The Pruritus Mediator Recombinant Mucunain Induced Apoptosis Through Caspase 8 Signaling Pathway in the Skin Epithelial Cells

Background Mucunain, a cysteine protease extracted from the spicules of cowhage, can induce intense itching as a pruritus mediator independent of histamine. It is well known that erythema, edema and vesicles on skin may occur immediately following contact with Mucunain. A recent study has demonstrated that Mucunain may trigger pruritus and inflammation on the left or right groin of each Maltese–Beagle atopic dog. The aim of this study was to investigate the cytotoxic effect of Mucunain on a cocultured skin epithelial cell model in vitro. Methods Functional recombination Mucunain (rtMucunain) were obtained following induction, purification, and renaturation. Human keratinocytes and dermal fibroblasts were cocultured to construct a cocultured cell model skin epithelium cell model. The growth inhibition was measured using an MTS test. Microscopy was used to observe morphological alterations in cell architecture. The DCFH-DA assay was used to evaluate reactive oxygen species production, the cells death type was identified using a Flow cytometry assay, and the pro-inflammatory cytokines IL-6 and −8 were detected using an ELISA assay. The ability to form an apoptosis network was investigated by a Western blot assay to assess apoptosis-related protein expression. Results Following purification and renaturation, rtMucunain was obtained with a yield of up to 50 mg/L. In the cocultured skin epithelial cells treated with rtMucunain, cell growth was inhibited in a dose- and time-dependent manner, with doses of 2.5 µM or higher resulting in significant cytotoxicity. Morphological analysis revealed increased cell dispersion and abnormal morphology, particularly following prolonged exposure. Levels of reactive oxygen species were elevated, indicating oxidative stress. Flow cytometry and Western-blot data demonstrated that the cells underwent apoptosis, with extrinsic apoptosis pathways (NF-κB and Caspase-8) being activated by rtMucunain. Furthermore, treatment with rtMucunain markedly enhanced levels of IL-6 and IL-8, suggesting a robust pro-inflammatory response. Conclusion rtMucunain showed a strong inflammation and apoptosis effect in a cocultured skin epithelium cell model. This effect may be due to the activated Caspase 8 signaling pathway, on which NF-κB might play an important role. It was the first time to clarify the cytotoxic effect of rtMucunain on the skin epithelial cell model, and the comprehensive understanding the damaged mechanisms of rtMucunain on normal skin cells can provide a data basis for the skin irritated experiment of rtMucunain and its analogues.