Downregulation of Aquaporin 4 Expression through Extracellular Signal-regulated Kinases1/2 Activation in Cultured Astrocytes Following Scratch-injury

Objective To investigate the role of extracellular signal-regulated kinase1/2（ERK1/2） pathway in the regulation of aquaporin 4（AQP4） expression in cultured astrocytes after scratch-injury. Methods The scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase（LDH） leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2（p-ERK1/2） expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 μmol/L U0126（ERK1/2 inhibitor） was incubated in the medium at 30 min before the scratch-injury in some groups. Results Increases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury. Conclusion These results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

Time-dependent effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in focal cerebral ischemia rats

BACKGROUND： The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells. OBJECTIVE： To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention. DESIGN： Randomized controlled study. SETTING： Department of Anatomy, Sun Yat-Sen University. MATERIALS： A total of 60 healthy adult male Wistar rats weighing （250±10） g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS： The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups： normal group （n = 6）, sham operation group （n = 18）, model group （n = 18）, and electroacupuncture group （n = 18）. Middle cerebral artery occlusion （MCAO） was performed in the model group and electroacupuncture group. Zea Longa＇s grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between l and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery （CCA） was isolated, and the external carotid artery （ECA） was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture （People＇s Publishing House; 1997; First Edition）. The Chengjiang, Qihai, and Guanyuan acupoints were labeled and connected to a G6805 electroacupuncture apparatus with sparse-dense waves （sparse waves were 30 Hz, dense waves were 100 Hz）, with a frequency of 6-15 V. The duration was 20 minutes. Two days after surgery, the model and sham operation groups were placed with their backs on the operating table, but they received no acupuncture. However, the normal group received acupuncture. The experimental animals under anesthesia were sacrificed on days 7, 14, and 28 post-surgery. Western blot analysis was used to measure expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall. Expression was measured in the normal group at time points corresponding to the sham operation group. MAIN OUTCOME MEASURES： Expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall at different time points after intervention. RESULTS： All 60 rats were included in the final analysis, without any loss. Seven days after MCAO, there was no significant difference in extracellular signal-regulated kinases 1/2 expression in the electroacupuncture group compared to the model group （P 〉 0.05）. However, extracellular signal-regulated kinases 1/2 expression significantly increased in the model group at 14 and 28 days after treatment （P 〈 0.05）. CONCLUSION： Electroacupuncture at the Ren channel can enhance extracellular signal-regulated kinasesl/2 expression in the inferior region of the lateral cerebral ventricle wall of rats with focal cerebral ischemia. However, this effect is not apparent until 14 days after electroacupuncture intervention.

Influence of Ren and Du meridian electro-acupuncture on neural stem cell proliferation and extracellular signal-regulated kinase pathway in a rat model of focal cerebral ischemia injury

BACKGROUND： Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However, there are few reports on the influence of electro-acupuncture at the Du meridian on neural stem cell proliferation. OBJECTIVE： To observe the influence of electro-acupuncture at Ren and Du meridians on neural stem cell proliferation in the subventricular zone and altered signal transduction in cerebral ischemia rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Human Anatomy, Medical College of Sun Yat-sen University from May 2006 to February 2008. MATERIALS： Mouse anti-rat bromodeoxyuridine （BrdU） monoclonal antibody was provided by Sigma, USA; mouse anti-rat nestin monoclonal antibody and extracellular signal-regulated protein kinase （ERK） specific inhibitor PD98059 were provided by Calbiochem, Germany; acupuncture needle was provided by Suzhou Acupuncture Supplies, China. METHODS： A total of 126 rats were randomly assigned to four groups： model （n = 36）, Du meridian （n = 36）, Ren/Du meridian （n = 36）, and Ren/Du meridian ＋ PD98059 （n = 18）. Rats in the Ren/Du meridian ＋ PD98059 group were observed on days 7 （n = 6） and 14 （n = 12） after cerebral ischemia injury. Rats in the model, Du meridian, and Ren/Du meridian groups were observed on days 7, 14, and 28 after cerebral ischemia injury, with 12 rats per group at each time point. Thread occlusion was used to establish middle cerebral artery occlusion models. Electro-acupuncture was performed at Renzhong （DU 26） and Baihui （DU 20） acupoints in the Du meridian group, as well as Chengjiang （RN 24）, Guanyuan （RN 4）, Renzhong, and Baihuiacupoints in the Ren/Du meridian and Ren/Du meridian ＋ PD98059 groups 2 days after model establishment. In addition, electro-acupuncture stimulation with disperse-dense waves was performed, with 30 Hz disperse wave, 100 Hz dense wave, and 5 V intensity for 20 minutes. Rats in the Ren/Du meridian ＋ PD98059 group were treated with 0.2 pg PD98059 injection into the subventricular zone, 2 pL per rat. Rats in the model group were not treated with electro-acupuncture. MAIN OUTCOME MEASURES： BrdU/nestin immunofluorescent staining was used to detect proliferating neural stem cells in the subventricular zone of cerebral ischemia rats; Western blot was used to determine phosphorylated ERK1 and 2 （pERK1/2） expression in the subventricular zone. RESULTS： On days 14 and 28 after cerebral ischemia, there were significantly more BrdU-positive and BrdU/nestin-positive cells in the Ren/Du meridian group compared with the Du meridian group （P 〈 0.05）. PD98059 decreased the number of BrdU-positive and BrdU/nestin-positive cells induced by electro-acupuncture at the/：ten and Du meridians （P 〈 0.05）. On days 7, 14, and 28 after treatment, pERK1/2 expression was significantly greater in the Du meridian and Ren/Du meridian groups compared with the model group （P 〈 0.05）. The promoting effect of electro-acupuncture at Ren and Du meridians on ERK1/2 phosphorylation was superior to electro-acupuncture at the Du meridian alone on day 14 after model induction （P 〈 0.05）. However, PD98059 completely abolished the promoting effect of electro-acupuncture at Ren/Du meridians on pERK1/2 expression （P 〈 0.05）. CONCLUSION： Electro-acupuncture at Ren and Du meridians increased proliferation of subventricular zone neural stem cells, which was related to activation of the ERK pathway in a rat model of cerebral ischemia injury.

An experimental study of extracellular signal-regulated kinase and its interventional treatments in hepatic fibrosis

BACKGROUND: The pathogenesis of hepatic fibrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a specific inhibitor of ERK, on the cell cycle, cell proliferation, secretion of type I collagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-beta 1 (TGF-beta 1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde. METHODS: Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by flow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-beta 1 was examined using the reverse transcriptase-polymerase chain reaction. Type I collagen in the culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: 20, 50 and 100 mu mol/L PD98059 significantly inhibited the proliferation and provoked a G0/G1-phase arrest of acetaldehyde-induced HSCs in a dose-dependent manner. The secretion of type I collagen and the expression of cyclin D1, CDK4 and TGF-beta 1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100 mu mol/L PD98059, respectively. CONCLUSIONS: The ERK pathway regulates the cell proliferation, secretion of type I collagen and the expression of TGF-beta 1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.

Regulation of extracellular signal-regulated kinase 1/2 influences hippocampal neuronal survival in a rat model of diabetic cerebral ischemia

Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor （U0126） was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and KuT0 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These findings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and ac- celerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/ reperfusion.

Pterygium epithelium abnormal differentiation related to activation of extracellular signal-regulated kinase signaling pathway in vitro

AIMTo investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase (ERK) signaling pathway in vitro.METHODSThe expression levels of phosphorylated ERK (P-ERK), keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays, the differences in clone morphology and in K10, K19, P-ERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency, differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis.RESULTSThe expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium. Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19. The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with 3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression.CONCLUSIONWe suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation.

Extracellular signal-regulated kinase, substance P and neurokinin-1 are involved in the analgesic mechanism of herb-partitioned moxibustion

Herb-partitioned moxibustion can effectively mitigate visceral pain, a major symptom in inflammatory bowel disease, but the analgesic lnechanism is still unclear. Moreover, extracellular signal-regulated kinase, substance P, and neurokinin-1 are involved in formation of central hyperalgesia. Thus, we postulated that the analgesic effect of herb-partitioned moxibustion may be associated with these factors. Accordingly, in this study, we established an inflammatory bowel disease visceral pain model in rat by enema with a mixed solution of 5% trinitrobenzenesulfonic acid and 50% ethanol. Bilateral Tianshu （ST25） and Qihai （CV6） points were selected for herb-partitioned moxi- bustion. Our results showed that herb-partitioned moxibustion improved visceral pain and down-regulated extracellular signal-regulated kinase, substance P, and neurokinin-1 protein and mRNA expression in dorsal root ganglia. These results indicate that down-regulation of extracellular signal-regulated kinase, substance E and neurokinin-1 protein and mRNA may be a central mechanism for the analgesic effect of herb-partitioned moxibustion.

Effect of extracellular signal-regulated kinase and nitric oxide on compressive neuralgia formation and maintenance

BACKGROUND： Previous studies have shown that extracellular signal-regulated kinase 1/2 （ERK1/2） and nitric oxide activation play a pivotal role in central sensitization and long-term neuronal plasticity induced by noxious stimulation. However, their effects on compressive neuralgia formation and maintenance remain poorly understood.OBJECTIVE： To investigate effects of the specific inhibitor of ERK1/2 signal pathway U0126 on neuronal nitric oxide synthase （nNOS） expression in the dorsal horn of the spinal cord in a compressive neuralgia rat model.DESIGN, TIME AND SETTING： A randomized, controlled experiment was performed at the Institute of Otolaryngology, Head and Neck Surgery, First Hospital of Jilin University from July 2008 to March 2009.MATERIALS： U0126 （Bio-Mol, USA） was used in this study.METHODS： A total of 84 rats were randomly assigned to two groups. In the first part of the experiment, 24 rats were used for behavioral testing, and they were randomly assigned to three sub-groups （n =8）： U0126, dimethyl sulfoxide （DMSO） and model control. In the second part of the experiment, 60 rats were used for immunofluorescence and Western blot analysis, and they were randomly assigned to six sub-groups （n = 10）： sham surgery, model control, U0126 post-injection at 0.5, 2, 12 and 24 hours. Neuropathic pain was produced by chronic compression to the dorsal root ganglion in rats from each sub-group. Rats in the U0126 group were administered a 5-ug U0126 intrathecal injection, and rats in the DMSO group were administered a 10-μL 5% DMSO intrathecal injection.MAIN OUTCOME MEASURES： Changes in mechanical and thermal hyperalgesia were observed using von Frey filaments and thermalqia stimular. Thermal and mechanical hyperalgesia were stimulated at different time points following intrathecal injection of U0126. nNOS activation and expression in the spinal cord dorsal horn were determined by immunofluorescence and Western blot analysis.RESULTS： Intrathecal injection of U0126 significantly attenuated chronic compression of dorsal root ganglion-induced mechanical and thermal hyperalgesia. Immunofluorescence staining results demonstrated that, compared to the sham surgery group, the number of nNOS-positive neurons was significantly increased in the injured spinal dorsal horn in the model control group （P〈0.01）. However, compared to the model control group, there were significantly decreasing numbers of nNOS-positive neurons in the U0126 post-injection groups at 0.5-hour, 2-hour, and 12-hour （P〈0.05）. Western blot analysis revealed similar results. CONCLUSION： Decreased activity in the ERK signal pathway resulted in down regulated nNOS expression in the dorsal horn of the spinal cord. These results suggested that ERK is involved in nitric oxide reaction to neuropathic pain.

Dexamethasone suppresses DU145 cell proliferation and cell cycle through inhibition of the extracellular signal-regulated kinase 1 /2 pathway and cyclin D1 expression

Aim： To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth. Methods： The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase （ERK）1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor （GR） antagonist RU486. Reverse transcription- polymerase chain reaction verified the expression of GR mRNA in DU145 cells. Results： Dexamethasone significantly inhibited DU 145 cell proliferation at the G0/G1 phase. Westem blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade. Conclusion： Dexamethasone suppresses DU145 cell proliferation and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy.

Neurotransmitter regulation of extracellular signal-regulated kinase expression following subarachnoid hemorrhage

BACKGROUND： Very few studies have addressed neuronal injury in cerebral vasospasm and subarachnoid hemorrhage （SAH）, and the role of neurotransmitters in the regulation of extracellular signal-regulated kinase 1/2 （ERK1/2） expression following SAH. OBJECTIVE： To analyze neurotransmitter regulation of ERK1/2 expression through the use of signal transduction, and to investigate cerebral injury mechanisms following SAH. DESIGN, TIME AND SETTING： A completely randomized grouping and controlled animal experiment was performed at the Experimental Center of Medical College of Xi＇an Jiaotong University from March to December 2008. MATERIALS： Extraceliular signal-regulated ERK1/2 polyclonal antibody and streptavidin-peroxidase method kits were purchased from Beijing Biosynthesis Biotechnology, China; DAB kit was purchased from Zhongshan Golden Bridge Biotechnology, China; TUNEL kit was purchased from Promega, USA. METHODS： A total of 114 male, Sprague Dawley rats, aged 55-63 days old, were randomly assigned to five groups： SAH （n = 30）, saline control （n = 30）, puncture control （n = 30）, normal control （n = 6）, and neurotransmitter-treated （n = 18）. The SAH model was established by twice injecting blood through the cisterna magna. The neurotransmitters-treated group was subdivided into three groups according to drugs injected into the lateral cerebral ventricle： acetylcholine chloride, norepinephrine, and saline, with six animals in each group. MAIN OUTCOME MEASURES： Rats from the SAH, saline control, and puncture control groups were respectively sacrificed at 6, 12, and 24 hours, as well as 3 and 5 days, with six rats at each time point. The normal control group rats were sacrificed at 6 hours, and the neurotransmitter group rats were sacrificed 3 days following neurotransmitter injection. Morphological cellular changes were observed by hematoxylin and eosin staining. Immunohistochemical SP method was used to detect expression of ERK1/2 in the cortex, and cortical apoptosis was detected using the TUNEL method. RESULTS： Neural tissue edema, apoptosis, and necrosis occurred in the cortex of the SAH group. ERK1/2-positive cells were first observed at 6 hours, peaked at 12 hours following SAH in the cortex, and gradually decreased thereafter. Cellular apoptosis was observed in the cortex at 6 hours and peaked at 24 hours following SAH. ERK1/2 distribution in the brain overlapped apoptotic cells to a great degree. The number of ERK1/2-positive and apoptotic cells was significantly greater in the SAH group compared with the three control groups （P 〈 0.05）. Compared to the number of ERK1/2-positive cells in the saline-treated group, acetylcholine chloride treatment resulted in decreased ERK1/2 expression and apoptosis （P 〈 0.05）. Norepinephrine resulted in increased ERK1/2 expression, but there was no significance in apoptosis compared to the saline-treated group （P 〉 0.05）. CONCLUSION： Apoptosis was observed early in the rat cortex following SAH. In addition, ERK1/2 was expressed earlier than apoptosis. Acetylcholine chloride treatment resulted in decreased numbers of apoptotic cells following SAH, possibly by down-regulating ERK1/2 expression.

OVER-EXPRESSION OF EXTRACELLULAR SIGNAL-REGULATED KINASE IN VASCULAR SMOOTH MUSCLE CELL OF HYPERTENSIVE RATS

Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery.The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively.The control group were sham operated age-matched Wistar rats.Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00±33.00 mm Hg at the end of experiment, significantly higher than that in the control rats (P<0.01).Blood pressure in SHR4w (108.00±11.25 mm Hg) was similar to that in the controls.However, it rose to 122.25±21.75 mm Hg in SHR8w, and even up to 201.75±18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P<0.01).The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P<0.05).Hyaline degeneration of the afferent arterioles was found in WHR.In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w.Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2.The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09%±1.75%, 14.57%±4.58%, 29.44%±7.35%, and 13.63%±3.85%, respectively) than that of the controls(P<0.01).The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09%±1.40%, 24.17%±6.92%, 32.44%±4.05%, and 18.61%±3.35%, respectively) than that of the controls (P<0.01), too.The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P<0.01).Conclusion Extracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR.Phospho-ERK1/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.

Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys

Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.

Hepatitis B virus X protein up-regulates tumor necrosis factor-α expression in cultured mesangial cells via ERKs and NF-κB pathways

Objective:To investigate the effects of hepatitis B virus(HBV)X protein(HBx)on the expression of tumor necrosis factor-α(TNF-α)in glomerular mesangial cells(GMCs)and the underlying intracellular signal pathways.Methods:The plasmid pCI-neo-X that carries the X gene of hepatitis B virus was transfected into cultured GMCs.HBx expression in the transfected GMCs was assessed by Western-blot.TNF-αprotein and mRNA were assessed by ELISA and semi-quantitative RT-PCR,respectively.Three kinase inhibitors-U0126,an inhibitor of extracellular signal-regulated kinases(ERKs);lactacvstin,an inhibitor of nuclear factor-κB(NF-κB);and SB203580,a selective inhibitor of p38 MAP kinase(p38 MAPK)were used to determine which intracellular signal pathways may underlie the action of HBx on TNF-αexpression in transfected GMCs.Results:A significant increase in HBx expression in pCI-neo-X transfected GMCs was detected at 36 h and 48 h,which was not affected by any of those kinase inhibitors mentioned above.A similar increase in the expression of both TNF-αprotein and mRNA was also observed at 36 h and 48 h,which was significantly decreased in the presence of U0126 or lactacytin,but not SB203580.Conclusions:HBx upregulates TNF-αexpression in cultured GMCs,possibly through ERKs and NF-κB pathway,but not p38 MAPK pathway.

Gan Shen Fu Fang ameliorates liver fibrosis in vitro and in vivo by inhibiting the inflammatory response and extracellular signalregulated kinase phosphorylation

BACKGROUND Liver fibrosis is a common health problem worldwide and there is still a lack of effective medicines.The Chinese herbal medicine,Gan Shen Fu Fang(GSFF)is composed of salvianolic acid B and diammonium glycyrrhizinate.In this study,we observed the effects of GSFF on liver fibrosis in vivo and in vitro in an attempt to provide some hope for the treatment.AIM To observe the effects of GSFF on liver fibrosis in vivo and in vitro and investigate the mechanism from the perspective of the inflammatory response and extracellular signal-regulated kinase(ERK)phosphorylation.METHODS Common bile duct-ligated rats were used for in vivo experiments.Hepatic stellate cells-T6(HSC-T6)cells were used for in vitro experiments.Hematoxylin and eosin staining and Masson staining,biochemical assays,hydroxyproline(Hyp)assays,enzyme-linked immunoasorbent assay and western blotting were performed to evaluate the degree of liver fibrosis,liver function,the inflammatory response and ERK phosphorylation.The CCK8 assay,immunofluorescence and western blotting were applied to test the effect of GSFF on HSC-T6 cell activation and determine whether GSFF had an effect on ERK phosphorylation in HSC-T6 cells.RESULTS GSFF improved liver function and inhibited liver fibrosis in common bile ductligated rats after 3 wk of treatment,as demonstrated by histological changes,hydroxyproline assays and collagen I concentrations.GSFF alleviated inflammatory cell infiltration and reduced the synthesis of pro-inflammatory cytokines[tumor necrosis factor-α(TNF-α)and interlukin-1β]and NF-κB.In addition,GSFF decreased ERK phosphorylation.In vitro,GSFF inhibited the viability of HSC-T6 cells with and without transforming growth factorβ1(TGF-β1)stimulation and decreased the synthesis of collagen I.GSFF had the greatest effect at a concentration of 0.5μmol/L.GSFF inhibited the expression ofα-smooth muscle actin(α-SMA),a marker of HSC activation,in HSC-T6 cells.Consistent with the in vivo results,GSFF also inhibited the phosphorylation of ERK and downregulated the expression of NF-κB.CONCLUSION GSFF inhibited liver fibrosis progression in vivo and HSC-T6 cell activation in vitro.These effects may be related to an alleviated inflammatory response and downregulated ERK phosphorylation.

利用FRET技术检测不同力学条件下T细胞中ERK活性的脉冲现象

目的探索Jurkat T细胞中胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)活性动力学以及基质刚度对ERK活性的影响。方法利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术实时观测Jurkat细胞中ERK活性的变化,或细胞处于I型胶原基质胶中检测其影响。结果部分Jurkat细胞中存在ERK活性脉冲现象,频率约为3次/h,FRET振幅变化约为20%。在抗体激活T细胞抗原受体(T-cell receptor,TCR)的条件下,ERK脉冲依然存在,频率和振幅无显著变化。当细胞处于I型胶原水凝胶中,随着胶基质刚度增加,脉冲频率有所下调。结论Jurkat T细胞中存在自发的ERK活性脉冲现象,初步实验显示其频率受基质刚度影响。而该信号波动的生理意义和分子机制仍有待探索。

Overexpression of mitogen-activated protein kinase phosphatase-1 in endothelial cells reduces blood-brain barrier injury in a mouse model of ischemic stroke

Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.

Role of phosphatase PTEN in the activation of extracellular signal-regulated kinases induced by estradiol in endometrial carcinoma cells

Objectives To study extracellular signal-regulated kinase (ERK) activation in the endometrial carcinoma cell line Ishikawa with stimulation by 17-β-estradiol, and to elucidate the role of phosphatase and tensin homologue (PTEN) and estrogen receptor (ER) subtype on the activation of ERKs.Methods Western blot was used to examine the expression of PTEN and PTEN (G129E) in Ishikawa cells after stable transfection as well as ERK activation in Ishikawa-EGFP, Ishikawa- PTEN and Ishikawa- PTEN (G129E) stimulated with various doses of 17-β-estradiol for different lengths of time. Western blot was also used for examining the expression of ERα and ERβ in NIH3T3 fibroblasts after transient transfection of pCXN2hERα and pCXN2hERβ. Then, ERK activation was examined after stimulation with 17-β-estradiol. Results 17-β-estradiol activated ERK cascades (mainly ERK2) in Ishikawa cells. The activation of ERK increased gradually as concentration of 17-β-estradiol also increased. The maximal activation of ERK2 took place 5 min after stimulation with 17-β-estradiol. The activation of ERK2 was inhibited markedly by PTEN, but not by PTEN (G129E). 17-β-estradiol activated ERK cascades in NIH3T3 fibroblasts after transient transfection of pCXN2hERα.Conclusions 17-β-estradiol activate ERK cascades in Ishikawa cells by integrating with ERα. Lipid phosphatase PTEN has an inhibitory role on the activation of ERK stimulated by 17-β-estradiol in Ishikawa cells.

Imbalanced expression of mitogen-activated protein kinase phosphatase-1 and phosphorylated extracellular signal-regulated kinases in lung squamous cell carcinoma

Objective:Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers.This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-1) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK 1/2),as the key regulatory mechanism of the MAPKs,in lung squamous cell carcinoma (SCC).Methods:We assessed the expressions of MKP-1 and p-ERK 1/2 in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis.Results:Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was negatively correlated with tumor differentiation (P<0.01).However,the expression of p-ERK 1/2 or ERK 1/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was positively correlated with tumor differentiation (P<0.01).Conclusions:Our data indicates the relevance of MKP-1 and p-ERK 1/2 in SCC as a potential positive and negative prognostic factor.The imbalanced expression of MKP-1 and p-ERK 1/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.

震荡流体剪切力通过ERK5信号通路促进成骨细胞增殖

目的探讨震荡流体剪切力(oscillatory shear stress,OSS)通过ERK5信号通路在诱导成骨细胞增殖中发挥的作用。方法对成骨MC3T3-E1细胞进行不同的处理,分为正常组、OSS组、XMD8-92组和OSS+XMD8-92组。采用MTT实验分别测定4组细胞的增殖活性并绘制生长曲线;蛋白免疫印迹法分别检测P-ERK5、ERK5和Cyclin D1等蛋白水平变化。结果 OSS可显著增加成骨MC3T3-E1细胞增殖活性,但此效应可被ERK5高选择性抑制剂XMD8-92阻断。OSS可显著上调Cyclin D1的表达,而XMD8-92可显著下调OSS诱导的Cyclin D1的表达。结论 OSS通过激活ERK5信号通路促进成骨细胞增殖,Cyclin D1是ERK5信号通路下游的重要靶点基因。

铝对大鼠海马ERK蛋白及mRNA表达影响

目的研究慢性铝中毒对大鼠海马细胞外信号调节蛋白激酶(ERK)蛋白及mRNA表达水平的变化,探讨铝中毒对学习记忆机制的影响。方法选择断乳后Wistar大鼠,用不同浓度AlCl3的水饲养3个月。测量大鼠海马长时程增强(LTP),用改良的Takai法测定丝裂原活化蛋白激酶(MAPK)活性的变化,蛋白印迹(Western blotting)方法检测ERK1/2的蛋白含量,RT-PCR方法检测ERK2的mRNA表达。结果(1)各染铝组MAPK活性降低且与对照组比较差异有统计学意义(P<0.05);(2)ERK1/2蛋白表达的比较:①ERK2在大鼠海马中的蛋白含量高于ERK1;②染铝组ERK1/2非磷酸水平与对照组比较均有所下降(P<0.05),但染铝组之间差异无统计学意义(P>0.05);③染铝组ERK1/2磷酸化水平与对照组的比较均有下降(P<0.05),染铝组之间的差异有统计学意义(P<0.05)。(3)染铝组ERK2的mRNA表达与对照组比较明显降低,但染铝组之间差异无统计学意义(P>0.05)。结论慢性铝中毒不仅影响大鼠海马中ERK2的mRNA表达水平,同时也影响ERK1/2的蛋白表达水平的降低,造成有丝分裂原活化蛋白激酶(MAPK)活性的下降,损害LTP的形成,从而影响学习记忆功能下降。