Cytotoxic effects of peroxynitrite, polymorphonuclear neutrophils, free-radical scavengers, inhibitors of myeloperoxidase, and inhibitors of nitric oxide synthase on bovine mammary secretory epithelial cells

OBJECTIVE: To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers. SAMPLE POPULATION: Polymorphonuclear neutrophils from 3 lactating cows. PROCEDURE: Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots. RESULTS: Lipopolysaccharide, PMA, and < or = 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, > or = 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MAC-T cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or > or = 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner. CONCLUSIONS AND CLINICAL RELEVANCE: Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability.     

Expression and function of TLR2, TLR4, and Nod2 in primary canine colonic epithelial cells

The gut maintains a delicate balance between the downregulation of inflammatory reactions to commensal bacteria and the capacity to respond to pathogens with vigorous cellular and humoral immune responses. Intestinal epithelial cells, including colonic epithelial cells (CECs) possess many properties of cells of the innate immune system, in particular the ability to recognize and respond to microbial antigens. Recognition of microorganisms by CECs is based upon their recognition of signature molecules, called microbe-associated molecular patterns (MAMP), by pattern recognition receptors (PRR) including membrane toll-like receptors (TLR) and cytosolic Nod2, an intracellular counterpart of TLRs. The purpose of this study was to determine whether primary CECs from normal dogs express a functional TLR2, TLR4, and Nod2 and whether they are regulated by inflammatory mediators. We show that canine primary CECs express TLR2, TLR4, and Nod2 that can be modulated in response to their respective MAMPs, lipopolysaccharides (LPS) or peptidoglycans (PGN). Furthermore, we demonstrate that these receptors are functional as evidenced by the induction of cytokine gene expression in response to LPS or PGN.     

Stable transduction of bovine TLR4 and bovine MD-2 into LPS-nonresponsive cells and soluble CD14 promote the ability to respond to LPS

The interaction of bovine cells with lipopolysaccharide (LPS) was explored using human embryo kidney (HEK) 293 cell line stably transduced with bovine toll-like receptor-4 (TLR4) alone or in combination with bovine MD-2. These lines and mock-transduced HEK293 cells were tested by flow cytometry for LPS-fluorescein isothiocyanate (LPS-FITC) binding, nuclear factor kappa B (NFkappaB) activation, interleukin-8 (IL-8) production and interferon-beta mRNA expression/interferon (IFN) type I production. Whereas bovine TLR4 was sufficient to promote binding of high concentrations of LPS-FITC, both bovine TLR4 and MD-2 were required for activation by LPS, as assessed by NFkappaB activation and IL-8 production. Induction of IFN bioactivity was not observed in doubly transduced HEK293 cells, and no evidence for IFN-beta mRNA induction in response to LPS was obtained, although cells responded by IFN-beta mRNA expression to stimulation by Sendai virus and poly-inosinic acid-poly-cytidylic acid (poly(I:C)). Cells stably transduced with both bovine TLR4 and bovine MD-2 responded to LPS by IL-8 production, in decreasing order, in the presence of fetal bovine serum (FCS), of human serum, and of human serum albumin (HSA). The reduced activity in the presence of HSA could be restored by the addition of soluble CD14 (sCD14) but not of LPS binding protein (LBP). This is in contrast to macrophages which show a superior response to LPS in the presence of HSA when compared with macrophages stimulated by LPS in the presence of FCS. This suggests that macrophages but not HEK293 cells express factors rendering LPS stimulation serum-independent. Stably double-transduced cells reacted, in decreasing order, to LPS from Rhodobacter sphaeroides, to LPS from Escherichia coli, to synthetic lipd-IVa (compound 406), to diphosphoryl-lipid-A (S. minnesota) and to monophosphoryl-lipid-A (S. minnesota). They failed to react to the murine MD-2/TLR4 ligand taxol. This resembles the reactivity of bovine macrophages with regard to sensitivity (ED(50)) and order of potency but is distinct from the reactivity pattern of other species. This formally establishes that in order to react to LPS, cattle cells require serum factors (e.g. sCD14) and cell-expressed factors such as MD-2 and TLR4. The cell lines described are the first of a series expressing defined pattern recognition receptors (PRR) of bovine origin. They will be useful in the study of the interaction of the bovine TLR4-MD-2 complex and Gram-negative bovine pathogens, e.g. the agents causing Gram-negative bovine mastitis.     

脂多糖诱导奶牛乳腺上皮细胞先天性免疫反应

采取荷斯坦奶牛乳腺,进行体外分离培养,并纯化细胞。用不同质量浓度(0、1、10、100mg/L)的脂多糖刺激乳腺上皮细胞,采用MTT法检测脂多糖对细胞增殖的影响,半定量PCR检测10mg/L的LPS对乳腺上皮细胞TLR4、TLR2、CD14、MD-2四个基因在不同时间(0、2、6h)mRNA表达水平的差异。结果表明,高剂量(100mg/L)的LPS对乳腺上皮细胞的增殖产生明显影响;LPS刺激乳腺上皮细胞后,导致TLR4、CD14、MD-2mRNA表达迅速升高,而TLR2mRNA弱表达。说明TLR4、CD14、MD-2参与LPS的识别,同时也说明脂多糖刺激乳腺上皮细胞后,乳腺上皮细胞能够产生先天性免疫反应。     

Involvement of connective tissue growth factor (CTGF) in insulin-like growth factor-I (IGF1) stimulation of proliferation of a bovine mammary epithelial cell line

The objective of this study was to determine the mechanism by which insulin-like growth factor-I (IGF1) stimulates proliferation of mammary epithelial cells, using the bovine mammary epithelial cell line MAC-T as a model. IGF1 significantly up- or down-regulated the expression of 155 genes in MAC-T cells. Among the most significantly suppressed was the gene for connective tissue growth factor (CTGF), a secretory protein that has both proliferative and apoptotic effects and is also a low-affinity binding protein of IGF1. IGF1 inhibited CTGF expression through the PI3K-Akt signaling pathway. Administration of growth hormone (GH), a strong stimulator of IGF1 production in vivo, decreased mammary CTGF mRNA in cattle; however, GH did not affect CTGF expression in MAC-T cells, suggesting that IGF1 may also inhibit CTGF expression in the mammary gland. Added alone CTGF stimulated proliferation of MAC-T cells, but in combination with IGF1 it attenuated IGF1's stimulation of proliferation of MAC-T cells. Excess IGF1 reversed this attenuating effect of CTGF. Despite being an IGF binding protein, CTGF did not affect IGF1-induced phosphorylation of IGF1 receptor (IGF1R) or IGF1R expression in MAC-T cells, indicating that the attenuating effect of CTGF on IGF1 stimulated proliferation of MAC-T cells was not mediated by decreasing IGF1's ability to bind to IGF1R or by decreasing IGF1R expression. Overall, these results suggest a novel biochemical and functional relationship between CTGF and IGF1 in the bovine mammary gland, where IGF1 may inhibit CTGF expression to reduce the attenuating effect of CTGF on IGF1 stimulated proliferation of epithelial cells.     

丁酸钠通过AMPK通路调控LPS造成牛乳腺上皮细胞脂代谢紊乱的作用机制

通过向脂多糖（LPS）诱导牛乳腺上皮细胞系（MAC-T）脂代谢紊乱模型中添加不同浓度（2、8、16 μmol·L^-1）的丁酸钠,探讨其对细胞脂代谢的调控机理及炎症损伤的修复作用。分别用1 000 ng·mL^-1 LPS刺激MAC-T细胞9 h后,检测细胞脂滴面积及三酰甘油（TG）含量;用不同浓度的丁酸钠刺激MAC-T细胞12 h后,流式细胞术检测细胞凋亡率;试验共分为5组：对照组、LPS处理组、2 μmol·L^-1丁酸钠+LPS处理组、8 μmol·L^-1丁酸钠+LPS处理组和16 μmol·L^-1丁酸钠+LPS处理组,分别对细胞TG含量、AMPK信号通路蛋白、脂代谢关键基因以及相关炎症因子进行检测。结果显示：LPS会造成MAC-T细胞总脂滴面积显著下降（P<0.05）,TG含量极显著下降（P<0.01）;不同浓度的丁酸钠对MAC-T细胞凋亡率没有影响;与对照组相比,LPS处理组TG含量极显著下降（P<0.01）、P-AMPK表达水平显著上升（P<0.05）、脂合成代谢相关基因ACC、SCD-1以及FAS mRNA表达水平均显著（P<0.05）或极显著（P<0.01）下降、脂分解代谢相关基因CPT-1、CPT-2以及ACO mRNA表达水平均显著上升（P<0.05）、炎症因子TNF-α和IL-6含量显著上升（P<0.05）;与LPS处理组相比,（2、8、16 μmol·L^-1）丁酸钠+LPS处理组TG含量有一定程度上升,P-AMPK表达水平下降,脂合成代谢相关基因表达水平上升,脂分解代谢相关基因表达水平有一定程度下降,炎症因子TNF-α和IL-6含量下降。本研究表明丁酸钠会通过AMPK通路激活脂合成代谢,调控TG的合成,并且对MAC-T细胞的炎症损伤具有一定的缓解作用。     

荷斯坦乳牛乳腺和乳腺上皮细胞中Toll样受体及辅助因子编码基因的检测

参照牛TLR4、TLR2、CD14、MD-2基因序列设计了相应基因的引物。采用RT-PCR技术检测了体外培养的荷斯坦乳牛乳腺和乳腺上皮细胞中Toll样受体TLR4、TLR2及辅助因子CD14、MD-2基因。结果显示,乳腺上皮细胞中存在TLR4、TLR2、CD14和MD-2四个基因的表达,而乳腺中除MD-2未检测到外,其余3个基因均扩增成功。说明该受体及辅助因子可能参与了乳腺的先天性免疫防御。该研究为探讨乳腺的先天性免疫及乳腺上皮细胞在乳腺先天性免疫中的作用奠定了基础。     

蜂胶对细菌脂多糖刺激下奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接蛋白的影响

本试验旨在研究中国蜂胶乙醇提取物（ethanol extract of Chinese propolis，EECP）对细菌脂多糖（lipopolysaccharide，LPS）刺激下体外培养奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接渗透性的影响。EECP中总酚酸和总黄酮含量测定采用福林酚法和硝酸铝法，并建立LPS诱导奶牛乳腺上皮细胞（bovine mammary epithelial cells，MAC-T）炎症模型，采用CCK-8法测定EECP对MAC-T相对增殖率的影响，利用实时荧光定量PCR（RT-qPCR）评估EECP对LPS诱导的MAC-T细胞炎症相关因子（IL-6、IL-8、TNF-α和IL-1β）相对mRNA转录水平；以及对紧密连接蛋白（occludin、ZO-1）相对mRNA转录水平进行检测，并进一步利用免疫荧光技术对紧密连接膜蛋白进行定位，确定EECP对LPS诱导MAC-T细胞炎症紧密连接渗透性的影响。结果显示：EECP中总酚酸含量为106.35 mg没食子酸当量（GAE）·g^-1、总黄酮含量为320.85 mg芦丁当量（RE）·g^-1；CCK-8结果显示EECP的安全浓度为0~15 μg·mL^-1，并可有效提高LPS刺激下MAC-T的活力；LPS刺激显著增加了细胞炎症相关因子IL-6、IL-8、TNF-α和IL-1β mRNA的转录量（P<0.001）；但2.5~15.0 μg·mL^-1 EECP预处理显著降低了IL-6、IL-8、TNF-α和IL-1β mRNA的转录量；与此类似，LPS刺激显著抑制了紧密连接蛋白基因（occludin、ZO-1）mRNA的转录量（P<0.01），而EECP预处理后紧密连接蛋白基因（occludin和ZO-1）mRNA的转录量显著增加（P<0.05）；免疫荧光染色试验也证实EECP能通过上调紧密连接蛋白（occludin、ZO-1）的表达，缓解LPS诱导的乳腺上皮细胞屏障功能紊乱。该结果证实，EECP对细菌脂多糖诱导奶牛乳腺上皮细胞炎症具有良好的保护作用，这为利用中国蜂胶预防奶牛乳腺炎提供了试验基础。     

Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells

ABSTRACT: Escherichia coli is a frequent cause of clinical mastitis in dairy cows. It has been shown that a prompt response of the mammary gland after E. coli entry into the lumen of the gland is required to control the infection, which means that the early detection of bacteria is of prime importance. Yet, apart from lipopolysaccharide (LPS), little is known of the bacterial components which are detected by the mammary innate immune system. We investigated the repertoire of potential bacterial agonists sensed by the udder and bovine mammary epithelial cells (bMEC) during E. coli mastitis by using purified or synthetic molecular surrogates of bacterial agonists of identified pattern-recognition receptors (PRRs). The production of CXCL8 and the influx of leucocytes in milk were the readouts of reactivity of stimulated cultured bMEC and challenged udders, respectively. Quantitative PCR revealed that bMEC in culture expressed the nucleotide oligomerization domain receptors NOD1 and NOD2, along with the Toll-like receptors TLR1, TLR2, TLR4, and TLR6, but hardly TLR5. In line with expression data, bMEC proved to react to the cognate agonists C12-iE-DAP (NOD1), Pam3CSK4 (TLR1/2), Pam2CSK4 (TLR2/6), pure LPS (TLR4), but not to flagellin (TLR5). As the udder reactivity to NOD1 and TLR5 agonists has never been reported, we tested whether the mammary gland reacted to intramammary infusion of C12-iE-DAP or flagellin. The udder reacted to C12-iE-DAP, but not to flagellin, in line with the reactivity of bMEC. These results extend our knowledge of the reactivity of the bovine mammary gland to bacterial agonists of the innate immune system, and suggest that E. coli can be recognized by several PRRs including NOD1, but unexpectedly not by TLR5. The way the mammary gland senses E. coli is likely to shape the innate immune response and finally the outcome of E. coli mastitis.     

猪TLR5基因表达水平与F18大肠杆菌侵染关系分析

为了探究猪Toll样受体（Toll-like receptor 5,TLR5） 基因表达水平与F18大肠杆菌抗性的关系,试验通过不同血清型产肠毒素大肠杆菌（F18ab和F18ac）侵染猪小肠上皮细胞（IPEC-J2）,同时通过脂多糖（LPS）分别诱导IPEC-J2细胞4和8 h,利用实时荧光定量PCR检测TLR5基因表达水平变化,并利用Western blotting进行蛋白表达分析。结果显示,不同血清型大肠杆菌（F18ab和F18ac）菌体侵染IPEC-J2细胞后,TLR5基因表达水平均极显著上调（P<0.01）;LPS诱导IPEC-J2细胞4和8 h后,TLR5基因表达水平均极显著上调（P<0.01）,且在LPS诱导IPEC-J2细胞8 h后,TLR5基因表达水平明显高于诱导4 h。与对照组相比,细胞中TLR5蛋白的表达水平极显著上调（P<0.01）,与LPS诱导及F18大肠杆菌菌体刺激IPEC-J2细胞后mRNA表达水平结果相一致。本研究在细胞水平上分析了TLR5表达水平和F18大肠杆菌侵染的相关性,进一步证实猪TLR5基因的表达水平在细胞抵抗F18大肠杆菌的侵染过程中发挥了重要的调控作用,为今后关于TLR5基因功能及其在大肠杆菌腹泻遗传育种应用的研究奠定基础。     

异体移植炎症因子1通过核因子κB信号促进牛乳腺上皮细胞炎症因子的释放

奶牛乳腺炎是指在不同理化因素刺激下奶牛乳腺的炎性反应,其严重影响了奶牛养殖业的健康发展。许多细胞因子是炎症调节剂,但与奶牛乳腺炎相关的关键细胞因子还未被鉴定。异体移植炎症因子1（allograft inflammatory factor-1,AIF-1）在免疫调节中扮演重要角色,并在多种炎性疾病中过量表达。因此,本研究探讨了牛AIF-1在乳腺炎中的可能作用。首先,利用ELISA试剂盒检测了乳腺炎牛奶中牛AIF-1的含量,并用RT-PCR方法克隆牛AIF-1基因,然后,用亲和层析纯化牛AIF-1蛋白。用此重组蛋白刺激牛乳腺上皮细胞,ELISA试剂盒检测肿瘤坏死因子α、白细胞介素6和单核细胞趋化蛋白1的分泌,Western blot测定IκBα的磷酸化。结果显示,患乳腺炎奶牛乳中AIF-1的平均含量显著高于健康奶牛,而抗生素治愈后的奶牛乳中AIF-1的含量回落到正常水平。这些结果提示,牛AIF-1是一个分泌型蛋白,可能与乳腺炎相关。为了进一步探索这种关系,本研究克隆了牛AIF-1基因并纯化了牛AIF-1蛋白。此蛋白上调了牛乳腺上皮细胞肿瘤坏死因子α、白细胞介素6和单核细胞趋化蛋白1的分泌,并刺激了IκBα的磷酸化。IκBα磷酸化抑制剂BAY 11-7085阻断了牛AIF-1刺激的炎性细胞因子上调。综上表明,牛AIF-1通过核因子κB信号促进了牛乳腺上皮细胞炎症因子的释放。     

Characterization of bovine ileal epithelial cell line for lectin binding,susceptibility to enteric pathogens,and TLR mediated immune responses

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.     

脂多糖对小鼠子宫内膜Toll样受体4介导的炎性信号通路的影响

革兰阴性菌感染可引起子宫内膜炎,机体的Toll样受体4(TLR4)可接受革兰阴性菌的细胞壁主要成分脂多糖(LPS)的刺激引发一系列炎症反应。为了研究LPS对TLR4介导的炎性信号通路的影响,本试验以小鼠为模型,分别用不同浓度的LPS对小鼠进行在体子宫灌注和处理体外培养的子宫内膜上皮细胞系。组织学观察显示,灌注不同浓度LPS后子宫内膜组织中炎性细胞增多。通过RT-PCR对各组中TLR4和核转录因子κB(NF-κB)、IL-6的mRNA表达水平进行检测,发现LPS刺激能够增强TLR4和NF-κB、IL-6 mRNA表达,影响TLR4介导的炎性信号通路。     

丁酸钠对脂多糖诱导的奶牛乳腺上皮细胞系炎性损伤的修复作用

本研究拟通过分析丁酸钠对脂多糖(LPS)诱导的奶牛乳腺上皮细胞系(MAC-T细胞)炎性损伤的修复作用,进一步从体外角度阐述丁酸钠对奶牛乳腺健康的调控机制。在MAC-T细胞中添加不同浓度(0、1、10、100、1 000、10 000 ng/mL)的LPS,检测细胞活力,以确定LPS的适宜浓度,建立细胞氧化损伤模型;并进一步在MAC-T细胞中添加不同浓度(0、2、4、8、16、32μmol/L)的丁酸钠,检测细胞凋亡率,以确定丁酸钠的适宜浓度。最终选用1 000 ng/mL LPS和16μmol/L丁酸钠用于本试验。试验分为3组,分别为对照组、LPS处理组和LPS+丁酸钠处理组,分别对其细胞形态、氧化应激指标及凋亡蛋白mRNA表达水平进行检测。结果表明：1)对照组MAC-T细胞呈扁平的无规则形态,贴壁状态良好;而LPS处理组MAC-T细胞核固缩、破裂,并出现大面积死亡脱落现象;LPS+丁酸钠处理组MAC-T细胞边缘清楚,胞内颗粒较少,死亡脱落现象明显减少。2)与对照组相比,LPS处理组MAC-T细胞中超氧化物歧化酶(SOD)活性和总抗氧化力(T-AOC)显著降低(P<0.05),丙...     

Characterisation of antibodies to bovine Toll-like receptor (TLR)-2 and cross-reactivity with ovine TLR2

Host recognition of conserved pathogen-associated molecular patterns (PAMPs) and their interactions with pattern-recognition receptors, including the Toll-like receptors (TLR) is essential for innate immune response induction. The TLR1 family (TLR1, 2, 6 and 10) is involved in the recognition of gram-positive and gram-negative bacteria and heterodimers of TLR1 or TLR6 with TLR2 are crucial for the identification of several PAMPs. Studies on cell surface expression of TLR in ruminants are hampered by the lack of specific antibodies and no convincingly cross-reactive anti-human antibodies have been described so far. We describe herein four antibodies which recognise bovine TLR2. Differences in TLR2 expression were evident on bovine antigen presenting cells with high level expression on peripheral blood monocytes and monocyte-derived macrophages. Lower levels of expression were evident on dendritic cell populations derived in vitro and ex vivo, and on alveolar macrophages. One of the antibodies recognised TLR2 expression on ovine peripheral blood monocytes. The identification of antibodies specific for bovine and ovine TLR2 will facilitate studies of the role of this important PRR in the initiation of immune responses to important pathogens.     

苜蓿黄酮对脂多糖诱导下奶牛乳腺上皮细胞凋亡的影响

研究苜蓿黄酮对脂多糖(LPS)诱导下奶牛乳腺上皮细胞凋亡的影响。将奶牛乳腺上皮细胞分成4个组,即基础培养基、基础培养基中加入1 μg·mL^-1的LPS、基础培养基中加入1 μg·mL^-1的LPS和75 μg·mL^-1苜蓿黄酮、基础培养基中加入75 μg·mL^-1苜蓿黄酮。细胞在37 ℃, 5% CO₂的培养箱中培养。结果表明:1)LPS刺激12 h后奶牛乳腺上皮细胞活性下降,而添加苜蓿黄酮能够极显著抑制LPS诱导下细胞活性的下降(P<0.01)。2)在LPS刺激下,细胞内的活性氧(ROS)浓度升高,而添加苜蓿黄酮能够显著降低其浓度(P<0.05)。3)LPS显著上调细胞的IL-1β、IL-6、TNF-α、TLR2、TLR4和MyD88表达(P<0.01),而苜蓿黄酮能够显著下调细胞的IL-1β、IL-6、TNF-α和TLR2表达(P<0.01或P<0.05)。4)在LPS刺激下,p53、Caspase3、p38和P-p38蛋白的表达显著升高(P<0.01或P<0.05),而添加苜蓿黄酮能够显著降低p53和p38蛋白的表达(P<0.05)。在LPS诱导下,苜蓿黄酮能够通过降低ROS浓度,抑制细胞凋亡,提高细胞活性;可能通过抑制TLR2/MyD88信号通路来降低细胞炎症因子的表达,从而保护细胞免受炎性损伤。