1.Effects of combination of dihydroartemisinin and Huobahua on expression of p-p38 MAPK and ICAM-1 in mice with delayed-type hypersensitivity.
Min-Xia ZHAN ; Du WEI ; Li HENG-HUA ; Jin-Ping LUO ; Xiao-Li MEI ; Tu RU-XIA ; Li ZHANG
China Journal of Chinese Materia Medica 2020;45(22):5561-5566
The aim of this paper was to investigate the immunosuppressive effects of dihydroartemisinin and Huobahua compatibility in mice with delayed hypersensitivity and explore its possible mechanism. The delayed-type hypersensitivity(DTH) model in mice was established to observe the immunosuppressive effects of dihydroartemisinin and Huobahua compatibility in DTH mice. ELISA assay was used to detect the contents of interferon(IFN-γ); histopathological changes and degree of mononuclear infiltration of right ear tissues were examined by HE staining; the expression level of intercellular cell adhesion molecule-1(ICAM-1) in the right ear of mice was detected by immunohistochemistry; the protein expression levels of p38 phospho mitogen activated protein kinase(p-p38 MAPK) was detected by Western blot analysis. As compared with the control group, the degree of ear swelling, thymus/spleen index, serum IFN-γ as well as the number and degree of infiltration of monocytes were significantly increased in the model group. As compared with the model group, the degree of ear swelling and thymus/spleen index of the mice in the combination group were significantly reduced; the number and degree of infiltration of monocytes were significantly relieved; the serum levels of IFN-γ and the expression levels of p-p38 MAPK and ICAM-1 proteins in the right ear were also significantly reduced. The combination of dihydroartemisinin and Huobahua can significantly inhibit the DTH response, and it may regulate the production and secretion of related inflammatory factor IFN-γ by inhibiting the phosphorylation activity of p38 MAPK, thereby further reducing the expression of ICAM-1 and thus exerting the immunosuppressive effect.
Animals
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Artemisinins
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Intercellular Adhesion Molecule-1/genetics*
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Mice
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Monocytes
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p38 Mitogen-Activated Protein Kinases/genetics*
3.Total flavonoids of Drynariae Rhizoma regulates ER-p38 MAPK signaling pathway to improve scopolamine-induced learning and memory impairments in model mice.
De-Ping ZHAO ; Da-Long LI ; Yan-Hang ZHANG ; Yue CUI ; Hong-Dan XU ; Bo YANG ; Xia LEI ; Ning ZHANG
China Journal of Chinese Materia Medica 2021;46(22):5922-5929
This study intended to explore the effect and mechanism of total flavonoids of Drynariae Rhizoma in improving scopola-mine-induced learning and memory impairments in model mice. Ninety four-month-old Kunming(KM) mice were randomly divided into six groups. The ones in the model group and blank group were treated with intragastric administration of normal saline, while those in the medication groups separately received the total flavonoids of Drynariae Rhizoma, Kangnaoshuai Capsules, donepezil, as well as total flavonoids of Rhizoma Drynariae plus estrogen receptor(ER) blocker by gavage. The mouse model of learning and memory impairments was established via intraperitoneal injection of scopolamine. Following the measurement of mouse learning and memory abilities in Morris water maze test, the hippocampal ERβ expression was detected by immunohistochemistry, and the expression levels of ERβ and phosphorylated p38(p-p38) in the hippocampus and B-cell lymphoma 2(Bcl-2), Bcl-2-associated death promoter(Bad), and cysteinyl aspartate-specific protease-3(caspase-3) in the apoptotic system were assayed by Western blot. The contents of malondia-ldehyde(MDA), superoxide dismutase(SOD), and nitric oxide(NO) in the hippocampus were then determined using corresponding kits. Compared with the control group, the model group exhibited significantly prolonged incubation period, reduced frequency of cros-sing the platform, shortened residence time in the target quadrant, lowered ERβ, Bcl-2 and SOD activity in the hippocampus, and increased p-p38/p38, Bad, caspase-3, MDA, and NO. Compared with the model group, the total flavonoids of Rhizoma Drynariae increased the expression of ERβ and SOD in the hippocampus, down-regulated the expression of neuronal pro-apoptotic proteins, up-re-gulated the expression of anti-apoptotic proteins, and reduced p-p38/p38, MDA, and NO. The effects of total flavonoids of Drynariae Rhizoma on the above indexes were reversed by ER blocker. It has been proved that the total flavonoids of Drynariae Rhizoma obviously alleviate scopolamine-induced learning and memory impairments in mice, which may be achieved by regulating the neuronal apoptotic system and oxidative stress via the ER-p38 mitogen-activated protein kinase(ER-p38 MAPK) signaling pathway.
Animals
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Flavonoids
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Hippocampus
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Maze Learning
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Mice
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Polypodiaceae
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Receptors, Estrogen
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Scopolamine/toxicity*
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Signal Transduction
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p38 Mitogen-Activated Protein Kinases/genetics*
4.Effects of sodium nitroprusside on P38MAPK/STAT3 activation and telomerase reverse transcriptase mRNA expression in inducing apoptosis of K562 cell line.
Yong-Lie ZHOU ; Ya-Ping LÜ ; Huo-Xiang LÜ ; Lian-Nu QIU ; Wen-Song WANG ; Hui-Jun LIN ; Jian-Dong LIU
Journal of Experimental Hematology 2006;14(4):686-691
This study was aimed to investigate the activation of P38MAPK/STAT3 and expression of telomerase reverse transcriptase during sodium nitroprusside (SNP) inducing apoptosis of human leukemia cell line K562 and to explore the molecular mechanisms of SNP-inducing apoptosis in K562 cells. The K562 cell were treated with different concentrations of SNP and were cultured for different time. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and Annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantitate the in situ cell apoptosis. The expressions of phosphorylated p38MAPK or STAT3 were analysed by flow cytometry, while the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that SNP inhibited K562 cell growth. The K562 cell apoptosis was confirmed by typical cell morphology and DNA fragment, peak of sub-G1 phase, TUNEL and Annexin-V/PI labeling. A majority of K562 cells were arrested in G0/G1 phase. After treatment with SNP at 0.5-3.0 mmol/L, the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 increased first and decreased afterwards. Incubation of K562 cell with SNP (2 mmol/L) could increase the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 at 12 hours and 24 hours respectively, and down-regulated at 72 hours and 48 hours. SNP could decrease the expression of hTERT-mRNA in time-and dose-dependent manner. It is concluded that SNP can significantly induce K562 cells apoptosis, its mechanism may be related to the activation of P38MAPK and suppression of phosphorylated-STAT3 and hTRET-mRNA.
Apoptosis
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drug effects
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Humans
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K562 Cells
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Nitroprusside
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pharmacology
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RNA, Messenger
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biosynthesis
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genetics
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STAT3 Transcription Factor
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genetics
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metabolism
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Telomerase
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biosynthesis
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genetics
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p38 Mitogen-Activated Protein Kinases
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genetics
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metabolism
5.Effects of MD2 gene silencing on high glucose-induced proliferation inhibition, apoptosis and inflammation in rat cardiomyocytes.
Zhong-Min LIN ; Guo-Rong CHEN ; Quan-Bo ZHANG ; Fang WANG ; Lan-Ting XIANG ; Qiong-Jie CAO
Chinese Journal of Applied Physiology 2019;35(3):273-278
OBJECTIVE:
To investigate the effects of myeloid differentiation-2 (MD2) gene silencing on high glucose-induced proliferation inhibition, apoptosis and inflammation in rat cardiomyocytes.
METHODS:
The immortalized rat cardiomyocyte cell line H9C2 were transfected with MD2 small interfering RNA (si-MD2) and negative control for 24 h, then stimulated with high glucose (HG) for 48 h. RT-qPCR was performed to detect the mRNA levels of MD2 and inflammatory factors TNF-α, IL-1β and IL-6. MTS and flow cytometry were used to evaluate cell proliferation, cell cycle and apoptosis rate. Western blot was used to detect protein expression levels and phosphorylation levels.
RESULTS:
The mRNA and protein levels of MD2 in H9C2 cells were dramatically decreased after transfected with si-MD2 (P<0.01). After stimulation of high glucose, the mRNA levels of inflammatory factors, the cells in G0/G1 phase , the cell apoptosis rate and the protein level of cleaved Caspase-3 were significantly increased, while the cell proliferation ability was decreased (P<0.01). MD2 gene silencing antagonized the effects of high glucose on cell proliferation, cell cycle, cell apoptosis and the mRNA levels of TNF-α, IL-1β , IL-6(P<0.05). Western blot analysis showed that the phosphorylation levels of extracellular signal-regulated kinase(ERK1/2), P38 mitogen-activated protein kinase(P38 MAPK) and C-Jun N-terminal kinase(JNK) protein were increased significantly in H9C2 cells treated with high glucose, which could be reversed by silencing of MD2 (P<0.01).
CONCLUSION
This study demonstrates that MD2 gene silencing reverses high glucose-induced myocardial inflammation, apoptosis and proliferation inhibition via the mechanisms involving suppression of ERK, P38 MAPK, JNK signaling pathway.
Animals
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Apoptosis
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Cell Proliferation
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Cells, Cultured
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Cytokines
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metabolism
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Gene Silencing
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Glucose
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Inflammation
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JNK Mitogen-Activated Protein Kinases
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metabolism
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Lymphocyte Antigen 96
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genetics
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Myocytes, Cardiac
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cytology
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Rats
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p38 Mitogen-Activated Protein Kinases
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metabolism
6.Jiawei Baitouweng Decoction affects intestinal mucosal tight junction proteins in rats with ulcerative colitis through p38 MAPK-MLCK signaling pathway.
Pei-Pei ZHANG ; Xin YANG ; Guo-Qiang LIANG ; Hui-Ping ZHU ; Hui ZHU ; Meng WANG ; Hong-Wen SUN
China Journal of Chinese Materia Medica 2021;46(21):5719-5726
The aim of this paper was to explore the effect and mechanism of Jiawei Baitouweng Decoction(JWBTW) against ulcerative colitis(UC) from the perspective of intestinal mucosal tight junction proteins. From 60 SPF-grade male SD rats, 10 were randomly selected as the blank control, and the remaining 50 were treated with 3% dextran sodium sulfate(DSS) solution to induce UC and then randomized into the model group, mesalazine group, and low-, medium-, and high-dose JWBTW( L-JWBTW, M-JWBTW and H-JWBTW) groups, with 10 rats in each group. After successive medication for 14 days, the rat general conditions like body weight and stool were observed and the disease activity index(DAI) was calculated. The pathological changes in colon tissue was observed under a microscope for injury severity scoring and histopathological scoring. The serum endotoxin content was determined by limulus assay, followed by the measurement of protein expression levels of ZO-1, occludin, claudin-1, p38 MAPK, MLCK, MLC2 and p-MLC in colon tissue by Western blot. The results showed that compared with the blank group, the model group exhibited significantly reduced body weight, elevated DAI, injury severity and histopathological scores and serum endotoxin content, up-regulated protein expression levels of p38 MAPK, MLCK, MLC2 and p-MLC, and down-regulated ZO-1, occludin and claudin-1. Compared with the model group,mesalazine and JWBTW at each dose obviously increased the body weight, lowered the DAI, injury severity and histopathological scores and serum endotoxin content, down-regulated the protein expression levels of p38 MAPK, MLCK, MLC2 and p-MLC, and up-regulated the ZO-1, occludin and claudin-1, with the most obvious changes noticed in the H-JWBTW group. All these have indicated that JWBTW exerts the therapeutic effect against UC by inhibiting the activation of p38 MAPK/MLCK pathway, reversing the protein expression levels of occludin, claudin-1 and ZO-1, decreasing the serum endotoxin content, promoting the repair of intestinal mucosal mechanical barrier, maintaining the integrity of tight junctions, and reducing the permeability of intestinal mucosa.
Animals
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Colitis, Ulcerative/genetics*
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Disease Models, Animal
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Intestinal Mucosa
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Male
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Rats
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Rats, Sprague-Dawley
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Signal Transduction
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Tight Junction Proteins/genetics*
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p38 Mitogen-Activated Protein Kinases/genetics*
7.Mek and p38 MAPK-dependant pathways are involoved in the positive effect of interleukin-6 on human growth hormone gene expression in rat MtT/S somatotroph cells.
Feng-Ying GONG ; Jie-Ying DENG ; Yi-Fan SHI
Chinese Medical Sciences Journal 2008;23(2):73-80
OBJECTIVETo investigate the effect of interleukin-6 (IL-6) on the human growth hormone (hGH) gene expression in a rat somatotropic pituitary cell line MtT/S.
METHODSThe plasmids containing various lengths of hGH gene 5'-promoter fragments were constructed. Stably transfected MtT/S cells were created by cotransfecting the above plasmids and pcDNA3. 1(+) with DMRIE-C transfection reagent After the administration of these cells with IL-6 and/or various inhibitors of signaling transduction pathways, the luciferase activities in MtT/S cells lysis were assayed to demonstrate the effects of IL-6 on hGH gene promoter activity and possibly involved mechanism.
RESULTSThe 10(3) U/mL IL-6 stimulated GH secretion and synthesis, and promoted the 5'-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.69 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 micromol/L) and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 micromol/L) completely blocked the stimulatory effect of IL-6. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected IL-6 induction of hGH promoter activity. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-6. The results showed that the stimulatory effect of IL-6 was abolished following deletion of the -196 to - 132 bp fragment.
CONCLUSIONSIL-6 promotes GH secretion and synthesis by rat MtT/S somatotroph cells. The stimulatory effect of IL-6 on hGH gene promoter appears to require the activation of MEK and p38 MAPK, and a fragment of promoter sequence that spans the - 196 to - 132 bp of the gene, but may be unlinked with Pit-1 protein.
Animals ; Cell Line ; Gene Expression Regulation ; Human Growth Hormone ; genetics ; metabolism ; Humans ; Interleukin-6 ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; MAP Kinase Signaling System ; physiology ; Mitogen-Activated Protein Kinase Kinases ; genetics ; metabolism ; Promoter Regions, Genetic ; Rats ; Somatotrophs ; cytology ; metabolism ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism
8.Gene profiling of MAPK pathway in human osteosarcoma.
Guo-dong LI ; Zheng-dong CAI ; Yin-quan ZHANG ; Hai-yang GONG ; Hao TANG ; Qiu-lin ZHANG
Chinese Journal of Oncology 2009;31(5):340-345
OBJECTIVETo explore the functional effects of MAPK pathway in the pathogenesis of human osteosarcoma.
METHODSGene microarray (Human Genome U133A, Affymetrix) was used to screen the differential expression of genes involved in MAPK pathway between osteosarcoma cell lines and 3 osteoblastic cell lines. KEGG metabolic pathway analysis was performed among significantly increased or decreased genes using the MATLAB software. Immunohistochemical technique was used to detect the expressions of ERK1/2, JNK and p38 proteins among 48 osteosarcoma and benign 24 osteoblastic tumor samples.
RESULTSUsing an entrance limit of > or = 2.0, 18 differentially expressed MAPK pathway-related genes were selected (10 up-regulated, 8 down-regulated) to mapped to the MAPK pathway of KEGG which are all important node genes. The positive rates of ERK1/2, JNK and p38 proteins were 83.3% (40/48), 72.9% (35/48) and 85.4% (41/48) in osteosarcomas,and 12.5% (3/24), 8.3% (2/24) and 16.7% (4/24) in the control group, respectively. The positive rates and expression intensities were statistically different between the 2 groups (P<0.01).
CONCLUSIONMAPK pathway plays an important role in the pathogenesis of osteosarcoma. ERK, JNK and p38 form an intercoordinating network and regulate the cell proliferation, differentiation, apoptosis, invasion and migration in osteosarcoma.
Adolescent ; Adult ; Aged ; Bone Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Child ; Female ; Gene Expression Profiling ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Middle Aged ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Oligonucleotide Array Sequence Analysis ; Osteoblastoma ; genetics ; metabolism ; pathology ; Osteosarcoma ; genetics ; metabolism ; pathology ; Signal Transduction ; Young Adult ; p38 Mitogen-Activated Protein Kinases ; metabolism
9.Pioglitazone inhibits the expression of nicotinamide adenine dinucleotide phosphate oxidase and p38 mitogen-activated protein kinase in rat mesangial cells.
Shan WANG ; Shan-Dong YE ; Wen-Jia SUN ; Yuan-Yuan HU
Chinese Medical Journal 2013;126(21):4054-4059
BACKGROUNDOxidative Stress and p38 mitogen-activated protein kinase (p38MAPK) play a vital role in renal fibrosis. Pioglitazone can protect kidney but the underlying mechanisms are less clear. The purpose of this study was to investigate the effect of pioglitazone on oxidative stress and whether the severity of oxidative stress was associated with the phosphorylation level of p38MAPK.
METHODSRat mesangial cells were cultured and randomly assigned to control group, high glucose group and pioglitazone group. After 48-hour exposure, the supernatants and cells were collected. The protein levels of p22(phox), p47(phox), phosphorylated p38MAPK, total p38MAPK were measured by Western blotting. The gene expressions of p22(phox), p47(phox) were detected by RT-PCR. The levels of intracellular reactive oxygen species (ROS) were determined by flow cytometry. The levels of superoxide dismutase (SOD) and maleic dialdehyde (MDA) in the supernatant were determined respectively.
RESULTSCompared with the control group, the expression levels of p22(phox), p47(phox), phospho-p38 and ROS significantly increased, activity of SOD decreased in high glucose group, while the level of MDA greatly increased (P < 0.01). Pioglitazone significantly suppressed p22(phox), p47(phox) expressions and oxidative stress induced by high glucose. The expressions of p22(phox), p47(phox), phospho-p38MAPK and ROS generation were markedly reduced after pioglitazone treatment (P < 0.05). The activity of SOD in the the supernatant increased (P < 0.05), while the level of MDA decreased greatly by pioglitazone (P < 0.05). The level of oxidative stress was associated with the phosphorylation level of p38MAPK (P < 0.01).
CONCLUSIONPioglitazone can inhibit oxidative stress through suppressing NADPH oxidase expression and p38MAPK phosphorylation.
Animals ; Blotting, Western ; Glucose ; pharmacology ; Mesangial Cells ; drug effects ; enzymology ; NADPH Oxidases ; genetics ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism ; Thiazolidinediones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism
10.P38MAPK pathway regulates COX-2 and caspase-3 expression in a mouse model of Parkinson disease.
Zi-feng WEI ; Yong-sheng WANG ; Li-ren MA ; Qian WANG ; Zuo-feng ZHANG ; Yu-xin ZHANG
Journal of Southern Medical University 2009;29(10):2010-2017
OBJECTIVETo investigate the effect of p38 mitogen-activated protein kinase (p38MAPK) on the expression of COX-2 and caspase-3 in the substania nigra (SN) of mice with MPTP-induced Parkinson disease (PD).
METHODSC57BL/CN mice were treated with MPTP to prepare a subacute PD model, and their behavioral changes following the treatment were observed. Immunohistochemistry and Western blotting were performed to detect the expression of tyrosine hydroxylase (TH), COX-2 and phosphorylation of P38MAPK in the SN and their changes following treatment with SB203580, a specific inhibitor of P38MAPK.
RESULTSThe 7-day model group showed typical symptoms of PD with decrements of TH-positive neurons and TH protein level in the SN of the midbrain by about 65% and 75%, respectively (P<0.01). In the 3-day model group, the COX-2-, caspase-3- and phosphorylated P38MAPK-immunoreactive cells and their protein levels in the SN increased markedly with obvious loss of TH-positive neurons. Administration of SB203580 obviously lessened the above changes (P<0.01).
CONCLUSIONP38MAPK regulates the inflammation and apoptosis in the SN of the mouse model of subacute PD, and SB203580 may provide some neuroprotective effect.
Animals ; Caspase 3 ; genetics ; metabolism ; Cyclooxygenase 2 ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Parkinson Disease ; metabolism ; Signal Transduction ; Substantia Nigra ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism