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Objective:To evaluate the effect of dexmedetomidine on the thioredoxin-interacting protein (TXNIP)/apoptosis signal-regulated kinase 1 (ASK1) signaling pathway in a mouse model of intestinal ischemia-reperfusion (I/R).Methods:Thirty-two SPF healthy adult male C57BL/6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 4 groups ( n=8 each) using a random number table method: sham operation group (Sham group), intestinal I/R group (I/R group), TXNIP inhibitor resveratrol group (Res group) and dexmedetomidine group (Dex group). The mouse model of intestinal I/R injury was developed by clamping the superior mesenteric artery for 45 min followed by 120-min reperfusion in anesthetized animals. Resveratrol 30 mg/kg was intraperitoneally injected before developing the model in Res group, and dexmedetomidine 25 μg/kg was intraperitoneally injected at 30 min before ischemia in Dex group. Blood samples were collected by cardiac puncture at the end of 120-min reperfusion, then the mice were sacrificed, and the small intestine tissues were removed for microscopic examination and for determination of the serum diamine oxidase (DAO) concentration (by enzyme-linked immunosorbent assay) and expression of TXNIP, ASK1 and cleaved-caspase-3 in small intestinal tissues (by Western blot). The apoptosis rate of intestinal epithelial cells was calculated. The intestinal damage was assessed and scored according to Chiu. Results:Compared with group Sham, the Chiu′s score, serum DAO concentrations and apoptosis rate of intestinal epithelial cells were significantly increased, and the expression of TXNIP, ASK-1 and cleaved-caspase-3 was up-regulated in group I/R ( P<0.05). Compared with group I/R, the Chiu′s score, serum DAO concentration and apoptosis rate of intestinal epithelial cells were significantly decreased, and the expression of TXNIP, ASK-1 and cleaved-caspase-3 was down-regulated in group Res ( P<0.05). Compared with I/R group, the Chiu′s score, serum DAO concentration and apoptosis rate of intestinal epithelial cells were significantly decreased, and the expression of TXNIP, ASK-1 and cleaved-caspase-3 was down-regulated in Dex group ( P<0.05). Conclusions:The mechanism by which dexmedetomidine alleviates intestinal I/R injury may be related to inhibition of the TXNIP/ASK1 signaling pathway and reduction of cell apoptosis in mice.
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Introducción: El síndrome de Noonan es un trastorno genético relacionado principalmente con la mutación del gen PTPN11. Reporte del caso: Recién nacido varón de 34 semanas de edad gestacional con ultrasonidos obstétricos que muestran higroma quístico, hidronefrosis renal bilateral, y polihidramnios. Al nacimiento, presentó edema nucal, puente nasal ancho, pabellón auricular de implantación baja, y criptorquidia derecha. Además, defecto del tabique auricular, ausencia de vena cava inferior, hipertensión pulmonar, conducto arterioso persistente y dificultad respiratoria. El resultado del análisis del panel de 14 genes mostró una mutación del gen MAP2K1 y una variante de significado incierto en el gen CBL, confirmando el diagnóstico del síndrome de Noonan negativo para PTPN11. Durante el seguimiento, también se le diagnosticó blefaroptosis izquierda y reflujo gastroesofágico. Conclusión: El presente caso destaca la amplia variedad de características fenotípicas en un paciente con síndrome de Noonan, con sospecha al nacimiento y confirmado durante el seguimiento.
Background: Noonan syndrome is a genetic disorder mostly related to PTPN11 gene mutation. Report Case: Newborn male of 34 weeks of gestational age with obstetric ultrasounds showing cystic hygroma, bilateral renal hydronephrosis, and polyhydramnios. At born, he presented nuchal edema, wide nose, low-set ears, and right cryptorchidism. Additionally, he presented atrial septum defect, absence of inferior vena cava, mild pulmonary hypertension, persistent ductus arteriosus, and respiratory distress. The result of the 14-gene panel analysis showed a MAP2K1 gene mutation and a variation of uncertain significance in the CBL gene, confirming the diagnosis of PTPN11- negative Noonan syndrome. During the follow-up, he was additionally diagnosed with blepharoptosis of left eye and gastroesophageal reflux disease. Conclusion:This report highlights the wide variety of phenotypical characteristics in a Noonan syndrome patient, which was suspected upon birth and developed during the follow-up.
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Objective:To investigate the neuroprotective effect of cerebroprotein hydrolysate (CH) -Ⅰ on cerebral ischemia-reperfusion injury in rats and its mechanism.Methods:Eighty adult healthy male SD rats were randomly divided into sham operation group, model group, CH-Ⅰ intervention group and cerebrolysin (CBL) positive control group. The model of ischemia-reperfusion injury was induced by temporarily occluding the left middle cerebral artery with suture-occluded method. The CH-Ⅰ and CBL groups intraperitoneally injected with CH-Ⅰ and CBL at 0, 3, 6 and 12 h after reperfusion at the dose of 20 mg/kg. The sham operation group and the model group were injected with the same volume of normal saline. At 24 h after reperfusion, the behavior changes of the rats were detected by the modified neurological severity score (mNSS). The volume of cerebral infarction was detected by TTC staining. The morphology and structure of neurons in ischemic cortex were observed by Nissl staining. The apoptosis of neurons in ischemic cortex was detected by TUNEL staining. The expression changes of phosphorylated extracellular signal-regulated kinase (pERK) 1/2, phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase (pMEK) 1/2, phosphorylated cAMP response element binding protein (pCREB) and brain-derived neurotrophic factor (BDNF) in the ischemic cortex were detected by Western blot.Results:At 24 h after reperfusion, the mNSS score and cerebral infarct volume in the model group were significantly higher and larger than those in the sham group (all P<0.001). The mNSS scores and cerebral infarct volumes in the CH-Ⅰ and CBL groups were significantly reduced compared with those in the model group (all P<0.05), but there was no significant difference between the CH-Ⅰ group and the CBL group. Nissl and TUNEL staining showed that the degenerative cell index and apoptotic cell index in the CH-Ⅰ group were significantly lower than those in the model group (all P<0.01), but there were no significant difference between the CH-Ⅰ group and the CBL group. Western blot analysis showed that compared with the sham operation group, the pMEK1/2, pERK1/2 and pCREB expressions in ischemic cortex were significantly enhanced and the BDNF expression was significantly attenuated in the model group ( P<0.05). Compared with the model group, pMEK1/2, pERK1/2, and pCREB expressions in the CH-Ⅰ group were significantly decreased (all P<0.05), and the BDNF expression was significantly increased ( P<0.05). Conclution:CH-Ⅰ can reduce cerebral infarct volume and improve neurological function, and its mechanism may be associated with the inhibition of the MEK-ERK-CREB pathway as well as the enhancement of BDNF expression.
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Objective:To investigate the role of Caveolin (Cav-3)/extracellular signal-regulated kinase (ERK) signaling pathway in reduction of myocardial ischemia-reperfusion (I/R) injury by morphine preconditioning in rats with chronic heart failure.Methods:Clean-grade healthy adult male Sprague-Dawley rats, weighing 200-250 g, were used in this study.Chronic heart failure was induced by ligating the left anterior descending coronary artery for 6 weeks.Thirty-six Langendorff-perfused hearts with chronic heart failure were divided into 4 groups ( n=9 each) by a random number table method: myocardial I/R group (group IR), morphine preconditioning group (group MP), morphine preconditioning plus methyl-β-cyclodextrin group (group MP+ MβCD), and methyl-β-cyclodextrin group (group MβCD). Global myocardial I/R was induced by 30 min ischemia followed by 120 min reperfusion.In group MP, after 15 min of equilibration, hearts were subjected to 3 cycles of 5 min perfusion with K-H solution containing 1 μmol/L morphine for preconditioning followed by 5 min perfusion with K-H solution, 30 min in total, and after the end of treatment, hearts were subjected to 30 min ischemia followed by 120 min reperfusion.In group MP+ MβCD, hearts were perfused with K-H solution containing 200 μmol/L methyl-β-cyclodextrin at 10 min before preconditioning with morphine, and the other treatments were similar to those previously described in group MP.In group MβCD, hearts were perfused with K-H solution containing 200 μmol/L methyl-β-cyclodextrin at 40 min before ischemia, and the other treatments were similar to those previously described in group IR.At the end of 15 min of equilibration (T 0) and 5 and 10 min of reperfusion (T 1, 2), coronary outflow was collected for determination of actate dehydrogenase (LDH) activity by chemical colorimetry.Myocardial infarct size (IS) and area at risk (AAR) were measured, and IS/AAR was calculated at the end of 120 min reperfusion.Myocardial tissues of left ventricle were taken to detect the expression of Cav-3, ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) by Western blot, and p-ERK1/2/ERK1/2 ratio was calculated. Results:Compared with group IR, IS, IS/AAR and LDH activity in coronary outflow were significantly decreased, the expression of Cav-3 was up-regulated, and p-ERK1/2/ERK1/2 ratio was increased in group MP ( P<0.05). Compared with group MP, IS, IS/AAR and LDH activity in coronary outflow were significantly increased, the expression of Cav-3 was down-regulated, and p-ERK1/2/ERK1/2 ratio was decreased in group MP+ MβCD ( P<0.05). Conclusions:The mechanism by which morphine preconditioning reduces I/R injury may be related to activation of Cav-3/ERK signaling pathway in rats with chronic heart failure.
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The MAPK signaling pathway can mediate a variety of cytokines to participate in the processes of inflammation, cancer, immune disorder, and neurodegenerative diseases, and it also plays an important role in the development and progression of hepatic echinococcosis. This article reviews the structure and regulation of the MAPK signaling pathway and elaborates on the role of the MAPK signaling pathway in hepatic echinococcosis. It is pointed out that the MAPK signaling pathway can activate both the cyst and the host in hepatic echinococcosis, participate in the development and progression of the disease, and exert an impact on its treatment. Drug therapy targeting the MAPK signaling pathway is expected to become a new strategy for the treatment of hepatic echinococcosis.
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ABSTRACT Purpose To demonstrate the effect of IL-33 on the macrophage pyroptosis in mice with sepsis through the NF-kB/p38 MAPK signal pathway. Methods In total, 24 C57BL/6 mice were divided into the sham operation group (sham) and the cecal ligation and puncture group (CLP). After CLP, 24 IL-33-/- mice were divided into the IL-33-/- group and the IL-33-/- intervention group. The latter group was intraperitoneally injected with IL-33. Mouse mortality was observed after CLP. Macrophage apoptosis in peritoneal lavage fluid was detected by flow cytometry. Serum inflammatory factor level was detected by ELISA. Apoptotic protein expression and NF-κB/p38 MAKP signaling pathway protein expression were detected by qRT-PCR and Western blot. Results Knocking out IL-33 significantly reduced the mortality of CLP mice, as well as the mRNA expression of IL-33 and the levels of serum inflammatory factors, including IL-33, IL-1β, and IL-18. It also reduced the rate of macrophage apoptosis and the expression of the apoptotic protein caspase-1 p10; increased the expression of IκBα; and reduced the protein expression of NF-κB and p38 MAPK. These effects were reversed after exogenous injection of IL-33. Conclusions IL-33 can increase the level of macrophage pyroptosis in mice with sepsis (by activating the NF-kB/p38MAPK signal pathway) and the mortality of these mice.
Subject(s)
Animals , Mice , NF-kappa B/metabolism , Sepsis , Signal Transduction , Tumor Necrosis Factor-alpha , p38 Mitogen-Activated Protein Kinases/metabolism , Interleukin-33 , Pyroptosis , Macrophages/metabolism , Mice, Inbred C57BLABSTRACT
The Ras family of small Guanosine Triphosphate (GTP)-binding proteins (G proteins) represents one of the main components of intracellular signal transduction required for normal cardiac growth, but is also critically involved in the development of cardiac hypertrophy and heart failure. The present review provides an update on the role of the H-, K- and N-Ras genes and their related pathways in cardiac diseases. We focus on cardiac hypertrophy and heart failure, where Ras has been studied the most. We also review other cardiac diseases, like genetic disorders related to Ras. The scope of the review extends from fundamental concepts to therapeutic applications. Although the three Ras genes have a nearly identical primary structure, there are important functional differences between them: H-Ras mainly regulates cardiomyocyte size, whereas K-Ras regulates cardiomyocyte proliferation. N-Ras is the least studied in cardiac cells and is less associated to cardiac defects. Clinically, oncogenic H-Ras causes Costello syndrome and facio-cutaneous-skeletal syndromes with hypertrophic cardiomyopathy and arrhythmias. On the other hand, oncogenic K-Ras and alterations of other genes of the Ras-Mitogen-Activated Protein Kinase (MAPK) pathway, like Raf, cause Noonan syndrome and cardio-facio-cutaneous syndromes characterized by cardiac hypertrophy and septal defects. We further review the modulation by Ras of key signaling pathways in the cardiomyocyte, including: (i) the classical Ras-Raf-MAPK pathway, which leads to a more physiological form of cardiac hypertrophy; as well as other pathways associated with pathological cardiac hypertrophy, like (ii) The SAPK (stress activated protein kinase) pathways p38 and JNK; and (iii) The alternative pathway Raf-Calcineurin-Nuclear Factor of Activated T cells (NFAT). Genetic alterations of Ras isoforms or of genes in the Ras-MAPK pathway result in Ras-opathies, conditions frequently associated with cardiac hypertrophy or septal defects among other cardiac diseases. Several studies underline the potential role of H- and K-Ras as a hinge between physiological and pathological cardiac hypertrophy, and as potential therapeutic targets in cardiac hypertrophy and failure. Highlights - The Ras (Rat Sarcoma) gene family is a group of small G proteins - Ras is regulated by growth factors and neurohormones affecting cardiomyocyte growth and hypertrophy - Ras directly affects cardiomyocyte physiological and pathological hypertrophy - Genetic alterations of Ras and its pathways result in various cardiac phenotypes? - Ras and its pathway are differentially regulated in acquired heart disease - Ras modulation is a promising therapeutic target in various cardiac conditions.
Subject(s)
Humans , Heart Defects, Congenital , Noonan Syndrome , Signal Transduction , Cardiomegaly , Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling SystemABSTRACT
Glioma is one of the most common primary intracranial tumors, accounting for 80% of malignant brain tumors. The conventional treatment of glioma is surgical resection followed by temozolomide chemotherapy, but the drug resistance will gradually appear that results in a poor prognosis of the patient. Berberine is an alkaloid extracted from Coptis Rhizoma, which has a wide range of pharmacological activities. It exerts its pharmacological effects on glioma such as inhibiting tumor growth through controlling different molecular and cellular pathways. In this article, the application of berberine in the treatment of glioma and the research progress of specific molecular mechanism are reviewed.
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Objective:To investigate the relationship between the mechanism of mental dependence of propofol and adenosine A2A receptor-neurotransmitter-extracellular signal-regulated kinase (ERK) pathway in rats.Methods:Forty-eight healthy male Sprague-Dawley rats, aged about 7 weeks, weighing 200-300 g, were used in this study.The model of propofol dependence was established by intraperitoneal injection of propofol 40 mg/kg for 14 consecutive days.The rats were divided into 6 groups ( n=8 each) using a random number table method: central control group (group c-C), central agonist group (group c-CGS), central antagonist group (group c-DMPX), peripheral control group (group p-C), peripheral agonist group (group p-CGS) and peripheral antagonist group (group p-DMPX). Adenosine A2A agonist CGS-21680 2.5 ng/0.5 μl was intracranially injected immediately after establishing the model in group c-CGS, while the equal volume of normal saline was given instead in c-C group.CGS-21680 0.1 mg/kg was intraperitoneally injected in group p-CGS, while the equal volume of normal saline was given instead in group p-C.Adenosine A2A receptor antagonist DMPX 50 ng/0.5 μl was intracranially injected at 20 min before each propofol injection in group c-DMPX, and DMPX 0.25 mg/kg was intraperitoneally injected in group p-DMPX.The position preference value (CPP value) was determined before establishing the model, immediately after establishing the model, and after administration of agonist or normal saline (after intervention). The animals were sacrificed at 1 day after establishing the model, and blood samples and brain tissues were obtained for determination of the levels of dopamine (DA) and glutamate (Glu) in plasma and hippocampus and content of serotonin (5-HT) in cerebral cortex (by enzyme-linked immunosorbent assay) and expression of phosphorylated ERK1/2 (p-ERK1/2) in cerebral cortex (by Western blot). Results:Compared with the baseline before establishing the model, CPP value was increased immediately after establishing the model in c-C, c-CGS, p-C and p-CGS groups ( P<0.05), and no significant change was found in CPP value immediately after establishing the model in c-DMPX and p-DMPX groups ( P>0.05). Compared with the value immediately after establishing the model, no significant change was found in CPP value after intervention in c-C and p-C groups ( P>0.05), and CPP value was increased after intervention in c-CGS and p-CGS groups ( P<0.05). Compared with group c-C, the contents of hippocampal DA and Glu were significantly increased in group c-CGS, and the contents of hippocampal Glu were decreased, the content of 5-HT in cerebral cortex was increased, and the expression of p-ERK1/2 was down-regulated in group c-DMPX ( P<0.05). Compared with group p-C, no significant change was found in levels of DA and glutamate (Glu) in plasma and hippocampus and 5-HT and p-ERK1/2 in cerebral cortex in group p-CGS ( P>0.05), and the contents of hippocampal DA and Glu were significantly decreased, the content of 5-HT in cerebral cortex was increased, and the expression of p-ERK1/2 was down-regulated in group p-DMPX ( P<0.05). Conclusion:The mechanism underlying the development of propofol mental dependence may be related to activating adenosine A2A receptors, increasing excitatory neurotransmitters in brain, and thus up-regulating ERK activity in rats.
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Abstract Purpose To investigate whether heat shock protein 90 (HSP90) is involved in complement regulation in ischemic postconditioning (IPC). Methods The left coronary artery of rats underwent 30 min of occlusion, followed by 120 min of reperfusion and treatment with IPC via 3 cycles of 30s reperfusion and 30s occlusion. The rats were injected intraperitoneally with 1 mg/kg HSP90 inhibitor geldanamycin (GA) after anesthesia. Eighty rats were randomly divided into four groups: sham, ischemia-reperfusion (I/R), IPC and IPC + GA. Myocardial infarct size, apoptosis index and the expression of HSP90, C3, C5a, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1β and c-Jun N-terminal kinase (JNK) were assessed. Results Compared with the I/R injury, the IPC treatment significantly reduced infarct size, release of troponin T, creatine kinase-MB, and lactate dehydrogenase, and cardiomyocyte apoptosis. These beneficial effects were accompanied by a decrease in TNF-α, IL-1β, C3, C5a and JNK expression levels. However, all these effects were abrogated by administration of the HSP90 inhibitor GA. Conclusion HSP90 exerts a profound effect on IPC cardioprotection, and may be linked to the inhibition of the complement system and JNK, ultimately attenuating I/R-induced myocardial injury and apoptosis.
Subject(s)
Animals , Rats , Complement System Proteins/metabolism , Myocardial Reperfusion Injury/metabolism , Benzoquinones/pharmacology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Lactams, Macrocyclic/pharmacology , JNK Mitogen-Activated Protein Kinases/metabolism , Myocardial Infarction/metabolism , RNA, Messenger/metabolism , Random Allocation , Tumor Necrosis Factor-alpha/metabolism , Rats, Sprague-Dawley , Inflammation Mediators , Creatine Kinase, MB Form/metabolism , Ischemic Postconditioning/methodsABSTRACT
Objective@#To investigate the drug resistance of kaempferol reversed adriamycin (ADM)-resistant K562/ADM cells in chronic myelogenous leukemia (CML) and its related mechanism.@*Methods@#Methyl thiazolyl tetrazolium (MTT) method was used to detect the toxicity of ADM on K562 and K562/ADM cells for 24 h. The half inhibitory concentration (IC50) of ADM and the drug resistance multiple for 24 h were calculated. MTT method was used to detect the toxicity of kaempferol on K562/ADM cells for 24 h. The 5% inhibitory concentration (IC5) and 10% inhibitory concentration (IC10) of kaempferol for 24 h were calculated to determine the concentration of kaempferol in the subsequent experiments. And the cells untreated by the kaempferol were selected as the control group. The cell inhibition after the treatment of ADM for 24 h of the blank control group and kaempferol intervention group was detected by using MTT method. And then the cell inhibition for 24 h and ADM IC50 for 24 h in the above groups were calculated. The ratio of IC50 in the blank control group and kaempferol group was the reversal drug resistance multiple of kaempferol. The fluorescence intensity of ADM in K562/ADM cells treated by kaempferol was detected by using flow cytometry. Western blotting was used to detect the expressions of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), phosphorylated p38 (p-p38), and total p38 (t-p38) protein in K562/ADM cells after the treatment of kaempferol, the specific inhibitor of p38-MAPK signaling pathway SB202190, and the combination of kaempferol and SB202190.@*Results@#After the treatment of ADM for 24 h, the IC50 value of K562 and K562/ADM cells was (0.9±0.6), (28.1 ±3.5) μg/ml, respectively. The drug resistance multiple of K562/ADM cells on the treatment of ADM for 24 h was 31.16 compared with the K562 cells. MTT method showed that kaempferol inhibited the proliferation of K562/ADM cells in a dose-dependent manner. According to the IC5 and IC10, 0.5 μmol/L and 1.0 μmol/L kaempferol were determined to do the subsequent experiments. After the combined interaction of kaempferol and ADM for 24 h, the ADM IC50 of K562/ADM cells in the blank control group, 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was (33.7±5.7), (21.4±0.6), (15.9±1.8) μg/ml, respectively (F = 30.85, P < 0.05), and there was a statistical difference of pairwise comparison (both P < 0.05). The reversal drug resistance multiple of K562/ADM cells for 24 h in 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was 1.58 and 2.12, respectively. Flow cytometry results showed that the mean fluorescence intensity (MFI) of ADM in the blank control group, 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was 138.4±8.9, 154.3±2.2, 165.7±4.8, respectively, and the difference was statistically significant (F = 161.48, P < 0.05). Compared with the blank control group, after treatment of K562/ADM cells with 0.5 μmol/L and 1.0 μmol/L kaempferol for 24 h, the relative expressions of P-gp, MRP1 and p-p38 protein were decreased in K562/ADM cells (all P < 0.05), but there was no statistical difference in the expression of t-p38 protein (P > 0.05); SB202190 could reduce the relative expressions of P-gp, MRP1 and p-p38 protein (all P < 0.05); after the treatment of SB202190 combined with different concentration of kaempferol, the relative expressions of P-gp, MRP1 and p-p38 protein in K562/ADM cells did not decrease (P > 0.05).@*Conclusions@#Kaempferol can decrease the relative expressions of P-gp and MRP1 in K562/ADM cells by inhibiting p38-MAPK pathway, so as to increase the concentrations of ADM and to reverse the drug resistance of K562/ADM cells.
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After binding to the specific insulin-like growth factor 1 receptor (IGF1R) on the surface of target tissue cells, IGF can regulate physiological processes such as apoptosis, proliferation and senescence, which are closely related to growth and development of the body, and occurrence and development of diseases. The binding between IGF1 and IGF1Rα can cause conformational changes of the beta subunit of IGF1R, lead to activation of receptor tyrosine kinase, initiation of the downstream phosphatidylinositol 3-kinase pathway and mitogen-activated protein kinase pathway, and finally participate in the occurrence of acne, psoriasis and other skin diseases. This review summarizes research advances in the role of the IGF1R signaling pathway in the pathogenesis of related skin diseases.
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Mter binding to the specific insulin-like growth factor 1 receptor (IGF1R) on the surface of target tissue cells,IGF can regulate physiological processes such as apoptosis,proliferation and senescence,which are closely related to growth and development of the body,and occurrence and development of diseases.The binding between IGF1 and IGF1Rα can cause conformational changes of the beta subunit of IGF1R,lead to activation of receptor tyrosine kinase,initiation of the downstream phosphatidylinositol 3-kinase pathway and mitogen-activated protein kinase pathway,and fina]ly participate in the occurrence of acne,psoriasis and other skin diseases.This review summarizes research advances in the role of the IGF1R signaling pathway in the pathogenesis of related skin diseases.
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This research is aimed to investigate the effect of ampelopsin on apoptosis and migration of human hepatoma SMMC-7721 cells and explore the molecular mechanism. SMMC-7721 cells were pretreated with different doses of ampelopsin and cells proliferation was detected by CCK8 kit. Cell morphology was observed under an inverted microscope. Nuclear morphology was detected by DAPI staining. Apoptotic rate was detected by Annexin V-FITC/PI flow cytometry. Migration and invasion were detected by Transwell and scratch healing test. Western blotting was used to detect cleavage of poly ADP-ribose polymerase (PARP), expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), E-cadherin, and N-cadherin, and phosphorylation of ERK, P38 and JNK in MAPKs pathway. Our results showed that ampelopsin significantly inhibited proliferation and induced apoptosis of SMMC-7721 cells, with half inhibition dose (IC50) for 24 h was 38.98 μg·mL-1. With 50 μg·mL-1 ampelopsin treatment, typical apoptotic morphological changes occurred, such as cell detachment, shrinkage and nuclear condensation. Apoptotic rate increased from 15% to 55.1%, with PARP cleavage significantly increased. In addition, treatment of ampelopsin reduced scratch healing of cells and transmembrane cells number. The expression levels of MMP-2 and MMP-9 were decreased. Further analysis of EMT-related proteins showed that after ampelopsin treatment, E-cadherin was up-regulated and N-cadherin was down-regulated. During ampelopsin treatment, ERK reached its peak of activation after 1 h, while the maximum activation time of JNK was 12 h. Meanwhile, P38 was activated within 4 h, with the highest point at 2 h. But after 4 h, ampelopsin inhibited phosphorylation of P38. These results indicated that ampelopsin induced apoptosis and reduced migration through activating MAPKs pathway and reversing EMT process in SMMC-7721 cells. This work provides a mechanistic basis for utilizing ampelopsin for anti-hepatocarcinoma treatment.
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Objective To evaluate the effect of curcumin on lidocaine-induced nerve injury and the role of protein kinase B ( Akt) and extracellular signal-regulated kinase 1∕2 ( ERK1∕2) signaling pathway in rats. Methods Eighty SPF male Sprague-Dawley rats, aged 8-10 weeks, weighing 220-250 g, were di-vided into 8 groups ( n=10 each) using a random number table method: control group ( group C) , sham operation group (group S), lidocaine group ( group L), dimethyl sulfoxide ( DMSO) group, low-dose curcumin group ( group CL ) , high-dose curcumin group ( group CH ) , curcumin plus ERK inhibitor PD98059 group (group P) and curcumin plus Akt inhibitor MK-2206 group (group M). The model of nerve injury was established by injecting 2％ lidocaine 10μl via the catheter in anesthetized rats in the other six groups except for C and S groups. Drugs were injected through a microcatheter for 14 consecutive days starting from 3rd day after IT catheterization as follows: DMSO 10μl, curcumin 100μg∕10μl and curcu-min 500 μg∕10 μl were injected once a day in D, CL and CH groups, respectively; curcumin 500 μg∕10μl was injected once a day, and D9805910μg and MK-220612μg ( in 5μl DMSO) were intraperitoneal-ly injected once a week in P and M groups, respectively. The mechanical paw withdrawal threshold ( MWT) and thermal paw withdrawal threshold ( TWL) on the operated side were measured on 1 day before IT catheterization, before administration on 3rd day after IT catheterization, and on 14th day after adminis-tration. The spinal cord was removed on 1st day after the end of administration for determination of the ex-pression of ERK1∕2, phosphorylated ERK1∕2 (p-ERK1∕2), Akt, phosphorylated Akt (p-Akt), c-fos, Bcl-2 and Bax ( by Western blot) and expression of ERK1∕2 and Akt mRNA ( by real-time polymerase chain reaction) . Results Compared with group C, the MWT was significantly decreased, the TWL was short-ened, the expression of ERK1∕2 protein and mRNA, p-ERK1∕2, c-fos, Akt protein and mRNA, p-Akt and Bcl-2 was down-regulated, and the expression of Bax was up-regulated in group L (P<0. 05). Com-pared with group L, the MWT was significantly increased, and the TWL was prolonged in CL, CH, P and M groups, the expression of p-ERK1∕2, c-fos, p-Akt and Bcl-2 was up-regulated, and the expression of Bax was down-regulated in CL and CH groups, the expression of p-Akt and Bcl-2 was up-regulated, and the expression of Bax was down-regulated in group P , and the the expression of p-ERK1∕2 and c-fos was up-regulated in group M ( P<0. 05) . Compared with group CH, the MWT was significantly decreased, and the TWL was shortened in P and M groups, the expression of p-ERK1∕2 and c-fos was down-regulated in group P, and the expression of p-Akt and Bcl-2 was down-regulated, and the expression of Bax was up-reg-ulated in group M ( P<0. 05) . Conclusion Curcumin can reduce lidocaine-induced nerve injury, and the mechanism may be partly related to enhancing the activity of Akt and ERK1∕2 signaling pathway in rats.
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BACKGROUND: Propofol is an intravenous anesthetic which has antioxidant effects due to its similarity in molecular structure to α-tocopherol. It has been reported that α-tocopherol increases osteoclast fusion and bone resorption. Here, we investigated the effects of propofol on signaling pathways of osteoclastogenic gene expression, as well as osteoclastogenesis and bone resorption using bone marrow-derived macrophages (BMMs). METHODS: BMMs were cultured with macrophage colony-stimulating factor (M-CSF) alone or M-CSF plus receptor activator of nuclear factor kappa B ligand (RANKL) in the presence of propofol (0–50 µM) for 4 days. Mature osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP) and the numbers of TRAP-positive multinucleated osteoclasts were counted. To examine the resorption activities of osteoclasts, a bone resorption assay was performed. To identify the mechanism of action of propofol on the formation of multinucleated osteoclasts, we focused on dendritic cell-specific transmembrane protein (DC-STAMP), a protein essential for pre-osteoclastic cell fusion. RESULTS: Propofol increased the formation of TRAP-positive multinucleated osteoclasts. In addition, the bone resorption assay revealed that propofol increased the bone resorption area on dentin discs. The mRNA expression of DC-STAMP was upregulated most strongly in the presence of both RANKL and propofol. However, SB203580, a p38 inhibitor, significantly suppressed the propofol/RANKL-induced increase in mRNA expression of DC-STAMP. CONCLUSION: We have demonstrated that propofol enhances osteoclast differentiation and maturation, and subsequently increases bone resorption. Additionally, we identified the regulatory pathway underlying osteoclast cell-cell fusion, which was enhanced by propofol through p38-mediated DC-STAMP expression.
Subject(s)
Acid Phosphatase , Antioxidants , Bone Resorption , Cell Fusion , Dentin , Gene Expression , Macrophage Colony-Stimulating Factor , Macrophages , Molecular Structure , Osteoclasts , p38 Mitogen-Activated Protein Kinases , Propofol , RANK Ligand , RNA, MessengerABSTRACT
The RASopathies are a group of syndromes that have in common germline mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway and have been a focus of study to understand the role of this pathway in development and disease. These syndromes include Noonan syndrome (NS), NS with multiple lentigines (NSML), neu-rofibromatosis type 1 (NF1), Costello syndrome (CS), cardio-facio-cutaneous (CFC) syndrome, neurofibromatosis type 1-like syndrome (NFLS) and capillary malformation-arteriovenous malformation syndrome (CM-AVM). These disorders affect multiple systems, including the craniofacial complex. Although the crani-ofacial features have been well described and can aid in clinical diagnosis, the dental phenotypes have not been analysed in detail for each of the RASopathies. In this review, we summarize the clinical features of the RASopathies, highlighting the reported craniofacial and dental findings.
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Objective To investigate the effects of WNK3 kinase on the regulation of large-conductance calcium-activated potassium channels (Maxi K channels) on African green monkey kidney fibroblast-like cells (Cos-7 cells) and its mechanisms.Methods (1) Cos-7 cells were transfected with 0,0.6,1.2,1.8 μg WNK3 plasmid+0.5 μg Maxi K plasmid.The total protein expression of Maxi K channel and the phosphorylation of mitogen-activated protein kinase (MAPK) extracellular regulated kinase-1 and-2 (ERK1/2) were detected by Western blotting.(2) Cos-7 cells were divided into the control group (2.5 μg Maxi K plasmid) and the experimental group (2.5 μg WNK3 plasmid+2.5 μg Maxi K plasmid).Cell surface biotinylation was used to investigate the cell surface protein expression of Maxi K channel in Cos-7 cells.Immunoprecipitation and Western blotting were used to detect the ubiquitination of Maxi K channel protein.(3) WNK3 kinase was knocked down by WNK3 siRNA.The lysosomal degradation pathway was blocked by the proton pump inhibitor (Baf-A1).Cos-7 cells were divided into Maxi K+negative control siRNA group,Maxi K+WNK3 siRNA group and Maxi K+WNK3 siRNA+Baf-A1 group.The protein expression of Maxi K channel protein was detected by Western blotting.Results (1) Compared with those in 0 μg WNK3 plasmid groups,in 0.6,1.2,1.8 μg WNK3 plasmid groups the total protein expression of the Maxi K channel increased and the phosphorylation level of MAPK ERK1/2 reduced on a dose-dependent manner (all P ＜ 0.01).(2)Compared with those in the control group,the total protein expression and cell surface membrane protein expression of the Maxi K channel increased in the experimental group (P ＜ 0.01),while the ubiquitination of the Maxi K channel protein reduced (P ＜ 0.01).(3) Compared with the Maxi K +negative control siRNA group,the expression of Maxi K protein reduced in the Maxi K+WNK3 siRNA group (P ＜ 0.01),but did not change in the Maxi K+WNK3 siRNA + Bar-A1 group (P ＞ 0.05).The expression of Maxi K protein in Maxi K+WNK3 siRNA+Baf-A1 group was higher than that in Maxi K+WNK3 siRNA group (P ＜ 0.01).Conclusions WNK3 kinase inhibits the lysosomal degradation pathway of Maxi K channel protein by reducing the ubiquitination of Maxi K channel,and promotes the expression of Maxi K channel protein in cells and on cell membrane.These effects may be achieved by suppressing MAPK ERK1/2 signal transduction pathway.
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Objective To evaluate the effect of dexmedetomidine on phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3 beta (GSK-3β) signaling pathway during apoptosis in cardiomyocytes of rats with severe scald.Methods Twenty-four healthy adult male SpragueDawley rats,weighing 220-280 g,were divided into 3 groups (n=8 each) using a random number table method:control group (group C),severe scald group (group S) and dexmedetomidine group (group D).Thirty percent of the total body surface area was shaved on the back and then exposed to 94 ℃ water (with 37 ℃ warm water in group C) for 12 s to establish the model of third degree scald in pentobarbital sodium-anesthetized rats.Dexmedetomidine 30 μg/kg (2 μg/ml) was intraperitoneally injected immediately after scald in group D.Rats received anti-shock treatment by intraperitoneal injection of isotonic saline according to Parkland formula,and group C received no injection.Rats were anesthetized using the method previously mentioned at 12 h after treatment,and myocardial specimens of the left ventricle were rapidly excised and stored at-80 ℃ for determination of cell apoptosis (by TUNEL) and expression of P13K,phosphorylated Akt (p-Akt) and phosphorylated GSK-3β (p-GSK-3β) (by Western blot).Apoptosis index (AI) was calculated.Results Compared with group C,AI was significantly increased,and the expression of P13K,p-Akt and p-GSK-3β was up-regulated in S and D groups (P＜0.05).Compared with group S,AI was significantly decreased,and the expression of P13K,p-Akt and p-GSK-3β was up-regulated in group D (P＜0.05).Conclusion Dexmedetomidine inhibits apoptosis in cardiomyocytes through activating PI3K/Akt/GSK-3β signaling pathway in the rats with severe scald.
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Objective To evaluate the role of reperfusion injury salvage kinase signaling pathway in reduction of myocardial ischemia-reperfusion (I/R) injury by sevoflurane postconditioning in rats.Methods Seventy SPF healthy adult male Sprague-Dawley rats,weighing 300-350 g,were divided into 7 groups (n =10 each) using a random number table:sham operation group (group S),group I/R,sevoflurane postconditioning group (group SP),phosphatidylinositol 3-kinase inhibitor LY294002 group (group LY),sevoflurane postconditioning plus LY294002 group (group SPLY),mitogen-activated protein kinase kinase 1/2 inhibitor U0126 group (group U) and sevoflurane postconditioning plus U0126 group (group SPU).Myocardial I/R was induced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120 min of reperfusion.In group SP,1.8％ sevoflurane was inhaled for 5 min starting from the beginning of reperfusion.In LY and U groups,LY294002 0.3 mg/kg and U0126 0.5 mg/kg were intravenously injected,respectively,at 10 min before reperfusion.In SPLY and SPU groups,LY294002 0.3 mg/kg and U0126 0.5 mg/kg were intravenously injected,respectively,at 10 min before reperfusion,and 1.8％ sevoflurane was inhaled for 5 min starting from the beginning of reperfusion.At 15 min of reperfusion,myocardial specimens were obtained from the left ventricular area at risk for determination of the phosphorylation of protein kinase B (Akt) and extra-cellular signal-regulated kinase 1/2 (ERK1/2) (by Western blot) and NAD+ content in myocardial tissues (by fluorescence spectrophotometry).At the end of reperfusion,blood samples were collected from the jugular vein for measurement of serum cardiac troponin Ⅰ (cTnI) concentrations (by photoelectric colorimetry),and myocardial specimens were obtained from the left ventricular area at risk for determination of myocardial infarct size (IS).Resuits Compared with group S,the IS and serum cTnI concentrations were significantly increased,the NAD+ content was decreased (P＜0.05),and no significant change was found in the phosphorylation of Akt or ERK1/2 in group I/R (P＞0.05).Compared with group I/R,the IS and serum cTnI concentrations were significantly decreased,and the NAD+ content and phosphorylation of Akt and ERK1/2 were increased in group SP (P＜0.05),and no significant change was found in the parameters mentioned above in LY,SPLY,U and SPU groups (P＞0.05).Compared with group SP,the IS and serum cTnI concentrations were significantly increased,and the NAD+ content was decreased in SPLY and SPU groups,the phosphorylation of Akt was significantly decreased in group SPLY,and the phosphorylation of ERK1/2 was significantly decreased in group SPU (P＜0.05).Conclusion The mechanism by which sevoflurane postconditioning reduces myocardial I/R injury may be related to activation of reperfusion injury salvage kinase signaling pathway in rats.