西格列汀激活基质细胞衍生因子-1/CXC趋化因子受体4信号通路对脂多糖诱导的人牙周膜干细胞增殖、凋亡、炎症和成骨分化的影响

doi:10.7518/hxkq.2024.2023213

华西口腔医学杂志 ›› 2024, Vol. 42 ›› Issue (1): 37-45.doi: 10.7518/hxkq.2024.2023213

西格列汀激活基质细胞衍生因子-1/CXC趋化因子受体4信号通路对脂多糖诱导的人牙周膜干细胞增殖、凋亡、炎症和成骨分化的影响

唐小雪(), 周政, 李启期, 姜丹丹

石河子大学第一附属医院口腔科，石河子 832008

收稿日期:2023-07-07 修回日期:2023-12-20 出版日期:2024-02-01 发布日期:2024-01-12
通讯作者: 唐小雪 E-mail:tangxiaoxue638@163.com
作者简介:唐小雪，副主任医师，硕士，E-mail：tangxiaoxue638@163.com
基金资助:
石河子大学自主资助支持校级科研立项项目(ZZZC20-1957A)

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide

Tang Xiaoxue(), Zhou Zheng, Li Qiqi, Jiang Dandan

Dept. of Stomatology, The First Affiliated Hospital of Shihezi University, Shihezi 832008, China

Received:2023-07-07 Revised:2023-12-20 Online:2024-02-01 Published:2024-01-12
Contact: Tang Xiaoxue E-mail:tangxiaoxue638@163.com
Supported by:
Shihezi University Provides Independent Funding to Support University Level Scientific Research Projects(ZZZC201957A)

摘要/Abstract

摘要：

目的探讨西格列汀对脂多糖（LPS）诱导的炎症微环境下人牙周膜干细胞（hPDLSCs）增殖、凋亡、炎症和成骨分化的影响及分子机制。方法体外培养hPDLSCs，用不同浓度的西格列汀处理后检测细胞活力，以确定后续西格列汀实验浓度。采用1 µg/mL LPS刺激诱导24 h建立hPDLSCs炎症模型并分为空白组、对照组、西格列汀低浓度组（0.5 µmol/L）、西格列汀中浓度组（1 µmol/L）、西格列汀高浓度组（2 µmol/L）、西格列汀高浓度+基质细胞衍生因子-1（SDF-1）/CXC趋化因子受体4（CXCR4）通路抑制剂（AMD3100）组（2 µmol/L+10 µg/mL）。细胞计数试剂盒-8检测培养24、48、72 h后的hPDLSCs增殖活性；流式细胞术检测培养72 h后hPDLSCs凋亡情况；诱导成骨分化21 d后茜素红染色检测hPDLSCs成骨分化能力，试剂盒测定hPDLSCs中碱性磷酸酶（ALP）活性；酶联免疫吸附检测hPDLSCs培养上清液中炎症因子肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-6水平；实时荧光定量聚合酶链反应（RT-qPCR）检测hPDLSCs中成骨分化相关基因Runt相关转录因子2（RUNX2）、骨钙素（OCN）、骨桥蛋白（OPN）及SDF-1和CXCR4 mRNA表达；Western blot检测hPDLSCs中SDF-1、CXCR4蛋白表达。结果与空白组比较，对照组hPDLSCs增殖活性、矿化结节数量、染色强度、ALP活性和RUNX2、OCN、OPN mRNA及SDF-1、CXCR4 mRNA和蛋白表达水平显著降低，凋亡率、TNF-α、IL-1β、IL-6水平显著升高（P<0.05）；与对照组比较，西格列汀低、中、高浓度组hPDLSCs增殖活性、矿化结节数量、染色强度、ALP活性和RUNX2、OCN、OPN mRNA及SDF-1、CXCR4 mRNA和蛋白表达水平依次升高，凋亡率、TNF-α、IL-1β、IL-6水平依次降低（P<0.05）；AMD3100可部分逆转高浓度西格列汀对LPS诱导的hPDLSCs的作用效果（P<0.05）。结论西格列汀可能通过激活SDF-1/CXCR4信号通路促进LPS诱导的炎症微环境下hPDLSCs的增殖和成骨分化，抑制hPDLSCs凋亡和炎症反应。

关键词: 西格列汀, 脂多糖, 人牙周膜干细胞, 成骨分化, 基质细胞衍生因子-1, CXC趋化因子受体4

Abstract:

Objective This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. Methods hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. Results Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1β, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1β, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). Conclusion Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

Key words: sitagliptin, lipopolysaccharide, human periodontal ligament stem cells, osteogenic differentiation, stromal cell derived factor-1, CXC chemokine receptor 4

中图分类号:

Q257

唐小雪, 周政, 李启期, 姜丹丹. 西格列汀激活基质细胞衍生因子-1/CXC趋化因子受体4信号通路对脂多糖诱导的人牙周膜干细胞增殖、凋亡、炎症和成骨分化的影响[J]. 华西口腔医学杂志, 2024, 42(1): 37-45.

Tang Xiaoxue, Zhou Zheng, Li Qiqi, Jiang Dandan. Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide[J]. West China Journal of Stomatology, 2024, 42(1): 37-45.

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基因名称	正向引物（5’-3’）	反向引物（5’-3’）
RUNX2	CGAATGGCTAGCACGCTATTAA	GTCGCCATAACAGATTCATCCG
OCN	TCACACTCCTCGCCCTATT	GATGTGGTCAGCCAACTCG
OPN	TCCTAGCCCCACAGACCCTT	CACACTATCACCTCGGCCAT
SDF-1	ATTCTCAACACTCCAAACTGTGC	CGGTATGAACTGAACTTGCAATG
CXCR4	AGCTGTTGGTGACACGCGTGGTCTATG	GCACTACTGGTGGCCCTTGGAGTATGA
GAPDH	GCACCGTCAAGGCTGAGAAC	TGGTGAAGACGCCAGTGGA

组别	增殖活性
组别	24 h	48 h	72 h
空白组	100.00±0.00	100.00±0.00	100.00±0.00
对照组	42.07±6.02^a	38.09±4.52^a	32.19±4.72^a
西格列汀低浓度组	60.13±8.11^b	53.38±6.90^b	48.25±5.96^b
西格列汀中浓度组	73.06±9.07^bc	67.94±8.57^bc	61.58±7.04^bc
西格列汀高浓度组	86.38±8.19^bcd	79.76±10.14^bcd	72.90±9.15^bcd
西格列汀高浓度+AMD3100组	51.74±7.08^e	45.21±6.35^e	41.55±6.03^e

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西格列汀激活基质细胞衍生因子-1/CXC趋化因子受体4信号通路对脂多糖诱导的人牙周膜干细胞增殖、凋亡、炎症和成骨分化的影响

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide

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