The Role of Toll-Like Receptors and Nuclear Factor κB p65 Protein in the Pathogenesis of Otitis Media

The role of Toll-like receptor 4 (TLR4) and nuclear factor κB p65 (NF-κB p65) proteins in the pathogenesis of otitis media is explored. In recent years, the incidence of otitis media has been rising globally, becoming a significant threat to human health. More and more studies have found that Toll-like receptor 4 (TLR4), as a member of the Toll-like receptor family, can promote the generation of inflammatory factors and is closely related to the body’s immune response and inflammatory response. Nuclear factor-κB p65 (NF-κB p65) is a nuclear transcription factor that can interact with various cytokines, growth factors, and apoptotic factors, participating in processes such as oxidative stress, apoptosis, and inflammation in the body [1]. This article elaborates on the structure, function, and signaling pathways of TLR4 and NF-κB p65 proteins in the pathogenesis of otitis media, aiming to provide more precise targets and better therapeutic efficacy for the diagnosis and treatment of otitis media. The role of inflammation in disease.

Nuclear Factor kappa B p65 Expression in Mouse Cochlea

Nuclear factor kappa B(NF-κB) is one of the best-characterized transcription factors playing important roles in many cellular responses to a large variety of stimuli,including inflammatory cytokines, phorbol esters, growth factors, and bacterial and viral products. The aim of this study is to demonstrate NF-κB expression in the mouse cochlea and its enhancement in response to lipopolysaccharides(LPS) and kanamycin(KA) treatment. Methods KA treatment consisted of subcutaneous KA injections at 700 mg/kg twice a day with an eight-hour interval between the two injections for 3 or 7 days. For animals in the LPS treatment group, a single dose of 0.3 mg LPS dissolved in 0.2 ml sterile saline were injected into both bullae through the tympanic membrane and kept there for 3 hours. Animals in the control group received subcutaneous saline injection for 7 days. Following immmunohistochemichal processing with rabbit polyclonal anti-NF-κB p65 antibodies, cryosections of the cochlea were examined for expression of NF-κB p65 in various structures in the cochlea. Results NF-κB p65 expression, identified by presence of brown reaction products characteristic of DAB immunohistochemistry, was visible in the spiral ligament, spiral prominence, tectorial membrane(TM), spiral ganglion and nerve fibers. Relatively weak NF-κB p65 expression was also visualized in the organ of Corti. Within the organ of Corti, the inner hair cells(IHC), outer hair cells(OHC), inner pillar cells(IP), outer pillar cells (OP), Deiter’s cells(DC), and Boettcher’s cells exhibited stronger staining than the inner sulcus cells, Hensen’s cells(HC) and Claudius’cells. No NF-κB p65 expression was seen in the nucleus of the IHC and OHC. NF-κB p65 expression was increased in animals exposed to LPS or KA, demonstrating significant differences in the staining between control animals and LPS/KA-treated animals. NF-κB p65 expression was not significantly different between LPS treated and KA treated animals or between 3 and 7 days in KA-treated animals. Conclusion LPS and KA exposure increases expression of NF-κB p65 in the mouse cochlea.

Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways

Background:G-protein coupled receptors(GPCRs)are recognized as attractive targets for drug therapy.However,it remains poorly understood how GPCRs,except for a few chemokine receptors,regulate the progression of liver fibrosis.Here,we aimed to reveal the role of GPR65,a proton-sensing receptor,in liver fibrosis and to elucidate the underlying mechanism.Methods:The expression level of GPR65 was evaluated in both human and mouse fibrotic livers.Furthermore,Gpr65-deficient mice were treated with either bile duct ligation(BDL)for 21 d or carbon tetrachloride(CCl4)for 8 weeks to investigate the role of GPR65 in liver fibrosis.A combination of experimental approaches,including Western blotting,quantitative real-time reverse transcription-polymerase chain reaction(qRT-PCR),and enzyme-linked immunosorbent assay(ELISA),confocal microscopy and rescue studies,were used to explore the underlying mechanisms of GPR65’s action in liver fibrosis.Additionally,the therapeutic potential of GPR65 inhibitor in the development of liver fibrosis was investigated.Results:We found that hepatic macrophage(HM)-enriched GPR65 was upregulated in both human and mouse fibrotic livers.Moreover,knockout of Gpr65 significantly alleviated BDL-and CCl4-induced liver inflammation,injury and fibrosis in vivo,and mouse bone marrow transplantation(BMT)experiments further demonstrated that the protective effect of Gpr65knockout is primarily mediated by bone marrow-derived macrophages(BMMs).Additionally,in vitro data demonstrated that Gpr65 silencing and GPR65 antagonist inhibited,while GPR65 overexpression and application of GPR65 endogenous and exogenous agonists enhanced the expression and release of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and transforming growth factor-β(TGF-β),all of which subsequently promoted the activation of hepatic stellate cells(HSCs)and the damage of hepatocytes(HCs).Mechanistically,GPR65 overexpression,the acidic pH and GPR65 exogenous agonist induced up-regulation of TNF-αand IL-6 via the Gαq-Ca^(2+)-JNK/NF-κB pathways,while promoted the expression of TGF-βthrough the Gαq-Ca^(2+)-MLK3-MKK7-JNK pathway.Notably,pharmacological GPR65 inhibition retarded the development of inflammation,HCs injury and fibrosis invivo.Conclusions:GPR65 is a major regulator that modulates the progression of liver fibrosis.Thus,targeting GPR65 could be an effective therapeutic strategy for the prevention of liver fibrosis.

人脑胶质瘤与人脑转移瘤中NF-κBP65的表达及意义

目的研究人脑胶质瘤(HBG)与人脑转移瘤(HBMC)中核转录因子κBP65(NF-κBP65)的表达及其在肿瘤生物学行为中的作用。方法应用SABC方法进行免疫组织化学染色,观察6例正常人脑组织、18例HBG组织与12例HBMC组织中NF-κBP65的表达及与肿瘤生物学行为之间的关系。结果正常脑细胞中NF-κBP65仅微量表达,在HBG细胞与HBMC细胞中呈不同程度的表达;在高级别HBG、HBMC和复发型HBG中,表达水平较低级别HBG细胞的高(P<0.01)。结论NF-κBP65表达的增强与HBG和HBMC恶性程度的增高、肿瘤浸润关系密切,并在HBG的复发中起重要作用。

Role of nuclear factor kappa B in central nervous system regeneration

Activation of nuclear factor kappa B （NF-κB） is a hallmark of various central nervous system （CNS） pathologies. Neuron-specific inhibition of its transcriptional activator subunit RelA, also referred to as p65, promotes neuronal survival under a range of conditions, i.e., for ischemic or excitotoxic insults. In macro- and microglial cells, post-lesional activation of NF-κB triggers a growth-permissive program which contributes to neural tissue inflammation, scar formation, and the expression of axonal growth inhibitors. Intriguingly, inhibition of such inducible NF-~B in the neuro-glial compartment, i.e., by genetic ablation of RelA or overexpression of a trans- dominant negative mutant of its upstream regulator IκBa, significantly enhances functional recovery and promotes axonal regeneration in the mature CNS. By contrast, depletion of the NF-κB subunit p50, which lacks transcriptional activator function and acts as a transcriptional repressor on its own, causes precocious neuronal loss and exacerbates axonal degeneration in the lesioned brain. Collectively, the data imply that NF-κB orchestrates a multicellular pro- gram in which κB-dependent gene expression establishes a growth-repulsive terrain within the post-lesioned brain that limits structural regeneration of neuronal circuits. Considering these subunit-specific functions, interference with the NF-κB pathway might hold clinical potentials to improve functional restoration following traumatic CNS injury.

Oxymatrine Improves TNBS-induced Colitis in Rats by Inhibiting the Expression of NF-κB p65

Inflammatory bowel disease is thought to be regulated by the balance between Th1 and Th2 cytokines secreted by T cells, and NF-κB p65 also plays a predominant role in the intestinal inflammation. We evaluated the potency of oxymatrine, one of active components of Sophora Root, in inhibiting the immune responses and inflammation in 2,4,6-trinitrobenzene sulfonic acid （TNBS）-induced colitis. The inflammation was markedly ameliorated in the oxymatrine-treated rats. The level of IL-2 was increased and that of IL-10 was decreased in colon tissue in the rat model, which was reversed by the treatment of oxymatrine. Moreover, the elevated expression of NF-κB p65 in colon tissue in the model was also improved by oxymatrine treatment. Our results suggest that oxymatrine might be beneficial for the abnormal immune responses and inflammation by regulating the unbalance of Th1 and Th2 cytokines secretion and inhibiting the expression of NF-κB p65 in colon tissue.

Clinical significance of SQSTM1/P62 and nuclear factor-κB expression in pancreatic carcinoma

BACKGROUND Overexpression of SQSTM1(sequestosome 1,P62)and nuclear factor-κB(NF-κB)plays an important role in the invasion and metastasis of a variety of malignant tumors.AIM To explore the expression of P62 and NF-κB in pancreatic cancer and their relationship with clinicopathological features.METHODS The expression levels of P62 and NF-κB were analyzed by immunohistochemistry with a tissue chip containing 40 cases of human pancreatic carcinoma.Then we analyzed the correlation among P62 expression,phospho-P65 expression,and clinicopathological features of pancreatic carcinoma samples.RESULTS P62 expression was mainly observed in the cytoplasm of pancreatic carcinoma cells.Phosphorylated P65(phospho-P65)was mainly expressed in the nucleus and cytoplasm of pancreatic carcinoma cells.There was a significant difference in P62 expression among T stages.And a significant difference in phosphor-P65 expression among pathology types was noted.In the cases with strongly positive P62 expression,significant differences were found in age.And there were significant differences in T stage and tumor-node-metastasis stage in the cases with strongly positive phosphor-P65 expression.CONCLUSION In pancreatic carcinoma,P62 expression is significantly correlated with T stage.It may be a valuable malignant indicator for human pancreatic carcinoma.

Effect of NF-κB p65 antisense oligodeoxynucleotide on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2

AIM：To study the inhibition of nuclear factor kappa-B p65（NF-κB p65）antisense oligodeoxynucleotide（ASODN）on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2（TGF-β2）.·M ETHODS：NF-κBp65ASODNand NF-κBp65missense oligodeoxynucleotide（MSODN）were designed and synthesized.Human lens epithelial cell line（HLE B-3）cells were prepared for study and divided into 7 groups.Control group was HLE B-3 cells cultured in dulbecco’s modified eagle medium（DMEM）.T1,T2,and T3 group were HLE B-3 cells cultured in DMEM with 10 ng/m L TGF-β2 for 6h,12h,24h respectively.A＋T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2for 24h after transfected by NF-κB p65 ASODN for 24h.M＋T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h.The negative control group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after cultured with transfer agent（Hi Per Fect）for 24h.Cell morphology was observed at different time points using an inverted microscope.The expression of NF-κB p65 m RNA was detected with reverse transcription-polymerase chain reaction（RT-PCR）,and the expression ofα-smooth muscle actin（α-SMA）protein was assayed with ELISA.·RESULTS：With the TGF-β2 stimulation prolongation,the expression of NF-κB p65 m RNA and a-SMA protein increased in T1,T2,T3 groups compared with the control group,and the difference was statistically significant（〈0.05）.NF-κB p65 ASODN lowered the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.NF-κB p65 MSODN and Hi Per Fect did not lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.The difference between control group and A＋T group was not statistically significant（〉0.05）,but the difference among A＋T group and other groups was statistically significant（〈0.05）.·CONCLUSION：NF-κB p65 ASODN could lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2,and antagonized TGF-β2-induced transdifferentiation of HLE B-3.NF-κB p65ASODN could be used as a new biological therapeutic target of posterior capsular opacification.

Isoflavone Attenuates the Nuclear Transcription Factor Kappa B (NF-<i>κ</i>B) Activation on MPP⁺-Induced Apoptosis of PC12 Cells

Objective: To explore the underlying molecular mechanisms of cellular response to the challenge by 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of PC12 cells, an in vitro cell model for Parkinson’s disease, and the effect of NF-κB activation on the protection of Parkinson’s disease by Isoflavone (I). Methods: PC12 cells were used to establish the cell model of Parkinson’s disease, and are divided into five groups: control group;MPP+ group;I (Isoflavone) + MPP+ group;I group;SN-50 + MPP+ group. The content of NF-κB in PC12 cells was determined by immunocytochemistry;The viability of PC12 cells after treated with cell-permeable NF-κB inhibitor SN-50 and cell viability were measured by MTT assay;the expression levels of NF-κB p65 in cytoplasm and nuclear fractions were evaluated by western blot analysis;the mRNA expression of NF-κB p65 was analyzed by in situ hybridization (ISH). Results: Compared with the control group, the protein of NF-κB p65 both in cytoplasm and in nuclei was significantly higher than in I + MPP+ and MPP+ groups;similarly, the mRNA expression level of NF-κB p65 gene was also significantly higher;moreover, the protein expression of NF-κB p65 was much lower in I group (P + group, the protein of NF-κB p65 was significantly lower in I + MPP+ group, the mRNA expression level of NF-κB p65 gene was also significantly lower, and the protein expression level of NF-κB p65 was much lower in I + MPP+ group (P + group (P > 0.05). Conclusion: NF-κB activation is essential to MPP+-induced apoptosis in PC12 cells;but Isoflavone can inhibit the cell damage to some extent to execute its protective function, which may be involved in nigral neurodegeneration in patients with Parkinson’s disease.

Homer1a reduces inflammatory response after retinal ischemia/reperfusion injury

Elevated intraocular pressure(IOP)is one of the causes of retinal ischemia/reperfusion injury,which results in NRP3 inflammasome activation and leads to visual damage.Homerla is repo rted to play a protective role in neuroinflammation in the cerebrum.However,the effects of Homerla on NLRP3inflammasomes in retinal ischemia/reperfusion injury caused by elevated IOP remain unknown.In our study,animal models we re constructed using C57BL/6J and Homer1^(flox/-)/Homerla^(+/-)/Nestin-Cre^(+/-)mice with elevated IOP-induced retinal ischemia/repe rfusion injury.For in vitro expe riments,the oxygen-glucose deprivation/repe rfusion injury model was constructed with M uller cells.We found that Homerla ove rexpression amelio rated the decreases in retinal thickness and Muller cell viability after ischemia/reperfusion injury.Furthermore,Homerla knockdown promoted NF-κB P65^(Ser536)activation via caspase-8,NF-κB P65 nuclear translocation,NLRP3 inflammasome formation,and the production and processing of interleukin-1βand inte rleukin-18.The opposite results we re observed with Homerla ove rexpression.Finally,the combined administration of Homerla protein and JSH-23 significantly inhibited the reduction in retinal thickness in Homer1^(flox/-)Homer1a^(+/-)/Nestin-Cre^(+/-)mice and apoptosis in M uller cells after ischemia/reperfusion injury.Taken together,these studies demonstrate that Homer1a exerts protective effects on retinal tissue and M uller cells via the caspase-8/NF-KB P65/NLRP3 pathway after I/R injury.

青藤碱对缺血再灌注大鼠脑保护的作用机制

目的探讨青藤碱(Sin)对缺血再灌注(IR)大鼠脑保护的作用机制。方法80只Wistar大鼠随机分为假手术组、IR组、Sin高剂量(60mg/kg)组和Sin低剂量(30mg/kg)组;Sin高剂量组和Sin低剂量组分别腹腔注射相应剂量Sin;30min后线栓法制备局灶性IR模型;IR后24h,应用2,3,5-三苯基氯化四氮唑(TTC)和HE染色观察脑梗死体积及脑组织病理学变化;干湿重法检测脑含水量;免疫组化法检测各组大鼠额顶部皮质核转录因子(NF)-κBp65、细胞间黏附分子(ICAM)-1表达及髓过氧化物酶(MPO)活性。结果与IR组比较,Sin预处理后脑组织病理学改变明显减轻,Sin高剂量组缺血性改变更轻;Sin高、低剂量组脑梗死体积明显缩小,脑含水量明显降低,且Sin高剂量组更明显(均P<0.05)。假手术组皮质可见少量NF-κBp65表达于胞质,IR组、Sin高、低剂量组NF-κBp65表达增加(均P<0.05),且表达于胞核;与IR组比较,Sin高、低剂量组NF-κBp65核表达明显减少,Sin高剂量组减少更显著(均P<0.05)。IR组及Sin高、低剂量组MPO活性较假手术组明显增加(均P<0.05);与IR组比较,Sin高、低剂量组ICAM-1表达和MPO活性明显降低,Sin高剂量组降低更显著(均P<0.05)。结论Sin通过抑制脑组织NF-κBp65及ICAM-1表达和MPO活性,减轻IR后脑组织的炎症反应和脑水肿;其脑保护作用呈剂量依赖性。

梓醇对脑缺血急性期及亚急性期的神经保护作用及作用机制

目的：探讨梓醇对永久性脑缺血模型急性期脑组织梗死体积、含水量及亚急性期炎性反应的影响。方法：采用化学刺激法建立局灶性脑缺血（MCAT）模型，检测急性期（24 h）神经行为学症状、脑梗死面积和脑含水量；线栓法制作永久性大鼠脑缺血（pMCAO）模型，酶联免疫吸附法（ELISA）测定缺血侧脑组织白细胞介素-6（IL-6）、白细胞介素-10（IL-10）和核转录因子Bp65（NF-κBp65）的含量。结果：大鼠MCAT后24 h，梓醇15-60 mg＆#183;kg-1剂量组可以显著改善模型动物神经症状损伤（P〈0.01或P〈0.001），梓醇15 mg＆#183;kg-1组可显著降低模型动物梗塞区面积（P〈0.05），30 mg＆#183;kg-1组和60 mg＆#183;kg-1组可显著降低脑水肿（P〈0.05）；大鼠pMCAO术后7天开始，梓醇30 mg＆#183;kg-1组或60 mg＆#183;kg-1组开始改善模型动物神经症状损伤；术后14天，与假手术组比较，缺血侧脑组织IL-10和核转录因子NF-κBp65的含量变化与模型组已经不明显，IL-6水平显著降低（P〈0.05），梓醇15 mg＆#183;kg-1灌胃14天可以降低模型动物缺血侧脑组织NF-κBp65含量（P〈0.05）。结论：梓醇能改善局灶性脑缺血模型动物急性期及亚急性期神经症状损伤，缩小梗死灶，减轻脑部水肿，其作用可能与抑制脑缺血引起的炎症损伤无关。

Influence of catgut implantation at acupoints on splenic lymphocyte nuclear factor (NF-κB p65) and correlated signaling molecules (β2AR) in rats with experimental colitis

Objective To investigate the mechanisms of catgut implantation at acupoints on ulcerative colitis. Methods Eighteen SD rats were randomly divided into a normal control group （NC）, a model group （MO） and a catgut implantation group （CI） with 6 rats in each group. Animals in group MO and group CI were treated by trinitro-benzene-sulfonic acid （TNBS） to establish model with colitis. No other treatment was given to the rats in group MO, but catgut was implanted at ＂Shàngjùxū＂ （上 巨虚 ST 37）, ＂Tiānshū＂ （天枢 ST 25） and ＂Dàchángshū＂ （大肠俞 BL 25） in the rats in group CI. The symptoms of diarrhea and bloody stool, and changes in histopathology were detected 15 days after the treatment. Expressions of splenic lymphocyte nuclear factor κB p65（NF-κB p65）and correlated signaling molecules（β2AR）were detected by the western blot method. Results Diarrhea and mucus bloody purulent stool were soon controlled, and mucous injures were obviously improved in group CI. The NF-κB p65 value of splenic lymphocytes was signifi cantly increased （P0.01） and expression of β2AR remarkably reduced in group MO （P0.01）, compared with group NC. But, the NF-κB p65 value was significantly decreased （P0.01） and expression of β2AR remarkably increased in group CI （P 0.01） , compared with group MO. Conclusion Catgut implantation at acupoints is obviously effective in treating experimental colitis. Modulation of NF-κB p65 and the correlated signaling molecules β2AR may be involved in the mechanisms.

Curcumin suppresses gastric NF-κB activation and macromolecular leakage in Helicobacter pylori-infected rats

AIM:To investigate whether curcumin could attenuate nuclear factor(NF)-κB p65 expression and macromolecular leakage in the gastric mucosa of Helicobacter pylori(H.pylori)-infected rats.METHODS:Twenty-five male Sprague-Dawley rats were equally divided into five groups:control rats(Control),control rats supplemented with 600 mg/kg curcumin,H.pylori-infected rats(Hp),H.pylori-infected rats supplemented with 200 mg/kg curcumin(Hp + curIn H.pylori-infected groups,rats were inoculated with H.pylori suspension twice a day at an interval of 4 h for 3 d.Two weeks later,200 or 600 mg/kg curcumin was given once daily to curcuminsupplemented groups for 7 d.On the day of the experiment,macromolecular leakage in gastric mucosa was examined by intravital fluorescence microscopy.The stomach tissue was removed to examine NF-κB p65 expression in gastric epithelial cells by immunohistochemistry.RESULTS:The expression of NF-κB p65 in gastric epithelial cells and the macromolecular leakage from gastric mucosal microcirculation significantly increased in the Hp group compared with the Control group.The percentages of NF-κB p65 immunoreactive cells in Control and Hp groups were 10.72% ± 2.10% vs 16.02% ± 2.98%,P = 0.004,respectively.The percentages of macromolecular leakage in Control and Hp groups were 10.69% ± 1.43% vs 15.41% ± 2.83%,P = 0.001,respectively.Curcumin supplementation in Hp + cur-CONCLUSION:H.pylori-induced gastric inflammation in rats is associated with increased NF-κB activation and macromolecular leakage which can be reduced by curcumin supplementation.

Role of moxibustion in inflammatory responses during treatment of rat ulcerative colitis

AIM: To investigate the efficacy of moxibustion in ulcerative colitis (UC) rats from morphological, immunological and molecular biological perspectives. METHODS: Thirty-two Sprague-Dawley rats were randomly assigned to a blank control group (normal rats, n = 6) and a model replication (MR) group (UC rats, n = 26). A UC model was established by 2,4,6-trinitrobenzenesulfonic acid/dextran sulfate sodium enema. Rats in the MR group were further randomly assigned to a 9-min moxibustion (9M) group (9 moxa-cone, n = 6), 6-min moxibustion (6M) group (6 moxa-cone, n = 6), 3-min moxibustion (3M) group (3 moxa-cone, n = 6), and a waiting list control (WLC) group (no moxibustion treatment, n = 6). Rats in the moxibustion treatment group were treated in 14 sessions over 28 d. Disease activity, local tissue morphology, serum level of interleukin (IL)-8 and IL-10, and expression of Toll-like receptor (TLR)9 as well as nuclear factor (NF)-kappa B p65 in colonic tissue were determined by disease activity index (DAI), hematoxylin and eosin staining, electron microscopy, enzyme-linked immunosorbent assay and Western blotting, respectively. RESULTS: DAI was lowest in the 9M group and highest in the WLC group. The differences in DAI between the moxibustion treatment (3M, 6M, 9M) and no treatment groups were significant for all one-to-one comparisons (0.60 +/- 0.54 vs 1.20 +/- 0.44, 0.60 +/- 0.54 vs 1.80 +/- 0.45, 0.60 +/- 0.54 vs 3.0 +/- 0.45, respectively, P < 0.05). Light and electron microscopy showed that the neatness of the glandular arrangement in colonic mucosal epithelia gradually increased in the WLC, 3M, 6M to 9M groups. IL-8 level successively decreased while IL-10 level increased from the WLC to 3M, 6M and 9M groups. The differences among these groups were significant for all comparisons (105.46 +/- 8.75 vs 76.61 +/- 3.58, 105.46 +/- 8.75 vs 69.78 +/- 1.87, 105.46 +/- 8.75 vs 67.41 +/- 1.84, respectively, P < 0.01 for IL-8; and 30.83 +/- 1.29 vs 75.64 +/- 1.90, 30.83 +/- 1.29 vs 80.90 +/- 3.16, 30.83 +/- 1.29 vs 83.46 +/- 2.37, respectively, P < 0.01 for IL-10), except comparison of 6M vs 9M. Expression of TLR9 and NF-kappa B p65 decreased in order: highest in the WLC group and lowest in the 9M group. In addition, the differences among the WLC, 3M, 6M and 9M groups were significant for all comparisons (0.492 +/- 0.026 vs 0.380 +/- 0.022, 0.492 +/- 0.026 vs 0.355 +/- 0.005, 0.492 +/- 0.026 vs 0.327 +/- 0.015, respectively, P < 0.05 for TLR9; and 0.436 +/- 0.041 vs 0.326 +/- 0.022, 0.436 +/- 0.041 vs 0.293 +/- 0.006, 0.436 +/- 0.041 vs 0.265 +/- 0.017, respectively, P < 0.05 for NF-kappa B p65). CONCLUSION: Moxibustion repairs damaged colonic mucosa, suppresses serum IL-8, activates serum IL-10 level, and decreases expression of TLR-9 and NF-kappa B p65 in UC rats. (C) 2014 Baishideng Publishing Group Inc. All rights reserved.

Expression and signifi cance of TLR4 and HIF-1α in pancreatic ductal adenocarcinoma

AIM:To investigate the expression of toll-like receptor(TLR) 4,nuclear factor-κB(NF-κB) p65 and hypoxiainducible transcription factor 1α(HIF-1α) in pancreatic ductal adenocarcinoma and their clinical significance.METHODS:The mRNA of TLR4 and HIF-1α were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues,and expression of TLR4,NF-κB p65 and HIF-1α protein were detected by immunohistochemistry in 65 cases of pancreatic ductal adenocarcinoma tissues and 38 cases of corresponding adjacent tissues.The relationship between TLR4 or HIF-1α and pathologic features,as well as the association between TLR4 and HIF-1α,were also analyzed.Kaplan-Meier method was used to assess the impact of expression of TLR4 and HIF-1α on survival of patients with pancreatic cancer.RESULTS:The relative quantif ication of TLR4 and HIF-1α mRNA in tumor tissues was 0.81±0.10 and 0.87±0.11,respectively,signif icantly higher than that in adjacent tissues(0.81±0.10 vs 0.70±0.16,P=0.002;0.87±0.11 vs 0.68±0.13,P=0.000).The protein expression of TLR4,NF-κB p65 and HIF-1α in tumor tissues was 69.20%,66.15% and 70.80%,respectively,being signif icantly higher than that in adjacent normal tissues(69.20% vs 39.50%,P=0.003;66.15% vs 31.58%,P=0.001;70.80% vs 36.80%,P=0.001).There was no signif icant correlation between TLR4 or HIF-1α expression and the age,gender,tumor location,the degree of tumor differentiation in the patients(P>0.05).However,there was signif icant correlation between the expression of TLR4 or HIF-1α and tumor size,lymph node metastasis,venous invasion and clinical staging(P<0.05).The expression of TLR4 and HIF-1α had a signif icant impact on survival of patients with pancreatic adenocarcinoma.CONCLUSION:TLR4,NF-κB p65 and HIF-1α are overexpressed in pancreatic adenocarcinoma,TLR4 may be partly involved in up-regulating HIF-1α,and both synergestically promote development of pancreatic adenocarcinoma.

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.

Losartan reduced connexin43 expression in left ventricular myocardium of spontaneously hypertensive rats

Objective:To assess the effect of angiotensin II type 1(AT1)receptor antagonist losartan on myocardium con- nexin43(Cx43)gap junction(GJ)expression in spontaneously hypertensive rats(SHRs)and investigate possible mechanisms. Methods:Sixteen 9-week-old male SHRs and 8 age-matched male Wistar-Kyoto(WKY)rats were included in this study.SHRs were randomly divided into two groups to receive losartan at 30 mg/(kg·d)by oral gavage once daily for 8 weeks(SHR-L)or vehicle(0.9%saline)to act as controls(SHR-V);WKY rats receiving vehicle for 8 weeks served as normotensive controls.At the end of the experiment,rats were sacrificed and the hearts were removed.Expressions of Cx43 and nuclear factor-kappaB p65 (NF-κB p65)proteins in all three groups were observed and further investigations on the effect of angiotensin II type 1 receptor antagonist losartan(30 mg/(kg·d),8 weeks)on Cx43 expression were conducted with Western blot and immunohistochemistry. NF-κB p65 protein in nuclear extracts was determined by Western blot.Results:Left ventricular(LV)hypertrophy was prominent in SHRs,Cx43 and NF-κB p65 protein expressions were obviously upregulated and Cx43 distribution was dispersed over the cell surface.Treatment with losarton reduced the over-expressions of Cx43 and NF-κB p65 in LV myocardium.The distribution of Cx43 gap junction also became much regular and confined to intercalated disk after losartan treatment.Conclu- sion:Cx43 level was upregulated in LV myocardium of SHR during early stage of hypertrophy.Angiotensin II type 1 receptor antagonist losartan prevented Cx43 gap junction remodeling in hypertrophied left ventricles,possibly through the NF-κB pathway.

Cardioprotective effect of erythropoietin on sepsisinduced myocardial injury in rats

BACKGROUND:Sepsis-induced myocardial injury is one of the major predictors of morbidity and mortality of sepsis.The cytoprotective function of erythropoietin(EPO) has been discovered and extensively studied.However,the cardioprotective effects of EPO on sepsis-induced myocardial injury in the rat sepsis model has not been reported.METHODS:The rat models of sepsis were produced by cecal ligation and perforation(CLP)surgery.Rats were randomly(random number) assigned to one of three groups(n=8 for each group):sham group,CLP group and EPO group(1000 lU/kg erythropoietin).Arterial blood was withdrawn at3,6,12,and 24 hours after CLP.cTnl,BNP,CK-MB,LDH,AST,TNF-a,IL-6,IL-10,and CRP were tested by the ELISA assay.Changes of hemodynamic parameters were recorded at 3,6,12,24 hours after the surgery.Histological diagnosis was made by hematoxylin and eosin.Flow cytometry was performed to examine cell apoptosis,myocardium mitochondrial inner membrane potential,and NF-κB(p65).Survival rate at 7 days after CLP was recorded.RESULTS:In the CLP group,myocardial enzyme index and inflammatory index increased at3,6,12 and 24 hours after CLP compared with the sham group,and EPO significantly blocked the increase.Compared with the CLP group,EPO significantly improved LVSP,LV +dpldt_(max) LV-dp/dt_(min),and decreased LVEDP at different time.EPO blocked the reduction of mitochondrial transmembrane potential,suppressed the cardiomyocyte apoptosis,inhibited the activation of NF-κB,and reduced the production of proinflmmatory cytokines.No difference in the survival rate at 7 days was observed between the CLP group and the EPO group.CONCLUSION:Exogenous EPO has cardioprotective effects on sepsis-induced myocardial injury.

Protective effect of Jiaweibugan decoction against diabetic peripheral neuropathy

Oxygen free radical damage is regarded as a direct or indirect common pathway associated with diabetic neuropathy and is the main cause of complications in peripheral neuropathies. We speculate that Jiaweibugan decoction has a significant effect in treating diabetic peripheral neuropathy through an anti-oxidative stress pathway. In this study, a diabetic rat model was established by intraperitoneal injection of streptozotocin. Rats were treated with Jiaweibugan decoction via intragastric administration. The levels of malondialdehyde and glutathione, which are indirect indexes of oxidative stress, in serum were determined using a colorimetric method. The expression levels of nuclear factor kappa B p65 mRNA and p38 mitogen-activated protein kinase, which are oxidative stress associated factors, in the dorsal root ganglion of spinal $4-6 segments were evaluated by reverse-transcriptase polymerase chain reaction and immunohistochemistry. Results showed that, Jiaweibugan decoction significantly ameliorated motor nerve conduction velocity in diabetic rats, effectively decreased malondialdehyde levels in serum and the expression of nuclear factor kappa B p65 mRNA and p38 mitogen-activated protein kinase mRNA in the dorsa root ganglion, and increased glutathione levels in serum. Therefore, our experimental findings indicate that Jiaweibugan decoction plays an anti-oxidative stress role in the diabetic peripheral neuropathy process, which has a protective effect on peripheral nerve injury.