丹参提取物对人牙周膜成纤维细胞的干预效果及其对CTGF、ALP水平的影响

Intervention Effects of Salvia Miltiorrhiza Extract on Human Periodontal Ligament Fibroblasts and Its Effect on CTGF and ALP Levels

目的研究丹参提取物对人牙周膜成纤维细胞结缔组织生长因子(connective tissue growth factor,CTGF)、碱性磷酸酶(alkaline phosphatase,ALP)水平的影响。方法取河北省中医院口腔科因正畸治疗拔除的第一前磨牙2颗,牙齿拔除后即刻放入含有青霉素和链霉素的无菌α-MEM培养基中,分离培养人牙周膜成纤维细胞,取第5代人牙周膜成纤维细胞用于后续试验,分为空白组、丹参提取物低浓度组、丹参提取物高浓度组,空白组加入100μL的基础培养基,丹参提取物低浓度组加入1μmol∙L^(−1)丹参提取物溶液,丹参提取物高浓度组加入2μmol∙L^(−1)丹参提取物溶液。测定细胞增殖、凋亡、成骨分化能力及磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun amino terminal kinase,p-JNK)、CTGF、骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)、成骨特异性转录因子(osteogenic specific transcription factor,Runx-2)、ALP表达量。结果丹参提取物高浓度组24,48,72 h细胞增殖率、ALP活性均高于丹参提取物低浓度组(P<0.05)。丹参提取物高浓度组24,48,72 h细胞凋亡率均低于丹参提取物低浓度组(P<0.05)。丹参提取物高浓度组p-JNK、CTGF、BMP-2、Runx-2、ALP表达量均高于丹参提取物低浓度组(P<0.05)。结论丹参提取物通过作用于JNK通路,上调CTGF表达,促进人牙周膜成纤维细胞增殖,通过调控BMP-2、Runx-2表达,增加ALP活性,促进人牙周膜成纤维细胞成骨分化,且具有一定浓度依赖性。

OBJECTIVE To study the effects of Salvia miltiorrhiza extract on the levels of connective tissue growth factor(CTGF)and alkaline phosphatase(ALP)in human periodontal ligament fibroblasts.METHODS Two first premolars extracted by orthodontic treatment in Hebei Hospital of Traditional Chinese Medicine were put into asepticα-MEM medium containing penicillin and streptomycin immediately after tooth extraction.Human periodontal ligament fibroblasts were isolated and cultured.The fifth generation of human periodontal ligament fibroblasts were used for follow-up test,which were divided into blank group,low and high concentration group of Salvia miltiorrhiza extract.The blank group was added with 100μL basic medium,low concentration group of Salwia miltiorrhiza extract was added to 1μmol∙L^(−1) Salvia miltiorrhiza extract solution,and 2μmol∙L^(−1) Salvia miltiorrhiza extract solution was added to high concentration group of Salvia miltiorrhiza extract.Cell proliferation,apoptosis,osteogenic differentiation and expression of phosphorylated c-Jun amino terminal kinase(p-JNK),CTGF,bone morphogenetic protein-2(BMP-2),osteogenic specific transcription factor(Runx-2)and ALP were measured.RESULTS The proliferation rate and ALP activity of high concentration group of Salvia miltiorrhiza extract in 24,48,72 h were higher than that of low concentration group of Salvia miltiorrhiza extract(P<0.05).The apoptosis rate of high concentration group of Salvia miltiorrhiza extract in 24,48,72 h was lower than that of low concentration group of Salvia miltiorrhiza extract(P<0.05).The expression levels of p-JNK,CTGF,BMP-2,Runx-2 and ALP in the high concentration group of Salvia miltiorrhiza extract were all higher than those in the low concentration group of Salvia miltiorrhiza extract(P<0.05).CONCLUSION Salvia miltiorrhiza extract by acting on JNK pathway,raise the CTGF expression,promote the periodontal ligament fibroblasts proliferation,through the regulation of BMP-2,Runx-2 expression,increase ALP activity,promote the periodontal ligament fibroblasts osteogenesis differentiation,and has a certain concentration dependence.