Role Of Mammary Serine Protease Inhibitor On The Inflammatory Response In Oral Lichen Planus

Objectives:Oral lichen planus(OLP)is a chronic inflammatory condition with an unclear pathological mechanism.I?B kinase ?(IKK?)-regulated mammary serine protease inhibitor(MASPIN)has been shown to mediate inflammation,particularly in cancers.But the role of MASPIN in the inflammatory response of OLP is unclear.Here,we explored the expression of MASPIN in OLP and its role in the inflammatory response.Materials and Methods:Immunohistochemistry staining and Real-time quantitative Polymerase Chain Reaction(Real-time PCR)assays were used to detect the subcellular localisation and expression of MASPIN and IKK? in OLP and healthy control(HC)tissues.Levels of the inflammatory factors(TNF-?,IL-6)were compared with enzyme-linked immunosorbent(ELISA)assays.Lipopolysaccharide(LPS)stimulates human oral keratinocytes(HOKs)to partially mimic the inflammatory environment of OLP.CCK8 and ELISA assays are used to detect the toxicity and the expression of inflammatory cytokines in LPS-induced HOKs,respectively,to determine the best conceration of LPS.Subsequently,the experiment was divided into four groups,namely experimental group(MASPIN plasmids?IKK? siRNA),negative control group(MASPIN-plasmids NC,IKK?-siRNA NC),fluorescent control group(MASPIN-pEX-1,IKK?-FAM NC)and LPS control group.After overexpressing MASPIN or silencing IKK? successfully,the experiment was divided into three groups,namely experimental group(MASPIN plasmids,IKK? siRNA),LPS control group and blank control group.MASPIN plasmid and IKK? siRNA were transfected into HOKs by liposome method and transfection efficiency was detected.Real-time PCR,Western Blot and ELISA were used to detect the expression of MASPIN,IKK? and inflammatory cytokines in LPS-stimulated HOKs.Results:Immunohistochemistry assay showed that MASPIN and IKK? were mainly localized in the cytoplasm of the epithelial layer of OLP tissues;MASPIN expression was down-regulated,whereas IKK? and inflammatory cytokines expression was up-regulated in OLP tissues compared to those of healthy controls(P < 0.01).CCK8 assay showed that 5ug/ml and 10ug/ml LPS had no toxic effect on HOKs.At the same time,ELISA result proved that the highest expression of inflammatory cytokines can be stimulated by 10ug/ml LPS in HOKs,and the difference was statistically significant(P<0.05).Therefore,we chosed 10ug/ml LPS as the finally concentration to HOKs.Real-time PCR result showed that MASPIN plasmids and IKK? siRNA were transfected into HOKs successfully and the transfection efficiency were 57% and 87%,respectively.In addition,lower expression of IKK? and inflammatory cytokines can be found in MASPIN-plasmids group compared to LPS control group and the differences were statistically significant(P<0.05).These results suggest MASPIN overexpression in LPS-stimulated HOKs could inhibit the levels of IKK? and the secretion of inflammatory cytokines.By contrast,higher expression of MASPIN and lower secretion of inflammatory cytokines can be found in IKK?-siRNA group compared to LPS control group and the differences were statistically significant(P<0.05).These results suggest IKK? silenced in LPS-stimulated HOKs could promoted the expression of MASPIN and inhibited the secretion of inflammatory cytokines.Conclusion:In this study,we found that MASPIN expression was down-regulated,whereas IKK? and inflammatory cytokines(TNF-?,IL-6)expression was up-regulated in OLP tissues.Overexpressing MASPIN or silencing IKK? could alleviate LPS-induced inflammatory response by inhibiting the expression of inflammatory cytokines.It is speculated that both MASPIN and IKK? may involve in and influence the inflammatory process of OLP,suggesting potential therapeutic targets of OLP.