The Expression Of Membrane-bound Complement Regulatory Proteins CD46,CD55 And CD59 In Oral Lichen Planus

Objective:1.To investigate the expression levels of membrane-bound complement regulatory proteins(mCRPs),CD46,CD55 and CD59 in oral lichen planus(OLP)local lesions and explore the correlation between mCRPs and OLP.2.To study the expression of mCRPs in primary OLP oral keratinocytes isolated and cultured in vitro,and explore the effects of different concentrations of Lipopolysaccharide(LPS)stimulation on the transcription levels of CD46,CD55 and CD59.To further explore the correlation between abnormal expression of mCRPs and OLP,and provide new insights for the pathogenesis,early diagnosis and treatment of OLP.Materials and Methods:1.Thirty-seven cases of OLP patients(20 non-erosive OLP and 17 erosive OLP)and twenty healthy controls were recruited in this study and the degree of oral lesions in OLP patients was scored by REU.The mRNA and proteins expression levels of CD46,CD55 and CD59 in OLP tissues were detected by RT-qPCR and western blotting respectively,and the expression levels of complement C3 and sC5b-9 in OLP patients' serum and saliva were detected by ELISA to evaluate the activation status of complement.Pearson correlation coefficient was used to analyze the correlation between the above indicators and REU score.2.In addition,mucosa tissues of 3 non-erosive OLP patients and 3 healthy controls were collected,and primary keratinocytes were separated and cultured in vitro by neutral protease digestion.The keratinocytes were identified by morphological observation and immunocytochemistry.Immunofluorescence was used to detect the expression levels of mCRPs at cellular level.The expression of CD46,CD55 and CD59 m RNA in HOK cells was detected by RT-qPCR after stimulated with LPS(0,0.1,1,10,20 ?g/mL)for 24 h and 48 h.Results:1.RT-qPCR showed that the levels of CD46,CD55 and CD59 mRNA in OLP tissues were significantly decreased compared with those of the healthy control group(P < 0.01).,but there was no significant difference between erosive and non-erosive cases(P > 0.05).Western blotting showed that the expression levels of CD46 and CD55 proteins in erosive OLP group were significantly reduced(P < 0.05),and the expression levels of CD46 and CD55 proteins in the non-erosive OLP group were not significantly different from those in the control group(P > 0.05).The expression of CD59 protein in erosive and non-erosive OLP group was not significantly different from the control group(P > 0.05).ELISA showed that the levels of C3 and sC5b-9 in serum were not significantly different from those in the healthy control group(P > 0.05).The level of complement C3 in the patients' saliva was significantly decreased(P < 0.05),while the level of sC5b-9 was increased(P < 0.05).Pearson correlation analysis showed that there was a negative correlation between salivary C3 and REU scores(r =-0.8098,P = 0.0148)in erosive OLP.There were no significant correlations between the other indicators and the REU scores(P > 0.05).2.OLP primary keratinocytes were successfully isolated and cultured in vitro.Morphological observation and keratin immunocytochemical staining showed that the cultured cells were single oral keratinocytes.Immunofluorescence showed that compared with the normal control group,the expression of m CRPs in OLP keratinocytes was significantly reduced(P < 0.01).The transcription level of CD46 in HOK cells decreased significantly after treated with LPS(20 ?g/mL)for 24 h(P < 0.05).The expression levels of CD55 and CD59 increased under the stimulated of low concentration of LPS for 24 h,and decreased slightly when the concentration reached to 20 ?g/m L,but there was no significant difference(P > 0.05).After treated with LPS for 48 h,the expression levels of CD46 and CD59 decreased significantly(P < 0.05),and the transcription level of CD55 decreased significantly only when stimulated with 0.1 ?g/mL LPS(P < 0.05).Conclusions:1.The results showed that there were decreased mCRPs expression levels and abnormal activation of local complement system in OLP.2.In vitro cell experiments further demonstrated that the expression levels of mCRPs decreased in cultured OLP primary keratinocytes.3.G-bacterial toxic substance LPS stimulation can down-regulate the expression of m CRPs,and the cells are more vulnerable to complement attack,which may be related to the apoptosis of OLP epithelial basal cell.