Screening Of HaCaT Cell Viability Inhibitor From Chinese Medicine Compounds And Analysis Of Its Anti-psoriasis Activity And Mechanisms

Psoriasis is a multifactorial inflammatory skin disease that inconveniences many patients.Considering the side effects and drug resistance of the current therapy,it is urgent to discover more effective and safer anti-psoriatic drugs.In the present study,we screened over 250traditional Chinese medicine compounds for their ability to inhibit the cell viability of cultured human HaCaT keratinocytes,a psoriasis-relevant in vitro model,and found that periplogenin was highly effective.Periplogenin significantly inhibited the viability of the HaCaT cells,but had a moderate cytotoxic effect on the normal human fibroblast cell line(Hs-68 cells)and the mouse subcutaneous connective tissue cell line A9 in the dose range we used.These results revealed that periplogenin has the potential to further develope as a candidate drug for the treatment of psoriasis.The forms of cell death induced by compounds include apoptosis,autophagy and necrosis.In order to clarify the mechanism underlying HaCaT cell viability inhibited by periplogenin,we first investigated the effect of periplogenin on the apoptosis of HaCaT cells.We observed the nuclear changes visualized by DAPI and TUNEL staining,analysed the caspase 3 activation detected by western blotting,the cell apoptosis rate detected by the flow cytometry and the rescue effect of the pan-caspase inhibitor z-VAD-fmk in HaCaT cells treated by periplogenin.The results indicated that the apoptotic features were not found in the periplogenin-treated HaCaT cells.Then,we studied the effect of periplogenin on the autophagy of HaCaT cells.The results revealed that there was no significant difference in the Beclin-1 and LC3-II levels in periplogenin-treated HaCaT cells.Even when the autophagy inhibitor CQ blocked autophagy,the levels of LC3-II did not increase following periplogenin exposure.Moreover,the autophagy inhibitors CQ,3-MA and LY294002 could not reversed HaCaT cell death triggered by periplogenin.These results revealed that apoptosis and autophagy were not induced by periplogenin in HaCaT cells.Then,we detected the effect of periplogenin on the necrosis of HaCaT cells.The results showed that periplogenin caused PI to permeate into HaCaT cells and increased lactate LDH release,suggesting that the permeability of HaCaT cells has elevated.Periplogenin could also decrease intracellular ATP levels and rapidly increase the number of necrotic cells.Additionally,the typical characteristics of necrosis were observed in the periplogenin-treated HaCaT cells by TEM.Notably,the necroptosis inhibitor Nec-1 and NSA were able to rescue the cells from necrotic cell death,supporting that necroptosis was involved in periplogenin-induced cell death.Furthermore,the ROS levels were elevated in the periplogenin-treated cells,NAC(an antioxidant)and Nec-1 could inhibit the ROS levels,and NAC could attenuate necroptotic cell death.These findings demonstrated that periplogenin inhibits the viability of HaCaT cells via increasing the intracellular ROS levels and inducing necroptotic cell death.To investigate the potential effect of periplogenin on psoriasis treatment in vivo,we generated TPA-and IMQ-induced psoriasis-like mouse models and studied the therapeutic effects of periplogenin on the psoriasis-like mouse models.Our results revealed that topical administration of periplogenin was able to ameliorate the skin lesions and reduce the inflammatory infiltrate of CD4~+Th cells,CD11b~+macrophages and CD11c~+DCs in the dermis tissues of skin lesion.In sum,our results indicate,for the first time,that periplogenin is a naturally occurring compound with potent anti-psoriatic effects in the cell model and animal models,making an important material foundation for the development of novel therapeutic drugs for psoriasis.