNF-αcontrol group were49.01%and50.73%respectively. The former in S phasewas30.38%, and MTX arrested cells in G1phase. When Qing Re Li Shi Yin were12.5g/Land15g/L, cells in G1phase was reduced evidently (38.06%and25.04%) and cells in Sphase increased significantly (56.98%and67.90%), compared with cells in TNF-α group.HaCaT cells were arrested in G1phase by MTX, and yet arrested in S phase by Qing Re LiShi Yin extract.6. Effect of Qing Re Li Shi Yin extract on HaCaT cells expression ofIL-6mRNA, IL-8mRNA and IL-10mRNA Expression of Qing Re Li Shi Yin oninflammatory cytokines IL-6mRNA and IL-8mRNA produced by HaCaT cells which wereactivated by TNF-α induced. The expression of IL-10mRNA in10g/L and12.5g/Lincreased obviously, and compared to TNF-α activation group, there was significantdifference (P <0.05and P <0.01).7.Effect of Qing Re Li Shi Yin extract on HaCaTcells secretion of IL-8and IL-10.Qing Re Li Shi Yin can reduce IL-8produced by thesecretion of HaCaT cells which were activated by TNF-α induced., but no obvious effecton IL-10.Conclusion: Chinese traditional medical Qing Re Li Shi Yin has obvious inhibitoryeffect on the proliferation of HaCaT cells, and the inhibition rate has time-and-dosedependent manner in a certain range of concentration. The effect on HaCaT cells caninduce the apoptosis of HaCaT cell; the cycle of HaCaT cells are interfered, in which cells are blocked in S phase; expression of IL-6mRNA, IL-8mRNA is weakened, and expressionof IL-10mRNA is enhanced with the increase of drug concentration, with which maybe theanti-psoriatic mechanism of Chinese traditional medical Qing Re Li Shi Yin hasrelationship.