Abstract：
OBJECTIVE: Periodontitis is a chronic infectious disease, characterized by loss of alveolar bone and supporting tissues. Cistanche deserticola(Cd), a local medicinal herb in Xinjiang, possesses favorable biological characteristics and potential applications. Our aim is to investigate the remodeling properties of Cd extract and elucidate the specific mechanisms underlying its therapeutic effects on periodontitis, by employing a combination of basic experimental and network pharmacology approaches.
METHODS: Firstly, UHPLC-QTOF-MS analysis was conducted on Cd extract to identify its main components, with several compounds were identified by standard. Subsequently, in vitro studies were performed using the Cd extract on MC3T3-E1 cells. Cell proliferation viability was assessed using CCK-8 and apoptosis assays, while ALP and ARS staining and quantitative experiments, qRT-PCR, and Western blot assays were employed to evaluate the osteogenic differentiation capability. Network pharmacology analysis was then carried out using the identified compounds to establish a database of Cd components and targets, along with a database of periodontitis. The intersection of these databases revealed the network relationship between Cd components-mapped genes-signaling pathways. KEGG/GO pathway analysis of the targets was performed to filter potential enriched pathways. PPI/CytoHubba protein interaction network analysis was utilized to identify hub genes. Molecular docking and molecular dynamics simulations were employed to analyze the docking and interaction between core gene and Cd components.
RESULTS: We detected 38 major components in the Cd extract, with Echinacoside, Acteoside, Tubuloside A, and Cistanoside A undergoing standard substance verification. In vitro studies indicated that the Cd, at concentrations below 100 μg/ mL, did not affect cell proliferation and inhibited apoptosis. Osteogenesis assays demonstrated that Cd at concentrations of 1 μg/ mL, 10 μg/ mL, and 100 μg/ mL significantly promoted the osteogenic differentiation ability of MC3T3-E1 cells. It also notably upregulated the mRNA and protein levels of Alp, Bmp2, Runx2, and Opn, and the optimal concentration was 10 μg/mL. Network pharmacology results revealed the network relationship between Cd\'s components, crossed targets and signaling pathways. Combined with KEGG/GO pathway analysis and PPI/CytoHubba protein interaction network analysis. The key pathway and hub genes of Cd regulating periodontitis are both related to hypoxia pathway and HIF-1α. Molecular docking results showed a strong binding affinity between Cd compounds and hub genes, and molecular dynamics simulation results indicated the stability of the complexes formed between HIF-1α and several Cd compounds.
CONCLUSIONS: Cistanche deserticola exhibits a notable capacity to promote bone regeneration, and its mechanism of action in regulating periodontitis is associated with the hypoxia signaling pathway. HIF-1α may serve as a potential core gene. Future research will focus on exploring the mechanism of Cd in intervene periodontitis and promoting bone remodeling in hypoxic environment.
METHODS: Firstly, UHPLC-QTOF-MS analysis was conducted on Cd extract to identify its main components, with several compounds were identified by standard. Subsequently, in vitro studies were performed using the Cd extract on MC3T3-E1 cells. Cell proliferation viability was assessed using CCK-8 and apoptosis assays, while ALP and ARS staining and quantitative experiments, qRT-PCR, and Western blot assays were employed to evaluate the osteogenic differentiation capability. Network pharmacology analysis was then carried out using the identified compounds to establish a database of Cd components and targets, along with a database of periodontitis. The intersection of these databases revealed the network relationship between Cd components-mapped genes-signaling pathways. KEGG/GO pathway analysis of the targets was performed to filter potential enriched pathways. PPI/CytoHubba protein interaction network analysis was utilized to identify hub genes. Molecular docking and molecular dynamics simulations were employed to analyze the docking and interaction between core gene and Cd components.
RESULTS: We detected 38 major components in the Cd extract, with Echinacoside, Acteoside, Tubuloside A, and Cistanoside A undergoing standard substance verification. In vitro studies indicated that the Cd, at concentrations below 100 μg/ mL, did not affect cell proliferation and inhibited apoptosis. Osteogenesis assays demonstrated that Cd at concentrations of 1 μg/ mL, 10 μg/ mL, and 100 μg/ mL significantly promoted the osteogenic differentiation ability of MC3T3-E1 cells. It also notably upregulated the mRNA and protein levels of Alp, Bmp2, Runx2, and Opn, and the optimal concentration was 10 μg/mL. Network pharmacology results revealed the network relationship between Cd\'s components, crossed targets and signaling pathways. Combined with KEGG/GO pathway analysis and PPI/CytoHubba protein interaction network analysis. The key pathway and hub genes of Cd regulating periodontitis are both related to hypoxia pathway and HIF-1α. Molecular docking results showed a strong binding affinity between Cd compounds and hub genes, and molecular dynamics simulation results indicated the stability of the complexes formed between HIF-1α and several Cd compounds.
CONCLUSIONS: Cistanche deserticola exhibits a notable capacity to promote bone regeneration, and its mechanism of action in regulating periodontitis is associated with the hypoxia signaling pathway. HIF-1α may serve as a potential core gene. Future research will focus on exploring the mechanism of Cd in intervene periodontitis and promoting bone remodeling in hypoxic environment.
摘要：
目的：牙周炎是一种慢性感染性疾病，以牙槽骨和支持组织丢失为特征。肉笋（Cd），一种新疆当地的草药，具有良好的生物学特性和潜在的应用。我们的目的是研究Cd提取物的重塑特性，并阐明其对牙周炎的治疗作用的具体机制。通过采用基础实验和网络药理学方法的结合。
方法：首先，对Cd提取物进行UHPLC-QTOF-MS分析，以鉴定其主要成分，与几种化合物的标准鉴定。随后，使用Cd提取物对MC3T3-E1细胞进行体外研究。使用CCK-8和凋亡测定法评估细胞增殖活力，而ALP和ARS染色和定量实验，qRT-PCR,和Western印迹分析用于评估成骨分化能力。然后利用已鉴定的化合物进行网络药理学分析,建立Cd组分和靶标的数据库,还有牙周炎的数据库.这些数据库的交集揭示了Cd成分映射的基因信号通路之间的网络关系。进行靶标的KEGG/GO途径分析以过滤潜在的富集途径。利用PPI/CytoHubba蛋白相互作用网络分析鉴定hub基因。利用分子对接和分子动力学模拟分析了核心基因与Cd组分的对接和相互作用。
结果：我们在Cd提取物中检测到38种主要成分，有了Echinacoside，Acteoside,TubulosideA,和CistanosideA正在进行标准物质验证。体外研究表明，Cd，浓度低于100μg/mL时，不影响细胞增殖，抑制细胞凋亡。成骨实验表明，Cd浓度为1μg/mL，10μg/mL,100μg/mL能显著促进MC3T3-E1细胞的成骨分化能力。它还显著上调了Alp的mRNA和蛋白质水平，Bmp2、Runx2和Opn，最佳浓度为10μg/mL。网络药理学结果揭示了Cd组分之间的网络关系，交叉靶标和信号通路。结合KEGG/GO通路分析和PPI/CytoHubba蛋白相互作用网络分析。Cd调控牙周炎的关键通路和hub基因均与缺氧通路和HIF-1α有关。分子对接结果显示Cd化合物与hub基因之间具有很强的结合亲和力,分子动力学模拟结果表明，HIF-1α与几种Cd化合物形成的配合物具有稳定性。
结论：肉桂显示出显著的促进骨再生的能力，其调控牙周炎的作用机制与缺氧信号通路有关。HIF-1α可能是一个潜在的核心基因。未来的研究将集中在探讨低氧环境下Cd干预牙周炎和促进骨重建的机制。
方法：首先，对Cd提取物进行UHPLC-QTOF-MS分析，以鉴定其主要成分，与几种化合物的标准鉴定。随后，使用Cd提取物对MC3T3-E1细胞进行体外研究。使用CCK-8和凋亡测定法评估细胞增殖活力，而ALP和ARS染色和定量实验，qRT-PCR,和Western印迹分析用于评估成骨分化能力。然后利用已鉴定的化合物进行网络药理学分析,建立Cd组分和靶标的数据库,还有牙周炎的数据库.这些数据库的交集揭示了Cd成分映射的基因信号通路之间的网络关系。进行靶标的KEGG/GO途径分析以过滤潜在的富集途径。利用PPI/CytoHubba蛋白相互作用网络分析鉴定hub基因。利用分子对接和分子动力学模拟分析了核心基因与Cd组分的对接和相互作用。
结果：我们在Cd提取物中检测到38种主要成分，有了Echinacoside，Acteoside,TubulosideA,和CistanosideA正在进行标准物质验证。体外研究表明，Cd，浓度低于100μg/mL时，不影响细胞增殖，抑制细胞凋亡。成骨实验表明，Cd浓度为1μg/mL，10μg/mL,100μg/mL能显著促进MC3T3-E1细胞的成骨分化能力。它还显著上调了Alp的mRNA和蛋白质水平，Bmp2、Runx2和Opn，最佳浓度为10μg/mL。网络药理学结果揭示了Cd组分之间的网络关系，交叉靶标和信号通路。结合KEGG/GO通路分析和PPI/CytoHubba蛋白相互作用网络分析。Cd调控牙周炎的关键通路和hub基因均与缺氧通路和HIF-1α有关。分子对接结果显示Cd化合物与hub基因之间具有很强的结合亲和力,分子动力学模拟结果表明，HIF-1α与几种Cd化合物形成的配合物具有稳定性。
结论：肉桂显示出显著的促进骨再生的能力，其调控牙周炎的作用机制与缺氧信号通路有关。HIF-1α可能是一个潜在的核心基因。未来的研究将集中在探讨低氧环境下Cd干预牙周炎和促进骨重建的机制。
