【作者】 周游；

【导师】 关树文；

【作者基本信息】 吉林大学 ， 生物学， 2025， 硕士

【摘要】 脓毒症(Sepsis),也称败血症,是一种严重感染引起的宿主免疫失调,严重时常导致多器官功能障碍,具有较高致死率。脓毒症时肺部是最先受损的器官,肺部出现炎性细胞浸润、微血管通透性增高、水肿严重等危害。过度活化的中性粒细胞等自然免疫细胞在肺部浸润会进一步加剧肺部炎症及组织损伤。目前,脓毒症的治疗存在许多挑战,例如诊断的复杂性、异质性,抗生素的耐药性以及免疫治疗的局限性等问题,迫切需要寻找新的药物和疗法以提高脓毒症患者的生存率和生活质量。近年来,人参皂苷Rd因其抗炎和免疫调节等特性被广泛用于炎症引起的疾病中,但人参皂苷Rd对脓毒症的改善及其机制还未明晰。因此,本研究旨在探索人参皂苷Rd对LPS诱导小鼠急性脓毒症模型的影响及其潜在的分子机制。结果:一、通过动物实验评估人参皂苷Rd对小鼠急性脓毒症模型的保护作用连续七天腹腔注射人参皂苷Rd,在第七天同时腹腔注射致死量LPS(10mg/kg)12小时构建急性小鼠脓毒症模型,结果显示:人参皂苷Rd显著提高小鼠急性脓毒症模型12小时存活率(p<0.01);显著降低小鼠急性脓毒症模型肺部谷氨酸释放及炎症因子IL-1β和TNF-α的释放(p<0.01);人参皂苷Rd显著减少小鼠急性脓毒症模型肺部组织中性粒细胞浸润、病理损伤评分(p<0.01);此外,人参皂苷Rd显著下调小鼠急性脓毒症模型肺部NMDAR以及p65、JNK蛋白磷酸化水平(p<0.01)。二、构建体外脓毒症细胞模型探索人参皂苷Rd改善脓毒症及其分子机制进一步构建脓毒症状态下中性粒细胞过度活化的细胞模型,利用1.25%DMSO诱导HL-60细胞分化形成中性粒细胞后用LPS刺激中性粒细胞过度活化进行实验。结果显示:LPS处理促进中性粒细胞NMDAR关键位点磷酸化,进而激活NMDAR;人参皂苷Rd能够抑制LPS诱导的中性粒细胞NMDAR激活,进而降低由此引起的谷氨酸释放和钙离子内流并进一步抑制中性粒细胞内ROS的产生以及炎症抑制因子IL-1β和TNF-α的分泌(p<0.01)。人参皂苷Rd对过度活化中性粒细胞的作用与NMDAR抑制剂MK-801类似;人参皂苷Rd能够显著下调LPS刺激后中性粒细胞表面NMDAR主要位点的磷酸化水平(p<0.01);此外,进一步的实验还发现人参皂苷Rd通过抑制JNK信号通路下调NMDAR磷酸化水平,进而抑制过度活化的中性粒细胞释放大量的谷氨酸、ROS、炎症因子IL-1β和TNF-α以及减少细胞钙离子内流降低炎症反应。综上,人参皂苷Rd改善急性脓毒症小鼠模型与降低炎症反应、减少中性粒细胞浸润与其下调NMDAR磷酸化水平有关;人参皂苷Rd通过抑制JNK信号通路下调NMDAR磷酸化水平进而抑制中性粒细胞过度活化降低炎症以改善脓毒症引发的炎症反应。这一结果可为开发新型脓毒症治疗药物提供理论依据,也为深入探究人参皂苷Rd的药理作用机制提供了新的视角。未来研究将进一步探索人参皂苷Rd在脓毒症治疗中的潜力,以及其在调节免疫反应中的具体作用。更多还原

【Abstract】 Sepsis is a severe infection-induced dysregulation of the host’s immunity,which often leads to multi-organ dysfunction in severe cases and has a high lethality rate.In sepsis,the lungs are the first organs to be damaged,and the lungs show hazards such as inflammatory cell infiltration,increased microvascular permeability,and severe edema.Natural immune cells such as over-activated neutrophils infiltrating the lungs will further exacerbate lung inflammation and tissue damage.Currently,there are many challenges in the treatment of sepsis,such as the complexity and heterogeneity of diagnosis,antibiotic resistance and limitations of immunotherapy,etc.There is an urgent need to find new drugs and therapies to improve the survival and quality of life of sepsis patients.In recent years,ginsenoside Rd has been widely used in inflammation-induced diseases due to its anti-inflammatory and immunomodulatory properties,but the amelioration of sepsis by ginsenoside Rd and its mechanism have not been clarified.Therefore,the present study aimed to explore the effects of ginsenoside Rd on LPS-induced acute sepsis model in mice and its potential molecular mechanisms.Results:Ⅰ.Evaluation of the protective effect of ginsenoside Rd on acute sepsis model in mice by animal experimentsGinsenoside Rd was injected intraperitoneally for seven consecutive days,and a lethal dose of LPS(10 mg/kg)was injected intraperitoneally for 12 hours on the seventh day to construct an acute mouse sepsis model.The results showed that ginsenoside Rd significantly increased the 12-hour survival rate of the mouse acute sepsis model(p<0.01);significantly reduced the release of glutamate and the release of inflammatory factors IL-1βand TNF-αin the lungs of the mouse acute sepsis model(p<0.01);ginsenoside Rd significantly reduced the neutrophil infiltration and pathological damage score of the lung tissues of the mouse acute sepsis model(p<0.01);moreover,ginsenoside Rd significantly reduced the neutrophil infiltration and pathological damage score of the lung tissues of the mouse acute sepsis model(p<0.01).0.01);in addition,ginsenoside Rd significantly down-regulated the phosphorylation levels of NMDAR,p65 and JNK proteins in the lungs of the mouse model of acute sepsis(p<0.01).Ⅱ.Construction of an in vitro sepsis cell model to explore ginsenoside Rd and its molecular mechanism to ameliorate sepsisA cell model of neutrophil over-activation in sepsis was further constructed,and the neutrophil over-activation was stimulated by LPS after inducing the differentiation of HL-60 cells to form neutrophils using 1.25%DMSO.The results showed that LPS promoted the phosphorylation of NMDAR at the key site of neutrophil NMDAR and activated NMDAR.Ginsenoside Rd was able to inhibit LPS-induced activation of NMDAR in neutrophils,which in turn reduced the resulting glutamate release and calcium in-flow,and further inhibited the production of ROS as well as the secretion of inflammatory inhibitory factors IL-1βand TNF-α(p<0.01).The effect of ginsenoside Rd on over-activated neutrophils was similar to that of the NMDAR inhibitor MK-801;ginsenoside Rd was able to significantly down-regulate the phosphorylation level of the main NMDAR site on the surface of neutrophils after LPS stimulation(p<0.01);further experiments revealed that ginsenoside Rd inhibited the JNK signaling pathway to down-regulate the phosphorylation level of NMDAR,and then inhibited the over-activated neutrophils(p<0.01).In addition,ginsenoside Rd was found to inhibit the JNK signaling pathway and down-regulate the phosphorylation level of NMDAR,thus inhibiting the release of glutamate,ROS,inflammatory factors IL-1βand TNF-αfrom the over-activated neutrophils,as well as decreasing the inward flow of cytosolic calcium.In conclusion,ginsenoside Rd improved the acute sepsis mouse model by lowering the inflammatory response,reducing neutrophil infiltration and down-regulating the phosphorylation level of NMDAR;ginsenoside Rd improved the sepsis-induced inflammation by inhibiting the JNK signaling pathway and down-regulating the phosphorylation level of NMDAR,thereby inhibiting the over-activation of neutrophils and lowering the inflammation.This result may provide a theoretical basis for the development of novel drugs for sepsis treatment,and also provides a new perspective for further investigation of the pharmacological mechanism of ginsenoside Rd.Future studies will further explore the potential of ginsenoside Rd in the treatment of sepsis and its specific role in regulating the immune response.更多还原