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.
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.
【Abstract】ObjectiveTo explore the effects of p38 mitogenactivated protein kinase (MAPK) on apoptosis of small intestinal epithelial cells after transplantation in rats. MethodsSmall intestinal transplantation was performed in SD and Wistar rats. The recipients were divided into three groups: isograft group (Wistar→Wistar group), allograft group (SD→Wistar group) and allograft+cyclosporine A group (SD→Wistar+CsA group). The grafts were harvested on day 1, 3, 5 and 7 after operation. All graft samples were subjected to histological examination. The apoptosis of graft epithelial cells was detected by TUNEL method. p38 MAPK was measured by Westernblotting method and serum TNFα was determined by ELISA. ResultsMild, moderate and severe rejection reaction occurred in the SD→Wistar group, it was showed that the number of apoptotic cells increased with the severity of the rejection reaction by TUNEL. In SD→Wistar group, the numbers of apoptotic cells were significantly higher than those of the other two groups （P＜0.01）. The severity of rejection reaction in SD→Wistar+CsA group was less than that of SD→Wistar group and the number of apoptotic cells increased with the severity of the rejection reaction (P＜0.01). The level of serum TNFα varied with the apoptotic degree of small intestinal epithelial cells in SD→Wistar group and SD→Wistar+CsA group （P＜0.01）. The expression of p38 MAPK increased with the number of the apoptotic cells in SD→Wistar group and SD→Wistar+CsA group （P＜0.01）, but there was no evident change in Wistar→Wistar group （Pgt;0.05）. The expression of p38 MAPK and the level of serum TNFα were positively correlated with apoptosis in small intestinal rejection after transplantation （r=0.875， P＜0.01； r=0.837， P＜0.01）. p38 MAPK and TNFα were also positively correlated （r=0.826，P＜0.01）. ConclusionApoptosis plays an important role in small intestinal rejection. p38 MAPK is involved in apoptosis and is an important regulator in signal pathway of cell apoptosis.
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 whether Chlamydia pneumoniae alters the expression of TLR2 mRNA and p38 MAPK mRNA in mice with Chlamydia pneumoniae infection in TLR2-p38 MAPK-dependent pathway, subsequently leading to the release of cytokines. Methods Seventy-two male C3H/HeJmice were randomly divided into three groups as follow: a normal control group, a C. pneumoniae-inoculated group, and a C. pneumoniae-inoculated with SB203580 treatment group. The mice in the three groups were sacrificed on 1st, 4th, 7th, 14th day separately, and lung tissues were sampled for measurement. The expression changes of TLR2 mRNA and p38 MAPK mRNA in the mice lung tissue were measured by semi-quantitative RT-PCR. The concentrations of TNF-αin the lung tissue were measured by ELISA.Results Compared with those in the normal group, the expressions of TLR2 mRNA and p38MAPK mRNA in the lung tissue increased quickly after C. pneumoniae infection, which was especially obvious on day 4 and on day 7, the expression level of TLR2 mRNA on day 7 was markedly higher than that of the normal group [ ( 7. 24 ±1. 78) mg/L vs.( 0. 64 ±0. 14) mg/L, P lt;0. 05] ; The expression level of p38 MAPK mRNA on day 4 was markedly higher than that of the normal group [ ( 9. 267 ±1. 813) mg/L vs. ( 3. 734 ±0. 946) mg/L, P lt;0. 05] . After 14 days, C. pneumoniae infection of mice was attenuated, the concentration of TNF-α in the lung tissue increased, and was clearly higher than that of the normal control group, peaking on day 4 [ ( 77. 29 ±9. 66) pg/mg] . Treatment with SB203580 could effectively inhibit TLR2 mRNA and p38 MAPK mRNA expression in lung, which was especially obvious on day 4 and on day 7. The expression level of TLR2 mRNA on day 7 was ( 0. 269 ±0. 09) mg/L, and the expression level of p38 MAPK mRNA on day 7 [ ( 0. 002 ±0. 001) mg/L] was even more obviously attenuated, the concentration of TNF-α in the lung tissue markedly decreased when compared with that in the infected group, and its concentration on day 4 [ ( 25. 76 ±3. 49) pg/mg] lowered more clearly. Conclusions The alteration of TLR2-p38 MAPKdependent signal pathway in lungs is closely connected with Chlamydia pneumoniae infection. SB203580 treatment can effectively controll the elevation of TLR2 mRNA and p38 MAPK mRNA expressions in lung. It can effectively control the TLR2-MAPK signal transduction pathway.
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.
ObjectiveTo investigate the effect of erigeron breviscapus (EBHM) on ocular hypertension and the protective effect of retinal ganglion cells (RGCs) in rats by regulating mitogen activated protein kinase (MAPK) signaling pathway.MethodsSixty male Sprague-Dawley rats were divided into control group, model group, low-dose EBHM group (group A), medium-dose EBHM group (group B), and high-dose EBHM group (group C) by random number table method. There were 12 rats in the group, the left eye was used as the experimental eye. The rats of model group, group A, group B, and group C were infused with normal saline through the anterior chamber to construct an acute ocular hypertension model; the control group was given general anesthesia only. Then, 2-30 days after modeling, rats in the control group and model group were given 3 ml of normal saline once a day; rats in group A, group B, and group C were given 0.30, 0.45, and 0.60 g/100 g EBHM by intragastric administration, respectively, 1 time/d. The rat intraocular pressure was measured before modeling and 1, 14, and 30 days after modeling, and the proportion of high intraocular pressure model was measured. Thirty days after modeling, hematoxylin-eosin (HE) staining was used to observe the pathological changes of retinal tissue; immunofluorescence staining was used to detect the changes in the number of RGCs; real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect p38 in the retinas of rats in each group. The relative expression of MAPK and Caspase-3 mRNA; western blot was used to detect p38MAPK and phosphorylation in the retina of rats in each group relative expression of phosphorylate-p38MAPK (p-p38MAPK) and Caspase-3 protein. One-way analysis of variance was used for multi-sample comparison, and SNK-q test was used for comparison between two samples.ResultsOne day after modeling, none of the rats in the control group developed acute ocular hypertension, and the other groups were successfully modeled. Compared with the model group, the rates of acute ocular hypertension at 14 days after modeling in groups B and C were lower (χ2=98.701, P＜0.05), and the rates of acute ocular hypertension at 30 days after modeling in groups A, B, and C were 0. There was no statistically significant difference in the rates of acute ocular hypertension between 14 and 30 days after modeling in the A, B, and C groups (P＞0.05). The results of HE staining showed that the structure of the retina in the control group was complete, and the layers were clearly visible; the RGCs count was not abnormal, and the morphology was plump and round. The retina of rats in the model group became thinner; the number of RGCs was greatly reduced, the morphology was vacuolated, and the arrangement was sparse. The retina of rats in groups A, B, and C became thicker, and the number of RGCs increased, and the retina structure in group C was better restored. The results of immunofluorescence staining showed that the RGCs counts of rats in groups A, B, and C were higher than those in the model group, and the difference was statistically significant (F=297.514, P＜0.05); pairwise comparison between groups, group A was lower than that of group B and C Group (q=2.842, 5.263), group B was lower than group C (q=2.457), the difference was statistically significant (P＜0.05). The results of RT-qPCR and Western blot showed that compared with the model group, the relative expression of Caspase-3 mRNA (F=267.912) and protein (F=692.279) and the relative expression of p-p38MAPK protein in the retina of rats in groups A, B and C. The expression level (F=150.061) all decreased, and the difference was statistically significant (P＜0.05); pairwise comparisons between groups showed that Caspase-3 mRNA (q=6.977, 15.642) and protein (q=6.997, 15.642) relative expression levels and p-p38MAPK protein (q=12.443, 24.358) relative expression levels are lower than groups A and B, group B was lower than group A (q=11.678, 12.471, 10.204), the difference was statistical academic significance (P＜0.05).ConclusionsEBHM can significantly reduce intraocular pressure in rats with acute ocular hypertension, increase RGCs counts, and reduce retinal damage. Its regulatory mechanism may be related to the MAPK pathway.
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.
ObjectiveTo investigate the regulatory effect of simvastatin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at middle/late stages by p38MAPK pathway under condition of osteoinductive environment. MethodsThe bone marrow of bilateral femur and tibia were harvested from 20 4-week-old female Sprague Dawley rats. BMSCs were isolated and cultured with whole bone marrow culture method; the second generation of cells were randomly divided into 5 groups: control group (complete medium, CM), simvastatin group (simvastatin medium, SIM), osteogenic induction group (osteogenic induction medium, OM), simvastatin and osteogenic induction group (simvastatin+osteogenic induction medium, OM+SIM), and blocker group (SB203580+simvastatin+osteogenic induction medium, OM+SIM+SB). MTT assay was used to detect the cell activity in CM group and SIM group at 2, 3, 4, 5, and 6 days, ELISA method to measure the content of alkaline phosphatase (ALP) in OM group and OM+SIM group at 7 and 14 days. The mRNA and protein expressions of osteocalcin (OCN) were detected by real-time quatitative PCR and Western blot after 1, 12, and 24 hours of osteogenic induction at 21 and 28 days. The protein expressions of phospho-p38 (p-p38) and p38 in OM group, OM+SIM group, and OM+SIM+SB group were detected by Western blot at the best induction time of simvastatin. ResultsMTT assay showed that no significant difference was found in absorbance (A) value between CM group and SIM group at each time point (P > 0.05), indicating no effect of 1×10-7 mol/L simvastatin on cell viability. ELISA results showed that ALP content significantly increased in OM+SIM group when compared with OM group at 7 and 14 days; the ALP content was significantly higher at 7 days than 14 days in OM group and OM+SIM group (P < 0.05). OCN mRNA and protein expressions at 12 hours were significantly higher than those at other time points in each group (P < 0.05), and the expressions of OM+SIM group was significantly higher than those of OM group (P < 0.05). The best induction time of simvastatin was 12 hours. At 12 hours after blocking intervention, the p-p38/p38 in OM+SIM+SB group was significantly lower than that in OM group and OM+SIM group (P < 0.05), and the p-p38/p38 in OM+SIM group was significantly higher than that in OM group (P < 0.05). ConclusionSimvastatin can increase the mRNA and protein expression levels of OCN and the protein of p-p38 in osteogenic differentiation of BMSCs at middle/ late stages, and its best induction time is 12 hours.