4’-Methoxyresveratrol Inhibits The Inflammatory Response Of Human Gingival Fibroblasts Stimulated By LPS With High Concentration Glucose And Its Mechanisms

Research background and purpose:Periodontitis,as a chronic inflammatory and destructive disease,is caused by many factors such as microbial,host,environmental and genetic factors.Periodontitis can cause loss of attachment,resorption of alveolar bone,and even loosening and loss of teeth.In human gingival connective tissue,gingival fibroblasts account for 60%～70%of the total tissue cells.They are involved in regulating the synthesis and degradation reaction of cell matrix and fiber tissue,and maintain the homeostasis of periodontal tissue.They are an important cell type in periodontal tissue.lipopolysaccharide(LPS)is the main component of the cell wall of Porphyromonas gingivalis(P.gingivalis).When cells are stimulated by LPS,nuclear factor kappa-B(NF-κB)signaling pathway can be activated,and a large number of inflammatory factors such as IL-1β and IL-6 can be synthesized and secreted to promote periodontal inflammation.Diabetes is a systemic disease of metabolic disorder,in which a variety of cells in the body are in an inflammatory state,leading to various complications of microvessels and macrovessels in the body.Many studies have reported the close relationship between diabetes and periodontal inflammation,and diabetes has been proved to be one of the main risk factors for periodontitis.Periodontal disease is also considered to be one of the "classic"complications of diabetes.4’-Methoxyresveratrol(4’-MR)is a polyphenol compound,which is a plant stilbene isolated from pteropteris and argyliaceae plants.Studies have shown that 4’-MR has significant anti-inflammatory and antioxidant activities.It has been suggested that the anti-inflammatory effect of 4’-MR may be achieved by inhibiting rage-mediated MAPK/NF-κB signaling pathway and NLRP3 inflammasome activation.In this study,human gingival fibroblasts were used as an in vitro experimental model to investigate the effect of 4’-MR on the expression of inflammatory factors in human gingival fibroblasts stimulated by LPS with high concentration glucose and explore its possible mechanism.Experimental diabetic periodontitis rats were used as experimental models to study the effects of 4’-MR on the inflammatory response of periodontal tissue in rats,in order to provide experimental basis and therapeutic strategies for the treatment of periodontitis,especially for the treatment of diabetes with periodontitis.Experimental method:Part 1 The effect of 4’-MR on the expression of inflammatory factors in human gingival fibroblasts stimulated by LPS with high concentration glucoseHuman gingival fibroblasts were isolated and cultured by tissue block method,and the 3～6 generations of HGFs were randomly grouped to detect the effects of 10、20、30、40、50μg/mL LPS and 10、20、30、40、50 μg/mL 4’-MR on cell proliferation by Cell counting kit-8(CCK-8)method.The effect of LPS on cell proliferation after 4’-MR Stimulation in high glucose culture was examined.The expression of inflammatory factor IL-1β was detected by ELISA under different concentrations of LPS,and the working concentration of LPS in subsequent experiments was determined.In order to detect the effect of 4’-MR on the expression of inflammatory factors in human gingival fibroblasts stimulated by LPS with high concentration glucose,the cells were divided into the following groups:Control group:normal medium without stimulation;LPS+HG group:high glucose culture medium,adding 10 μg/mL LPS for 24 h;LPS+HG+4’-MR group:high glucose culture medium was added with 10 μg/mL LPS and 10 μg/mL 4’-MR solution for 24 h.The expression of inflammatory factors IL-1β and IL-6 were detected by qRT-PCR and ELISA.Part 2 The mechanism of 4’-MR on the expression of inflammatory factors in human gingival fibroblasts stimulated by LPS with high concentration glucoseTo further investigate the effects of 4’-MR on the expression of TLR4 and NF-κB pathway related factors in human gingival fibroblasts stimulated by LPS with high concentration glucose.The 3rd to 6th generation cells were selected and randomly divided into the following groups:Control group:normal culture medium without stimulation;LPS+HG group:high concentration glucose culture medium,adding 10 μg/mL LPS for 24 h;LPS+HG+4’-MR group:high concentration glucose culture medium was added with 10μg/mL LPS and 10μg/mL 4’-MR solution for 24 h.The mRNA and protein expression levels of TLR4,p65,p50 and IκBα,and the expression levels of phosphorylated proteins P-p65,P-p50 and P-IκBα were detected by qRT-PCR and Western-Blot.Part 3 The effect of 4’-MR on the inflammation of periodontal tissues and blood glusoe in experimental diabetic periodontitis ratsTwenty male Wistar rats were randomly divided into 4 groups:healthy group was given normal saline intragastric administration as blank control;CP group:experimental periodontitis rat model,normal saline gavage;DM+CP group:diabetic rats with experimental periodontitis were given normal saline intragastric administration;4’-MR+DM+CP group:diabetic rats with experimental periodontitis were given 4’-MR solution by intragastric administration.Rats with experimental diabetes were stimulated by intraperitoneal injection of 30 mg/kg STZ solution.once every other day,three times in total.The tail venous blood was taken 3 days after the last injection for blood glucose measurement.The rats with blood glucose≥16.7 mmol/L were determined as experimental diabetic rats.If the blood glucose level is not up to standard,additional injection is given.The stimulation of experimental periodontitis was ligation of the right maxillary first molar and injection of 20 μL P.gingivalis bacterial solution into the buccal-lingual gingival groove.The periodontitis model established in diabetic rats is the experimental periodontitis model of diabetic rats.2 weeks after modeling,HE staining was used to observe the periodontal inflammation of rats,and qRT-PCR was used to detect the expression of IL-1β、IL-6 in periodontal tissues.Experiment results:Part 1 The effect of 4’-MR on the expression of inflammatory factors in human gingival fibroblasts stimulated by LPS with high concentration glucoseIn HGFs cultured by tissue block method,a small number of spindle cells could be seen to crawl out of the tissue block about 7～10 days later,and the cells showed high refractive index,no obvious bifurcation and the cell structure could not be distinguished.After cells passage,the cells showed radial growth centered on the tissue block and showed long spindle shape,which had the standard morphology of human gingival fibroblasts.The results of CCK-8 showed that compared with Control group,10～50 μg/mL LPS and 4’-MR of 10 and 20 μg/mL had no obvious inhibition on cell proliferation.After the use of high glucose culture medium,the cell proliferation activity was not significantly inhibited,so the lowest concentration of 4’-MR(10 μg/mL)was selected as the working concentration for subsequent experiments.ELISA results showed that LPS 10 μg/mL could significantly enhance IL-1β expression at both 24 h and 48 h,so 10 μg/mL was selected as the working concentration of LPS in subsequent experiments.ELISA and qRT-PCR results showed that compared with the Control group,the expressions of inflammatory factors IL-6 and IL-1β were significantly increased after LPS stimulation,and the expressions were further increased after high concentration glucose culture medium was used compared with LPS group.4’-MR Significantly reduced the expression of the above inflammatory factors.Part 2 The mechanism of 4’-MR on the expression of inflammatory factors in human gingival fibroblasts stimulated by LPS with high concentration glucoseThe results of qRT-PCR showed that compared with the Control group,LPS significantly increased the mRNA expression levels of NF-κB pathway related factors p65 and TLR4 at 24 and 48 h after stimulation,and the expression levels were further significantly increased compared with the LPS group after the use of high concentration glucose culture medium.4’-MR can significantly reduce the mRNA expression of factors related to NF-κB pathway after LPS stimulation in high glucose culture.The addition of different concentrations of 4’-MR had different effects on the increase of TLR4 mRNA level in LPS-stimulated cells under high concentration glucose culture.5 μg/mL 4’-MR treatment showed significant inhibition,while 10 and 15 μg/mL 4’-MR treatment had no inhibitory effect on the up-regulation of TLR4 mRNA expression.Western-Blot assay showed that,compared with the Control group,LPS induced HGFs to up-regulate the protein expression of NF-κB pathway related factors p65,IκBa,P-p65,P-p50,P-IκBa and TLR4.4’-MR can significantly reduce the protein expression of NF-κB pathway related factors,but has no inhibitory effect on TLR4 protein expression.Part 3 The effect of 4’-MR on the inflammation of periodontal tissues and blood glusoe in experimental diabetic periodontitis ratsHE staining showed that compared with the healthy group,the CP and CP+DM group rats had obviously inflammatory histological manifestations.The inflammatory manifestations of 4’-MR+DM+CP group rats were reduced compared with CP and CP+DM group rats.qRT-PCR results showed that the expression level of inflammatory factor IL-1β、IL-6 mRNA in periodontal tissues of CP、CP+DM group was significantly up-regulated compared with that of healthy group,and 4’-MR could inhibit the expression of inflammatory factor IL-1β IL-6 mRNA in periodontal tissues.Conclusion:(1)LPS significantly enhanced the expression and release of HGFs inflammatory factors IL-1β and IL-6,and LPS stimulated the expression of inflammatory factors further significantly under the high glucose environment.10 μg/mL 4’-MR significantly decreased the expression of inflammatory factors IL-1β and IL-6 in HGFs stimulated by high glucose culture.(2)10 μg/mL 4’-MR can significantly inhibit the activation of p65,p50,IκBa,P-p65,P-p50,P-IκBa of HGFs NF-κB pathway under high glucose culture stimulated by LPS,which may not be mediated by TLR4.(3)4’-MR can significantly reduce inflammatory response of periodontal tissues in experimental diabetic periodontitis rats,and reduce IL-1β、IL-6 mRNA expression in periodontal tissues,indicating potential value in the treatment of diabetes and periodontitis.