Effect Of Ginsenoside Rg1 On Biobehavior Of Human Periodontal Ligament Cells And Expression Of Serum Cytokine In Periodontal Tissues In Rats

Objectives:1.To study the primary culture of human periodontal ligament fibroblasts(HPDLFs) by covering coverslip and to investigate the influences of ginsenoside Rglon the biobehaviors of HPDLFs in primary culture.2.To establish an experimental periodontitis model in Wistar rats by prednisolone acetate injection and ligature of their maxillary molars with steel-wire.Study the effects of ginsenoside Rglon the expression of IL-6 and BGP in periodontal tissues of rat periodontitis model.Methods:1.Morphological and immunocytochemical methods were used to identify the cell origin and the efficiency of the primary culture by covering coverslip.2.A series of experiments were performed to investigate the effects of ginsenoside Rglon the biobehaviors of HPDLFs in vitro.Cell proliferation was determined by MTT assay,the total protein synthesis was measured by Coumassie Bright Blue method and the alkaline phosphatase(ALP) activities were detected by spectrophotometric assay.3.Cell cycle of HPDLFs were examined by cell culture and flow cytometry(FCM).4.The intracellular calcium levels of HPDLFs were measured by the fluorescent Ca²⁺ indicator Fluo-3 and laser scanning confocal microscopy(LSCM).5.One hundred 160-200g Wistar rats were randomized into ten groups.Group A used as the normal group was killed at the beginning of the experiment.Group B-E received the therapy of ginsenoside Rg1 were killed at 1st,2nd,3rd and 4th weeks.Group F-J were experimental periodontitis models and were killed at 0,1st,2nd,3rd and 4th weeks.The level of BGP and IL-6 in the periodontium were measured with immunohistochemistry after the rats were killed.Result:1.Covering coverslip significantly increased the yield of primarily cultured HPDLFs.2.The effect of ginsenoside Rg1 on the proliferation of HPDLCsCompared with the control group,the proliferative abilities of PDLC were increased when treated with 0.005-1μmol/L of ginsenoside Rg1,cells treated with 0.01-0.05μmol/L were significantly higher than other group(P＜0.05).3.The effect of ginsenoside Rg1 on the total amount of protein of HPDLCsCompared with the control group,the total amount of protein of HPDLCs treated with ginsenoside Rg1 in 0.01μmol/L,0.05μmol/L,0.1μmol/L was increased dramatically for 72 hours(P＜0.05).4.The effect of ginsenoside Rg1 on the alkaline phosphatase(ALP)activity of HPDLCsCompared with the control group,ALP activities of PDLCs treated with 0.01μmol/L,0.05μmol/L, 0.1μmol/L of ginsenoside Rg1 for 72h were enhanced significantly(P＜0.05).5.The effect of ginsenoside Rg1 on intracellular free calcium concentration in human periodontal ligament FibroblastsAfter being treated with Ginsenoside Rg1(0.01μmol/L),intracellular calciumin human periodontal ligament fibroblasts decreased.At last,it came to lower level of resting intracellular calcium.6.The effect of ginsenoside Rg1 on cell cycle of HPDLCsAfter 48 hours of ginsenoside Rg1(0.1μmol/L),the FCMs demonstrated that the G1 phase cells reduce (P＜0.05),the S phase cells and(S+G2/M)%significanctly increased(P＜0.05). 7.The successful establishment of rat periodontitis modelThe rats in experimental and control groups showed bad appetite and retardation in movement after the fourth day of injection.These symptoms were accordance with those of "shen xu" in traditional Chinese medicine.The osteoporotic changes in alveolar bone,the absorption of alveolar ridge,the formation of periodontal pocket.None of above pathological changes occurred in periodontal tissues of the normal group.The levels of IL-6 in the periodontitis tissues were significantly higher and BGP much lower than those in the normal groups.(P＜0.05).8.Effects of the ginsenoside Rg1 on the expressions of serum cytokines in periodontal tissues of rats The levels of BGP in the group treated were significantly higher than that in the group periodontitis.The levels of IL-6 in the group treated were much lower than that in the group periodontitis(P＜0.05),and the general physical conditions of rats were improved.Conclusion:Covering coverslip can significantly increase the yield of primarily cultured HPDLCs.Ginsenoside Rgl can promote cell proliferation and the total amount of proteins and ALP activity of HPDLCs.The number of S stage cells and muhiplieation index S+G₂M%markedly increased byGinsenoside Rg1,Calcium participates in the process of Ginsenoside Rg1 message transduction.Experimental periodontitis model may be successfully established in Wistar rats by ligature with steel-wire plus prednisolone injection,and this animal model offers a good means for the study of periodontitis.Ginsenoside Rg1 can down-regulate the expressions of IL-6,up-regulate the expression of BGP in periodontal tissues of rat periodontitis model, promote the regeneration of the periodontal tissues,our studies suggest that ginsenoside Rg1 can promote wound healing and tissue regeneration,and provide scientific evidence for the rational drug administration of periodontal diseases.