人参皂苷Rb3调节磷酸化细胞外信号调节激酶通路减轻牙周炎大鼠炎症反应促进成骨  

Ginsenoside Rb3 regulates the phosphorrylated extracellular signal-regulated kinase signaling pathway to alleviate inflammatory responses and promote osteogenesis in rats with periodontitis

作　　者：张雪颖 孟鑫 刘志臻 张康 冀洪海 孙敏敏 Zhang Xueying;Meng Xin;Liu Zhizhen;Zhang Kang;Ji Honghai;Sun Minmin(School of Stomatology,Shandong Second Medical University,Weifang 261053,China;Dept.of Stomatology,Affiliated Hospital of Shandong Second Medical University,Weifang 261000,China)

机构地区：[1]山东第二医科大学口腔医学院,潍坊261053 [2]山东第二医科大学附属医院口腔科,潍坊261000

出　　处：《华西口腔医学杂志》2025年第2期236-248,共13页West China Journal of Stomatology

基　　金：山东省自然科学基金青年项目(ZR2022QH273)。

摘　　要：目的探究人参皂苷Rb3(Rb3)在牙周炎症环境中对成骨的促进作用,并阐述其机制。方法组织块法培养人牙周膜干细胞(hPDLSCs),通过流式细胞术进行细胞干性鉴定。细胞计数试剂盒(CCK-8)、钙黄绿素乙酰氧基甲酯(Calcein-AM)与碘化丙啶(PI)检测Rb3对hPDLSCs活力的影响。体外细胞实验分组:对照组、10μg/mL脂多糖(LPS)组、10μg/mL LPS+100μmol/L Rb3组和10μg/mL LPS+200μmol/L Rb3组。碱性磷酸酶(ALP)染色检测成骨诱导后各组hPDLSCs的ALP活性。定量逆转录聚合酶链反应(qRT-PCR)法检测成骨诱导后各组hPDLSCs中白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、runt相关转录因子2(RUNX2)、转化生长因子-β(TGF-β)基因的表达情况。蛋白质免疫印迹(Western blot)检测各组hPDLSCs内磷酸化细胞外信号调节激酶(p-ERK)相关蛋白表达情况。Sprague-Dawley大鼠随机分为对照组、结扎组、结扎+Rb3组。对左侧磨牙-上颌骨组织行显微计算机断层扫描(micro-CT)检测;检测完成后,将左侧磨牙-上颌骨制作牙周组织切片。苏木精-伊红(HE)染色检测炎细胞浸润、附着丧失情况。马松(Masson)染色检测牙龈胶原纤维的破坏情况。免疫荧光染色检测RUNX2蛋白、p-ERK蛋白表达情况。qRT-PCR法检测大鼠牙龈组织中TGF-β的表达情况。酶联免疫吸附试验(ELISA)检测大鼠外周血清IL-6蛋白表达情况。大鼠心脏血行流式细胞术,检测Treg细胞占比。采用GraphPad Prism 10.1.2软件对实验数据进行统计学分析。结果Rb3对hPDLSCs活性无影响。hPDLSCs成骨诱导后的qRT-PCR与ALP染色结果显示Rb3可抑制炎症性hPDLSCs内IL-6、IL-8基因表达,促进TGF-β基因表达,同时促进炎症性hPDLSCs成骨分化。Western blot结果显示Rb3抑制炎症性hPDLSCs p-ERK蛋白表达。micro-CT、Masson染色、HE染色结果显示Rb3可促进牙周炎大鼠牙槽骨的形成,同时抑制牙周纤维组织破坏、附着丧失及炎症细胞浸润。流式细胞术结果Objective To explore the promoting effect of ginsenoside Rb3(Rb3)on osteogenesis in periodontitis environment,and to explain its mechanism.Methods Human periodontal ligament stem cells(hPDLSCs)were cultured by tissue block method and identified by flow cytometry.Cell counting kit-8(CCK8)method and calcein acetoxymethyl ester/propidium iodide staining were used to detect the effect of Rb3 on the viability of hPDLSCs cells.In vitro cell experiments were divided into control group,10μg/mL lipopolysaccharides(LPS)group,10μg/mL LPS+100μmol/L Rb3 group and 10μg/mL LPS+200μmol/L Rb3 group.Alkaline phosphatase(ALP)staining was used to detect the ALP activity of hPDLSCs in each group after osteogenesis induction.The expression of hPDLSCs interleukin-6(IL-6),interleukin-8(IL-8),runt-related transcription factor 2(RUNX2)and transforming growth factor-β(TGF-β)genes in each group after osteogenesis was detected by quantitative reverse transcription polymerase chain reaction(qRT-PCR)method.Western blot was used to detect the protein expression of hPDLSCs phosphorrylated extracellular signal-regulated kinase(p-ERK)in each group.Sprague-Dawley rats were randomly divided into the control group,ligation group and ligation+Rb3 group.The left molar-maxillary tissue was subjected to micro-computed tomography(micro-CT)scanning.After the scanning,the left molar-maxilla was made into periodontal tissue sections.Hematoxylin-eosin(HE)staining was used to detect the infiltration and loss of adhesion of inflammatory cells.Masson staining was used to detect the destruction of gingival collagen fibers.Immunofluorescence staining was used to detect the protein expression of RUNX2 and p-ERK.The expression of TGF-βin rat gingival tissue was detected by qRT-PCR.The protein expression of IL-6 in peripheral serum of rats was detected by enzyme-linked immunosorbent assay(ELISA).Flow cytometry was used to detect the proportion of Treg cells in rat heart blood.The experimental data were statistically analyzed by Graph Pad Prism10.1.2 software.

关 键 词：人参皂苷RB3 磷酸化细胞外信号调节激酶 成骨分化 牙周炎 牙槽骨吸收 

分 类 号：R781.4[医药卫生—口腔医学]