| Periodontitis is one of the most common health issues all over the world,which can lead to a series of symptoms such as halitosis,gingival pain and swelling,gingival recession,as well as tooth loss.Apart from severely impacting the quality of human life,periodontitis is also an important risk factor related to many systemic diseases.As we known,the dysbiosis between periodontal pathogens and host immune responses leads to the disease occurrence,and both of the pathogen and host response play critical roles in the tissue destruction.However,the current clinical therapy for periodontitis has focused on the mechanical removal of plaques and antimicrobial medications,but has been lack of the host-modulated treatment.Besides,antibiotics and surfactants,which have been used commonly in the medication of periodontitis,exert several side effects such as leading to bacterial resistance,irritation to mucosa,and taste disorder.In recent years,attentions have been widely attached to natural herbs,which have high security and abundant biological activities.Ginseng is a traditional Chinese herb which has a medicinal history for thousands of years,and the ginsenosides are the main active ingredient derived from ginseng.Based on previous sutdies,anti-inflammatory and antibacterial effects of ginsenosides have been demonstrated in the stomatology field,and we have found that many active monomers belong to the protopanaxadiol type.Therefore,antibacterial effects of 4 protopanaxadiol ginsenosides were investigated in the preliminary assay,and ginsenoside Rd(GSRd)showed best efficiency.Then we further investigated inhibitory effects of GSRd on P.gingivalis,periodontal inflammation and bone resorption in vitro and in vivo,in order to assess the applicable potential of GSRd in periodontitis.Part 1 Antibacterial effects of GSRd in vitroObjective:P.gingivalis is the dominant pathogenic bacterium of periodontitis,which has the ability to invade host cells and destroy the epithelial barrier,and can form biofilms to obtain better vitality and virulence.This study aims to explore the antibacterial and anti-biofilm effects of GSRd on P.gingivalis in vitro.Methods:The antibacterial effects of GSRd on planktonic P.gingivalis were detected by the half dilution method,plate counting method and scanning electron microscope.The effects on virulent factors of P.gingivalis were detected by qRT-PCR and the MATH method.The anti-biofilm effects of GSRd on P.gingivalis biofilm were detected by the crystal violet assay and confocal scanning microscope.Results:The minimum inhibitory concentration of GSRd on P.gingivalis was 400 μM,while lower GSRd concentrations of 200 and 100 μM significantly inhibited bacterial growth,expression of virulent genes,and cell surface hydrophobicity,as well as destroyed the morphology of P.gingivalis.On the other hand,200 and 100 μM GSRd also showed well inhibitory effects on the biomass and quantity of dead bacteria in P.gingivalis biofilms.Conclusion:GSRd exerts excellent antibacterial and anti-biofilm effects on P.guingivalis.Part 2 Anti-inflammatory and anti-osteoclastogenesis effects of GSRd in vitroObjective:Under bacterial stimulation,the inflammatory cytokines produced by host cells also play an indispensable role in the destruction of periodontal tissues.Meanwhile,some inflammatory cytokines also serve as promoting factors towards osteoclastogenesis,which mediates the bone resorption subsequently.This study aims to investigate inhibitory effects of GSRd on inflammatory responses and osteoclastogenesis in vitro.Methods:The in vitro inflammation model was established in human gingival fibroblasts(HGFs)using P.gingivalis-originated lipopolysaccharide(LPS).Then gene expressions and extracellular secret levels of inflammatory cytokines(IL-1β,IL-6 and IL-8)were detected by qRT-PCR and ELISA.The osteoclastogenesis was induced by RANKL in osteoclast precursor cells(RAW264.7)and BMDMs respectively,and TRAP staining was performed to observe the quantity and volume of osteoclasts,while expressions of osteoclast related genes were detected by qRT-PCR.Results:100 μM GSRd significantly down-regulated gene expressions of IL-1β,IL-6 and IL-8,as well as reduced the extracellular secret levels in HGFs.On the other hand,100 μM GSRd effectively inhibited the osteoclastogenesis both in RAW264.7 and BMDMs,and also down-regulated expressions of osteoclast related genes.Conclusion:GSRd exerted good anti-inflammatory efficiency,while the inhibitory effects on osteoclastogenesis were satisfying as well.Part 3 Effects of GSRd on experimental periodontitis in a mice modelObjective:To further verify the biological activity of GSRd in vivo,this study conducted a animal experiment to explore the treatment effects of GSRd on experimental periodontitis.Methods:The experimental periodontitis of mice was established by ligaturing the maxillary second molars with sterile sutures,and P.gingivalis was inoculated by spreading.Next,the GSRd treated group was given local injection of GSRd every other day,while sterile PBS was instead of drugs in the control group.After 8-day modeling,gingival crevicular fluid samples were collected for bacteria counting using the plate spreading method,and the relative quantity of P.gingivalis was detected by qRT-PCR.What’s more,maxillary bone samples were prepared for methylene blue staining,micro-CT scanning and tissue sections,in order to evaluate levels of periodontal inflammation and bone resorption.Results:Firstly,both the photographs and CT reconstructed images showed that GSRd treatments alleviated the alveolar bone resorption of periodontitis mice.Secondly,the total amount of bacteria and the relative quantity of P.gingivalis in mice GCFs showed significantly decreased in the GSRd-treated group,compared with the periodontitis group.On the other hand,the immunohistochemical staining revealed that GSRd effectively reduced the infiltration of inflammatory cytokines in periodontal tissues.What’s more,the TRAP staining showed that the number of osteoclasts in the GSRd-treated group was less than the periodontitis group,although there has no significant difference between groups.Conclusion:GSRd showed integrated inhibitory efficiency towards P.gingivalis,periodontal inflammation and alveolar bone resorption in mice experimental periodontitis,which supported and verified the previous results. |
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