Daphnetin Ameliorates Neuropathic Pain via Regulation of Microglial Responses and Glycerophospholipid Metabolism in the Spinal Cord.

Neuropathic pain (NP) is a common type of chronic pain caused by a lesion or disease of the somatosensory nervous system. This condition imposes a considerable economic burden on society and patients. Daphnetin (DAP) is a natural product isolated from a Chinese medicinal herb with various pharmacological activities, such as anti-inflammatory and analgesic properties. However, the underlying mechanisms of these effects are not fully understood. In the present study, we aimed to investigate DAP's anti-inflammatory and analgesic effects and explore the underlying mechanisms of action. The NP model was established as chronic constrictive injury (CCI) of the sciatic nerve, and pain sensitivity was evaluated by measuring the mechanical withdrawal threshold (MWT) and thermal withdrawal threshold (TWT). The activation of microglia in the spinal dorsal horn was measured via immunofluorescence staining. Protein levels were measured using a western blot assay. Using a mass-spectrometry proteomics platform and an LC-MS/MS-based metabolomics platform, proteins and metabolites in spinal cord tissues were extracted and analyzed. DAP treatment ameliorated the MWT and TWT in CCI rats. The expression of IL-1ß, IL-6, and TNF-α was inhibited by DAP treatment in the spinal cords of CCI rats. Moreover, the activation of microglia was suppressed after DAP treatment. The elevation in the levels of P2X4, IRF8, IRF5, BDNF, and p-P38/P38 in the spinal cord caused by CCI was inhibited by DAP. Proteomics and metabolomics results indicated that DAP ameliorated the imbalance of glycerophospholipid metabolism in the spinal cords of CCI rats. DAP can potentially ameliorate NP by regulating microglial responses and glycerophospholipid metabolism in the CCI model. This study provides a pharmacological justification for using DAP in the management of NP.

RNA polymerase I subunit D activated by Yin Yang 1 transcription promote cell proliferation and angiogenesis of colorectal cancer cells.

This study aims to explore possible effect of RNA polymerase I subunit D (POLR1D) on proliferation and angiogenesis ability of colorectal cancer (CRC) cells and mechanism herein. The correlation of POLR1D and Yin Yang 1 (YY1) expressions with prognosis of CRC patients in TCGA database was analyzed. Quantitative realtime polymerase chain reaction (qRT-PCR) and Western blot were applied to detect expression levels of POLR1D and YY1 in CRC cell lines and CRC tissues. SW480 and HT- 29 cells were transfected with si-POLR1D or pcDNA3.1-POLR1D to achieve POLR1D suppression or overexpression before cell migration, angiogenesis of human umbilical vein endothelial cells were assessed. Western blot was used to detect expressions of p38 MAPK signal pathway related proteins and interaction of YY1 with POLR1D was confirmed by dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP). TCGA data showed that both POLR1D and YY1 expressions were up-regulated in CRC patients. High expression of POLR1D was associated with poor prognosis of CRC patients. The results showed that POLR1D and YY1 were highly expressed in CRC cell lines. Inhibition or overexpression of POLR1D can respectively suppress or enhance proliferation and angiogenesis of CRC cells. YY1 inhibition can suppress CRC progression and deactivate p38 MAPK signal pathway, which can be counteracted by POLR1D overexpression. JASPAR predicted YY1 can bind with POLR1D promoter, which was confirmed by dual luciferase reporter gene assay and ChIP. YY1 transcription can up-regulate POLR1D expression to activate p38 MAPK signal pathway, thus promoting proliferation and angiogenesis ability of CRC cells.

Dexmedetomidine alleviates blood-brain barrier disruption in rats after cerebral ischemia-reperfusion by suppressing JNK and p38 MAPK signaling.

Dexmedetomidine displays multiple mechanisms of neuroprotection in ameliorating ischemic brain injury. In this study, we explored the beneficial effects of dexmedetomidine on blood-brain barrier (BBB) integrity and neuroinflammation in cerebral ischemia/reperfusion injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1.5 h and reperfusion for 24 h to establish a rat model of cerebral ischemia/reperfusion injury. Dexmedetomidine (9 􀁐g/kg) was administered to rats 30 min after MCAO through intravenous injection, and SB203580 (a p38 MAPK inhibitor, 200 􀁐g/kg) was injected intraperitoneally 30 min before MCAO. Brain damages were evaluated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, Nissl staining, and brain water content assessment. BBB permeability was examined by Evans blue staining. Expression levels of claudin-5, zonula occludens-1, occludin, and matrix metalloproteinase-9 (MMP-9) as well as M1/M2 phenotypes-associated markers were assessed using immunofluorescence, RT-qPCR, Western blotting, and gelatin zymography. Enzyme-linked immunosorbent assay was used to examine inflammatory cytokine levels. We found that dexmedetomidine or SB203580 attenuated infarct volume, brain edema, BBB permeability, and neuroinflammation, and promoted M2 microglial polarization after cerebral ischemia/reperfusion injury. Increased MMP-9 activity by ischemia/reperfusion injury was inhibited by dexmedetomidine or SB203580. Dexmedetomidine inhibited the activation of the ERK, JNK, and p38 MAPK pathways. Moreover, activation of JNK or p38 MAPK reversed the protective effects of dexmedetomidine against ischemic brain injury. Overall, dexmedetomidine ameliorated brain injury by alleviating BBB permeability and promoting M2 polarization in experimental cerebral ischemia/reperfusion injury model by inhibiting the activation of JNK and p38 MAPK pathways.

Blockage of the fractalkine pathway reduces hyperalgesia and prevents morphological glial alterations-Comparison between inflammatory and neuropathic orofacial pain in male rats.

This study aimed to evaluate the effects of inhibitors of the fractalkine pathway in hyperalgesia in inflammatory and neuropathic orofacial pain in male rats and the morphological changes in microglia and satellite glial cells (SGCs). Rats were submitted to zymosan-induced arthritis of the temporomandibular joint or infraorbital nerve constriction, and treated intrathecally with a P2 X7 antagonist, a cathepsin S inhibitor or a p-38 mitogen-activated protein kinase (MAPK) inhibitor. Mechanical hyperalgesia was evaluated 4 and 6 h following arthritis induction or 7 and 14 days following nerve ligation. The expression of the receptor CX3 CR1 , phospho-p-38 MAPK, ionized calcium-binding adapter molecule-1 (Iba-1), and glutamine synthetase and the morphological changes in microglia and SGCs were evaluated by confocal microscopy. In both inflammatory and neuropathic models, untreated animals presented a higher expression of CX3 CR1 and developed hyperalgesia and up-regulation of phospho-p-38 MAPK, which was prevented by all drugs (p < .05). The number of microglial processes endpoints and the total branch length were lower in the untreated animals, but the overall immunolabeling of Iba-1 was altered only in neuropathic rats (p < .05). The mean area of SGCs per neuron was significantly altered only in the inflammatory model (p < .05). All morphological alterations were reverted by modulating the fractalkine pathway (p < .05). In conclusion, the blockage of the fractalkine pathway seemed to be a possible therapeutic strategy for inflammatory and neuropathic orofacial pain, reducing mechanical hyperalgesia by impairing the phosphorylation of p-38 MAPK and reverting morphological alterations in microglia and SGCs.

Effect of Guipitang on ERK1/2 and p38 MAPK in Rats with Myocardial Ischemia / 中国实验方剂学杂志

ObjectiveTo explore the therapeutic effect and mechanism of Guipitang on rats with myocardial ischemia. MethodFifty SD rats were divided into five groups： a control group， a model group， low and high-dose Guipitang （7.52， 15.04 g·kg-1） groups， and a trimetazidine group （0.002 g·kg-1）. By intragastric administration of vitamin D3 and feeding rats with high-fat forage and injecting isoproterenol， the rat model of myocardial ischemia was established. After drug treatment of 15 d， an electrocardiogram （ECG） was performed to analyze the degree of myocardial injury. A fully automatic biochemical analyzer was used to detect the changes in the serum levels of total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C）. Hematoxylin-eosin （HE） staining and Masson staining were used to observe myocardial histopathological changes. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect cardiomyocyte apoptosis. Western blot was adopted to detect the protein levels of extracellular signal-regulated kinase 1/2 （ERK1/2）， phospho-ERK1/2 （p-ERK1/2）， p38 mitogen-activated protein kinase （p38 MAPK）， phospho-p38 MAPK （p-p38 MAPK）， B-cell lymphoma-2 （Bcl-2）-associated X （Bax）， Bcl-2， and cleaved cysteine aspartate proteolytic enzyme （cleaved Caspase-3）. ResultCompared with the control group， the ECG S-T segment decreased in the model group. The serum levels of TC， TG， and LDL-C were increased significantly （P<0.05）. The arrangement of myocardial tissue was disordered， and the proportion of cardiomyocyte apoptosis increased. The protein levels of cleaved Caspase-3， Bax， and p-p38 MAPK in the heart were increased， and the Bcl-2 expression was decreased （P<0.05）. Compared with the model group， the S-T segment downward shift was restored in the low and high-dose Guipitang groups and trimetazidine group， and the levels of TC， TG， and LDL-C were decreased. The protein expression of cleaved Caspase-3 and Bax in the heart dropped， and p-p38 MAPK and p-ERK1/2 protein expressions increased significantly （P<0.05）. The degree of myocardial injury was alleviated， and the proportion of cardiomyocyte apoptosis decreased. Bcl-2 protein expression was increased significantly in the low-dose Guipitang group （P<0.05）. ERK1/2 and p38 MAPK proteins had no significant difference among different groups. ConclusionGuipitang could alleviate myocardial injury and inhibit cardiomyocyte apoptosis in rats by activating the expression of ERK1/2 and p38 MAPK.

Spinosin protects Neuro-2a/APP695 cells from oxidative stress damage by inactivating p38.

OBJECTIVE: To explore the protective mechanism of spinosin (SPI) on Alzheimer's disease (AD) model cells, Neuro-2a/APP695 (N2a/APP695), against HO-induced oxidative stress damage, to reflect the influence of oxidative stress on the development of AD, and to provide a valuable basis for the research and development of therapeutic drug for AD. METHODS: N2a/APP695 cells were exposed to HO and then treated with spinosin. Firstly, the secretion level of amyloid ß (Aß) and the production of malondialdehyde (MDA) and lactate dehydrogenase (LDH) were detected by enzyme linked immunosorbent assay kits. Secondly, the oligomerization degree of Aß was performed by Thioflavin T staining. Thirdly, the expression levels of p-Tau (Ser199/202/396), synaptophysin (SYP), postsynaptic density protein 95 (PSD95), and mitogen-activated protein kinase (MAPK) family-related proteins were detected by Western blot analysis. In addition, FITC-labeled phalloidin was used in cytoskeleton staining to reflect synaptic function. RESULTS: This study showed that HO stimulated N2a/APP695 cells to produce excessive MDA and LDH and secrete a large amount of Aß, promoted the aggregation of Aß, induced Tau protein hyperphosphorylation, and led to synaptic dysfunction. Spinosin reversed these changes caused by HO by inactivating p38, which was verified by treatment with the p38 inhibitor BIRB796. CONCLUSION: Spinosin protects N2a/APP695 cells from oxidative stress damage caused by HO through inactivating p38.

Iris Koreana NAKAI Inhibits Osteoclast Formation via p38-Mediated Nuclear Factor of Activated T Cells 1 Signaling Pathway.

BACKGROUND: Iris Koreana NAKAI (IKN) is a flowering perennial plant that belongs to the Iridaceae family. In this study, we aimed to demonstrate the effects of IKN on osteoclast differentiation in vitro and in vivo. We also sought to verify the molecular mechanisms underlying its anti-osteoclastogenic effects. METHODS: Osteoclasts were formed by culturing mouse bone marrow macrophage (BMM) cells with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). Bone resorption assays were performed on dentin slices. mRNA expression levels were analyzed by quantitative polymerase chain reaction. Western blotting was performed to detect protein expression or activation. Lipopolysaccharide (LPS)-induced osteoclast formation was performed using a mouse calvarial model. RESULTS: In BMM cultures, an ethanol extract of the root part of IKN suppressed RANKL-induced osteoclast formation and bone resorptive activity. In contrast, an ethanol extract of the aerial parts of IKN had a minor effect on RANKL-induced osteoclast formation. Mechanistically, the root part of IKN suppressed RANKL-induced p38 mitogen-activated protein kinase (MAPK) activation, effectively abrogating the induction of c-Fos and nuclear factor of activated T cells 1 (NFATc1) expression. IKN administration decreased LPS-induced osteoclast formation in a calvarial osteolysis model in vivo. CONCLUSIONS: Our study suggested that the ethanol extract of the root part of IKN suppressed osteoclast differentiation and function partly by downregulating the p38 MAPK/c-Fos/NFATc1 signaling pathways. Thus, the root part.

Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein.

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic ß loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

Role of P2X4R in maintenance of trigeminal neuralgia in rats: relationship with p38 MAPK/BDNF signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of P2X4 receptor (P2X4R) in the maintenance of trigeminal neuralgia and the relationship with p38 mitogen-activated protein kinase (p38 MAPK)/brain-derived neurotrophic factor (BDNF) signaling pathway in rats.Methods:Forty-eight clean-grade healthy adult male Sprague-Dawley rats, weighing 190-230 g, aged 2-3 months, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (S group), trigeminal neuralgia group (TN group), trigeminal neuralgia+ dimethylsulfoxide (DMSO) group (TN+ DMSO group), and trigeminal neuralgia+ P2X4R specific antagonist 5-BDBD group (TN+ 5-BDBD group). The model was developed by chronic constriction of the infraorbital nerve. The infraorbital nerve was only exposed without ligation in group S. At 3, 7, 10 and 14 days after developing the model, 5 μg/μl 5-BDBD 10 μl was intrathecally injected in TN+ 5-BDBD group, and 2% DMSO 10 μl was intrathecally injected in TN+ DMSO group. The facial mechanical pain withdrawal threshold (MWT) was measured at 1 day before developing the model and 1, 3, 7, 10, 14 and 28 days after developing the model (T 0-6). The rats were sacrificed and the trigeminal ganglia were taken for determination of the expression of P2X4R, p38 MAPK, phosphorylated p38 MAPK (p-p38 MAPK) and BDNF (by Western blot) and contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β and IL-6 (by enzyme-linked immunosorbent assay). Results:Compared with group S, the MWT was significantly decreased at T 1-6, the expression of P2X4R, p-p38 MAPK and BDNF in trigeminal ganglion was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in TN group ( P<0.05). Compared with TN group, the MWT was significantly increased at T 3-6, and the expression of P2X4R, p-p38 MAPK and BDNF in trigeminal ganglion was down-regulated, and the contents of TNF-α, IL-1β and IL-6 were decreased in TN+ 5-BDBD group ( P<0.05), and no significant change was found in the indexes mentioned above in TN+ DMSO group ( P>0.05). Conclusions:P2X4R is involved in the maintenance of trigeminal neuralgia in rats, which may be related to the activation of p38 MAPK/BDNF signaling pathway and the increase in inflammatory mediator release.

Phenotype expansion of variants affecting p38 MAPK signaling in hypospadias patients.

BACKGROUND: Hypospadias is a congenital anomaly of the male urogenital system. Genetics factors play an important role in its pathogenesis. To search for potential causal genes/variants for hypospadias, we performed exome sequencing in a pedigree with three patients across two generations and a cohort of 49 sporadic patients with hypospadias. RESULTS: A novel BRAF variant (NM_004333.6: c.362C > A) was found to co-segregate with the hypospadias phenotype in the disease pedigree. In cells overexpressing the BRAF mutant, the phosphorylation level of p38 MAPK was significantly increased as compared with the cells overexpressing the wild-type BRAF or RASopathy-related BRAF mutant. This variant further led to a reduced transcription level of the SRY gene, which is essential for the normal development of the male reproductive system. In the cohort of sporadic patients, we identified two additional variants in p38 MAPK signaling-related genes (TRIM67 and DAB2IP) potentially associated with hypospadias. CONCLUSION: Our study expands the phenotypic spectrum of variants affecting p38 MAPK signaling toward the involvement of hypospadias.

Modified Gexiazhuyu decoction alleviates chronic salpingitis p38 signaling pathway.

OBJECTIVE: To investigate pharmacodynamic effects of modified Gexiazhuyu decoction (MGXZYD) and explore the underlying mechanism in the treatment of chronic salpingitis METHODS: Chronic salpingitis model rats were firstly constructed and the blood was collected to detect the whole blood viscosity and plasma viscosity. Rat oviduct were collected to evaluate the macroscopic damage and the pathological injury and fibrosis of oviduct by hematoxylin-eosin (HE) and Masson staining. Elisa assay was to detect the production interleukin-1 ß (IL-1ß) in serum and collagen I (COL-1), matrix metalloprotein 9 (MMP-9), tissue inhibitor of metalloproteinases 1 (TIMP-1) in oviduct tissue. And immunohistochemical staining with MMP-9 and TIMP-1 in oviduct tissue were examined. Western blot was used to detect the expressions of p38 mitogen-activated protein kinases (p38MAPK), phospho-p38MPAK (p-p38MPAK), transforming growth factor-ß1 (TGF-ß1) in oviduct. The expression of α-smooth muscle actin (α-SMA), p-p38MPAK, in oviduct tissue were detected by immunofluorescence method. The mRNA of p-p38MAPK, α -SMA, COL-1, MMP-9, TIMP-1 was measured by reverse transcription-polymerase chain reaction. RESULTS: Rats administrated with MGXZYD demonstrated decreased the whole blood viscosity and plasma viscosity. MGXZYD obviously improved the tubal wall thickening, swelling and pelvic adhesion. And HE and Masson staining showed MGXZYD improved the pathological injury and fibrosis of oviduct. The results of MTT assay and flow cytometry indicated that MGXZYD could decreased the NIN-3T3 cells viability and improved the apoptosis. Besides, MGXZYD inhibited the protein and / or mRNA of TGF-ß1, IL-1ß, COL-1, α-SMA, p-p38MAPK expressions and increased the production of MMP-9/TIMP-1. CONCLUSION: MGXZYD could prevent the progression of chronic salpingitis by inhibited the fibrocyte and inflammation which inhibited the p38 MAPK signaling pathway.

Impact of Myeloid p38α/MAPK on Orthodontic Tooth Movement.

Objectives: Myeloid p38α/MAPK regulate and coordinate osteoclastogenesis. The present study was conducted to investigate the role of myeloid p38α/MAPK during orthodontic tooth movement. Methods: Orthodontic tooth movement was performed in wildtype and p38αΔmyel mice lacking p38α/MAPK expression in myeloid cells. First, bone parameter as well as osteoblast and osteoclast number were determined in tibiae. RNA was isolated from the untreated and orthodontically treated maxillary jaw side and expression of genes involved in inflammation and bone remodelling were analysed. Finally, periodontal bone loss, alveolar bone density and extent of orthodontic tooth movement were assessed. Results: Bone density was increased in p38αΔmyel mice compared to wildtype mice in tibiae (p = 0.043) and alveolar bone (p = 0.003). This was accompanied by a reduced osteoclast number in tibiae (p = 0.005) and TRAP5b in serum (p = 0.015). Accordingly, expression of osteoclast-specific genes was reduced in p38αΔmyel mice. Extent of tooth movement was reduced in p38αΔmyel mice (p = 0.024). This may be due to the higher bone density of the p38αΔmyel mice. Conclusions: Myeloid p38α/MAPK thus appears to play a regulatory role during orthodontic tooth movement by regulating osteoclastogenesis.

Yomogin, Isolated from Artemisia iwayomogi, Inhibits Neuroinflammation Stimulated by Lipopolysaccharide via Regulating MAPK Pathway.

Neuroinflammation causes various neurological disorders, including depression and neurodegenerative diseases. Therefore, regulation of neuroinflammation is a promising therapeutic strategy for inflammation-related neurological disorders. This study aimed to investigate whether yomogin, isolated from Artemisia iwayomogi, has anti-neuroinflammatory effects. First, we evaluated the effects of yomogin by assessing pro-inflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The results showed that yomogin inhibited the increase in neuroinflammatory factors, including nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, interleukin-6, and tumor necrosis factor-α, and suppressed phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase and p38, which participate in the mitogen-activated protein kinase (MAPK) pathway. To confirm these effects in vivo, we measured the activation of astrocyte and microglia in LPS-injected mouse brains. Results showed that yomogin treatment decreased astrocyte and microglia activations. Collectively, these results suggest that yomogin suppresses neuroinflammation by regulating the MAPK pathway and it could be a potential candidate for inflammation-mediated neurological diseases.

Effects of neutrophil extracellular traps on proliferation and apoptosis of human amniotic epithelial cells and its possible mechanisms / 中华围产医学杂志

Objective:To investigate the effects of neutrophil extracellular traps (NETs) on the proliferation and apoptosis of human amniotic epithelial cells.Methods:NETs were induced in vitro from the neutrophil cells obtained from the peripheral blood of normal pregnant women before elective cesarean section at full-term. Human amniotic epithelial cell lines (WISH cells) were cultured in vitro, and were divided into four groups:(1) control group: without any stimulus; (2) NETs group: WISH cells were stimulated with NETs (500 ng/ml); (3) NETs+SB203580 (p38 kinase inhibitor) group: WISH cells were pretreated with SB203580 (5 μmol/L) for 30 min and then NETs (500 ng/ml) was added; (4) SB203580 group: only SB203580 was added. After stimulating for 48 h, cell proliferation assay, lactate dehydrogenase(LDH) assay, and flow cytometry assay were used to detect the cell proliferation rate, LDH level of cell supernatant, and cell apoptosis rate among different groups. The results were analyzed and compared using one-way analysis of variance and LSD- t test. Results:(1) Cell proliferation: The cell proliferation ratio in the NETs group was lower than that in the control group [(9.379±0.775)% vs (36.560±1.208)%, LSD- t=20.78, P<0.001]; and the figure in the NETs+SB203580 group [(27.920±0.926)%] was higher than that in the NETs group (LSD- t=14.18, P<0.001). (2)LDH: There was an increased LDH level in the cell supernatant of the NETs group compared with the control group (1.518±0.038 vs 0.274±0.004, LSD -t=44.25, P<0.05), and the LDH level in the NETs+SB203580 group (0.857±0.009) was decreased than that in the NETs group (LSD -t=23.51, P<0.001). (3) Apoptosis: Compared with the control group, the cell apoptosis level of the NETs group was increased [(14.290±0.141)% vs (10.110±0.044)%, LSD- t=21.76, P<0.001]; but that in the NETs+SB203580 group [(10.500±0.218)%] was lower than in the NETs group (LSD- t=19.70, P<0.001). Conclusion:p38/mitogen-activated protein kinases signaling pathway may be involved in the process of NETs, inhibiting proliferation and promoting apoptosis of human amniotic epithelial cells.

Role of AMPK/p38 MAPK/Nrf2 signaling pathway in myocardial ischemia-reperfusion injury in diabetic rats / 中华麻醉学杂志

Objective:To evaluate the role of adenosine monophosphate-dependent protein kinase/p38 mitogen-activated protein kinase/nuclear factor E2-associated factor 2 (AMPK/p38 MAPK/Nrf2) pathway in myocardial ischemia-reperfusion (I/R) injury in diabetic rats.Methods:Clean-grade healthy Sprague-Dawley male rats, aged 2-3 months, weighing 220-280 g, were fed with a high fat diet, and 1% streptozotocin 50 mg/kg was intraperitoneally injected for 4 consecutive days to develop the model of diabetes mellitus.Thirty diabetic rats were divided into 3 groups ( n=10 each) using the random number table method: sham operation group (sham group), myocardial I/R group (I/R group), and AMPK inhibitor compound C+ myocardial I/R group (C+ I/R group). The model of myocardial I/R injury was developed by ligation of the left anterior descending coronary artery for 30 min followed by 120 min reperfusion.Compound C 0.5 mg/kg was injected via the caudal vein at 30 min before ischemia in C+ I/R group, while the equal volume of normal saline was given instead in Sham group and I/R group.At 120 min of reperfusion, the percentage of myocardial infarct size was calculated, the serum concentrations of creatine kinase isoenzymes (CK-MB) and lactic dehydrogenase (LDH) were determined by enzyme-linked immunosorbent assay, the levels of glutathione (GSH), superoxide dismutase (SOD) and reactive oxygen species (ROS) in myocardial tissues were measured by enzyme-linked immunosorbent assay, and the expression of AMPK, phosphorylated AMPK (p-AMPK), phosphorylated p38 MAPK (p-p38 MAPK), Nrf2 and heme oxygenase-1 (HO-1) in myocardium was determined by Western blot. Results:Compared with Sham group, the percentage of myocardial infarct size and serum CK-MB and LDH levels were significantly increased, the levels of GSH and SOD in myocardial tissues were decreased, ROS level was increased, and the expression of AMPK, p-AMPK, p-p38 MAPK, Nrf2 and HO-1 was up-regulated in I/R group ( P<0.05). Compared with I/R group, the percentage of myocardial infarct size and serum CK-MB and LDH levels were significantly increased, the levels of GSH and SOD in myocardial tissues were decreased, ROS level was increased, and the expression of AMPK, Nrf2 and HO-1 was down-regulated in C+ I/R group ( P<0.05). Conclusions:AMPK/p38 MAPK/Nrf2 signaling pathway is involved in the mechanism of endogenous antioxidant stress during myocardial I/R in diabetic rats.

Chlorella vulgaris is an effective supplement in counteracting non-alcoholic fatty liver disease-related complications through modulation of dyslipidemia, insulin resistance, and inflammatory pathways.

This study was aimed to investigate the effect of microalgae Chlorella vulgaris (C. vulgaris) on nonalcoholic fatty liver disease (NAFLD)-related complications induced by high-fat diet (HFD). Fifty adult male rats were divided into six groups. Control group and HFD group treated with or without C. vulgaris 5% and 10%. Biochemical parameters in serum were measured by spectrophotometric and enzyme-linked immunosorbent assay (ELISA) methods. The relative gene expression levels of Tumor Necrosis Factor-alpha (TNF-α), NF-kappa B (NF-ƙB), and p38 Mitogen-Activated Protein Kinases (p38 MAPK) in the liver were assessed by using quantitative real-time PCR, while the protein levels of NF-ƙB and TNF-α in the liver homogenate were determined by ELISA. The effects of HFD significantly were reversed by C. vulgaris, especially at a 10% dose. Therefore, it can be concluded that C. vulgaris therapeutically could be useful to improve NAFLD and its complications. PRACTICAL APPLICATIONS: It is established that NAFLD is associated with the resistance to insulin, dyslipidemia, and inflammation. Accordingly, modulating of these conditions may be useful in the management of NAFLD. Our results showed the effectiveness of C. vulgaris against NAFLD-related complication through the alleviating insulin resistance, dyslipidemia and also down-regulation of inflammatory genes in p38 MAPK/TNF-α/NF-ƙB pathway. The results of our study may be useful for scientist to prepare an effective supplement from C. vulgaris to overcoming NAFLD-related complications.

Rapamycin reduces orofacial nociceptive responses and microglial p38 mitogen-activated protein kinase phosphorylation in trigeminal nucleus caudalis in mouse orofacial formalin model.

The mammalian target of rapamycin (mTOR) plays a role in various cellular phenomena, including autophagy, cell proliferation, and differentiation. Although recent studies have reported its involvement in nociceptive responses in several pain models, whether mTOR is involved in orofacial pain processing is currently unexplored. This study determined whether rapamycin, an mTOR inhibitor, reduces nociceptive responses and the number of Fos-immunoreactive (Fos-ir) cells in the trigeminal nucleus caudalis (TNC) in a mouse orofacial formalin model. We also examined whether the glial cell expression and phosphorylated p38 (p-p38) mitogen-activated protein kinases (MAPKs) in the TNC are affected by rapamycin. Mice were intraperitoneally given rapamycin (0.1, 0.3, or 1.0 mg/kg); then, 30 min after, 5% formalin (10 µl) was subcutaneously injected into the right upper lip. The rubbing responses with the ipsilateral forepaw or hindpaw were counted for 45 min. High-dose rapamycin (1.0 mg/kg) produced significant antinociceptive effects in both the first and second phases of formalin test. The number of Fos-ir cells in the ipsilateral TNC was also reduced by high-dose rapamycin compared with vehicle-treated animals. Furthermore, the number of p-p38-ir cells the in ipsilateral TNC was significantly decreased in animals treated with high-dose rapamycin; p-p38 expression was co-localized in microglia, but not neurons and astrocytes. Therefore, the mTOR inhibitor, rapamycin, reduces orofacial nociception and Fos expression in the TNC, and its antinociceptive action on orofacial pain may be associated with the inhibition of p-p38 MAPK in the microglia.

PSD-95 protects the pancreas against pathological damage through p38 MAPK signaling pathway in acute pancreatitis.

Acute pancreatitis is one of the leading causes of gastrointestinal disorder-related hospitalizations, yet its pathogenesis remains to be fully elucidated. Postsynaptic density protein-95 (PSD-95) is closely associated with tissue inflammation and injury. We aimed to investigate the expression of PSD-95 in pancreatic acinar cells, and its function in regulating the inflammatory response and pancreatic pathological damage in acute pancreatitis. A mouse model of edematous acute pancreatitis was induced with caerulein and lipopolysaccharide in C57BL/6 mice. Tat-N-dimer was injected to inhibit the PSD-95 activity separately, or simultaneously with SB203580, inhibitor of p38 MAPK phosphorylation. Rat pancreatic acinar cells AR42J were cultured with 1 µM caerulein to build a cell model of acute pancreatitis. PSD-95-knockdown and negative control cell lines were constructed by lentiviral transfection of AR42J cells. Paraffin-embedded pancreatic tissue samples were processed for routine HE staining to evaluate the pathological changes of human and mouse pancreatic tissues. Serum amylase and inflammatory cytokine levels were detected with specific ELISA kits. Immunofluorescence, immunohistochemical, Western-blot, and qRT-PCR were used to detect the expression levels of PSD-95, p38, and phosphorylated p38. Our findings showed that PSD-95 is expressed in the pancreatic tissues of humans, C57BL/6 mice, and AR42J cells, primarily in the cytoplasm. PSD-95 expression increased at 2 h, reaching the peak at 6 h in mice and 12 h in AR42J cells. IL-6, IL-8, and TNF-α increased within 2 h of disease induction. The pancreatic histopathologic score was greater in the PSD-95 inhibition group compared with the control (P < 0.05), while it was lesser when phosphorylation of p38 MAPK was inhibited compared with the PSD-95 inhibition group (P < 0.05). Moreover, phosphorylation of p38 MAPK increased statistically after PSD-95 knocked-down. In conclusion, PSD-95 effectively influences the pathological damage of the pancreas in acute pancreatitis by affecting the phosphorylation of p38 MAPK.

p38 Mitogen-Activated Protein Kinase Is Involved in Interleukin-6 Secretion from Human Ligamentum Flavum-Derived Cells Stimulated by Tumor Necrosis Factor-α.

STUDY DESIGN: Human ligamentum flavum-derived cells (HFCs) were obtained from surgical samples for a basic experimental study. PURPOSE: We sought to evaluate the inflammatory response of human ligamentum flavum cells to investigate hypertrophic changes occurring in the ligamentum flavum. OVERVIEW OF LITERATURE: Lumbar spinal stenosis (LSS) is a disease commonly observed in the elderly. The number of patients with LSS has increased over time, yet the pathomechanisms of LSS still have not been fully elucidated. One of the clinical features of LSS is hypertrophy of the ligamentum flavum, which results in narrowing of the lumbar spinal canal. Some reports have suggested that ligamentum flavum hypertrophy is associated with inflammation and fibrosis; meanwhile, the p38 mitogen-activated protein (MAP) kinase is involved in the hypertrophy of human ligamentum flavum cells. METHODS: HFCs were obtained from patients with LSS who underwent surgery. HFCs were stimulated by tumor necrosis factor-α (TNF-α) and a p38 MAP kinase inhibitor, SB203580. Phosphorylation of the p38 MAP kinase was analyzed by western blotting. The concentration of interleukin-6 (IL-6) in the conditioned medium was measured by enzyme-linked immunoassay and IL-6 messenger RNA expression levels were determined by real-time polymerase chain reaction. RESULTS: TNF-α induced the phosphorylation of p38 MAP kinase in a time-dependent manner, which was suppressed by the p38 MAP kinase inhibitor, SB203580. TNF-α also stimulated IL-6 release in both a time- and dose-dependent manner. On its own, SB203580 did not stimulate IL-6 secretion from HFCs; however, it dramatically suppressed the degree of IL-6 release stimulated by TNF-α from HFCs. CONCLUSIONS: This is the first report suggesting that TNF-α stimulates the gene expression and protein secretion of IL-6 via p38 MAP kinase in HFCs. A noted association between tissue hypertrophy and inflammation suggests that the p38 MAP kinase inflammatory pathway may be a therapeutic molecular target for LSS.

Proof of pharmacology of Org 48775-0, a p38 MAP kinase inhibitor, in healthy volunteers.

AIM: To investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of the highly selective oral p38alpha/beta mitogen-activated protein (MAP) kinase inhibitor Org 48,775-0 in a first-in-human study. METHODS: In the single ascending dosing (SAD) study, an oral dose of Org 48,775-0 (0.3-600 mg) was evaluated in healthy males. In the multiple ascending dosing (MAD) study, levels of 30, 70 and 150 mg were dosed for six consecutive days, twice daily. Both studies were performed in a double-blind, randomized, placebo-controlled, cross-over fashion and evaluated pharmacokinetics, pharmacodynamics (ex vivo inhibition of lipopolysaccharide [LPS]-induced tumor necrosis factor (TNFα) release) and routine clinical and laboratory data. Pharmacokinetic and pharmacodynamic parameters of Org 48,775-0 were compared between healthy males and postmenopausal females, and the effect of a standardized fat meal was evaluated. RESULTS: All adverse events observed in the SAD (16; dizziness and headache, diarrhoea and catheter-related phlebitis) and MAD (43; mainly somnolence, dizziness, headache and nasopharyngitis) cohorts were mild, transient and completely reversible. Pharmacokinetics were linear up to single doses of 400 mg. Median Tmax ranged from 0.5 to 1.8 hours, geometric mean for T1/2 from 7.0 to 14.4 hours. Org 48,775-0 doses equal to and greater than 30 mg significantly inhibited LPS-induced TNFα release (42.3%; 95% CI = -65.2, -4.3) compared to placebo. In the MAD study, Org 48,775-0 treatment inhibited LPS-induced TNFα release during the entire steady-state period. Levels of inhibition amounted 30-75% for 30 mg, 53-80% for 70 mg and 77-92% for 150 mg Org 48,775-0. CONCLUSION: Org 48,775-0 has the capacity to significantly inhibit MAP kinase activity in humans without safety concerns.