Effects Of Puerarin On Periodontitis As A Host Modulatory Agent

Periodontitis occurs as a consequence of the host immune response to periodontal pathogens, mainly involving periodontal support tissues, leading to progressive alveolar bone loss and connective tissue damage, resulting in the loss of teeth. Bacteria in dental plaque is the initial factor, and its role in periodontitis has been widely recognized and accepted. In recent years, with a better understanding of the pathogenesis of periodontitis, the role of host factors has attracted more and more attention.Periodontal pathogens and their virulence factors stimulate host responses, which is the determinant for the outcome of the disease. In persons who are not susceptible to periodontitis, defense mechanisms including recruitment of neutrophils, production of protective antibodies, and release of cytokines control the infection, disease is limited in gingiva and is considered reversible. However, in susceptible individuals or when persistent bacterial challenge exists, perpetuation inflammation can extend into deeper connective tissues and alveolar bone, resulting in irreversible tissue damage, that is periodontitis.Host modulatory therapies (HMT) is a new term in dental therapy. The purpose of HMT is to modulate host immune response and to restore the balance between pro-inflammatory or destructive mediators and anti-inflammatory or protective mediators. Recent studies have demonstrated that HMT brought significant adjunctive benefits to scaling and root planning in the treatment of chronic periodontitis. Combination of convential antimicrobial therapy and adjuctive HMT may be a new choice for periodontitis patients, especially in susceptible patients who do not respond adequately to conventional therapeutic approaches. Three categories of host-modulating agents have been investigated in the periodontal therapy. The first one is anti-inflammatory drugs which inhibit the production of proinflammatory or destructive mediators to reduce periodontal destructions. The second one is bone-sparing drugs, which reduce alveolar bone loss by improving bone metabolism and interfering with osteoclasts function to. The third one is antiproteinases, which target MMPs to decrease connective tissue damage.Puerarin (C21H20O9) is the major isoflavone in kudzu root, has been recently demonstrated to possess all the three bioactivities, including anti-inflammatory, bone-sparing and antiproteinase properties. We thus hypothesized that puerarin is a promising host modulatory agent for periodontitis. In this study, we first verified the effect of puerarin on alveolar bone loss and collagen destruction, which are the major hallmarks of periodontitis, using rat models with ligature-induced periodontitis. Then the mechanisms underlying such effects were assessed through three aspects, including anti-inflammatory, bone-sparing, and antiproteinase properties of puerarin.Part Ⅰ Effects of puerarin as a host modulatory agent on experimental periodontitis in ratsObjective:To evaluate the effects of puerarin on alveolar bone loss and collagen destruction, which are the major hallmarks of periodontitis, using rat models with ligature-induced periodontitis.Materials and methods:Forty male SD rats were randomly divided into five groups: negative control group (CON), ligature group with vehicle (L), ligature groups treated with puerarin at 100 mg/kg (PR100),200 mg/kg (PR200) and 400 mg/kg (PR400). Puerarin was administrated daily by gavage at three doses, and only the vehicle (0.5% carboxymethylcellulose) was administered to the CON and L groups. Rat models of periodontitis were developed by bilaterally placing ligatures around the first mandibular molars. Rats were humanely killed 7 d after the induction of periodontitis. Micro-CT and sirius red staining were used to evaluate alveolar bone loss and collagen destruction, respectively. Histomorphometrical analysis was used to assess the inflammatory cell infiltration.Results:Puerarin at doses of 200 and 400 mg/kg significantly reduced the alveolar bone loss compared with the L group. Collagen destruction and inflammatory cell infiltration were significantly less in the puerarin-treated group (200 mg/kg) compared with that of the L group.Conclusions:Puerarin reduced the alveolar bone loss, collagen destruction and inflammatory infiltration in rats with ligature-induced periodontitis, thus can be considered a promising agent for adjunctive therapy of periodontitis.Part Ⅱ Mechanism underlying the effects of puerarin on periodontitis as a host modulatory agentExperiment one Effects of puerarin on MMPs in periodontal tissuesObjective:To evaluate the effects of puerarin on MMP-2 and MMP-9 in periodontal tissues of rats with ligature-induced periodontitis and the underlying mechanism.Materials and methods:Protein was gained from the two millimeter-wide gingival tissues below the gingival margin surround the first molars of the rats in CON, L, PR200 groups from experiment one. The production of MMP-2 and MMP-9, and the glycosylation of EMMPRIN were assessed by western blot.Results:EMMPRIN was detected at two molecular masses by western blot, representing high-glycosylated (HG)-EMMPRIN and lowglycosylated (LG)-EMMPRIN, respectively. The ratio of total EMMPRIN to GAPDH was not significantly different among the three groups, whereas the HG/LG-EMMPRIN ratio was significantly increased in the L group compared with the CON group, which was lower in the puerarin-treated group than in the L group. In accordance with the changes of HG/LGEMMPRIN among the three groups, the MMP-2 and MMP-9 levels increased in the vehicle group, and such an increase was inhibited by puerarinConclusions:Puerarin reduced MMP-2 and MMP-9 in periodontal tissues of rats with ligature-induced periodontitis by inhibiting the glycosylation of EMMPRIN.Experiment two The anti-inflammatory effects of puerarinObjective:To test the anti-inflammatory effects of puerarin in the pathogenesis of periodontitis.Materials and method:In vitro, porphyromonas gingivalis lipopolysaccharide (Pg-LPS) stimulated RAW264.7 cells were treated with various concentrations of puerarin (25μM,50μM, 100μM,200μM), the effects of puerarin on TNF-α and IL-1β Mrna were tested using realtime-PCR. Furthermore, the influences of puerarin on MAPK and NF-κB pathways in Pg-LPS stimulated RAW264.7 cells were tested using western blot. Then the effects of puerarin on TNF-α and IL-1β were verified in vivo. Briefly, protein was gained from the two millimeter-wide gingival tissues below the gingival margin surround the first molars of the rats in CON, L, PR200 groups from experiment one, and TNF-α、IL-1β levels were tested by western blot.Results:Puerarin significantly inhibited TNF-α、IL-1β mRNA production in Pg-LPS stimulated RAW264.7 cells in dose-dependent manner. Moreover, puerarin reduced the phosphorylation of p38 MAPK, JNK MAPK and NF-κB p65 in Pg-LPS stimulated RAW264.7 cells. In vivo, puerarin treated group presented significantly less TNF-α、IL-1β compared with the L group.Conclusions:Puerarin inhibited TNF-α、IL-1β production by suppression of NF-κB activation and blockade of MAPK signal pathway. Puerarin might be a good anti-inflammatory agent for periodontitis.Experiment three Bone sparing effects of puerarin in periodontitisObjective:To evaluate the possible effects of puerarin on bone metabolism in periodontitis.Materials and methods:In vitro, after treated with puerarin (25μM,50μM, 100μM, 200μM) for 1-4 days, RANKL and OPG in human periodontal ligament cells (PDLCs) were examined on transcription levels using realtime-PCR. In vivo, sections from the CON, L, PR200 groups in experiment one were selected, and immunohistochemistry and tartrate-resistant acid phosphatase were used to detect RANKL and OPG expressions, and osteoclast activity in the gingiva and alveolar bone.Results:In vitro, RANKL mRNA was not detected in any groups. Puerarin did not change OPG mRNA in PDLCs on first day; and on the second and third day, puerarin increased OPG mRNA in dose-dependent manner; on the fourth day, puerarin reduced OPG mRNA in PDLCs. In. vivo, puerarin reduced RANKL and OPG in periodontal tissues, leading to a significantly lower ratio of RANKL/OPG and a significantly lower number of active osteoclasts than the L group.Conclusions:Puerarin could regulate the expressions of RANKL/OPG and the activity of osteoclast in periodontitis, thus reduced alveolar bone loss.In conclusion, we verified that puerarin could decrease bone loss and collagen destruction in rats with ligature-induced periodontitis in Part Ⅰ, and postulated that puerarin was a promising host modulatory agent for periodontitis. In Part Ⅱ, we tested the host modulatory mechanism of puerarin involved in periodontitis through three aspects, including antiproteinase, anti-inflammatory and bone sparing effects. We verified that puerarin reduced MMP-2 and MMP-9 production via inhibiting the glycosylation of EMMPRIN, and that puerarin inhibited osteoclast formation by reducing the ratio of RANKL to OPG, and that puerarin reduced TNF-α、IL-1β production through blocking MAPK and NF-κB pathways.