| ObjectiveThis study explored the potential therapeutic effect of LIRA on periodontitis.The classical Gram-negative bacteria E.coli LPS and hPDLCs were co-cultured to simulate the pathological process of periodontitis.The effects of LIRA on the proliferation,migration,inflammation and osteogenic differentiation of hPDLCs under LPS were analyzed.MethodsPrimary culture of hPDLCs were done by explants with enzymatic digestion.The cells were identified by immunohistochemical staining,and alizarin red staining identified whether the cells have the potential of bone differentiation.QRT-PCR was used to identify whether hPDLCs express GLP-1R;MTT method was used to screen the optimal concentration of LIRA and E.coli LPS.The effects of LIRA on the proliferation of hPDLCs were studied by LIRA.The effects of LIRA on the migration of hPDLCs were analyzed by healing assay.QRT-PCR and Western Blot were used to analyze the effect of LIRA on inflammatory factors(IL-6 and TNF-?)and osteogenic factors(ALP and Runx2)in hPDLCs.Results1.The hPDLCs were successfully cultured by explants with enzymatic digestion.The results showed that the expression of hPDLCs was confirmed by immunohistochemical staining: anti-vimentin protein was positive and anticytokeratin was negative.The results showed that the cells were derived from mesenchymal and alizarin red staining confirmed the osteogenic differentiation of hPDLCs.2.The results of qRT-PCR showed that GLP-1R mRNA was present on hPDLCs,and LIRA increased the expression of GLP-1R mRNA.3.MTT: cultured with 25,50,75,100,125 n M LIRA for 24 h,the proliferation activity of hPDLCs are enhanced in a dose-dependent(P <0.05);and 100 nM is the best drug concentration of LIRA.Low concentration of LPS(0.1,1,10 ?g / ml)could promote the proliferation of hPDLCs,while high concentration of LPS(100 ?g / ml)inhibited the proliferation of hPDLCs in a time-dependent manner(P <0.05).10 ?g / ml LPS was co-cultured with hPDLCs to simulate the pathogenesis of periodontitis.Furthermore,LIRA attenuated the inhibitory effect of high concentration LPS on the proliferation of hPDLCs(P <0.05).4.Healing assay: LIRA can promote the migration ability of hPDLCs in a time-dependent manner.5.qRT-PCR: inflammatory factors: compared with the control group,LPS could significantly increase the expression of IL-6 and TNF-? mRNA(P <0.01),LIRA group could inhibit the expression of IL-6 and TNF-? mRNA(P <0.05).Compared with LPS group,LPS + LIRA group could significantly decrease the expression of IL-6 and TNF-? mRNA(P <0.05).Osteogenesis factors : compared with the control group,LPS group and LIRA group could increase the expression of ALP and Runx2 mRNA(P <0.05),and compared with LPS group,LPS+LIRA could slightly suppress the expression of ALP and Runx2 mRNA(P <0.05).6.Western Blot: The expression of inflammatory factors was consistent with the results of qRT-PCR.The results of osteogenic factors were slightly different from qRT-PCR: LIRA significantly promoted the expression of ALP and Runx2 protein(P <0.05),while LPS group could inhibit the expression of ALP and Runx2 protein(P <0.05).Compared with LPS group,LPS + LIRA group increased the expression of ALP and Runx2 protein(P <0.05).Conclusions1.The expression of GLP-1R was present in hPDLCs.LIRA promoted the expression of GLP-1R and the proliferative activity and migration ability of h PDLCs.2.LPS can induce the inflammatory response of hPDLCs,and inhibit osteogenic differentiation of hPDLCS.It is suggested that LPS and hPDLCs were co-cultured to simulate the pathological process of periodontitis.3.LIRA can not only inhibit the inflammatory response of hPDLCs,promote the osteogenic differentiation potential of hPDLCs,but also inhibit the LPS-induced inflammatory response and restore the osteogenic differentiation potential of hPDLCs.This indicates that LIRA has a potential therapeutic effect on periodontitis and provides a theoretical basis for LIRA as an adjunct agency to periodontitis. |
PDF Full Text Request