程序性坏死特异性抑制剂-1对高糖环境下牙周膜干细胞增殖和成骨分化的影响

目的探讨程序性坏死特异性抑制剂-1（Nec-1）对高糖环境下牙周膜干细胞（PDLSC）的增殖和成骨分化的影响。 方法体外克隆培养的PDLSC,按照如下处理方法分为3组：对照组（5 mmol/L葡萄糖）、高糖组（25 mmol/L葡萄糖）和高糖+Nec-1组（25 mmol/L葡萄糖+30 μmol/L Nec-1）。通过蛋白免疫印迹（Western blot）检测细胞坏死性凋亡相关分子RIP1、RIP3的表达,噻唑蓝（MTT）比色法检测各组细胞增殖活力,通过茜素红染色、碱性磷酸酶（ALP）定量检测和实时荧光定量PCR等方法分析PDLSC的成骨分化情况。使用SPSS 26.0软件进行统计分析,采用单因素方差分析比较各组间细胞增殖活力（MTT吸光度A值）、ALP表达含量及成骨相关基因（COL1、RUNX2、OCN）相对表达水平,并用LSD-t检验进行组间多重比较。 结果Western blot结果显示,高糖组PDLSC的RIP1和RIP3的表达较对照组明显增加,而高糖+Nec-1组的RIP1和RIP3的表达明显弱于高糖组。PDLSC培养7 d后,高糖组增殖活力较对照组明显降低,其吸光度A值（0.67 ± 0.06）显著低于对照组（1.23 ± 0.12）,差异有统计学意义（t = 9.652,P<0.001）;而Nec-1抑制后,PDLSC的增殖活力明显增加,高糖+Nec-1组吸光度A值（1.12 ± 0.11）显著高于高糖组（0.67 ± 0.06）,差异有统计学意义（t = 8.185,P<0.001）。经矿化诱导后,高糖组PDLSC 14 d时形成的矿化结节、7 d时ALP表达含量（3.42 ± 0.37）和成骨相关基因COL1（1.86 ± 0.16）、RUNX2（1.55 ± 0.23）、OCN（1.08 ± 0.20）的相对表达量均较对照组均显著减少,差异均有统计学意义（t_ALP = 13.149,t_COL1 = 14.257,t_RUNX2 = 7.593,t_OCN = 8.606,P均<0.001）;而Nec-1抑制后,PDLSC的成骨分化增加,高糖+Nec-1组PDLSC 14 d时形成的矿化结节、7 d时ALP表达含量（6.06 ± 0.26）和成骨相关基因COL1（3.64 ± 0.30）、RUNX2（2.53 ± 0.26）、OCN（2.14 ± 0.30）的相对表达量较高糖组均显著升高,差异均有统计学意义（t_ALP = 13.033,t_COL1 = 11.636,t_RUNX2 = 6.332,t_OCN = 6.573,P均<0.001）。 结论体外培养条件下,高糖环境抑制了PDLSC的增殖和成骨分化,而Nec-1明显改善了高糖环境下PDLSC的增殖和成骨分化。

Necrostatin-1 promotes the proliferation and osteogenic differentiation of periodontal ligament stem cells in high-glucose environment

ObjectiveTo investigate the effect of necrostatin-1 (Nec-1) on the biological characteristics of periodontal ligament stem cells (PDLSCs) in the high-glucose environments in vitro. MethodsPDLSCs were successfully cultured by single colony and divided into three groups according to the following treatments: control group (5 mmol/L glucose) , high-glucose group (25 mmol/L glucose) , high-glucose + Nec-1 group (25 mmol/L glucose + 30 μmol/L Nec-1) . Western blot was used to detect the expression of necroptosis related molecules (RIP1 and RIP3) and the cell proliferation of PDLSCs were evaluated by MTT assay in the above three groups. The osteogenic differentiation of PDLSCs were evaluated by alizarin red staining, the quantitative detection of alkaline phosphatase (ALP) and real-time quantitative PCR assay. All statistical analyses were used SPSS 26.0 software. One way ANOVA was used to compare the cell proliferation activity (A value of the MTT absorbance) , the expression of ALP and the relative levels of osteogenesis related genes (COL1, RUNX2, OCN) among the groups, and the LSD-t test was used for multiple comparisons between the groups. ResultsWestern blot showed that the expression levels of RIP1 and RIP3 in high-glucose group were significantly higher than those in control group, while significantly lower in high-glucose + Nec-1 group than in high-glucose group. The proliferation activity of PDLSCs at day 7 in high-glucose group was significantly lower than that in the control group, and its MTT absorbance (0.67 ± 0.06) was significantly lower than that of the control group (1.23 ± 0.12) (t = 9.652, P<0.001) . After the inhibition of Nec-1, the proliferation activity of PDLSCs increased significantly, and the MTT absorbance at day 7 of high-glucose + Nec-1 group (1.12±0.11) was significantly higher than that of high-glucose group (0.67±0.06) (t = 8.185, P<0.001) . Compared with the control group, the mineralized nodules formed by PDLSCs at day 14, the levels of ALP (3.42 ± 0.37) and the relative expression of osteogenesis related genes COL1 (1.86 ± 0.16) , RUNX2 (1.55 ± 0.23) , OCN (1.08 ± 0.20) at day 7 were significantly lower in high-glucose group (t_ALP = 13.149, t_COL1 = 14.257, t_RUNX2 = 7.593, t_OCN = 8.606, all P<0.001) . After the inhibition of Nec-1, the osteogenic differentiation of PDLSCs increased, and the mineralized nodules formed by PDLSCs at day 14, the levels of ALP (6.06±0.26) and the relative expression of osteogenesis related genes COL1 (3.64 ± 0.30) , RUNX2 (2.53 ± 0.26) , OCN (2.14 ± 0.30) at day 7 of high-glucose + Nec-1 group were significantly higher than those of high-glucose group (t_ALP = 13.033, t_COL1 = 11.636, t_RUNX2 = 6.332, t_OCN = 6.573, all P<0.001) . ConclusionsThe proliferation and osteogenic differentiation of PDLSCs were inhibited in the high-glucose environments in vitro. Nec-1 significantly improved the proliferation and osteogenic differentiation of PDLSCs in the high-glucose environments.