【作者】 陈凯；

【导师】 李术；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2025， 硕士

【摘要】 槲皮素(Quercetin,QUE)是一种天然黄酮类化合物,在多种植物性食品和药用植物中含量丰富。现代药理学研究表明,该化合物具有多重生物活性,包括但不限于抗氧化、抑制炎症反应以及调节免疫功能等。在氧化应激状态下,QUE可通过提升细胞内源性抗氧化防御系统的效能,降低促炎因子的表达水平,进而发挥细胞保护作用。坏死性凋亡(Necroptosis)是一种受严格调控的细胞死亡方式,其典型特征为细胞膜完整性丧失及胞内物质外泄。该过程主要由TNF受体家族或干扰素等信号分子触发,并伴随强烈的炎症反应。研究表明,革兰氏阴性菌细胞壁成分脂多糖(Lipopolysaccharide,LPS)作为一种强效炎症刺激物,可通过特异性激活坏死性凋亡信号转导途径,导致免疫器官结构和功能的异常。胸腺作为禽类重要的中枢免疫器官,是LPS引发机体损伤的主要靶器官之一,其功能受损会显著影响机体的免疫反应。Nrf2信号系统是调控细胞氧化还原平衡和炎症反应的重要分子枢纽。然而,关于QUE能否通过调控Nrf2依赖性信号网络来拮抗LPS诱导的禽类胸腺坏死性凋亡,进而改善炎症相关病理改变目前尚未明确。这一机制的阐明将为QUE在禽类免疫保护中的应用提供重要的理论依据。本课题选择80只1日龄AA肉鸡为试验动物,并常规饲养至21天,随机分为:对照组(CON组)、脂多糖组(LPS组)、槲皮素组(QUE组)及脂多糖和槲皮素共同处理组(LPS+QUE组)。QUE组和LPS+QUE组鸡连续7天按10 mg/kg·BW灌服QUE,CON组和LPS组鸡按动物体重灌服相应体积玉米油。在试验的第7天,LPS组和LPS+QUE组鸡腹腔注射0.15 mg/kg·BW LPS。CON组和QUE组注射相应体积的生理盐水,24小时后,对鸡实施安乐死,采集胸腺组织用于后续试验。体外以鸡淋巴瘤细胞(MSB-1)为研究对象,分别使用10μM QUE和/或30μg/m L LPS处理细胞。此外,为了阐明Nrf2/PERK在缓解鸡胸腺损伤中的作用,在体外分别增加了Nrf2抑制剂ML385和PERK激动剂MK-28处理组。本试验通过H&E染色观察胸腺组织的病理变化。采用免疫荧光染色法检测鸡胸腺组织和MSB-1细胞中内质网应激相关因子(PERK)和坏死性凋亡相关因子(RIPK3和MLKL)的表达水平。对鸡胸腺组织和不同组别MSB-1细胞的氧化应激水平进行检测,并使用实时荧光定量PCR(Quantitative Real-time PCR,q RT-PCR)及蛋白免疫印迹方法(Western Blot,WB)检测Nrf2通路、内质网应激、坏死性凋亡、炎症等基因的表达水平,进一步阐明Nrf2/PERK通路在QUE缓解LPS诱导鸡胸腺细胞坏死性凋亡中的调控机制。研究结果如下:(1)鸡胸腺组织的病理学结构观察结果显示:CON组和QUE组中,细胞形态清晰正常,细胞分布正常,排列规则。在LPS处理的胸腺组织中,细胞排列呈现明显的紊乱状态,皮质与髓质的分界变得模糊不清,淋巴细胞减少。胸腺小叶出现萎缩现象,组织内存在明显的充血和出血迹象。此外,观察到细胞破碎甚至裂解、炎性细胞的浸润以及局灶性的细胞死亡现象。这些病理变化表明LPS对胸腺组织造成了严重的损伤,影响了其正常的结构和功能。在LPS+QUE组中,细胞损伤较LPS组有所缓解。以上结果提示QUE可以缓解LPS诱导的鸡胸腺组织炎性损伤。(2)氧化应激的检测结果显示:鸡胸腺组织和MSB-1细胞ROS染色结果显示,LPS组的ROS荧光强度显著高于CON组和QUE组(P<0.05);加入QUE共处理后,ROS荧光强度显著下降(P<0.05)。与CON组相比,LPS组鸡胸腺组织和MSB-1细胞的抗氧化相关指标如CAT、SOD、GSH-Px和T-AOC活性均发生显著下降(P<0.05),MDA含量则显著上升(P<0.05)。而QUE与LPS共处理组较LPS组抗氧化相关指标的活性提高(P<0.05),MDA含量降低(P<0.05)。(3)Nrf2通路的检测结果显示:鸡胸腺组织和MSB-1细胞中LPS组Nrf2通路相关指标(Keap1、HO-1和NQO1)的m RNA和蛋白表达水平降低(P<0.05);QUE的加入逆转了上述结果(P<0.05)。在体外试验中,与LPS+QUE组相比,使用LPS+QUE+ML385组的Nrf2信号通路相关因子的表达显著降低(P<0.05),揭示QUE通过调控Nrf2通路显著增强了抗氧化基因的表达水平。(4)内质网应激的检测结果显示:在鸡胸腺组织和MSB-1细胞中,LPS组与CON组相比,内质网应激指标(GRP78、PERK、ATF-4和CHOP)的m RNA和蛋白表达水平升高(P<0.05),QUE显著降低LPS诱导的内质网应激相关基因的表达水平(P<0.05)。PERK的体内外荧光结果也与上述结果一致(P<0.05)。在体外试验中使用ML385和MK-28的组别,同LPS+QUE组相比,均加剧了内质网应激发生(P<0.05),降低了钙离子转运相关基因(SERCA和NCX)的表达(P<0.05)。以上结果表明,QUE通过调节Nrf2/PERK通路抑制内质网应激缓解LPS诱导的鸡胸腺损伤。(5)坏死性凋亡的检测结果显示:在鸡胸腺组织和MSB-1细胞中,LPS组较CON组相比,MLKL和RIPK3荧光强度增强(P<0.05)。与LPS组相比,QUE+LPS组MLKL和RIPK3的荧光强度减弱(P<0.05)。LPS组RIPK1、RIPK3和MLKL的m RNA和蛋白水平较CON组显著升高(P<0.05),Caspase8的m RNA和蛋白水平显著降低(P<0.05),QUE+LPS组较LPS组Caspase8 m RNA和蛋白的表达水平发生了显著上调(P<0.05),RIPK1、RIPK3和MLKL的表达下调(P<0.05)。在体外试验中使用MK-28后,同LPS+QUE组相比,坏死性凋亡相关因子的表达显著升高(P<0.05)。以上结果表明QUE可以通过Nrf2/PERK通路缓解LPS诱导的坏死性凋亡。(6)炎性因子的检测结果显示:在鸡胸腺组织和MSB-1细胞中,与CON组相比,LPS组的炎症指标(TNF-α、IL-1β、IL-6、IFN-γ和COX-2)的m RNA和蛋白的表达水平均显著上升(P<0.05);QUE的加入显著降低了炎性因子的表达水平(P<0.05)。在体外试验中使用MK-28后,同LPS+QUE组相比,炎性因子的表达显著升高(P<0.05)。以上结果表明,QUE通过Nrf2/PERK通路缓解LPS导致的炎症反应。综上所述,在LPS诱导的鸡胸腺坏死性凋亡中,QUE能通过上调Nrf2通路因子的表达、减轻氧化应激、抑制内质网应激,尤其是PERK通路的变化、从而有效抑制坏死性凋亡,缓解胸腺组织的炎症反应。本研究阐明了QUE抑制LPS诱发的鸡胸腺坏死性凋亡的作用机制,并明确了Nrf2/PERK通路在这一过程中的重要性,为槲皮素在家禽养殖生产中的应用提供了理论依据。更多还原

【Abstract】 Quercetin(QUE),a flavonoid widely found in vegetables,fruits,and traditional herbal extracts,possesses significant antioxidant,anti-inflammatory,and immunomodulatory biological activities.When cells are subjected to oxidative stress,QUE is able to protect them from damage by enhancing their antioxidant capacity and reducing inflammatory responses.Necroptosis is a form of programmed necrosis that is usually triggered by mediator stimuli such as tumor necrosis factor receptor(TNFR)or interferon.This process leads to the rupture of cell membranes and the release of cellular contents,which triggers a significant inflammatory response.Lipopolysaccharide(LPS),a common inflammatory inducer,is able to cause immune organ damage by activating necro-apoptotic pathways.The thymus,as an important central immune organ in avian species,is one of the main target organs for LPS-induced injury,and its impaired function significantly affects the immune response of the body.The Nrf2pathway is a key regulatory mechanism for cellular antioxidant and anti-inflammatory activities,however,whether QUE can inhibit LPS-induced necroptosis of chicken thymus by activating the Nrf2-associated signaling pathway to reduce the inflammatory injury has not yet been clarified.has not yet been clarified.The elucidation of this mechanism will provide an important theoretical basis for the application of QUE in avian immunoprotection.In this study,80-day-old AA broiler chickens were selected as the experimental animals.They were conventionally raised until day 21 and then randomly divided into:control group(CON group),lipopolysaccharide group(LPS group),quercetin group(QUE group)and lipopolysaccharide and quercetin co-treated group(LPS+QUE group).Chickens of the QUE group and the LPS+QUE group were dosed with QUE at 10 mg/kg-BW for 7 consecutive days,and chickens of the CON and LPS groups were dosed with corn oil in corresponding volumes according to the animal’s The chickens in CON and LPS groups were dosed with the corresponding volume of corn oil according to the body weight of the animals.On day 7,chickens in the LPS and LPS+QUE groups were injected intraperitoneally with 0.15 mg/kg-BW LPS,and chickens in the CON and QUE groups were injected with the corresponding volume of saline,and then euthanized after 24 h.The thymus tissues were collected for the subsequent experiments.In vitro,chicken lymphoma cells(MSB-1)were used for the study,and the cells were treated with the addition of 10μM QUE and/or 30μg/m L LPS,respectively.In addition,to elucidate the role of Nrf2/PERK in alleviating chicken thymic injury,Nrf2 inhibitor ML385and PERK agonist MK-28 treatment groups were added in vitro,respectively.In this experiment,pathological changes in thymic tissues were observed through H&E staining.Immunofluorescence staining was used to detect the expression levels of endoplasmic reticulum stress-related factor(PERK)and necroptosis-related factors(RIPK3 and MLKL)in chicken thymic tissues and MSB-1 cells.The oxidative stress levels in chicken thymic tissues and MSB-1 cells from different groups were measured.Additionally,Quantitative Real-time PCR(q RT-PCR)and Western Blot(WB)were employed to detect the expression levels of genes related to the Nrf2 pathway,endoplasmic reticulum stress,necroptosis,and inflammation.These approaches aimed to further elucidate the regulatory mechanism of the Nrf2/PERK pathway in QUE-mediated alleviation of LPS-induced necroptosis in chicken thymic cells.The results of the study are as follows:(1)Observations on the pathological structure of chicken thymus tissues showed that:In the CON and QUE groups,the cell morphology was clear and normal,the cell distribution was normal and the arrangement was regular.In the LPS-treated thymus tissue,the arrangement of cells showed obvious disorder,the demarcation between cortex and medulla became blurred,and lymphocytes were reduced.Thymic lobules showed atrophy,and there were obvious signs of congestion and hemorrhage in the tissue.In addition,cell disruption and even lysis,infiltration of inflammatory cells,and focal cell death were observed.These pathological changes indicated that LPS had caused severe damage to the thymic tissue,affecting its normal structure and function.In the LPS+QUE group,cell damage was alleviated compared with the LPS group.The above results suggested that QUE could alleviate LPS-induced inflammatory injury in chicken thymus tissue.(2)Detection of oxidative stress showed that ROS staining of chicken thymus tissue and MSB-1cells demonstrated that the ROS fluorescence intensity in the LPS group was significantly higher compared to both the CON and QUE groups(P<0.05);the addition of the QUE co-treatment resulted in a significant decrease in ROS fluorescence intensity(P<0.05).Compared with the CON group,antioxidant-related indexes such as CAT,SOD,GSH-Px and T-AOC activities in chicken thymus tissues and MSB-1 cells in the LPS group underwent a significant decrease(P<0.05),while MDA content increased significantly(P<0.05).In contrast,the activities of antioxidant-related indices were increased(P<0.05)and MDA content was decreased(P<0.05)in the QUE and LPS co-treated group compared to the LPS group.(3)The results of Nrf2 pathway showed that the m RNA and protein expression levels of Nrf2pathway-related indicators(Keap1,HO-1,and NQO1)were reduced in chicken thymus tissues and MSB-1 cells in the LPS group(P<0.05);the addition of QUE reversed these results(P<0.05).In the in vitro assay,the expression of factors related to the Nrf2 signaling pathway was significantly reduced in the group using LPS+QUE+ML385 compared with the LPS+QUE group(P<0.05),revealing that QUE significantly enhanced the expression level of antioxidant genes by regulating the Nrf2 pathway.(4)The results of endoplasmic reticulum stress assay showed that the m RNA and protein expression levels of endoplasmic reticulum stress indicators(GRP78,PERK,ATF-4,and CHOP)were elevated in the LPS group compared with the CON group in chicken thymus tissues and MSB-1 cells(P<0.05),and the expression levels of endoplasmic reticulum stress-related genes induced by QUE were significantly reduced by LPS(P<0.05).The ex vivo and in vivo fluorescence results of PERK were also consistent with the above results(P<0.05).The groups using ML385 and MK-28 in the in vitro assay both exacerbated the occurrence of endoplasmic reticulum stress(P<0.05)and decreased the expression of calcium ion transport-related genes(SERCA and NCX)compared with the LPS+QUE group(P<0.05).The above results suggest that QUE inhibits endoplasmic reticulum stress by regulating the Nrf2/PERK pathway to alleviate LPS-induced injury in chicken thymus.(5)Detection of necrotic apoptosis showed that MLKL and RIPK3 fluorescence intensities were enhanced in chicken thymus tissues and MSB-1 cells in the LPS group compared with the CON group(P<0.05).The fluorescence intensity of MLKL and RIPK3 was weakened in the QUE+LPS group compared with that in the LPS group(P<0.05).m RNA and protein levels of RIPK1,RIPK3 and MLKL were significantly higher in the LPS group compared with that in the CON group(P<0.05),and m RNA and protein levels of Caspase8 were significantly lower in the QUE+LPS group compared with that in the CON group(P<0.05).The expression levels of Caspase8 m RNA and protein in the LPS group underwent significant up-regulation(P<0.05),and the expression of RIPK1,RIPK3 and MLKL was down-regulated(P<0.05).The expression of necrotic apoptosis-associated factors was significantly elevated(P<0.05)after the use of MK-28 in the in vitro assay compared with the LPS+QUE group.The above results suggest that QUE can alleviate LPS-induced necrotic apoptosis through the Nrf2/PERK pathway.(6)The results of inflammatory factors showed that in chicken thymus tissues and MSB-1 cells,the expression levels of m RNA and protein Levels of inflammatory indexes,including TNF-α,IL-1β,IL-6,IFN-γ,and COX-2,showed significant increases in the LPS group compared with the CON group(P<0.05);the addition of QUE significantly decreased the expression of inflammatory factors levels(P<0.05).After the use of MK-28 in the in vitro assay,the expression of inflammatory factors was significantly elevated compared with that in the LPS+QUE group(P<0.05).The above results indicated that QUE alleviated the inflammatory response caused by LPS through the Nrf2/PERK pathway.In summary,in LPS-induced necroptosis in chicken thymus,QUE effectively inhibited necrotic apoptosis and alleviated the inflammatory response of thymus tissue by up-regulating the expression of Nrf2 pathway factors,alleviating oxidative stress,and suppressing endoplasmic reticulum stress,especially the changes in the PERK pathway.In this study,we elucidated the mechanism of action of QUE in inhibiting LPS-induced necroptosis in chicken thymus,and clarified the importance of Nrf2/PERK pathway in this process,which provides a theoretical basis for the application of quercetin in poultry production.更多还原