Effects of TDP-43 on mouse gingival fibroblasts stimulated by LPS:An experimental study
Objective:To explore the effects of TARDNA binding protein-43(TDP-43)on mouse gingival fibroblasts(MGFs)in lipopolysaccharide(LPS)-induced inflammatory microenvironment.Methods:The gingival fibroblasts of wild-type C57BL/6J mice were cultured.LPS of different concentrations and treatment time were set,mRNA expression levels of related inflammatory factors were detected by real-time quantitative polymerase chain reaction(RT-qPCR),and the optimal treatment conditions were selected.The cells were stained with immunofluorescence to observe the changes of TDP-43 in MGFs under inflammatory microenvironment.TDP-43 in MGFs was knocked down by small interfering RNA(siRNA)transfection technique,and transfection efficiency was measured.Three experimental groups were set up:Negative control group(NC group),LPS induced control group(NL group)and TDP-43 knockdown+LPS induced group(SL group).RT-qPCR was used to detect the mRNA expression of some inflammatory factors,matrix metalloproteinases(MMPs)and adhesion related factors.Western blotting was used to detect the expression of some inflammatory factors and adhesion related factors.Picro sirius red staining was used to investigate intracellular collagen deposition.Results:The optimal concentration of LPS stimulation was 100 ng/mL for 6 h.In the absence of LPS stimulation,TDP-43 in MGFs was basically in the nucleus,and it appears in the nucleus and the cytoplasm after LPS stimulation.The cell model with TDP-43 knockdown was successfully constructed by siRNA transfection with MGFs,and the transfection efficiency was nearly 50％.Compared with the control group(NL group),the mRNA expressions of inflammatory factors,MMPs and adhesion-related factors were significantly down-regulated in the TDP-43 down-knocked experimental group(SL group)under LPS stimulation(P＜0.05).The protein expressions of some adhesion related factors and inflammatory factors were also significantly down-regulated.Collagen deposition in SL group and NL group was decreased compared with NC group(P＜0.05),but there was no striking difference between the NL group and SL group(P＞0.05).Conclusion:When MGFs were in the inflammatory microenvironment induced by LPS,TDP-43 showed a tendency to move from inside the nucleus to outside.TDP-43 positively regulates the expression of inflammatory factors,adhesion related factors and MMPs in the LPS-induced inflammatory microenvironment of MGFs,but has no significant effect on collagen deposition.
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