| Psoriasis is a proliferative,inflammatory and chronic skin disease.The incidence of psoriasis is high and easy to recur.At present,the pathogenesis of psoriasis is still not completely clear,and it has the characteristics of long onset period and high cure difficulty,which make the patients suffer both mental and physical pressure and pain,and obviously interfere with their daily life and work.Therefore,psoriasis is considered as one of the most important diseases in the field of dermatology.Clinically,drugs widely used in the treatment of psoriasis include methotrexate,dexamethasone,retinoic acid,vitamin D3analogues and glucocorticoids,and some biological agents are also used in the treatment of psoriasis,such as etanercept.However,these drugs have some limitations and different degrees of toxicity and side effects,so it is urgent to develop more effective and safer drugs for psoriasis treatment.1.Screening compounds for inhibiting the HaCaT cell viabilityIn the present study,we first screened more than 250 traditional Chinese medicine compounds using a psoriasis-relevant cell model-HaCaT keratinocytes,and found that periplogenin could significantly inhibit the viability of HaCaT cells,but had little effect on the viability of human fibroblast Hs-68 in the dose range used.The results suggest that periplogenin may have potential as a candidate drug for psoriasis treatment.Subsequently,in order to obtain more efficient and low toxic compounds that inhibit the viability of HaCaT cells,we examined the effects of six structural analogues of periplogenin on the viability of HaCaT cells.Finally,we selected the structural analogue of periplogenin,convallatoxin,which had strong inhibitory effect on HaCaT cells but low toxicity to normal HS-68 cells,as follow-up drugs,and compared its activity with that of periplogenin.2.Inhibitory mechanism of periplogenin and its structural analogue,convallatoxin,on the viability of HaCaT cellsIt has been reported that apoptosis of keratinocytes is blocked in psoriasis lesions.Therefore,in order to investigate the mechanism of inhibition of HaCaT cells by periplogenin and convallatoxin,we first examined the effects of periplogenin or convallatoxin on the proliferation of HaCaT cells.The results showed that periplogenin or convallatoxin induced G1-phase cell-cycle arrest by inhibiting DNA synthesis;decreasing the levels of ki67,cyclinD1and cyclinE;while increasingthe expression of the p21 protein,thereby inhibiting the proliferative of HaCaT.Then,we analyzed the effect of periplogenin or convallatoxin on the apoptosis of HaCaT cells by examining the morphological changes of HaCaT cells,the activation of apoptosis-related protein casapase-3,the rate of cell apoptosis and the rescue effect of inhibitor z-VAD-fmk in HaCaT cells treated with periplogenin or convallatoxin.The results showed that apoptotic characteristics of HaCaT cells treated with periplogenin or convallatoxin were not obvious.However,it was found that the nuclei of HaCaT cells which treated with periplogenin or convallatoxin were sparse and reticulated,and the chromatin aggregated to the edge,then the cells swelled and finally burst.This morphological change is similar to the characteristics of cell necrosis,suggesting that periplogenin or convallatoxin may induce HaCaT cells necrosis.Subsequently,PI staining and increased lactate LDH release in cell culture medium showed that the treatment of periplogenin or convallatoxin made the cell membrane of HaCaT cells lose integrity.In addition,periplogenin or convallatoxin could also decreased ATP levels in HaCaT cells and promote cell necrosis.Additionally,the typical features of cell necrosis were also clearly observed by transmission electron microscopy in periplogenin or convallatoxin treated cells.Importantly,the necroptosis inhibitor Nec-1could effectively block the inhibitory effect of periplogenin or convallatoxin on cell viability,confirming that periplogenin or convallatoxin inhibited cell viability by induceing necroptosis of HaCaT cells.To further explore the mechanism of necroptosis,we found that periplogenin or convallatoxin increased the level of ROS,but active oxygen scavenger NAC and necroptosis inhibitor Nec-1 could inhibit the production of ROS,and NAC could also block the necroptosis induced by periplogenin or convallatoxin.These results suggest that periplogenin or convallatoxin can inhibite the proliferation and promote necroptosis by inducing ROS production in HaCaT cells,thus inhibiting the viability of HaCaT cells.3.The therapeutic effects of periplogenin and its structural analogue,convallatoxin,on psoriasis-like mice models.The previous results show that periplogenin or convallatoxin have obvious anti-psoriasis effects at the cellular level.In order to explore the therapeutic effects of two compounds on psoriasis,we generated two psoriasis-like mice models which induced by IMQ and TPA,and discussed the therapeutic effects of two compounds on psoriasis-like lesions in model mouses.The results showed that the application of periplogenin or convallatoxin could significantly alleviate psoriasis-like skin lesions induced by IMQ or TPA,and significantly reduced the neovascularization and infiltration of inflammatory cells around blood vessels,such as CD4~+T cells,CD8~+T cells,CD11b~+/CD11c~+dendritic cells,CLA~+T cells and V?4~+T.Simultaneously,periplogenin or convallatoxin also down-regulated the expression levels of related inflammatory factors,such as IL-17A,IL-17F,IL-22,TNF-?,IFN-?.In conclusion,our study found that periplogenin and its structural analogue,convallatoxin,can ameliorate skin lesions and alleviate the symptoms of psoriasis by inhibiting the proliferation and inducing necroptosis of keratinocytes,inhibiting infiltration of immune cell and abnormal expression of inflammatory factors.This study revealed the anti-psoriasis effects of two natural compounds with high efficiency and low toxicity at the cell level and in animal models,which provided novel leading compounds for the development of new drugs for psoriasis treatment. |
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