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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.
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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
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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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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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.
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PURPOSE: We investigated the effects of laminin on the fraction of cells with self-renewing capacity in the estrogen-dependent, tamoxifen-sensitive LM05-E breast cancer cell line. We also determined whether laminin affected the response to tamoxifen. MATERIALS AND METHODS: The LM05-E breast cancer cell line was used as a model for all experiments. Aldehyde dehydrogenase (ALDH) activity, clonogenic and mammosphere assays were performed to measure the effects of laminin on modulation of the stem cell subpopulation. Pluripotent gene expression was analyzed by reverse transcriptase–polymerase chain reaction. The involvement of the mitogen-activated protein kinase (MAPK)/ERK pathway was determined using specific inhibitors. The effects of laminin on the response to tamoxifenwere determined and the involvement of α6 integrin was investigated. RESULTS: We found that pretreatment with laminin leads to a decrease in cells with the ability to form mammospheres that was accompanied by a decrease in ALDH activity. Moreover, exposure of mammospheres to laminin reduced the capacity to form secondary mammospheres and decreased the expression of Sox-2, Nanog, and Oct-4. We previously reported that 4-OH-tamoxifen leads to an increase in the expression of these genes in LM05-E cells. Treatment with signaling pathway inhibitors revealed that the MAPK/ERK pathway mediates the effects of laminin. Finally, laminin induced tamoxifen resistance in LM05-E cells through α6 integrin. CONCLUSION: Our results suggest that the final number of cells with self-renewing capacity in estrogen-dependent breast tumors may result from the combined effects of endocrine treatment and microenvironmental cues.
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Aldehyde Dehydrogenase , Breast Neoplasms , Breast , Cell Line , Cues , Estrogen Receptor alpha , Gene Expression , Laminin , MAP Kinase Signaling System , Protein Kinases , Stem Cells , TamoxifenABSTRACT
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)
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Animals , Rats , Bone Marrow Transplantation , Extracellular Signal-Regulated MAP Kinases/metabolism , Liver Failure, Acute/therapy , Disease Models, Animal , Enzyme Activation/physiology , Hepatectomy , Survival RateABSTRACT
Abnormal activation of the mitogen-activated protein kinase (MAPK) signaling pathway is closely associated with the development, progression, and metastasis of liver cancer. This article introduces the expression of MAPK proteins in liver cancer and its role in the proliferation, differentiation, and metastasis of liver cancer, and elaborates on the value of the MAPK signaling pathway in the treatment and prognostic evaluation of liver cancer. It is pointed out that the MAPK signaling pathway plays an important role in the development/progression and treatment of liver cancer and is a potential molecular target for the treatment and prognostic evaluation of liver cancer.
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Objective To investigate whether the new mechanically-activated (MA) cation channel Piezo 1 protein can cause the apoptosis of human chondrocytes under compressive loading,using a Flexercell unit by activating MAPK/ERK5 signal pathway.Methods Primary human chondrocytes were isolated,cultured,and then subjected to the static compressive loading for 2 h,12 h,24.h and 48 h,respectively.The GsMTx4,which is the special inhibitor of the Piezo1 protein and the BIX02188,which is the inhibitor of the ERK5 served as the inhibitor group.The immunofluorescence was used to locate the expression of the Piezo 1 protein.The expressions of Piezo1 and ERK5 were assessed by reverse transcription-polymerase chain reaction (RT-PCR),as well as the apoptosis gene B cell lymphoma/leukemia-2 (Bcl-2),Bcl-associated X protein (Bax) and Bcl-associated death promoter (BAD).In addition,Piezo1 inhibitor,GsMTx4,was used to block the MA cation channel Piezo1,served as the inhibitor group.AVPI was used to detect the apoptosis of the OA chondrocytes.Results The location of the Piezo1 was expressed in nucleus and cytoplasm of chondrocytes.The expression of the Piezo1,ERK5,BAD,and Bax mRNA in the OA chondrocytes is weak.The 12 h stretch force group was significant increased,and the 24 h stretch force group was the highest expression.However,the expression of the 48 h group was decreasing.The expression of the Bcl-2 in the 12 h group was decreasing,and the 12 h stretch force group was the lowest expression while the 24 h stretch force group was increasing.In the GsMTx4 group,the expression of the Piezo1,ERK5,BAD,Bax was decreasing while the Bcl-2 was increasing.In the BIX02188 group,the expression of the ERKS,BAD,Bax was increasing while the Bcl-2 was decreasing,while the expression of the Piezo1 was not change.The result of AV-PI shown that the 2 h stretch force group increased early stage of apoptosis.The 12 h stretch force group increased late stage of apoptosis,and the 24 h stretch force group's apoptotic rate was the highest.However,the apoptotic rate of the 48 h group was lower than the 24 h stretch force group.The GsMTx4 could inhibit the late stage of apoptosis.Conclusion Piezo 1 plays an important role in the apoptosis of human chondrocyte through the MAPK/ERK5 signal pathway.
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Objective:To investigate the expression of MEK/ERK signaling pathways in renal cell car-cinoma with bone metastasis,and to analyze the differences of expressions of VEGFR-2,MEK,ERK on the primary and metastasis tissue and its mechanism.Methods:The tissue samples were obtained from 7 renal cell carcinoma patients kindly provided by Department of Urology,Peking University People’s Hos-pital from January 1,2009 to January 1,2010.The expression of MEK/ERK signaling pathways was de-tected in the 7 renal cell carcinoma patients`primary and matched metastatic tissues with ICH,The anti-body concentrations were 1 ∶200,1 ∶25,and 1 ∶250,respectively.The mutation of the twentieth exon of the PDGFRA gene,the second exon of the K-ras gene,the fifteenth exon of the Brafgene and the se-cond exon of the MEK1 gene were detected with PCR.Results:The expression intensities of VEGFR-2, MEK,and ERK were measured by H-score [intensity (1,2,3,or 4)multiplied by the distribution (%)].VEGFR-2,MEK,and ERK expressions were divided into 3 groups according to the positive dis-tribution of the tumor cells:1,0 -5%;2,6% -50%;and 3,>50%,To assess intratumor heteroge-neity,three distinct microscopic fields (×200)from each specimen were used to evaluate the expres-sions,Subsequently,the scores were averaged to obtain a single concatenated score for each tissue. VEGFR-2,MEK,and ERK expressions were assessed by 2 independent pathologists who were blinded to the clinicopathological data.The data were expressed as the mean value of the triplicate experiments.The expressions of MEK,and ERK were higher in the metastatic tissues than in the matched RCC tissues (6.10 ±4.10 vs.1.33 ±0.51,P =0.015;9.10 ±2.24 vs.4.43 ±2.84,P =0.021 )while the ex-pression of VEGFR-2 was not different between the primary and metastatic tissues (P =0.901).No mu-tation was detected on the twentieth exon of the PDGFRA gene,the second exon of the K-ras gene,the fifteenth exon of the Brafgene and the second exon of the MEK1 gene.Conclusion:MEK/ERK signa-ling pathways may play an important role in the metastasis and the resistance of sunitinib in RCC patients with bone metastasis.
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Objective To investigate the role of transforming growth factor-β activated kinase-1 (TAK1) signaling pathway in the activation of bone marrow derived macrophages (BMDM) induced by high glucose.Methods Purity of mouse BMDM was detected by flow cytometry.The mice macrophages cultured in vitro were stimulated by high glucose and treated with TAK1 specific inhibitor 5Z-7-oxozeaenol.Cells were divided into normal control group (RPMI 1640),osmolality control group (25 mmol/L mannitol),high glucose group (33 mmol/L D-glucose) and inhibitor group (33 mmol/L D-glucose+300 nmol/L 5Z-7-oxozeaenol).Immunocytochemistry and flow cytometry were used to detect macrophage subtype.The expression of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis Factor-α (TNF-α) mRNA were determined by real time PCR.Expressions of p-TAK1,TAK1 binding protein (TAB1),p-JNK,p-p38 MAPK and NF-κB p65 proteins were analyzed by Western blotting.Results The purity of BMDM was about 99.36％.Compared with normal control group,high glucose group had increased percentage of M1 macrophages,increased expression of MCP-1 and TNF-α mRNA (all P ＜ 0.05).Moreover,p-TAK1,TAB1,p-JNK,p-p38 MAPK and NF-κB p65 proteins expression also increased significantly in high glucose group (all P ＜ 0.05).After treatment with inhibitor 5Z-7-oxozeaenol,the effects induced by high glucose were inhibited (P ＜ 0.05).Conclusions High glucose can induce M1 macrophage activation and expression of inflammatory cytokine of BMDM,which can be inhibited 5Z-7-oxozeaenol through inhibiting TAK1/MAPK and TAK1/NF-κB pathway.
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Objective To establish adriamycin-induced focal segmental glomerular sclerosis(FSGS) mice model,and observe the expressions of and relation between oxidative stress and p38 MAPK signal pathway in renal injury.Methods Eight-week-old male Balb/c mice were randomly divided into FSGS group (n=20) and control group (n=20).In FSGS group mice were intravenously injected with a single dose of adriamycin (0.01 rag/g),and mice in control group were received saline with the same dose.At day 3,7,14,22 and 32,urine protein-to-urine creatinine ratio (P/C) was detected.At day 22 and 32,serum creatinine,blood urea nitrogen,nitric oxide (NO) and reactive oxygen species (ROS) in blood and urine,and ROS in kidney tissues were detected;changes of pathological morphology in renal tissue were analyzed by HE stain;the expressions of NF-κB,CD36,IL-13,BAX and Bcl-2 mRNA were detected by real time quantitative PCR;the expressions of NF-κB,p-p38 and p-ERK1/2 protein were detected by Western blotting.Results Compared with that in control group,P/C was gradually increasing in FSGS group,and peaked at day 22 (P ＜ 0.05).At day 22 and 32,mice had higher creatinine,serum creatinine,urea nitrogen,ROS and NO in FSGS group than those in control group (all P ＜ 0.05).There were mild hyperplasia of mesangial cells and mesangial matrix,segment with moderate exacerbations,podocytes with significant proliferation,and the capillary loops of the stenosed in the glomerular in FSGS group at day 32.Compared with those in control group,the mRNA expression of NF-κB,BAX,IL-13 and CD36,and the protein expressions of NF-κB and p-p38 MAPK were gradually increased in FSGS group,all showed statistical differences at day 32 (all P＜ 0.05);the expression of p-ERK1/2 was increased at day 22 (P ＜ 0.05) but was reduced at day 32 (P ＜ 0.05).Conclusions Adriamycin has induced FSGS in mice successfully,which may through oxidative stress activating p38,up-regulating NF-κB,increasing the inflammatory cytokines and inducing apoptosis pathways.
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Objective To investigate the effects and mechanisms of prostaglandin E2 receptor subtype 3 (EP3) on transforming growth factor β1 (TGF-β1)-induced mouse mesangial cells damage. Methods Primary mouse mesangial cells were separated and cultured. Three siRNAs were synthesized and transfected into mesangial cells for silencing EP3 by LipofectamineTM 2000 and the best one was chosen. MCs were grouped into: (1)control group; (2)TGF-β1 (10 μg/L) group; (3)NC-siRNA plus TGF-β1 (10 μg/L) group; (4) EP3-siRNA group; (5)EP3-siRNA plus TGF-β1 (10 μg/L). Then the proliferation of MCs was evaluated by CCK-8 assay. The expression of PGE2 and cAMP in cell supernatant were detected by ELISA. The mRNA and protein expression of fibronectin (FN), connective tissue growth factor (CTGF), cyclooxygenase-2 (COX2), membrane-bound prostaglandin E2 synthase 1 (mPGES1) were detected by real - time quantitative PCR and Western blotting. The phosphorylation of p38 MAPK and ERK1/2 was decected by Western blotting. Results Compared with control group, the cell proliferation induced by TGF-β1 was increased (P<0.05), the expression of PGE2 and cAMP were improved, mRNA and protein expression of FN, CTGF, COX2 and mPGES1 were up-regulated (all P<0.05). Compared with TGF-β1 group, the cell proliferation in EP3-siRNA plus TGF-β1 group was reduced, the expression of FN, CTGF, COX2 and mPGES1 mRNA and protein were downregulated (all P<0.05), the phosphorylation of ERK1/2, p38 MAPK were also declined (P<0.05). Conclusion EP3-siRNA may reduce TGF-β1-induced cell damage through upregulating the expression of cAMP, repressing the activity of ERK1/2 and p38 MAPK, inhibiting the expression of COX2 mPGES1 and PGE2 by feedback, then decreased the expression of FN and CTGF.