RESUMEN
To explore the effect of Hei Xiaoyaosan on autophagy levels in Alzheimer's disease(AD). A total of 100 4-month-old Wistar male rats were randomly selected as a blank group, and 10 rats were taken as a sham operation group and injected with 1 µL of normal saline on both sides of the hippocampus. The other rats were injected with Aß_(1-42) solution in the hippocampus to replicate the AD model. Fifty successfully modeled rats were selected and randomly divided into the model group, Aricatio group(0.5 mg·kg~(-1)), and high, medium, and low dose groups of Hei Xiaoyaosan(15.30, 7.65, and 3.82 g·kg~(-1)), with 10 rats in each group. The rats were administered by continuous gavage for 42 days. Morris water maze was used to detect the learning and memory ability of rats, and Hoechst staining was used to observe the pathological changes of nerve cells in the hippocampal CA1 region. The mRNA expression of p38MAPK, Beclin-1, and Bcl-2 was detected by RT-qPCR.Western blot was used to detect the expressions of p38MAPK, Beclin-1, Bcl-2, APP, and related proteins. The level of Aß_(1-42) in the hippocampus was detected by ELISA, and the expression level of LC3â ¡ in the hippocampus was detected by immunohistochemistry. The experimental results showed that compared with the blank group, the learning and memory ability of rats in the model group decreased(P<0.01). The nuclei in the CA1 region of the hippocampus showed blue bright spots and were closely arranged. The mRNA expression of p38MAPK was up-regulated, and the mRNA expressions of Beclin-1 and Bcl-2 were down-regulated(P<0.01). The expressions of p38MAPK, p-p38MAPK, and APP were increased, while those of Beclin-1, Bcl-2, and p-Bcl-2 were decreased(P<0.01). The expression of Aß_(1-42) was increased(P<0.01). The relative expression of LC3â ¡ decreased(P<0.01). Compared with the model group, the learning and memory ability of rats in each administration group was improved(P<0.05 or P<0.01). The nuclei in the CA1 region of the hippocampus gradually became clear, showing light blue. The mRNA expression of p38MAPK was down-regulated(P<0.01), and that of Beclin-1 and Bcl-2 was increased(P<0.05 or P<0.01). The expressions of p38MAPK, p-p38MAPK, and APP were down-regulated, while those of Beclin-1, Bcl-2, and p-Bcl-2 were up-regulated(P<0.05 or P<0.01). The expression of Aß_(1-42) was decreased(P<0.01). The relative expression of LC3â ¡ was increased(P<0.01). It can be concluded that Hei Xiaoyaosan can improve the cognitive ability of AD model rats, and its potential mechanism may be related to regulating the p38MAPK/Beclin-1/Bcl-2 signaling pathway, increasing the level of autophagy, and reducing the accumulation of Aß_(1-42).
Asunto(s)
Enfermedad de Alzheimer , Autofagia , Beclina-1 , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos , Proteínas Proto-Oncogénicas c-bcl-2 , Ratas Wistar , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Masculino , Ratas , Medicamentos Herbarios Chinos/administración & dosificación , Medicamentos Herbarios Chinos/farmacología , Autofagia/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Beclina-1/metabolismo , Beclina-1/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Humanos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Macrophages polarize into alternatively activated M2 macrophages through interleukin (IL)-4, and they express high levels of arginase-1, which promotes anti-inflammatory responses. Several studies have confirmed the anti-inflammatory effects of cyclin-dependent kinase (CDK) 8/19 inhibition, and hence, numerous CDK8/19 inhibitors, such as BRD6989, have been developed. However, the effects of CDK8/19 inhibitors on arginase-1 expression in macrophages have not yet been elucidated. This study investigated the effects of CDK8/19 inhibitor on arginase-1 expression in IL-4-activated macrophages. The results showed that BRD6989 increased arginase-1 expression transcriptionally in murine peritoneal macrophages and the murine macrophage cell line RAW264.7 in an IL-4-dependent manner. In addition, the results indicated that BRD6989 enhances signal transducer and activator of transcription (STAT) 6 phosphorylation. Meanwhile, BRD6989 exhibited the capability to activate p38 mitogen-activated protein kinase (MAPKï¼ even in the absence of IL-4 stimulation. Moreover, we observed that a p38 MAPK inhibitor suppressed the BRD6989-induced increase in arginase-1 expression. Besides, BRD6989 increased the surface expression of CD206, an M2 macrophage marker. Thus, this study demonstrated for the first time that CDK8/19 inhibition increases arginase-1 expression, suggesting that this mechanism involves the activation of STAT6 and p38 MAPK. This finding implies that CDK8/19 inhibition may facilitate the production of anti-inflammatory M2 macrophages.
Asunto(s)
Arginasa , Quinasa 8 Dependiente de Ciclina , Quinasas Ciclina-Dependientes , Interleucina-4 , Factor de Transcripción STAT6 , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Arginasa/metabolismo , Arginasa/antagonistas & inhibidores , Factor de Transcripción STAT6/metabolismo , Ratones , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Células RAW 264.7 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Interleucina-4/metabolismo , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Fosforilación/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Activación Enzimática/efectos de los fármacos , Flavonoides , Piperidinas , Quinasa 9 Dependiente de la CiclinaRESUMEN
OBJECTIVE: To explore the effect and mechanism of Dahuang Zhechong Pill (DHZCP) on liver fibrosis. METHODS: Liver fibrosis cell model was induced by transforming growth factor-ß (TGF-ß) in hepatic stellate cells (HSC-T6). DHZCP medicated serum (DMS) was prepared in rats. HSC-T6 cells were divided into the control (15% normal blank serum culture), TGF-ß (15% normal blank serum + 5 ng/mL TGF-ß), DHZCP (15% DMS + 5 ng/mL TGF-ß), DHZCP+PDTC [15% DMS + 4 mmol/L ammonium pyrrolidine dithiocarbamate (PDTC)+ 5 ng/mL TGF-ß], and PDTC groups (4 mmol/L PDTC + 5 ng/mL TGF-ß). Cell activity was detected by cell counting kit 8 and levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the cell supernatant were determined by enzyme-linked immunosorbnent assay. Western blot was used to measure the expressions of p38 mitogen-activated protein kinase/nuclear factor kappa B/transforming growth factor-ß1 (p38 MAPK/NF-κ B/TGF-ß1) pathway related proteins, and the localization and expressions of these proteins were observed by immunofluorescence staining. RESULTS: DHZCP improves the viability of cells damaged by TGF-ß and reduces inflammatory cytokines and ALT and AST levels in the supernatant of HSC-T6 cells induced with TGF-ß (P<0.05 or P<0.01). Compared with the TGF-ß group, NF-κ B p65 levels in the DHZCP group were decreased (P<0.05). p38 MAPK and NF-κ B p65 levels in the DHZCP+PDTC were also reduced (P<0.01). Compared with the TGF-ß group, the protein expression of Smad2 showed a downward trend in the DHZCP, DHZCP+PDTC, and PDTC groups (all P<0.01), and the decreasing trend of Samd3 was statistically significant only in DHZCP+PDTC group (P<0.01), whereas Smad7 was increased (P<0.05 or P<0.01). CONCLUSION: DHZCP can inhibit the process of HSC-T6 cell fibrosis by down-regulating the expression of p38 MAPK/NF-κ B/TGF-ß1 pathway.
RESUMEN
Objective: To investigate the feasibility of selenium-methylselenocysteine (SMC) to promote peripheral nerve regeneration and its mechanism of action. Methods: Rat Schwann cells RSC96 cells were randomly divided into 5 groups, which were group A (without any treatment, control group), group B (adding 100 µmol/L H 2O 2), group C (adding 100 µmol/L H 2O 2+100 µmol/L SMC), group D (adding 100 µmol/L H 2O 2+200 µmol/L SMC), group E (adding 100 µmol/L H 2O 2+400 µmol/L SMC); the effect of SMC on cell proliferation was detected by MTT method, and the level of oxidative stress was detected by immunofluorescence for free radicals [reactive oxygen species (ROS)] after determining the appropriate dose group. Thirty-six 4-week-old male Sprague Dawley rats were randomly divided into 3 groups, namely, the sham operation group (Sham group), the sciatic nerve injury group (PNI group), and the SMC treatment group (SMC group), with 12 rats in each group; the rats in the PNI group were fed with food and water normally after modelling operation, and the rats in the SMC group were added 0.75 mg/kg SMC to the drinking water every day. At 4 weeks after operation, the sciatic nerves of rats in each group were sampled for neuroelectrophysiological detection of highest potential of compound muscle action potential (CMAP). The levels of inflammatory factors [interleukin 17 (IL-17), IL-6, IL-10 and oxidative stress factors catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA)] were detected by ELISA assay. The luxol fast blue (LFB) staining was used to observe the myelin density, fluorescence intensity of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) was observed by immunofluorescence staining, and myelin morphology was observed by transmission electron microscopy with measurement of axon diameter. Western blot was used to detect the protein expressions of p38 mitogen-activated protein kinases (p38MAPK), phosphorylated p38MAPK (p-p38MAPK), heme oxygenase 1 (HO-1), and nuclear factor erythroid 2-related factor 2 (Nrf2). Results: MTT assay showed that the addition of SMC significantly promoted the proliferation of RSC96 cells, and the low concentration could achieve an effective effect, so the treatment method of group C was selected for the subsequent experiments; ROS immunofluorescence test showed that group B showed a significant increase in the intensity of ROS fluorescence compared with that of group A, and group C showed a significant decrease in the intensity of ROS fluorescence compared with that of group B ( P<0.05). Neuroelectrophysiological tests showed that the highest potential of CMAP in SMC group was significantly higher than that in PNI and Sham groups ( P<0.05). ELISA assay showed that the levels of IL-6, IL-17, and MDA in PNI group were significantly higher than those in Sham group, and the levels of IL-10, SOD, and CAT were significantly lower; the levels of IL-6, IL-17, and MDA in SMC group were significantly lower than those in PNI group, and the levels of IL-10, SOD, and CAT were significantly higher ( P<0.05). LFB staining and transmission electron microscopy showed that the myelin density and the diameter of axons in the SMC group were significantly higher than those of the PNI group and the Sham group ( P<0.05). Immunofluorescence staining showed that the fluorescence intensity of GFAP and MBP in the SMC group were significantly stronger than those in the PNI group and Sham group ( P<0.05). Western blot showed that the relative expressions of Nrf2 and HO-1 proteins in the SMC group were significantly higher than those in the PNI group and Sham group, and the ratio of p-p38MAPK/p38MAPK proteins was significantly higher in the PNI group than that in the SMC group and Sham group ( P<0.05). Conclusion: SMC may inhibit oxidative stress and inflammation after nerve injury by up-regulating the Nrf2/HO-1 pathway, and then inhibit the phosphorylation of p38MAPK pathway to promote the proliferation of Schwann cells, which ultimately promotes the formation of myelin sheaths and accelerates the regeneration of peripheral nerves.
Asunto(s)
Regeneración Nerviosa , Estrés Oxidativo , Ratas Sprague-Dawley , Células de Schwann , Nervio Ciático , Selenio , Selenocisteína , Animales , Regeneración Nerviosa/efectos de los fármacos , Ratas , Masculino , Selenocisteína/análogos & derivados , Selenocisteína/farmacología , Células de Schwann/metabolismo , Células de Schwann/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Nervio Ciático/efectos de los fármacos , Selenio/farmacología , Proliferación Celular/efectos de los fármacos , Traumatismos de los Nervios Periféricos/metabolismoRESUMEN
Upregulation of soluble tumor necrosis factor (sTNF) cytokine signaling through TNF receptor 1 (TNFR1) and subsequent neuronal hyperexcitability are observed in both animal models and human chronic neuropathic pain (CNP). Previously, we have shown that estrogen modulates sTNF/TNFR1 signaling in CNP, which may contribute to female prevalence of CNP. The estrogen-dependent role of TNFR1-mediated supraspinal neuronal circuitry in CNP remains unknown. In this study, we interrogated the intersect between supraspinal TNFR1 mediated neuronal signaling and sex specificity by selectively removing TNFR1 in Nex + neurons in adult mice (NexCreERT2::TNFR1f/f). We determined that mechanical hypersensitivity induced by chronic constriction injury (CCI) decreases over time in males, but not in females. Subsequently, we investigated two downstream pathways, p38MAPK and NF-κB, important in TNFR1 signaling and injury response. We detected p38MAPK and NF-κB activation in male cortical tissue; however, p38MAPK phosphorylation was reduced in NexCreERT2::TNFR1f/f males. We observed a similar recovery from acute pain in male mice following CCI when p38αMAPK was knocked out of supraspinal Nex + neurons (NexCreERT2::p38αMAPKf/f), while chronic pain developed in female mice. To explore the intersection between estrogen and inflammation in CNP we used a combination therapy of an estrogen receptor ß (ER ß) inhibitor with a sTNF/TNFR1 or general p38MAPK inhibitor. We determined both combination therapies lends therapeutic relief to females following CCI comparable to the response evaluated in male mice. These data suggest that TNFR1/p38αMAPK signaling in Nex + neurons in CNP is male-specific and lack of therapeutic efficacy following sTNF inhibition in females is due to ER ß interference. These studies highlight sex-specific differences in pathways important to pain chronification and elucidate potential therapeutic strategies that would be effective in both sexes.
Asunto(s)
Dolor Crónico , Estrógenos , Neuralgia , Neuronas , Receptores Tipo I de Factores de Necrosis Tumoral , Transducción de Señal , Animales , Neuralgia/metabolismo , Masculino , Femenino , Ratones , Estrógenos/metabolismo , Estrógenos/farmacología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Neuronas/metabolismo , Dolor Crónico/metabolismo , Transducción de Señal/fisiología , FN-kappa B/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Hiperalgesia/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVES: To observe the effect of catgut embedding at "Feishu"(BL13), "Dingchuan" (EX-B1) and "Danzhong" (CV17) on expression of phosphorylated p38 mitogen activated protein kinase (p-p38 MAPK), interleukin-4 (IL-4), interferon-γ (IFN-γ) and changes of airway epithelial cells (AEC) in the lung tissue of bronchial asthma (BA) rats, so as to explore its mechanisms underlying improvement of BA. METHODS: Forty male Wistar rats were randomly and equally divided into blank control, model, dexamethasone (DEX) and catgut embedding groups. The BA model was established by intraperitoneal injection of suspension of ovalbumin and aluminum hydroxide. Rats of the DEX group received intraperitoneal injection of DEX (1.5 mg/kg), once daily for 2 weeks, and those of the catgut embedding group received catgut embedding at BL13, EX-B1 and CV17 only one time. The rats' sneezing times per miniute in each group were recorded. H.E. staining was used to observe the histopathological changes of the lung tissue under light microscope. A transmission electron microscope (TEM) was used to observe the ultrastructural changes of AEC in the lung tissue, including the thickness of bronchial wall and bronchial smooth muscle by using an image analysis software. The protein expressions of p-p38 MAPK, IL-4 and INF-γ in the lung tissue were determined using Western blot. RESULTS: Morphological observation revealed that in the model group, light microscope showed deformed and swollen bronchial tube wall with increased folds and thickened bronchial smooth muscleï¼and TEM showed a large number of autophagy vesicles containing swollen and deformed organelles in the AEC, and apparent reduction of intracellular mitochondria, these situations were obviously milder in both DEX and catgut embedding groups. Compared with the blank control group, the sneezing times, thickness of bronchial wall and bronchial smooth muscle in the model group were significantly increased (P<0.01), and the expressions of p-p38 MAPK and IL-4 in lung tissue were significantly increased (P<0.01), while the expression of IFN-γ was significantly decreased (P<0.01) in the model group. In comparison with the model group, the sneezing times, thickness of bronchial wall and bronchial smooth muscle, protein expressions of p-p38 MAPK and IL-4 were significantly decreased (P<0.01), while the expression of IFN-γ was obviously increased (P<0.01) in both the DEX and catgut embedding groups. CONCLUSIONS: Acupoint catgut embedding can reduce the expression of IL-4 and increase the expression of IFN-γ by inhibiting p38 MAPK signal pathway of lung tissues in BA rats, which may contribute to its effect in alleviating the degree of airway epithelial cells damage.
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Asma , Interleucina-4 , Ratas , Masculino , Animales , Ratas Wistar , Interleucina-4/genética , Catgut , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Puntos de Acupuntura , Estornudo , Pulmón , Asma/genética , Asma/terapiaRESUMEN
Chronic allergen exposure can significantly induce p38 mitogen-activated protein kinase (MAPK) activation in asthma. p38 MAPK is involved in steroid resistance through phosphorylation of glucocorticoid receptors (GR) at S226. This study aims to investigate whether chronic allergen exposure can induce steroid resistance and whether it is associated with p38 MAPK activation in asthma. A mouse model of asthma was prepared by sensitizing and challenging mice with chronic ovalbumin (OVA) exposure. Key features of allergic asthma, encompassing bronchial hyperresponsiveness, pathology of lung tissues, cytokine profiles of inflammation in bronchoalveolar lavage fluid (BALF), and serum immunoglobulin (Ig)E concentration were evaluated. Furthermore, suppressive effects of corticosteroid on the splenocytes under stimulation of lipopolysaccharides, glucocorticoid receptor (GR) DNA binding ability of splenocytes, expression of GRα and phosphorylation of GR s226 in splenocytes, and p38 MAPK phosphorylation in splenocytes and lung tissues were determined. Chronic OVA exposure substantially induced airway hypersensitivity, leading to increased inflammatory infiltration in lung tissues. Additionally, it resulted in elevated levels of interleukin (IL)-4, IL-5, and IL-6 in BALF, as well as heightened levels of IgE in serum. Furthermore, OVA exposure substantially enhanced p38 MAPK phosphorylation in lung tissues. It also weakened the suppressive impacts of corticosteroids on splenocytes, impaired the GR DNA binding ability, and led to an enhanced phosphorylated state of GR S226 and p38 MAPK in splenocytes. Taken together, chronic allergen exposure contributes to steroid resistance in asthma, which is linked to an increased phosphorylated state of GR S226 and p38 MAPK.
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Asma , Receptores de Glucocorticoides , Animales , Ratones , Receptores de Glucocorticoides/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/farmacología , Fosforilación , Asma/metabolismo , Pulmón/metabolismo , Líquido del Lavado Bronquioalveolar , Corticoesteroides , Esteroides/metabolismo , Esteroides/farmacología , Alérgenos/metabolismo , ADN , Ovalbúmina , Modelos Animales de Enfermedad , Ratones Endogámicos BALB CRESUMEN
Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside-out and integrin αIIbß3-initiated outside-in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα-/- mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin αIIbß3, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα-/- platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα-/- platelets and the p38 inhibitor blocked the increased integrin-mediated outside-in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα-/- mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis.
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Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Trombosis , Ratones , Animales , Proteína Fosfatasa 1/metabolismo , Dominio Catalítico , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Isoformas de Proteínas/metabolismo , Colágeno , Fibrinógeno/metabolismoRESUMEN
Bladder cancer is a urothelial malignancy. Bladder cancer starts in the urothelial cells lining the inside of the bladder. The 5-year recurrence rate for bladder cancer ranges from 31% to 78%, and the progression rate is approximately 45%. To treat bladder cancer, intravesical drug therapy is often used. Leonurus artemisia extract (LaE) was obtained from medicinal samples of Chinese motherwort Scientific Chinese Medicine; L. artemisia has various biological effects. This study investigated the impact of LaE on human bladder cancer cells (the BFTC-905 cell line) and the molecular mechanism underlying apoptosis resulting from the activation of cell signal transduction pathways in bladder cancer cells. A cell counting kit-8 (CCK-8) assay was used to determine the effect of LaE on cell growth. The effect of LaE on migration ability was observed using a wound healing assay. The effects of LaE on the cell cycle, reactive oxygen species production, and apoptosis were investigated. Western blot analysis detected apoptosis-related and mitogen-activated protein kinase signaling pathway-related protein concentrations. At non-toxic concentrations, LaE inhibited the proliferation of BFTC-905 cells in a concentration-dependent manner, and the half-maximal inhibitory concentration (IC50) was 24.08172 µg/µL. LaE impaired the migration ability of BFTC-905 cells. LaE arrested the cell cycle in the G1 and G0 phases, increased reactive oxygen species production, and induced apoptosis. LaE increased Bax and p-ERK concentrations and decreased Bcl-2, cleaved caspase-3, and p-p38 concentrations. No differences in PARP, C-PARP, vimentin, e-cadherin, p-JNK, or TNF-alpha concentrations were observed. These results suggest that LaE inhibits the proliferation of human bladder cancer cells. Moreover, the mitogen-activated protein kinase signaling pathway is involved in the inhibition of the proliferation of BFTC-905 cells.
RESUMEN
Transforming growth factor-beta (TGF-ß) is a multifunctional cytokine that controls various cellular processes. Protein phosphatase 6 (PP6) is an evolutionarily conserved serine/threonine protein phosphatase with diverse functions in cell signaling. However, it has not been linked to TGF-ß signaling. We found that TGF-ß treatment increased PP6 protein levels via transcriptional and post-translational regulation. Loss of the Ppp6c gene suppressed TGF-ß-induced canonical Smad3 phosphorylation and its transcriptional activity. PP6 knockout also inhibited non-canonical p38 mitogen-activated protein kinase (MAPK) pathway. Moreover, PP6 depletion suppressed cell migration induced by TGF-ß. These findings uncovered the role of PP6 as a positive regulator for TGF-ß signaling.
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Proteínas Quinasas Activadas por Mitógenos , Factor de Crecimiento Transformador beta , Animales , Ratones , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fibroblastos/metabolismo , Transducción de Señal , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Fosforilación , Factores de Crecimiento Transformadores/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Upregulation of soluble tumor necrosis factor (sTNF) cytokine signaling through TNF receptor 1 (TNFR1) and subsequent neuronal hyperexcitability are observed in both animal models and human chronic neuropathic pain (CNP) [1-4]. To test the hypothesis that supraspinal circuitry is critical to pain chronification, we studied the intersect between supraspinal TNFR1 mediated neuronal signaling and sex specificity by selectively removing TNFR1 in Nex + neurons in adult mice (NexCreERT2::TNFR1f/f). We determined that following chronic constriction injury (CCI), pain resolves in males; however, female acute pain transitions to chronic. Subsequently, we investigated two downstream pathways, p38MAPK and NF-κB, important in TNFR1 signaling and injury response. We detected p38αMAPK and NF-κB activation in male cortical tissue; however, p38αMAPK phosphorylation was reduced in NexCreERT2::TNFR1f/f males. We observed similar behavioral results following CCI in NexCreERT2::p38αMAPKf/f mice. Previously, we established estrogen's ability to modulate sTNF/TNFR1 signaling in CNP, which may contribute to female prevalence of CNP [5-9]. To explore the intersection between estrogen and inflammation in CNP we used a combination therapy of an estrogen receptor ß (ER ß) inhibitor with a sTNF/TNFR1 or general p38MAPK inhibitor. We determined both combination therapies lend "male-like" therapeutic relief to females following CCI. These data suggest that TNFR1/p38αMAPK signaling in Nex + neurons in CNP is male-specific and lack of therapeutic efficacy following sTNF inhibition in females is due to ER ß interference. These studies highlight sex-specific differences in pathways important to pain chronification and elucidate potential therapeutic strategies that would be effective in both sexes.
RESUMEN
Methylmercury (MeHg) is a toxic metal that causes irreversible damage to the nervous system, making it a risk factor for neuronal degeneration and diseases. MeHg activates various cell signaling pathways, particularly the mitogen-activated protein kinase (MAPK) cascades, which are believed to be important determinants of stress-induced cell fate. However, little is known about the signaling pathways that mitigate the neurotoxic effects of MeHg. Herein, we showed that pretreatment with a p38 MAPK-specific inhibitor, SB203580, attenuates MeHg toxicity in human neuroblastoma SH-SY5Y cells, whereas pretreatment with the extracellular signaling-regulated kinase inhibitor U0126 and the c-Jun N-terminal kinase inhibitor SP600125 does not. Specifically, we quantified the levels of intracellular mercury (Hg) and found that pretreatment with SB203580 reduced Hg levels compared to MeHg treatment alone. Further analysis showed that pretreatment with SB203580 increased multidrug resistance-associated protein 2 (MRP2) mRNA levels after MeHg treatment. These results indicate that detoxification of MeHg by p38 MAPK inhibitors may involve an efflux function of MeHg by inducing MRP2 expression.
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Mercurio , Compuestos de Metilmercurio , Neuroblastoma , Humanos , Compuestos de Metilmercurio/toxicidad , Proteínas Quinasas p38 Activadas por Mitógenos , Transporte BiológicoRESUMEN
OBJECTIVES: This study was aimed at exploring whether klotho improved neurologic function in rats with cerebral infarction by inhibiting P38 mitogen-activated protein kinase (MAPK) activation and thus down-regulating aquaporin 4 (AQP4). METHODS: In this study, we induced intracerebral Klotho overexpression in 6-week-old Sprague Dawley rats by injecting lentivirus carrying full-length rat Klotho cDNA into the lateral ventricle of the brain, followed by middle cerebral artery occlusion (MCAO) surgery after three days. Neurologic function was evaluated by neurological deficit scores. Infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. The expressions of Klotho, AQP4, and P38 MAPK were detected by Western blot and Immunofluorescence. RESULTS: when rats were subjected to cerebral ischemia, their neurologic function was impaired, the protein expressions of klotho downregulated, the protein expressions of AQP4 and P38 MAPK increased, and the ratios of AQP4 and P-P38-positive area were significantly increased compared with the sham group rats. LV-KL-induced Klotho overexpression greatly improved neurobehavioral deficits and reduced infarct volume in MCAO rats. Klotho overexpression significantly reduced AQP4 and P38 MAPK pathway-related protein expression levels and the ratios of P-P38 and AQP4-positive area in MCAO rats. In addition, SB203580, a P38 MAPK signal pathway inhibitor, improved neurobehavioral deficits, reduced infarct volume, downregulated the expressions levels of AQP4 and P38 MAPK, and reduced the size of P-P38 and AQP4-positive area in MCAO rats. CONCLUSION: Klotho could alleviate the infraction volume and neurological dysfunction in MCAO rats, and its mechanism may involve AQP4 expression downregulation by suppressing P38-MAPK activation.
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Proteínas Klotho , Transducción de Señal , Accidente Cerebrovascular , Animales , Ratas , Acuaporina 4/metabolismo , Infarto Cerebral/tratamiento farmacológico , Infarto Cerebral/metabolismo , Infarto Cerebral/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Ratas Sprague-Dawley , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Proteínas Klotho/genéticaRESUMEN
Activating transcription factor 6α (ATF6α) is an endoplasmic reticulum protein known to participate in unfolded protein response (UPR) during ER stress in mammals. Herein, we show that in mouse C2C12 myoblasts induced to differentiate, ATF6α is the only pathway of the UPR activated. ATF6α stimulation is p38 MAPK-dependent, as revealed by the use of the inhibitor SB203580, which halts myotube formation and, at the same time, impairs trafficking of ATF6α, which accumulates at the cis-Golgi without being processed in the p50 transcriptional active form. To further evaluate the role of ATF6α, we knocked out the ATF6α gene, thus inhibiting the C2C12 myoblast from undergoing myogenesis, and this occurred independently from p38 MAPK activity. The expression of exogenous ATF6α in knocked-out ATF6α cells recover myogenesis, whereas the expression of an ATF6α mutant in the p38 MAPK phosphorylation site (T166) was not able to regain myogenesis. Genetic ablation of ATF6α also prevents the exit from the cell cycle, which is essential for muscle differentiation. Furthermore, when we inhibited differentiation by the use of dexamethasone in C2C12 cells, we found inactivation of p38 MAPK and, consequently, loss of ATF6α activity. All these findings suggest that the p-p38 MAPK/ATF6α axis, in pathophysiological conditions, regulates myogenesis by promoting the exit from the cell cycle, an essential step to start myoblasts differentiation.
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OBJECTIVE: To observe the effects of electroacupuncture (EA) on the expression levels of N-methyl-D-aspartate receptor (NMDAR), extracellular signal-regulated kinase (ERK)1/2, p38 mitogen activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) in the spinal cord of rats with primary dysmenoramia (PDM), so as to explore the underlying mechanism of EA treating PDM. METHODS: Thirty female SD rats were randomly divided into normal group, model group and EA group, with 10 rats in each group. The PDM rat model was established by subcutaneous injection of estradiol benzoate and oxytocin into the thigh. At the same time of modeling, rats in the EA group were treated with EA (50 Hz) at "Sanyinjiao" (SP36) and "Guanyuan" (CV4) once daily, 20 min each time, for 10 consecutive days. The writhing times, writhing score and writhing latency were observed within 30 min after oxytocin injection. The uterine pathological morphology was observed by HE staining, and pathological score was calculated. Serum prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) were determined by ELISA. The protein expression levels of NMDAR, ERK1/2, p38MAPK and JNK in spinal cord were detected by Western blot. RESULTS: Compared with the normal group, the writhing times and writhing score were significantly increased (P<0.05); the endometrial epithelial cells showed vacuolar degeneration, death and hyperemia, the uterine pathological score was increased (P<0.05); the content of serum PGF2α and the ratio of PGF2α/PGE2 were significantly increased (P<0.01), while the content of serum PGE2 was significantly decreased (P<0.01); the expression levels of NMDAR, ERK1/2, p38MAPK and JNK in spinal cord were significantly increased (P<0.05, P<0.01) in the model group. Compared with the model group, the writhing times and writhing score were significantly decreased (P<0.05), the writhing latency was prolonged (P<0.05); the endometrial epithelial cells still showed vacuolar degeneration, death and hyperemia, and the uterine pathological score was decreased (P<0.01); the content of serum PGF2α and the ratio of PGF2α/PGE2 were significantly decreased (P<0.01), while the content of serum PGE2 was significantly increased (P<0.01); the protein expression levels of ERK1/2 and JNK in spinal cord were significantly decreased (P<0.01) in the EA group. CONCLUSION: EA intervention at SP36 and GV4 has obvious analgesic effect on PDM rats, and its mechanisms may be related to reducing serum prostaglandin, alleviating uterine inflammation, and inhibiting the protein expressions of NMDAR, ERK1/2, p38 MAPK and JNK in spinal cord.
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Electroacupuntura , Hiperemia , Animales , Femenino , Ratas , Puntos de Acupuntura , Dinoprost , Dinoprostona , Dismenorrea/terapia , Proteínas Quinasas Activadas por Mitógenos , Oxitocina , Proteínas Quinasas p38 Activadas por Mitógenos , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Médula EspinalRESUMEN
Background: Tumor necrosis factor (TNF)-α induces changes in the glucocorticoid receptor (GR) isoforms' expression in human nasal epithelial cells (HNECs) in chronic rhinosinusitis (CRS). Objective: However, the underlying mechanism of TNF-α induced GR isoforms' expression in HNECs remains unclear. Here, we explored changes in inflammatory cytokines and glucocorticoid receptor alpha isoform (GRα) expression in HNECs. Materials and Methods: To explore the expression of TNF-α in nasal polyps and nasal mucosa of CRS, fluorescence immunohistochemical analysis was employed. To investigate changes in inflammatory cytokines and GRα expression in HNECs, RT-PCR and western blotting were performed following the cells' incubation with TNF-α. Cells were pretreated with the nuclear factor-κB gene binding (NF-κB) inhibitor QNZ, the p38 inhibitor SB203580, and dexamethasone for one hour, then a TNF-α. Western blotting, RT-PCR, and immunofluorescence had been utilized for the cells' analysis and the ANOVA for the data analysis. Results: The TNF-α fluorescence intensity was mainly distributed in nasal epithelial cells of nasal tissues. TNF-α prominently inhibited the expression of GRα mRNA from 6 to 24 h in HNECs. GRα protein was decreased from 12 to 24 h. Treatment with QNZ, SB203580, or dexamethasone inhibited the TNF-α and interleukin (IL)-6 mRNA expression and increased the GRα levels. Conclusion: TNF-α induced changes in the GR isoforms' expression in HNECs, and it was mediated through p65-NF-κB and p38-MAPK signal transduction pathways, which could be considered a promising neutrophilic CRS treatment.
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Oxaliplatin (OXA) is a usual chemotherapeutic agent applied in the colorectal cancer (CRC) clinical treatment. Interferon-alpha inducible protein 6 (IFI6) has been proved to promote proliferation and suppress apoptosis in several tumor cells, while the impacts of IFI6 on OXA resistance in CRC still need exploration. HCT116 and SW620 cells were used as the parental to obtain OXA-resistant cells. The influence of IFI6 on OXA sensitivity, cell proliferation and apoptosis were evaluated by overexpression or knockdown IFI6 in cells. In this work, we found that the level of IFI6 was significantly enhanced in HCT116/OXA and SW620/OXA cells as compared to the parental cells. Overexpression of IFI6 decreased the sensitivity of HCT116 and SW620 cells to OXA. However, knockdown of IFI6 enhanced the sensitivity of HCT116/OXA and SW620/OXA cells to OXA. And upregulated IFI6 promoted the proliferation and repressed apoptosis in HCT116 cells, while suppressed IFI6 markedly reduced proliferation and increased apoptosis in HCT116/OXA cells. Additionally, IFI6 suppressed the phosphorylation level of p38, and silenced IFI6 enhanced it. The addition of the p38 kinase inhibitor, SB203580, alleviated the decreased cell proliferation and increased apoptosis in HCT116/OXA cells. Suppressed IFI6 enhanced the reactive oxygen species (ROS) level in HCT116/OXA cells, and blockade of ROS with N-acetyl-L-cysteine (NAC) decreased the enhancement level of ROS and the phosphorylation level of the p38, which was induced by IFI6 down-regulation. We, therefore, implied that suppressed IFI6 reverses OXA-resistance of CRC cells via promoting the ROS-induced p38 mitogen-activated protein kinase (MAPK) signaling pathway.
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Neoplasias Colorrectales , Humanos , Oxaliplatino/farmacología , Oxaliplatino/uso terapéutico , Regulación hacia Abajo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Colorrectales/patología , Apoptosis , Células HCT116 , Línea Celular Tumoral , Transducción de Señal , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/uso terapéuticoRESUMEN
Sepsis is a systemic inflammatory response syndrome, mainly caused by infection or suspected infectious factors. The intestine is not only one of the most easily involved organs in the course of sepsis, but also the dynamic organ for the course of sepsis. The present study investigated the protective effect and mechanism of salidroside on intestinal barrier dysfunction of septic mice. Briefly, C57BL/6 mice were used to establish a septic model and then administered with salidroside. The ileum tissues of mice were examined by histopathological examination. Fluorescein isothiocyanate-dextran concentration was measured. IL-17, IL-6, IL-13 and TNF-α levels in ileum tissues and NF-κB and p38 MAPK activations were detected by ELISA and the expressions of NF-κB p65 and p38 MAPK protein with their phosphorylation and intestinal tight junction proteins were gauged by western blotting. The above assays were performed again to investigate the effect of anti-IL-17A and salidroside (160 mg/kg) alone or in combination. The septic model induced the ileum tissue injury, increased intestinal permeability and TNF-α, IL-17 and IL-6 levels, activated NF-κB and p38 MAPK pathways, promoted the expressions of NF-κB p65 and p38 MAPK and their phosphorylation, while suppressing the levels of IL-13 and intestinal tight junction proteins. Salidroside and anti-IL-17A partially reversed the above effects of septic model, which in combination further strengthened the reversing effect. Collectively, salidroside protected against intestinal barrier dysfunction in septic mice by downregulating IL-17 level to inhibit NF-κB and p38 MAPK signaling pathways, thus providing a new treatment direction.
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In recent decades, studies have reported that inflammation serves key roles in epilepsy and that high mobility group box protein1 (HMGB1) may be involved in status epilepticus. However, it has not been reported whether HMGB1 participates in the pathogenesis of status epilepticus through the regulation of the p38 mitogenactivated protein kinase (p38MAPK) signalling pathway. In the present study, SpragueDawley rats were randomly divided into four groups as follows: Control, status epilepticus (SE), dimethyl sulfoxide treatment (DMSO + SE), and glycyrrhizin treatment (GL + SE) groups. Behavioural changes were then evaluated using the Racine score. In the hippocampus, the protein expression levels of HMGB1 were assessed using western blotting, the neuronal damage was evaluated using haematoxylin and eosin staining and transmission electron microscopy, and the activation of microglia was assessed using immunochemistry and immunofluorescence. The results demonstrated that, in the hippocampal region, HMGB1 existed in neurons and astrocytes and the protein expression levels of HMGB1, p38MAPK and phosphorylatedp38MAPK were significantly inhibited after treatment with GL. Furthermore, GL could alleviate neuronal injury in the CA1 region of the hippocampus and prevented HMGB1 translocation from the nucleus into the cytoplasm in these areas. These findings expand the understanding of how HMGB1 may participate in SE and lay a foundation for evaluation of HMGB1 as a drug target.