Effect And Mechanism Of Carbon Monoxide Releasing Molecule 3 On Expression Of Inflammatory Factors In Human Periodontal Ligament Cells Stimulated By LPS And High Glucose

Research background and purpose:Periodontitis is a chronic inflammatory disease initiated with bacterial biofilm,in which multiple factors are also involved.Periodontitis has been reported to be associated with systemic diseases,such as diabetes mellitus.Lipopolysaccharide(LPS),as an important virulence factor of Porphyromonas gingivalis,can bind to TLR2 and TLR4 of cells involved in innate immunity and then activate NF-κB pathway,which triggers the release of inflammatory factors.Advanced glycation end products(AGEs)can bind to RAGE and eventually activate NF-κB.Therefore,for patients with periodontitis and diabetes,TLR2,TLR4 and RAGE can be regarded as potential therapeutic targets.Carbon monoxide releasing molecule 3(CORM-3)is a water-soluble metal carbonyl compound,which can release CO controllably.As CO carrier,CORM-3 has the same regulatory effect as CO,such as improving ischemia-reperfusion injury,anti-inflammatory effect and so on.Our research group reported that CORM-3 down-regulates the expression of TLR2 and TLR4 in human periodontal ligament cells induced by LPS and reduce the release of inflammatory factors.The purpose of this study was to explore the effect and possible mechanism of CORM-3 on the expression of inflammatory factors released by human periodontal ligament cells induced by LPS and high glucose.We also studied the effect of CORM-3 on the inflammatory response in periodontal tissues in experimental periodontitis in diabetic rats.Experimental method:Chapter 1 The effect of CORM-3 on the expression of inflammatory factors released by human periodontal ligament cells stimulated by LPS and high glucose.Human periodontal ligament cells were isolated and cultured by tissue block method,and the effect of CORM-3 on the viability of human periodontal ligament cells was detected by CCK-8 method.The third to sixth generation hPDLCs were randomly divided into four groups:blank control group:without stimulation;LPS group:10 μg/mL LPS stimulation for 24 h;LPS+HG group:high glucose medium,10 μg/mL LPS stimulation for 24 h;LPS+HG+CORM group:high glucose medium,400 μmol/L CORM-3 and 10 μg/mL LPS stimulation for 24 h.IL-1β,IL-6 and IL-8 in each group were detected by ELISA.Chapter 2 The mechanism of CORM-3 in the expression of inflammatory factors released by human periodontal ligament cells stimulated by LPS and high glucose.The experimental groups were the same as before.Expression of RAGE,TLR-2,TLR-4,MyD88,IκBα,p65,p50,P-IκBα,P-p65 and P-p50 was detected by RT-qPCR and Western blot.Silent expression or overexpression of RAGE on the expression of the factors described above was studied.Cells were divided into 5 groups as following.Control group:high glucose medium without transfection,10 μg/mL LPS stimulation for 24 h;NC group:high glucose medium,siRNA NC transfection,10 μg/mL LPS stimulation for 24 h;NC+CORM group:high glucose medium,siRNA NC transfection for 24 hours,treated with 10 μg/mL LPS and 400 μmol/L CORM-3 for 24 hours;Silence group:high glucose medium,RAGE siRNA transfection,10 μg/mL LPS stimulation for 24 h;Silence+CORM group:high glucose medium,RAGE siRNA transfection for 24 hours,treated with 10 μg/mL LPS and 400 μmol/L CORM-3 for 24 hours.RT-qPCR and western blot were used to detect the expression levels of the above related molecules,and immunofluorescence was used to observe NF-κB p65 entry.Then,RAGE overexpression was transfected and cells were randomly divided into 5 groups.Control group:high glucose medium without transfection,10 μg/mL LPS stimulation for 24 h;empty body group:high glucose medium,empty body transfection,10 μg/mL LPS stimulation for 24 h;empty+CORM group:high glucose medium,empty body transfection for 24 hours,treated with 10 μg/mL LPS and 400 μmol/L CORM-3 for 24 hours;overexpression group:high glucose medium,RAGE overexpression transfection,10 μg/mL LPS stimulation for 24 h;overexpression+CORM group:high glucose medium,RAGE overexpression transfection for 24 h,treated with 10 μg/mL LPS and 400 μmol/L CORM-3 for 24 hours.The detection is the same as the silence experiment.Chapter 3 The effect of CORM-3 on periodontal tissue inflammation in experimental periodontitis in diabetic rats.Twenty Wistar male rats were randomly divided into four groups:healthy group as blank control,intraperitoneal injection of normal saline;CP group:experimental periodontitis rat model,intraperitoneal injection of normal saline;DM+CP group:diabetic rats of experimental periodontitis model were injected intraperitoneal saline;CORM+DM+CP group:diabetic rats of experimental periodontitis model,intraperitoneal injection with CORM-3.The induction of experimental diabetes in rats was through injecting intraperitoneally with 30 mg/kg STZ every other day.Three days after the last injection,the tail vein blood was taken for blood glucose detection.The blood glucose was more than 16.65 mmol/L,that was determined as experimental diabetic rats.If the blood glucose concentration did not meet the standard,additional injection would be given.Experimental periodontitis was induced by the ligation of the right maxillary second molars and injecting into the buccal and lingual gingival sulcus for 20 μL P.g solution.Periodontitis models were established in diabetic rats,which are models of experimental periodontitis in diabetic rats.Two weeks after modeling,the models were obtained.The samples were taken for HE staining and Masson staining for the detection of the inflammation in periodontal tissues.RAGE and NF-κB p65 were examined by immunohistochemistry and immunofluorescence.Experimental results:Chapter 1 The effect of CORM-3 on the expression of inflammatory factors released by human periodontal ligament cells stimulated by LPS and high glucose.Within the safe concentration range of CORM-3,the most obvious concentration to inhibit the expression of inflammatory factors was 400 μmol/L,800 μmol/L CORM-3 solution has toxic effect.The expressions of the inflammatory factor IL-1β,IL-6 and IL-8 in hPDLCs stimulated by LPS were significantly higher than those in the control group.With high glucose costimulation with LPS,the expression of these factors was further increased significantly,compared with that of LPS group.In the presence of 400 μmol/L CORM-3,the expression of the inflammatory factors decreased significantly.Chapter 2 The mechanism of CORM-3 in the expression of inflammatory factors released by human periodontal ligament cells stimulated by LPS and high glucose.The expression of TLR2,TLR4,RAGE and NF-κB pathway related molecules MyD88,IκBα,p65,p50,P-IκBα,P-p65 and P-p50 increased significantly in hPDLCs stimulated with LPS.With high glucose costimulation with LPS,these expressions were significantly increased than that of LPS group.Treatment with 400 μmol/L CORM-3,the expression of pathway related molecules in the cells decreased significantly.Silent expression or overexpression of RAGE decreased or increased the expression of the factors mentioned above.While treatment with 400 μmol/L CORM-3 significantly suppressed the expression of the factors.Chapter 3 The effect of CORM-3 on periodontal tissue inflammation in experimental periodontitis in diabetic rats.The expression of RAGE and NF-κB p65 in the periodontal tissues in healthy rats was less than in CP group and DM+CP group.CORM-3 reduced the expression of RAGE and NF-κB p65 in the periodontal tissues of experimental periodontitis in diabetic rats.Conclusion:(1)CORM-3 significantly decreases the expression of inflammatory factors IL-1β,IL-6,and IL-8 in hPDLCs stimulated by LPS and high glucose.(2)CORM-3 significantly down-regulates the expression of TLR2,TLR4 and RAGE in hPDLCs stimulated by LPS and high glucose.CORM-3 inhibits the activation of NF-κB pathway in LPS and high glucose stimulated cells.(3)CORM-3 suppresses the inflammatory response in experimental periodontitis in diabetic rats,and reduces the expression of RAGE and NF-κB p65 in periodontal tissues.