Measurement of serum lipopolysaccharide binding protein for diagnosis and prognosis prediction in septic patients

AIM: To investigate the role of lipopolysaccharide binding protein (LBP) for diagnosis and prognosis prediction in the septic patients. METHODS: A total number of 80 ICU patients were enrolled. The patients were divided into systemic inflammatory response syndrome (SIRS) group and sepsis group, the patients in sepsis group were divided into non-survivor sub-group and survivor sub-group. We collected the serum samples and analyzed acute physiology and chronic health evaluation (APACHE) II score on the first day of the patients hospitalized in ICU. In addition, we also selected 10 healthy volunteers and collected their serum samples. The serum concentrations of LBP, C-reactive protein (CRP) and procalcitonin (PCT) were measured by ELISA. ROC analysis of LBP, CRP, PCT and APACHE II score was conducted to discriminate among critically ill patients with sepsis and predict the prognosis of the patients with sepsis. RESULTS: The levels of the 4 indicators in the septic patients were higher than those in the patients of SIRS (P<0.05). In addition, serum LBP and APACHE II score in the non-survivor sub-group were higher than those in the survivor sub-group (P<0.05), whereas no difference of the PCT and CRP levels between survivors and non-survivors with sepsis was observed. LBP levels greater than 26.84 mg/L had 97.1% sensitivity and 95.9% specificity to discriminate between SIRS and sepsis. LBP levels greater than 54.16 mg/L had 85.2% sensitivity and 80.0% specificity for prognosis of unfavorable outcome.CONCLUSION: LBP level was more accurately correlated with diagnosis or prognosis prediction than CRP or PCT in patients with sepsis.     

Lipoic acid protects dopaminergic neurons in LPS-induced model of Parkinson's disease

AIM: To evaluate the effect of lipoic acid(LA) on LPS-induced Parkinson disease(PD) model of mice. METHODS: Female C57BL/6 mice of 10-month-old were randomly divided into saline control group, PD group and LA group. The PD mouse model was induced by intranasal instillation of LPS. Assays of tyrosine hydroxylase, microglia and nuclear factor kappa B(NF-κB) were performed by the methods of immunohistochemistry and Western blotting. RESULTS: Intranasal LPS instillation exhibited basic characteristics of PD model. However, LA administration significantly improved motor dysfunction, protected dopaminergic neurons from damage, and inhibited NF-κB activation in inflammatory microglia in the substantia nigra area of the brain. CONCLUSION: LA may exert a profound neuroprotective effect by anti-neuroinflammatory reaction to arrest the progression of PD.     

Altered monocyte and macrophage numbers in blood and organs of chickens injected i.v. with lipopolysaccharide

Lipopolysaccharide (LPS) is a Gram-negative bacteria cell wall component that activates monocytes and macrophages to produce nitric oxide (NO) from inducible nitric oxide synthase. Nitric oxide production in the plasma of chickens peaks 5–6-h post-i.v. LPS injection reflecting iNOS activation. To determine monocyte responsiveness after an i.v. LPS injection, a time course study was conducted examining the concentrations among peripheral blood leukocytes post-i.v. LPS injection in male and female chickens, the proportions among peripheral mononuclear leukocyte (PBMC; containing lymphocytes, thrombocytes, and monocytes) populations isolated from the blood samples collected at various times post-i.v. LPS treatment, and the ability of monocytes to produce NO with and without further LPS stimulation in vitro using the PBMC NO production assay. Additionally, monocyte extravasation activity was determined by analyzing macrophage proportions after the i.v. LPS injection in spleen, lung, and liver tissues. Blood was collected from male and female chickens at 0 h (pre-LPS injection control) and at 1, 3, 6, 24, and 48 h post-LPS injection, and additionally, at 72 h from female chickens. Tissues were collected 0, 1, 6, and 48 h post-i.v. LPS injection from male chickens. Monocyte concentrations dropped substantially by 1 h in both males and females. In males, monocyte concentrations returned to control concentrations by 6 h and increased at 24- and 48-h post-LPS injection, whereas in females, monocyte concentrations recovered more slowly, returning to near control concentrations by 24–48-h and increasing above control levels by 72 h. Lipopolysaccharide stimulated NO production by PBMC cultures established from blood samples obtained at various times post-LPS injection in vivo followed the same pattern as monocyte concentrations in the blood. Hence, NO concentrations within PBMC cultures were dependent upon the number of monocytes that were in the PBMC cultures isolated at different times post-i.v. LPS injection. Furthermore, macrophage proportions in spleen tissues responded similarly to monocyte concentrations in the blood, decreased in lung tissue, and varied widely in liver tissue throughout 48 h after an LPS injection. Monocytes and other leukocytes may attach to the endothelium post-i.v. LPS injection preventing the monocytes from entering the needle during blood collection resulting in what seems to be leukopenia in blood and in PBMC cultures attenuating NO production in PBMC cultures. Furthermore, monocyte differentiation and recruitment from the bone marrow is a likely contributor to the reconstitution and rise of monocyte concentrations in blood samples post-i.v. LPS injection.     

Escherichia coli Lipopolysaccharide-Induced Immunomodulation Along With Oxytocin Administration After Mating as a Treatment Protocol for Persistent Endometritis in Mares

Endometritis is accepted as a major hindrance to achieve optimal reproductive efficiency in mares. The objective of the present study was to evaluate the therapeutic efficacy of combined therapy of immunomodulator and ecbolic as an alternative stand-alone therapy for mares with persistent endometritis. On the basis of history, culture, endometrial cytology, and per rectal and/or ultrasonographic genital examinations, 76 subfertile mares were selected and assigned to three age groups and four treatment (G-1, 2, 3) and control (G-4) groups. At estrus, all the mares were bred once naturally. Thereafter, the mares of G-1 (n = 28) were aseptically treated at 6-hours after natural service with intrauterine infusion (in 50 mL normal saline solution) of 100 μg of Escherichia coli lipopolysaccharide (LPS). Additionally, these mares received two injections of 20 IU of oxytocin (IV) at 12 and 24-hours after infusion. Mares in group G-2 (n = 11) were treated with LPS as mares of G-1, whereas mares in G-3 (n = 12) received oxytocin injections only. The mares of G-4 (n = 23) did not receive any treatment. Pregnancy rates at day 21 and foaling rates were higher (P < .001) in group G-1 than in G-4. In G-1, higher percentage of mares at ages 6–10 years conceived and foaled than mares aged ≥16 years. On re-swabbing of mares that remained nonpregnant, the majority of G-1 and G-2 mares demonstrated sterile cultures and negative cytology, whereas uterine inflammation persisted in mares of G-3 and G-4. In conclusion, the combined therapy was effective for the elimination of persistent endometritis and improved reproductive performance of subfertile mares.     

Curcumin inhibits Porphyromonas gingivalis LPS-induced inflammatory response in human gingival fibroblasts by up-regulating miR-124

AIM To investigate the effects of curcumin （Cur） on the inflammatory response of human gingival fibroblasts （HGFs） induced by Porphyromonas gingivalis （Pg） lipopolysaccharide （LPS） and the role of microRNA-124 （miR-124） in this process. METHODS The HGFs were divided into control group, LPS group （10 mg/L LPS） and LPS+Cur （20, 40 and 80 μmol/L） groups （10 mg/L LPS+corresponding dose of Cur）. After treatment for 24 h, CCK-8 assay was used to measure the cell viability. ELISA was used to measure the levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in the supernatant. The level of miR-124 in the cells was detected by RT-qPCR. The protein levels of nuclear factor kappa B （NF-κB） p-p65 in cytoplasm and nucleus were determined by Western blot, and the nuclear translocation of NF-κB p-p65 was evaluated by laser confocal microscopy. After transfection with mimic-NC or miR-124 mimic, the expression of miR-124 and NF-κB p-p65 protein in the cytoplasm and nucleus of the cells were also detected. RESULTS The cell viability, the level of miR-124 in the cells and NF-κB p-p65 protein level in cytoplasm of LPS group were lower than those in control group （P<0.05）, while the levels of IL-1β and TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were higher than those in control group （P<0.05）. The cell viability, the level of miR-124 in cells and NF-κB p-p65 protein level in the cytoplasm of LPS+Cur （40 and 80 μmol/L） groups were higher than those in LPS group （P<0.05）, while the level of TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were lower than those in LPS group （P<0.05）. The level of IL-1β in the supernatant of LPS+80 μmol/L Cur group was lower than that in LPS group （P<0.05）. The levels of miR-124 and NF-κB p-p65 protein level in the cytoplasm of miR-124 mimic group were higher than those in LPS group and mimic-NC group （P<0.05）, while the level of NF-κB p-p65 proteinlevel in the nucleus was lower than that in LPS group and mimic-NC group （P<0.05）. CONCLUSION Curcumin inhibits the inflammatory response of HGFs induced by Pg LPS, which may be achieved by up-regulating miR-124 and then inhibiting the nuclear translocation of NF-κB p-p65.     

Scutellarin affects LPS-induced oxidative stress and apoptosis of glomerular epithelial cells by up-regulating miR-7-5p

AIM To investigate the effect of scutellarin （SCU） on oxidative stress and apoptosis induced by lipopolysaccharide （LPS） in human glomerular epithelial cells and its mechanism. METHODS Human glomerular epithelial cells were cultured in vitro, and were treated with LPS （1.0 mg/L） to establish a cell injury model. The cells were divided into normal control （NC） group, LPS group, NC+SCU group, LPS+SCU group, LPS+miR-NC group, LPS+microRNA-7-5p （miR-7-5p） group, LPS+SCU+anti-miR-NC group and LPS+SCU+anti-miR-7-5p group. Cell viability was detected by CCK-8 assay. Apoptosis was detected by flow cytometry. The intracellular malondialdehyde （MDA） content and superoxide dismutase （SOD） activity, and lactate dehydrogenase （LDH） activity in the cell culture supernatant were determined by kit. RT-qPCR was used to detect the expression level of miR-7-5p. RESULTS Compared with NC group, the cell viability, miR-7-5p expression and SOD activity in LPS group were significantly reduced, and the apoptotic rate, MDA content and LDH activity were significantly increased （P<0.05）. Compared with LPS group, the cell viability, miR-7-5p expression and SOD activity in LPS+SCU group were significantly increased, and the apoptotic rate, MDA content and LDH activity were significantly reduced （P<0.05）. Compared with LPS+miR-NC group, the cell viability and SOD activity in LPS+miR-7-5p group were significantly increased, and the apoptotic rate, MDA content and LDH activity were significantly reduced （P<0.05）. Compared with LPS+SCU+anti-miR-NC group, the cell viability and SOD activity in LPS+SCU+anti-miR-7-5p group were significantly reduced, and the apoptotic rate, MDA content and LDH activity were significantly increased （P<0.05）. CONCLUSION Scutellarin inhibits LPS-induced oxidative stress damage and apoptosis in glomerular epithelial cells via up-regulating miR-7-5p expression.     

脂多糖对细菌生物被膜形成及厚壳贻贝幼虫变态的影响

为探究脂多糖（LPS）对海洋细菌生物被膜形成、海洋贝类幼虫变态所产生的作用,深入了解影响厚壳贻贝附着变态的因素,本实验使用不同浓度的LPS直接刺激厚壳贻贝幼虫,观察其对幼虫附着变态的直接作用;同时在海假交替单胞菌形成生物被膜的过程中添加不同浓度的LPS,分析生物被膜生物学特性的变化,及变化后的生物被膜对厚壳贻贝幼虫附着变态的影响。结果显示,3种浓度的LPS均可直接诱导厚壳贻贝幼虫的变态;10.0mg/L浓度LPS处理后的生物被膜其细菌密度、膜厚度明显降低,且膜厚度降低了12.1%,胞外产物中多糖、脂类显著增加,其中代表性多糖可拉酸的含量增加了35.4%,同时其对幼虫变态的诱导作用也提高了53.3%。因而,在厚壳贻贝幼虫的附着变态过程中,LPS具有直接诱导作用,同时还可以通过调控生物被膜胞外物质,特别是可拉酸的生成,间接影响厚壳贻贝幼虫的附着变态。本研究成果对海洋细菌互作关系研究、厚壳贻贝养殖产业的改善以及海洋防污技术研发具有重要推动价值。     

miR-486 targeted regulation of PTEN on LPS-induced apoptosis of alveolar epithelial cell A549

AIM: To investigate the effect of microRNA-486 (miR-486) on lipopolysaccharide (LPS)-induced apoptosis of alveolar epithelial cell A549. METHODS: A549 cells were treated with LPS, and the expression of miR-486 was detected by RT-qPCR. miR-486 mimics were transfected into LPS-induced A549 cells, and RT-qPCR was used to detect the up-regulation effect. The apoptotic rate was analyzed by flow cytometry and the protein levels of cleaved caspase-3 (C-caspase-3) and C-caspase-9 were determined by Western blot. The target gene prediction software was used to predict the target gene PTEN of miR-486. Luciferase reporter vector was used to identify the target relationship. pcDNA 3.1-PTEN and miR-486 mimics were co-transfected into A549 cells to detect the effect of PTEN up-regulation on apoptosis of miR-486 mimics transfected A549 cells stimulated with LPS. RESULTS: After LPS treatment, the expression of miR-486 in A549 cells was significantly decreased (P<0.05). Transfection of miR-486 mimics significantly up-regulated the expression of miR-486 in A549 cells stimulated with LPS (P<0.05). The apoptotic rate of A549 cells and the protein levels of C-caspase-3 and C-caspase-9 were significantly increased after LPS treatment (P<0.05). Up-regulation of miR-486 significantly down-regulated LPS-induced apoptosis of A549 cells (P<0.05). The expression of PTEN was negatively regulated by miR-486. Transfection of pcDNA 3.1-PTEN significantly increased the expression of PTEN, promoted the apoptosis and increased the protein levels of C-caspase-3 and C-caspase-9 in A549 cells stimulated with LPS after co-transfection with miR-486 mimics(P<0.05). CONCLUSION: miR-486 inhibits PTEN expression and reduces LPS-induced apoptosis of A549 cells.     

阴沟肠杆菌B29菌株脂多糖合成基因（waaL,waaG）缺失突变株的构建及分析

脂多糖（LPS）在革兰氏阴性细菌中的功能作用在不同菌株中已有研究,但尚未有对肠杆菌属LPS结构与功能的报道。本研究试图构建能引起肥胖的阴沟肠杆菌B29菌株waaL和waaG基因的缺失突变株,以了解突变体菌株产生的LPS结构与活性与野生菌株产生的LPS的差异。根据同源重组技术原理,利用广宿主自杀性质粒构建成敲除载体pKNG101△waaL和pKNG101△waaG,转化E.coli SM10菌株,并与B29菌株进行双亲杂交,再以抗生素和蔗糖筛选条件筛选waaL和waaG基因缺失突变株;利用多重PCR、ERICPCR、PCR-DGGE、DNA测序结果均证实突变株B29△waaL和B29△waaG构建成功;最后,对野生菌株B29与两个突变株生物学特性的差异进行了比较。银染实验结果表明野生型B29菌株的脂多糖为光滑型结构,突变株的LPS结构明显缺失O抗原,B29△waaG突变株同时还缺失了外部核心部分;用鲎试剂法检测LPS的内毒素活性显示,突变株的内毒素活性与野生型相比有较大幅度下降;B29△waaL突变株的生长速率与野生型相当,而B29△waaG突变株的生长速率则相对降低。本研究成功构建两种内毒素活性下降的突变株,为下一步利用突变株研究B29菌株在肥胖发生中的机制奠定基础。