Epilepsy is a disorder of the brain in which sudden abnormal discharges of neurons cause transient dysfunction and is a common disorder of the nervous system. Although most patients experience remission of symptoms with medication, about 20 ~ 30% of patients still have poor outcomes with medication and progress to refractory epilepsy. The etiology of epilepsy is complex and the exact pathogenesis is not yet clear. Current research has explored the pathophysiological mechanisms underlying epileptogenesis, thus providing a basis for identifying potential therapeutic targets for epilepsy and advancing the precision treatment of epilepsy. p38 Mitogen-activated protein kinase (MAPK) signalling pathway is a conserved class of kinases involved in many physiological/pathological processes by regulating intracellular gene expression levels, cell division, differentiation and apoptosis in response to various extracellular stimuli in order to mediate intracellular signalling cascades. The p38 MAPK signalling pathway is one of the subfamilies of MAPK that mediates inflammatory responses, apoptosis, tissue edema and other biological processes involved in the development of central nervous system diseases. The p38 MAPK signalling pathway is now reviewed for its involvement in the development of epilepsy through unused pathways, in order to identify new potential targets for epilepsy treatment and provide clinical precision.
Objective To investgate the expression of p38 mitogen-activated protein kinase （p38MAPK） in lung tissue of rats with severe acute pancreatitis （SAP）， and to explore the relationship between p38MAPK and pulmonary capillary barrier injury. Methods Forty male and healthy Sprague-Dawley （SD） rats were randomly （random number method） divided into sham operation （SO） group and SAP group， then rats of SAP group were sub-divided into 3， 6， 12， and 24 h group， each group enrolled 8 rats， respectively. SAP model rats were established by injecting 5% sodium taurocholate solution retrograde into the biliopancreatic duct. ELISA method was used to test the serum tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β）， and pathological changes in lung and pancreas tissues were observed by HE staining. Immunohischemistry method was used to detect phosphorylated p38 （p-p38） protein and aquaporin 1 （AQP1） protein of lung tissues. The expression level of AQP1 mRNA was measured by quantitative real-time PCR. Results Hyperemia， edema， and inflammatory cell infiltration were observed in lung tissues， abundance of necrosis， part gland structure fuzzy or even disappear were observed in pancreas tissues of all 4 time point groups. Compared with SO group， levels of serum TNF-α and IL-1β were significantly higher in 4 time point groups （P＜0.05）. Lower expression level of p-p38 protein was detected in lung tissues of SO group， while in the early stage of SAP （SAP 3 h group）， the expression level of p-p38 protein significantly increased， which peaked in 6 h group and was still higher than SO group in 24 h group （P＜0.05）. Compared with SO group， the expression levels of AQP1 mRNA and protein were significantly lower in all 4 time point groups （P＜0.05）， which had negative correlation with the levels of serum TNF-α，IL-1β， and the expression level of p-p38 protein （r=-0.87， P＜0.05；r=-0.88， P＜0.05；r=-0.78， P＜0.05）. Conclusion The decrease of AQP1 protein in lung tissue is one of the vital causes for pulmonary capillary barrier injury in SAP， which probably works by the activation of p38MAPK and the excessive release of inflammatory cytokines.
Myocardial remodeling is one of the important pathological basis when myocardial infarction or pressure overload occurs, whereas mangiferin which is a naturally occurring xanthone has a broad range of therapeutic effect on postinfarction myocardial remodeling. Mangiferin attenuates myocardial infarction by preventing the accumulation of myocardial collagen and the development of intercellular fibrosis. Mangiferin's inhibition to p38 mitogen activated protein kinases plays an important role in the cardioprotective effect. Inhibition of p38 mitogen activated protein kinases significantly decreases TNF-α and then brings the cardioprotective effect. Similarly, p38 mitogen activated protein kinases in pressure overload disease also play a very important role. Understanding of these has direct implications for clinical therapy.
ObjectiveTo investigate whether the miR-33s negatively regulates LPS-induced production of inflammatory cytokines by targeting p38 MAPK. MethodsHuman monocytes THP-1 cells were cultured in vitro and transfected with miR-33s mimic (25 nmol/L) or miR-33s inhibitor (25 nmol/L)by TransIT-X2® Dynamic Delivery System for 24 h. Then the transfected THP-1 cells were stimulated by LPS of 10.0 ng/mL for 24 h. The expression of miR-33s and p38 MAPK protein were measured by semi-quantitative RT-PCR. The concentrations of TNF-α,IL-6 and IL-1β in the cultured supernatant were assessed by ELISA. ResultsThe transfection of miR-33s mimic significantly increased the release of TNF-α,IL-6 and IL-1β(P<0.05). The expression of p38 MAPK protein was also significantly reduced(P<0.05). However,the pre-treatment of miR-33s inhibitor reversed the LPS-induced release of TNF-α,IL-6,and IL-1β,and the expression of p38 MAPK protein of THP-1 cells. ConclusionmiR-33s may play an important role in the regulation in inflammatory factors released from THP-1 cells by targeting p38 MAPK.
ObjectiveTo investigate the effects and mechanisms of G protein-coupled receptor 91 (GPR91) on blood-retinal barrier (BRB) in diabetic rats. MethodsA lentiviral vector of shRNA targeting rat GPR91 and scrambled shRNA were constructed. Healthy male Sprague-Dawley (SD) rats were selected in this study. The 60 rats were randomized into 4 groups and treated as follows:(1) control group (Group A, n=15), the rats received injections of an equal volume of 0.1% citrate buffer; (2) streptozocin (STZ) group (Group B, n=15), the rats received injections of STZ; (3) LV.shScrambled group (Group C, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml scrambled shRNA lentiviral particles at 2 weeks after the induction of diabetes; (4) LV.shGPR91 group (Group D, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml pGCSIL-GFP-shGPR91 lentiviral particles. At 12 weeks after intravitreal injection, immunohistochemistry and Western blot were used to assess the expression of GPR91, p-extracellular signal-regulated kinase(ERK)1/2, t-ERK1/2, p-Jun N-terminal kinase (JNK), t-JNK, p-p38 mitogen-activated protein kinase (MAPK) and t-p38 MAPK. Haematoxylin and eosin (HE) staining and Evans blue dye were used to assess the structure and function of the retinal vessel. Immunohistochemistry enzyme-linked immunosorbent assay (ELISA) was used to test the protein level of VEGF. ResultsImmunohistochemistry staining showed that GPR91 was predominantly localized to the cell bodies of the ganglion cell layer. Western blot showed that GPR91 expression in Group D decreased significantly compared with Group C (F=39.31, P < 0.01). HE staining showed that the retina tissue in Group B and C developed telangiectatic vessels in the inner layer of retina, while the telangiectatic vessels attenuated in Group D. It was also demonstrated in Evans blue dye that the microvascular leakage in Group D decreased by (33.8±4.11)% compared with Group C and there was significant difference (F=30.35, P < 0.05). The results of ELISA showed the VEGF secretion of Group B and C increased compared with Group A and the VEGF expression in Group D was significantly down regulated after silencing GPR91 gene (F=253.15, P < 0.05).The results of Western blot indicated that compared with Group A, the expressions of p-ERK1/2, p-JNK and p-p38 MAPK were significantly upregulated (q=6.38, 2.94, 3.45;P < 0.05). Meanwhile, the activation of ERK1/2 was inhibited by GPR91 shRNA and the difference was statistically significant (F=22.50, P < 0.05). ConclusionsThe intravitreal injection of GPR91 shRNA attenuated the leakage of BRB in diabetic rats. GPR91 regulated the VEGF release and the leakage of BRB possibly through the ERK1/2 signaling pathway.
Abstract： Objective To investigate the acute cardioprotective effect of 17b-estradiol (17b-E2) against severe myocardial ischemia/reperfusion (I/R) injury in rabbits and the mechanism of the effect. Methods We established the model of myocardial I/R in vivo by occluding the left anterior descending coronary artery of the rabbits (who underwent coronary occlusion for 40 minutes followed by 3 hours of reperfusion). Twentyfour New Zealand white male rabbits were randomly divided into two groups with 12 in each group. Before coronary occlusion, 1 ml of ethanol or 17b-E2 at 10 μg/kg was administered intravenously to the rabbits in the control group and the experimental group respectively. The serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by enzymelinked immunosorbent assay (ELISA) at the following time points: before occlusion, 40 minutes after occlusion, 1 hour, 2 hours and 3 hours after reperfusion. Activation of p38 mitogen activated protein kinase(MAPK) was determined by Western blotting analysis, and apoptosis of cardiocytes was identified by terminal deoxynucleotidlyl transferase mediated deoxyuridinebiotin dUTP Nick End Labeline (TdT)mediated dNTP nick end labeling (TUNEL) staining. Results During myocardial ischemia, TNF-α decreased significantly in the experimental group compared with the control group (F=0.007,P=0.001), while there was no difference in IL-6 between the two groups (F=0.616,P=0.095). During the process of reperfusion, the levels of TNF-α and IL-6 in the experimental group were significantly lower than those in the control group (Plt;0.01). Besides, the activation of p38 MAPK and apoptotic index for the experimental group were also lower (45.07%±2.73% vs. 61.25%±2.41%, t＝－15.398, P=0.000; 11.21%±3.85% vs. 22.02%±4.49%, t＝－6.332, P=0.000). Conclusion The cardioprotective effect of 17b-E2 against myocardial I/R may be attributed to its antiinflammatory and antiapoptotic properties, which is probably associated with the inhibition of 17bE2 on p38MAPK activity.
Objective To investigate changes of TLR2 mRNA expression in lung of a mouse model of Chlamydia Pneumoniae pneumonitis, and to explore the possible mechanism of signal transduction. Methods Ninety-six male C3H/HeJ mice were randomly divided into four groups as follows: a control group, a model group, a SB203580 intervened group, and a pyrrolidine dithiocarbamate( PDTC) intervened group. Chlamydia Pneumoniae pneumonitis was induced by intranasally inoculated with 4. 0 ×106 IFU/mL of C. Pneumoniae per mouse in the model group and two intervened groups. Then the intervened groups were intraperitoneally injected with the p38MAPK inhibitor SB203580 and nuclear factor kappa B ( NF-κB)inhibitor PDTC, respectively. Six mice in each group were randomly killed in 1st, 4th, 7th and 14th day. The expressional changes of TLR2 mRNA in the mice lung tissue were measured by semi-quantitative RT-PCR. The concentrations of TNF-α in the lung homogenate were measured by ELISA. Results TLR2 mRNA expression in the lung tissue significantly increased after C. Pneumoniae infection, peaking at 4th and 7th days, then decreased after 14th day. Tumor necrosis factor-α( TNF-α) was also elevated in the lung tissue after C. Pneumoniae challenging. Both SB203580 and PDTC treatment effectively inhibited TNF-αand TLR2 mRNA expressions in lung. The inhibitory effect was more obvious by SB203580 treatment. Conclusion C. Pneumoniae can upregulate the expressions of TLR2 and TNF-α in lung, and TLR2/MAPK and TLR2 /NF-κB signal pathways may be involved in Chlamydia Pneumoniae pneumonitis.
Objective To investigate whether p38 mitogen activated protein kinase (p38MAPK) inhibitor can reduce acute lung injury (ALI) caused by lipopolysaccharide (LPS) by regulating Th17/Treg balance. Methods Balb/c mice were randomly divided into a control group, an ALI group and an intervention group. The mice in the control group were injected with phosphate-buffered saline, the mice in the ALI group were intraperitoneally injected with 40 mg/kg LPS, and the mice in the intervention group were injected with SB203580 (0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg) intraperitoneally 1 h prior to the intraperitoneal injection of LPS. All mice were killed on 12 h later respectively. Hematoxylin-eosinstin staining was used to observe the pathological changes of lung tissue, and cell classification, counting, and total protein levels in bronchoalveolar lavage fluid (BALF) were detected. Transcript expression of forkhead box p3 (Foxp3) and retinoic acid receptor-related orphan receptor-γt (RORγt) was detected by real-time polymerase chain reaction. Interleukin (IL)-6, IL-10, IL-17, IL-23 and transforming growth factor-β (TGF-β) in lung tissue and IL-6, tumor necrosis factor-α (TNF-α) in serum were measured by enzyme-linked immunosorbent assay. The Th17 and Treg subset distribution in spleen was determined by flow cytometry. Results Histopathological examination showed that LPS induced inflammatory cell infiltration in lung tissue, increased cell count and protein levels in BALF (P<0.05), and increased proportion of neutrophils and monocytes in the ALI mice. SB203580 significantly attenuated tissue injury of the lungs in LPS-induced ALI mice. Serum levels of IL-6 and TNF-α in the ALI group were significantly higher than those in the control group, and inflammatory cytokines were decreased after SB203580 intervention. Compared with the ALI group, the production of inflammatory cytokines associate with Th17, including IL-17, IL-23, RORγt was inhibited, and the production of cytokines associate with Treg, such as IL-10 and Foxp3 in lung tissue was increased in the intervention group in a concentration-dependent manner with SB203580. After SB203580 intervention, Th17/Treg ratio was significantly decreased compared with the LPS group (P<0.05). Conclusion p38MAPK inhibitor can reduce LPS-induced ALI by regulating the imbalance of Treg cells and Th17 cells.
Objective To investigate the feasibility of selenium-methylselenocysteine (SMC) to promote peripheral nerve regeneration and its mechanism of action. Methods Rat Schwann cells RSC96 cells were randomly divided into 5 groups, which were group A (without any treatment, control group), group B (adding 100 μmol/L H2O2), group C (adding 100 μmol/L H2O2+100 μmol/L SMC), group D (adding 100 μmol/L H2O2+200 μmol/L SMC), group E (adding 100 μmol/L H2O2+400 μmol/L SMC); the effect of SMC on cell proliferation was detected by MTT method, and the level of oxidative stress was detected by immunofluorescence for free radicals [reactive oxygen species (ROS)] after determining the appropriate dose group. Thirty-six 4-week-old male Sprague Dawley rats were randomly divided into 3 groups, namely, the sham operation group (Sham group), the sciatic nerve injury group (PNI group), and the SMC treatment group (SMC group), with 12 rats in each group; the rats in the PNI group were fed with food and water normally after modelling operation, and the rats in the SMC group were added 0.75 mg/kg SMC to the drinking water every day. At 4 weeks after operation, the sciatic nerves of rats in each group were sampled for neuroelectrophysiological detection of highest potential of compound muscle action potential (CMAP). The levels of inflammatory factors [interleukin 17 (IL-17), IL-6, IL-10 and oxidative stress factors catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA)] were detected by ELISA assay. The luxol fast blue (LFB) staining was used to observe the myelin density, fluorescence intensity of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) was observed by immunofluorescence staining, and myelin morphology was observed by transmission electron microscopy with measurement of axon diameter. Western blot was used to detect the protein expressions of p38 mitogen-activated protein kinases (p38MAPK), phosphorylated p38MAPK (p-p38MAPK), heme oxygenase 1 (HO-1), and nuclear factor erythroid 2-related factor 2 (Nrf2). ResultsMTT assay showed that the addition of SMC significantly promoted the proliferation of RSC96 cells, and the low concentration could achieve an effective effect, so the treatment method of group C was selected for the subsequent experiments; ROS immunofluorescence test showed that group B showed a significant increase in the intensity of ROS fluorescence compared with that of group A, and group C showed a significant decrease in the intensity of ROS fluorescence compared with that of group B (P<0.05). Neuroelectrophysiological tests showed that the highest potential of CMAP in SMC group was significantly higher than that in PNI and Sham groups (P<0.05). ELISA assay showed that the levels of IL-6, IL-17, and MDA in PNI group were significantly higher than those in Sham group, and the levels of IL-10, SOD, and CAT were significantly lower; the levels of IL-6, IL-17, and MDA in SMC group were significantly lower than those in PNI group, and the levels of IL-10, SOD, and CAT were significantly higher (P<0.05). LFB staining and transmission electron microscopy showed that the myelin density and the diameter of axons in the SMC group were significantly higher than those of the PNI group and the Sham group (P<0.05). Immunofluorescence staining showed that the fluorescence intensity of GFAP and MBP in the SMC group were significantly stronger than those in the PNI group and Sham group (P<0.05). Western blot showed that the relative expressions of Nrf2 and HO-1 proteins in the SMC group were significantly higher than those in the PNI group and Sham group, and the ratio of p-p38MAPK/p38MAPK proteins was significantly higher in the PNI group than that in the SMC group and Sham group (P<0.05). Conclusion SMC may inhibit oxidative stress and inflammation after nerve injury by up-regulating the Nrf2/HO-1 pathway, and then inhibit the phosphorylation of p38MAPK pathway to promote the proliferation of Schwann cells, which ultimately promotes the formation of myelin sheaths and accelerates the regeneration of peripheral nerves.
Objective To investigate the gene expression of p38mitogen-activated protein kinase (p38MAPK) and its upstream signaling molecule (mkk3 and mkk6) in fetal skin at different developmental stages and postnatal skin and its potential biological significance. Methods The fetal skin biopsies were obtained from human embryo of spontaneous abortion at gestational ages from 13 to 32 weeks and postnatal skin specimens were collected from patients(4-16 years) undergoing plastic surgery. After the morphological characteristics of skins at different developmental stages were detected with pathological methods, the gene expressions of p38MAPK, mkk3 and mkk6 in skins were examined with reverse transcriptionpolymerase chain reaction analysis (RT-PCR). Results The gene expressions of p38MAPK, mkk3 and mkk6 could all be detected in fetal and postnatal skins. In fetal skins, these 3 genes were bly expressed. Along with fetal growth and development, the gene expressions of p38MAPK and its upstream signaling molecules were faded gradually. In postnatal skin, the mRNA contents of these 3 genes were significantly decreased in comparison with those in fetal skin (Plt;0.01). Conclusion p38 MAPK mediated signal pathways might be involved in the skin developmentat embryonic stage and in the determination of cutaneous structure and function, and also in wound healing at postnatal stage. The relative increment of these gene transcription in younger fetal skin might be one of the reasons why cutaneous cells proliferate rapidly and the wounds heal without scar.