Study On The Effect Of PD On The Proliferation And Osteogenic Activity Of Human Inflammatory Periodontal Membrane Cells

Objective: Periodontitis leads to periodontal tissue defect and subsequent repair and regeneration is still a difficult problem in clinical work.At present,the treatment methods of periodontitis mainly include periodontal basic treatment,surgical debridement and antibiotic drugs.Among them,antibiotic use will produce drug resistance and efficacy uncertainty,long-term use can lead to oral microflora disorder.Moreover,it is very difficult to regenerate and repair periodontal defects under the special oral inflammatory microenvironment.Traditional Chinese medicine（TCM）has multiple biological effects such as low toxicity,high efficiency,high drug resistance,anti-inflammatory,immune-promoting and anti-tumor,and has been widely used in clinical treatment.Peucedanum praeruptorum Dunn（PD）is a pinocoumarin compound isolated from eriothecum.It has been previously reported that it has significant anti-inflammatory and antitumor activities.Therefore,this study intends to investigate the effects of PD on the proliferation and osteogenic activity of periodontal membrane cells in patients with periodontitis,so as to provide a low-toxicity and efficient strategy for clinical drug treatment of periodontitis.Methods:First,Primary culture and identification of human periodontal membrane cells:1.Healthy periodontal membrane cells（H-PDLCs）were extracted from the affected teeth of healthy patients（orthodontic reduction/maxillary wisdom teeth）and inflammatory periodontal membrane cells（P-PDLCs）were extracted from the affected teeth of periodontitis patients（severe alveolar bone absorption /III° loosening）for primary culture.2.The sources of H-PDLCs and P-PDLCs were identified by immunofluorescence.3.Alizarin red S（ARS）staining was used to identify the osteogenic differentiation ability of H-PDLCs and P-PDLCs.4.The identified cells were divided into healthy cells group（negative control group-H）,inflammatory cells group（inflammatory group-P）,PD group with different concentrations（experimental group）and minocycline hydrochloride group（positive control group-YS）according to experimental requirements.CCK-8 was used to detect the cell proliferation of healthy cells and inflammatory cells at 1,2,3 and 5 days,and minocycline hydrochloride（100ug/ml）was used as positive control.Effects of PD concentrations of 10ug/m L,20ug/ml,30ug/ml and 40ug/m L on the toxicity and proliferation of P-PDLCs.Second,Evaluation of anti-inflammation and osteogenic differentiation ability of PD on P-PDLCs:1.The expression levels of inflammation-related factors TNF-ɑ and IL-1β in each experimental group were detected by ELISA.2.Real-time polymerase chain reaction（RT-q PCR）was used to detect the m RNA expression of inflammatory factors（TNF-ɑ and IL-1β）and osteogenic factors（RUNX-2,OCN and ALP）in each experimental group.3.Alizarin red S（ARS）staining were used to analyze the effect of PD on osteogenic differentiation induced by P-PDLCs.4.The cytoskeleton changes of P-PDLCs under PD were observed by immunofluorescence.5.Western blotting（WB）was used to detect the expression of osteogenic associated protein RUNX-2 and OCN in P-PDLCs under PD.Result:Human periodontal membrane cells from healthy patients and patients with periodontitis were mainly in long spindle shape,similar to fibroblasts.After culture for a period of time（21 days）,microscopic observation showed that P-PDLCs were longer and larger than H-PDLCs.The growth rate of P-PDLCs was slower than that of H-PDLCs in primary culture period.After passage,the growth rate of P-PDLCs was faster than that of H-PDLCs.Immunohistochemical results showed that vimentin was positive and keratin was negative in the two kinds of cells,indicating that the two kinds of cells were derived from mesenchymal tissue and periodontal tissue.Alizarine red S（ARS）staining showed small orange-red calcified nodules of different sizes,indicating that both cells had the ability to induce osteogenic differentiation.The calcified nodules in H-PDLCs group were more than those in P-PDLCs group,indicating that the osteogenic differentiation ability of H-PDLCs group was stronger than that of P-PDLCs group CCK-8 results showed that the proliferation of the two kinds of cells was time-dependent,and the proliferation of H-PDLCs was faster than that of P-PDLCs（P<0.05）,PD concentration below 40ug/m L had no significant effect on the cell viability of P-PDLCs,and PD could promote the proliferation of P-PDLCs to a certain extent（P<0.05）.ELISA results showed that the contents of inflammatory factors TNF-ɑ and IL-1β in periodontal ligament cells extracted from periodontitis patients were significantly higher than those extracted from healthy periodontal ligament cells（P<0.05）,indicating that the cells extracted from periodontitis patients showed inflammatory expression,which could be classified as inflammatory periodontal membrane cells,that is,P-PDLCs treated with PD decreased the expression of inflammatory factor TNF-ɑ and IL-1β,and decreased linearly with the increase of PD concentration（P<0.05）,indicating that PD had significant anti-inflammatory activity on P-PDLCs,but the expressions of inflammatory factors TNF-ɑ and IL-1β in minocycline hydrochloride group were lower than those in PD group,indicating that although PD had certain anti-inflammatory function,the anti-inflammatory effect was still not as good as that in minocycline hydrochloride group PCR results showed that PD significantly inhibited the inflammatory factors TNF-ɑ and IL-1β of P-PDLCs（P<0.05）,Alizarin red S（ARS）staining RT-q PCR and WB staining showed that PD significantly promoted the expression of P-PDLCs osteogenesis and osteogenic related genes RUNX-2,ALP and OCN,but P-PDLCs had weaker osteogenic differentiation ability than H-PDLCs（P<0.05）m RNA expression of osteogenic factors such as RUNX-2,ALP and OCN were significantly increased at 20μg/m L and30μg/m L PD（P<0.05）.The m RNA expressions of RUNX-2,ALP and OCN were the highest when PD concentration was 20μg/m L.These results indicated that PD concentration of 20μg/m L might be the best drug concentration for osteogenesis immunofluorescence staining results showed that in the absence of PD,P-PDLCs showed typical fibroblast morphology,with long fusiform cells,relatively elongated cells,and tiny pseudopodia protruded around the cells under the action of PD,the cell morphology began to change from long spindle to squat shape,and the peripheral pseudopodia were reduced.The cells became more round and smooth,and the nucleus was located in the center of the field of vision more clearly.When treated with minocycline hydrochloride（100ug/m L）,the cell morphology was similar to that of PD（20ug/ml）,which became smooth and round,and the pseudopodia were reduced.The experimental results showed that both PD and minocycline hydrochloride had certain changes in cell morphology.This discovery should be the first discovery of such phenomenon,which may provide a certain theoretical basis for subsequent experimental research.Conclusion: In this study,it was found that PD significantly promoted the proliferation and induced osteogenic differentiation of P-PDLCs,and up-regulated the expressions of osteogenic genes RUNX-2,ALP and OCN,showing a certain osteogenic effect.Meanwhile,PD also inhibited the expressions of TNF-ɑ and IL-1βrelated inflammatory factors in P-PDLCs.This indicates that PD has significant anti-inflammatory activity.Obviously,PD,as a kind of low toxicity and high efficiency Traditional Chinese medicine,may have positive application value for clinical drug-assisted treatment of periodontitis.