Semaphorin 7A impairs barrier function in cultured human corneal epithelial cells in a manner dependent on nuclear factor-kappa B

●AIM:To evaluate the role of semaphorin 7A(Sema7A)and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells(HCEs).●METHODS:Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0,125,250,or 500 ng/mL for 24,48,or 72h in vitro.Transepithelial electrical resistance(TEER)as well as Dextran-fluorescein isothiocyanate(FITC)permeability assays were conducted to assess barrier function.To quantify tight junctions(TJs)such as occludin and zonula occludens-1(ZO-1)at the mRNA level,reverse transcriptionpolymerase chain reaction(RT-PCR)analysis was performed.Immunoblotting was used to examine the activity of the nuclear factor-kappa B(NF-κB)signaling pathway and the production of TJs proteins.Immunofluorescence analyses were employed to localize the TJs.Enzyme-linked immunosorbent assay(ELISA)and RT-PCR were utilized to observe changes in interleukin(IL)-1βlevels.To investigate the role of NF-κB signaling activation and IL^(-1)βin Sema7A’s anti-barrier mechanism,we employed 0.1μmol/L IκB kinase 2(IKK2)inhibitor IV or 500 ng/mL IL^(-1)receptor(IL-1R)antagonist.●RESULTS:Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time-and dose-dependent manner,as well as altering the localization of TJs.Furthermore,Sema7A stimulated the activation of inhibitor of kappa B alpha(IκBα)and expression of IL-1β.The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists.●CONCLUSION:Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins,as well as the expression of IL-1β.These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.

Delayed hepatocarcinogenesis through antiangiogenic intervention in the nuclear factor-kappa B activation pathway in rats

BACKGROUND: The active form of nuclear factor-kappa B (NF-kappa B) is involved in the initiation, generation, and development of hepatocellular carcinoma (HCC), and is up-regulated in inflammation-associated malignancies. We investigated the dynamic expression of NF-kappa B and its influences on the occurrence of HCC through antiangiogenic (thalidomide) intervention in NF-kappa B activation. METHODS : Hepatoma models were induced with 2-fluorenylacetamide (2-FAA, 0.05%) in male Sprague-Dawley rats, and thalidomide (100 mg/kg body weight) was administered intragastrically to intervene in NF-kappa B activation. The pathological changes in the liver of sacrificed rats were assessed after hematoxylin and eosin staining. NF-kappa B mRNA was amplified by RT-nested PCR. The alterations of NF-kappa B and vascular endothelial growth factor (VEGF) expression were analyzed by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blotting. RESULTS: Rat hepatocytes showed denatured, precancerous, and cancerous stages in hepatocarcinogenesis, with an increasing tendency of hepatic NF-kappa B, NF-kappa B mRNA, and VEGF expression, and their values in the HCC group were higher than those in controls (P<0.001). In the thalidomide-treated group, the morphologic changes generated only punctiform denaturation and necrosis at the early or middle stages, and nodular hyperplasia or a little atypical hyperplasia at the final stages, with the expression of NF-kappa B (chi(2)=9.93, P<0.001) and VEGF (chi(2)=8.024, P<0.001) lower than that in the 2-FAA group. CONCLUSION: NF-kappa B is overexpressed in hepatocarcinogenesis and antiangiogenic treatment down-regulates the expression of NF-kappa B and VEGF, and delays the occurrence of HCC. (Hepatobiliary Pancreat Dis Int 2010; 9: 169-174)

Urinary trypsin inhibitor attenuates hepatic ischemia-reperfusion injury by reducing nuclear factor-kappa B activation

BACKGROUND: Urinary trypsin inhibitor (UTI) inhibits the inflammatory response and protects against ischemia-reperfusion (I/R) injury. The inflammatory response is mediated by nuclear factor-kappa B (NF-kappa B) and its related target genes and products such as vascular endothelial cell adhesion molecule and CXC chemokines. We aimed to assess the roles of those mediators in a UTI-treated mouse model of hepatic I/R injury. METHODS: Treatment group 1 (UTI given 5 minutes prior to liver ischemia), treatment group 2 (UTI given 5 minutes after the anhepatic phase) and a control group were investigated. Blood and liver samples were obtained and compared at 1, 3, 6 and 24 hours after reperfusion. RESULTS: Attenuation of pathological hepatocellular damage was greater in the treatment groups than in the control group (P < 0.05). Compared with the control group, the UTI treatment groups showed significantly lower serum alanine aminotransferase and aspartate aminotransferase levels, decreased myeloperoxidase activity, and reduced NF-kappa B activation. Also downregulated was the expression of tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein-2 at the mRNA level. P-selectin protein and intercellular adhesion molecule-1 protein expression were also downregulated. In addition, the treatment group I showed a better protective effect against I/R injury than the treatment group 2. CONCLUSIONS: UTI reduces NF-kappa B activation and downregulates the expression of its related mediators, followed by the inhibition of neutrophil aggregation and infiltration in hepatic I/R injury. The protective role of UTI is more effective in prevention than in treatment.

Characteristics of hepatic nuclear-transcription factor-kappa B expression and quantitative analysis in rat hepatocarcinogenesis

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. We analyzed the expression of miclear-transcription factor-kappa B (NF-kappa B) during hepatocarcinogenesis in order to evaluate its dynamic expression and its clinical value in the development and diagnosis of HCC. METHODS: Hepatoma models were induced by oral administration of 2-acetamidoflurene (2-FAA) to male Sprague-Dawley rats. Morphological changes were observed after hematoxylin and eosin staining. The cellular distribution of NF-kappa B expression during different stages of cancer development was investigated by immunohistochemistry, and the level of NF-kappa B expression in liver tissues was quantitatively analyzed by ELISA. The gene fragments of hepatic NF-kappa B were amplified by nested-polymerase chain reaction assay. RESULTS: Hepatocytes showed vacuole-like degeneration during the early stages, then had a hyperplastic nodal appearance during the middle stages, and finally progressed to tubercles of cancerous nests with high differentiation. The NF-kappa B-positive material was buff-colored, fine particles localized in the nucleus, and the incidence of NF-kappa B-positive cells was 81.8% in degeneration, 83.3% in precancerous lesions, and 100% in cancerous tissues. All of these values were higher than those in controls (P<0.01). Hepatic NF-kappa B expression and hepatic NF-kappa B-mRNA were also higher during the course of HCC development (P<0.01). CONCLUSION: The NF-kappa B signal transduction pathway is activated during the early stages of HCC development, and its abnormal expression may be associated with the occurrence of HCC.

Testosterone alleviates tumor necrosis factor-alpha-mediated tissue factor pathway inhibitor downregulation via suppression of nuclear factor-kappa B in endothelial cells

We have observed earlier that testosterone at physiological concentrations can stimulate tissue factor pathway inhibitor(TFPI)gene expression through the androgen receptor in endothelial cells.This study further investigated the impact of testosterone on TFPI levels in response to inflammatory cytokine tumor necrosis factor-alpha(TNF-α).Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-α.TFPI protein and mRNA levels were assessed by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction.To study the cellular mechanism of testosterone’s action,nuclear factor-kappa B(NF-κB)translocation was confirmed by electrophoretic mobility shift assays.We found that after NF-κB was activated by TNF-α,TFPI protein levels declined significantly by 37.3%compared with controls(P<0.001),and the mRNA levels of TFPI also decreased greatly(P<0.001).A concentration of 30 nmol L-1 testosterone increased the secretion of TFPI compared with the TNF-α-treated group.NF-κB DNA-binding activity was significantly suppressed by testosterone(P<0.05).This suggests that physiological testosterone concentrations may exert their antithrombotic effects on TFPI expression during inflammation by downregulating NF-κB activity.

Inhibitory effects of Shuanghuanglian injection on nuclear factor-kappa B expression in mice with viral encephalitis in a time-and dose-dependent manner

Previous studies have confirmed that the anti-virus effects of Shuanghuanglian injection may be associated with nuclear factor-kappa B activity. This study observed nuclear factor-kappa B expression in mice with viral encephalitis, and showed significant decreases in nuclear factor-kappa B protein and mRNA levels following Shuanghuanglian injection. The inhibitory effect was more significant with prolonged intervention duration and increased treatment dose. These findings verify that Shuanghuanglian injection plays a therapeutic role in viral encephalitis by reducing expression of nuclear factor-kappa B in a time- and dose-dependent manner.

Shuanghuanglian injection downregulates nuclear factor-kappa B expression in mice with viral encephalitis

A mouse model of viral encephalitis was induced by intracranial injection of a Coxsackie virus B3 suspension. Quantitative real-time reverse transcription-PCR and western blot assay were applied to detect mRNA and protein expression of intelectin-2 and nuclear factor-kappa B in the viral encephalitis and control groups. Nuclear factor-kappa B and intelectin-2 mRNA and protein expression were significantly increased in mice with viral encephalitis. After intraperitoneal injection of Shuanghuanglian at a dose of 1.5 mg/kg for 5 successive days, intelectin-2 and nuclear factor-kappa B protein and mRNA expression were significantly decreased. To elucidate the relationship between intelectin-2 and nuclear factor-kappa B, mice with viral encephalitis were administered an intracerebral injection of 107 pfu recombinant lentivirus expressing intelectin shRNA. Both protein and mRNA levels of intelectin and nuclear factor-kappa B in brain tissue of mice were significantly decreased. Experimental findings suggest that Shuanghuanglian injection may downregulate nuclear factor-kappa B production via suppression of intelectin production, thus inhibiting inflammation associated with viral encephalitis.

Effects of Five Chitosan Oligosaccharides on Nuclear Factor-kappa B Signaling Pathway

The effects of five chito-oligomers, from dimer to hexamer （chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose） separated from chitosan oligosaccharides, on nuclear factor -kappaB （NF-rd3） signaling pathway were investigated by using luciferase assay and laser scanning microscopy. The expression of NF-rd3 downstream genes （cyclin DI, TNFa and IL-6） were tested by real time PCR. We found that all five chitosan oligosaccharides increased NF-KB-dependent luciferase gene expression and NF-KB downstream genes transcription, and the most significant were chitotetraose and chitohexaose. In addition, laser scanning microscopy experiments showed that chitotetraose and chitohexaose also activated the p65 subunite of NF-kB translocating from cytoplasm to nucleus, which suggested that they were the most potent activators of NF-kB signaling pathway.

Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney,and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

BACKGROUND Jianpi Gushen Huayu Decoction(JPGS)has been used to clinically treat diabetic nephropathy(DN)for many years.However,the protective mechanism of JPGS in treating DN remains unclear.AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN.METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model.We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics.Furthermore,we examined the effects of JPGS on c-Jun N-terminal kinase(JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3).RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress.Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice.A total of 51 differential metabolites were screened.Pathway analysis results indicated that nine pathways significantly changed between the control and model groups,while six pathways significantly altered between the model and JPGS groups.Pathways related to cysteine and methionine metabolism;alanine,tryptophan metabolism;aspartate and glutamate metabolism;and riboflavin metabolism were identified as the key pathways through which JPGS affects DN.Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-κB/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors.CONCLUSION JPGS could markedly treat mice with streptozotocin(STZ)-induced DN,which is possibly related to the regulation of several metabolic pathways found in kidneys.Furthermore,JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-κB/NLRP3 pathway and inhibit JNK/P38 pathwaymediated apoptosis in DN mice.

Effects of transplantation of microencapsulated rabbit sciatic nerve on nuclear factor-kappa B expression after spinal cord injury in rats

BACKGROUND： It has been reported that nuclear factor-kappa B （NF- κB）, activated after spinal cord injury in rats, plays a key role in inflammatory responses in the central nervous system. OBJECTIVE： To investigate the effects of transplantation of microencapsulated rabbit sciatic nerve on NF- κB expression and motor function after spinal cord injury in rats, and to compare the results with the transplantation of rabbit sciatic nerve alone. DESIGN, TIME AND SETTING： This completely randomized, controlled study was performed at the Department of Neurobiology, Medical College of Nanchang University between December 2007 and July 2008. MATERIALS： A rabbit anti-NF- κB P65 monoclonal antibody was made by the Santa Cruz Company, USA and a streptavidin peroxidase immunohistochemical kit was provided by the Sequoia Company, China. METHODS： Eight rabbits were used to prepare a sciatic nerve cell suspension that was divided into two parts： one stored for transplantation, and the other mixed with a 1.5% sodium alginate solution. One hundred and twenty adult Sprague Dawley rats weighing 220-250 g were randomly divided into four groups： the microencapsulated cell group （n = 36）, the non-encapsulated cell group （n = 36）, the saline group （n = 36） and the sham operation group （n = 12）. The first three groups underwent a right hemisection injury of the spinal cord at the T10 level, into which was transplanted a gelatin sponge soaked with 10 μL of a microencapsulated nerve tissue/cell suspension （microencapsulated cell group）, a tissue/cell suspension （non-encapsulated cell group） or physiological saline （saline group）. In the sham operation group the vertebrae were exposed, but the spinal cord was not injured, and no implantation was given. MAIN OUTCOME MEASURES： Pathological changes were detected using hematoxylin-eosin staining; NF- κB expression was quantified using immunohistochemical staining; motor function was assessed using the Basso, Beattie and Bresnahan （BBB） scale. RESULTS： Spinal cord injuries, such as neuronal death and inflammatory cell infiltration, were found in the microencapsulated cell group, the non-encapsulated cell group and the saline group. However, the damage in the microencapsulated cell group was milder than in the non-encapsulated cell or saline groups. NF- κB expression in the microencapsulated cell group, the non-encapsulated cell group and the saline group was increased after spinal cord injury; it reached a peak after 24 hours, gradually decreased after 3 days, and was close to normal levels after 7 days. NF- κB expression in the microencapsulated cell group was significantly lower than in the saline group and the non-encapsulated cell group （P 〈 0.05）. With time, the motor function of the animals in each group improved to a certain extent, but did not reach normal levels. There were no significant differences in BBB scores between the different groups on post-operative day 3; however, the BBB scores for the microencapsulated cell group and the non-encapsulated cell group were significantly higher than the saline group on post-operative day 7 （P 〈 0.05）. In addition, the motor function recovered better in the microencapsulated cell group than in the non-encapsulated cell group （P 〈 0.05）. CONCLUSION： The transplantation of microencapsulated rabbit sciatic nerve can inhibit NF- κB expression and inflammatory reactions and promote recovery of motor function after spinal cord injury in rats. The effects of microencapsulated cell transplantation are superior to those of transplantation of cells alone.

葫芦巴碱调节PI3K/Akt/NF-κB信号通路对变应性接触性皮炎大鼠免疫反应的影响

目的探讨葫芦巴碱对变应性接触性皮炎(ACD)大鼠免疫、炎性反应及PI3K/Akt/NF-κB信号通路的影响。方法60只SD大鼠随机分为正常组、ACD组、葫芦巴碱(低、中、高)剂量组(20、40、80 mg/kg)和PI3K抑制剂(LY294002)组(40 mg/kg),每组10只。除正常组外,其余组大鼠采用2,4二硝基氟苯(DNFB)诱导ACD模型。给药结束后,通过录像观察大鼠挠痒行为;HE染色检测大鼠耳皮肤组织病理学变化;ELISA检测大鼠血清IgE及Th1、Th2、Th17型细胞因子(IFN-γ、IL-4、IL-17)水平;Western blot检测大鼠耳皮肤组织中炎性因子(IL-1β、IL-6)蛋白及PI3K/Akt/NF-κB信号通路相关蛋白表达。结果与正常组比较,ACD组大鼠挠痒次数增加,血清IgE、IFN-γ、IL-4、IL-17水平及耳皮肤组织中IL-1β、IL-6蛋白表达和p-PI3K/PI3K、p-Akt/Akt、p-NF-κB/NF-κB比值升高(P<0.05),耳皮肤组织角化过度且可见大量炎性细胞浸润;与ACD组比较,葫芦巴碱(低、中、高)剂量组和LY294002组大鼠挠痒次数减少,血清IgE、IFN-γ、IL-4、IL-17水平及耳皮肤组织中IL-1β、IL-6蛋白表达和p-PI3K/PI3K、p-Akt/Akt、p-NF-κB/NF-κB比值降低(P<0.05),耳皮肤组织病理损伤均有所改善,且葫芦巴碱各给药组呈剂量依赖效应;葫芦巴碱高剂量组和LY294002组大鼠上述指标比较,差异无统计学意义(P>0.05)。结论葫芦巴碱可抑制ACD大鼠皮肤组织中PI3K/Akt/NF-κB信号通路激活,调控Th1、Th2、Th17型免疫应答并减轻皮肤局部和全身炎性反应。

Salvianic acid A inhibits induction of inflammatory mediators by blocking Nuclear Factor-κB activation in macrophages

Objective To investigate the anti-inflammation effect and possible mechanism of Salvianic acid A(SAA)in mouse peritoneal macrophages.Methods Peritoneal macrophages were obtained from BALB/c mice.LPS induced nitric oxide(NO),tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)in supernatant,protein expression of inducible nitric oxide synthase(iNOS),matrix metalloproteinase-9(MMP-9)and activation of nuclear factor-kappa B(NF-κB)in the extract were measured.Results SAA strongly inhibited the excessive production of NO,TNF-α and IL-6 in LPS-induced peritoneal macrophages in a concentration-dependent manner and blocked the expression of iNOS and MMP-9.Treatment with LPS alone increased the translocation of NF-κB(p65)from cytosol to the nucleus,but the SAA inhibited the translocation of NF-κB(p65).Conclusions The results showed that SAA had strong anti-inflammatory effects in LPS-stimulated peritoneal macrophages.The important mechanism is due to its inhibition of NF-κB activation.

L-and T-type Ca^(2+)channels dichotomously contribute to retinal ganglion cell injury in experimental glaucoma

Retinal ganglion cell apoptotic death is the main pathological characteristic of glaucoma,which is the leading cause of irreversible blindness.Disruption of Ca^(2+)homeostasis plays an important role in glaucoma.Voltage-gated Ca^(2+)channel blockers have been shown to improve vision in patients with glaucoma.However,whether and how voltage-gated Ca^(2+)channels are involved in retinal ganglion cell apoptotic death are largely unknown.In this study,we found that total Ca^(2+)current densities in retinal ganglion cells were reduced in a rat model of chronic ocular hypertension experimental glaucoma,as determined by whole-cell patch-clamp electrophysiological recordings.Further analysis showed that L-type Ca^(2+)currents were downregulated while T-type Ca^(2+)currents were upregulated at the later stage of glaucoma.Western blot assay and immunofluorescence experiments confirmed that expression of the Ca_(V)1.2 subunit of L-type Ca^(2+)channels was reduced and expression of the Ca_(V)3.3 subunit of T-type Ca^(2+)channels was increased in retinas of the chronic ocular hypertension model.Soluble tumor necrosis factor-α,an important inflammatory factor,inhibited the L-type Ca^(2+)current of isolated retinal ganglion cells from control rats and enhanced the T-type Ca^(2+)current.These changes were blocked by the tumor necrosis factor-αinhibitor XPro1595,indicating that both types of Ca^(2+)currents may be mediated by soluble tumor necrosis factor-α.The intracellular mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and nuclear factor kappa-B signaling pathway mediate the effects of tumor necrosis factor-α.TUNEL assays revealed that mibefradil,a T-type calcium channel blocker,reduced the number of apoptotic retinal ganglion cells in the rat model of chronic ocular hypertension.These results suggest that T-type Ca^(2+)channels are involved in disrupted Ca^(2+)homeostasis and apoptosis of retinal ganglion cells in glaucoma,and application of T-type Ca^(2+)channel blockers,especially a specific CaV3.3 blocker,may be a potential strategy for the treatment of glaucoma.

Ketogenic diet alleviates cognitive dysfunction and neuroinflammation in APP/PS1 mice via the Nrf2/HO-1 and NF-κB signaling pathways

Alzheimer's disease is a progressive neurological disorder characterized by cognitive decline and chronic inflammation within the brain.The ketogenic diet,a widely recognized therapeutic intervention for refractory epilepsy,has recently been proposed as a potential treatment for a variety of neurological diseases,including Alzheimer's disease.However,the efficacy of ketogenic diet in treating Alzheimer's disease and the underlying mechanism remains unclear.The current investigation aimed to explore the effect of ketogenic diet on cognitive function and the underlying biological mechanisms in a mouse model of Alzheimer's disease.Male amyloid precursor protein/presenilin 1(APP/PS1)mice were randomly assigned to either a ketogenic diet or control diet group,and received their respective diets for a duration of 3 months.The findings show that ketogenic diet administration enhanced cognitive function,attenuated amyloid plaque formation and proinflammatory cytokine levels in APP/PS1 mice,and augmented the nuclear factor-erythroid 2-p45 derived factor 2/heme oxygenase-1 signaling pathway while suppressing the nuclear factor-kappa B pathway.Collectively,these data suggest that ketogenic diet may have a therapeutic potential in treating Alzheimer's disease by ameliorating the neurotoxicity associated with Aβ-induced inflammation.This study highlights the urgent need for further research into the use of ketogenic diet as a potential therapy for Alzheimer's disease.

siRNA阻断NF-κB信号通路抑制胃癌SGC-7901细胞的增殖及侵袭

目的采用RNA干扰(RNA interference,RNAi)技术下调胃癌细胞株SGC-7901中NF-κB p65基因的表达,探讨其对肿瘤细胞增殖活性和侵袭能力的影响。方法利用阳离子脂质体LipofectamineTM2000将化学合成的人NF-κB p65的小干扰RNA(small interference RNA,siRNA)转染入胃癌细胞株SGC-7901中。采用RT-PCR法测定细胞内NF-κB p65mRNA的表达;酶联免疫吸附测定法(ELISA)检测NF-κB亚单位p65的DNA结合活性的改变;采用MTT法测定细胞增殖活性的变化;利用Transwell侵袭实验检测细胞体外侵袭能力的变化。结果与对照组比较,化学合成的人NF-κB p65siRNA能有效地抑制SGC-7901细胞中NF-κB p65mRNA的表达(P<0.05);RelAsiRNA组的p65亚单位与DNA结合活性明显低于对照组(P<0.05),并且RelA siRNA组中SGC-7901细胞的体外侵袭力减弱,增殖活性降低。结论特异的siRNA可以有效阻断NF-κB信号通路,影响人胃癌细胞的增殖活性和体外侵袭能力。

TLR4 upregulates CBS expression through NF-κB activation in a rat model of irritable bowel syndrome with chronic visceral hypersensitivity

AIM:To investigate the roles of toll-like receptor 4(TLR4) and nuclear factor(NF)-κB on cystathionine βsynthetase(CBS) expression and visceral hypersensitivity in rats.METHODS:This study used 1-7-wk-old male SpragueDawley rats.Western blot analysis was employed to measure the expression of TLR4,NF-kB and the endogenous hydrogen sulfide-producing enzyme CBS in colon dorsal root ganglia(DRG) from control and "irritable bowel syndrome" rats induced by neonatal colonic inflammation(NCI).Colon-specific DRG neurons were labeled with Dil and acutely dissociated to measure excitability with patch-clamp techniques.Immunofluorescence was employed to determine the co-expression of TLR4,NF-kB and CBS in Dil-labeled DRG neurons.RESULTS:NCI significantly upregulated the expression of TLR4 in colon-related DRGs(0.34 ± 0.12 vs 0.72 ±0.02 for the control and NCI groups,respectively,P <0.05).Intrathecal administration of the TLR4-selective inhibitor CLI-095 significantly enhanced the colorectal distention threshold of NCI rats.CLI-095 treatment also markedly reversed the hyperexcitability of colonspecific DRG neurons and reduced the expression of CBS(1.7 ± 0.1 vs 1.1 ± 0.04,p < 0.05) and of the NF-kB subunit p65(0.8 ± 0.1 vs 0.5 ± 0.1,P< 0.05).Furthermore,the NF-KB-selective inhibitor pyrrolidine dithiocarbamate(PDTC) significantly reduced the upregulation of CBS(1.0 ± 0.1 vs 0.6 ± 0.1,P< 0.05)and attenuated visceral hypersensitivity in the NCI rats.In vitro,incubation of cultured DRG neurons with the TLR4 agonist lipopolysaccharide significantly enhanced the expression of p65(control vs 8 h:0.9 ± 0.1 vs1.3 ± 0.1;control vs 12 h:0.9 ± 0.1 vs 1.3 ± 0.1,P< 0.05;control vs 24 h:0.9 ± 0.1 vs 1.6 ± 0.1,P <0.01) and CBS(control vs 12 h:1.0 ± 0.1 vs 2.2 ±0.4;control vs 24 h:1.0 ± 0.1 vs 2.6 ± 0.1,P< 0.05),whereas the inhibition of p65 via pre-incubation with PDTC significantly reversed the upregulation of CBS expression(1.2 ± 0.1 vs 0.6 ± 0.0,P< 0.01).CONCLUSION:Our results suggest that the activation of TLR4 by NCI upregulates CBS expression,which is mediated by the NF-kB signaling pathway,thus contributing to visceral hypersensitivity.

IL-36 Cytokine Expression and Its Relationship with p38 MAPK and NF-κB Pathways in Psoriasis Vulgaris Skin Lesions

Summary： This study examined the correlation of the expression of interleukin-36 （IL-36）, a novel member of interleukin-1 （IL-1） family, with p38 mitogen-activated protein kinase （p38 MAPK） and nu clear factor-kappa B （NF-kB） pathways in psoriasis vulgaris skin lesions. The expression levels of IL-36a, IL-3613, IL-367, phosphorylated p38 MAPK, and NF-id3p65 were detected in the skin tissues of 38 psoriasis patients and 17 healthy control subjects by real-time quantitative reverse transcription po lymerase chain reaction （qRT-PCR） and Western blotting. The cytokine expression levels were com pared between the psoriasis group and the control group. A correlation analysis between cytokine pro teins was performed in the psoriasis group. Results showed that the expression levels of IL-36a, IL-3613, IL-36y, phosphorylated p38 MAPK and NF-rh3p65 in the psoriasis group were Significantly higher than those in the control group （P〈0.001）. In the psoriasis group, the IL-36 cytokine expression was positively correlated with phosphorylated p38 MAPK and NF-kBp65 expression （P〈0.05）. A significant positive correlation was also found between the phosphorylated p38 MAPK and NF-v,.Bp65 expression （P〈0.01）. It was concluded that the increased IL-36 expression is correlated with p38 MAPK and NF-kB pathways in psoriasis vulgaris skin lesions. All the three factors may be jointly involved in the pathogenesis and local inflammatory response of psoriasis.

I_κB kinase-beta inhibitor attenuates hepatic fibrosis in mice

AIM: To investigate the anti-fibrosis effect of IκB kinase-beta inhibitor (IKK2 inhibitor IMD0354) in liver fibrosis. METHODS: Twenty male C57BL6 mice were divided into four groups. Five high-fat fed mice were injected with lipopolysaccharide (LPS, 10 mg/kg) intraperitoneally and five high-fat fed mice were without LPS injection to build models of liver injury, and the intervention group (five mice) was injected intraperitoneally with IKK2 inhibitor (IMD 30 mg/kg for 14 d), while the remaining five mice received a normal diet as controls. Hepatic function, pathological evaluation and liver interleukin-6 (IL-6) expression were examined. Western blotting and real-time polymerase chain reaction were used to detect the expressions of nuclear factor-κB (NF-κB), alpha-smooth muscle actin (α-SMA), tumor growth factor-beta1 (TGF-β1), tumor necrosis factor-alpha (TNF-α), typeⅠand type Ⅲ collagen proteins and mRNA. RESULTS: A mouse model of liver injury was successfully established, and IMD decreased nuclear transloca-tion of NF-κB p65 in liver cells. In the IMD-treated group, the levels of alanine aminotransferase (103 ± 9.77 μ/L vs 62.4 ± 7.90 μ/L, P < 0.05) and aminotransferase (295.8 ± 38.56 μ/L vs 212 ± 25.10 μ/L, P < 0.05) were significantly decreased when compared with the model groups. The histological changes were significantly ameliorated. After treatment, the expressions of IL-6 (681 ± 45.96 vs 77 ± 7.79, P < 0.05), TGF-β1 (Western blotting 5.65% ± 0.017% vs 2.73% ± 0.005%, P < 0.05), TNF-α (11.58% ± 0.0063% vs 8.86% ± 0.0050%, P < 0.05), typeⅠcollagen (4.49% ± 0.014% vs 1.90% ± 0.0006%, P < 0.05) and type Ⅲ collagen (3.46% ± 0.008% vs 2.29% ± 0.0035%, P < 0.05) as well as α-SMA (6.19 ± 0.0036 μ/L vs 2.16 ± 0.0023 μ/L, P < 0.05) protein and mRNA were downregulated in the IMD group compared to the fibrosis control groups (P < 0.05). CONCLUSION: IKK2 inhibitor IMD markedly improved non-alcoholic fatty liver disease in mice by lowering NF-κB activation, which could become a remedial target for liver fibrosis.

Magnolol protects against acute gastrointestinal injury in sepsis by down-regulating regulated on activation,normal T-cell expressed and secreted

BACKGROUND Sepsis is a major medical challenge.Magnolol is an active constituent of Houpu that improves tissue function and exerts strong anti-endotoxin and anti-inflammatory effects,but the mechanism by which it reduces intestinal inflammation in sepsis is yet unclear.AIM To assess the protective effect of magnolol on intestinal mucosal epithelial cells in sepsis and elucidate the underlying mechanisms.METHODS Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,and regulated on activation,normal T-cell expressed and secreted(RANTES)levels in serum and ileal tissue in animal studies.The histopathological changes of the ileal mucosa in different groups were observed under a microscope.Cell Counting Kit-8 and cell permeability assays were used to determine the concentration of drug-containing serum that did not affect the activity of Caco2 cells but inhibited lipopolysaccharide(LPS)-induced decrease in permeability.Immunofluorescence and Western blot assays were used to detect the levels of RANTES,inhibitor of nuclear factor kappa-B kinaseβ(IKKβ),phosphorylated IKKβ(p-IKKβ),inhibitor of nuclear factor kappa-B kinaseα(IκBα),p65,and p-p65 proteins in different groups in vitro.RESULTS In rats treated with LPS by intravenous tail injection in the presence or absence of magnolol,magnolol inhibited the expression of proinflammatory cytokines,IL-1β,IL-6,and TNF-αin a dose-dependent manner.In addition,magnolol suppressed the production of RANTES in LPS-stimulated sepsis rats.Moreover,in vitro studies suggested that magnolol inhibited the increase of p65 nucleation,thereby markedly downregulating the production of the phosphorylated form of IKKβin LPS-treated Caco2 cells.Specifically,magnolol inhibited the translocation of the transcription factor nuclear factor-kappa B(NF-κB)from the cytosol into the nucleus and down-regulated the expression level of the chemokine RANTES in LPS-stimulated Caco2 cells.CONCLUSION Magnolol down-regulates RANTES levels by inhibiting the LPS/NF-κB signaling pathways,thereby suppressing IL-1β,IL-6,and TNF-αexpression to alleviate the mucosal barrier dysfunction in sepsis.

Sodium butyrate protects against toxin-induced acute liver failure in rats

BACKGROUND: Acute liver failure(ALF) is a serious clinical syndrome with high mortality. Sodium butyrate has been shown to alleviate organ injury in a wide variety of preclinical models of critical diseases. The aim of this study was to investigate the protective effect of sodium butyrate on ALF in rats.METHODS: All rats were randomly divided into control,model and sodium butyrate treatment groups. Except the control group, the rats were induced ALF animal model by subcutaneous injection of human serum albumin+D- galactosamine+lipopolysaccharide. After induction of ALF,the rats in the treatment group received sodium butyrate(500mg/kg) at 12-hour or 24-hour time point. Fourty-eight hours after ALF induction, the animals were sacrificed and samples were harvested. Serum endotoxin, high mobility group box-1(HMGB1), liver function parameters, tumor necrosis factoralpha(TNF-α) and interferon-gamma(IFN-γ) were measured.The expression of HMGB1 and nuclear factor-kappa B(NF-κB)p65 protein in liver tissue was detected by Western blotting. The histological changes of liver and intestine were examined. The survival duration was also observed.RESULTS: Serum endotoxin, alanine aminotransferase, HMGB1,TNF-α and IFN-γ were significantly increased and the liver histology showed more severe histopathological injury in the model group compared with the control group(P<0.05).Compared to the model group, sodium butyrate treatment significantly improved the histopathological changes in the liver and intestine, reduced serum endotoxin and inflammatory cytokines, suppressed HMGB1 and NF-кB p65 proteins in liver tissue, and prolonged the survival duration regardless of treatment at 12 hours or 24 hours after induction of ALF(P<0.05).CONCLUSIONS: Sodium butyrate protected the liver from toxin-induced ALF in rats. The mechanisms may be due to direct hepatoprotection and decreased intestinal permeability.