Experimental Study On IKK? Regulation Of Maspin Expression In Human Periodontal Ligament Cells And Regulation Of Baicalin On Maspin

Section 1 Objective :To observe the expression of Maspin which regulated by NF-?B Non-classical Pathway IKK? in LPS-induced HPDLCs.Methods :The abruption,culture of HPDLCs was carried out using tissue culture method.Cells were identified by inverted microscope and SP immunohistochemistry.Immunocytochemical was used to observe the localization of maspin expression in HPDLCs and the effect of LPS on the proliferation of HPDLCs was detected by CCK-8.Then the experiment was divided into blank control group(NC),LPS1-4 groups(LPS:0.1,1,10,50 mg/L).Protein and mRNA levels of Maspin and IKK? were measured by Western Blot and RT-PCR respectively,and the secretions of TNF-? and IL-1? were tested using ELISA assay.Results:Maspin showed cytoplasmic expression in HPDLCs.The results of Western Blot showed that with the increasing of LPS concentrations,the expression of Maspin protein compared with NC group was down-regulated in LPS1-4 groups(P<0.05);and the expression of IKK? protein was significantly up-regulated than that in NC group(P<0.05);two-variable correlation analysis indicated a negative relationship between Maspin and IKK?(r=-0.959,P<0.05).The expression of mRNA was similar to protein.Secretions of TNF-? and IL-1? were increased accompanied with growing concentrations of LPS,and negatively correlated with Maspin.Conclusion:Maspin showed cytoplasmic expression in HPDLCs.IKK? involved in NF-?B Non-classical Pathway can down-regulate the expression of Maspin in LPS-induced HPDLCs,at the same time Maspin showed a negative correlation with TNF-? and IL-1?.Section 2 Objective :This study aims to test the effect of Baicalin on the expression of Maspin regulated by IKK? in human periodontal ligament cells.Methods :The effects of Baicalin on the proliferation of HPDLCs was detected by CCK-8.The experiment was divided into blank control group(NC: Baicalin:0mg/L,LPS: 0mg/L),LPS group(Baicalin: 0mg/L,LPS:10mg/L)and Baicalin + LPS1-4 groups(Baicalin: 0.01,0.1,1,10 mg/L,LPS:10mg/L);The methods were consistent with the first part.Protein and mRNA levels of Maspin and IKK? were measured by Western Blot and RT-PCR respectively,and the secretions of TNF-? and IL-1? were tested using method by ELISA assay.Results:The expression of Maspin protein was downregulated and the IKK? protein was up-regulated in LPS group compared with NC group(P<0.01).At the same time,compared with the LPS group,the expression of Maspin protein was up-regulated with the increasing of Baicalin concentrations in Bai + LPS 1-4 groups(P<0.05);and IKK? protein was significantly down-regulated(P<0.01);two-variable correlation analysis indicated a negative relationship between Maspin and IKK?(r=-0.886,P<0.05).The expression of mRNA was similar with protein.The results of ELISA showed that the expression of TNF-? and IL-1? were increased in LPS group than that in NC group,however,the expression of TNF-? and IL-1? in Bai+LPS1-4 groups were declined with the increasing of Baicalin concentrations than that in LPS group,as well as negatively correlated with Maspin.Conclusion:Baicalin can up-regulate the expression of Maspin which regulated by IKK? involved in NF-?B Non-classical Pathway in LPS-induced HPDLCs.Furthermore it also inhibits the secretions of TNF-? and IL-1?,protecting human periodontal ligament cells from injury induced by LPS.