Corticosterone inhibits LPS-induced expression of NLRP3 in mouse macrophages and its relation with XO

AIM: To investigate the inhibitory effect of corticosterone (CORT) on lipopolysaccharide (LPS)-induced expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and its relation with xanthine oxidase (XO). METHODS: An inflammatory model of mouse macrophage RAW 264.7 was established by stimulating with LPS. Total cellular protein was extracted after the macrophages were treated with CORT at different concentrations (0~900 μg/L). The protein levels of NLRP3 and caspase-1 were determined by Western blot. According to the treatments, the macrophages were divided into control group, LPS group, LPS+CORT group and LPS+allopurinol group. Cell components were extracted at 0, 0.5, 1, 1.5 and 2 h. The protein levels of NLRP3 and XO were determined by Western blot,and the mRNA expression of NLRP3 and XO was detected by real-time PCR. RESULTS: CORT at 700 μg/L and above significantly inhibited the expression of NLRP3 and the activation of caspase-1 in the macrophages induced by LPS (P<0.05). Compared with LPS group, the expression of NLRP3 and XO in LPS+CORT group was inhibited (P<0.05), and the expression of NLRP3 in LPS+allopurinol group was also reduced (P<0.05).CONCLUSION: High concentration of CORT inhibits the expression of NLRP3 in LPS-induced mouse macrophages, which is associated with XO. The inhibitory effect of CORT may be related to the reduction of XO expression.     

紫锥菊多糖对LPS损伤后IEC-6分泌TNF-αmRNA的影响

[目的]研究紫锥菊多糖(EPS)在内毒素(LPS)损伤小肠上皮细胞(IEC-6)时对肿瘤坏死因子(TNF)表达的影响,以探讨 EPS对损伤细胞的作用机制。[方法]采用 TRIzon试剂提取总RNA,RT-PCR扩增 TNF-α mRNA,琼脂糖凝胶电泳,并进行电泳及图像分析。[结果]50μg/ml EPS 可以部分抑制 LPS 刺激 IEC-6产生的TNF-α mRNA水平,而200、500μg/ml EPS随着浓度的增加,其抑制 TNF-α mRNA的水平逐渐增加;将 IEC-6分别用50、100、200及500μg/ml EPS预处理24 h,然后用10μg/ml LPS刺激达1、4 h,采用 RT-PCR方法分析得, LPS诱导 TNF-α mRNA表达被 EPS有效地抑制,4 h的抑制率高于1 h的抑制率。[结论]EPS通过抑制 LPS刺激细胞分泌 TNF-α mRNA的产生而起到肠道粘膜的保护作用,且 EPS对这种抑制作用具有浓度及时间依赖性。     

Effect of maternal restraint stress during gestation on temporal lipopolysaccharide-induced neuroendocrine and immune responses of progeny

The impact of gestational dam restraint stress on progeny immune and neuroendocrine temporal hormone responses to lipopolysaccharide (LPS) challenge was assessed. Maternal stress (5-min snout snare restraint stress during days 84 to 112 of gestation) increased (P < 0.05) the magnitude of tumor necrosis factor (TNF)-α, interleukin-6, epinephrine (E), norepinephrine, and serum amyloid A (SAA) production following LPS infusion in the offspring. Moreover, these effects appear to be dependent on gender for TNF-α, E, and cortisol production. However, maternal stress did not affect (P > 0.05) the normalization of proinflammatory cytokines or neuroendocrine hormones produced following LPS. Collectively, these results indicate that maternal stress impacts aspects of the proinflammatory cytokine and stress hormone response in their progeny following LPS dosing of the offspring. This response is potentially responsible in part for the resultant changes to SAA production. Because several of the changes observed here are dependent on pig gender, these results are also the first evidence that inherent epigenetic factors coupled with maternal stress impact the cumulative response to stress and LPS in young pigs.     

AHV-PI attenuates endothelial cell injury induced by LPS

AIM:To investigate the effect of platelet inhibitor from Agkistrodon halys venom (AHV-PI) on lipopolysaccharide (LPS) induced human umbilical vein endothelial cells (HUVECs) injury in vitro, and to explore its mechanism. METHODS:Cultured HUVECs were induced inflammatory injury by LPS (1 mg/L). The experiment was divided into blank control group, LPS group, AHV-PI group and AHV-PI+LPS group. The viability of HUVECs was measured by MTT assay. The morphological changes of HUVECs were observed under inverted microscope. The optimum concentration of AHV-PI at 5 mg/L was selected. Flow cytometry was used to detect apoptosis of HUVECs. Immunohistochemical method was used to observe the expression of tissue type plasminogen activator (t-PA) and plasminogen activator indhibitor-1 (PAI-1) of HUVECs. ELISA was used to detect the concentrations of intercellular adhesion molecule-1 (ICAM-1) and tissue factor (TF) in the supernatant. The activation and translocation of NF-κB subunit p65 were observed by immunofluorescence staining. RESULTS:The HUVECs were spindle shaped, the ratio of length to width was increased, the cells were fibroblast-like, and granular substance appeared in the cytoplasm in LPS group. The viability and morphological changes of HUVECs were not significantly affected as treated with AHV-PI at concentration of 0~5 mg/L, but the viability of HUVECs induced by LPS was inhibited and the morphological changes were alleviated. Compared with the blank control group, the levels of TF and ICAM-1 in the supernatant increased, and the expression of t-PA and PAI-1 in the HUVECs was decreased in LPS group. Compared with the LPS group, the contents of TF and ICAM-1 in the supernatant were significantly decreased, the expression of t-PA and PAI-1 in the HUVECs was increased and the expression of nucleus NF-κB p65 was decreased in AHV-PI+LPS group (P<0.05). CONCLUSION:AHV-PI reduces HUVECs damage. The protective mechanism is related to the inhibition of cytokine secretion and NF-κB activation.     

复合植物精油对脂多糖刺激仔猪肝脏的保护作用

试验旨在研究复合植物精油(OCT)对脂多糖(LPS)刺激仔猪肝脏的保护作用。选取18头仔猪,随机分为对照组、LPS组和OCT+LPS组,每组6个重复。对照组与LPS组饲喂基础日粮,OCT+LPS组在基础日粮中添加50 mg/kg OCT。试验期21 d。于试验第21天,LPS组与OCT+LPS组仔猪腹腔注射LPS,对照组注射等量的生理盐水,3 h后采血,6 h后屠宰。结果表明:与对照组相比,LPS刺激提高了血浆谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、谷酰转肽酶(GGT)、肝脏诱导型一氧化氮合酶(i-NOS)的活力(P0.05),肝脏热休克蛋白70(HSP70)基因的相对表达量显著提高(P0.05);仔猪肝脏表皮生长因子(EGF)、白细胞介素10(IL-10)、胰岛素样因子(IGF-1)、丝氨酸蛋白激酶(mTOR)基因的相对表达量显著降低(P0.05)。在日粮中添加50 mg/kg的OCT,可缓解LPS导致的仔猪血浆ALT、AST、GGT活力的升高以及肝脏i-NOS活力的升高(P0.05),且有缓解LPS导致的仔猪肝脏MPO活力升高的趋势(P0.1),另外可缓解LPS导致的仔猪肝脏HSP70基因相对表达量的升高以及EGF、IL-10、IGF-1、mTOR等基因相对表达量的降低(P0.05)。综上可知,日粮中添加50 mg/kg的OCT可缓解LPS刺激引起的肝脏炎症反应,提高仔猪肝脏细胞的生长和增殖,进而缓解LPS刺激导致的仔猪肝脏氧化应激损伤。     

Supplementation of diets for gestating sows with zinc amino acid complex and gastric intubation of suckling pigs with zinc-methionine on mineral status, intestinal morphology and bacterial translocation in lipopolysaccharide-challenged early-weaned pigs

Eighty male pigs from 20 litters were used to evaluate dietary addition of 250 mg/kg of Zn from zinc amino acid complex (ZnAA) to sows during the last trimester of gestation and gastric intubation of 40 mg Zn from soluble zinc methionine (ZnMet) to suckling pigs at birth and on day 7 and 14 (weaning) on small intestinal morphology, Zn status and bacterial translocation in early-weaned pigs. At weaning, pigs were challenged with an intramuscular injection of saline without or with 120 μg/kg BW of lipopolysaccharide (LPS; from Escherichia coli O26:B6) and were euthanized 24 h later prior to collection of intestinal lymph nodes and small intestinal sections. Zinc concentration in serum 7 days after birth and at weaning were higher in pigs from ZnAA-supplemented sows and those receiving gastric intubation with ZnMet (P = 0.05 and P < 0.0001, respectively). Post-weaning liver tissue concentrations for Zn (P < 0.0001) and Fe (P = 0.04) were higher and for Cu lower (P < 0.0001) in pigs intubated with ZnMet. Pigs from ZnAA-supplemented compared with control-fed sows tended (P < 0.1) to have increased villi height and villus:crypt ratio in the jejunum and higher (P = 0.1) goblet cell counts in the ileum. Goblet cell counts of ZnMet-intubated (P = 0.03) and LPS-challenged pigs (P = 0.05) were also higher in the jejunum. Supplementation of ZnAA to gestating sows increased (P = 0.04) E. coli colony forming unit counts in the small intestinal mesenteric lymph nodes of early-weaned pigs. In conclusion, dietary addition of ZnAA to gestating sows and gastric intubation of ZnMet improved Zn status of suckling pigs.     

Effect of menhaden fish oil supplementation and lipopolysaccharide exposure on nursery pigs. I. Effects on the immune axis when fed diets containing spray-dried plasma

The objective of the present study was to evaluate the potential immunological benefit of adding menhaden fish oil to the diet of weaned pigs. Twenty-four crossbred male pigs were weaned at approximately 18 days of age and placed on a complex nursery diet containing 30% lactose and 7% plasma protein with 6% corn oil as the fat source (Cont, n=12) or with 5% menhaden fish oil and 1% corn oil as the fat source (MFO, n=12) for a period of 15 days. Body weights did not differ (P>0.78) between dietary groups either at the beginning or end of the 15 days feeding period. On day 15, all pigs were non-surgically fitted with an indwelling jugular catheter. On d 16, pigs received an i.v. injection of either saline (n=6/dietary group) or lipopolysaccharide (LPS; 150 μg/kg body weight; n=6/dietary group) and blood samples were collected at 30 min intervals for a period of 5 h. Serum was harvested and stored at −80 °C for analysis of cortisol (CS), corticosteroid-binding globulin (CBG), tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-γ). There was no significant effect of diet on basal concentrations (Time 0) of any of the blood parameters analyzed. A Time×Treatment×Diet interaction (P<0.03) was observed for serum CS such that those pigs which consumed the MFO diet followed by LPS treatment had a reduced CS response as compared to the LPS-treated pigs on the Cont diet. A Time×Treatment interaction (P<0.01) was observed for serum CBG such that LPS treatment reduced circulating CBG as compared to the saline-treated pigs. Time×Treatment×Diet interactions were also observed for serum concentrations of TNF- (P=0.084) and IFN-γ (P=0.022) such that both the TNF- and IFN-γ response to the LPS challenge was lower in those pigs receiving the MFO diet as compared to the LPS-treated pigs on the Cont diet. Overall, serum CS was negatively correlated with the CBG response (r=−0.40, P<0.001), however, the strongest negative correlation was observed in the LPS-treated pigs which consumed the MFO diet (r=−0.63, P<0.001). While further studies are needed to evaluate the immunological response of including MFO in the nursery pig diet, the present study demonstrates that supplementation with MFO does indeed alter the immunological response to an LPS challenge.     

Influence of glycine on LBP mRNA expression induced by LPS in the liver of rats

AIM:To study the influence of glycine(GLY) on lipopolysaccharide-binding protein(LBP) mRNA expression induced by LPS.METHODS:The level of LBP mRNA expression in liver tissues of rats was examined by RT-PCR, and the effects of glycine on LBP mRNA expression in liver tissues of rats induced by LPS were investigated.RESULTS: The level of LBP mRNA expression in hepatic tissue of rats in the LPS group was significantly higher than that in the control group(P＜0.01), the level of LBP mRNA expression in the hepatic tissue of rats in the LPS+GLY group was lower than that in the LPS group(P＜0.01).CONCLUSION:LPS can induce LBP mRNA expression in the hepatic tissue of rats, glycine can inhibit LBP mRNA expression in the hepatic tissue of rats treated by LPS.     

Vinpocetine injection attenuates lipopolysaccharide-induced acute lung injury in rats

AIM:To observe the inhibitory effects of vinpocetine injection on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in the rats and to explore the underlying mechanisms.METHODS:Male Wistar rats (n=50) were randomly divided into 5 groups:control group,ALI model group,and low,medium and high doses of vinpocetine treatment groups.The rats in control group were injected with 0.9% NaCl at 5 mL/kg through femoral vein.The rats in ALI model group received LPS at 10 mg/kg through femoral vein.After injected with LPS,the rats in vinpocetine treatment groups received vinpocetine at 0.2 mg/kg,0.7 mg/kg or 1.2 mg/kg via intraperitoneal injection.The pathological changes of the lung tissues were observed under microscope with HE staining.The cell apoptosis in the lung tissues was detected by TUNEL staining.Myeloperoxidase (MPO) activity was measured by the method of spectrophotometry.The protein expression of NF-κB,ICAM-1,VCAM-1,Bax and Bcl-2 was determined by Western blot.RESULTS:Compared with ALI group,administration of vinpocetine significantly attenuated the structural injury of the lung and the infiltration of inflammatory cells.Moreover,vinpocetine decreased cell apoptosis and MPO activity in the lung tissues of ALI rats.In addition,the protein expression of NF-κB,ICAM-1,VCAM-1 and Bax was inhibited after vinpocetin treatment,whereas Bcl-2 expression was increased.CONCLUSION:Vinpocetine attenuates LPS-lung injury by reducing MPO activity and regulating NF-κB,ICAM-1,VCAM-1,Bax and Bcl-2 protein expression.     

MiR-155在脂多糖诱导的RAW264.7巨噬细胞炎症反应中的表达研究

[目的]探讨miR-155在脂多糖(LPS)诱导的RAW264.7巨噬细胞炎症反应中的表达及糖皮质激素(GCs)的干预影响。[方法]体外培养RAW 264.7巨噬细胞,分别用浓度为10.0、1.0、0.1μg/ml LPS刺激RAW 264.7巨噬细胞,于2、6、12、24、36 h 5个时间点收集上清用ELISA检测白介素-6(IL-6)蛋白浓度;实时定量-PCR检测miR-155在2、6、12、36 h 4个时间点的表达变化。[结果]IL-6在各浓度LPS处理组、各时间点其含量均高于对照组(CK);miR-155的表达在各时间点LPS组均高于LPS+GCs和CK。[结论]LPS可以诱导RAW264.7巨噬细胞的炎症反应,炎症反应时miR-155高表达,糖皮质激素可抑制miR-155的表达。