Electroacupuncture reduces apoptotic index and inhibits p38 mitogen-activated protein kinase signaling pathway in the hippocampus of rats with cerebral ischemia/reperfusion injury

Electroacupuncture attenuates cerebral hypoxia and neuronal apoptosis induced by cerebral ischemia/reperfusion injury.To further identify the involved mechanisms,we assumed that electroacupuncture used to treat cerebral ischemia/reperfusion injury was associated with the p38 mitogen-activated protein kinase（MAPK） signaling pathway.We established rat models of cerebral ischemia/reperfusion injury using the modified Zea-Longa＇s method.At 30 minutes before model establishment,p38 MAPK blocker SB20358 was injected into the left lateral ventricles.At 1.5 hours after model establishment,electroacupuncture was administered at acupoints of Chize（LU5）,Hegu（LI4）,Zusanli（ST36）,and Sanyinjiao（SP6） for 20 minutes in the affected side.Results showed that the combination of EA and SB20358 injection significantly decreased neurologic impairment scores,but no significant differences were determined among different interventional groups.Hematoxylin-eosin staining also showed reduced brain tissue injuries.Compared with the SB20358 group,the cells were regularly arranged,the structures were complete,and the number of viable neurons was higher in the SB20358 ＋ electroacupuncture group.Terminal deoxynucleotidyl transferase（Td T）-mediated d UTP nick-end labeling assay showed a decreased apoptotic index in each group,with a significant decrease in the SB20358 ＋ electroacupuncture group.Immunohistochemistry revealed reduced phosphorylated p38 expression at 3 days in the electroacupuncture group and SB20358 ＋ electroacupuncture group compared with the ischemia/reperfusion group.There was no significant difference in phosphorylated p38 expression between the ischemia/reperfusion group and SB20358 group.These findings confirmed that the electroacupuncture effects on mitigating cerebral ischemia/reperfusion injury are possibly associated with the p38 MAPK signaling pathway.A time period of 3 days could promote the repair of ischemic cerebral nerves.

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

Hippocampal activation of c-Jun N-terminal kinase,protein kinase B,and p38 mitogen-activated protein kinase in a chronic stress rat model of depression

Recent studies have shown that varied stress stimuli activate c-Jun N-terminal kinase （JNK）, protein kinase B （Akt）, and p38 mitogen-activated protein kinase （p38） signal transduction pathway, and also regulate various apoptotic cascades. JNK and p38 promote apoptosis, but Akt protects against apoptosis, in hippocampal neurons. However, changes in the transduction pathway in different regions of brain tissues in a chronic stress rat model of depression remain poorly understood. Results from this study showed that JNK phosphorylation levels were significantly greater in the stress group hippocampus compared with the control group （P 〈 0.05）. No significant difference in JNK phosphorylation levels was detected in the rat cerebral cortex between stress and control groups, and no significant difference in Akt and p38 phosphorylation levels was detected in the rat hippocampus and cerebral cortex between stress and control groups （P 〉 0.05）. These results suggested that the JNK signal pathway is activated by JNK phosphorylation and participates in pathophysiological changes in rat models of depression.

Inhibiting p38 mitogen-activated protein kinase attenuates cerebral ischemic injury in Swedish mutant amyloid precursor protein transgenic mice

Cerebral ischemia was induced using photothrombosis 1 hour after intraperitoneal injection of the p38 mitogen-activated protein kinase （MAPK） inhibitor $B239063 into Swedish mutant amyloid precursor protein （APP/SWE） transgenic and non-transgenic mice. The number of surviving neurons in the penumbra was quantified using Nissl staining, and the activity of p38 MAPKs was measured by western blotting. The number of surviving neurons in the penumbra was significantly reduced in APP/SWE transgenic mice compared with non-transgenic controls 7 days after cerebral ischemia, but the activity of p38 MAPKs was significantly elevated compared with the non-ischemic hemisphere in the APP/SWE transgenic mice. SB239063 prevented these changes. The APP/SWE mutation exacerbated ischemic brain injury, and this could be alleviated by inhibiting p38 MAPK activity.

“Three Methods and Three Points” regulates p38 mitogen-activated protein kinase in the dorsal horn of the spinal cord in a rat model of sciatic nerve injury

Tuina is a traditional Chinese treatment for sensory disturbances caused by peripheral nerve injury and related diseases. Our previous studies showed that tuina regulates relevant regions and indices of the spinal dorsal horn using the Dian, Bo, and Rou method in Yinmen（BL37）, Yanglingquan（GB34）, and Weizhong（BL40）. Treatment prevents muscle atrophy, protects spinal cord neurons, and promotes sciatic nerve repair. The mechanisms of action of tuina for treating peripheral nerve injury remain poorly understood. This study established rat models of sciatic nerve injury using the crushing method. Rats received Chinese tuina in accordance with the principle of ＂Three Methods and Three Points,＂ once daily for 20 days. Tuina intervention reduced paw withdrawal latency and improved wet weight of the gastrocnemius muscle, as well as promoting morphological recovery of sciatic nerve fibers, Schwann cells, and axons. The protein expression levels of phospho-p38 mitogen-activated protein kinase, tumor necrosis factor-α, and interleukin-1β also decreased. These findings indicate that ＂Three Methods and Three Points＂ promoted morphological recovery and improved behavior of rats with peripheral nerve injury.

p38 mitogen-activated protein kinase regulates type-Ⅰ vs type-Ⅱ phenotyping of human vascular endothelial cells

AIM: To identify kinases involved in phenotype regulation of vascular endothelial cells(VECs): Proproliferative G-protein signaling 5(RGS5)^(high)(typeⅠ) vs anti-proliferative RGS5^(low)(typeⅡ) VECs.METHODS: Proteomic kinase assays were performed to identify the crucial kinase involved in the phenotype regulation of human VECs using typeⅠ VECs, which promotes the proliferation of human vascular smooth muscle cells(VSMCs), and typeⅡ VECs, which suppress the proliferation of human VSMCs. The assays were performed using multiple pairs of typeⅠ and typeⅡ VECs to obtain the least number of candidates. The involvement of the candidate kinases was verified by evaluating the effects of their specific inhibitors on the phenotype regulation of human VECs as well as the expression levels of regulator of RGS5, which is the causative gene for the "typeⅡ to typeⅠ" phenotype conversion of human VECs. RESULTS: p38α mitogen-activated protein kinase(p38α MAPK) was the only kinase that showed distinctive activities between typeⅠ and typeⅡ VECs: p38α MAPK activities were low and high in type-Ⅰand typeⅡ VECs, respectively. We found that an enforced expression of RGS5 indeed lowered p38α MAPK activitiesin typeⅡ VECs. Furthermore, treatments with a p38α MAPK inhibitor nullified the anti-proliferative potential in typeⅡ VECs. Interestingly, MAPK inhibitor treatments enhanced the induction of RGS5 gene. Thus, there is a vicious cycle between "RGS5 induction" and "p38α MAPK inhibition", which can explain the unidirectional process in the stress-induced "typeⅡ to typeⅠ" conversions of human VECs. To understand the upstream signaling of RGS5, which is known as an inhibitory molecule against the G protein-coupled receptor(GPCR)-mediated signaling, we examined the effects of RGS5 overexpression on the signaling events from sphingosine-1-phosphate(S1P) to N-cadherin, because S1 P receptors belong to the GPCR family gene and N-cadherin, one of their downstream effectors, is reportedly involved in the regulation of VEC-VSMC interactions. We found that RGS5 specifically bound with S1P1. Moreover, N-cadherin localization at intercellular junctions in typeⅡ VECs was abolished by "RGS5 overexpression" and "p38α MAPK inhibition".CONCLUSION: p38α MAPK plays crucial roles in "type-Ⅰ vs type-Ⅱ" phenotype regulations of human VECs at the downstream of RGS5.

Neuroprotective mechanisms of rutin for spinal cord injury through anti-oxidation and anti-inflammation and inhibition of p38 mitogen activated protein kinase pathway

Rutin has anti-inflammatory, antioxidant, anti-viral, anti-tumor and immune regulatory effects. However, the neuroprotective effects of rutin in spinal cord injury are unknown. The p38 mitogen activated protein kinase （p38 MAPK） pathway is the most important member of the MAPK family that controls inflammation. We assumed that the mechanism of rutin in the repair of spinal cord injury is associated with the inhibition of p38 MAPK pathway. Allen’s method was used to establish a rat model of spinal cord injury. The rat model was intraperitoneally injected with rutin （30 mg/kg） for 3 days. After treatment with rutin, Basso, Beattie and Bresnahan locomotor function scores increased. Water content, tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 levels, p38 MAPK protein expression and caspase-3 and -9 activities in T8–9 spinal cord decreased. Oxidative stress related markers superoxide dismutase and glutathione peroxidase levels increased in peripheral blood. Rutin exerts neuroprotective effect through anti-oxidation, anti-inflammation, anti-apoptosis and inhibition of p38 MAPK pathway.

Xuebijing alters tumor necrosis factor-alpha, interleukin-1beta and p38 mitogen activated protein kinase content in a rat model of cardiac arrest following cardiopulmonary resuscitation

We established a rat model of cardiac arrest by clamping the endotracheal tube of adult rats at expiration. Twenty-four hours after cardiopulmonary resuscitation, nerve cell injury and expression of tumor necrosis factor-α, interleukin-1β, and p38 mitogen activated protein kinase content were increased. Rats injected with Xuebijing, a Chinese herb compound preparation, exhibited normal cellular structure and morphology, dense neuronal cytoplasm, and decreased tumor necrosis factor-α, interleukin-1β, and p38 mitogen activated protein kinase expression at 24 hours following cardiopulmonary resuscitation. These data suggest that Xuebijing can attenuate neuronal injury induced by hypoxia and reperfusion during cardiopulmonary resuscitation.

N-methyl-D-aspartate receptor effects on p38 mitogen activated protein kinase and its role in a rat model of diabetic neuropathic pain

BACKGROUND： Activated N-methyl-D-aspartate （NMDA） receptor is involved in the formation of chronic neuropathic pain, and its antagonist, ketamine, exhibits effective amelioration of diabetic neuropathic pain （DNP）. However, the mechanisms of NMDA receptor participation in the formation and maintenance of DNP remain poorly understood. OBJECTIVE： To evaluate the role NMDA receptor plays in DNP and effects on p38 mitogen activated protein kinase （p38 MAPK） in a rat model of DNP. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Human Embryonic Stem Cell Research Institute of Yunyang Medical College Affiliated Taihe Hospital between July 2005 and September 2007. MATERIALS： Streptozotocin was provided by Sigma, USA; p38 MAPK inhibitor （SB203580） was provided by Shanghai KangChen Biotech, China; NMDA receptor antagonist （MK-801） was purchased from Shanghai Yope Biotech, China. METHODS： A total of 128 healthy, Wistar rats of clean grade, aged 3 months and weighing 180- 220 g, were randomly assigned to 4 groups： control, DNP model, p38 MAPK, and NMDA receptor. Each group contained 32 rats. DNP was established in all groups except for the control group by intraperitoneal injection of streptozocin （65 mg/kg）. Subsequently, 1 mg/kg SB203580 and 1 mg/kg MK-801 were injected once each week via intraperitoneal injection in the p38 MAPK and NMDA receptor groups, respectively. MAIN OUTCOME MEASURES： At the end of 2, 4, 6, and 8 weeks following streptozotocin injection, mechanical withdrawal threshold was measured in 8 animals from each group following von Frey filament stimulation. The rats were anesthetized and nerve conduction velocity of the left sciatic nerve was measured. Subsequently, the right sciatic nerve, the lumbar segment of the spinal cord, and dorsal root ganglia were removed from the L3-6 segment for microscopic examination, p38 MAPK expression was determined using immunohistochemistry and Western blot analysis. Expression of NMDA receptor 1 mRNA in dorsal root ganglion and spinal cord neurons was detected using RT-PCR. RESULTS： Mechanical withdrawal threshold and nerve conduction velocity were significantly reduced, and p38 MAPK and NMDA receptor 1 mRNA expression in the spinal cord and dorsal root ganglia were significantly increased, in the model, p38 MAPK, and NMDA receptor groups compared with the control group at all time points （P 〈 0.05）. At 4-8 weeks following successful DNP model establishment, SB203580 and MK-801 increased mechanical withdrawal threshold, accelerated nerve conduction velocity, and attenuated p38 MAPK expression, compared with the model group. The NMDA receptor group exhibited downregulated mRNA expression of NMDA receptor 1 compared with the model and p38 MAPK groups （P 〈 0.05）. CONCLUSION： NMDA receptor was highly expressed in the brains of DNP rats and was involved in DNP development via activation of the p38 MAPK signal pathway.

Cross-talk between Smad4 and P38 Proteins in Non-small Cell Lung Cancer

Objective： Impaired signal transduction is associated with tumorigenesis and progression of various kinds of human cancers. Transforming growth factor （TGF）-beta/Smad and ras-mitogen activated protein kinase （MAPK） are two major signal transduction pathways for adjusting cell proliferation and differentiation. Little is known about TGF-beta/Smad4 in non-small cell lung cancer （NSCLC）. Hereby, we investigated the expression of Smad4 in NSCLC, its correlation with MAPK proteins （including p38, ERK1 and JNK1 proteins） and their clinical significance in NSCLC. Methods： The expressions of Smad4, p38, ERK1 and JNK1 were detected at protein level with Western blotting and immunohistochemistry, at transcription level with RT-PCR. Statistical analysis was performed for the comparisons of expressions of Smad4, p38, ERK1 and JNK1, and their correlation with various clinicopathological parameters and the prognosis of NSCLC. Results： The levels of protein and mRNA expression of Smad4 in lung cancer tissues were significantly lower than in normal tissues （P〈0.05）. All these four proteins were associated with TNM staging. There was a strongly negative correlation between p38 and Smad4. Expressions of Smad4, p38 and JNK1, as well as tumor differentiation and staging were significantly correlated with the prognosis of NSCLC by univariate analysis. By multivariate analysis, only Smad4, p38, tumor differentiation and staging were correlated with the prognosis. Taken together, the negative expression of p38 and positive expression of Smad4 were associated with a better prognosis of NSCLC. Conclusion： Smad4 could be of vital importance for the initiation and development of NSCLC. The expression of Smad4 might be inhibited by p38, supporting a cross-talk between main proteins of TGF-beta/Smad and ras-MAPK signal transduction pathways. Smad4 and p38 could be possible prognostic factors for NSCLC.

Effect of oxymatrine on the p38 mitogen-activated protein kinases signalling pathway in rats with CCl4 induced hepatic fibrosis

Background Recent studies have suggested that p38 mitogen-activated protein kinases （MAPK） signalling pathway plays an important role in hepatic fibrosis. This study explored the antifibrotic effect of oxymatrine on tetrachloromethane induced liver fibrosis in rats and its modulation on the p38 MAPK signalling pathway. Methods One hundred and twenty healthy male Sprague-Dawley rats were randomly assigned to six groups： normal （n=20）, induced fibrosis （n=20）, colchicine （n=20） and three treatment groups of oxymatrine （n=20x3）. We obesrved changes in deposition of collagen, hyaluronic acid （HA）, laminin （LN）, collagen type IV （CIV）, procollagen III （PCIll） and hydroxyproline （Hyp）, a-smooth muscle actin （α-SMA） and phosphor-p38 （pp38）. Results The relative indicators of changes in histopathology, HA, LN, CIV, PCIII, Hyp, a-SMA and pp38 were raised significantly in the induced fibrosis group （P〈0.01 vs normal group）. The semiquantitative hepatic fibrosis staging scores of middle dose group and high dose group were decreased （P 〈0.05 and P 〈0.01 respectively vs the induced fibrosis group）, as was the average area of collagen in rats＇ liver, the concentrations of serum HA, LN, CIV, PCIII and liver tissue homogenate Hyp. The gene expression of α-SMA mRNA was considerably decreased in the treated animals, as was the protein espression of pp38 protein. Conclusions Oxymatrine is effective in reducing the production and deposition of collagen in the liver tissue of experimental rats in ways which relate to modulating the fibrogenic signal transduction via p38 MAPK signalling pathway.

Role of p38 mitogen-activated protein kinases in cardioprotection of morphine preconditioning

Background p38 mitogen-activated protein kinases （MAPK） in ischemic preconditioning （IPC） may be essential to cardioprotection. We assessed whether protective effect of morphine-induced preconditioning （MPC） on myocardial ischemia and reperfusion injury in rat hearts involved p38 MAPK activation.Methods Male Spargue-Dawley rats （weighing 300--350 g） were randomly assigned to 1 of the following 8 groups： control （CON, saline vehicle, n=9）, SB 203580 （SB, a p38 MAPK inhibitor, n=6）, MPC （n=6）, IPC （n=9）, SB＋MPC, SB＋IPC, MPC＋SB, and IPC＋SB （n=6）. Infarct sizes （IS）, a percentage of the area at risk （AAR）, were determined by triphenyltetrazolium （TTC） staining. Hssue samples were processed from the entire AAR of left ventricle for the determination of p38 MAPK protein expression （5 hearts/group）. The bands representing the proteins were visualized using an enhanced chemiluminescence detection system. Results The IS/AAR was significantly reduced by I PC （12.9±1.6）% or MPC （25.3±2.9）% compared to the control （52.7±5.5）%. SB 203580 administered prior to preconditioning abolished the effect of IPC （SB＋IPC： （43.8±2.6）%, P〉0.05 vs CON, P〈0.01 vs IPC）, but not MPC （SB＋MPC： （30.7±0.9）%, P〈0.01 vs CON, P〉0.05 vs MPC）. Treatment with SB 203580 prior to sustained ischemia diminished the protective effect of both MPC （MPC＋SB： （42.4±2.9）%, P〉0.05 vs CON） and IPC （IPC＋SB： （52.0±2.5）%, P〉0.05 vs CON） on IS/AAR. In the IPC group, phospho-p38 MAPK protein increased significantly within 5 minutes into ischemia and remained elevated at 30 minutes into reperfusion, while phospho-p38 MAPK protein in the MPC group only increased significantly at 30 minutes into reperfusion.Conclusion The activation of p38 MAPK just acts as a mediator of MPC,whereas it acts as both a trigger and a mediator in IPC.

Activation of p38 mitogen-activated protein kinase contribute to BMP4-induced alkaline phosphatase expression in MC3T3-E1 preosteoblast

Bone morphogenetic proteins （BMPs） induce ectopic bone formation and promote osteoblast differentiation. It has been documented that Smad transcriptional factors function as primary mediators of BMPs activity. Receptor-regulated Smad （Smad1, 5, 8） could be phosphorylated by activated BMPR-I and form complex with Smad4. The Smad complex translocates to the nucleus and regulate target gene transcription. Recently, several reports suggested that Mitogen-Activated Protein Kinase （MAPK） signaling pathways could be initiated downstream of the BMP receptor complex. Alkaline phosphatase （ALP） is an early marker of osteoblast differentiation Both ALP activity and its mRNA expression level could be increased by BMP4 treatment. Previously, we demonstrated that mutation of ERK1/2 phosphorylation sites in Smad5 partially rescued Smad transcriptional activity. However, fibroblast growth factor2-suppressed ALP activity could not be rescued similarly by introduction of Smad5 mutant in MC3T3-E1. These results prompted us to further evaluate the effect of BMP4-stimulated Smad transcriptional activity on ALP expression in this study.

Involvement of the p38 mitogen-activated protein kinase signal transduction pathway in burns-induced lung injury

Acute lung injury (ALI) is a leading complication in extensively burnedpatients, especially those with inhalation injury. It can cause hypoxia resulting in injury ofremote organs and dysfunction. p38 mitogen-activated protein kinase (p38 MARK) is a stress activatedprotein kinase in the MARK family. Most of the previous studies have demonstrated that p38 MARKsignal transduction pathway mediated All in rats with acute severe pancreatitis, sepsis etc.However, there is little information regarding the role of p38 MARK signal transduction pathway inALI after severe burn trauma.

Coronary microembolization induced myocardial contractile dysfunction and tumor necrosis factor-α mRNA expression partly inhibited by SB203580 through a p38 mitogen-activated protein kinase pathway

Background The microemboli produced during spontaneous plaque rupture and ulceration and during coronary intervention will reduce coronary reserve and cause cardiac dysfunction. It is though that inflammation caused by the microinfarction induced by the microembolization may play an essential role. It is known that the activation of p38 mitogen-activated protein kinases （MAPK） in both infected and non-infected inflammation in myocardium may cause a contractile dysfunction. But the relation between the activation of p38 MAPK and microembolization is still unknown. Methods Sprague-Dawley rats were randomly divided into three groups： Sham group, coronary microembolization （CME） group and SB203580 group （n=-10 per group）. CME rats were produced by injection of 42 pm microspheres into the left ventricle with occlusion of the ascending aorta. SB203580, a p38 MAPK inhibitor, was injected into the femoral vein after the injection of microspheres to make the SB203580 group. Left ventricular ejection fraction （LVEF） was determined by echocardiography. The protein concentration of P38 MAPK in the myocardium was assessed by Western blotting. The relative expression of mRNA for tumor necrosis factor （TNF）-a was assessed by the technique of semi-quantitative polymerase chain reaction amplification. Results LVEF was depressed at three hours up to 12 hours in the CME group. Increased p38 MAPK activity and TNF-a mRNA expression were observed in the CME group. The administration of SB203580 partly inhibited p38 MAPK activity, but did not fully depress the TNF-α expression, and partly preserved cardiac contractile function. Conclusions p38 MAPK is significantly activated by CME and the inhibition of p38 MAPK can partly depress the TNF-a expression and preserve cardiac contractile function.

A new cell death program regulated by toll-like receptor 9 through p38 mitogen-activated protein kinase signaling pathway in a neonatal rat model with sepsis associated encephalopathy

Background:Sepsis,a serious condition with high mortality,usually causes sepsis associated encephalopathy(SAE)that involves neuronal cell death.However,the cell death programs involved and their underlying mechanisms are not clear.This study aimed to explore the regulatory mechanisms of different cell death programs in SAE.Methods:A neonatal rat model of SAE was established by cecal ligation and perforation.Survival rate and vital signs(mean arterial pressure and heart rate)were monitored,nerve reflexes were evaluated,and cortical pathological changes were observed by hematoxylin and eosin staining.The expression of pyroptosis,apoptosis,and necroptosis(PANoptosis)-related proteins,mitogen-activated protein kinase(MAPK),and its upstream regulator toll-like receptor 9(TLR9)were detected.The expression of TLR9 in neurons was observed by immunofluorescence staining.The ultrastructure of neurons was observed by transmission electron microscope.Results:First,PANoptosis was found in cortical nerve cells of the SAE rats.Meanwhile,the subunits of MAPKs,p38 MAPK,Jun N-terminal kinase,and extracellular signal-regulated kinase(ERK)were activated.After pharmacologically inhibiting each of the subunits,only p38 MAPK was found to be associated with PANoptosis.Furthermore,blocking the p38 MAPK signaling pathway activated necroptosis but inhibited apoptosis and pyroptosis.When necroptosis was pharmacologically inhibited,apoptosis and pyroptosis were reactivated.Finally,we found that the expression of TLR9,a regulator of MAPKs,was significantly increased in this model.After down-regulation of TLR9,p38 MAPK,and ERK signaling pathways were inhibited,which led to the inhibition of PANoptosis.Further analysis found that down-regulation of TLR9 improved the survival rate and reduced the pathological changes in SAE rats.Conclusions:Our study showed that the programs comprising PANoptosis are activated simultaneously in SAE rats.TLR9 activated PANoptosis through the p38 MAPK signaling pathway.TLR9 may work as a potential target for SAE treatment.

Ammonia induces upregulation of aquaporin-4 in neocortical astrocytes of rats through the p38 mitogen-activated protein kinase pathway

Background Astrocyte swelling is an important consequence of hepatic encephalopathy, and aquaporin-4 has been reported to play a vital role in this swelling. Ammonia causes astrocyte swelling and is also known to modulate aquaporin-4 expression in the astrocyte foot processes. The purpose of this study was to explore the mechanism of ammonia-induced aquaporin-4 expression, which has been suggested to involve the p38 mitogen-activated protein kinase pathway. Methods We exposed cultured astrocytes to ammonium chloride, an in vitro model of hepatic encephalopathy. The purity of cultured astrocytes was evaluated by fluorescent glial fibrillary acidic protein labeling; cell morphology was assessed by light microscopy; the expression of aquaporin-4, phospho-p38, and p38 were detected by Western blotting analysis. Statistical analysis was performed by one-way factorial analysis of variance, and the relationship between variables was calculated by linear regression using SPSS version 13.0 program for Windows （SPSS, Chicago, IL, USA）. Results The purity of cultured astrocytes was （96.6±1.4）%. Astrocytes swelled significantly when exposed to 5 mmol/L ammonium chloride for 24 hours as compared to non-exposed astrocytes. Co-treatment with 10 μmol/L SB203580 （an inhibitor of p38） attenuated the degree of ammonium chloride induced astrocyte swelling. Western blotting analysis revealed that the expression levels of phospho-p38 and aquaporin-4 in ammonium chloride treated cells were significantly increased relative to the control group （P 〈0.001）; SB203580 co-treatment inhibited the increased expression of phospho-p38 and aquaporin-4 relative to the ammonium chloride treated group （P=0.002 and P=0.015 respectively）. The phosphorylation of p38 and upregulation of aquaporin-4 were highly correlated （r=0.909）. There were no significant differences in total p38 expression among the groups （P=0.341）. Conclusions Ammonium chloride induced upregulation of aquaporin-4 in astrocytes is regulated by the p38 mitogen-activated protein kinase pathway. Inhibiting p38 activation prevented ammonium chloride induced aquaporin-4 protein upregulation.

Electroacupuncture Attenuates Spinal Nerve Ligation-Induced Microglial Activation Mediated by p38 Mitogen-Activated Protein Kinase

Objective： To investigate whether analgesic effect of electroacupuncture（EA） is affected by p38 mitogen-activated protein kinase（p38 MAPK） on microglia. Methods： There were two experiments. The experiment 1： 40 male Sprague-Dawley（SD） rats were randomly divided into the normal, surgery, EA and sham EA groups, and the L5 spinal nerve ligation（SNL） on the right side was used to establish neuropathic pain model. EA was applied to bilateral Zusanli（ST36） and Kunlun（BL60） at 24, 48 and 72 h after SNL for 30 min, once per day. The paw withdrawal thresholds（PWTs） were measured before surgery（as base） and at 24, 25, 49 and 73 h after surgery. Phospho-p38 MAPK（p-p38 MAPK）, oxycocin-42（OX-42, marker of microglia）, and glial fibrillary acidic protein（GFAP, marker of astrocyte） in bilateral spinal cord dorsal horn（SCDH） were detected by immunofluorescence, respectively. The experiment 2： 40 male SD rats were cannulated for SNL-induced neuropathic pain, and then were randomly divided into the dimethyl sulfoxide（DMSO）, EA plus DMSO, 4-（4-fluorophenyl）-2-（4-methylsulfonylpheny）-5-（4-pyridyl）-1H-imidazole（SB203580） and EA plus SB203580 groups. SB203580（30 nmol/L） was administered 5 min prior to EA treatment. The PWTs and OX-42 in bilateral SCDH were measured as mentioned above. Results： SNL-induced neuropathic pain reduced PWTs and increased the expression of p-p38 MAPK and OX-42 in bilateral lumbar SCDH of rats（P〈0.01）. Spinal p-p38 MAPK was only co-localized with OX-42 in our study. EA treatment significantly alleviated SNL-mediated mechanical hyperalgesia, and suppressed the expression of p-p38 MAPK and OX-42 in lumbar SCDH（P〈0.05 or P〈0.01）. Intrathecal injection of low dose SB203580 had no influence on PWTs（P〉0.05）, but significantly inhibited the expression of OX-42 positive cells in bilateral SCDH（P〈0.01 or P〈0.05）. EA plus SB203580 synergistically increased PWTs, and reduced the expression of bilateral spinal OX-42（P〈0.01 or P〈0.05）. Conclusions： The central mechanism of EA-induced anti-hyperalgesia may be partially associated with the reduced expression of p-p38 MAPK, and subsequently reducing the activation of OX-42 in neuropathic pain. Therefore, EA may be a new complementary and alternative therapy for neuropathic pain.

Efficacy of needling acupoints of Guanyuan(CV4), Sanyinjiao(SP6), Zusanli(ST36), Pishu(BL20), Shenshu(BL23), Zigong(EX-CA1) on expression of p38 mitogen-activated protein kinase in ovarian tissue in rats with premature ovarian failure induced by cyclophosp

OBJECTIVE:To investigate the efficacy of needling acupoins of Guanyuan(CV4),Sanyinjiao(SP6),Zusanli(ST36),Pishu(BL20),Shenshu(BL23),Zigong(EX-CA1)on the expression of p38 mitogen-activated protein kinase(p38MAPK)in ovarian tissue in rats with premature ovarian failure induced by cyclophosphamide,and to study the underlying mechanism.METHODS:Forty specific pathogen free female Sprague-Dawley rats were randomly divided into the blank group,the model group,the acupuncture group,the Western Medicine group and the Western Medicine combined with acupuncture group.Except the blank group,the rest of the rats were given with cyclophosphamide for 14 d to establish premature ovarian failure model.No intervention was conducted in the blank group and model group;the acupuncture group was given with acupuncture daily;the Western Medicine group was given with estradiol valerate(0.09 mg/kg)by intragastrical gavage daily;the combination group was given with acupuncture combined with estradiol valerate(0.09 mg/kg)daily.Each group was intervened in continuously for 14 d.After the last treatment,the levels of estradiol(E2),follicle stimulating hormone(FSH)and luteinizing hormone(LH)were detected by enzyme-linked immunosorbent assay,then the ovarian tissue was dissected.Western blot was used to detect the expression of p38 MAPK protein.RESULTS:Compared with the blank group,E2 in the serum of rats in the model group was significantly decreased(P<0.05),FSH and LH were significantly increased(P<0.05),and the expression of p38 MAPK protein in the ovarian tissue of the rats was significantly increased(P<0.05).Compared with the model group,E2 in the serum of the acupuncture group,Western Medicine group and the combination group were significantly increased(P<0.05),FSH and LH levels were significantly decreased(P<0.05),and the expression of p38MAPK protein in the ovarian tissue of the rats was significantly decreased(P<0.05).However,there was no statistically significant difference between the Western Medicine group and the acupuncture group(P>0.05).CONCLUSIONS:Acupuncture has the same effect as estrogen in interfering POF caused by cyclophosphamide,and its mechanism may be related to inhibiting the expression of p38MAPK protein in ovarian tissue and affecting the activation of p38MAPK signaling pathway.

Cyclovirobuxinum D Alleviates Cardiac Hypertrophy in Hyperthyroid Rats by Preventing Apoptosis of Cardiac Cells and Inhibiting the p38 Mitogen-Activated Protein Kinase Signaling Pathway

Objective： To investigate the underlying mechanisms of cyclovirobuxinum D（Cvb-D） on alleviating cardiac hypertrophy in rats. Methods： Sprague-Dawley rats were randomly divided into 5 groups： control group; levothyroxine-induced cardiac hypertrophy group（model）; levothyroxine-induced cardiac hypertrophy ＋ Cvb-D group（Cvb-D）; levothyroxine-induced cardiac hypertrophy ＋ captopril group（captopril）; levothyroxine-induced cardiac hypertrophy ＋ SB203580 group（SB203580）, n=10 for each group. Rats were daily administered the respective drugs continuously for14 days by gastric gavage. A rat model of cardiac hypertrophy was established by intraperitoneal injection of levothyroxine to investigate whether Cvb-D protects against cardiac hypertrophy by inhibiting the p38 mitogen-activated protein kinase（MAPK） signaling pathway and preventing apoptosis of cardiac cells. Results： Treatment with Cvb-D significantly deceased left ventricle hypertrophy, improved the histopathology, hemodynamic conditions, and cardiac function in rats with cardiac hypertrophy. Compared with the normal control group, in rats with cardiac hypertrophy, expression of bax in the heart and phospho-p38 MAPK protein levels were significantly up-regulated（P〈0.01 or 0.05）, whereas the bcl-2 protein level was downregulated（P〈0.01）. In contrast, Cvb-D treatment reversed the changes in bax and phospho-p38 MAPK protein levels but increased the bcl-2 protein level（P〈0.01 or 0.05）, and these effects were similar to those of captopril and SB203580（a specific p38 MAPK inhibitor） treatment. Furthermore, both Cvb-D, captopril and SB203580 reduced m RNA expression of p38α, p38β, c-fos, and c-jun m RNA, and Cvb-D had a stronger effect（P〈0.01）. Conclusion： These results demonstrate that Cvb-D protects against cardiac hypertrophy, which is possibly mediated by prevention of cardiac cell apoptosis and inhibition of the p38 MAPK signaling pathway.