Effect Of Liuweidihuang Wan On The Proliferation And The Expression Of OPG/RANKL Of The Human Periodontal Ligment Fibroblasts Intervened By IL-1β、TNF-α

BackgroundPeriodontal disease refers to diseases only occur in the periodontal supporting tissue (gingiva, the periodontal ligament, alveolar bone and cementum)[1].It is one of the two major oral diseases. In our country, the incidence rate of periodontal disease is higher than caries disease. Along with our country entering an aging society, periodontal disease become a more prominent health care problem.Alveolar bone is an important part of the periodontal support tissues and contributes to maintaining the teeth stable with gingiva, periodontal membrane and teeth cementum. Alveolar bone absorption is one important pathological change of the periodontitis.This could make the loss of the supportive groups even cause the tooth away from the mouth. If the serious influence happened, patient’s chewing function and beautiful appearance could be affected gravely. Therefore, experiment and clinical research aiming at the restriction of bone resorption is deepening.Bone resorption is mainly caused by osteoclast (OC). In recent years, some researches have shown that osteoclast functions are regulated by a series of local factors, such as, IL-6, IL-1,TNF-α, PGE2and lymphotoxin. The bacteria from the dental plaque can release lipopolysaccharide and other products, and these substances stimulate the immune cells and osteoblast of the tissue to release inflammatory mediators.These mediators promote the macrophages and fibroblasts to secret cell factors which induce the formation of osteoclast and cause alveolar bone resorption.In recent years,some researches have found that OPG/RANK/RANKL system is the main factor of osteoclast formation and activation.Various cytokines promoting the activation of bone resorption could be found in the affected site of periodontal disease.Although these factors have synergistic effect on the formation and activation of OC,the key factor is the change of the local expression of RANKL[2]. It’s confirmed that the expression of RANKL increases in the process of alveolar bone resorption, and the ratio of RANKL/OPG fortifies in the patients who are during the active stage of periodontitis[3]. The periodontal ligament cell（PDLCs）is the most abundant and essential in the periodontal ligament. Furthermore it is the main mesenchymal cells in periodontal membrane.It plays an important role in the physiology and pathology changes of periodontal tissue.One of its subgroups owning the osteoblast phenotype could express OPG and RANKL mRNA and regulates the differentiation of osteoclast bidirectionally [4].In the research of drug on inhibiting bone resorption, traditional chinese medicine attracts attention for its lower side effects. Liuweidihuang, represention of enriching yin and nourishing kidney,is favored and admired by past dynasties doctors. It promotes bone growing. According the history,we find that it originates from "Xin kai bu he" written by the song dynasty Qian Yi and it was used to treat non-closure of fontanels in infants originally. Modern researches have proved that this prescription could regulate the metabolism of calcium and phosphate in vivo and promote fracture healing. In recent years,strengthening teeth paste and pill,which are used to treat periodontitis along with alveolar bone absorption as the main pathological change,are based on Liuweidihuang Wan. Otherwise some studies have shown that Liuweidihuang decoction could regulate human body’s immune function through two-way,meantime it could inhibit rats’ abdominal macrophage secreting TNF-αand reduce the level of IL-6in serum after empyrosis.It also have obvious inhibitory effect on swelling of joint from adjuvant arthritis rats[5]. IL-1and PGE2produced in the process of inflammation can influence the expression of RANKL and OPG[6].One argue about whether Liuweidihuang Wan inhibits bone absorption and promote bone formation through lowering the expression of RANKL is not so far seen. If we start from OPG/RANK/RANKL system to research Liuweidihuang’s molecular mechanism of alveolar bone absorption, Liuweidihuang Wan is helpful to explore the essence of the theory of "Guchi".At the same time, it provides experiment basis for treatment of periodontitis by traditional chinese medicine.It will be of great significance on the absorption and prevention of periodontal.Purpose1Study of Liuweidihuang Wan on the proliferation and expression of OPG/RANKL of periodontal ligament fibroblasts.2Study of Liuweidihuang Wan on the proliferation and the expression of OPG/RANKL of periodontal ligament fibroblasts intervened by IL-1β，TNF-α.3Explore the essence of "Bushen Guchi" theory.Method1Enzymatic digestion method was used to cultivate human periodontal ligament fibroblasts in vitro, then the cells were gained by serial subcultivation and identified by immunohistochemistry. Growth curve was drawn as well.2We used MTT assay to detect the proliferation of periodontal ligament fibroblasts after the influence of Liuweidihuang Wan, TNF-αand IL-1β.3We used immunohistochemical method to detect the expression of OPG/RANKL intervened by Liuweidihuang Wan, TNF-αand IL-1β.Result1Primary and subcultured periodontal ligament fibroblasts were gained and proven to derive from mesenchymal cells through immunohistochemistry method and growth curve was drawn.2The results of MTT method showed that when the concentrations of Liuweidihuang Wan water extraction liquid increased from0.001mg/ml to0.1mg/ml, the proliferation of hPDLFs increased along with the concentration of drug, also along with the time (1-4day),but from1to10mg/ml,the value was lower than the control group, the difference had statistically significance (P <0.05);Water extraction liquid+IL-1β group compared with IL-1β group, and the value of water extraction liquid+IL-1β group at each time point was higher than IL-1β group, then the difference was statistically significant (P <0.05); The water extraction liquid+TNF-α group compared with TNF-α group, and the value of water extraction liquid+TNF-α group was higher than the TNF-α group, the difference was statistically significant (P <0.05).3The results of immunohistochemistry showed that when water extraction liquid group compared with normal cells group,the estimated value of OPG in the former group was higher than the latter and RANKL’s value is lower than the normal group, and there was statistically significant difference (P <0.05); water extraction liquid+IL-1β group compared with IL-1β group and the water extraction liquid+TNF-α group compared with the TNF-α group,the estimated value of OPG in the group containing Liuweidihuang Wan was higher than the group without Liuweidihuang Wan and RANKL’s value in the group containing Liuweidihuang Wan was lower than the group without Liuweidihuang Wan,and the difference had statistically significance (P <0.05).Conclusion1Water extraction liquid from Liuweidihuang Wan could stimulate the proliferation of hPDLFs in the range of certain concentrations.2Water extraction liquid from Liuweidihuang Wan can modify IL-1β and TNF-α’ inhibition on hPDLFs proliferation and expression.3Liuweidihuang Wan can reduce inflammatory reaction through inhibiting IL-1β and TNF-α’effect on the periodontal ligament fibroblasts and inhibit alveolar bone absorption by up-regulating the expression of OPG and down-regulating the expression of RANKL.The results illustrate that traditional Chinese medicine may achieve the effect of "Guchi" through inhibiting inflammatory factors’ effect on the OPG/RANK/RANKL system.This study provides important experimental basis for "Bushen Guchi" theory in traditional chinese medicine,also provides a foundation for the treatment of alveolar bone resorption in periodontitis with chinese medicine.