Metformin Moderates Inflammatory Status In Periodontitis By Inhabiting HMGB1 Expression And Releasing

Background: Periodontitis is a chronic inflammatory disease,which affects the periodontal tissues supporting the teeth and accelerates progressive resorption of the alveolar bone.However,current therapeutic methods which involve periodontal basic treatment and the guided tissue regeneration(GTR),were not fully satisfied by the patients and dentists.Metformin is the preferred oral hypoglycemic drug for T2 DM.Recent studies have found that metformin has significant effects on polycystic ovary syndrome,cancer,heart and cardiovascular diseases,nonalcoholic fatty liver disease and puberty precocity.in addition to hypoglycemic effects and has protective effects on anti-inflammation.However,the effect of periodontitis is still unclear.High Mobility Group Protein(HMG)is a non-group DNA bind protein,wildly distributed in the nucleus of higher eukaryotes,and can be divided in HMGA,HMGB,HMGBN families.There are three members of HMGB family,namely HMGB1,HMGB2 and HMGB3,among which HMGB1 is the most abundant.Due to its highly conserved sequence in the evolutionary process,it exists in almost all eukaryotic cells with up to 99%homology,and is the only protein that can be released to play its extracellular biological activities.Human HMGB1 gene exist in the long aim of cliromosome 13 1 zone 2,relative molecular weight of 25 KD,distribution is very extensive,a variety of organs,such as lymphoid tissue,brain,liver,lung,heart,spleen,kidney etc,in addition to mainly exist in the cytoplasm in the liver,brain,in the most organizations exist in nucleus.Under normal physiological condition,the binding of HMGB1 to DNA in the nucleus is loose,but when the cell suffers mechanical damage or necrosis,HMGB1 is easy to be released from the damaged nucleus to the outside of the cell,but when the cell,thus inducing inflammatory response.Because HMGB1 plays an important role in the pathogenesis of inflammatory disease.Objectives: Animal models of experimental periodontitis were established by Ligature in vivo and Lipopolysaccharide(LPS)induced human periodontal membrane cells(h PDLCs)in vitro.To observe the effect of Met on experimental periodontitis and explore its mechanism.At the same time,the expression and function of HMGB1 under the action of inflammatory stimulation and Met were observed.Methods:1.Effects of Met on bone resorption in mice with periodontitis:The experimental periodontitis model was established by placing 6-0silk thread in the maxillary first and second molar space of mice for 9 days.Meanwhile,the rats were treated with 200mg/kg Met by gavage once a day for 14 days,and the mice were sacrificed after 14 days.Micro-ct was used to observe the alveolar bone resorption of the first and second maxillarygrinding teeth in mice.Bone volume fraction(BV/TV)and trabecular thickness(Tb.Th)were measured at the bifurcation of the root of the maxillary first molar.The distance between the cementoenamel junction to the alveolar bone crest(CEJ-ABC)was calculated to monitor the resorption of alveolar bone.2.Effects of Met on inflammatory factors in mouse periodontitis model:After decalcification,dehydration,transparency,waxing,embedding and slicing of the mouse maxilla,the infiltration level of inflammatory cells in the alveolar bone was observed by H&E staining.Immunohistochemistry of il-6 and il-8 was used to observe the anti-inflammatory effect of Met on experimental periodontitis in mice.The expression and localization of HMGB1 under the combined action of inflammation and Met were detected by immunohistochemistry.3.Inflammatory model of h PDLCs induced by LPS in vitro:HPDLCs were treated with LPS of 0,0.5,1,5,10,and 20,respectively,and cell count kit-8(cck-8)was used to detect cell viability.Real-time PCR(rt-pcr)and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression of inflammatory cytokines il-6and il-8,so as to screen out the optimal concentration of LPS that did not significantly inhibit the activity of h PDLCs and significantly increased inflammatory cytokines.4.Expression and localization of HMGB1 in the inflammatory model of h PDLCs:After treating h PDLCs with the same concentration of LPS,real-time rt-pcr and ELISA were used to detect HMGB1 expression and extracellular release of h PDLCs under the action of different concentrations of LPS.The intracellular localization of HMGB1 was determined by Western blot.5.The impact of Met on the inflammatory model of h PDLCs:h PDLCs were treated with Met of 0,50,100,150 and 200,respectively.Cell count kit-8(CCK-8)was used to detect cell viability.HPDLCs were pretreated with different concentrations of Met for24 h,and then treated with the optimal concentration of LPS for 24 h.RT-PCR and ELISA were used to detect the expression changes of IL-6 and IL-8 inflammatory factors.6.HMGB1 and AMPK/NF-?B pathway in the mechanism research of Met in h PDLCs periodontal inflammation model:With the same concentration of Met and LPS h PDLCs,RT-PCR and ELISA to detect the changes of gene expression of HMGB1 and extracellular release quantity;Intracellular localization of HMGB1 was determined by intracellular and extracellular Western blot.Immunofluorescence clear HMGB1 intracellular localization by Western blot experiments to detect AMPK/NF-? B related protein expressionResults:1.In the fibro-induced periodontitis model treated with Met,micro-ct observed a significant decrease in alveolar bone resorption,a significant increase in trabecular bone mass and thickness,and a significant decrease in cej-abc distance.2.After the decalcification,embedding and immunohistochemical staining of the maxillary bone of the mice,the expression of IL-6 and IL-8decreased,the expression of HMGB1 in extracellular and cytoplasmic decreased,and the expression of HMGB1 in nucleus increased in the Met+Ligature group.3.Establish an inflammatory model of LPS-induced h PDLCs.Cell viability was slightly reduced after LPS treatment of h PDLCs.When the LPS concentration was greater than 1?M,the expression of inflammatorycytokines(IL-6,IL-8)increased significantly and increased in a dose-dependent manner with the LPS concentration.4.After LPS treatment with h PDLCs,the results of RT-PCR,ELISA and Western blot showed that LPS could promote the expression,nucleosomal transport and release of HMGB1.5.Cells showed no inhibitory effect after 24 h of Met treatment of h PDLCs.After the inflammatory model of h PDLCs induced by LPS with Met pretreatment of 200?M,the expression of pro-inflammatory factors IL-6 and IL-8 decreased,and the content of supernatant decreased.6.After the inflammatory model of h PDLCs induced by LPS pretreatment with Met,the results of RT-PCR,ELISA,Western blot and immunofluorescence showed that Met could reverse the high expression of HMGB1 in the inflammatory process,inhibit nuclear transport to the nucleus and prevent extracellular release.Met on the role of HMGB1 may be associated with AMPK/NF-? B.Conclusions: In conclusion,these results demonstrated that metformin alleviated periodontitis by inhabiting HMGB1 expression,translocation and releasing.Thus,due to favorable prognosis depending on ideal inflammation control,this study provides novel evidence that HMGB1 may be a potential target of intervention in periodontitis,while metformin is an anti-inflammatory drug in periodontitis treatment.