Effect Of ICT-CMC On CD59 Gene Expression In Oral Lichen Planus

Objective:Oral lichen planus(OLP)is a common oral mucosal disease with high malignant potential.The pathogenesis of OLP is unknown,and there is no specific drug treatment at present.The aim of this study is to investigate the correlation between abnormal expression of cell differentiation antigen 59(CD59)and oral lichen planus(OLP)using molecular biology techniques.Icaritin(ICT-CMC),an extract of Epimedium plant,was combined with chitosan(CMC),a Marine extract,to prepare a new drug-loaded microspheres(ICT-CMC).The regulatory effect of ICT-CMC on the expression of CD59 and its downstream molecules in keratinocytes of OLP was explored,so as to provide new ideas for the molecular mechanism and targeted therapy of OLP.Open new avenues.Methods:1.Clinical case selection: the experimental group consisted of 15 OLP patients and the control group consisted of 15 healthy controls.Normal mucosa keratinocytes(HOK)and OLP primary keratinocytes were isolated and cultured in vitro by enzyme digestion.The cultured keratinocytes were identified by immunocytochemical staining and morphological observation.2.The expression levels of CD59 protein,CD59 m RNA,PI3K/AKT protein and s CD59 in HOK and OLP keratinocytes were detected by real-time fluorescent quantitative RTqPCR,Western blotting and enzyme-linked immunosorbent assay,respectively.3.Icaritin and chitosan were combined by emulsification crosslinking method to form a new drug-loaded microspheres ICT-chitosan microspheres(ICT-CMC),and the morphology,drug loading and stability of the microspheres were characterized by scanning electron microscopy,UV-Vis,FT-IR and other technologies.4.CD59 m RNA,CD59 protein and JAK/STAT-6 m RNA were detected by immunocytochemical staining,immunochemical fluorescence technology,real-time fluorescent quantitative PCR(RT-qPCR),Western blotting,enzyme-linked immunosorbent assay(ELISA)and flow cytometry,PI3K/AKT protein,s CD59 expression in the culture supernatant,and apoptosis of keratinocytes before and after OLP treatment;CCK-8 method was used to explore the inhibitory effect of different concentrations of ICT-CMC microspheres on primary keratinocytes of OLP.Results:1.HOK and OLP keratinocytes were isolated and cultured in vitro.The extracted keratinocytes were strongly positive for keratin by immunocytochemical staining The results showed that the cultured cells were single keratinocytes.2.The results of RT-qPCR and Western blotting showed that the expression of CD59 m RNA and PI3K/AKT protein in OLP keratinocytes were significantly lower than those in normal HOK cells(P<0.001).Enzyme-linked immunosorbent assay results showed that s CD59 in the culture supernatant of OLP keratinocytes was significantly reduced.3.The Angle of reposition measured by funnel method showed that the Angle of reposition of ICT-CMC microspheres was 36.34°,indicating that ICT-CMC microspheres had good fluidity.The bulk density of the microspheres was determined by the cylinder method,and the bulk density of the microspheres was 0.547g/m L.The swelling rate and swelling rate of ICT-CMC microspheres were 29.7% and 74.1%respectively.The morphology of the microspheres was observed by scanning electron microscopy,and the microspheres showed a uniform spherical shape with a diameter of about 50μm.Fourier transform infrared spectroscopy(FTIR)analysis showed that the ICT-CMC microspheres presented another new phase property and had a new chemical bond,which confirmed that the ICT-CMC microspheres were prepared successfully.4.The results of immunocytochemistry and immunofluorescence showed that the expression of CD59 in primary keratinocytes of OLP was significantly increased after ICT-CMC microspheres treatment(P<0.05).After treatment,the supernatant of HOK cells and OLP primary keratinocytes was collected.Elisa results showed that s CD59 in the supernatant of OLP primary keratinocytes was significantly higher than that before treatment(P<0.001).The results of RT-qPCR showed that the expression of CD59 m RNA in OLP cells was significantly increased after microsphere treatment,and the difference of JAK/STAT-6 m RNA between the groups was very significant(P< 0.001).Western blotting results showed that the expression of PI3K/AKT protein was increased in OLP keratinocytes after drug treatment(P<0.001);CCK-8 and flow cytometry results showed that ICT-CMC microspheres had good biocompatibility and could promote the growth of primary keratinocytes of OLP and promote the apoptosis of keratinocytes of OLP(P<0.01).Conclusions:1.In vitro cell experiments confirmed that the isolated and cultured cells were positive for keratin and negative for vimentin,which proved that the extracted cells were single keratinocytes,and the expression of CD59 and PI3K/AKT was decreased in OLP keratinocytes.2.This project successfully developed two new drug-loaded microspheres ICT-CMC with good morphology and biocompatibility,which greatly improved the drawbacks of icaritin being insoluble in water and improved the utilization rate of icaritin.3.ICT-CMC microspheres could up-regulate the gene expression levels of CD59 and JAK/STAT-6,activate the PI3K/AKT phosphorylation pathway,and inhibit the proliferation and promote the apoptosis of OLP keratinocytes at the gene and molecular levels.Normal keratinocytes were induced to divide and proliferate.