Lipopolysaccharide Stimulation Group Research Articles

Temporal and spatial expression analysis of periostin in mice periodontitis model.

This study aimed to investigate the temporal and spatial changes in the expression of periostin during periodontal inflammation in mice. A periodontitis model was constructed using silk thread ligation. Mice were randomly divided into five groups including control group, 4-day ligation group, 7-day ligation group, 14-day ligation group, and self-healing group (thread removal for 14 days after 14-day ligation). Micro-CT and histological staining were performed to characterize the dynamic changes in the mouse periodontal tissue in each group. RNAscope and immunohistochemical staining were used to analyze the pattern of changes in periostin at various stages of periodontitis. The cell experiment was divided into three groups: control group, lipopolysaccharide (LPS) stimulation group (treated with LPS for 12 h), and LPS stimulation removal group (treated with LPS for 3 h followed by incubation with medium for 9 h). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of periostin, transforming growth factor-β1 (TGF-β1), and matrix metalloproteinase 2 (MMP2). Significant alveolar bone resorption was observed 7 days after ligation. With increasing duration of ligation, the damage to the mouse periodontal tissue was aggravated, which manifested as increased osteoclasts, widening of the periodontal membrane space, and decreased alveolar bone height. Some degree of periodontal tissue repair was observed in the self-healing group. Periostin expression decreased at 4 and 7 days compared with the control group and increased at 14 days compared with 4 and 7 days. A significant recovery was found in the self-healing group. The qRT-PCR results showed that the expression of periostin and TGF-β1 in the LPS stimulation group decreased compared with that in the control group but significantly recovered in the LPS removal group. Periostin expression in the PDL of mice showed a downward and upward trend with inflammation progression. The significant recovery of periostin expression after removing inflammatory stimuli may be related to TGF-β1, which is crucial to maintain the integrity of the PDL.

Rosiglitazone alleviates LPS-induced endometritis via suppression of TLR4-mediated NF-κB activation.

The aim of this study was to investigate the anti-inflammatory effect of Rosiglitazone (RGZ) on lipopolysaccharide (LPS) -induced Endometritis and explore its possible mechanism. The preventive and therapeutic effects of RGZ on Endometritis were studied in vivo and in vitro. A total of 40 female C57BL/6 mice were randomly divided into the following 4 groups: RGZ+LPS, RGZ control, LPS and DMSO control. The mice uterine tissue sections were performed with HE and immunohistochemical staining. Human endometrial stromal cells (HESCs) were cultured, and different concentrations of LPS stimulation groups and RGZ and/or a TLR4 signaling inhibitor TAK-242 pretreatment +LPS groups were established to further elucidate the underlying mechanisms of this protective effect of RGZ. The HE results in mice showed that RGZ+LPS group had less tissue loss than LPS group. Immunohistochemical staining (IHC) results showed that the expression of TLR4 after RGZ treatment was significantly lower than that in LPS group. These findings suggested that RGZ effectively improves the pathological changes associated with LPS-induced endometritis by inhibiting TLR4. Reverse transcription-polymerase chain reaction and western blot analysis demonstrated that RGZ pretreatment suppresses the expression of Toll-like receptor 4 (TLR4) and its downstream activation of nuclear factor-κB (NF-κB). In vitro, RGZ inhibited LPS-stimulated expression of proinflammatory cytokines in a dose-dependent manner and also downregulated LPS induced toll-like receptor 4 (TLR4) expression and inhibited phosphorylation of LPS-induced nuclear transcription factor-kappa B (NF-κB) P65 protein. These results suggest that RGZ may inhibit LPS-induced endometritis through the TLR4-mediated NF-κB pathway.

Influence of organism stimulation with bacterial lipopolysaccharide on nitric oxide production and metabolism in rat heart on the background of metabolic syndrome

Aim. The aim of the study was to establish the changes in nitric oxide production and metabolism in rat heart during combined influence of organism stimulation with bacterial lipopolysaccharide (LPS) and modeling of metabolic syndrome (MetS).
 Materials and methods. The study was conducted on 24 mature male Wistar rats weighing 200–260 g. Experiment lasted 60 days. The animals were divided into 4 groups of 6 animals each: control group, MetS group, LPS stimulation group, LPS + MetS group. MetS was reproduced by using a 20 % fructose solution as the only source of drinking water. LPS of Salmonella typhi was administered at a dose of 0.4 μg/kg intraperitoneally. Animals from LPS + MetS group received a 20 % fructose solution as the only source of drinking water and were administered LPS. In 10 % tissue homogenate of rat heart we studied: total activity of NO-synthases (NOS), activity of constitutive (cNOS) and inducible (iNOS) isoforms, activity of nitrate (NaR) and nitrite (NiR) reductases, concentration of peroxynitrites (ONOO-), nitrites, nitrosothiols and hydrogen sulfide.
 Results. Combination of MetS and stimulation of organism with LPS led to increase in total NOS activity by 32.72 % compared to control group. Activity of cNOS did not change compared to control group. Activity of iNOS increased by 33.76 %. Arginase activity decreased by 23.53 %. NaR activity and NiR activity were increased by 86.67 % and by 149.29 %, respectively. Combination of MetS and stimulation of organism with LPS led to decrease in nitrite and nitrosothiols concentration by 38.73 % and by 54.79 %, respectively. Under these conditions concentration of ONOOelevated by 398.0 % compared to control group. Concentration of H S decreased by 27.56 %.
 Conclusions. Combination of metabolic syndrome and stimulation of organism with bacterial lipopolysaccharide leads to prevalence of peroxynitrite formation during increased nitric oxide production NO-synthase-dependent and nitrate-nitrite-NO pathways in rat heart.

Dietary gamma-aminobutyric acid (GABA) improves non-specific immunity and alleviates lipopolysaccharide (LPS)-induced immune overresponse in juvenile Chinese mitten crab (Eriocheir sinensis)

Inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is an immunomodulator to inhibit immune-mediated pro-inflammatory response and has been used to treat various immune-related diseases in mammals. However, the immunoregulatory effect of GABA in crustaceans has not been reported. This study evaluates the regulatory effect of dietary GABA supplementation on the innate immune status and immunoregulatory potential in lipopolysaccharide (LPS)-induced immune response in juvenile Eriocheir sinensis. Juvenile crabs were fed with six diets supplemented with graded GABA levels (0, 40, 80, 160, 320 and 640 mg/kg dry matter) for 8 weeks and then 24 h LPS challenge test was carried out. The results showed that dietary GABA supplementation significantly decreased mortality at 4 and 8 weeks. Moreover, the hemocyanin content, acid phosphatase, and alkaline phosphatase activities significantly increased in the crabs fed GABA supplementation compared with the control. On the contrary, the alanine aminotransferase and alanine aminotransferase activities in serum decreased significantly in the GABA supplementation groups compared with the control. Similarly, superoxide dismutase activity, glutathione content, and the transcriptional expression of the antioxidant-related genes and immune-related genes were significantly higher in the GABA supplementation groups than in the control. In addition, the mRNA expressions of anti-lipopolysaccharide factors (ALF 1, ALF 2, ALF 3) and inflammatory signaling pathways related genes (TLR, Myd88, Relish, LITAF, P38-MAPK, ADAM17) were significantly up-regulated in LPS stimulation groups compared with PBS treatment. Meanwhile, pro-apoptosis-related genes' mRNA expressions were significantly up-regulated, and anti-apoptosis-related genes were significantly down-regulated under LPS stimulation compared with PBS treatment. However, GABA pretreatment effectively alleviated LPS-induced immune overresponse and apoptosis. Therefore, this study demonstrates that dietary GABA supplementation could be used as an immunomodulator to improve the non-specific immunity and antioxidant capacity and alleviate the immune-mediated immune overresponse of juvenile E. sinensis.

The changes of MRP2 expression in three kinds of pulmonary inflammation models: the downregulation occurred in cigarette smoke extract (CSE) stimulation group and CSE plus LPS stimulation group, unchanged in LPS stimulation group

The transporter multidrug resistance protein 2 (MRP2) can transport some tobacco carcinogens and plays an important role in the transport of mediators related to pulmonary inflammatory diseases. However, it is not fully understood whether the pulmonary inflammation caused by cigarette smoke extract (CSE) and lipopolysaccharide (LPS) is related to the regulation of MRP2. In this study, CSE and LPS were used alone and in combination as stimuli to induce pulmonary inflammation. In addition, the establishment of a pulmonary inflammation model was verified by animal experiments in vivo. We found that compared with those in the control group, the expression of MRP2 protein was downregulated and the expression of inflammatory cytokines was upregulated in pulmonary inflammation in the CSE group and the CSE combined with LPS group. However, there was almost no change in the expression of MRP2 stimulated by LPS alone. Our results show that CSE and CSE combined with LPS downregulate the expression of MRP2 under inflammatory conditions, while LPS has almost no effect on the expression of MRP2 under inflammatory conditions. The in vivo experimental results of CSE combined with LPS were consistent with the cellular results of CSE combined with LPS, which provides a model and basis for other studies of the role of MRP2 in pulmonary inflammation.

Extracellular histones are involved in lipopolysaccharide-induced alveolar macrophage injury by activating the TWIK2-NLRP3 pathway

To explore the role and mechanism of extracellular histones involved in lipopolysaccharide (LPS)-induced alveolar macrophage injury. The mouse alveolar macrophage cell line (MH-S) was cultured in vitro and passaged, and the cells were cultured to 80% of cells for cell proliferation. The cells were stimulated with 1 mg/L LPS for 3 hours and 50 mg/L exogenous histones for 3, 6, 12, and 24 hours, respectively (LPS+histones 3, 6, 12, 24 h groups), and other groups included phosphate buffered saline (PBS) control group (PBS group), LPS alone stimulation group (LPS group), the exogenous histones alone stimulation group (histones group) and heparin pretreatment histones group (heparin+LPS+histones group). The cells in each group were challenged with different reagent, the expression of lactate dehydrogenase (LDH) and inflammatory factors in the supernatant were detected by enzyme linked immunosorbent assay (ELISA), and the change of intracellular K+ concentration was detected by FluxORTM II green potassium channel. The proteins such as potassium channel protein (TWIK2), inflammasome (NLRP3), and apoptosis associated speck like protein containing a CARD (ASC) were determined by Western Blot. Compared with the PBS group, the levels of LDH and inflammatory factors such as interleukin (IL-1β, IL-18) and tumor necrosis factor-α (TNF-α) were significantly increased after LPS stimulation group. Compared with the LPS group, the levels of LDH and inflammatory factors were significantly increased after the treatment with exogenous histones, and reached a peak after 3 hours of the histones stimulation [LDH (U/L): 123.10±1.83 vs. 85.32±1.66, IL-1β (mg/L): 40.75±2.60 vs. 18.78±1.37, IL-18 (mg/L): 49.94±2.45 vs. 30.19±1.82, TNF-α (mg/L): 36.51±1.56 vs. 20.84±1.61, all P < 0.01]. Western Blot results showed that compared with the LPS group, NLRP3, ASC and TWIK2 protein expression were significantly up-regulated in the LPS+histones group (NLRP3/GAPDH: 0.80±0.02 vs. 0.57±0.02, ASC/GAPDH: 0.57±0.02 vs. 0.38±0.01, TWIK2/GAPDH: 0.65±0.01 vs. 0.41±0.01, all P < 0.01), and the expression of the above proteins were significantly down-regulated after heparin pretreatment (NLRP3/GAPDH: 0.28±0.02 vs. 0.80±0.02, ASC/GAPDH: 0.25±0.02 vs. 0.57±0.02, TWIK2/GAPDH: 0.35±0.01 vs. 0.65±0.01, all P < 0.01), indicating that histones could activate NLRP3 through TWIK2 to participate in inflammatory reaction. In addition, intracellular K+ concentration in LPS+histones group decreased significantly compared with the LPS group (fluorescence intensity: 35.48±2.53 vs. 83.92±3.11, P < 0.01). Compared with LPS+histones group, K+ concentration increased significantly after pretreatment with heparin (fluorescence intensity: 72.10±1.78 vs. 35.48±2.53, P < 0.01), indicating that extracellular histones could cause K+ massive efflux through TWIK2, and thus mediate NLRP3 activation and participate in inflammatory injury of alveolar macrophages. Extracellular histones can cause inflammatory damage in alveolar macrophages, and its mechanism may be related to the activation of NLRP3 by extracellular histones activation of TWIK2 channel to promote K+ efflux.

Effect of autophagy on expression of neutrophil programmed death ligand-1 in mice with sepsis

To investigate the effect and mechanism of autophagy on the expression of neutrophil programmed death ligand-1 (PD-L1) in mice with sepsis. (1) In vivo experiment: male C57BL/6 mice aged 6-8 weeks were divided into sham operation group (Sham group), cecum ligation and perforation (CLP) group, and rapamycin (RAP)+CLP group by random number table with 10 mice in each group. The sepsis model was reproduced by CLP, and the cecum and perforation were not ligated in Sham group, and other operations were the same as CLP group. The mice in RAP+CLP group were intraperitoneally injected with autophagy agonist RAP 4 mg×kg-1×d-1 7 days before modeling, while the mice in Sham group and CLP group were not treated. Lung, liver, spleen and pancreas tissues were harvested for immunohistochemical staining 4 days after the operation, and the infiltration of neutrophils in various organs was observed under light microscope. Meanwhile, the expressions of immunosuppressive molecule PD-L1 and autophagy marker microtubule-associated protein 1 light chain 3 (LC3) in lung neutrophils were determined by immunofluorescence staining. (2) In vitro experiment: mouse bone marrow neutrophils were extracted and re-suspended to 1×1010/L, and they were divided into blank control group (without any treatment), RAP control group (RAP 100 μmol/L), autophagy inhibitor Bafilomycin A1 (Baf) control group (Baf 10 μmol/L), lipopolysaccharide (LPS) stimulation group (LPS 1 mg /L), RAP+LPS group, and Baf+LPS group. The latter two groups were pretreated with 100 μmol/L RAP or 10 μmol/L Baf 30 minutes before LPS stimulation, respectively. The expression of PD-L1 mRNA of neutrophils was determined by reverse transcription-polymerase chain reaction (RT-PCR) at 0, 4, 12 hours after LPS stimulation. At the same time, the expressions of PD-L1, LC3 and p62 at the protein level were determined by Western Blot. (1) In vivo experiment: according to immunohistochemical experiments, a large amount of infiltration of neutrophils in lung, liver, spleen and pancreas was found at 4 hours after CLP. In the immunofluorescence, with the time extension after CLP, the positive expression of LC3 in the lung tissue showed a decreased tendency, and PD-L1 expression was significantly increased. RAP pretreatment could promote the expression of LC3 and reduce the expression of PD-L1 in CLP mice. (2) In vitro experiment: in terms of mRNA levels, with the extension of LPS stimulation time, the expression of PD-L1 mRNA in mouse neutrophils was increased continuously, and peaked at 12 hours, it was significantly higher than that in the blank control group (2-ΔΔCT: 72.2±10.0 vs. 13.0±0.8, P < 0.01). Compared with LPS stimulation group, the expression of PD-L1 mRNA in RAP+LPS group was significantly down-regulated [12-hour PD-L1 mRNA (2-ΔΔCT): 47.4±7.3 vs. 72.2±10.0, P < 0.01]. In Baf+LPS group, PD-L1 mRNA expression was significantly up-regulated as compared with that in LPS stimulation group [12-hour PD-L1 mRNA (2-ΔΔCT): 109.1±7.4 vs. 72.2±10.0, P < 0.01]. At the protein levels, at 4 hours after LPS stimulation, the positive expressions of PD-L1, LC3 and p62 were increased significantly as compared with those in the blank control group, and PD-L1 and p62 were increased continuously with time. Compared with the LPS stimulation group, the expressions of PD-L1 and p62 in the RAP+LPS group were significantly down-regulated, while the expression of LC3 was continually increased, indicating that the level of autophagy was increased, and autophagy was circulated smoothly. On the contrary, the expressions of PD-L1, LC3 and p62 in the Baf+LPS group were significantly up-regulated, indicating that the binding of autophagy and lysosome was blocked, and autophagy was not smooth. In sepsis, the infiltration of neutrophils in all organs increased, and the expression of PD-L1 of neutrophils in lungs was increased significantly, while the expression level of autophagy was decreased. The expression of PD-L1 stimulated by LPS can be inhibited by autophagy agonists, and promoted by autophagy inhibitors. PD-L1 has a negative regulatory effect on sepsis. It can reduce the expression of PD-L1 molecule in sepsis by targeting autophagy, so as to improve sepsis.

Interleukin-1 receptor-associated kinase M regulates lipopolysaccharide-induced respiratory burst in peritoneal macrophages of mouse through inhibiting p22phox

Objective To investigate whether interleukin-1 receptor-associated kinase M (IRAK-M) regulates p22phox-mediated lipopolysaccharide (LPS)-induced macrophage respiratory burst. Methods Mouse peritoneal macrophages were isolated and cultured in votro. The cells were divided into endotoxin tolerance (ET) group and control group (NC group). The ET group was stimulated with 10 μg/L lipopolysaccharide (LPS) for 2 h, then with 100 μg/L LPS re-stimulation. The NC group was not pretreated, directly stimulated by 100 μg/L LPS. Supernatant was collected 3 h later, TNF-α and IL-6 levels were detected by enzyme-linked immunosorbent assay (ELISA). H2O2 and O2- levels were also detected. Western blot was used to detect the expression levels of IRAK-M and p22phox proteins. IRAK-M small interfering RNA (siRNA) was used to interfere with IRAK-M. Groups are set as follows: NC-control group, the interfering with IRAK-M+LPS stimulation group (NC-si-IRAK-M group), the interfering with endotoxin tolerance group (ET-control group), and the interfering with endotoxin tolerance group (ET-si-IRAK-M group). The interference efficiency was detected by polymerase chain reaction (PCR). The protein expression levels of IRAK-M and p22phox were also measured. After successful IRAK-M interference, p22phox was inhibited by apocynin. The groups were: dimethyl sulfoxide (DMSO)+endotoxin tolerance group (control-placebo group), apocynin+endotoxin tolerance group (control-apocynin group), DMSO+interference IRAK-M+endotoxin tolerance group (si-IRAK-M-placebo group) and interfering with IRAK-M+apocynin+endotoxin tolerance group (si-IRAK-M-apocynin group). The supernatant was taken for detection of TNF-α, IL-6, H2O2 and O2- levels. Results Comparing with ET-control group, after interference with IRAK-M, the protein content of ET-si-IRAK-M was significantly decreased. In contrast, the protein content of p22phox was significantly increased (P<0.05); after interfering with IRAK-M and inhibiting p22phox, the levels of TNF-α, IL-6 and H2O2 O2- in the IRAK-M-apocynin group were significantly lower than the levels of these proteins in si-IRAK-M-placebo group (P<0.05). Compared with NC group, TNF-α, IL-6, H2O2 and O2- levels were decreased in ET group. The ET group has low expression of p22phox protein and high expression of IRAK-M protein (P<0.05). Compared with the ET-control group, the expression of IRAK-M in the ET-si-IRAK-M group was significantly decreased while the expression of p22phox was significantly increased after interference IRAK-M(P<0.05). Compared with the si-IRAK-M-placebo group, the levels of TNF-α, IL-6 and H2O2, O2-in the si-IRAK-M-apocynin group were significantly decreased after interfering with IRAK-M and inhibiting p22phox(P<0.05). Conclusions IRAK-M exerts a negative regulatory effect on endotoxin-induced respiratory outbursts of macrophages in mice by inhibiting p22phox. Key words: Interleukin-1 receptor-associated kinase M; p22phox; Immunosuppression; Macrophage; Respiratory burst; Oxidative stress; Sepsis

Expression of TLR4 and its effect on Treg cells in early pregnancy decidual stromal cells after lipopolysaccharide treating

ObjectiveTo investigate the expression of TLR4 in human early pregnancy decidual stromal cells (DSCs) induced by lipopolysaccharide (LPS) and its effect on the peripheral blood regulatory T (Treg) cells subgroup in women of childbearing age. MethodIsolating and cultivating normal human early pregnancy DSCs followed by treatment with 0, 25, 50, 100 and 200 ng/ml LPS, and the expression level of TLR4 mRNA was detected by RT-PCR. After 3 or 4 generation we divide the DSCs into 5 groups: ①Control group: Cultivation of peripheral blood lymphocyte (PBLC); ②Co-cultivation group: Co-cultivation of PBLC and DSCs; ③LPS stimulation group: PBLC + DSCs + LPS; ④PDTC blocking-up group: PBLC + DSCs + LPS + PDTC; ⑤TLR4 blocking-up group: PBLC + DSCs + LPS + TLR4mAb. In ①–④ groups, western blot was used to detect the expression of inhibitory factor-κB (IκB-α) protein and RT-PCR was used to detect the expression of FoxP3 mRNA. In ①–⑤ groups, flow cytometry was applied to detect the percentage of Treg cells subgroup. ResultsThe purity of primary cultured DSCs was more than 95%. RT-PCR results showed that the expression level of TLR4 mRNA increased gradually with the augment of LPS concentration. Western blot and RT-PCR showed that the expression of IκBα protein and FoxP3 mRNA in the other 3 groups was significantly higher than that in the control group (P < 0.05), and the expression of IκBα protein and FoxP3 mRNA in LPS stimulation group was lower than that in the co-cultivation group (P < 0.05). Compared with the LPS stimulation group, the expression of IκBα protein and FoxP3 mRNA in PDTC blocking-up group was higher than that in the LPS stimulation group (P < 0.05), but still lower than the co-cultivation group (P < 0.05). The proportion of Treg cells in the other 4 groups detected by flow cytometry was significantly higher than that in the control group (P < 0.05). Compared with the co-cultivation group, the Treg cells ratio of the LPS stimulation group was significantly decreased (P < 0.05). The proportions of Treg cells in PDTC blocking-up group and TLR4 blocking-up group were higher than that in the LPS stimulation group, but still lower than that in the co-cultivation group (P < 0.05). There was no significant difference between the PDTC blocking-up group and the TLR4 blocking-up group (P > 0.05). ConclusionHuman early pregnancy DSCs can promote the differentiation of Treg cells. LPS can stimulate the expression of TLR4 in early pregnancy DSCs and decrease the proportion of Treg cells in PBLC, with NF-κB signaling pathway being the potential underlying mechanisms.

The inflammatory effect of exosomes derived from alveolar epithelial cells stimulated by lipopolysaccharide on the alveolar macrophages

Objective To explore the inflammatory effect of exosomes derived from alveolar epithelial cells stimulated by lipopolysaccharide (LPS) on the alveolar macrophages (NR8383). Methods The alveolar epithelial cells disposed with different treatments were co-cultured with alveolar macrophages by using a Transwell system separately. Alveolar epithelial cells (RLE-6TN) were randomly divided into 4 groups: normal group, LPS-stimulated group, exosome inhibitor group, and exosome inhibitor pretreatment + LPS stimulation group. NR8383 cultured alone was considered as a blank control. After the 12-h co-culture, the real-time PCR (qPCR) was performed to examine the mRNA relative expression of IL-6, TNF-α, and IL-1β in NR8383 cells. To further explore the role of exosomes derived from RLE-6TN on alveolar macrophages mediated inflammationary response, the experimental exosomes (exosomes derived from LPS-induced RLE-6TN) and control exosomes exosomes derived from normal RLE-6TN were extracted by gradient ultracentrifugation. Transmission electron microscopy and Western blotting analyses was performed to identify the exosomes, and qNano particle diameter analyzer was conducted to measure the particle diameter of exosomes. In vitro, NR8383 cells were divided into 3 groups which were cultured with exosomes derived from LPS-stimulated RLE-6TN at a concentration of 10 μg/mL (experimental group), exosomes derived from untreated RLE-6TN at the same concentration of 10 μg/mL (control group), and the PBS at the same volume with experimental group (PBS group), respectively for 12 h. After the treatment, the phagocytosis of NR8383 cells was observed by laser confocal microscope and the release of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in supernatants of NR8383 was detected by enzyme-linked immunosorbent assay ELISA Results (1)In the co-culture experiment, the mRNA relative expression of pro-inflammatory cytokine in the LPS group was significantly increased compared with the blank control group (P 0.05). Conclusions Exosomes derived from LPS-disposed alveolar epithelial cells activate the alveolar macrophage-mediated inflammatory response. Key words: Lipopolysaccharide; Exosomes; Alveolar epithelial cells; Alveolar macrophages; Inflammation

Doses Lactobacillus reuteri depend on adhesive ability to modulate the intestinal immune response and metabolism in mice challenged with lipopolysaccharide.

The objective of this study was to evaluate the modulatory effects of Lactobacillus reuteri ZJ617 and ZJ615, which have high and low adhesive abilities, respectively, and Lactobacillus rhamnosus GG (LGG) on immune responses and metabolism in mice stimulated with lipopolysaccharide (LPS). Six C57BL/6 mice per group were orally inoculated with ZJ617, ZJ615 or LGG for one week (1 × 108 CFU/mouse) and i.p. injected with LPS (10 mg/kg) for 24 h. Compared with the LPS stimulation group, ZJ615, ZJ617 and LGG significantly decreased TNF-α levels in the sera of mice stimulated by LPS. ZJ615 and LGG significantly down-regulated mRNA levels of cytokines and Toll-like receptors, and suppressed activation of MAPK and NF-κB signaling, while ZJ617 up-regulated anti-inflammatory cytokine IL-10 mRNA levels in the ilea of mice stimulated by LPS. Correlation analysis confirmed that adhesive ability is relative with the immunomodulation in the ilea of mice. There were 24, 7 and 10 metabolites and 10, 9 and 8 major metabolic pathways with significant differences (VIP > 1, P < 0.05) between the LPS and ZJ617 + LPS groups, the LPS and ZJ615 + LPS groups, and the ZJ617 + LPS and ZJ615 + LPS groups, respectively. The results indicated that both ZJ617 and ZJ615 could modulate the intestinal immune responses and metabolism in LPS-stimulated mice.

Protective effect of insulin on lipopolysaccharide-induced impairments of H9c2 cells and corresponding mec-hanism

Objective To research the protective effect of insulin(IN)on lipopolysaccharide(LPS)-induced impairments of rat cardiomyocytes H9c2, and the role of uncoupling protein 2(UCP2)in this process. Methods Using randomized controlled grouping, after cultured for 24 h, H9c2 cells were randomly divided into 5 groups as follows: control group, LPS stimulation group (LPS group), LPS+ 70 IU/L IN group(IN 70 IU/L group), LPS+ 350 IU/L IN group(IN 350 IU/L group), and LPS+ 700 IU/L IN group(IN 700 IU/L group). H9c2 cells in IN group were treated with 70 IU/L, 350 IU/L or 700 IU/L IN 15 min before LPS stimulation, and H9c2 cells in control group were treated with an equal volume of saline.After that, cells in group LPS and IN were treated with LPS for 24 h. Lactate dehydrogenase (LDH)in the culture was determined with LDH detecting assay kit.The activity of reactive oxygen species (ROS)and superoxide dismutase (SOD), and content of malonaldehyde (MDA)were determined by colorimetric detection.Cell viability was evaluated by cell count kit-8.The expressions of UCP2 in transcription and translation levels were detected through transcription polymerase chain reaction and Western blot respectively. Results The levels of LDH, MDA, and intracellular ROS in LPS group significantly increased compared with control group[LDH: (829.3±75.3)U/L vs (223.5±23.6)U/L, MDA: (60.90±5.73) nmol/mgprot vs (19.70±1.99)nmol/mgprot, ROS: (410.2±81.6)U/well vs (94.3±18.5)U/well, all P<0.05)], while the cell viability and SOD activity significantly decreased[cell viability: 0.822±0.058 vs 1.012±0.023, SOD: (49.20±5.81)U/mgprot vs (89.80±2.57)U/mgprot, all P<0.05]. And the mRNA and protein expressions of UCP2 in LPS stimulation group were up-regulated(1.867±0.130 vs 1.028±0.097, 0.288±0.018 vs 0.180±0.008, all P<0.05).350 IU/L and 700 IU/L IN intervention significantly decreased the levels of LDH, MDA and intracellular ROS[LDH: (568.2±35.7)U/L, (622.8±27.6)U/L vs (829.3±75.3)U/L, MDA: (29.20±4.20)nmol/mgprot, (42.10±2.32)nmol/mgprot vs (60.90±5.73)nmol/mgprot, ROS: (270.3±46.8)U/well, (301.5±16.9)U/well vs (410.2±81.6)U/well, all P<0.05], increased the cell survival and the levels of SOD activity[cell viability: 0.960±0.029, 0.906±0.039 vs 0.822±0.058, SOD: (75.20±2.21)U/mgprot, (61.20±3.38)U/mgprot vs (49.20±5.81)U/mgprot, all P<0.05]. And IN with 350 IU/L and 700 IU/L increased the mRNA and protein expression of UCP2(3.830±0.265, 2.855±0.215 vs 1.867±0.130, 0.464±0.215, 0.355±0.006 vs 0.288±0.018, all P<0.05). Compared with 70 IU/L and 700 IU/L IN group, 350 IU/L IN group had better results. Conclusions IN attenuates LPS-induced oxidative injury in H9c2 cells, which is probably mediated through up-regulating the expression of UCP2. Key words: Insulin; Lipopolysaccharide; H9c2 cell; UCP2; Oxidative stress

High glucose enhances permeability of human pulmonary microvascular endothelial cells by lipopolysaccharide stimulation in vitro and effect of DDAH/NOS/NO imbalance on its pathogenesis

To investigate the damage to endothelial cells incubated in high concentration of glucose challenged by lipopolysaccharide (LPS), and the likely mechanisms of injury. Human pulmonary microvascular endothelial cells (PMVECs) were divided into the following groups: normal glucose group (NG), normal glucose + LPS stimulation group (NGL), high glucose stimulation group (HG), and high glucose + LPS stimulation group (HGL). The cells were incubated with normal glucose (5.5 mmol/L, contained 10% calf serum) or high glucose (33 mmol/L) for 5 days to form a monolayer of cells before LPS stimulation (10 mg/L) for 24 hours. The microfilaments (F-actin) were investigated by immuno-fluorescence, and the number and size change in fenestrae were examined by scanning electron microscopy. The permeability of vascular endothelial cell was assessed by trans-PMVEC horseradish peroxidase (HRP) flux. Western blotting was used to determine the expressions of dimethylarginine dimethylaminohydrolase 2 (DDAH2), inducible nitricoxide synthase (iNOS) and endothelial nitricoxide synthase (eNOS). Nitric oxide (NO) was assessed by Griess method. When stimulated with LPS, cells incubated with high glucose showed obvious microfilament rearrangement, a larger average diameter and increased number of F-actin, as well as higher HRP permeability on the hyperglycemic PMVECs compared with PMVECs cultured with normal glucose [(53.62±6.70)% vs. (23.63±3.92)%, P<0.01]. Furthermore, high glucose down-regulated DDAH2 expression (arbitrary units, AU, 0.33±0.08 vs. 0.77±0.14 , P<0.01) and up-regulated LPS-stimulated iNOS production (1.40±0.29 vs. 1.04±0.09, P<0.01), as well as increased LPS-stimulated nitrite/nitrate and stable NO end products compared with normal (20.36±2.25 μmol/L vs. 7.99±0.33 μmol/L, P<0.01) and reduction of eNOS levels was observed (0.67±0.09 vs. 0.91±0.17, P<0.05). It demonstrated that, in vitro high glucose deteriorate LPS-stimulated F-actin rearrangement and hyperpermeability of an endothelial monolayer, and the worsened imbalance of the NO pathway may lead to endothelial damage in microcirculation.

The effects of activated protein C on the von Willebrand factor and von Willebrand factor cleaving protease of rat aortic endothelial cell induced by lipopolysaccharide

To investigate the activated protein C (APC) on the von Willebrand factor antigen (vWFAg) and von Willebrand factor cleaving protease (ADAMTS-13) protein expression in rat aortic endothelial cells (RAECs) induced by lipopolysaccharide (LPS). RAECs from Wistar rats were cultured with the tissue explants adherence method. RAECs were cultured for one week, After one week culture, RAECs in 4-5 generations were divided into control group, LPS stimulation groups (1 mg/L) and APC intervention groups (0.1, 1 and 10 mg/L APC was added after LPS stimulation). The supernatants were obtained at 12, 24, 48, and 72 hours after LPS stimulated to determine the vWFAg and protein of ADAMTS-13 expression by enzyme-linked immunoadsorbent assay (ELISA). In the control group, RAECs expressed little vWFAg and protein of ADAMTS-13. With stimulation of LPS, the vWFAg was significantly increased at 12 hours, and reached the peak at 48 hours [(285.45±30.13)%], and the level of ADAMTS-13 (μg/L) was gradually decreased, and reached the nadir at 72 hours (13.32±2.37), there was significant difference compared with control group [vWFAg: (94.53±7.83)%, ADAMTS-13: 115.76±2.36, both P<0.01). The effects on vWFAg promoting and ADAMTS-13 inhibition after LPS stimulation could be dose-dependently reversed by APC. 10 mg/L of APC could decrease the peak of vWFAg at 48 hours of LPS stimulation [(198.43±17.92)% vs. (285.45±30.13)%], and increase the minimize of ADAMTS-13 (μg/L) at 72 hours of LPS stimulation (125.25±2.70 vs. 13.32±2.37), with significant difference (both P<0.01). After stimulation with LPS, the level of vWFAg was time-dependent increased, as the protein of ADAMTS-13 was decreased. APC could attenuate the effect of LPS on vWFAg and protein of ADAMTS-13 with dose-dependent and time-dependent patterns.

Effect of Shenfu injection on expression of lipopolysaccharide --induced microRNA-146a in alveolar macrophages

To study the effects of Shenfu injection (SF) on the expression of lipopolysaccharide(LPS)-induced microRNA-146a (miR-146a) in rat alveolar macrophages (AMs), and to extrapolate its potential anti-inflammatory mechanisms. In vitro cultured rat AMs (NR8383 cells) were randomly divided into control group, LPS stimulation group, and SF stimulation group. The LPS stimulation group was challenged with a final concentration of 1 mg/L LPS, and to the control group an equal volume of phosphate buffer solution (PBS) was added instead. For SF treated group, SF in different concentrations (1 ml/L or 10 ml/L) was used during incubation of AMs for half an hour, and then LPS was added (1 mg/L final concentration). After 6 hours, the cells and were collected. MiRNA-146a expression [reverse transcription-polymerase chain reaction (RT-PCR)] in cells and tumor necrosis factor-α (TNF-α ) content [enzyme-linked immunosorbent assay (ELISA)] in culture supernatant were determined for each group. Both the expression of miR-146a and TNF-α content in LPS stimulation group were significantly elevated compared with control group [miR-146a (expression folds): 5.92 + 1.57 vs. 1.04 +0.38; TNF-α (ng/L): 636.93 _ 30.21 vs. 20.46 + 2.81; both P<0.05]. Compared with LPS stimulation group, the expression of miR-146a was significantly upregulated in cells in both 1 ml/L and 10 ml/L SF stimulation groups, but TNF- α content was significantly reduced in the supernatant [miR-146a (expression folds): 7.02 + 0.91, 8.11 ± 1.07 vs. 5.92 -1.57; TNF-α (ng/L): 447.24 +21.29, 357.83 +19.73 vs. 636.93 +30.21, all P<0.05] in a dose-dependent manner (both P<0.05). SF could up-regulate miR-146a expression in AMs in a dose-dependent manner, and it was speculated that miR-146a might be involved in the anti-inflammatory processes with SF treatment.

Induction of expression of von Willebrand factor cleaving protease of rat aortic endothelial cell by lipopolysaccharide challenge

To investigate the dose response effect and time response effect of lipopolysaccharide(LPS) on von Willebrand factor cleaving protease (ADAMTS-13, Vwf-cp) mRNA expression and protein in rat aortic endothelial cells (RAECs). RAECs were grown by culturing of aortic tissue. When ARECs were cultured for one week, it was co-cultured by 1:3 to reach 4-5 generations. ARECs were randomly divided into five groups: control group and four LPS stimulation groups (0.01, 0.1, 1 and 5 μg/ml) . The RAECs and supernatants were obtained at 12, 24, 48, and 72 hours after being stimulated by LPS. ADAMTS-13 mRNA expression of RAECs was assessed by quantitation reverse transcription-polymerase chain reaction (RT-PCR), and protein of ADAMTS-13 in supernatants was determined by enzyme linked immunoadsorbent assay (ELISA). In the control group RAECs were shown to express ADAMTS-13 at both protein and mRNA levels. With the increase of concentration of LPS, or increase in stimulus duration, expression of ADAMTS-13 mRNA and protein were gradually lowered. Compared with the control group (25.22 ±1.41), the level of ADAMTS-13 mRNA in 0.01 μg/ml LPS stimulation group was markedly decreased at 48 hours (18.78±0.86, P<0.01). At 24 hours, the levels of ADAMTS-13 mRNA (23.43±0.63, 22.41±0.76) were markedly decreased in 0.1 μg/ml and 1 μg/ml LPS stimulation groups (P<0.05 and P<0.01). The level of ADAMTS-13 mRNA (20.01±2.47) in 5 μg/ml LPS stimulation group was markedly decreased at 12 hours (P<0.01). Compared with the control group [(115.76±2.36) ng/ml], protein level of ADAMTS-13 [(113.43±1.07) ng/ml] was markedly decreased at 12 hours in 0.01 μg/ml LPS stimulation group (P<0.05). The protein level of ADAMTS-13 [(7.63±2.64) ng/ml] was lowest in 5 μg/ml LPS stimulation group at 72 hours (P<0.01). Normal RAECs can express ADAMTS-13 at both mRNA and protein to certain extent. The expression of ADAMTS-13 mRNA and protein are decreased after LPS challenge in different concentrations for different duration in dose dependent and time dependent manner.

Effect of inhibition of cytosolic phospholipase A₂ on Leptin release from human umbilical vein endothelial cells induced by lipopolysaccharide

To determine Leptin levels in supernatant fluid of culture of human umbilical vein endothelial cells (ECV-304) after being challenged by lipopolysaccharide (LPS) and calcium ion vector A23187, and to explore the possible relation between Leptin release and cytosolic phospholipase A(2) (cPLA(2)) activity in an inflammatory cell model. ECV-304 cells were cultured in vitro. Experiment 1: the cells were divided into seven groups: blank control group, LPS 5, 10, 20 μg/ml stimulation groups, A23187 0.1, 1.0, 10.0 μmol/L stimulation groups. The supernatants were collected at 6, 12 and 24 hours.Experiment 2: according to the results of experiment 1, the cells were divided into eight groups: blank control group, LPS 20 μg/ml stimulation group, the inhibitor of cPLA2 AACOCF3 0.1, 1.0, 10.0 μmol/L plus LPS stimulation groups, the inhibitor of mitogen-activated protein/extracellular signal-regulated protein kinase kinase 1/2 (MEK1/2) U0126 0.1, 1.0, 5.0 μmol/L plus LPS stimulation groups, with AACOCF3 or U0126 added 1 hour before the addition of LPS, and the supernatants were collected 24 hours after the addition of LPS. Leptin level was determined by radioimmunoassay. Experiment 1: with increase in LPS concentration and prolongation of time, Leptin release was decreased gradually. After 24 hours of interaction the concentration of Leptin (ng/ml) in LPS 20 μg/ml group was decreased significantly compared with the blank control group (0.540±0.109 vs. 0.823±0.048,P<0.05). However, A23187 had no significant effect on Leptin release. Experiment 2: LPS rendered cells to release less Leptin (ng/ml: 0.558±0.069 vs. 0.825±0.067,P<0.05); by adding AACOCF3 or U0126 in different concentration before adding LPS rendered the cells to release more Leptin (ng/ml), and it showed concentration-dependent (the AACOCF3 0.1, 1.0, 10.0 μmol/L groups were 0.673±0.135, 0.723±0.055, 0.797±0.062, respectively; the U0126 0.1, 1.0, 5.0 μmol/L groups were 0.698±0.112, 0.862±0.184, 0.935±0.145, respectively). The release of Leptin in AACOCF3 1.0 μmol/L, 10.0 μmol/L and U0126 1.0 μmol/L, 5.0 μmol/L groups was significantly higher than LPS 20 μg/ml stimulation group (all P<0.05). There is a possible relation between Leptin release and cPLA 2 activity in inflammatory cells induced by LPS.

Effect of Xuebijing injection on expression of endothelial protein C receptor and protease activated receptor 1 mRNA and protein in endothelial cells induced by lipopolysaccharide

To investigate the effect of Xuebijing injection on expression of endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1) mRNA and protein in rat aortic endothelial cells (RAECs) after lipopolysaccharide (LPS) challenge. RAECs were cultured for one week, and the purity was determined with flow cytometry. ARECs were randomly divided into four groups: control group, LPS stimulation group (1 mg/L LPS), Xuebijing injection treatment group (LPS: 1 mg/L, Xuebijing injection ; 10 g/L) and activated protein C (APC) treatment group (LPS: 1 mg/L, APC: 0.1 mg/L). The RAECs were collected at 12, 24, 48, and 72 hours after being stimulated by LPS. EPCR and PAR1 mRNA of RAECs were assessed by reverse transcription-polymerase chain reaction (RT-PCR). EPCR and PAR1 protein were assessed by flow cytometry. At 12 hours, EPCR and PAR1 protein expressions were not significantly different among groups (all P>0.05). The level of EPCR and PAR1 mRNA were decreased in LPS stimulation group, but they were elevated in both APC and Xuebijing injection treatment groups (P<0.05 or P<0.01). From 24 to 72 hours, compared to control group, the levels of EPCR mRNA and protein expression were significantly decreased after LPS stimulation (P<0.05 or P<0.01). The levels of EPCR expression were decreased and negatively correlated with the time of LPS treatment. Also, compared to LPS stimulation group, treatment with Xuebijing markedly elevated the levels of EPCR (P<0.01). The levels of PAR1 expression were significantly decreased by LPS stimulation compared with those of control group (P<0.05 or P<0.01). After the treatment with Xuebijng, the expression of PAR1 was gradually increased (all P<0.01). Compared with APC treatment group, Xuebijing could increase the PAR1 expression better. Xuebijing could raise EPCR and PAR1 mRNA and protein expression of RAECs after LPS challenge, and it may be related to its protection of endothelial cell from undergoing apoptosis.

Biochemical pathways in the antiatherosclerotic effect of berberine

This study investigated the inhibitory effect of berberine (BBR) on lipopolysaccharide (LPS) induced cyclooxygenase-2 (COX-2) expression via the mitogen activated protein kinase (MAPK) signalling cascade pathways in human peripheral blood monocytes (PBMC). PBMC from whole blood were isolated and cultured for up to 24 hours after division into 5 groups treated with LPS, LPS + BBR 25 micromol/L, LPS + BBR 50 micromol/L or LPS + BBR 100 micromol/L and untreated. Monocytes were extracted for RT-PCR and Western blot analyses to examine COX-2 mRNA and protein activated expression of p38 mitogen activated protein kinase (p38MAPK), Jun N-terminal kinase (JNK) and extracellular regulated kinases 1/2 (ERK1/2) signalling pathways. COX-2 mRNA and protein expression decreased to a minimum at 12 hours after BBR treatment (P < 0.05). With the increasing concentration of BBR treatment, the COX-2 expression decreased progressively (P < 0.01). With BBR treatment for 6, 12 or 24 hours at three doses, ERK1/2 protein expression was significantly inhibited. For the JNK pathway, only with the treatment of BBR at the concentration of 100 micromol/L was JNK protein expression inhibited compared with the LPS stimulation group (P < 0.01). Irrespective of the BBR concentration, no difference was shown between the BBR group and the LPS group for p38MAPK protein expression. Human monocytes COX-2 mRNA, by RT-PCR, and protein expression, by Western blot analysis, were inhibited when incubated with PD98059, SP600125 and SB203580 (P < 0.05). Berberine inhibits COX-2 expression via the ERK1/2 signalling pathway and, possibly, at a high dosage via the JNK pathway. P38MAPK may have no relationship with the effect of BBR in PBMC. Berberine inhibited COX-2 mRNA and protein expression in a dose dependent manner and suppressed COX-2 expression to a minimal level after 12 hours of berberine treatment.