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.

Influence of electroacupuncture on mitogen-activated protein kinase signal transduction in a rat model of cerebral ischemia/reperfusion

Following electroacupuncture at Baihui （DU 20） and Dazhui （DU 14） in a rat model of cerebral ischemia/reperfusion, extracellular-signal-regulated kinase expression in cerebral cortex and corpus striatum, serum glutathione reductase, glutathione peroxidase activity, and serum glutathione content were elevated, and neurobehavioral scores improved. However, these effects were antagonized by mitogen-activated protein kinase inhibitor PD98059. Results indicated that electroacupuncture reversed free radical chain reactions and oxidative stress injury caused by cerebral ischemia/reperfusion, thereby providing neuroprotection. This process could correlate with the mitogen-activated protein kinase signal transduction pathway.

Inhibition of p38 mitogen-activated protein kinase may decrease intestinal epithelial cell apoptosis and improve intestinal epithelial barrier function after ischemia- reperf usion injury

AIM: To investigate the role of p38 mitogen-activated protein kinase in rat small intestine after ischemia-reperfusion (I/R)insult and the relationship between activation of p38 MAPK and apoptotic cell death of intestine.METHODS: Ninety Wistar rats were divided randomly into three groups, namely sham-operated group (C), I/R vehicle group (R) and SB203580 pre-treated group(S).In groups R and S, the superior mesenteric artery(SMA)was separated and occluded for 45 min, then released for reperfusion for0.25, 0.5, 1, 2, 6, 12 and 24 h. In group C, SMA was separated without occlusion. Plasma D-lactate levels were examined and histological changes were observed under a light microscope. The activity of p38 MAPK was determined by Western immunoblotting and apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL).RESULTS: Intestinal ischemia followed by reperfusion activated p38 MAPK, and the maximal level of activation (7.3-fold vs sham-operated group) was reached 30 min after I/R. Treatment with SB 203580, a p38 MAPK inhibitor,reduced intestinal apoptosis (26.72±3.39% vs62.50±3.08%in I/R vehicle, P＜0.01) and decreased plasma D-lactate level (0.78±0.15 mmol/L in I/R vehicle vs0.42±0.17 mmol/L in SB-treated group) and improved post-ischemic intestinal histological damage.CONCLUSION: p38 MAPK plays a crucial role in the signal transduction pathway mediating post-ischemic intestinal apoptosis, and inhibition of p38 MAPK may attenuate ischemia-reperfusion injury.

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.

INVOLVEMENT OF p38 MITOGEN-ACTIVATED PROTEIN KINASE IN E.coli-INDUCED U937 APOPTOSIS

Objective To investigate whether the effect of E. coli on U937 cell fines apoptosis is mediated via p38 mitogen-activated protein kinase （MAPK） activation. Methods The U937 cell lines were treated with E. coli at different time or together with SB203580, an inhibitor for p38. Cell apoptosis was analyzed by flow cytometry, p38 activities were detected by Western blotting. Results E. coli induced apoptosis in cultured U937 cell lines in a time-dependent manner. The phosphorylation of p38 was induced after 10 minutes infection, reached the peak after 20 minutes, and started to decline after 30 minutes. In contrast, the level of total p38 protein was not changed in whole experimental period. Inhibition of p38 with SB203580 significantly inhibited E. coli induced apoptosis in U937 cells. Conclusion The activation of the p38 MAPK in U937 cell lines by E. coli is a major pathway to mediate the apoptosis.

Functional study of p38 mitogen-activated protein kinase based on cell-penetrating peptide delivery system

Objective p38 Mitogen-activated protein kinase （MAPK） is a crossing center of various pathways. In this study, protein transduction system based on human immunodeficiency virus （HIV）-1 transactivator of transcription （TAT）, which is an efficient delivery peptide of the foreign proteins into cells, was employed to study p38 MAPK functions in eukaryotic cells. Methods p38 And its dominant negative form, p38AF, were constructed into pET-His-TAT vector correctly to verify that the recombinant plasmids were well-founded through restriction enzyme digestion and DNA sequencing. The two proteins, His-TAT-p38 and His-TAT-p38AF, were expressed and purified in Escherichia coli by SDS-PAGE. Then they were incubated with ECV304 cells respectively and readily transduced into cells in a time-dependent and dose-dependent manner. The cells were stimulated by sorbitol. Activating transcription factor （ATF） 2 phosphorylation level was checked using Western blot to assess the activity of endogenous p38. Results Compared with controls, it was found that His-TAT-p38 increased the level ofATF2 phosphorylation in sorbitol-stimulated ECV304 cells, while His-TAT-p38AF inhibited it, indicating p38 MAPK protein delivery system based on TAT was constructed successfully. TAT-p38 and its dominant negative form possessed high biological activity after transduction into ECV304 cells by TAT protein delivery system. The results showed that p38AF fused with TAT could inhibit the transduction of endogenous p38 signal pathway in part, and other pathway might regulate p38 phosphorylation. Conclusions Our study provides a novel pathway to inhibit p38 signal pathway and establish a new method to study p38 function.

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.

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.

Increased expression of mitogen-activated protein kinase and its upstream regulating signal in human gastric cancer

AIM: To investigate the expression of mitogen-activated protein kinases (MAPKs) and its upstream protein kinase in human gastric cancer and to evaluate the relationship between protein levels and clinicopathological parameters. METHODS: Western blot was used to measure the expression of extracellular signal-regulated kinase (ERK)-1, ERK-2, ERK-3, p38 and mitogen or ERK activated protein kinaseMEK-1 proteins in surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 42 patients. Immunohistochemistry was employed for their localization. RESULTS: Compared with normal tissues, the protein levels of ERK-1 (integral optical density value 159 526?5 760 vs 122 807±65 515, P= 0.001), ERK-2 (168 471±95 051 vs 120 469±72 874, P<0.001), ERK-3 (118 651±71 513 vs 70 934±68 058,P<0.001), P38 (104 776±51 650 vs 82 930±40 392, P= 0.048) and MEK-1 (116 486±45 725 vs 101 434±49 387, P = 0.027) were increased in gastric cancer tissues. Overexpression of ERK-3 was correlated to TNM staging [average ratio of integral optic density (IOD)tumor: IODnormal in TNM I, II, III, IV tumors was 1.43±0.34, 5.08±3.74, 4.99±1.08, 1.44±1.02, n = 42, P= 0.023] and serosa invasion (4.31±4.34 vs 2.00±2.03, P = 0.037). In poorly differentiated cancers (n = 33), the protein levels of ERK-1 and ERK-2 in stage III and IV tumors were higher than those in stage I and II tumors (2.64+3.01 vs 1.01±0.33, P= 0.022; 2.05±1.54 vs1.24±0.40, P= 0.030). Gastric cancer tissues with either lymph node involvement (2.49±2.91 vs1.03±0.36, P= 0.023; 1.98±1.49vs1.24±0.44, P= 0.036) or serosa invasion (2.39±2.82 vs 1.01±0.35, P= 0.022; 1.95±1.44 vs1.14±0.36, P=0.015) expressed higher protein levels of ERK-1 and ERK-2. In Borrmann II tumors, expression of ERK-2 and ERK-3 was increased compared with Borrmann III tumors (2.57±1.86 vs1.23±0.60, P= 0.022; 5.50±5.05 vs1.83±1.21, P= 0.014). Borrmann IV tumors expressed higher p38 protein levels. No statistically significant difference in expression of MAPKs was found when stratified to tumor size or histological grade (P>0.05). Protein levels of ERK-2, ERK-3 and MEK-1 in metastatic lymph nodes were 2-7 folds higher than those in adjacent normal mucosa. The immunohistochemistry demonstrated that ERK-1, ERK-2, ERK-3, p38 and MEK-1 proteins were mainly localized in cytoplasm. The expression of MEK-1 in gastric cancer cells metastasized to lymph nodes was higher than that of the primary site. CONCLUSION: MAPKs, particularly ERK subclass are overexpressed in the majority of gastric cancers. Overexpression of ERKs is correlated to TNM staging, serosa invasion, and lymph node involvement. The overexpression of p38 most likely plays a prominent role in certain morphological subtypes of gastric cancers. MEK-1 is also overexpressed in gastric cancer, particularly in metastatic lymph nodes. Upregulation of MARK signal transduction pathways may play an important role in tumorigenesis and metastatic potential of gastric cancer.

Interleukin-1 beta up-regulates tissue inhibitor of matrix metalloproteinase-1 mRNA and phosphorylation of c-jun N-terminal kinase and p38 in hepatic stellate cells

AIM： To study the relationship between interleukin-lbeta （IL-1β） up-regulating tissue inhibitor of matrix metalloproteinase-1 （TIMMP-1） mRNA expression and phosphorylation of both c-jun N-terminal kinase （INK） and p38 in rat heffatic stellate cells （HSC）. METHODS： RT-PCR was performed to measure the expression of TIMMP-1 mRNA in rat HSC. Western blot was performed to measure IL-1β-induced JNK and p38 activities in rat HSC. RESULTS： TIMMP-1 mRNA expression （1.191± 0.079） was much higher after treatment with IL-1β （10 ng/mL） for 24 h than in control group （0.545±0.091） （P〈0.01）. IL-1β activated INK and p38 in a time-dependent manner. After stimulation with IL-1β for 0, 5, 15, 30, 60 and 120 min, the INK activity was 0.982±0.299, 1.501±0.720, 2.133±0.882, 3.360±0.452, 2.181±0.789, and 1.385 ± 0.368, respectively. There was a significant difference in JNK activity at 15 min （P〈 0.01）, 30 min （P〈 0.01） and 60 min （P〈0.01） in comparison to that at 0 min. The p38 activity was 1.061±0.310, 2.050±0.863, 2.380±0.573, 2.973±0.953, 2.421±0.793, and 1.755 ± 0.433 at the 6 time points （0, 5, 15, 30, 60 and 120 min） respectively. There was a significant difference in p38 activity at 5 min （P〈0.05）, 15 min （P〈0.01）, 30 min （P〈0.01） and 60 min （P〈0.01） compared to that at 0 min. TIMMP-1 mRNA expression trended to decrease in 3 groups pretreated with different concentrations of SP600125 （10 μmol/L, 1.022±0.113; 20 μmol/L, 0.869±0.070; 40 μmol/L, 0.666±0.123）. Their decreases were all significant （P〈0.05, P〈0.01, P〈0.01） in comparison to control group （without SP600125 treatment, 1.163±0.107）. In the other 3 groups pretreated with different concentrations of SB203580 （10 μmol/L, 1.507±0.099; 20 μmol/L, 1.698±0.107; 40 μmol/L, 1.857±0.054）, the expression of TIMMP-1 mRNA increased. Their levels were higher than those in the control group （without SB203580 treatment, 1.027 ± 0.061） with a significant statistical significance （P〈 0.01）. CONCLUSION： IL-1β has a direct action on hepatic fibrosis by up-regulating TIMMP-1 mRNA expression in ratessionin in rate HSC.JNK and p38 mitogen-activated protein kinases （MAPKs） are involved in IL-1β-induced TIMMP-1 gene expression, and play a distinct role in this process, indicating that p38 and .INK pathways cooperatively mediate TIMP-1 mRNA expression in rat HSC.

Effect of cigarette smoke extract on lipopolysaccharide-activated mitogen-activated protein kinase signal transduction pathway in cultured cells

Background Lipopolysaccharide （LPS） forms outer membrane of the wall of Gram-negative cells. LPS can directly cause damage to epithelia of respiratory tract and is the major factor responsible for the chronic inflammation of respiratory passage. The mitogen-activated protein kinase （MAPK） signal transduction pathway of the airway epithelia is intimately associated with the action of LPS. The chronic inflammation of respiratory tract and smoking are interrelated and entwined in the development and progression of chronic lung diseases. This study was designed to examine the effects of cigarette smoke extract （CSE） and LPS on MAPK signal transduction pathway in order to further understand the roles CSE and LPS play in chronic lung inflammation. Methods Cultured primary human epithelial cells of airway were divided into four groups according to the stimulants used： blank control group, LPS-stimulation group, CSE-stimulation group and CSE plus LPS group. Western blotting was employed for the detection of phosphorylation level of extracellular-signal-regulated-kinase （ERK1/2）, p38 MAPK and c-Jun N-terminal kinase （JNK）. The expression of cytokines of MAPK transduction pathway （granulocyte-macrophage colony stimulating factor （GM-CSF） and mRNA of IL-8） in the primary epithelial cells of respiratory tract was also determined. Results Western blotting revealed that the phosphorylation levels of ERK1/2, p38 MAPK and JNK were low and 2 hours after the LPS stimulation, the phosphorylation of ERK1/2, p38 MAPK and JNK were all increased. There was a significant difference in the phosphorylation between the LPS-stimulation group and blank control group （P〈0.05）; no significant difference was found between CSE-stimulation group and blank control group （P〉0.05）; there was a significant difference between CSE ＋ LPS group and blank control group and between CSE ＋ LPS group and LPS group （P〈0.05）. The phosphorylation of CSE-LPS group was higher than that of blank control group but lower than that of LPS group. In blank control group, the expression of IL-8 and GM-CSF mRNA was low in the epithelial cells of airway and the release of IL-8 and GM-CSF was also at a low level. One hour after LPS stimulation, the level of IL-8 mRNA increased （P〈0.05） and reached a peak after 2 hours. On the other hand, GM-CSF mRNA level increased 2 hours after the stimulation （P〈0.05） and reached the highest level 4 hours after the stimulation. Two hours after LPS stimulation, IL-8 and GM-CSF protein level began to rise （P〈0.05）, and the level was the highest 8 hours after the stimulation （P〈0.01）. Stimulation with CSE alone had no effect on the release of IL-8 and GM-CSF and expression of IL-8 mRNA （P〉0.05）, but pre-treatment with CSE could delay the LPS-induced release of IL-8 and GM-CSF and the expression of IL-8 mRNA and its peak was lower. Conclusions LPS stimulation can significantly increase the phosphorylation of ERK1/2, p38 MAPK and JNK in the epithelial cells of airway and activate the MAPK transduction pathway, thereby can activate the downstream signal transduction pathway, and can ultimately result in the release of cytokines by the epithelial cells of airway. CSE can partially abolish the LPS-induced activation of MAPK signal transduction pathway and the expression of cytokines of the pathway, which might contribute to the development and progression of the inflammatory reactions in COPD patients.

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.

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.

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.

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.

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.

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.

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.

Involvement of p38 mitogen-activated protein kinase in the regulation of platelet-derived growth factor -induced cell migration

The aim of this study was to investigate the role of p38 mitogen-activated protein kinase(MAPK)in cell migration induced by platelet-derived growth factor(PDGF).Western blot was performed to detect the phosphorylation of p38 in NIH3T3 cells treated with PDGF.A Transwell cell migration system was used to determine the effects of PDGF treatment on the migration of NIH3T3 cells and the influence of p38 deficiency on this process in a p38 gene knockout(p38^(−/−))mouse embryonic fibroblast cell line.On the stimulation of PDGF,the migration of NIH3T3 cells was significantly increased(P<0.001)compared to the control and p38 MAP kinase was simultaneously phosphorylated.Furthermore,the PDGF-induced cell migration was significantly blocked in p38 gene knockout(p38^(−/−))mouse embryonic fibroblasts(MEFs)(P<0.001)as compared with the wild type cells(p38+/+).p38 MAPK plays an important role in the regulation of cell migration induced by PDGF.