Mechanism of paeoniflorin inhibiting apoptosis of hippocampal neurons of rats induced by lead acetate / 中华劳动卫生职业病杂志

Objective: To investigate the effect and underlying mechanism of paeoniflorin on hippocampal neuron apoptosis induced by lead acetate. Methods: In September 2020, primary hippocampal neuronal cells were isolated and cultured from fetal rats, and identified using cellular immunofluorescent. MTT assay was used to measure the cell viability to determine the concentration and time of lead acetate-induced hippocampal neuron apoptosis. MTT was also used to evaluate the effect of paeoniflorin concentration on the apoptosis of hippocampal neurons induced by lead acetate. According to the results, different concentrations of paeoniflorin were selected to intervene hippocampal neuron cells, after 24 h, lead acetate was added to the cells, meanwhile, blank and model groups were set up, the content of reactive oxygen species (ROS) , superoxide dismutase (SOD) , lactate dehydrogenase (LDH) , malondialdehyde (MDA) and Caspase-3 were measured. Extracellular signal regulated kinase (ERK) , phosphorylated ERK (p-ERK) , p38 mitogen -activated protein kinases (p38MAPK) , phosphorylated p38MAPK (p-p38MAPK) , c-Jun N-terminal kinase (JNK) and phosphorylated JNK (p-JNK) protein expression in hippocampal neuronal cells were determined by Western blotting. Results: The isolated and cultured hippocampal neurons were identified by immunofluorescence chemical staining and then treated with lead acetate, MTT results showed that lead acetate had the best toxicity effect when treated for 24 h at a concentration of 25 μmol/L. Paeoniflorin showed no cytotoxic effect on hippocampal neuronal cells when the concentrations below 80 μmol/L. Compared with the model group, the activity of hippocampal neuronal cells was significantly increased after treating with 20, 40 or 80 μmol/L paeoniflorin (P<0.05) . Compared with the blank group, the ROS activity, LDH release level, MDA content and caspase-3 content were significantly increased (P<0.01) , and the SOD activity was significantly decreased (P< 0.01) in the hippocampal neuronal cells of the model group. Compared with the model group, the ROS activity, LDH release level, MDA content and caspase-3 content were obviously decreased (P<0.05) , SOD activity was significantly increased (P <0.01) after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin. Relative to the model group, the ratio of p-ERK/ERK were significantly up-regulated (P<0.01) , while the ratios of p-p38MAPK/p38MAPK and p-JNK/JNK were significantly down-regulated after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin (P<0.05) . Conclusion: Paeoniflorin may down-regulate the expression of p-p38MAPK and p-JNK protein, up-regulate the expression of p-ERK protein, and inhibit the apoptosis of hippocampal neurons induced by lead acetate through the MAPK signaling pathway.

Electroacupuncture Attenuated Phenotype Transformation of Vascular Smooth Muscle Cells via PI3K/Akt and MAPK Signaling Pathways in Spontaneous Hypertensive Rats / 中国结合医学杂志

OBJECTIVE@#To investigate whether the antihypertensive mechanism of electroacupuncture (EA) is associated with attenuating phenotype transformation of vascular smooth muscle cells (VSMCs) via phosphoinositide3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) signaling pathways.@*METHODS@#Eight Wistar-ktoyo (WKY) rats were set as normal blood pressure group (normal group). A total of 32 spontaneous hypertensive rats (SHRs) were randomly divided into 4 groups using random number tables: a model group, an EA group, an EA+PI3K antagonist group (EA+P group), and an EA+p38 MAPK agonist+extracellular signal-regulated kinase (ERK) agonist group (EA+M group) (n=8/group). SHRs in EA group, EA+P group and EA+M group received EA treatment 5 sessions per week for continuous 4 weeks, while rats in the normal and model groups were bundled in same condition. The systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) of each rat was measured at 0 week and the 4th week. After 4-week intervention, thoracic aorta was collected for hematoxylin-eosin (HE) staining, immunohistochemistry [the contractile markers α-smooth muscle actin (α-SMA) and calponin and the synthetic marker osteopontin (OPN)] and Western blot [α-SMA, calponin, OPN, PI3K, phosphorylated-Akt (p-Akt), Akt, p-p42/44 ERK, total p42/44 ERK, p-p38 MAPK and total p38 MAPK].@*RESULTS@#EA significantly reduced SBP, DBP and MAP (P<0.01). HE staining showed that the wall thickness of thoracic aorta in EA group was significantly decreased (P<0.01). From results of immunohistochemistry and Western blot, EA increased the expression of α-SMA and calponin, and decreased the expression of OPN (P<0.01). In addition, the expression of PI3K and p-Akt increased (P<0.01), while the expression of p-p42/44 ERK and p-p38 MAPK decreased in EA group (P<0.01). However, these effects were reversed by PI3K antagonist, p38 MAPK agonist and ERK agonist.@*CONCLUSIONS@#EA was an effective treatment for BP management. The antihypertensive effect of EA may be related with inhibition of phenotypic transformation of VSMCs, in which the activation of PI3K/Akt and the repression of MAPK pathway were involved.

Effect of periapical inflammation on calcium binding proteins and ERK in the trigeminal nucleus / Efecto de la inflamación periapical sobre las proteínas fijadoras de calcio y ERK en el núcleo trigeminal

Peripheral inflammation induces plastic changes in neurons and glia which are regulated by free calcium and calcium binding proteins (CaBP). One of the mechanisms associated with the regulation of intracellular calcium is linked to ERK (Extracellular Signal-Regulated Kinase) and its phosphorylated condition (pERK). ERK phosphorylation is important for intracellular signal transduction and participates in regulating neuroplasticity and inflammatory responses. The aim of this study is to analyse the expression of two CaBPs and pERK in astrocytes and neurons in rat trigeminal subnucleus caudalis (Vc) after experimental periapical inflammation on the left mandibular first molar. At seven days post-treatment, the periapical inflammatory stimulus induces an increase in pERK expression both in S100b positive astrocytes and Calbindin D28k positive neurons, in the ipsilateral Vc with respect to the contralateral side and control group. pERK was observed coexpressing with S100b in astrocytes and in fusiform Calbindin D28k neurons in lamina I. These results could indicate that neural plasticity and pain sensitization could be maintained by ERK activation in projection neurons at 7 days after the periapical inflammation.

La inflamación periférica induce cambios plásticos en las neuronas y en la glía, los cuales están regulados por el calcio libre y las proteínas fijadoras calcio (CaBP). Uno de los mecanismos asociados con la regulación del calcio intrace-lular está vinculado con la fosforilación de la pro teína quinasa ERK. Asimismo, ERK fosforilado es importante para la trans-ducción de señales intracelulares y participa en la regulación de la neuroplasticidad y las respuestas inflamatorias. El objetivo de este estudio es analizar la expresión de dos CaBPs y pERK en astrocitos y neuronas del subnúcleo caudal del trigémino (Vc) después de una inflamación periapical experimental en el primer molar inferior izquierdo en ratas. A los siete días posteriores al tratamiento, el estímulo inflamatorio periapical induce un aumento en la expresión de pERK, en el número de astrocitos positivos para la proteína marcadora astroglial S100b y en neuronas positivas para Calbindina D28k, en el Vc ipsilateral respecto del lado contralateral y el grupo de control. Además, se observó coexpresión de pERK tanto en astrocitos S100b positivos, como en neuronas fusiformes Calbindin D28k positivas, de la lámina I. Estas observaciones podrían indicar que la neuroplasticidad y la sensibilización al dolor podrían mantenerse mediante la activación de ERK en las neuronas de proyección a los 7 días de la inflamación periapical.

Testosterone and Voluntary Exercise, Alone or Together Increase Cardiac Activation of AKT and ERK1/2 in Diabetic Rats / Testosterona e Exercício Voluntário, Individualmente ou em Conjunto, Aumentam a Ativação Cardíaca de AKT e ERK1/2 em Ratos Diabéticos

Abstract Background: Impaired angiogenesis in cardiac tissue is a major complication of diabetes. Protein kinase B (AKT) and extracellular signal regulated kinase (ERK) signaling pathways play important role during capillary-like network formation in angiogenesis process. Objectives: To determine the effects of testosterone and voluntary exercise on levels of vascularity, phosphorylated Akt (P- AKT) and phosphorylated ERK (P-ERK) in heart tissue of diabetic and castrated diabetic rats. Methods: Type I diabetes was induced by i.p injection of 50 mg/kg of streptozotocin in animals. After 42 days of treatment with testosterone (2mg/kg/day) or voluntary exercise alone or in combination, heart tissue samples were collected and used for histological evaluation and determination of P-AKT and P-ERK levels by ELISA method. Results: Our results showed that either testosterone or exercise increased capillarity, P-AKT, and P-ERK levels in the heart of diabetic rats. Treatment of diabetic rats with testosterone and exercise had a synergistic effect on capillarity, P-AKT, and P-ERK levels in heart. Furthermore, in the castrated diabetes group, capillarity, P-AKT, and P-ERK levels significantly decreased in the heart, whereas either testosterone treatment or exercise training reversed these effects. Also, simultaneous treatment of castrated diabetic rats with testosterone and exercise had an additive effect on P-AKT and P-ERK levels. Conclusion: Our findings suggest that testosterone and exercise alone or together can increase angiogenesis in the heart of diabetic and castrated diabetic rats. The proangiogenesis effects of testosterone and exercise are associated with the enhanced activation of AKT and ERK1/2 in heart tissue.

Resumo Fundamento: Angiogênese prejudicada em tecido cardíaco é uma das principais complicações das diabetes. As vias de sinalização da proteína-quinase B (AKT) e a quinase regulada por sinal extracelular (ERK) exercem um importante papel durante a formação de uma rede similar à capilar no processo de angiogênese. Objetivos: Determinar os efeitos da testosterona e exercícios voluntários sobre os níveis de vascularidade, AKT fosforilada (P- AKT) e ERK fosforilada (P-ERK) sobre o tecido cardíaco de ratos diabéticos e castrados diabéticos. Métodos: A diabetes tipo 1 foi induzida através de injeção intraperitoneal de 50 mg/kg de estreptozotocina em animais. Após 42 dias de tratamento com testosterona (2mg/kg/dia) ou exercícios voluntários, individualmente ou em conjunto, as amostras de tecidos cardíacos foram coletadas e usadas para avaliação histológica e determinação de níveis de P-AKT e P-ERK através do método ELISA. Resultados: Os nossos resultados mostraram que a testosterona ou os exercícios aumentaram a capilaridade, os níveis de P-AKT, e P-ERK nos corações de ratos diabéticos. O tratamento de ratos diabéticos com testosterona e exercícios obteve um efeito sinérgico sobre a capilaridade, níveis de P-AKT, e P-ERK no coração. Além disto, na capilaridade do grupo diabético castrado, os níveis de P-AKT e P-ERK diminuíram significativamente no coração, ao passo que o tratamento com testosterona ou o treinamento com exercícios reverteu tais efeitos. O tratamento simultâneo de ratos diabéticos castrados com testosterona e exercícios obteve um efeito aditivo sobre os níveis de P-AKT e P-ERK. Conclusão: Nossas descobertas sugerem que a testosterona e exercícios, em conjunto ou individualmente, podem aumentar a angiogênese nos corações de ratos diabéticos e castrados diabéticos. Os efeitos favoráveis à angiogênese da testosterona e dos exercícios estão associados à ativação reforçada de AKT e ERK1/2 no tecido cardíaco.

ERK 1/2 activation does not contribute to increased survival mediated by bone marrow cells after 90% partial hepatectomy in Wistar rats

We investigated the influence of bone marrow cells upon activation of ERK 1/2 in an animal model of 90% PH. Phosphorylated ERK 1/2 was evaluated by western blot. No differences were found between the groups. Thus, increased survival does not appear to be mediated by ERK 1/2 activation (AU)

ß-ARRESTIN/CONNEXIN 43 (Cx43) complex anchors ERKS outside the nucleus: a prerequisite for bisphosphonate antiapoptotic effect mediated by Cx43/ERK in osteocytes / El complejo ß-ARRESTINA/CONEXINA 43 (Cx43) retiene ERKS fuera del núcleo: un paso necesario para el efecto anti-apoptótico de los bisfosfonatos mediado por Cx34/ERK en osteocitos

Bisphosphonates (BPs) anti-fracture efficacy may be due in part to inhibition of osteocyte apoptosis. This effect requires opening of connexin (Cx) 43 hemichannels and phosphorylation of the extracellular signal regulated kinases (ERKs). However, unlike ERK activation by other stimuli, the Cx43/ERK pathway activated by BPs does not result in nuclear ERK accumulation. Instead, the anti-apoptotic effect of BPs depends on phosphorylation of cytoplasmic ERK targets and is abolished by forced nuclear retention of ERKs. We now report that ERKs and the scaffolding protein ß-arrestin co-immuno-precipitate with Cx43 in MLO-Y4 osteocytic cells and that the BP alendronate increases this association. Moreover, ERK2 fused to red fluorescent protein (ERK2-RFP) co-localizes with Cx43 fused to green fluorescent protein outside the nucleus in cells untreated or treated with alendronate. Alendronate does not induce ERK nuclear accumulation in cells transfected with wild type ß-arrestin (wtARR) or vector control, whereas it does in cells expressing a dominant negative ß-arrestin mutant (dnARR) consisting of the ß-arrestin-clathrin binding domain that competes with endogenous ß-arrestin for binding to clathrin. Alendronate activates ERKs in dnARRtransfected cells as effectively as in cells transfected with wtARR, demonstrating that dnARR only interferes with subcellular localization but not with activation of ERKs by BPs. Further, whereas alendronate inhibits apoptosis in cells expressing wtARR or vector control, it is ineffective in cells expressing dnARR. Thus, BPs induce the formation of a complex comprising Cx43, ß-arrestin, and clathrin, which directs ERKs outside the nucleus and is indispensable for osteocyte survival induced by BPs. (AU)

La efectividad de los bisfosfonatos (BPs) en la prevención de fracturas puede deberse en parte a la inhibición de la apoptosis de osteocitos. Este efecto depende de la apertura de hemicanales de conexina (Cx) 43 y la fosforilación de quinasas reguladas por señales extracelulares (ERKs). Sin embargo, a diferencia de la activación de ERKs debida a otros estímulos, la vía de señalización Cx43/ERK activada por BPs no conlleva la acumulación de ERKs en el núcleo. El efecto anti-apoptótico de los BPs depende de la fosforilación de blancos citoplasmáticos de ERKs y es inhibido cuando las quinasas son retenidas en el núcleo. En este estudio hemos demostrado que ERKs y la proteína "scaffolding" ß-arrestina co-inmunoprecipitan con Cx43 en células osteocíticas MLO-Y4 y que alendronato aumenta esta asociación. Más aún, ERK2 fusionada a la proteína roja fluorescente (ERK2-RFP) co-localiza con Cx43 fusionada con la proteína verde fluorescente fuera del núcleo en células tratadas con vehículo o alendronato. Alendronato no indujo la acumulación nuclear de ERK en células transfectadas con ß-arrestina nativa (wtARR) o con un vector control, pero si lo hizo en células que expresan una forma dominante negativa de ß-arrestina (dnARR), consistente en el dominio de interacción entre ß-arrestina y clatrina, y que compite con ß-arrestina endógena por la unión a clatrina. Alendronato activa ERKs con la misma eficiencia en células transfectadas con dnARR o wtARR, demostrando que dnARR sólo interfiere con la localización subcelular de ERKs, pero no con su activación inducida por los BPs. Más aún, mientras alendronato inhibe apoptosis en células que expresan wtARR o vector control, es inefectivo en células que expresan dnARR. En conclusión, los BPs inducen la formación de un complejo que incluye Cx43, ß-arrestina y clatrina, el cual retiene ERKs fuera del núcleo y es indispensable para la sobrevida de los osteocitos inducida por estas drogas. (AU)

STAT3 and ERK Signaling Pathways Are Implicated in the Invasion Activity by Oncostatin M through Induction of Matrix Metalloproteinases 2 and 9

PURPOSE: Our previous studies have shown that oncostatin M (OSM) promotes trophoblast invasion activity through increased enzyme activity of matrix metalloproteinase (MMP)-2 and -9. We further investigated OSM-induced intracellular signaling mechanisms associated with these events in the immortalized human trophoblast cell line HTR8/SVneo. MATERIALS AND METHODS: We investigated the effects of OSM on RNA and protein expression of MMP-2 and -9 in the first-trimester extravillous trophoblast cell line (HTR8/SVneo) via Western blot. The selective signal transducer and activator of transcription (STAT)3 inhibitor, stattic, STAT3 siRNA, and extracellular signal-regulated kinase (ERK) siRNA were used to investigate STAT3 and ERK activation by OSM. The effects of STAT3 and ERK inhibitors on OSM-induced enzymatic activities of MMP-2 and -9 and invasion activity were further determined via Western blot and gelatin zymography. RESULTS: OSM-induced MMP-2 and -9 protein expression was significantly suppressed by STAT3 inhibition with stattic and STAT3 siRNA silencing, whereas the ERK1/2 inhibitor (U0126) and ERK silencing significantly suppressed OSM-induced MMP-2 protein expression. OSM-induced MMP-2 and MMP-9 enzymatic activities were significantly decreased by stattic pretreatment. The increased invasion activity induced by OSM was significantly suppressed by STAT3 and ERK1/2 inhibition, though to a greater extent by STAT3 inhibition. CONCLUSION: Both STAT3 and ERK signaling pathways are involved in OSM-induced invasion activity of HTR8/SVneo cells. Activation of STAT3 appears to be critical for the OSM-mediated increase in invasiveness of HTR8/SVneo cells.

Anti-Proliferative and Apoptotic Activities of Mullerian Inhibiting Substance Combined with Calcitriol in Ovarian Cancer Cell Lines

PURPOSE: This study aimed to investigate whether Mullerian inhibiting substance (MIS) in combination with calcitriol modulates proliferation and apoptosis of human ovarian cancer (OCa) cell lines (SKOV3, OVCAR3, and OVCA433) and identify the signaling pathway by which MIS mediates apoptosis. MATERIALS AND METHODS: OCa cell lines were treated with MIS in the absence or presence of calcitriol. Cell viability and proliferation were evaluated using the Cell Counting Kit-8 assay and apoptosis was evaluated by DNA fragmentation assay. Western blot and enzyme-linked immunosorbent assay were used to determine the signaling pathway. RESULTS: The cells showed specific staining for the MIS type II receptor. Treatment of OCa cells with MIS and calcitriol led to dose- and time-dependent inhibition of cell growth and survival. The combination treatment significantly suppressed cell growth, down-regulated the expression of B-cell lymphoma 2 (Bcl-2), and up-regulated the expressions of Bcl-2 associated X protein, caspase-3, and caspase-9 through the extracellular signal-regulated kinase signaling pathway. CONCLUSION: These results, coupled with a much-needed decrease in the toxic side effects of currently employed therapeutic agents, provide a strong rationale for testing the therapeutic potential of MIS, alone or in combination with calcitriol, in the treatment of OCa.

Association of junk food consumption with high blood pressure and obesity in Iranian children and adolescents: the CASPIAN-IV Study / Associação entre o consumo de junk food e a pressão arterial alta e obesidade em crianças e adolescentes iranianos: o Estudo Caspian-IV

OBJECTIVE: This study aimed to evaluate the association of junk food consumption with hypertension and obesity in a national sample of Iranian children and adolescents. METHODS: This nationwide study was conducted in 2011-2012 among 14,880 students, aged 6-18 years, selected by cluster sampling from 30 provinces. Weight, height, waist circumference (WC), hip circumference (HC), waist-to-hip ratio (WHR), waist-to-height ratio (WHtR), as well as systolic and diastolic blood pressure (BP) were measured. Junk food was divided into four categories, including salty snacks, sweets, sweetened beverages, and fast food. Subjects reported how many times they had consumed each item (daily, weekly, and seldom). RESULTS: The intake of sweets was significantly associated with anthropometric indices and BP levels. Moreover, a significant association was found between fast food consumption, BP levels, and anthropometric indices (except for WHtR and WHR). Sweet beverages consumption was significantly associated with anthropometric indices; however, the consumption of salty snacks was only significantly associated with height, HC, and WHR. The risk of general obesity (OR: 0.75, 95% CI: 0.65-0.87) and abdominal obesity (OR: 0.81, 95% CI: 0.72-0.92) among participants who seldom consumed sweets was less than those who consumed daily. Also, the risk of general obesity (OR: 0.85, 95% CI: 0.74-0.97) among students that seldom consumed sweetened beverages was less than subjects who consumed them on a daily basis. CONCLUSION: It was found that junk food consumption increased the risk of both general and abdominal obesity; therefore, consumption of junk food should be reduced via restricting TV advertisements and increasing taxes on junk foods. .

OBJETIVO: Avaliar a associação entre o consumo de junk food e a hipertensão e obesidade em uma amostra nacional de crianças e adolescentes iranianos. MÉTODOS: Este estudo nacional foi feito entre 2011 e 2012 com 14.880 estudantes com seis-18 anos, selecionados por amostra em bloco em 30 províncias. Foram medidos o peso, a estatura, a circunferência da cintura (CC), a circunferência do quadril (CQ), a razão cintura/quadril (RCQ), a razão cintura/estatura (RCE) e a pressão arterial sistólica e diastólica (PAS e PAD). A junk food foi dividida em quatro categorias, incluindo lanches salgados, doces, bebidas açucaradas e fast food. Os indivíduos relataram quantas vezes consumiam cada um dos itens (diariamente, semanalmente, raramente). RESULTADOS: O consumo de doces foi associado significativamente aos índices antropométricos e níveis de pressão arterial (PA). Além disso, havia uma associação significativa entre o consumo de fast food e os níveis de PA e os índices antropométricos (exceto RCE e RCQ). O consumo de bebidas açucaradas foi associado significativamente aos índices antropométricos, porém o consumo de lanches salgados foi associado significativamente apenas a estatura, CQ e RCQ. O risco de obesidade geral (RC: 0,75, IC de 95%: 0,65-0,87) e obesidade abdominal (RC: 0,81, IC de 95%: 0,72-0,92) entre participantes que raramente consumiam doces era menor do que naqueles que os consumiam diariamente. Além disso, o risco de obesidade geral (RC: 0,85; IC de 95%: 0,74-0,97) entre estudantes que raramente consumiam bebidas açucaradas era menor do que entre indivíduos que os consumiam diariamente. CONCLUSÃO: Constatamos que o consumo de junk food aumentou o risco de obesidade geral e abdominal; portanto, o consumo de junk food deve ser reduzido por meio da restrição de comerciais de TV e do aumento de impostos sobre esse tipo de alimento. .

Antiangiogenic Activity of Acer tegmentosum Maxim Water Extract in Vitro and in Vivo

Angiogenesis, the formation of new blood vessels, is critical for tumor growth and metastasis. Notably, tumors themselves can lead to angiogenesis by inducing vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors. Inhibition of angiogenesis is currently perceived as one of the most promising strategies for the blockage of tumor growth. In this study, we investigated the effects of Acer tegmentosum maxim water extract (ATME) on angiogenesis and its underlying signal mechanism. We studied the antiangiogenic activity of ATME by using human umbilical vein endothelial cells (HUVECs). ATME strongly inhibited VEGF-induced endothelial cell proliferation, migration, invasion, and tube formation, as well as vessel sprouting in a rat aortic ring sprouting assay. Moreover, we found that the p44/42 mitogen activated protein (MAP) kinase signaling pathway is involved in the inhibition of angiogenesis by ATME. Moreover, when we performed the in vivo matrigel plug assay, VEGF-induced angiogenesis was potently reduced when compared to that for the control group. Taken together, these results suggest that ATME exhibits potent antiangiogenic activity in vivo and in vitro and that these effects are regulated by the extracellular regulated kinase (ERK) pathway.

Fucoidan promotes osteoblast differentiation via JNK- and ERK-dependent BMP2-Smad 1/5/8 signaling in human mesenchymal stem cells

Fucoidan has attracted attention as a potential drug because of its biological activities, which include osteogenesis. However, the molecular mechanisms involved in the osteogenic activity of fucoidan in human alveolar bone marrow-derived mesenchymal stem cells (hABM-MSCs) remain largely unknown. We investigated the action of fucoidan on osteoblast differentiation in hABM-MSCs and its impact on signaling pathways. Its effect on proliferation was determined using the crystal violet staining assay. Osteoblast differentiation was evaluated based on alkaline phosphatase (ALP) activity and the mRNA expression of multiple osteoblast markers. Calcium accumulation was determined by Alizarin red S staining. We found that fucoidan induced hABM-MSC proliferation. It also significantly increased ALP activity, calcium accumulation and the expression of osteoblast-specific genes, such as ALP, runt-related transcription factor 2, type I collagen-alpha 1 and osteocalcin. Moreover, fucoidan induced the expression of bone morphogenetic protein 2 (BMP2) and stimulated the activation of extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase by increasing phosphorylation. However, the effect of fucoidan on osteogenic differentiation was inhibited by specific inhibitors of ERK (PD98059) and JNK (SP600125) but not p38 (SB203580). Fucoidan enhanced BMP2 expression and Smad 1/5/8, ERK and JNK phosphorylation. Moreover, the effect of fucoidan on osteoblast differentiation was diminished by BMP2 knockdown. These results indicate that fucoidan induces osteoblast differentiation through BMP2-Smad 1/5/8 signaling by activating ERK and JNK, elucidating the molecular basis of the osteogenic effects of fucoidan in hABM-MSCs.

MAPK immunoreactivity in streptozotocin-induced diabetic rat testis

PURPOSE: To evaluate the alterations of two mitogen-activated protein kinases (MAPK)s, extracellular signal regulated kinase (ERK) and c-Jun NH2 terminal kinase (JNK), in the testes of male rats with experimental diabetes. METHODS: Twenty males Sprague-Dawley rats were randomly divided into a control group (n=8) and a diabetes group (administration of 40 mg/kg/day streptozotocin (STZ) for five sequential days, n=12). After six weeks, testicular biopsy samples were obtained for light microscopy and immunohistochemical methods. RESULTS: The PCNA (proliferating cell nuclear antigen) index was significantly decreased in the diabetes group (p=0.004) when compared to the control group. Both total (t)-ERK and phosphor (p)-ERK immunoreactivities were significantly decreased in the diabetes group (p=0.004, p<0.001, respectively). The t-JNK immunoreactivity was unchanged in both groups (p=0.125), while p-JNK immunoreactivity was significantly increased in the diabetic group (p=0.002). CONCLUSIONS: The decrease of androgen levels in the course of diabetes may contribute to the decrease of the immunoreactivities of t-ERK and p-ERK. JNK may be activated due to the changes in various cytokines and chemochines that participate in the oxidative stress process of diabetes. Therefore, testicular apoptosis may occur and lead to infertility associated with diabetes. .

Effects of Antioxidant on Reduction of Hindlimb Muscle Atrophy Induced by Cisplatin in Rats

PURPOSE: The purpose of this study was to examine the effects of Cu/Zn SOD on reduction of hindlimb muscular atrophy induced by cisplatin in rats. METHODS: Forty-two rats were assigned to three groups; control group, Cisplatin (CDDP) group and cisplatin with Cu/Zn SOD (CDDP-SOD) group. At day 35 hindlimb muscles were dissected. Food intake, activity, withdrawal threshold, muscle weight, and Type I, II fiber cross-sectional area (CSA) of dissected muscles were measured. Relative SOD activity and expression of MHC and phosphorylated Akt, ERK were measured after dissection. RESULTS: Muscle weight and Type I, II fiber CSA of hindlimb muscles in the CDDP group were significantly less than the control group. Muscle weight and Type I, II fiber CSA of hindlimb muscles, food intake, activity, and withdrawal thresholds of the CDDP-SOD group were significantly greater than the CDDP group. There were no significant differences in relative SOD activities of hindlimb muscles between the CDDP-SOD and CDDP groups. MHC expression and phosphorylated Akt, ERK of hindlimb muscles in the CDDP-SOD group were significantly greater than the CDDP group. CONCLUSION: Cu/Zn SOD attenuates hindlimb muscular atrophy induced by cisplatin through increased food intake and activity. Increment of phosphorylated Akt, ERK may relate to attenuation of hindlimb muscular atrophy.

Synergistic Effect of Interleukin-6 and Hyaluronic Acid on Cell Migration and ERK Activation in Human Keratinocytes

Wound healing is initiated and progressed by complex integrated process of cellular, physiologic, and biochemical events, such as inflammation, cell migration and proliferation. Interleukin 6 (IL-6) is a multifunctional cytokine, and it could regulate the inflammatory response of wound healing process in a timely manner. Hyaluronic acid (HA) is an essential component of the extracellular matrix, and contributes significantly to cell proliferation and migration. The purpose of this study was to investigate the effects of IL-6 or/and HA on the cell migration process in human keratinocytes. Combining IL-6 and HA significantly increased the cell migration in scratch based wound healing assay. The phosphorylation of extracellular-signal-regulated kinase (ERK) was significantly increased after 1 hr of IL-6 and HA treatment, but the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was not. We also found that significant increase of the NF-kappaB translocation from cytoplasm into nucleus after 1 hr of IL-6 or/and HA treatments. This study firstly showed that synergistic effects of combining IL-6 and HA on the cell migration of wound healing by activation of ERK and NF-kappaB signaling. Further studies might be required to confirm the synergistic effects of HA and IL-6 in the animal model for the development of a novel therapeutic mixture for stimulation of wound healing process.

Phospholipase D inhibitor enhances radiosensitivity of breast cancer cells

Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.

Activated protein C differentially regulates both viability and differentiation of osteoblasts mediated by bisphosphonates

Activated protein C (APC) is a cytoprotective anticoagulant that can promote cutaneous healing. We examined the effect of APC on viability and differentiation of the osteoblastic line, MG63, in the presence and absence of bisphosphonates (BPs). Osteoblasts were cultured and treated for 24 or 48 h with Alendronate (Aln), Zoledronate (Zol) or Pamidronate (Pam) at concentrations ranging from 10-4 to 10-6 M. Cell differentiation was measured using type 1 collagen production, Alizarin red staining and alkaline phosphatase activity, whereas cell viability was assessed using MTT and crystal violet assays. All three BPs induced MG63 cell death in a dose- and time-dependent manner. Pam- and Zol-related cell death was prevented by APC treatment; however, cell death induced by Aln was accelerated by APC. APC induced MG63 cell differentiation that was enhanced by Aln, but inhibited by Pam or Zol. Endothelial protein C receptor (EPCR) was expressed by MG63 cells and mediated the protective effect of APC on Zol-induced viability. In summary, we have demonstrated that (1) APC favorably regulates MG63 viability and differentiation toward bone growth, (2) APC differentially regulates the effects of specific BPs and (3) at least part of the effects of APC is mediated through EPCR. These findings highlight the potential importance of the PC pathway in bone physiology and provide strong evidence that APC may influence bone cells and has potential to be a therapeutic drug for bone regeneration, depending on concurrent BP treatment.

Wnt5a stimulates chemotactic migration and chemokine production in human neutrophils

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (beta-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.

Taurine inhibits serum deprivation-induced osteoblast apoptosis via the taurine transporter/ERK signaling pathway

Taurine has positive effects on bone metabolism. However, the effects of taurine on osteoblast apoptosis in vitro have not been reported. The aim of this study was to investigate the activity of taurine on apoptosis of mouse osteoblastic MC3T3-E1 cells. The data showed that 1, 5, 10, or 20 mM taurine resulted in 16.7, 34.2, 66.9, or 63.75 percent reduction of MC3T3-E1 cell apoptosis induced by the serum deprivation (serum-free α-MEM), respectively. Taurine (1, 5, or 10 mM) also reduced cytochrome c release and inhibited activation of caspase-3 and -9, which were measured using fluorogenic substrates for caspase-3/caspase-9, in serum-deprived MC3T3-E1 cells. Furthermore, taurine (10 mM) induced extracellular signal-regulated kinase (ERK) phosphorylation in MC3T3-E1 cells. Knockdown of the taurine transporter (TAUT) or treatment with the ERK-specific inhibitor PD98059 (10 μM) blocked the activation of ERK induced by taurine (10 mM) and abolished the anti-apoptotic effect of taurine (10 mM) in MC3T3-E1 cells. The present results demonstrate for the first time that taurine inhibits serum deprivation-induced osteoblast apoptosis via the TAUT/ERK signaling pathway.

Toll-Like Receptor 4 Signaling is Involved in IgA-Stimulated Mesangial Cell Activation

PURPOSE: Deposition of polymeric IgA1 in the kidney mesangium is the hallmark of IgA nephropathy, but the molecular mechanisms of IgA-mediated mesangial responses and inflammatory injuries remain poorly understood. We hypothesize that Toll-like receptor 4 (TLR4) is involved in IgA-induced mesangial cell activation. MATERIALS AND METHODS: Mouse mesangial cells were stimulated with lipopolysaccharide (LPS) (1 microg/mL), IgA (20 microg/mL), or both, and TLR4 expression was measured by real time RT-PCR and Western blot. Intracellular responses to LPS or IgA were assessed by Western blot for ERK1/2, JNK, p38 MAP kinases (MAPKs), Ikappa-Balpha degradation and fibronectin secretion. MCP-1 secretion was assessed by ELISA. Small interfering RNA (siRNA) of TLR4 was used to confirm that the effects were caused by TLR4 activity. RESULTS: LPS- or IgA-treatment upregulated the levels of TLR4 mRNA and protein in cultured MMC at 24 h. LPS and IgA induced rapid phosphorylation of MAPKs, but degradation of Ikappa-Balpha was observed only in LPS-treated MMC. LPS, but not IgA, induced increased secretion of MCP-1 and fibronectin at 24 h or 48 h. Combined LPS and IgA treatment did not cause additional increases in TLR4 mRNA and protein levels or Ikappa-Balpha degradation, and MCP-1 and fibronectin secretions were less than with LPS alone. LPS- or IgA-induced TLR4 protein levels and MAPK activation were inhibited by transfection with TLR4 siRNA. CONCLUSION: These results indicate that the activation of MAPKs and MCP-1 secretion are mediated by TLR4, at least in part, in IgA-treated mesangial cells. TLR4 is involved in mesangial cell injury by induction of pro-inflammatory cytokines in IgA nephropathy.