经靶向OPN激活的IKBα/NF-κB信号通路对细胞自噬、细胞增殖和细胞周期的影响

目的探讨经靶向骨桥蛋白(OPN)激活的IKBα/NF-κB信号通路对低氧环境中肺动脉平滑肌细胞(PASMCs)的自噬、增殖和周期的影响。方法培养大鼠PASMCs;观察低氧组PASMCs中OPN的表达情况和IKBα/NF-κB的表达情况。将带有OPN(过表达或敲低)的慢病毒与PASMCs共培养,在OPN过表达的PASMCs中加入NF-κB抑制剂QNZ干扰IKBα/NF-κB信号通路,随后在基因和蛋白层面观察OPN对自噬的影响,并在电镜下观察自噬体数量。用5-乙炔基-2-脱氧嘧啶核苷法和流式细胞术检测OPN对细胞增殖能力和细胞周期的影响。结果低氧组PASMCs表达的OPN高于常氧组(P<0.05),低氧组NF-κB高于常氧组(P<0.05)。常氧组可通过PASMCs过表达OPN激活NF-κB来抑制自噬蛋白LC3B、Beclin1,加入NF-κB抑制剂后可以促进PASMCs的自噬蛋白表达(P<0.05);干扰低氧组PASMCs中OPN的表达可以进一步促进自噬蛋白和自噬小体的表达(P<0.05)。抑制PASMCs中OPN和NF-κB的表达可以阻断细胞从G0/G1期向S期过渡并减弱由OPN增多引起的增殖(P<0.05)。结论经靶向OPN可以激活IKBα/NF-κB信号通路;激活的IKBα/NF-κB信号通路对低氧环境中PASMCs的自噬、增殖和细胞周期产生影响。

Downregulation of MUC1 Inhibits Proliferation and Promotes Apoptosis by Inactivating NF-κB Signaling Pathway in Human Nasopharyngeal Carcinoma

Objective To investigate the effect of mucin 1(MUC1)on the proliferation and apoptosis of nasopharyngeal carcinoma(NPC)and its regulatory mechanism.Methods The 60 NPC and paired para-cancer normal tissues were collected from October 2020 to July 2021 in Quanzhou First Hospital.The expression of MUC1 was measured by real-time quantitative PCR(qPCR)in the patients with PNC.The 5-8F and HNE1 cells were transfected with siRNA control(si-control)or siRNA targeting MUC1(si-MUC1).Cell proliferation was analyzed by cell counting kit-8 and colony formation assay,and apoptosis was analyzed by flow cytometry analysis in the 5-8F and HNE1 cells.The qPCR and ELISA were executed to analyze the levels of TNF-αand IL-6.Western blot was performed to measure the expression of MUC1,NFкB and apoptosis-related proteins(Bax and Bcl-2).Results The expression of MUC1 was up-regulated in the NPC tissues,and NPC patients with the high MUC1 expression were inclined to EBV infection,growth and metastasis of NPC.Loss of MUC1 restrained malignant features,including the proliferation and apoptosis,downregulated the expression of p-IкB、p-P65 and Bcl-2 and upregulated the expression of Bax in the NPC cells.Conclusion Downregulation of MUC1 restrained biological characteristics of malignancy,including cell proliferation and apoptosis,by inactivating NF-κB signaling pathway in NPC.

Apatinib reduces liver cancer cell multidrug resistance by modulating NF-κB signaling pathway

Objectives:This investigation aimed to elucidate the inhibitory impact of apatinib on the multidrug resistance of liver cancer both in vivo and in vitro.Methods:To establish a Hep3B/5-Fu resistant cell line,5-Fu concentrations were gradually increased in the culture media.Hep3B/5-Fu cells drug resistance and its alleviation by apatinib were confirmed via flow cytometry and Cell Counting Kit 8(CCK8)test.Further,Nuclear factor kappa B(NF-κB)siRNA was transfected into Hep3B/5-Fu cells to assess alterations in the expression of multidrug resistance(MDR)-related genes and proteins.Nude mice were injected with Hep3B/5-Fu cells to establish subcutaneous xenograft tumors and then categorized into 8 treatment groups.The treatments included oxaliplatin,5-Fu,and apatinib.In the tumor tissues,the expression of MDRrelated genes was elucidated via qRT-PCR,immunohistochemistry,and Western blot analyses.Results:The apatinibtreated mice indicated slower tumor growth with smaller size compared to the control group.Both the in vivo and in vitro investigations revealed that the apatinib-treated groups had reduced expression of MDR genes GST-pi,LRP,MDR1,and p-p65.Conclusions:Apatinib effectively suppresses MDR in human hepatic cancer cells by modulating the expression of genes related to MDR,potentially by suppressing the NF-κB signaling pathway.

Kuicolong-yu enema decoction retains traditional Chinese medicine enema attenuates inflammatory response ulcerative colitis through TLR4/NF-κB signaling pathway

BACKGROUND Ulcer colitis(UC)is a chronic,nonspecific,and noninfectious inflammatory bowel disease.Recently,Toll-like receptors(TLRs)have been found to be closely associated with clinical inflammatory diseases.Achieving complete remission in patients with intermittent periods of activity followed by dormancy is challenging.Moreover,no study has explored the mechanism by which Kuicolong-yu enema decoction retains traditional Chinese medicine enemas to attenuate the inflammatory response in UC.AIM To explore the mechanism by which Kuicolong-yu enema decoction retains traditional Chinese medicine enemas to attenuate the inflammatory response in UC.METHODS This prospective clinical study included patients who met the exclusion criteria in 2020 and 2021.The patients with UC were divided into two groups(control and experimental).The peripheral blood of the experimental and control groups were collected under aseptic conditions.The expression of TLR4 protein,NF-κB,IL-6,and IL-17 was detected in the peripheral blood of patients in the experimental group and control group before and 1 month after taking the drug.Linear co rrelation analysis was used to analyze the relationship between the expression level of TLR4 protein and the expression levels of downstream signal NF-κB and inflammatory factors IL-6 and IL-17,and P<0.05 was considered statistically significant.RESULTS There were no significant differences in the patient characteristics between the control and experimental groups.The results showed that the expression levels of TLR4 and NF-κB in the experimental group were significantly lower than those in the control group(P<0.05).The levels of IL-6 and IL-17 in the experimental group were significantly lower than those in the control group(P<0.05).The TLR4 protein expression in the experimental group was positively correlated with the expression level of downstream signal NF-κB and was positively correlated with the levels of downstream inflammatory cytokines IL-6 and IL-17(r=0.823,P<0.05).CONCLUSION Kuicolong-yu enema decoction retains traditional Chinese medicine enema attenuates the inflammatory response of UC through the TLR4/NF-κB signaling pathway.

Parthenolide enhances the metronomic chemotherapy effect of cyclophosphamide in lung cancer by inhibiting the NF-kB signaling pathway

BACKGROUND Parthenolide(PTL),a sesquiterpene lactone derived from the medicinal herb Chrysanthemum parthenium,exhibits various biological effects by targeting NF-kB,STAT3,and other pathways.It has emerged as a promising adjunct therapy for multiple malignancies.AIM To evaluate the in vitro and in vivo effect of PTL on cyclophosphamide(CTX)metronomic chemotherapy.METHODS The cytotoxicity of PTL and CTX on Lewis lung cancer cells(LLC cells)was assessed by measuring cell activity and apoptosis.The anti-tumor efficiency was evaluated using a tumor xenograft mice model,and the survival of mice and tumor volume were monitored.Additionally,the collected tumor tissues were analyzed for tumor microenvironment indicators and inflammatory factors.RESULTS In vitro,PTL demonstrated a synergistic effect with CTX in inhibiting the growth of LLC cells and promoting apoptosis.In vivo,metronomic chemotherapy com-bined with PTL and CTX improved the survival rate of tumor-bearing mice and reduced tumor growth rate.Furthermore,metronomic chemotherapy combined with PTL and CTX reduced NF-κB activation and improved the tumor immune microenvironment by decreasing tumor angiogenesis,reducing Transforming growth factorβ,andα-SMA positive cells.CONCLUSION PTL is an efficient compound that enhances the metronomic chemotherapy effects of CTX both in vitro and in vivo,suggesting its potential as a supplementary therapeutic strategy in metronomic chemotherapy to improve the chemotherapy effects.

Aszonapyrone A Isolated from Neosartorya spinosa IFM 47025 Inhibits the NF-κB Signaling Pathway Activated by Expression of the Ependymoma-Causing Fusion Protein ZFTA-RELA

Ependymoma is a rare and chemotherapy-resistant brain tumor, which has resulted in a delay in the development of drugs to treat it. A subclass of supratentorial ependymomas (ST-EPN), designated ST-EPN-zinc finger-translocation-associated (ZFTA, ST-EPN-ZFTA), exhibits the expression of a fusion protein comprising ZFTA and v-rel reticuloendotheliosis viral oncogene homolog A (RELA), an effector transcription factor of the nuclear factor-kappa B (NF-κB) pathway (ZFTA-RELA). The expression of ZFTA-RELA results in the hyperactivation of the oncogenic NF-κB signaling pathway, which ultimately leads to the development of ST-EPN-ZFTA. To identify inhibitors of the NF-κB signaling pathway activated by the expression of ZFTA-RELA, we used a doxycycline-inducible ZFTA-RELA-expressing NF-κB reporter cell line and found that extracts of the fungus Neosartorya spinosa IFM 47025 exhibited NF-κB inhibitory activity. We identified eight compounds [aszonapyrone A (2), sartorypyrone A (3), epiheveadride (4), acetylaszonalenin (5), (R)-benzodiazepinedione (6), aszonalenin (7), sartorypyrone E (8) and (Z, Z)-N,N’-(1,2-bis[(4-methoxyphenyl)methylene]-1,2-ethanediyl)bis-formamide (9)] from N. spinosa IFM 47025 culture extract using a variety of chromatographic techniques. The structures of these compounds were identified through the analysis of various instrumental data (1D, 2D-NMR, MS, and optical rotation). The NF-κB responsive reporter assay indicated that compounds 2, 3, 5, 7, and 9 exhibited inhibitory activity. We further evaluated the inhibitory activity of these compounds against the expression of endogenous NF-κB responsive genes (CCND1, L1CAM, ICAM1, and TNF) and found that compound 2 showed significant inhibitory activity. Further studies are required to elucidate the mechanism of action of compound 2, which may serve as a lead compound for the development of a novel therapy for ST-EPN-ZFTA.

Quercetin regulates depression-like behavior in CUMS rat models via TLR4/NF-κB signaling

Background:Depression is becoming increasingly prevalent around the world,imposing a substantial burden on individuals,families,as well as society.Quercetin is known to be highly effective in treating depression.However,additional research is needed to dissect the mechanisms of its anti-depressive effects.Methods:For this study,Sprague-Dawley(SD)rats were randomized into the control,model,quercetin,or fluoxetine group.The latter three groups were exposed to chronic unpredictable mild stress(CUMS)for 42 d.The first two groups received saline solution daily via oral gavage.Meanwhile,the quercetin group was orally administered a quercetin suspension(52.08 mg/kg)every day,while the fluoxetine group was orally administered a fluoxetine solution(2.08 mg/kg).Here,fluoxetine served as the positive control drug to compare the therapeutic effects of quercetin.The experimental period was 6 weeks.Depressive behaviors in rats were assessed through various physiological and behavioral measures.Additionally,pathological changes in hippocampal tissues were examined using Nissl staining.Serum cytokines were detected using an enzymelinked immunosorbent assay(ELISA),and immunohistochemistry was employed to quantify the levels and integral optical density(IOD)values of ionized calcium binding adaptor molecule-1(Iba-1)expression in the brain.Real-time fluorescence quantitative PCR(RT-qPCR)was utilized to evaluate the mRNA levels of inflammatory indicators as well as toll-like receptor 4(TLR4),and nuclear factor-κappa B P65(NF-κB P65)in hippocampus.Western blot(WB)technique was employed to observe the protein levels of TLR4,NF-κB P65,and phospho-NF-κB P65(p-NF-κB P65).Results:After 42 d of exposure to CUMS,rats exhibited a slow increase in body weight,a reduction in food intake,an abnormal preference for sugar water,and aberrant open-field behaviors.Pathological analysis revealed the disintegration,rupture,interruption,and disorganization of hippocampal neuronal cells after CUMS exposure,along with a decrease in Nissl bodies in the CA1 region.This was accompanied by the elevated expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)in the serum and the upregulation of IL-1β,IL-6,and TNF-αmRNA expression in the hippocampus.Increases in Iba-1-positive cells and the IOD values of Iba-1 were detected in hippocampal microglia.Furthermore,TLR4 and NF-κB P65 mRNA and protein levels were upregulated in hippocampal tissues.Quercetin,an antidepressant,could alleviate depression-like symptoms in rats and downregulate inflammatory factors associated with the TLR4/NF-κB signaling pathway in hippocampal microglia,and its therapeutic effect was comparable to fluoxetine.Conclusion:In rat models of CUMS,quercetin may act as an antidepressant by inhibiting inflammation in hippocampal microglia via TLR4/NF-κB signaling pathway.These results offer experimental and theoretical support for applying quercetin in the clinical management of depression.

Huangqin decoction alleviates lipid metabolism disorders and insulin resistance in nonalcoholic fatty liver disease by triggering Sirt1/NF-κB pathway

BACKGROUND Nonalcoholic fatty liver disease(NAFLD)is a clinicopathological entity characterized by intrahepatic ectopic steatosis.As a consequence of increased consumption of high-calorie diet and adoption of a sedentary lifestyle,the incidence of NAFLD has surpassed that of viral hepatitis,making it the most common cause of chronic liver disease globally.Huangqin decoction(HQD),a Chinese medicinal formulation that has been used clinically for thousands of years,has beneficial outcomes in patients with liver diseases,including NAFLD.However,the role and mechanism of action of HQD in lipid metabolism disorders and insulin resistance in NAFLD remain poorly understood.AIM To evaluate the ameliorative effects of HQD in NAFLD,with a focus on lipid metabolism and insulin resistance,and to elucidate the underlying mechanism of action.METHODS High-fat diet-induced NAFLD rats and palmitic acid(PA)-stimulated HepG2 cells were used to investigate the effects of HQD and identify its potential mechanism of action.Phytochemicals in HQD were analyzed by highperformance liquid chromatography(HPLC)to identify the key components.RESULTS Ten primary chemical components of HQD were identified by HPLC analysis.In vivo,HQD effectively prevented rats from gaining body and liver weight,improved the liver index,ameliorated hepatic histological aberrations,decreased transaminase and lipid profile disorders,and reduced the levels of pro-inflammatory factors and insulin resistance.In vitro studies revealed that HQD effectively alleviated PA-induced lipid accumulation,inflammation,and insulin resistance in HepG2 cells.In-depth investigation revealed that HQD triggers Sirt1/NF-κB pathwaymodulated lipogenesis and inflammation,contributing to its beneficial actions,which was further corroborated by the addition of the Sirt1 antagonist EX-527 that compromised the favorable effects of HQD.CONCLUSION In summary,our study confirmed that HQD mitigates lipid metabolism disorders and insulin resistance in NAFLD by triggering the Sirt1/NF-κB pathway.

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways

BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.

Acupuncture at Back-Shu point improves insomnia by reducing inflammation and inhibiting the ERK/NF-κB signaling pathway

BACKGROUND Insomnia is a disease where individuals cannot maintain a steady and stable sleep state or fail to fall asleep.Western medicine mainly uses sedatives and hypnotic drugs to treat insomnia,and long-term use is prone to drug resistance and other adverse reactions.Acupuncture has a good curative effect and unique advantages in the treatment of insomnia.AIM To explore the molecular mechanism of acupuncture at Back-Shu point for the treatment of insomnia.METHODS We first prepared a rat model of insomnia,and then carried out acupuncture for 7 consecutive days.After treatment,the sleep time and general behavior of the rats were determined.The Morris water maze test was used to assess the learning ability and spatial memory ability of the rats.The expression levels of inflammatory cytokines in serum and the hippocampus were detected by ELISA.qRTPCR was used to detect the mRNA expression changes in the ERK/NF-κB signaling pathway.Western blot and immunohistochemistry were carried out to evaluate the protein expression levels of RAF-1,MEK-2,ERK1/2 and NF-κB.RESULTS Acupuncture can prolong sleep duration,and improve mental state,activity,diet volume,learning ability and spatial memory.In addition,acupuncture increased the release of 1L-1β,1L-6 and TNF-αin serum and the hippocampus and inhibited the mRNA and protein expression of the ERK/NF-κB signaling pathway.CONCLUSION These findings suggest that acupuncture at Back-Shu point can inhibit the ERK/NF-κB signaling pathway and treat insomnia by increasing the release of inflammatory cytokines in the hippocampus.

Mechanism of Sanshi decoction inhibits macrophage pyroptosis by inhibiting BRD4/NF-κB/NLRP3 pathway in the treatment of gouty arthritis

Objective:To observe the effect of Sanshi decoction on BRD4/NF-κB/NLRP3 pathwaymediated macrophage pyroptosis,so as to elucidate the molecular mechanism of Sanshi decoction in the treatment of gouty arthritis.Methods:THP-1 was induced into macrophages with foboside and the divided into the control group,model group,low-dose,medium-dose,high-dose group of Sanshi decoction,and BRD4 inhibitor group.Except for the control group,the remaining groups were induced with monosodium urate crystals to construct a gouty arthritis cell model.The activity of macrophages was detected by CCK8,the level of macrophage pyroptosis was detected by flow cytometry,the activity of LDH,the content of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay,and the expression of related proteins in the BRD4/NF-κB/NLRP3 pathway was detected by Western blot.Results:Compared with the control group,macrophage activity was decreased in the model group,and the level of pyroptosis,LDH activity,contents of IL-1β and IL-18,expression levels of BRD4,p-NF-kB p65,NLRP3,Caspase-1 p20,and IL-1β protein were significantly up-regulated,the differences were statistically significant(P<0.05 and P<0.01).Compared with the model group,macrophage activity was up-regulated in the Sanshi Decoction,and the level of pyroptosis,LDH activity,IL-1β and IL-18 contents,expression levels of BRD4,p-NF-kB p65,NLRP3,Caspase-1 p20,and IL-1β protein were significantly decreased with statistically significant differences(P<0.05 and P<0.01).Conclusion:Sanshi decoction inhibits macrophage pyroptosis by inhibiting BRD4/NF-κB/NLRP3 pathway activation,thus improving the inflammation level of gouty arthritis.

人参二醇组皂苷对内毒素休克大鼠心肌组织CD14、IkB和NF-κ B p65表达的影响

目的通过观察内毒素诱导的休克大鼠心肌CD14、核因子-κB抑制蛋白(IκB)和核因子-κB亚基p65(NF-κB p65)的表达变化,探讨人参二醇组皂苷(panaxadiolsaponin,PDS)对休克大鼠心肌保护作用的相关机制。方法 40只Wistar大鼠分为空白对照组(CTR)、内毒素休克模型组(LPS)、地塞米松治疗组(DEX)和人参二醇组皂苷治疗组(PDS)。LPS(5 mg/kg)舌下静脉注射复制休克模型,当动脉血压降至初始血压的2/3时作为判定休克的标准。记录不同时间段平均动脉压(mean arterial pressure,MAP),各组大鼠分别于休克后2 h和4 h测定血清谷草转氨酶(aspartate aminotransferase,AST)、肌酸激酶(creatinekinase,CK)和乳酸脱氢酶(lactate dehydrogenase,LDH)活性,采用R T-PCR方法检测心肌组织CD14和IκB mR NA水平,Western blotting检测CD14和NF-κBp65蛋白质表达水平。结果各组大鼠注入LPS 5min后进入休克状态,并在10min后MAP代偿性回升,但LPS组从休克第45 min MAP进行性下降,而PDS和DEX组在LPS组进入可逆性失代偿阶段后,仍维持代偿性变化;LPS组血清中AST、CK和LDH活性最强,PDS及DEX组酶活性明显低于LPS组(P<0.05);R T-PCR结果显示,LPS组IκB mR NA表达明显减弱,CD14 mR NA表达增强,给予DEX治疗后IκB和CD14 mRNA表达均降低;给予PDS治疗后IκB mRNA表达较LPS组明显增高,而CD14表达明显降低;Western blotting证实,LPS组CD14及NF-κB表达明显增高,给予DEX和PDS治疗后CD14和NF-κB表达受到抑制,其表达量接近CTR组。结论 PDS对休克大鼠心肌具有保护作用,能减轻内毒素血症时心肌表达CD14、IκB和NF-κB p65。

通心络胶囊对糖尿病小鼠视网膜IKKβ/IKBα/NF-κB通路的作用

目的观察通心络胶囊对糖尿病小鼠视网膜IKKβ/IKBα/NF-κB通路的影响。方法 40只KK/Upj-Ay小鼠随机分为模型组、通心络低(1 g·kg-1)、中(2 g·kg-1)、高(4 g·kg-1)剂量组,每组各10只,10只C57BL/6小鼠为对照组。通心络组按相应剂量灌胃给药,对照组和模型组给予等体积的蒸馏水,每天1次,连续12周。测定体质量、空腹血糖值(fasting blood glucose,FBG),伊文思蓝法(evans blue method,EB)测定血-视网膜屏障的通透性,HE染色法观察视网膜形态学变化,Real-time PCR(qPCR)和Western blot法测定IKKβ、IKBα、NF-κB mRNA和蛋白的表达及P-IKBα和核NF-κB蛋白的表达。结果通心络高剂量组较中、低剂量组细胞结构更致密,排列更有序。通心络低、中、高剂量组EB含量分别(20.82±3.88)ng·mL-1、(16.43±2.60)ng·mL-1、(16.14±2.42)ng·mL-1,较模型组(25.53±3.57)ng·mL-1明显下降(均为P<0.05)。通心络中、高剂量组IKKβmRNA和蛋白表达量较模型组显著下降(均为P<0.01);通心络组IKBαmRNA,通心络中、高剂量组IKBα蛋白以及PIKBα蛋白的表达均较模型组显著下降(均为P<0.01),通心络低、中、高剂量组核NF-κB蛋白表达量为0.52±0.05、0.43±0.05、0.34±0.04,显著低于模型组(0.61±0.07)(均为P<0.05)。结论通心络胶囊可明显改善KK/Upj-Ay小鼠视网膜病变,其机制与抑制IKKβ/IKBα/NF-κB通路相关。

艾灸对腹泻型肠易激综合征模型大鼠海马与结肠组织中IKKβ/IKBα/NF-κB通路的影响

目的观察艾灸对腹泻型肠易激综合征(diarrhea predominant irritable bowel syndrome,IBS-D)模型大鼠海马与结肠组织中核因子κB抑制蛋白(inhibitor of nuclear factor kappa B,IκB)激酶β(IκB kinase beta,IKKβ)、核因子κB抑制蛋白α(NF-kappa-B inhibitor alpha,IKBα)、核因子κB(nuclear factor kappa B,NF-κB)(P65)相对表达水平的影响,探讨艾灸治疗IBS-D的机制。方法将24只健康雄性SD大鼠随机分为空白组、模型组和艾灸组,每组8只。采用慢性束缚联合番泻叶灌胃方法建立IBS-D大鼠模型。艾灸组予以艾灸"天枢""上巨虚",每日30min,每日1次,共治疗7d。治疗后,观察大鼠稀便率和直肠扩张所引起腹部回缩反射(abdominal withdrawal reflex,AWR)的最小容量阈值;蛋白质印迹法检测海马和结肠组织中IKKβ、IKBα、NF-κB(P65)蛋白表达水平。结果与空白组比较,模型组AWR的最小容量阈值明显下降(P<0.05),稀便率明显升高(P<0.05);海马和结肠组织中IKKβ、IKBα、NF-κB(P65)蛋白表达水平明显增加(P<0.05)。与模型组比较,艾灸组AWR的最小容量阈值明显升高(P<0.05),稀便率明显下降(P<0.05);海马和结肠组织中IKKβ、IKBα、NF-κB(P65)蛋白表达水平显著降低(P<0.05)。结论艾灸"天枢""上巨虚"改善IBS-D大鼠腹泻症状和内脏高敏感性可能与艾灸抑制海马与结肠组织IKKβ/IKBα/NF-κB信号通路有关,且其作用机制可能是通过脑-肠轴途径实现的。

柴胡皂苷A对溃疡性结肠炎大鼠IKK/IKB/NF-κB信号通路及肠上皮细胞凋亡的影响

目的探讨柴胡皂苷(SS)A对溃疡性结肠炎(UC)大鼠核因子(NF)-κB抑制物激酶(IKK)/抑制因子κB(IKB)/NF-κB信号通路及肠上皮细胞凋亡的影响。方法制备UC大鼠模型,造模后随机分为模型组、SSA(低、中、高)剂量组、阳性对照组,每组10只;正常组10只大鼠作为空白对照。常规饲养2 d,第3天时SSA(低、中、高)剂量组分别灌胃(12.5、25.0、50.0)mg/kg SSA,阳性对照组灌胃0.5 g/kg柳氮磺吡啶(SASP),正常组和模型组灌胃等体积生理盐水,1次/d,连续5 d。苏木素-伊红(HE)染色观察肠黏膜组织形态;酶联免疫吸附试验(ELISA)检测血清中白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α水平;TUNEL染色观察肠上皮细胞凋亡情况;Western印迹检测肠黏膜组织中IKKα、IKB、NF-κB p65、天冬氨酸特异性半胱氨酸蛋白酶(caspase)-3、B细胞淋巴瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白水平。结果正常组大鼠实验结束后毛色有光泽,大便正常;肠黏膜组织完整,肌层结构正常。模型组大鼠毛色暗淡无光泽,大便出现不同程度的便血现象,便稀且黏;肠黏膜处出现明显炎症现象,细胞排列紊乱,部分结肠腺体被浸润的炎症细胞替代,黏膜层变薄。SSA(低、中、高)剂量组、阳性对照组大鼠毛色较暗淡,大便出现不同程度的缓和;出现不同程度的炎症浸润和肠腺溃疡现象,但较模型组有所降低。与正常组相比,模型组血清中IL-1β、IL-6、TNF-α水平,肠上皮细胞凋亡率,肠黏膜组织中IKKα、IKB、NF-κB p65、caspase-3、Bax蛋白水平显著升高,Bcl-2蛋白水平显著降低(均P<0.05);与模型组相比,SSA(低、中、高)剂量组、阳性对照组血清中IL-1β、IL-6、TNF-α水平,肠上皮细胞凋亡率,肠黏膜组织中IKKα、IKB、NF-κB p65、caspase-3、Bax蛋白水平显著降低,Bcl-2蛋白水平显著升高(均P<0.05);随着SSA给药剂量的增加,SSA各剂量组血清中IL-1β、IL-6、TNF-α水平,肠上皮细胞凋亡率,肠黏膜组织中IKKα、IKB、NF-κB p65、caspase-3、Bax蛋白水平逐渐降低,Bcl-2蛋白水平逐渐升高(均P<0.05),呈剂量依赖性。结论SSA可抑制IKK/IKB/NF-κB信号通路及肠上皮细胞凋亡,实现对UC大鼠的保护。

Blautia producta displays potential probiotic properties against dextran sulfate sodium-induced colitis in mice

Blautia has attracted attention because of its potential efficacy in ameliorating host energy metabolism and inflammation.This study aims to investigate the influences of Blautia producta D4 on colitis induced by dextran sulfate sodium(DSS)and to reveal the underlying mechanisms.Results showed that B.producta D4 intervention significantly relieved body weight loss,and suppressed the elevation of pro-inflammatory cytokines(including interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and interleukin-1β(IL-1β))and excessive oxidative stress(myeloperoxidease(MPO)activity,superoxide dismutase(SOD)activity,glutathione peroxidase(GSH-Px)activity,and malondialdehyde(MDA)level)in colitis mice.Moreover,the concentrations of tight junction proteins(occludin,claudin-1,and ZO-1)related to the intestinal barrier were obviously elevated,and colitis-related TLR4/NF-κB pathway activation was remarkably inhibited after B.producta D4 intervention.The intestinal microbial disorder was evidently ameliorated by increasing the relative abundance of Clostridium sensu stricto 1,Bifidobacterium,GCA-900066225,Enterorhabdus,and reducing the relative abundance of Lachnospiraceae NK4A136 group.In conclusion,oral administration of B.producta D4 could ameliorate DSS-induced colitis by suppressing inflammatory responses,maintaining the intestinal barrier,inhibiting TLR4/NF-κB pathway,and regulating intestinal microbiota balance.These results are conducive to accelerate the development of B.producta D4 as a functional probiotic for colitis.

Anti-inflammation Effects of Sinomenine on Macrophages through Suppressing Activated TLR4/NF-kB Signaling Pathway

Sinomenine(SN)has been used in the clinical treatment of systemic lupus erythematosus and rheumatoid arthritis for many years.Studies showed that SN held protective effects such as anti-inflammation,scavenging free radicals and suppressing immune response in many autoimmune diseases.The purpose of the present study is to explore the mechanism of anti-inflammation of SN on lipopolysaccharide(LPS)-induced macrophages activation and investigate whether the TLR4/NF-κB signaling pathway participated in.Macrophages isolated from mouse peritoneal cavity were stimulated by 1 pg/mL LPS for 24 h.And then the cells were treated with various concentrations of SN,TLR4 inhibitor respectively for additional 48 h.Drug toxicity was detected by MTT assay and Transwell experiment was used to assess chemotaxis.Furthermore,TLR4 and MyD88 mRNA levels were detected by real-time PCR.Western blotting was used to examine TLR4,MyD88 and phosphorylated IκB protein expression in macrophages.Immunofluorescence assay was applied to observe p65 NF-κB protein expression in macrophage nucleus.We extracted macrophages with high purity and activity from the abdominal cavity of mice.SN remarkably inhibited the chemotaxis and secretion function of LPS-stimulated macrophages.It also down-regulated both the protein levels of inflammatory cytokines(TNF-α,IL-β and IL-6)and the RNA and protein levels of the key factors(TLR4,MyD88,p-IkB)in TLR4 pathway.The expression of p65 NF-κB protein in nuclei was down-regulated,which was correlated with a similar decrease in p-IκB protein level.In conclusion,SN can inhibit the LPS induced immune responses in macrophages by blocking the activated TLR4/NF-κB signaling pathway.These results may provide a therapeutic approach to regulate inflammatory responses.

β-arrestin 2 attenuates lipopolysaccharide-induced liver injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation in mice

AIM To study the role and the possible mechanism of β-arrestin 2 in lipopolysaccharide(LPS)-induced liver injury in vivo and in vitro.METHODS Male β-arrestin 2^(+/+) and β-arrestin 2^(-/-)C57 BL/6 J mice were used for in vivo experiments, and the mouse macrophage cell line RAW264.7 was used for in vitro experiments. The animal model was established via intraperitoneal injection of LPS or physiological sodium chloride solution. Blood samples and liver tissues were collected to analyze liver injury and levels of pro-inflammatory cytokines. Cultured cell extracts were collected to analyze the production of pro-inflammatory cytokines and expression of key molecules involved in the TLR4/NF-κB signaling pathway.RESULTS Compared with wild-type mice, the β-arrestin 2 knockout mice displayed more severe LPS-induced liver injury and significantly higher levels of proinflammatory cytokines, including interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α, and IL-10. Compared with the control group, pro-inflammatory cytokines(including IL-1β, IL-6, TNF-α, and IL-10) produced by RAW264.7 cells in the β-arrestin 2 si RNA group were significantly increased at 6 h after treatment with LPS. Further, key molecules involved in the TLR4/NF-κB signaling pathway, including phosphoIκBα and phosho-p65, were upregulated.CONCLUSION β-arrestin 2 can protect liver tissue from LPS-induced injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation.

Recombinant adeno-associated virus serotype 9 with p65 ribozyme protects H9c2 cells from oxidative stress through inhibiting NF-κB signaling pathway

Background Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. It can trigger inflammatory cascades which are primarily mediated via nuclear factor-κB （NF-κB）. The NF-κB transcription factor family includes several subunits （p50, p52, p65, c-Rel, and Rel B） that respond to myocardial ischemia. It has been proved that persistent myocyte NF-κB p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-κB p65 ribozyme （AAV9-R65-CMV-eGFP） was constructed. The cells were assessed by MTT assay, Annexin V–propidium iodide dual staining to study apoptosis. The expression of P65 and P50 were assessed by Western blot to investigate the under-lying molecular mechanisms. Results After stimulation with H2O2 for 6 h, H9c2 cells viability decreased significantly, a large fraction of cells underwent apoptosis. We observed a rescue of H9c2 cells from H2O2-induced apoptosis in pretreatment with AAV9-R65-CMV-eGFP. Moreover, AAV9-R65-CMV-eGFP decreased H2O2-induced P65 expression. Conclusions AAV9-R65-CMV-eGFP protects H9c2 cells from oxidative stress induced apoptosis through down-regulation of P65 expression. These observations indicate that AAV9-R65-CMV-eGFP has the potential to exert cardioprotective effects against oxidative stress, which might be of great importance to clinical efficacy for cardiovascular disease.

Non-SMC condensin Ⅰ complex subunit D2 and non-SMC condensin Ⅱ complex subunit D3 induces inflammation via the IKK/NF-κB pathway in ulcerative colitis

BACKGROUND Ulcerative colitis(UC)is a chronic,nonspecific intestinal inflammatory disease with undefined pathogenesis.Non-SMC condensin I complex subunit D2(NCAPD2)and non-SMC condensin II complex subunit D3(NCAPD3)play pivotal roles in chromosome assembly and segregation during both mitosis and meiosis.To date,there has been no relevant report about the functional role of NCAPD2 and NCAPD3 in UC.AIM To determine the level of NCAPD2/3 in intestinal mucosa and explore the mechanisms of NCAPD2/3 in UC.METHODS Levels of NCAPD2/3 in intestinal tissue were detected in 30 UC patients and 30 healthy individuals with in situ hybridization(ISH).In vitro,NCM60 cells were divided into the NC group,model group,si-NCAPD2 group,si-NCAPD3 group and si-NCAPD2+si-NCAPD3 group.Inflammatory cytokines were measured by ELISA,IKK and NF-κB were evaluated by western blot,and IKK nucleation and NF-κB volume were analyzed by immunofluorescence assay.RESULTS Compared with expression in healthy individuals,NCAPD2 and NCAPD3 expression in intestinal tissue was significantly upregulated(P<0.001)in UC patients.Compared with levels in the model group,IL-1β,IL-6 and TNF-αin the si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 groups were significantly downregulated(P<0.01).IKK and NF-κB protein expression in the si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 groups was significantly decreased(P<0.01).Moreover,IKK nucleation and NF-κB volume were suppressed upon si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 transfection.CONCLUSION NCAPD2/3 is highly expressed in the intestinal mucosa of patients with active UC.Overexpression of NCAPD2/3 promotes the release of pro-inflammatory cytokines by modulating the IKK/NF-κB signaling pathway.