Effect of IL-33 on pyroptosis of macrophages in mice with sepsis via NF-κB/p38 MAPK signaling pathway

ABSTRACT Purpose To demonstrate the effect of IL-33 on the macrophage pyroptosis in mice with sepsis through the NF-kB/p38 MAPK signal pathway. Methods In total, 24 C57BL/6 mice were divided into the sham operation group (sham) and the cecal ligation and puncture group (CLP). After CLP, 24 IL-33-/- mice were divided into the IL-33-/- group and the IL-33-/- intervention group. The latter group was intraperitoneally injected with IL-33. Mouse mortality was observed after CLP. Macrophage apoptosis in peritoneal lavage fluid was detected by flow cytometry. Serum inflammatory factor level was detected by ELISA. Apoptotic protein expression and NF-κB/p38 MAKP signaling pathway protein expression were detected by qRT-PCR and Western blot. Results Knocking out IL-33 significantly reduced the mortality of CLP mice, as well as the mRNA expression of IL-33 and the levels of serum inflammatory factors, including IL-33, IL-1β, and IL-18. It also reduced the rate of macrophage apoptosis and the expression of the apoptotic protein caspase-1 p10; increased the expression of IκBα; and reduced the protein expression of NF-κB and p38 MAPK. These effects were reversed after exogenous injection of IL-33. Conclusions IL-33 can increase the level of macrophage pyroptosis in mice with sepsis (by activating the NF-kB/p38MAPK signal pathway) and the mortality of these mice.

The Expression of NF-kB Signaling Pathway Was Inhibited by Silencing TGF-b4 in Chicken IECs Infected with E. tenella

Chicken coccidiosis is a parasitic disease caused by one or more species of Eimeria infection with severe consequences. The NF-B signaling pathway and TGF-4 play an important role in the inflammation and immune response caused by coccidiosis infection. This study was designed to investigate the changes of NF-B signaling pathway and the effects of TGF-4 silencing on the expression of NF-B signaling pathway in chickens intestinal epithelial cells (IECs) after infecting with E. tenella sporozoites. TGF-4 small interfering RNA (TGF-4 siRNA) sequences were transfected into chicken IECs for reducing TGF-4 expression. The results showed that compared with uninfected control group (Con.), the proinflammatory factors of NF-B, IL-6, IL-2, IL-1 increased significantly in the E. tenella infected group (ET) (p 0.05). In comparison with the IECs in the ET, the expression level of NF-B, IL-6, IL-2, IL-1 dropped significantly in the group infected both by E. tenella and TGF-4 siRNA vector (ET+siRNA) (p 0.05). The results of this experiment indicate that NF-B signaling pathway is closely correlated with TGF-4 in IECs infected with coccidia sporozoites. TGF-4 plays an important role in regulating the immune function of coccidia-infected chicken epithelial cells through NF-B signaling pathway and preventing immune-mediated pathological changes.(AU)

The Expression of NF-kB Signaling Pathway Was Inhibited by Silencing TGF-b4 in Chicken IECs Infected with E. tenella

Chicken coccidiosis is a parasitic disease caused by one or more species of Eimeria infection with severe consequences. The NF-B signaling pathway and TGF-4 play an important role in the inflammation and immune response caused by coccidiosis infection. This study was designed to investigate the changes of NF-B signaling pathway and the effects of TGF-4 silencing on the expression of NF-B signaling pathway in chickens intestinal epithelial cells (IECs) after infecting with E. tenella sporozoites. TGF-4 small interfering RNA (TGF-4 siRNA) sequences were transfected into chicken IECs for reducing TGF-4 expression. The results showed that compared with uninfected control group (Con.), the proinflammatory factors of NF-B, IL-6, IL-2, IL-1 increased significantly in the E. tenella infected group (ET) (p 0.05). In comparison with the IECs in the ET, the expression level of NF-B, IL-6, IL-2, IL-1 dropped significantly in the group infected both by E. tenella and TGF-4 siRNA vector (ET+siRNA) (p 0.05). The results of this experiment indicate that NF-B signaling pathway is closely correlated with TGF-4 in IECs infected with coccidia sporozoites. TGF-4 plays an important role in regulating the immune function of coccidia-infected chicken epithelial cells through NF-B signaling pathway and preventing immune-mediated pathological changes.

Preconditioning with L-Ala-Gln reduces the expression of inflammatory markers (TNF-alpha, NF-kbeta, IL-6 and HO-1) in an injury animal model of cerebrovascular ischemia in Meriones unguiculatus (gerbils)

Purpose. To evaluate the neuroprotective effect of L-alanyl-glutamine in a gerbil model of brain ischemia-reperfusion injury based on immunohistochemical quantification of pro-inflammatory and cell activation biomarkers (TNF-α, NF-κB, IL-6 and HO-1).. Methods. Male gerbils weighing 100-180 g were pretreated with either 0.75 g/kg L-Ala-Gln (n=18) or 2.0 mL saline (n=18) administered i.v. 30 minutes before the bilateral ligation of the common carotid artery during 15 min and then the ligation was removed. Under anesthesia with urethane, brain tissue was harvested at 0 min (T0), 30 min (T30) and 60 min (T60) after reperfusion. The tissue was embedded in 10% formalin overnight and 4-μm sections were prepared for immunostaining with monoclonal antibodies. Immunostained cells were counted by optical microscopy. The statistical analysis used mean values based on 4 sections.. Results. The pretreatment with L-Ala-Gln animal group 1 demonstrated significantly lower levels of TNF-α, NF-κB and IL-6. On the other hand, the levels of HO-1 were significantly higher, suggesting a protective role in model of brain ischemia-reperfusion injury.. Conclusion. These findings suggest a protective effect of L-Ala-Gln by decreasing levels of TNF-alpha, IL-6 and NF-κB and Increasing levels of HO-1.(AU)

Milk fat globule-epidermal growth factor 8 (MFG-E8) attenuates sepsis-induced acute kidney injury by inhibiting NF-kappa B signaling pathway

Purpose:To explore the effect of milk fat globule-epidermal growth factor 8 (MFG-E8) on sepsis-induced acute kidney injury (SAKI).Methods:Male C57BL/6 mice were randomized to control, sham, CLP, CLP+PBS, and CLP+rmMFG-E8 groups. SAKI was induced by cecal ligation and puncture (CLP). Recombinant mouse MFG-E8 (rmMFG-E8) (20 μg/kg) or PBS (vehicle) was administered intraperitoneally. Blood, urine and renal tissue were collected at 24 h after CLP. Blood samples were tested for serum kidney injury biomarker and cytokines. Urine samples were collected to detect KIM-1, and NGAL. Real-time PCR was tested for Bax and Bcl-2. TUNEL staining was used to determine renal apoptosis. Western blot was used to detect the expression of Bax, Bcl-2, and proteins in the NF-κB pathway.Results:MFG-E8 alleviated SAKI by decreasing serum Cre, BUN, urine KIM-1 and NGAL and by mitigating renal pathological changes significant (p < 0.05). IL-1β, IL-6, TNF-α were significantly inhibited by MFG-E8 (p < 0.05). Apoptosis induced by SAKI was markedly suppressed by MFG-E8. Finally, MFG-E8 attenuated the activation of the NF-𝜅B signaling pathway in SAKI.Conclusion:MFG-E8 has beneficial effects on SAKI, which may be achieved by inhibiting the NF-κB pathway.(AU)

Therapeutic effect of adenosine on experimentally induced acute ulcerative colitis model in rats

Purpose To examine the therapeutic effect of external adenosine on an acetic acid-induced acute ulcerative colitis model in rats. Methods Thirty male mature rats were divided into three groups as control, acute colitis (AC) and AC+adenosine group (AC+AD). AC was induced by rectal administration of 4% acetic acid (AA). 5mg/kg/day adenosine was performed i.p for 4 weeks to AC+AD group. Rectum and colon were excised for microscopic and histopathological histopathologic evaluations, and immunohistochemical analysis of nuclear factor kappa B (NF-kB). Blood samples were collected for biochemical detection of TNF-α, Pentraxin-3 and malondialdehyde (MDA) levels. Results AC group had generalized hyperemia and hemorrhage with increased macroscopic and histopathological scores compared with control (P <0.0001) while adenosine treatment decreased these scores significantly (P <0.001), with reduced distribution of disrupted epithelium, leukocyte infiltrates, and focal hemorrhage. AC group showed significantly increased immunoexpression of NF-kB in rectum, plasma and tissue levels of TNF-α, plasma Pentraxin-3 and MDA levels (P <0.0001) while adenosine reduced these levels (P < 0.05). Conclusion Adenosine appears to promote healing of colon and rectum exposed to AA-induced AC, suggesting a boosting effect of adenosine on the intestinal immune system to cure ulcerative colitis.(AU)

Epigallocatechin Gallate (EGCG) Inhibited the Alv-J-Induced Apoptosis in Df-1 Cells by Inactivation of Nuclear Factor b Pathway

Avian leukosis virus subgroup J (ALV-J), a member of the retroviridae family, can infect both broilers and layers and induce a spectrum of different neoplasms, resulting in serious economic losses in poultry production. Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has demonstrated remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays, cell culture systems and epidemiological studies. To assess the antiviral effects of EGCG on ALV-J-induced cell apoptosis in vitro, DF-1 cells were treated with different EGCG concentrations (0, 5, 10, 20 and 40 µg/mL), and their antiviral effects were examined at different time points (0, 24, 48, 72 and 96 h) using a variety of assays. EGCG alleviated the ALV-J-induced apoptosis in a dose-dependent manner. Because high concentrations (20 and 40 µg/mL) inhibited DF-1 cell growth, and low concentration (5 µg/mL) did not suppress the ALV-J virus, 10 µg/mL was the most appropriate concentration. After 96 h of incubation, 10 µg/mL EGCG improved the ALV-J-triggered suppression of the nuclear transcription factor system by enhancing cytoplasmic NF-B p50/p65 expression and inhibiting nuclear NF-B p50/p65 expression, resulting in decreased cell apoptosis. These results demonstrated that EGCG inhibited ALV-J-induced apoptosis in DF-1 cells in a dose-dependent manner via the NF-B signaling pathway, and that 10 µg/mL EGCG is the optimal concentration, which may be useful for therapeutic drug design.(AU)

Epigallocatechin Gallate (EGCG) Inhibited the Alv-J-Induced Apoptosis in Df-1 Cells by Inactivation of Nuclear Factor b Pathway

Avian leukosis virus subgroup J (ALV-J), a member of the retroviridae family, can infect both broilers and layers and induce a spectrum of different neoplasms, resulting in serious economic losses in poultry production. Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has demonstrated remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays, cell culture systems and epidemiological studies. To assess the antiviral effects of EGCG on ALV-J-induced cell apoptosis in vitro, DF-1 cells were treated with different EGCG concentrations (0, 5, 10, 20 and 40 µg/mL), and their antiviral effects were examined at different time points (0, 24, 48, 72 and 96 h) using a variety of assays. EGCG alleviated the ALV-J-induced apoptosis in a dose-dependent manner. Because high concentrations (20 and 40 µg/mL) inhibited DF-1 cell growth, and low concentration (5 µg/mL) did not suppress the ALV-J virus, 10 µg/mL was the most appropriate concentration. After 96 h of incubation, 10 µg/mL EGCG improved the ALV-J-triggered suppression of the nuclear transcription factor system by enhancing cytoplasmic NF-B p50/p65 expression and inhibiting nuclear NF-B p50/p65 expression, resulting in decreased cell apoptosis. These results demonstrated that EGCG inhibited ALV-J-induced apoptosis in DF-1 cells in a dose-dependent manner via the NF-B signaling pathway, and that 10 µg/mL EGCG is the optimal concentration, which may be useful for therapeutic drug design.

Immunhistochemical analysis of Nuclear Factor Kappa Beta expression in etiopathogenesis of ovarian tumors

Purpose: To investigate the place of the transcription factor nuclear kappa B (NF-kB), which is a marker of chronic inflammation, in the etiology of the ovarian carcinoma. Methods: NFkB analysis with the immunohistochemical method has been performed. To evaluate immunohistochemical NF-kB expression in the ovarian tissue, the H-score method. H-score = Pi (i+1), where Pi is the percentage of stained cells in each intensity category (0-100%) and i is the intensity indicating weak (i=1), moderate (i=2) or strong staining (i=3). Results: It has been seen that, the mean H score is statistically significantly higher in the patient group with serous and musinous adenocarcinoma diagnosis than the two other patient groups (p 0.005). Conclusions: Factor nuclear kappa B is an important mediator that acts in the chronic inflammation. The highest expression rates are determined by the immunohistochemical method in the ovarian cancer group.(AU)

Effects of baicalin on inflammatory reaction, oxidative stress and PKDl and NF-kB protein expressions in rats with severe acute pancreatitis

Purpose: To investigate the effects of baicalin on inflammatory reaction, oxidative stress and protein kinase D1 (PKD1) and nuclear factor-kappa B (NF-B) protein expressions in severe acute pancreatitis (SAP) rats. Methods: Sixty rats were divided into sham operation, model, and low-, medium- and high-dose baicalin group. SAP model was established in later 4 groups. The later 3 groups were injected with 0.1, 0.2 and 0.4 ml/100 g 5% baicalin injection, respectively. At 12 h, the serum SAP related indexes and inflammatory factors, peripheral blood CD3 and T cell percentages, wet/dry ratio and pancreas ascites volume, oxidative stress indexes and PKD1 and NF-B protein expressions in pancreatic tissue were determined. Results: Compared with model group, in high-dose baicalin group the wet/dry ratio and ascites volume, serum amylase level, phospholipase A2 activity, TNF-, IL-1 and IL-6 levels, and pancreatic malondialdehyde level and PKD1 and NF-B protein expression were significantly decreased (P 0.05), and peripheral blood CD3 and T cell percentages and pancreatic superoxide dismutase and glutathione peroxidase levels were significantly increased (P 0.05). Conclusion: Baicalin can resist the inflammatory reaction and oxidative stress, and down-regulate protein kinase D1 and nuclear factor-kappa B protein expressions, thus exerting the protective effects on severe acute pancreatitis in rats.(AU)

NF-kB mediated CX3CL1 activation in the dorsal root ganglion contributes to the maintenance of neuropathic pain induced in adult male Sprague Dawley rats

Purpose: To evaluate the role of CX3CL1 and NF-B in the lumbar disc herniation induced neuropathic pain. Methods: After LDH induced by implantation of autologous nucleus pulposus (NP) on the left L5 nerve root was established, mechanical thresholds and thermal hyperalgesia were tested at relevant time points during an observation period of 28 days. Expression of CX3CL1 and NF-Bin the dorsal root ganglion (DRG) were performed by using Western blotting and RT-PCR. Results: Implantation of autologous nucleus pulposus (NP) induced neuropathic pain, associated with increased mRNA and protein expression of CX3CL1 in the DRG. Moreover, intrathecal injection of neutralizing antibody against CX3CL1 could attenuates LDH-induced persistent pain hypersensitivity. Interestingly, NF-B activation in the DRGs were found in LDH-induced neuropathic pain. Furthermore, NF-B downregulation by p65 inhibitor PDTC markedly alleviated LDH-induced mechanical allodynia and thermal hyperalgesia in rat. Importantly, CX3CL1 neutralizing antibody (10 g/10 l, i.t.) reduces p-p65 protein level in DRG Conclusions: CX3XL1 could regulate LDH-induced neuropathic pain through NF-B pathway. Targeting CX3CL1 and NF-B may represent a potential treatment for neuropathic pain caused by LDH.(AU)

Resveratrol attenuates LPS-induced apoptosis via inhibiting NF-kB activity in chicken peripheral lymphocyte cultures

This study aims to investigate whether resveratrol (RES) could inhibit NF-κB activity and protect in vitro cultured chicken lymphocytes from apoptosis induced by continuous LPS stimulation. Blood lymphocytes of chickens were collected and cultured. LPS was added to the samples of treatment group, while the same volume of normal saline (NS) was supplemented to those of the control group. In the treatment groups, four different concentrations of RES (0, 20, 40, and 80 µg/ml) were admixed. Then, the following indicators were tested: lymphocyte apoptotic rate, lymphocyte viability, NF-κB activity, TNF-a level, reactive oxygen species (ROS), and expression of apoptosis-related proteins (Fas/FasL and Caspase-8). The results indicated that the application of different concentrations of RES could significantly increase the viability of lymphocytes and decrease the apoptotic rate (p<0.05) after continuous stimulations by LPS. The activity of NF-κB levels of TNF-a and ROS in the RES treatment subgroups was significantly decreased in comparison with that in the RES-blank subgroup (p<0.05). The cells in the treatment group that had been treated with 20, 40 and 80 μl/ml of RES exhibited a significantly lower Fas and Caspase-8 expression level than that in the RES-blank subgroup (p<0.05). These findings revealed that RES could substantially diminish the concentrations of ROS and TNF-a, down-regulate NF-κB activity, decrease the expression of Fas and Caspase-8, stimulate the activity of peripheral lymphocytes, and lower the rate of their apoptosis, induced by continuous LPS stimulation in chicken lymphocyte cultures.(AU)

Resveratrol attenuates LPS-induced apoptosis via inhibiting NF-kB activity in chicken peripheral lymphocyte cultures

This study aims to investigate whether resveratrol (RES) could inhibit NF-κB activity and protect in vitro cultured chicken lymphocytes from apoptosis induced by continuous LPS stimulation. Blood lymphocytes of chickens were collected and cultured. LPS was added to the samples of treatment group, while the same volume of normal saline (NS) was supplemented to those of the control group. In the treatment groups, four different concentrations of RES (0, 20, 40, and 80 µg/ml) were admixed. Then, the following indicators were tested: lymphocyte apoptotic rate, lymphocyte viability, NF-κB activity, TNF-a level, reactive oxygen species (ROS), and expression of apoptosis-related proteins (Fas/FasL and Caspase-8). The results indicated that the application of different concentrations of RES could significantly increase the viability of lymphocytes and decrease the apoptotic rate (p<0.05) after continuous stimulations by LPS. The activity of NF-κB levels of TNF-a and ROS in the RES treatment subgroups was significantly decreased in comparison with that in the RES-blank subgroup (p<0.05). The cells in the treatment group that had been treated with 20, 40 and 80 μl/ml of RES exhibited a significantly lower Fas and Caspase-8 expression level than that in the RES-blank subgroup (p<0.05). These findings revealed that RES could substantially diminish the concentrations of ROS and TNF-a, down-regulate NF-κB activity, decrease the expression of Fas and Caspase-8, stimulate the activity of peripheral lymphocytes, and lower the rate of their apoptosis, induced by continuous LPS stimulation in chicken lymphocyte cultures.

Identification of therapeutic target genes with DNA microarray in multiple myeloma cell line treated by IKK beta/NF-kappa B inhibitor

PURPOSE:To explore the mechanism of resistance to IKKβ inhibitor in multiple myeloma (MM) cells and uncover novel therapeutic targets for MM.METHODS:We downloaded the microarray data (GSE8476) from GEO (Gene Expression Omnibus) database. The data were derived from the human MM cells lines (L363 cells) treated with IKKβ inhibitor MLN120b (MLN) for eight, 12 and 24 hours. Furthermore, we applied the Search Tool for the Retrieval of Interacting Genes (STRING) and Expression Analysis Systematic Explorer (EASE) database to construct protein-protein interaction networks and identified over-represented pathway among DEGs (differentially expressed genes).RESULTS:We obtained 108 DGEs in 8h vs. 12h group and 101 ones in 8h vs. 24h group. Most of DGEs were found to be involved in biological regulation. The significant pathways were Ig A pathway and the CAMs pathways. In addition, 24 common DGEs were found in the networks of the two groups such as ICAM 3 and SELL.CONCLUSION:Intercellular adhesion molecule 3 and SELL may be potential targets in multiple myeloma treatment in the future.(AU)