人参皂苷Rh2衍生物体外促进IFN-γ分泌及CTLL-2细胞增殖的作用研究
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摘要
背景：人参皂苷Rh2是中国传统中药人参中的主要活性成分，具有优异的免疫调节、抗炎、抗疲劳、抗肿瘤等药理作用。然而因其难溶于水,限制了体外实验中可应用的最大浓度，人参皂苷Rh2的生物活性在实际应用和生产研究中受到严重的制约。为了提高Rh2的水溶性和生物活性，本室制备了人参皂苷Rh2的硫酸化修饰产物Rh2-B1和Rh2-B2。两种衍生物的水溶性都获得大幅提高。在本论文中，我们研究了Rh2-B1对细胞毒性T细胞系CTLL-2细胞的作用，细胞毒性T细胞因其能够保护机体免受病毒感染而成为近年来研究的热点，此外，我们还探讨了Rh2影响CTLL-2细胞增殖和发挥免疫功能的分子机制。
目标：我们的目标是调查Rh2-B1对干扰素-（γInterferon-γ, IFN-γ）的分泌和细胞增殖的影响，并研究其可能的机制。
材料与方法：酶联免疫吸附实验（Enzyme Linked ImmunosorbentAssay,ELISA）被用于检测IFN-γ在全血培养上清和CTLL-2细胞培养上清液中的浓度。噻唑蓝染色法（MTT Cell Proliferation Assay，MTT）被用来检测CTLL-2细胞的增殖情况。蛋白免疫印迹试验（Western blot）被用来评价CTLL-2细胞中信号转导通路相关蛋白表达量及其活化程度的变化。
结果：我们的研究证明，Rh2-B1能够显著提高Balb/c小鼠全血培养上清中IFN-γ的水平。在此之后，我们评估了Rh2-B1对CTLL-2这种细胞毒性T细胞的增殖、IFN-γ的分泌情况的影响，并研究了其作用机制。Rh2-B1刺激了CTLL-2细胞的增殖和IFN-γ的分泌，还能够介导丝裂原活化蛋白激酶p38（p38mitogen-activated protein kinase，p38MAPK）和胞外信号调节激酶（extracellular regulated proteinkinases，ERK）的表达和活化，但对淋巴细胞特异性蛋白酪氨酸激酶（Lymphocyte-specific protein tyrosine kinase,Lck or p56Lck）和信号转导及转录激活因子（Signal transducers and activators oftranscription5，STAT5）的表达呈现抑制作用。这种作用能够被特异性的p38MAPK抑制剂SB203580和特异性ERK抑制剂U0126阻断。
结论：Rh2-B1能够通过激活p38MAPK和ERK依赖的信号通路进而刺激细胞毒性T细胞的增殖和IFN-γ的分泌。这可能成为一种新的病毒感染治疗潜在药物。
Context: Ginsenoside Rh2is the major active component of Chinesetraditional medicine ginseng. Ginsenoside Rh2exerts excellentimmunoregulatory, anti-inflammatory, anti-fatigue and anti-tumor activity.However, the bio-activity of Ginsenoside Rh2is limited by the poorwater solubility. Low water solubility not only decreases the maximalRh2concentration that can be used for in vitro studies, but also causespoor oral absorption of Rh2. In order to enhance the water solubility andbio-activity of the Ginsenoside Rh2, two sulfated derivative, Rh2-B1andRh2-B2were prepared. Both of them were demonstrated to have higherwater solubility. In this study, we investigated the effect of Rh2-B1onCTLL-2, a CD8+cytotoxic cell line, which is known for protectingagainst viral infection. In addition, the molecular mechanism responsiblefor Rh2-B1enhancing CTLL-2cell proliferation and IFN-gammaprodution.
Objective: We aimed to investigate the effect of Rh2-B1onIFN-gamma production and cell proliferation and its possible mechanism.
Materials and methods: Enzyme-linked-immunosorbent assay(ELISA) was employed to analyze the IFN-γ concentration of the blood whole blood and the supernatant of CTLL-2cell culture. Cellproliferation assay was conducted using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) assay. Western blots were usedto evaluate changes in signal transduction pathways in CTLL-2cells.
Results: Rh2-B1was able to enhance IFN-γ production from bloodwhole blood of Balb/c mice. We then evaluated the effect of Rh2-B1on acytotoxic T cell line, CTLL-2for cell proliferation, IFN-γ production andits molecular mechanism. Rh2-B1promoted cell proliferation and IFN-γproduction of CTLL-2cells. It aslo induced activation of p38mitogen-activated protein kinase (MAPK) and extracellular-signalregulated kinases (ERK), but inhibited p56Lck and transducer andactivator of transcription5(STAT5) expression. The effect was blockedby the specific p38MAPK inhibitor SB203580and ERK inhibitorU0126.
Conclusion: Rh2-B1could stimulate cell proliferation and IFN-γproduction by activating the p38MAPK-and ERK-dependent signalingpathways in cytotoxic T cells. This may be a novel medicine for treatmentof viral infections.
目标：我们的目标是调查Rh2-B1对干扰素-（γInterferon-γ, IFN-γ）的分泌和细胞增殖的影响，并研究其可能的机制。
材料与方法：酶联免疫吸附实验（Enzyme Linked ImmunosorbentAssay,ELISA）被用于检测IFN-γ在全血培养上清和CTLL-2细胞培养上清液中的浓度。噻唑蓝染色法（MTT Cell Proliferation Assay，MTT）被用来检测CTLL-2细胞的增殖情况。蛋白免疫印迹试验（Western blot）被用来评价CTLL-2细胞中信号转导通路相关蛋白表达量及其活化程度的变化。
结果：我们的研究证明，Rh2-B1能够显著提高Balb/c小鼠全血培养上清中IFN-γ的水平。在此之后，我们评估了Rh2-B1对CTLL-2这种细胞毒性T细胞的增殖、IFN-γ的分泌情况的影响，并研究了其作用机制。Rh2-B1刺激了CTLL-2细胞的增殖和IFN-γ的分泌，还能够介导丝裂原活化蛋白激酶p38（p38mitogen-activated protein kinase，p38MAPK）和胞外信号调节激酶（extracellular regulated proteinkinases，ERK）的表达和活化，但对淋巴细胞特异性蛋白酪氨酸激酶（Lymphocyte-specific protein tyrosine kinase,Lck or p56Lck）和信号转导及转录激活因子（Signal transducers and activators oftranscription5，STAT5）的表达呈现抑制作用。这种作用能够被特异性的p38MAPK抑制剂SB203580和特异性ERK抑制剂U0126阻断。
结论：Rh2-B1能够通过激活p38MAPK和ERK依赖的信号通路进而刺激细胞毒性T细胞的增殖和IFN-γ的分泌。这可能成为一种新的病毒感染治疗潜在药物。
Context: Ginsenoside Rh2is the major active component of Chinesetraditional medicine ginseng. Ginsenoside Rh2exerts excellentimmunoregulatory, anti-inflammatory, anti-fatigue and anti-tumor activity.However, the bio-activity of Ginsenoside Rh2is limited by the poorwater solubility. Low water solubility not only decreases the maximalRh2concentration that can be used for in vitro studies, but also causespoor oral absorption of Rh2. In order to enhance the water solubility andbio-activity of the Ginsenoside Rh2, two sulfated derivative, Rh2-B1andRh2-B2were prepared. Both of them were demonstrated to have higherwater solubility. In this study, we investigated the effect of Rh2-B1onCTLL-2, a CD8+cytotoxic cell line, which is known for protectingagainst viral infection. In addition, the molecular mechanism responsiblefor Rh2-B1enhancing CTLL-2cell proliferation and IFN-gammaprodution.
Objective: We aimed to investigate the effect of Rh2-B1onIFN-gamma production and cell proliferation and its possible mechanism.
Materials and methods: Enzyme-linked-immunosorbent assay(ELISA) was employed to analyze the IFN-γ concentration of the blood whole blood and the supernatant of CTLL-2cell culture. Cellproliferation assay was conducted using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) assay. Western blots were usedto evaluate changes in signal transduction pathways in CTLL-2cells.
Results: Rh2-B1was able to enhance IFN-γ production from bloodwhole blood of Balb/c mice. We then evaluated the effect of Rh2-B1on acytotoxic T cell line, CTLL-2for cell proliferation, IFN-γ production andits molecular mechanism. Rh2-B1promoted cell proliferation and IFN-γproduction of CTLL-2cells. It aslo induced activation of p38mitogen-activated protein kinase (MAPK) and extracellular-signalregulated kinases (ERK), but inhibited p56Lck and transducer andactivator of transcription5(STAT5) expression. The effect was blockedby the specific p38MAPK inhibitor SB203580and ERK inhibitorU0126.
Conclusion: Rh2-B1could stimulate cell proliferation and IFN-γproduction by activating the p38MAPK-and ERK-dependent signalingpathways in cytotoxic T cells. This may be a novel medicine for treatmentof viral infections.
引文
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