Study on protective effects of platelet-leukocyte aggregation on myocardial ischemia reperfusion injury and postischemic recovery in rats / 天津医药

Objective To observe the effect of postconditioning (PostC) on the expression of platelet-leukocyte aggregation (PLA) during the process of myocardial ischemia and reperfusion in rats, and to explore the mechanisms of ischemic postconditioning (PostC) alleviating myocardial ischemia-reperfusion injury (MIRI). Methods Sixty rats were randomly divided into six groups:sham,reperfusion injury(I/R),postconditioning(PostC),SP600125(inhibition of c-Jun N-terminal kinase,I-JNK),anisomycin and postconditioning(Ani+PostC)and anisomycin(Ani)groups.After constructing the model of myocardial ischemia reperfusion in rats,the levels of myocardial injury markers were detected by using the CK-MB kits and TnI kits. The levels of PLA at different time points were detected by using flow cytometry.The myocardial infarction area were measured by using 2.3.5-Triphenyte-trazoliumchloride(TTC)staining,and the level of phosphorylation of JNK(P-JNK) was determined by using Western blot method. Results (1) The levels of CK-MB, TnI and the infarct size were significantly higher in the I/R group than those in the Sham group(P<0.05).The levels of CK-MB,TnI and the infarct size were significantly lower in the PostC group and I-JNK group than those in the I/R group(P<0.05).Compared with the PostC group,the levels of CK-MB,TnI and the infarct size were significantly higher in the Ani+PostC group and Ani group(P<0.05).(2)Compared with the Sham group,the expression levels of PLA significantly increased in the I/R group at different time points after ischemia (P<0.05). At different time points of MIRI, the expressions of PLA increased gradually in I/R group, Ani+PostC group and Ani group (P<0.05). At the time point of reperfusion for 60 minutes and reperfusion for 3 hours,the expressions of PLA were significantly lower in the PostC group and I-JNK group compared with those of I/R group (P<0.05).Compared with the PostC group,the expressions of PLA were significantly higher in the Ani+PostC group and Ani group (P<0.05). (3) Compared with the Sham group, the expression levels of P-JNK were significantly higher in the I/R group(P<0.05).PostC and I-JNK inhibited the production of P-JNK(P<0.05),while Ani promoted the increase of P-JNK (P<0.05).Compared with the PostC group,the expression levels of P-JNK were significantly higher in the Ani+PostC group and Ani group (P<0.05). Conclusion PostC can reduce the expression of PLA during reperfusion by inhibiting the phosphorylation of JNK,thereby reducing myocardial ischemia-reperfusion injury.

Studies on the mechanism of Syk and JNK in the heart tissue of type 1 diabetic rats / 天津医药

Objective To investigate the expressions of spleen tyrosine kinase (Syk), c-Jun amino terminal kinase (JNK) and nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in the heart tissue in SD rat model of diabetic cardiomyopathy, and to explore the relationship between Syk, JNK and NLRP3. Methods Clean male SD rats were randomly divided into the control (Ctrl) group and diabetic cardiomyopathy model (DCM) group. Rats of DCM group were treated with a single intraperitoneal injection of streptozotocin (STZ), while rats of Ctrl group were injected with the same dose of citrate buffer. The random blood glucose level and body weight were monitored every week until 20 weeks after STZ or citrate buffer injection, then all the rats were killed and their hearts were obtained. Rat H 9c2 cardiomyocytes were randomly divided into normal glucose treatment (NG) group, high glucose treatment (HG) group, Syk inhibitor control (BAY) group and Syk inhibitor high glucose (HG+BAY) group. The Syk and JNK phosphorylations and NLRP3 protein expression were detected by Western blot assay in the heart tissue of SD rats and H9c2 cardiomyocytes. The NLRP3, cysteine-containing aspartate specific protease 1(caspase-1) and interleukin (IL)-1β expressions at mRNA level were detected by reverse transcription-polymerase chain reaction (RT-PCR). Results The random blood glucose level was significantly increased (P<0.05) and the body weight was significantly decreased (P<0.05) in DCM group compared with those of Ctrl group. The expressions of cardiac p-Syk, p-JNK and NLRP3 at protein level were significantly increased in DCM group compared with those of Ctrl group (P<0.05). Furthermore, the mRNA levels of NLRP3, caspase-1 and IL-1β were significantly up-regulated (P < 0.05). BAY treatment significantly inhibited the high glucose-induced NLRP3, caspase-1 and IL-1β mRNA expressions and p-JNK, NLRP3 protein expressions in H9c2 cardiomyocytes (P < 0.05). Conclusion JNK phosphorylation and NLRP3 inflammasome activation induced by Syk play an important role in the pathogenesis of diabetic cardiomyopathy.

Effect of Toll-like receptor 7 agonist on JNK signaling pathway during liver injury in septic mice / 中华麻醉学杂志

Objective To evaluate the effect of Toll-like receptor 7 (TLR7) agonist on c-Jun Nterminal kinase (JNK) signaling pathway during liver injury in the septic mice.Methods One hundred eighty pathogen-free adult male C57BL/6 mice,aged 10-14 weeks,weighing 20-26 g,were randomly divided into 3 groups (n=60 each) using a random number table:sham operation group (group S);sepsis group (group Sep);TLR7 agonist group (group GDQ).Sepsis was induced by cecum ligation and puncture.In group GDQ,TLR7 agonist 1.5 g/kg was injected intraperitoneally at 24 h before establishment of the model.At 6,12 and 24 h after operation,10 mice in each group were sacrificed,and the livers were removed to detect the expression of interleukin-6 (IL-6) and IL-10 by Western blot.The expression of JNK was determined by immuno-histochemistry,and the histopathologic changes of livers were examined with a light microscope at 24 h after operation.The survival of mice was observed at 14 days after operation,and the 14-day survival rates were calculated.Results Compared with group S,the 14-day survival rates were significantly decreased,and the expression of IL-6,IL-10 and JNK was significantly up-regulated at 6,12 and 24 h after operation in Sep and GDQ groups (P＜0.05).Compared with group Sep,the 14-day survival rates were significantly increased,and the expression of IL-6,IL-10 and JNK was significantly down-regulated at 6,12 and 24 h after operation in group GDQ (P＜0.05).The pathological changes of livers were significantly attenuated in group GDQ as compared with group Sep.Conclusion TLR7 agonist can reduce the liver injury through blocking the JNK signaling pathway in the septic mice.

Protective effect of SP600125 against liver cell injury in rats under repeated and sustained high +Gz exposure / 解放军医学杂志

Objective To explore the effect of JNK inhibitor SP600125 on expression of JNK/c-jun in liver cells of rats under repeated and sustained high +Gz exposure and its mechanism of the effect. Methods Eighteen inbred adult male Wistar rats were randomly divided into control group, +10Gz group and SP600125 group (n=6). The rats in +10Gz group and SP600125 group were fixed to the rotating arm of a centrifuge with head towards the axis. The increase rate of acceleration was 1G/s with a peak-time of 3 minutes, and the +Gz exposure was repeated 5 times with an interval of 30 minutes. SP600125 was given to rats of SP600125 group 30 minutes before the first centrifugation by intraperitoneal injection. All of the animals were sacrificed 30 minutes after centrifugation. Blood samples were collected from inferior vena cava to determine the plasma level of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The expression of c-jun mRNA was determined by quantitative real-time RT-PCR (qRT-PCR). The expressions of p-JNK, JNK, p-c-jun and c-jun protein were determined by Western blotting. The morphological change in the liver tissue was observed after HE staining. Results The plasma level of ALT and AST, expression level of c-jun mRNA and p-JNK, p-c-jun, c-jun protein in the liver tissue of SP600125 group were significantly higher than those of control group (P0.05). HE staining revealed disorganized hepatic cords, irregular liver cells, vacuolar changes, and marked edema of hepatocytes, and collapsed hepatic sinusoids in +10Gz group, but these changes were alleviated obviously in SP600125 group. Conclusion SP600125 could alleviate the liver cell injury in rats under repeated and sustained high +Gz exposure.

Regulation mechanism of autophagy-related protein LC3 by c-Jun in methotrexate resistant human choriocarcinoma JEG-3 cells / 中华妇产科杂志

Objective To explore the regulation mechanism of autophagy-related protein, microtubule-associated protein 1 light chain 3 (LC3), via c-Jun in methotrexate resistant human choriocarcinoma JEG-3 cell lines. Methods Human choriocarcinoma JEG-3 cell lines, and methotrexate resistant choriocarcinoma JEG-3 (JEG-3/MTXR) cell lines were used in our present study. Phosphorylation c-Jun (p-c-Jun) was evaluated after exposure to 0.02 ng/ml methotrexate for 72 hours in both cells by western blot. c-Jun gene was knockdown by small interference RNA (siRNA) in JEG-3/MTXR cells, and LC3 was evaluated by western blot and reverse transcription-PCR. The binding of LC3 promoter with c-Jun protein was detected via chromatin immunoprecipitation assay (ChIP) with or without 0.02 ng/ml methotrexate exposure. Results The results showed that p-c-Jun was up-regulated after methotrexate treatment for 72 hours (1.99±0.20, versus 0.20±0.06 at 0 hour;P<0.05) by western blot analysis in JEG-3/MTXR cell lines. Further investigation demonstrated that c-Jun-siRNA could inhibit the up-regulation of LC3 formation and after methotrexate exposure (LC3 mRNA:1.24±0.17 versus 3.03±0.43;LC3 protein:0.52±0.07 verus 1.20± 0.15; all P<0.05). The binding of LC3 promoter by c-Jun protein was up-regulated after methotrexate treatment by the method of ChIP in methotrexate resistant JEG-3/MTXR cells [(2.95 ± 0.35) times]. Conclusion Autophagy-related gene LC3 expression regulated by c-Jun protein may be involved in the effect mechanism of the development of methotrexate resistance in choriocarcinoma JEG-3 cells.

Potential role of HMG CoA reductase inhibitor on oxidative stress induced by advanced glycation endproducts in vascular smooth muscle cells of diabetic vasculopathy

Advanced glycation endproducts (AGEs)-induced vascular smooth muscle cell (VSMCs) proliferation and formation of reactive oxygen species (ROS) are emerging as one of the important mechanisms of diabetic vasculopathy but little is known about the antioxidative action of HMG CoA reductase inhibitor (statin) on AGEs. We hypothesized that statin might reduce AGEs-induced intracellular ROS of VSMCs and analyzed the possible mechanism of action of statin in AGEs-induced cellular signaling. Aortic smooth muscle cell of Sprague-Dawley rat (RASMC) culture was done using the different levels of AGEs stimulation in the presence or absence of statin. The proliferation of RASMC, ROS formation and cellular signaling was evaluated and neointimal formation after balloon injury in diabetic rats was analyzed. Increasing concentration of AGEs stimulation was associated with increased RASMC proliferation and increased ROS formation and they were decreased with statin in a dose-dependent manner. Increased NF-kappaB p65, phosphorylated ERK, phosphorylated p38 MAPK, cyclooxygenase-2, and c-jun by AGEs stimulation were noted and their expression was inhibited by statin. Neointimal formation after balloon injury was much thicker in diabetic rats than the sham-treated group but less neointimal growth was observed in those treated with statin after balloon injury. Increased ROS formation, subsequent activation of MAPK system and increased VSMC proliferation may be possible mechanisms of diabetic vasculopathy induced by AGEs and statin may play a key role in the treatment of AGEs-induced diabetic atherosclerosis.

Construction and identification of dominant-negative c-Jun N-terminal kinase(DN-JNK)recombinant adenovirus / 中国医师杂志

Objective To construct and identify replication deficient recombinant adenovirus expressing human c-Jun N-terminal kinase(JNK)by homologous recombination adenovirus dominant-negative type JNK(Ad-DN-JNK).Methods The linearized recombinant shuttle vector pAdTrack-CMV-DN-JNK Was co-transformed with backbone vector pAdEasy-l into bacteria BJ5183 for recombinant adenoviral vector.The recombinant adenoviral vector was transfected into HEK293 packing cells tO construct replication deficient recombinant adenovirus,and then the recombinant edenovirns WaS detected by PCR and DNA sequencing.Western blot analysis was utilized to detect the Cxpression of Ad-DN-JNK and the level of insulin receptor substrate l Serine307 phosphorylation.Results JNK recombinant adenoviral vectorcould be effectively transfeeted into HEK 293 cell and successfully packed by intracellular enzyme.The expression of green fluorescent protein(GFP)Was observed on the 5th day after transfection.The fragment of JNK gene waS amplified by PCR and identified by sequencing.The titer of the prepared Ad-DN-JNK is 2.5×1010 pfu/ml.The animal experiment confirmed that constructed Ad-DN-JNK could be effectively expressed in liver tissue.Conclusion The research successfully constructed recombinant adenoviral vector and recombinant adenoviral particle.Animal experiment demonstrated the Ad-DN-JNK could effectively mediated the expression of DN-JNK gene and down-regulated the level of IRSlscfine307 phosphorylation.The achievement laid a foundation for further investigation of the function and application of JNK.

Transcription of the protein kinase C-delta gene is activated by JNK through c-Jun and ATF2 in response to the anticancer agent doxorubicin

Expression of protein kinase C-delta (PKC delta) is up-regulated by apoptosis-inducing stimuli. However, very little is known about the signaling pathways that control PKC delta gene transcription. In the present study, we demonstrate that JNK stimulates PKC delta gene expression via c-Jun and ATF2 in response to the anticancer agent doxorubicin (DXR) in mouse lymphocytic leukemia L1210 cells. Luciferase reporter assays showed that DXR-induced activation of the PKC delta promoter was enhanced by ectopic expression of JNK1, c-Jun, or ATF2, whereas it was strongly reduced by expression of dominant negative JNK1 or by treatment with the JNK inhibitor SP600125. Furthermore, point mutations in the core sequence of the c-Jun/ATF2 binding site suppressed DXR-induced activation of the PKC delta promoter. Our results suggest an additional role for a JNK signaling cascade in DXR-induced PKC delta gene expression.

Influence of jiaweibugan decoction on c-jun expression of sciatic nerve in experimental diabetic rats / 中国医师杂志

Objective To observe the effect of Jiaweibugan decoction on c-jun mRNA expression of sciatic nerve in experimental di-abetic rat and explore the preventive and therapeutic effects of Jiaweibugan decoction on peripheral neuropathy in experimental diabetic rats. Methods The diabetic rat model, was established by streptozotocin (STZ). The rots were killed on the 4th or 8th week from the beginning of treatment, and c-jun mRNA of sciatic nerve was detected by reverse-transcriptase polymerase chain reaction (RT-PCR). Serum SOD was determined by xanthine oxidase method. Results The results of RT-PCR showed that c-jun mRNA expression of the model group on the 4th or 8th week was higher than that of the normal control group (P <0.05) , while those in model rats treated by Jiaweibugan decoction were markedly lower than those in non-treated model rats(P <0.05). The results showed that SOD of the model group on the 4th or 8th week was lower than that of the normal control group (P < 0.05) , while those in model rats treated by Jiaweibugan decoction were markedly higher than those in non-treated model rats(P < 0.05). Conclusion Jiaweibugan decoction has a preventive and therapeutic effect on peripheral neuropathy in experimental diabetic rats.

Activation of the intrinsic mitochondrial apoptotic pathway in swine influenza virus-mediated cell death

The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK-stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.

Catechins inhibit angiotensin II-induced vascular smooth muscle cell proliferation via mitogen-activated protein kinase pathway

Catechins, components of green tea, reduce the incidence of cardiovascular diseases such as atherosclerosis. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMC), resulting in atherosclerosis. The acting mechanisms of the catechins remain to be defined in the proliferation of VSMC induced by Ang II. Here we report that catechin, epicatechin (EC), epicatechingallate (ECG) or epigallocatechingallate (EGCG) significantly inhibits the Ang II-induced [3H]thymidine incorporation into the primary cultured rat aortic VSMC. Ang II increases the phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK 1/2), c-jun-N-terminal kinase 1/2 (JNK 1/2), or p38 mitogen-activated protein kinases (MAPKs) and mRNA expression of c-jun and c-fos. The EGCG pretreatment inhibits the Ang II-induced phosphorylation of ERK 1/2, JNK 1/2, or p38 MAPK, and the expression of c-jun or c-fos mRNA. U0126, a MEK inhibitor, SP600125, a JNK inhibitor, or SB203580, a p38 inhibitor, attenuates the Ang II-induced [3H]thymidine incorporation into the VSMC. In conclusion, catechins inhibit the Ang II-stimulated VSMC proliferation via the inhibition of the Ang II-stimulated activation of MAPK and activator protein-1 signaling pathways. The antiproliferative effect of catechins may be associated with the reduced risk of cardiovascular diseases by the intake of green tea. Catechins may be useful in the development of prevention and therapeutics of vascular diseases.

EARLY EXPRESSION OF JUN PROTEIN IN CRANIOCOREBRAL GUNSHOT WOUND IN DOGS AND ITS SIGNIFICANCE / 解放军医学杂志

Objective To explore the rule of early Jun’ expression in craniocerebral gunshot injury and its significance. Methods After a direct shot of the dog′s head with a small calibre rifle, the expression of Jun ′ protein was assayed by immunohistochemistry method at different periods and in different regions, and the water contents and the ultrastructural changes in brain tissue were also observed. Results Nearly no Jun expression was found in cerebral tissues of the control group. However, the Jun expression was first observed begun at 30min postinjury both at the regions of contusion and concussion ( P

Effects of diazoxide on ?-calpain activation, c-Fos and c-Jun expres sion in neonatal hypoxic-ischemic rat brain / 中国病理生理杂志

AIM: To investigate the effects of diazoxide, an ATP-sensitive K + channel opener on the ?-calpain activation, c-Fos and c- Jun expression in neonatal hypoxic-ischemic rat brain. METHODS:The animal model of hypoxic-ischemic brain injury (HIBI) was made in the 7-day -old SD rats. Diazoxide was injected into the left lateral ventricle prior or po st hypoxic-ischemia (HI) insults. Western blot was applied to detect the integra ted density (ID) of the nuclear c-Fos and c-Jun at 4h, and the cleavage of cytos olic ?-calpain at 24 h after HI insults. RESULTS: Low c-Fos and c-Jun expressions from cortical and hippocampal samples were observed in the tw o diazoxide groups, and significant differences in their expressions were found by comparison with the HI controls (P