The Molecular Mechanism Underlying The Inhibition Of HaCaT Cells Proliferation By Triptolide

Tripterygium wilfordii polyglycoside,the mixture of lipophilic components extracted from Tripterygium wilfordii Hook F,showing anti-inflammatory,anti-fertility,anti-bacterial and other activities.It has been used for the treatment of psoriasis as a non-steroidal immunosuppressive agent.As the most important active substance of Tripterygium wilfordii Hook F extract,Triptolide has shown potent anti-inflammatory and anti-tumor effects both in vitro and in vivo.In a variety of tumor cells,triptolide can inhibit cell proliferation,induce cell cycle arrest and cause cell apoptosis.Some recent studies have shown that triptolide can inhibit the proliferation of normal human keratinocytes as well as the HaCaT cells stimulated by EGF.Although it is known that triptolide can inhibit the proliferation of keratinocytes,the underlying mechanism remains unclear.HaCaT cells were used to explore the mechanism involved in the inhibition of keratinocytes proliferation by Triptolide.The effects of Triptolide on proliferation,cell cycle and apoptosis of HaCaT cells were studied by MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide),RTCA(Real Time Cellular Analysis),Flow cytometry and Western Blot.The results showed that Triptolide inhibited the proliferation of HaCaT cells in a concentration-dependent manner,with an IC50 of about 50 nM.20 nM Triptolide could significantly induce cell arrest of HaCaT cells in S phase.When the concentration of Triptolide reached 50 nM,HaCaT cells were induced to apoptosis and nuclear shrinkage.In apoptotic cells,both Caspase 8 and Caspase 9 were activated..showing that apoptosis was associated with receptor-mediated apoptotic pathway and mitochondrial apoptosis pathway.The results from Western Blot showed that the phosphorylation of ERK in HaCaT cells was up-regulated after Triptolide treatment,while the level of phosphorylated JNK and p38 did not change.U0126,the inhibitor of MEK1/2 could partially revert the inhibition of HaCaT cells proliferation by TriptolideBecause keratinocytes are activated by IFN-y and other cytokines.,we studied the effects of Triptolide on the proliferation of IFN-y-activated HaCaT cells.The results showed that Triptolide could inhibit the proliferation of IFN-?-activated HaCaT cells Further analysis showed that JAK2 and STAT3 were activated in HaCaT cells after IFN-?stimulation.Triptolide could inhibit the phosphorylation of JAK2 and STAT3 in HaCaT cells and down-regulate the expression of the downstream genes of STAT3 such as VEGF and PD-L1.We tried to study the mechanism underlying the inhibition of keratinocytes proliferation by Triptolide.The data demonstrate that Triptolide can inhibit proliferation of HaCaT cells,induce S phase cell cycle arrest and Caspase-dependent apoptosis.For IFN-? activated HaCaT cells,Triptolide can also inhibit its proliferation and the activation of the JAK2/STAT3 signaling pathway.Our results may be instructive for the treatment of diseases such as psoriasis.