Protective effect of Liujing Toutong Tablets on rats with permanent cerebral ischemia via NF-κB signaling pathway / 中国中药杂志

This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1β, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(β-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1β, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and β-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1β, IL-6, CGRP, and NO in rat serum, increased VEGF and β-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and β-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1β. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing β-EP levels.

Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway / 中国中药杂志

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1β, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

Advances in the Action Mechanism of Classical Pathways IKKα and IKKβ in Hematological Tumors and Drug Therapy Blocking Their Effect--Review / 中国实验血液学杂志

In recent years, it is found that the classical IKKα and IKKβ pathway were closely relates with hematological tumors, except the classical pathogenesis, moreover the classical IKKβ pathway is deeply studied. The studies indicated that the IKKβis activated to phosphorylate the NF-κB through multiple cascades under the effect of extracellular IL-6, TNF-α and other stimulating factors. At the cellular level, the classical IKKβcan promote the tumor cell survival and proliferation, reduce the cell apoptosis, and promote the angiogenesis and cell transfer. Although the classical IKKα plays a role in regulating IKKβ activity, but its role in non-classical pathway is more prominent. This review briefly summarizes the latest advance of researches on the pathogenesis of hematological malignancies in term of IKKα and IKKβpathway, so as to provide the theoretic basis for deeply understanding and studying the pathogenesis of hematologic tumors. At present, blocking the classical IKKα and IKKβ pathway has become a new target for treatment of hematological tumors, moreover, some specific inhibitor for IKKα and IKKβpathway have been developed, for example, LY2409881, BMS 345541 and so on. Most of these drugs are in clinical trials and display some good anti-tumor effects.

Incontinentia pigmenti: genodermatosis multisistémica / Incontinentia pigmenti: multisistemic genodermatosis

Resumen La incontinentia pigmenti es una genodermatosis ligada al cromosoma X, generalmente letal en los hombres. Está causada por una mutación con pérdida de función en el gen IKBKG (inhibitor of kappa polypeptide gene enhancer in B cells kinase gamma), que impide que la proteína NFкβ (nuclear factor kappa-light-chain-enhancer of activated B cells) migre al núcleo y comience la transcripción de factores que amplifican la respuesta inmunitaria y previenen la apoptosis. Por tanto, las células mutantes se vuelven vulnerables a la apoptosis cuando son expuestas a citocinas y provocan vaso-oclusión e isquemia de tejidos como la piel, el sistema nervioso central y la retina. Las lesiones dermatológicas son características; se distribuyen a lo largo de las líneas de Blaschko, las cuales siguen el patrón de migración de las células de la piel en la embriogénesis, y ocurren en el 100% de los pacientes. Las manifestaciones cutáneas aparecen en una secuencia de cuatro fases que inicia desde el nacimiento: vesicular, verrucosa, hiperpigmentada e hipopigmentada. Estas lesiones son relevantes, puesto que orientan al clínico hacia el diagnóstico. Además, se acompañan de anomalías neurológicas, como crisis convulsivas, y múltiples manifestaciones oftalmológicas, como el desprendimiento de la retina. Los pacientes con incontinentia pigmenti, pero sin compromiso oftalmológico o neurológico clínicamente significativo, tienen un pronóstico bueno y una esperanza de vida normal. Las anomalías que se presentan son permanentes, lo que puede generar preocupación en los pacientes.

Abstract Incontinentia pigmenti is an X-linked genodermatosis generally lethal in males; thus, it presents almost exclusively in females. It is caused by a loss-of-function mutation in the IKBKG (inhibitor of kappa polypeptide gene enhancer in B cells, kinase gamma) gene that prevents the NFкβ (nuclear factor kappa-light-chain-enhancer of activated B cells) protein from migrating to the nucleus to begin the transcription of factors that amplify the immune response and prevent apoptosis. Consequently, mutant cells become vulnerable to apoptosis when exposed to cytokines and, in turn, lead to vaso-occlusion and ischemia of tissues, such as the skin, the central nervous system and the retina. Dermatological lesions are characteristic and occur in 100% of patients; they are distributed along Blaschko lines, which follow the pattern of migration of skin cells in embryogenesis. The cutaneous manifestations follow a sequence of four phases since birth: vesicular, verrucous, hyperpigmented and hypopigmented. These lesions are relevant for the disease because they guide the clinician towards the diagnosis. Additionally, they are accompanied by neurological abnormalities, such as seizures, and multiple ophthalmological manifestations, such as retinal detachment. Incontinentia pigmenti patients with no clinically significant ophthalmic or neurological compromise have a good prognosis and a normal life expectancy. The abnormalities present are permanent, which can be a cause of concern for the patients.

Application value of whole exome sequencing in critically ill neonates with inherited diseases / 中国当代儿科杂志

OBJECTIVE@#To study the application value of whole exome sequencing (WES) in critically ill neonates with inherited diseases.@*METHODS@#A total of 66 critically ill neonates with suspected inherited diseases or unclear clinical diagnosis who were admitted to the neonatal intensive care unit were enrolled as subjects. The clinical data of the neonates were collected, and venous blood samples were collected from the neonates and their parents for WES. The clinical manifestations of the neonates were observed to search for related pathogenic gene mutations.@*RESULTS@#Among the 66 critically ill neonates with suspected inherited diseases or unclear clinical diagnosis (34 boys and 32 girls), 14 (21%) were found to have gene mutations by WES. One neonate had no gene mutation detected by WES but was highly suspected of pigment incontinence based on clinical manifestations, and multiplex ligation-dependent probe amplification detected a heterozygous deletion mutation in exons 4-10 of the IKBKG gene. Among the 15 neonates with gene mutations, 10 (67%) had pathogenic gene mutation, 1 (7%) was suspected of pathogenic gene mutation, and 4 (27%) had gene mutations with unknown significance. Among the 15 neonates, 13 underwent chromosome examination, and only 1 neonate was found to have chromosome abnormality.@*CONCLUSIONS@#Chromosome examination cannot be used as a diagnostic method for inherited diseases, and WES detection technology is an important tool to find inherited diseases in critically ill neonates with suspected inherited diseases or unclear clinical diagnosis; however WES technology has some limitation and it is thus necessary to combine with other sequencing methods to achieve an early diagnosis.

Investigação da quinase IKKß como um alvo terapêutico anti-metastático em câncer de pulmão associado à ativação do oncogene KRAS / Exploring IKKß as an anti-metastatic therapeutic target in KRAS-induced lung câncer

O câncer de pulmão é o tipo de câncer que apresenta o maior índice de mortalidade em todo o mundo. As alterações genéticas mais frequentes em câncer de pulmão são as mutações pontuais no oncogene que codifica a GTPase KRAS. Apesar destas mutações estarem diretamente ligadas à oncogênese, terapias que visam inibir diretamente a proteína Ras falharam em ensaios clínicos. Uma das propriedades mais importantes na oncogênese é a aquisição de capacidade metastática tumoral. Desta forma, o objetivo deste projeto é identificar alvos terapêuticos que inibam as metástases tumorais induzidas pelo oncogene KRAS no pulmão. Com base em relatos recentes mostrando que a forma oncogênica de KRAS promove, não só a iniciação tumoral, mas também promove a aquisição de um fenótipo metastático, a hipótese deste projeto é que (1) a capacidade mestastática tumoral induzida por KRAS no pulmão é potencializada pela quinase IKKß; e (2) que a inibição desta quinase reduzirá a capacidade invasiva celular e metastática tumoral. Esta hipótese foi formulada com base em estudos anteriores, os quais demonstraram que o principal substrato da IKKß, o fator de transcrição NF-κB, é ativado por KRAS em tumores pulmonares in situ de forma dependente da IKKß, que o NF-κB é capaz de promover metástase em diferentes modelos tumorais, e que a inibição da atividade da IKKß com um inibidor farmacológico em um modelo animal de câncer de pulmão induzido por KRAS, diminui o crescimento tumoral e a progressão tumoral para graus histológicos mais avançados. Nosso objetivo era avaliar se a inibição de IKKß é capaz de afetar a migração e invasão de células portadoras de mutação em KRAS in vitro e se a inibição de IKKß é capaz de afetar a capacidade metatática dessas células in vivo. Primeiramente, avaliamos a expressão de enzimas relacionadas ao fenótipo metastático, as metaloproteinases de matriz 2 e 9 (MMP-2 e MMP-9) e, também uma molécula intimamente relacionada ao processo de adesão mediado por integrinas, FAK (quinase de adesão focal), frente a inibição de IKKß através de um inibidor farmacológico altamente especifico (Composto A) e frente a inibição genética de IKKß por interferência de RNA (siRNA) em células A549 e H358. Avaliamos também a atividade das MMPs frente inibição genética de KRAS (siKRAS) e IKKß (siIKKß) e vimos que IKKß parece modular a expressão ou atividade de MMP-9 e reduz a expressão de FAK. Já a expressão de MMP-2 não apresentou alteração. Posteriormente avaliamos migração na célula A549 e invasão nas células A549 e H358 com inibição de IKKß, por ensaios Transwell, e observamos uma redução da migração e invasão celular in vitro. Em seguida, fomos gerar linhagens celulares paraa expressar luciferase, as linhagens A549 pLUC e H358 pLUC. Os clones A549 pLUC B4 e H358 pLUC F1 com inibição de KRAS e IKKß por interferência de RNA, foram injetados pela veia da cauda nesses camundongos e as metástases foram monitoradas por imageamento in vivo. Houve metástases em 20% dos animais com siIKKß na região anatômica da boca. Os animais que receberam siControle e siKRAS não apresentaram nenhuma metástase visível no equipamento, mas foi observado micrometástases nas análises histológicas dos pulmões. O resultado do experimento de metástase in vivo é inesperado, não só pelo fato de ocorrer no grupo experimental siIKKß, mas também pelo local anatômico do tumor, sendo necessária uma maior investigação do papel de IKKß nesse processo, podendo ser um resultado aleatório. Quando avaliamos em conjunto, nossos resultados sugerem que a quinase IKKß desempenha um papel importante no fenótipo migratório e invasivo de células pulmonares portadoras de KRAS oncogênica, contribuindo para a capacidade metastática

Lung cancer is the leading cause of cancer deaths worldwide. The most frequent genetic changes found in lung cancer are driver mutations in the KRAS proto-oncogene. Even though KRAS mutations have been causally linked to the oncogenic process, therapies targeted to oncogenic RAS have failed in clinical trials. One of the main characteristics in oncogenesis is the ability of tumors to acquire metastatic capability. The objective of this project is to identify therapeutic targets that reduce KRASinduced lung cancer metastasis. Based on previous reports that oncogenic KRAS, drives not only tumor initiation, but also promotes a metastatic phenotype, the hypothesis of this project is that (1) the acquisition of metastatic ability induced by KRAS in the lung is potentiated by the IKK kinase; and (2) that IKKß inhibition will reduce KRAS-induced cell invasive properties and KRAS-induced tumor metastasis. This hypothesis has been formulated on the basis of previous studies showing that the main IKKß substrate, the transcription factor NF-κB, is activated by KRAS in lung tumors in situ in an IKKß-dependent manner, that NF-κB is known to promote metastasis in different tumor models, and that pharmacological IKKß inhibition in a KRAS-induced lung cancer mouse model reduces tumor growth and progression to higher histological tumor grades. Our goal was evaluate how inhibition of IKKß affects migration and invasion of KRAS-positive lung cells in vitro and whether inhibition of IKKß is capable of affecting the metatactic capacity of these cells in vivo. First, we evaluated the expression of enzymes involved in the metastatic phenotype, matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and also a molecule involved in the integrinmediated adhesion, FAK (focal adhesion kinase), we targeted IKKß by a highly specific IKK inhibitor (Compound A) or with RNA interference in A549 and H358 cells. We also used colorimetric Matrix Biotrak Activity Assay System to measure the activity of MMPs with RNA interference for KRAS (siKRAS) and IKKß (IKKß) and we have seen that IKKß appears to modulate the expression or activity of MMP-9 and decreases the expression of FAK. The expression of MMP-2 did not change. Then we evaluated migration in A549 cell and invasion in A549 and H358 cells with inhibition of IKK by RNA interference or with Compound A treatment in Transwell assays, and observed a significantly reduced cell migration and invasion in vitro. We then generated cell lines to express luciferase, the A549 pLUC and H358 pLUC lines. A549 pLUC B4 and H358 pLUC F1 cells with RNA interference for KRAS and IKKß were injected in the tail vein in nude (balb/c) mice and metastases were monitored by in vivo imaging. There were metastases in 20% of IKKß animals in the anatomical region of the mouth. Animals that received siControl and siKRAS had no visible metastasis in the live imaging, but micrometastases were observed in the histological analyzes of the lungs. The result of this experiment is unexpected, not only due to the fact that it occurs in the IKKß experimental group, but also due to the anatomical site of the tumor, and a further investigation of the role of IKKß in this process, can be a random result. When evaluated together, our results suggest that the IKKß kinase plays an important role in the migratory and invasive phenotype of in KRAS positive lung cancer cells, contributing to metastatic capacity

Explorando a quinase IKK como um alvo terapêutico para células iniciadoras de tumor pulmonares induzidas pelo oncogene KRAS / Exploring IKKb kinase as a therapeutic target for KRAS-driven lung tumour-initiating cells

As alterações genéticas mais frequentes em câncer de pulmão são mutações pontuais que ativam o oncogene KRAS. Embora estas mutações estejam causalmente relacionadas à oncogênese, até hoje diferentes abordagens para inibir as proteínas RAS diretamente não obtiveram sucesso. Portanto, para que melhores alvos terapêuticos para o câncer de pulmão se tornem disponíveis é necessário identificar os mecanismos moleculares ativados por KRAS que estão diretamente envolvidos com a aquisição de propriedades malignas importantes, como o desenvolvimento e a manutenção de um fenótipo tronco-tumoral pelas células iniciadoras de tumor (CITs). CITs, também conhecidas como células tronco-tumorais, são definidas como uma subpopulação de células tumorais capazes de se autorrenovar, iniciar a formação de tumores e sustentar o crescimento tumoral. O desenvolvimento de estratégias terapêuticas dirigidas a estas células é imprescindível para melhorar a eficácia da terapia antitumoral. Uma vez que KRAS está associada a manutenção de um fenótipo tronco-tumoral e ativa o fator de transcrição NF-kB através da quinase IKKß para promover a tumorigênese pulmonar, nós hipotetizamos que a quinase IKKß contribui para o fenótipo tronco-tumoral induzido por KRAS em câncer de pulmão. Nós utilizamos ensaios de formação de tumoresferas para enriquecer e avaliar a função de CITs das linhagens pulmonares positivas para KRAS A549 e H358. As células A549 e H358 formaram tumoresferas em cultura de baixa aderência e, quando comparadas às células derivadas da cultura aderente, as células oriundas da cultura de tumoresferas apresentaram maior crescimento clonogênico, maior expressão de genes associados ao fenótipo tronco por qPCR e maior atividade da quinase IKKß. A inibição da atividade de IKKß através de um inibidor farmacológico altamente específico (Composto A) diminuiu levemente a proliferação de células A549 e H358, sem resultar em morte celular significativa. Entretanto, a inibição da atividade ou da expressão de IKKß por interferência de RNA reduziu a expressão de genes associados ao fenótipo tronco e diminuiu a formação de tumoresferas. A inibição da expressão de IKKß em células A549 reduziu também a capacidade de autorrenovação de CITs. Estes resultados sugerem que IKKß desempenha um papel importante na manutenção do fenótipo tronco-tumoral de CITs pulmonares induzidas por KRAS. Em seguida, nós demonstramos que a inibição da atividade de IKKß afetou preferencialmente a proliferação celular e o crescimento clonogênico de células oriundas da cultura de tumoresfera, sugerindo que IKKß desempenha um papel mais importante em CITs do que em células derivadas da cultura aderente. A análise por citometria de fluxo identificou que células derivadas da cultura de tumoresfera apresentam um enriquecimento para células CD24+ na linhagem A549 e células CD44+ na linhagem H358, sugerindo que estes possam ser marcadores promissores para purificação de CITs nestas linhagens. Adicionalmente, demonstramos, por ensaios de wound-healing de células A549 e H358, que a inibição da atividade de IKKß reduziu a migração celular, uma outra uma propriedade aumentada em CITs. Além disso, mostramos que a atividade da quinase IKKß em células A549 e H358 não depende das vias da MAPK ou PI3K/Akt. Interessantemente, a inibição combinada de IKK (um efetor downstream de KRAS) e de EGFR/ERRB2 (reguladores upstream de KRAS que ativam as vias MAPK e PI3K/Akt) reduziu de forma aditiva a formação de tumoresferas, proliferação e migração celular. Quando avaliados em conjunto, nossos resultados sugerem que a quinase IKKß desempenha um papel importante na biologia de CITs pulmonares portadoras de KRAS oncogênica e que a inibição desta quinase sozinha ou em combinação com a inibição de outras vias pode representar uma estratégia terapêutica promissora a ser explorada para reduzir a recidiva e metástase no câncer de pulmão induzido por KRAS

The most frequent genetic alterations in lung cancer are point mutations that activate the KRAS oncogene. Although these mutations are causally related to oncogenesis, different approaches to inhibit RAS proteins directly have not been successful to date. Therefore, for better therapeutic targets for lung cancer to become available, it is necessary to identify the molecular mechanisms activated by KRAS that are directly involved with important malignant features, such as the development and maintenance of a cancer stem-like phenotype by the tumour-initiating cells (TICs). TICs, also known as cancer stem cells, are defined as a subpopulation of tumour cells able to self-renew, promote tumour initiation, and sustain tumour growth. The development of therapeutic strategies to target these cells is imperative to improve the efficacy of antitumor therapy. Since KRAS is associated with the maintenance of a cancer stem-like phenotype and activates the transcription factor NF-kB through the IKKß kinase to promote lung tumourigenesis, we hypothesised that IKKß kinase contributes to the cancer stem-like phenotype induced by KRAS in lung cancer. We used tumoursphere formation assays to enrich and evaluate the function of TICs of KRAS-mutant cell lines A549 and H358. A549 and H358 cells formed tumourspheres in low adhesion culture and, when compared to cells grown in adherent culture, sphere-derived cells displayed increased clonogenic growth, higher expression of stemness genes by qPCR, and increased IKKß kinase activity . Inhibition of IKKß activity through a highly specific pharmacological inhibitor (Compound A) slightly decreased proliferation of A549 and H358 cells without inducing significant cell death. On the other hand, inhibition of IKKß activity or expression by RNA interference reduced the expression of stemness genes and decreased tumoursphere formation. Inhibition of IKKß expression in A549 cells also reduced TICs self-renewal . These results suggest that IKKß plays an important role in maintaining the cancer stem-like phenotype of KRAS-driven lung TICs. Next, we demonstrated that IKKß inhibition preferentially reduced cell proliferation and clonogenic growth of sphere-derived cells, suggesting that IKKß plays a more important role in TICs than in adherent culture-derived cells. Flow cytometry analysis identified that sphere-derived cells display an enrichment for the surface marker CD24 in A549 cells and CD44 in H358 cells, indicating that these could be promising markers for the purification of TICs in these cell lines. Furthermore, we have shown by wound-healing assays of A549 and H358 cells that IKKß inhibition reduced cell migration , another feature increased in TICs. In addition, we have shown that IKKß activity in A549 and H358 cells does not depend on the MAPK or PI3K/Akt pathways. Interestingly, combined inhibition of IKKß (a downstream effector of KRAS) and EGFR/ERBB2 (upstream regulators of KRAS that activate the MAPK and PI3K/Akt pathways) additively reduced tumoursphere formation, cell proliferation and migration. Taken together, our results suggest that IKKß kinase plays an important role in the biology of KRAS-driven lung TICs, and that inhibition of this kinase alone or in combination with inhibition of other signalling pathways may represent a promising therapeutic strategy to be explored in order to reduce tumour recurrence and metastasis in KRAS-driven lung cancer

Aldosterone induces inflammatory cytokines in penile corpus cavernosum by activating the NF-κB pathway / 亚洲男科学杂志(英文版)

Emerging evidence indicates that aldosterone and mineralocorticoid receptors (MRs) are associated with the pathogenesis of erectile dysfunction. However, the molecular mechanisms remain largely unknown. In this study, freshly isolated penile corpus cavernosum tissue from rats was treated with aldosterone, with or without MRs inhibitors. Nuclear factor (NF)-kappa B (NF-κB) activity was evaluated by real-time quantitative PCR, luciferase assay, and immunoblot. The results demonstrated that mRNA levels of the NF-κB target genes, including inhibitor of NF-κB alpha (IκB-α), NF-κB1, tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6), were higher after aldosterone treatment. Accordingly, phosphorylation of p65/RelA, IκB-α, and inhibitor of NF-κB kinase-β was markedly increased by aldosterone. Furthermore, knockdown of MRs prevented activation of the NF-κB canonical pathway by aldosterone. Consistent with this finding, ectopic overexpression of MRs enhanced the transcriptional activation of NF-κB by aldosterone. More importantly, the MRs antagonist, spironolactone blocked aldosterone-mediated activation of the canonical NF-κB pathway. In conclusion, aldosterone has an inflammatory effect in the corpus cavernosum penis, inducing NF-κB activation via an MRs-dependent pathway, which may be prevented by selective MRs antagonists. These data reveal the possible role of aldosterone in erectile dysfunction as well as its potential as a novel pharmacologic target for treatment.

Effect of IKK2 Inhibitor LY2409881 on Diffuse Large B-Cell Lymphoma Cell Apoptosis and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects and its potential mechanism of IKK2 inhibitor LY2409881 on proliferation, cell cycle and apoptosis of diffuse large B-cell lymphoma (DLBCL) cell lines.</p><p><b>METHODS</b>CCK8 assay was used to detect the effect of LY2409881 on proliferation of DLBCL cell lines; Flow cytometry was used to analyze cell cycle; Western blot was used to analyze apoptosis and its potential mechanism.</p><p><b>RESULTS</b>LY2409881 inhibited the proliferation of multiple DLBCL cell lines obviously, and caused G cell arrest. Furtherly, LY2409881 inhibited the expression of c-FLIP, induced the activation of DR4 and caspase 8. Meanwhile, LY2409881 induced the expression of pro-apoptotic protein BAX and inhibited the expression of anti-apoptotic protein MCL-1 and BCL-2.</p><p><b>CONCLUSION</b>LY2409881 inhibits the proliferation of DLBCL cell lines, causes G cell arrest and induces apoptosis via the endogenous and exogenous apoptotic pathways.</p>

IκB kinase b Mediating the Downregulation of p53 and p21 by Lipopolysaccharide in Human Papillomavirus 16Cervical Cancer Cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cervical cancer is the second most common cancer of woman in the world, and human papillomavirus (HPV) infection plays an important role in the development of most of the cases. IκB kinase β (IKKβ) is a kinase-mediating nuclear factor kappa B (NF-κB) activation by phosphorylating the inhibitor of NF-κB (IκB) and is related by some diseases caused by virus infection. However, there is little known about the correlation between IKKβ and HPV infection in cervical cancer. This study aimed to investigate the expression of IKKβ protein in cervical cancer tissues and effects of inflammation on HPV positive or negative cervical cancer cells through detecting the expression of IKKβ, IκBα, p53, and p21 proteins after treated with lipopolysaccharide (LPS) to mimic bacterial infection. We also examined the effects of LPS on cervical cancer cells after blocking IKKβ with pharmacological inhibitor.</p><p><b>METHODS</b>Thirty-six matched specimens of cervical cancer and adjacent normal tissues were collected and analyzed in the study. The expression of IKKβ in the tissue specimens was determined by immunohistochemical staining. In addition, Western blot was used to detect the expression level changes of IKKβ, IκBα, p53, and p21 after LPS stimulated in the HPV16+ (SiHa) and HPV16- (C33A) cervical cancer cell lines. Furthermore, the effects of IKKβ inhibitor SC-514 on LPS-induced expression change of these proteins were investigated.</p><p><b>RESULTS</b>The expression of IKKβ was higher in cervical cancer than adjacent normal tissues, and there was no significant difference between tumor differentiation, size, and invasive depth with IKKβ expression. The LPS, which increased the expression level of IKKβ protein but decreased in the IκBα, p53 and p21 proteins, was illustrated in HPV16+ (SiHa) but not in HPV16- (C33A) cells. Moreover, IKKβ inhibitor SC-514 totally reversed the upregulation of IKKβ and downregulation of p53 and p21 by LPS in SiHa cells.</p><p><b>CONCLUSIONS</b>IKKβ may mediate the downregulation of p53 and p21 by LPS in HPV16+ cervical cancer cells.</p>

Loss of IκB kinase β promotes myofibroblast transformation and senescence through activation of the ROS-TGFβ autocrine loop

Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.

Interaction between polymorphisms of TLR4 gene G11367C in 3' untranslated region and IκB-α Hae III in acute pancreatitis and the degree of severity / 中南大学学报(医学版)

OBJECTIVE@#To investigate the interaction between polymorphism of Toll-like receptor 4 (TLR4) gene G11367C in 3' untranslated region (UTR) and inhibitor of nuclear factor kappaB (IκB)-α 
Hae III in acute pancreatitis (AP) and the degree of severity.
@*METHODS@#A total of 450 patients with confirmed AP (AP group), who came from the First Affiliated Hospital of Xinxiang Medical College from May 2013 to June 2015, were divided into a mild AP subgroup (MAP subgroup), a moderately severe AP (MSAP subgroup), and a severe acute AP (SAP subgroup) (n=150 in each group). One hundred fifty healthy persons were served as a control group. There was no significant difference in age, gender, ethnicity and birthplace among all groups. The genetic polymorphisms of TLR4 gene G11367C in 3' untranslated region and IκB-α Hae III were analyzed by polymerase chain reaction (PCR). Eligible participants were personally interviewed by a questionnaire. Unconditional logistic regression model and single factor analysis were performed to calculate the adjusted odds ratios (OR) and 95% confidence intervals (95% CI) of G11367C and IκB-α Hae III polymorphisms, respectively. The interaction of nucleotide polymorphisms was analyzed.
@*RESULTS@#The frequencies of G11367C (GC), IκB-α Hae III (AG) and IκB-α Hae III (GG) were 69.56%, 33.78% and 36.22% in the AP group; 49.33%, 24.67% and 26.00% in the MAP subgroup; 70.67%, 34.67% and 36.67% in the MSAP subgroup; 88.67%, 42.00% and 46.00% in the SAP subgroup and 26.67%, 14.00% and 14.67% in the control group, respectively. There was significant difference in the frequencies betweenc the AP group and the control group, or among each AP subgroup (all P1). Similarly, there were also positive interactions in the pathogenesis of AP between G11367C (GC) and IκB-α Hae III (AG) (All γ>1). 
@*CONCLUSION@#These carriers of G11367C(GC), IκB-α Hae III(AG) and IκB-α Hae III (GG) genotypes may have a high risk of AP occurency, and there are significant interactions between genetic polymorphisms of G11367C and IκB-α Hae III, which increaes the risk of the occurrence and development of AP.

Berberine relieves insulin resistance via the cholinergic anti-inflammatory pathway in HepG2 cells / 华中科技大学学报（医学）（英德文版）

Berberine (BBR) is an isoquinoline alkaloid extracted from Rhizoma coptidis and has been used for treating type 2 diabetes mellitus (T2DM) in China. The development of T2DM is often associated with insulin resistance and impaired glucose uptake in peripheral tissues. In this study, we examined whether BBR attenuated glucose uptake dysfunction through the cholinergic anti-inflammatory pathway in HepG2 cells. Cellular glucose uptake, quantified by the 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2-deoxy-D-glucose (2-NBDG), was inhibited by 21% after HepG2 cells were incubated with insulin (10(-6) mol/L) for 36 h. Meanwhile, the expression of alpha7 nicotinic acetylcholine receptor (α7nAChR) protein was reduced without the change of acetylcholinesterase (AChE) activity. The level of interleukin-6 (IL-6) in the culture supernatant, the ratio of phosphorylated I-kappa-B kinase-β (IKκβ) Ser181/IKKβ and the expression of nuclear factor-kappa B (NF-κB) p65 protein were also increased. However, the treatment with BBR enhanced the glucose uptake, increased the expression of α7nAChR protein and inhibited AChE activity. These changes were also accompanied with the decrease of the ratio of pIKKβ Ser181/IKKβ, NF-κB p65 expression and IL-6 level. Taken together, these results suggest that BBR could enhance glucose uptake, and relieve insulin resistance and inflammation in HepG2 cells. The mechanism may be related to the cholinergic anti-inflammatory pathway and the inhibition of AChE activity.

The Anti-Inflammatory Effect of Arginine-Vasopressin on Lipopolysaccharide-Induced IkappaBalpha/Nuclear Factor-kappaB Cascade / 대한구급학회지

BACKGROUND: Arginine vasopressin (AVP) is widely used as a vasopressor agent. Some recent studies have suggested that AVP may exert an immunomodulatory effect. However, the mechanism about the anti-inflammatory effect of AVP is not well known. We investigated the effect of AVP on the ihibitor of kappa B (IkappaBalpha)/nuclear factor-kappa B (NF-kappaB) pathway in RAW 264.7 cells. METHODS: Cultured RAW 264.7 cells were pretreated with AVP and stimulated with lipopolysaccharide (LPS). To evaluate the effect of AVP on inflammatory cytokines, the concentration of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were assessed by an enzyme-linked immunosorbent assay technique. The expression of IkappaBalpha and nuclear translocation of NF-kappaB p65 were measured by Western blotting, and IkappaB kinase (IKK) activity was analyzed by an in vitro immune complex kinase assay. To confirm the AVP effect on IkappaBalpha/NF-kappaB cascade and via V2 receptor, we added tolvaptan (V2 receptor antagonist) after AVP pretreatment. RESULTS: The increase of IL-6 and TNF-alpha in LPS-stimulated RAW 264.7 cells was suppressed by a treatment with AVP. Pretreatment of AVP inhibited increasing of IKK activity and IkappaBalpha degradation induced by LPS in RAW 264.7 cells. Furthermore, LPS induced and NF-kappaB transcription was inhibited by AVP pretreatment. The observed changes in IKK activity, IkappaBalpha degradation and NF-kappaB transcription by AVP was abolished by tolvaptan treatment. CONCLUSIONS: Our results suggest that AVP showed anti-inflammatory effect on LPS-induced IkappaBalpha/NF-kappaB cascade in mouse macrophages via V2 receptors.

Inhibitory effect of kaempferol on inflammatory response of lipopolysaccharide-stimulated human mast cells / 药学学报

This study is to investigate the inhibitory effect of kaempferol on inflammatory response of lipopolysaccharide(LPS)-stimulated HMC-1 mast cells. The cytotoxicity of kaempferol to HMC-1 mast cells were analyzed by using MTT assay and then the administration concentrations of kaempferol were established. Histamine, IL-6, IL-8, IL-1β and TNF-α were measured using ELISA assay in activated HMC-1 mast cells after incubation with various concentrations of kaempferol (10, 20 and 40 µmol.L-1). Western blot was used to test the protein expression of p-IKKβ, IκBα, p-IκBα and nucleus NF-κB of LPS-induced HMC-1 mast cells after incubation with different concentrations of kaempferol. The optimal concentrations of kaempferol were defined as the range from 5 µmol.L-1 to 40 µmol.L-1. Kaempferol significantly decreased the release of histamine, IL-6, IL-8, IL-1β and TNF-α of activated HMC-1 mast cells (P<0.01). After incubation with kaempferol, the protein expression of p-IKKβ, p-IKBa and nucleus NF-κB (p65) markedly reduced in LPS-stimulated HMC-1 mast cells (P<0.01). Taken together, we concluded that kaempferol markedly inhibit mast cell-mediated inflammatory response. At the same time, kaempferol can inhibit the activation of IKKβ, block the phosphorylation of IκBα, prevent NF-KB entering into the nucleus, and then decrease the release of inflammatory mediators.

Suppression of Kiss-1 gene inhibits HCT116 human colorectal carcinoma cell migration in vitro via nuclear factor-κB signaling pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of Kiss-1 gene suppression on the metastatic capacity of HCT116 human colorectal carcinoma cells in vitro and the involvement of nuclear factor-κB (NF-κB) signaling pathway.</p><p><b>METHODS</b>A recombinant lentiviral vector of Kiss-1 gene pGC-LV-Kiss-1-EGFP or the empty vector was transfected in HCT116 cells. Cell Counting Kit-8 (CCK8) and Transwell chamber assay were used to detect the changes in cell proliferation, invasion and migration ability after the transfection. Western blotting was used to detect the expression of I-κB, the inhibitive protein of NF-κB signal pathway, and the expression of the downstream effector MMP-9 before and after transfection.</p><p><b>RESULTS</b>In cells over-expressing Kiss-1, I-κB expression increased and MMP-9 expression decreased significantly compared to those in the blank control and vector-transfected cells (P<0.05). Kiss-1 gene over-expression resulted in significant inhibition of HCT116 cell proliferation, invasion, and migration as compared to the control cells (P<0.05).</p><p><b>CONCLUSION</b>Lentivirus-mediated Kiss-1 gene over-expression can inhibit the proliferation, invasion, and migration of HCT116 cells via the NF-B signaling pathway.</p>

The Anti-Inflammatory Effect of Arginine-Vasopressin on Lipopolysaccharide-Induced IkappaBalpha/Nuclear Factor-kappaB Cascade / 대한중환자의학회지

BACKGROUND: Arginine vasopressin (AVP) is widely used as a vasopressor agent. Some recent studies have suggested that AVP may exert an immunomodulatory effect. However, the mechanism about the anti-inflammatory effect of AVP is not well known. We investigated the effect of AVP on the ihibitor of kappa B (IkappaBalpha)/nuclear factor-kappa B (NF-kappaB) pathway in RAW 264.7 cells. METHODS: Cultured RAW 264.7 cells were pretreated with AVP and stimulated with lipopolysaccharide (LPS). To evaluate the effect of AVP on inflammatory cytokines, the concentration of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were assessed by an enzyme-linked immunosorbent assay technique. The expression of IkappaBalpha and nuclear translocation of NF-kappaB p65 were measured by Western blotting, and IkappaB kinase (IKK) activity was analyzed by an in vitro immune complex kinase assay. To confirm the AVP effect on IkappaBalpha/NF-kappaB cascade and via V2 receptor, we added tolvaptan (V2 receptor antagonist) after AVP pretreatment. RESULTS: The increase of IL-6 and TNF-alpha in LPS-stimulated RAW 264.7 cells was suppressed by a treatment with AVP. Pretreatment of AVP inhibited increasing of IKK activity and IkappaBalpha degradation induced by LPS in RAW 264.7 cells. Furthermore, LPS induced and NF-kappaB transcription was inhibited by AVP pretreatment. The observed changes in IKK activity, IkappaBalpha degradation and NF-kappaB transcription by AVP was abolished by tolvaptan treatment. CONCLUSIONS: Our results suggest that AVP showed anti-inflammatory effect on LPS-induced IkappaBalpha/NF-kappaB cascade in mouse macrophages via V2 receptors.

Honokiol ameliorates endothelial dysfunction through suppression of PTX3 expression, a key mediator of IKK/IkappaB/NF-kappaB, in atherosclerotic cell model

Pentraxin 3 (PTX3) was identified as a marker of the inflammatory response and overexpressed in various tissues and cells related to cardiovascular disease. Honokiol, an active component isolated from the Chinese medicinal herb Magnolia officinalis, was shown to have a variety of pharmacological activities. In the present study, we aimed to investigate the effects of honokiol on palmitic acid (PA)-induced dysfunction of human umbilical vein endothelial cells (HUVECs) and to elucidate potential regulatory mechanisms in this atherosclerotic cell model. Our results showed that PA significantly accelerated the expression of PTX3 in HUVECs through the IkappaB kinase (IKK)/IkappaB/nuclear factor-kappaB (NF-kappaB) pathway, reduced cell viability, induced cell apoptosis and triggered the inflammatory response. Knockdown of PTX3 supported cell growth and prevented apoptosis by blocking PA-inducted nitric oxide (NO) overproduction. Honokiol significantly suppressed the overexpression of PTX3 in PA-inducted HUVECs by inhibiting IkappaB phosphorylation and the expression of two NF-kappaB subunits (p50 and p65) in the IKK/IkappaB/NF-kappaB signaling pathway. Furthermore, honokiol reduced endothelial cell injury and apoptosis by regulating the expression of inducible NO synthase and endothelial NO synthase, as well as the generation of NO. Honokiol showed an anti-inflammatory effect in PA-inducted HUVECs by significantly inhibiting the generation of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1. In summary, honokiol repaired endothelial dysfunction by suppressing PTX3 overexpression in an atherosclerotic cell model. PTX3 may be a potential therapeutic target for atherosclerosis.