| Background and objectiveChronic periodontitis（CP）is a pathogenic microorganisms-Induced chronic inflammatory disease predominantly presenting as gingival inflammatory response,inflammatory response in gingiva,alveolar bone resorption and periodontal ligament destruction as the main clinical manifestation of Chronic periodontitis.Microbial plaque and their metabolites are major pathogenic factors for Chronic periodontitis during the pathogenesis of periodontitis.Inflammatory response in periodontal tissues by microbial plaque and their metabolites is also a significant factor in periodontal tissue injury.Periodontal tissues release massive inflammatory factors upon stimulation of microbial plaque and their metabolites,which exacerbates the severity of periodontal tissues destruction.At present,in addition to the physical approach to dental splines,it is necessary to use local or systemic auxiliary drugs to inhibit inflammatory response.Early scholars screened for the treatment of antibiotics such as penicillin,large cyclo-ester,etc.,and scholars have also used non-steroidal anti-inflammatory drugs and tetracycline drugs to regulate the inflammatory response of the host to treat the periodontal cycle.Inflammation,but the efficacy of these drugs is general,and long-term use can cause side effects such as human flora tolerance,and thus is limited in clinical use.At present,Chinese herbal medicine has attracted many scholars due to the advantages of adjustable body immunity and small side effects,wide resource distribution,low cost,etc.In these Chinese herbal extracts,some anti-inflammatory properties can effectively inhibit IL-1β and IL-6 and other inflammatory factors.Extraction of the effective ingredients of inhibiting inflammation from Chinese herbal medicine have become one of the new ways to study the treatment of tooth pass.Oroxylin A is a naturally bioactive flavonoid compound,molecular formula C16H12O5,molecular weight 284.2635.Oroxylin A can interfere with multiple molecular targets during the inflammatory response,which makes it in the treatment of multiple inflammatory diseases such as chronic gastroenteritis,prostatitis.Its antiinflammatory activity is inconsistent with steroidal anti-inflammatory drugs,but there is no side effects of these drugs.So does Oroxylin A have inhibitory effect on periodontitis?Is there any adjustment of inflammatory response to the inflammatory fibroblast（HGF）under inflammation stimulation?What is the mechanism of action?Focusing on these problems,this topic has studied from both in vivo and in vitro.The first chapter establishes model of Periodontitis for rats with wire ligating and neck,and is partially injected with Oroxylin A for treatment.The role of the inflammatory response of Oroxylin A on the model of rat teeth.The second three chapters were preprocessed by establishing TNF-α-induced gum fibroblast model,and the Oroxylin A was prepared,and the inflammatory response of Oroxylin A on the inflammation of inflammatory cells was adjusted.Preliminary discussion on its mechanism.Materials and Methods1.The rat periodontitis model is constructed by a wire ligation rat teeth,and the gums are partially injected on the gums,and the changes in teeth circumference of each group are observed,and IL-1β is used to use immunohistochemistry.The expression and semi-quantitative analysis of IL-6 and COX2 in periodontal tissue were used to detect the gene levels and protein expression levels of rats IL-1β,IL-6 and COX2 of each group of rats and protein expression.2.Initial culture HGF The inhibitory effect of Oroxylin A was influencing apoptosis under inflammation of inflammation,and the RT-qPCR and Western-blot method were detected by inflammatory factor IL-1β,IL-6 and COX2 gene levels and protein expression levels.3.The HGF is divided into three groups of blank groups（Model）and Oroxylin A,using RNA-Seq technology to sequencing the three sets of genes,find different genes,enrichment,and further explore the mechanism of action of Oroxylin A on HGF Inflammatory Model.4.In vitro of HGF differential genes,further verification of gene and protein levels,using gene silencing and overexpression techniques,further study the mechanism of action of inflammatory cell models on the model of inflammatory cell model by gene silencing and overexpression technology.Results1.The stagnation of the rats in the rats in the rats,the gums are retired,and the degree of gingival is reduced,and the degree of gingival retreat is reduced.The immunohistochemical results showed that IL-1β,IL-6 and COX2 have varying degrees of expression in different groups of rats,and weak positive expression in the teeth of the model group,in the gum skin in the experimental group.Strong positive expression,the degree of positive expression of IL-1β,IL-6 and COX2 in the periodontitis tissue of Oroxylin A was reduced.Western-blot shows that the level of IL-1β,IL-6 and COX2 protein in Oroxylin A group is significantly reduced by the periodontitis group（p<0.05）.2.CCK-8 results show that there is no statistical difference between 24 h and 48 h cells in group A group（p>0.05）,but there is a significant difference in absorbance after experiments 3,4 and 5d（p<0.05）;400 mmol/L The concentration is the most obvious in day 4.After the inflammatory induction of TNF-α,RT-qPCR results showed that IL-1β,IL-6 and COX2 expressed significantly（p<0.05）,suggesting that inflammatory induction model was successfully constructed.Flow cytometry detection results show that Oroxylin A pretreatment can significantly reduce HGF cell apoptosis（p<0.05）.Elisa results show that the protein expression of IL-1β,IL-6 and COX2 of the experimental group was significantly lower than that of the periodontitis group（p<0.05）.RT-qPCR and Western-blot results showed that IL-1β,IL-6 and COX2 mRNA and protein expression of the experimental group were significantly reduced by the periodontitis（p<0.05）.3.Transcription group sequencing results show that Oroxylin A is associated with TNF-α-induced HGF inflammatory reaction inhibitors,which may be related to different genes in the MAPK signaling pathway,and related to DUSP5.4.DUSP5 decreases in the expression of TNF-α-induced gum fibroblasts treated with Oroxylin A.After knocking down DUSP5,the effect of Oroxylin A on the inflammatory cell model is lowered;after expressing DUSP5,the effect of Oroxylin A on the inflammatory cell model is lowered.Conclusions1.Oroxylin A can significantly inhibit inflammatory response in a rat model of TNF-α-Induced periodontitis.2.Oroxylin A can inhibit TNF-α-induced IL-1β,IL-6 and COX2 production in HCG.3.The inhibitory effect of inflammation Oroxylin A-induced is mediated by MAPK signaling pathway.4.DUSP5 is a target for OroxylinA-induced inhibitory effect of inflammation in HGF.Innovations1.The role of OroxylinA in oral field is a less explored area of research.This study confirms that Oroxylin A can inhibit TNF-α-Induced IL-1β,IL-6 and COX2 production in HCG.2.Regulatory network of microRNAs and genes on Chronic periodontitis is complex.The study makes a research about the role of Oroxylin A in inhibiting TNFα-Induced IL-1β,IL-6 and COX2 production in HCG based on RNA-Seq technology.3.The study gives the first presentation that DUSP5 is regulated by OroxylinA,and DUSP5 plays a significant role in TNF-α-induced chronic periodontitis,which offers a new direction for treating Chronic periodontitis. |
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