The Effect Of Salidroside On The Expression Of Reactive Oxygen And Species Inflammatory Cytokines In Human Periodontal Ligament Cells Stimulated By Lipopolysaccharide Under Hypoxia

Objective: Periodontitis is a persistent inflammation and immune response caused by pathogens and their metabolic products,especially LPS.It is one of the two major chronic diseases threating human oral health and a major cause of the loosen teeth and teeth loss.The survey of epidemiology shows that the blood in body becomes "sticky、 thick、gathering and clotting",the oxygen concentration in the periodontal tissue becomes more lower,oxidative stress becomes more higher,subgingival bacterial species become more richer,including the G-anaerobic bacteria.These make microenvironment change and host resistance to bacteria or their metabolic products decline in periodontal tissue.In conclusion,plateau periodontal disease is more particular than ordinary periodontitis in pathogenesis,so anti-inflammatory and antioxidant are the important strategies.However,there is no specific treatments for plateau periodontal disease,the mostly treatment is basic treatment with drug therapy or periodontal surgery,which not only have long treatment period、poor prognosis 、 potentially unsafety of synthetic drugs,but also psychological shadow of aggressive treatment to patients.Therefore it is important to find safe and effective drugs for plateau periodontal disease.In recent years,more and more attentions were paied to the medicinal plant rhodiola from scholars.Salidroside(SAL)is a kind of main active ingredient extracted from rhodiola,which has immunorregulation 、anti-fatigue、antidepressant、anti-inflammatory、 antioxidant and other benefits.However,the therapeutic of SAL on plateau periodontal disease is very little.With reference to domestic and foreign research,the periodontal ligament cells were stimulated by Lipopolysaccharide(LPS)to set up a cell model of periodontitis,which was cultured under hypoxia.The purpose of this experiment is examining the expression of intracellular reactive oxygen species(ROS),testing the expression of TNF-a、IL-1βand IL-6 inside or outside the periodontal ligament cells,examining the phosphorylation of NF-κB and IκBa,so we could preliminary study whether SAL have the protection on periodontal ligament cells stimulated by LPS under hypoxia and the related mechanism,providing a new thought for the prevention or treatment of plateau periodontal disease.Method: 1.Modified enzyme digestion tissue adherent explants method cultured human periodontal ligament cells.2.The fifth hPDLCs were cultivated with 0、30、60and 100 ug/ml SAL,the effects of SAL on proliferation of hPDLCs were examined by MTT at the same time of the first day to the sixth day;3.Model establishment and grouping: Then the fifth generation of hPDLCs were randomly divided into 5 groups: DMEM without LPS and SAL was served as control group(A),LPS stimulated group was 10ug/mLLPS+0ug/mLSAL(B),and other three SAL treatment groups : 10ug/mL LPS+30ug/mL SAL(C)、 10ug/ml LPS+60ug/mL SAL(D)、10ug/mL LPS+100ug/mL SAL(E).These groups were cultured in low oxygen incubator,Supernatant and cells were collected for experiments after 6 hours.The level of reactive oxygen species(ROS)in hPDLCs were tested by DCFH-DA,the mRNA and protein level of the TNF-a、IL-1β、IL –6 of hPDLCs were detected by RT-PCR and ELISA,the phosphorylation of NF-κB signaling pathways were detected by Western blot.But there were three groups in Western blot: control group(A)、LPS stimulated group(B)and 10ug/mL LPS+100ug/mL SAL(C).Results: 1.The original periodontal ligament cells could be successfully obtained by modified enzyme digestion tissue adherent explants method in vitro.2.These doses of SAL did not affect the proliferation of the h PDLCs stimulated by LPS under hypoxia(P>0.05).3.DCFH-DA results: Compared with the control group,the expression of intracellular ROS in the h PDLCs stimulated by 10ug/ml LPS significantly increased under hypoxia(P<0.05);compared with the LPS stimulated group,three SALgroups could reduce the expression of intracellular ROS(P<0.05).4.RT-PCR and ELISA results: Compared with the control group,the expression of TNF-a、IL-1β and IL-6 in the hPDLCs stimulated by 10 ug/ml of LPS significantly increased at the level of mRNA and protein(P<0.05);compared with the LPS stimulated group,three SALgroups could reduce the expression of TNF-a、IL-1β and IL-6(P<0.05).5.Western blot results: Compared with the control group,the phosphorylation of NF-κB and IκBa in the hPDLCs stimulated by 10 ug/ml of LPS under hypoxia significantly increased(P<0.05),compared with the LPS stimulated group,100ug/mL SAL treated group could reduce the phosphorylation of NF-κB and IκBa(P<0.05).Conclusion: 1.Herbal source of SAL did not affect the hPDLCs proliferation,which is safe and non-toxic.2.SAL reduced the expression of ROS、TNF-a、IL-1β and IL-6 in the hPDLCs stimulated by 10 ug/ml LPS under hypoxia,the protective mechanism might be that SAL reduced the phosphorylation of NF-κB signaling pathway in the h PDLCs.However,further studies are necessary to investigate the exact mechanisms 3.SAL could be used as a new treatment for plateau periodontal disease.