Anti-inflammatory Effect Of Paeoniflorin Combined With Baicalin In Oral Inflammatory Diseases

Objective:Oral inflammatory diseases are among the most serious diseases affecting humans’ daily life,including oral lichen planus(OLP)and periodontitis.There are challenges in the treatment of oral mucosa inflammatory diseases now.In recent years,the study found that Paeoniflorin(PF)extracted from the root of Paeonia lactiflora pall and Baicalin(BAI)extracted from Scutellaria baicalensis showed good anti-inflammatory effects in the treatment of oral inflammatory diseases.However,the anti-inflammatory of PF combined with BAI in oral inflammatory diseases is not yet clear.The purpose of this study was to investigate the effect of PF combined with BAI,and to compare the effect with PF or BAI alone,so as to provide a theoretical basis for PF combined with BAI in the treatment of oral inflammatory diseases.Materials and Methods:The subcellular localization of Nuclear factor-κB p65(NF-κB p65)and IKKα in OLP and healthy control(HC)tissues was detected by immunohistochemical staining.Enzymelinked immunosorbent assay(ELISA)was used to detect the levels of inflammatory cytokines TNF-α and IL-6.Human oral keratinocytes(HOKs)were treated with lipopolysaccharide to mimic the inflammatory environment of OLP in vitro.Cell Counting Kit-8(CCK-8)are used to detect the proliferative capacity of HOKs treated with PF and BAI.The expression of inflammatory cytokines(TNF-α and IL-6)in LPS-stimulated HOKs after different concentrations of PF and BAI were detected by ELISA.The optimal concentration of PF and BAI was determined by CCK-8 and ELISA.Real-time PCR and Western Blot were used to investigate the m RNA levels of inflammatory cytokines and the levels of NF-κB pathway related proteins(phosphorylated-IκBα,phosphorylated-NF-κB p65)in HOKs when PF and BAI were used alone or in combination with PF and BAI,respectively.Immunofluorescence assay was used to detect PF combined with BAI on nuclear translocation of NF-κB p65.Results:Immunohistochemistry showed that NF-κB p65 and IKKα were positively expressed in OLP tissues and negatively expressed in HC tissues.Moreover,ELISA showed significant TNF-α and IL-6 overexpression in OLP tissues compared to that in HC tissues(P < 0.01).In a cultured cell model,HOKs mimicked an inflammatory response of OLP induced by LPS(10μg/ml)according to previous research method.CCK-8 result showd that PF(5μg/m L and10μg/m L)and BAI(2.5μg/m L and 5μg/m L)could significantly increase the survival rate of HOKs.The combination of PF and BAI(5 μg/ml + 2.5μg/ml,5μg/ml + 5μg/ml,10μg/ml +2.5μg/ml,10μg/ml + 5μg/ml)also significantly increased the survival rate of LPS-induced HOKs(P < 0.05).Therefore,5μg/m L and 10μg/m L PF,2.5μg/m L and 5μg/m L BAI were selected for ELISA assay.The results showed that the expression of TNF-α and IL-6 was significantly decreased after PF and BAI combined treatment compared with PF or BAI alone,especially when both PF and BAI were 5μg/m L.The factorial analysis of variance showed that there was an interaction between PF(5μg/m L)and BAI(5μg/m L).RT-PCR results showed that combined treatment with 5μg/ml PF and 5μg/ml BAI could significantly reduce the m RNA expression levels of TNF-α and IL-6 in LPS-stimulated HOKs.Western blotting and immunofluorescence results showed that PF(5μg/m L)combined with BAI(5μg/m L)significantly decreased the expression of NF-κB p65 and IκBα phosphorylation,and reduced the nuclear translocation of NF-κB p65 in HOKs,indicating that PF and BAI combined inhibited the activation of NF-κB signaling pathway.Conclusion:The results of this study showed high expression of NF-κB p65,IKKα,TNF-α and IL-6 in OLP tissue.Compared with PF and BAI alone,the combination of PF and BAI may better inhibit the activation of NF-κB signaling pathway by inhibiting the phosphorylation of NF-κB p65 and IκBα,thus inhibiting the inflammatory response of LPS-induced HOKs,which provides an experimental basis for the combination of PF and BAI in the treatment of oral inflammatory diseases.