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Aim To study the mechanism of quereetin (Que) inhibiting mitochondrial damage induced by Aβ
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Aim To investigate the effect of echinatin on the non-alcoholic fatty liver disease model of free fatty acids ( FFA) -induced HepG2 cells and its mechanism. Methods The experimental groups were divided into control group, FFA model group and echinatin group (0.3 , 1, 3 μmol • L
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Background It has been found that fluoride may cause cardiomyocyte damage. c-Jun N-terminal kinases (JNK) signaling pathway plays an important role in apoptosis, but its role in fluorosis-induced cardiomyocyte damage is still unknown yet. Objective To explore the toxic effect of sodium fluoride (NaF) on H9c2 cardiomyocytes of rats and whether NaF affects cardiomyocyte apoptosis through the JNK signaling pathway. Methods According to the concentrations of sodium fluoride and whether sp600125 (JNK inhibitor) was added, cardiomyocytes of rats were divided into six groups, including control group, SP600125 group (SP group), 0.24, 0.48, and 0.96 mmol·L−1 NaF groups, and 0.96 mmol·L−1 NaF+SP600125 group (NaF+SP group). Cardiomyocytes exposed to NaF for 24 h were observed using a fluorescence inverted microscope. The changes of cell viability at 24, 48, and 72 h after the treatment were detected by CCK-8 method. The levels of reactive oxygen species (ROS) at 24 h after the treatment in H9c2 cardiomyocytes were determined by fluorescent probe method. The expression levels of Bcl-2, Bax, Caspase-3, and JNK mRNA at 24 h after the treatment were detected by real-time PCR. The protein expression levels of Bcl-2, Bax, Caspase-3, and p-JNK at 24 h after the treatment were detected by Western blotting. Results Compared with the control group, after being exposed to 0.48 and 0.96 mmol·L−1 NaF for 24 h, the cell growth density decreased. With the increase of NaF concentration, rounded cells and some suspended dead cells appeared. At 24h after exposure to NaF, the cell viability of the 0.48 and 0.96 mmol·L−1 NaF groups decreased compared with the control group (P<0.05). At 48h and 72h after exposure to NaF, the cell viability levels of the NaF treated groups were significantly lower than that of the control group (P<0.05). After NaF exposure for 24 h, compared with the control group, the intracellular ROS levels were increased (P<0.05); the mRNA expression levels of Bcl-2 were decreased to varying degrees, especially in the 0.48 and 0.96 mmol·L−1 NaF groups (P<0.05); the mRNA expression levels of Bax, Caspase-3, and JNK were increased (P<0.05); the protein expression levels of Bcl-2 were reduced (P<0.05); the protein expression levels of Bax, Caspase-3, and p-JNK were elevated (P<0.05). Compared with the 0.96 mmol·L−1 NaF group, the cell viability of the NaF+SP group was increased, the intracellular ROS level was decreased, the mRNA expression levels of Bax, Caspase-3, and JNK were decreased, the protein expression level of Bcl-2 was increased, and the protein expression levels of Bax, Caspase-3, and p-JNK were decreased (P<0.05); the expression level of Bcl-2 mRNA had a rising trend but showed no statistical significance (P>0.05). Conclusion Cardiomyocyte damage after excessive fluoride exposure may result from fluoride inducing excessive ROS production in cardiomyocytes, which may activate the JNK signaling pathway and induce cardiomyocyte apoptosis.
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ObjectiveTo investigate the mechanism of Huazhuo Jiedu Huoxue Tongluo prescription in alleviating cerebral ischemia-reperfusion injury via regulating nerve cell autophagy based on c-Jun N-terminal kinase（JNK）signaling pathway . MethodSixty SD rats were randomly divided into 6 groups： sham group， middle cerebral artery occlusion/reperfusion （MCAO/R） group （model group）， Huazhuo Jiedu Huoxue Tongluo prescription group ［traditional Chinese medicine （TCM） group（25.0 g·kg-1）］， JNK inhibitor SP600125 （SP） group（5 mg·kg-1）， TCM+SP group and JNK agonist Anisomycin （Ani） group（15 mg·kg-1）. After 24 h of modeling， TCM group and TCM+SP group were given TCM decoction （ig） for 3 consecutive days， and the other groups were given equal volume of normal saline （ig）. Neurological deficit was evaluated by neurological function score and cerebral infarct volume was determined by 2，3，5-triphenyltetrazole chloride （TTC） staining. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe the structural changes of brain tissue and the damage of neurons， respectively. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） was performed to detect cell apoptosis. The ultrastructure of autophagosomes was observed by transmission electron microscope. Western blot was employed to detect the protein expressions of microtubule-associated protein 1 light chain 3A/B （LC3A/B）， autophagy related 5 （Atg5）， the ortholog of yeast Atg6 （Beclin1）， p62， B-cell lymphoma 2 （Bcl-2）， JNK， phosphorylated （p）-JNK and c-Jun in brain tissue. The mRNA expressions of LC3A/B， Beclin1， p62， Atg5， Bcl-2， JNK and c-Jun were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham group， the model group had elevated neurological deficit score （P<0.05）， enlarged cerebral infarct volume （P<0.05）and typical infarction manifestations formed in hippocampal region and its surrounding brain tissue. In addition， there were a large number of neuronal cell degeneration， necrosis， liquefaction， nucleus pyknosis and deep staining， and inflammatory cell infiltration in the cortex in the model group， and severe swelling of mitochondria， lysosomes， autophagosomes and autophagolysosomes were clearly seen under electron microscope. TUNEL positive cells were increased （P<0.05）， and cell apoptosis was severe. The nuclear membrane and nucleolus of neurons in brain tissue were blurred with discontinuous processes， and Nissl bodies in cytoplasm were stained light with reduced number. Western blot revealed that the model group had up-regulated protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in brain tissue （P<0.05）， while down-regulated protein expressions of p62 and Bcl-2 （P<0.05）as compared with the sham group. Real-time PCR indicated that the mRNA expressions of LC3A/B， Beclin1， Atg5， JNK and c-Jun in the model group were higher （P<0.05） while the mRNA expressions of p62 and Bcl-2 were lower （P<0.05） than those in the sham group. Compared with the conditions in model group， the neurological deficit scores of TCM， SP and TCM+SP groups were lowered （P<0.05）， and the cerebral infarct volume was reduced （P<0.05）， with improved pathological status of brain tissue， especially in the TCM group. Furthermore， there were abundant and basically normal mitochondrial cristae， slightly dilated endoplasmic reticulum， slightly swollen golgi apparatus， slightly fused nuclear membrane， and few visible lysosomes， autophagosomes and autophagolysosomes. TUNEL positive cells were decreased （P<0.05）， displaying reduced apoptosis， especially in the TCM group. The nucleolus and nuclear membrane of neurons in brain tissue were discernible， and Nissl bodies in cytoplasm was reduced to a certain degree as compared with those in the model group. Western blot showed a decrease in the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in the TCM group ，the SP group，and the TCM+SP group（P<0.05），while an increase in the protein expressions of p62 in the TCM group and SP group（P<0.05），and an increase in the protein expressions of Bcl-2 in the TCM group and TCM+SP group. By Real-time PCR， the mRNA expressions of LC3A， LC3B， Beclin1， Atg5， JNK and c-Jun had a down-regulation（P<0.05） while the mRNA expression of p62 a up-regulation in the TCM group ，the SP group，and the TCM+SP group（P<0.05），and the mRNA expression of Bcl-2 a up-regulation in the TCM group and the TCM+SP group（P<0.05）.Scores of the Ani group were raised （P<0.05）， and infarct volume was increased significantly， with aggravated neuronal cell necrosis and obvious inflammatory infiltration. Moreover， there were neuronal nuclear membrane fusion with abnormal protrusion， increased heterochromatin aggregation in edge， severe mitochondrial swelling， endoplasmic reticulum expansion， increased lysosomes， increased intracytoplasmic vesicles， and visible autophagosomes and autophagolysosomes. TUNEL positive cells were increased （P<0.05）， representing severe apoptosis. The number of Nissl bodies dropped with light staining， and the nucleolus and nuclear membrane were blurred. Real-time PCR found that the mNRA expressions of Atg5， c-Jun， JNK were up-regulated （P<0.05），while Beclin1， p62， Bcl-2 were were down-regulated in the Ani group （P<0.05）. Compared with the TCM group and SP group，the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK， c-Jun were decreased，and p62， Bcl-2 were increased in the Ani group（P<0.05）. Compared with the TCM group，the mRNA expressions of JNK mRNA had a down-regulation in the SP group and TCM+SP group，while LC3A， LC3B， Atg5， c-Jun， JNK had an up-regulation（P<0.05） and Bcl-2 had a down-regulation in the Ani group（P<0.05）. Compared with the SP group，the mRNA expressions of Atg5， c-Jun， JNK had an up-regulation（P<0.05）， and Beclin1， p62， Bcl-2 had a down-regulation in the Ani group（P<0.05）. ConclusionHuazhuo Jiedu Huoxue Tongluo prescription significantly up-regulates the protein and mRNA expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun， and down-regulates the protein and mRNA expressions of p62 and Bcl-2， suggesting that the prescription can inhibit autophagy through JNK signaling pathway to reduce ischemia/reperfusion injury in rats.
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This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P<0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P<0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P<0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P<0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P<0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P<0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P<0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.
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Masculino , Animales , Ratones , Cisplatino/farmacología , Carcinoma Hepatocelular/genética , Sistema de Señalización de MAP Quinasas , Beclina-1 , Apoptosis , Neoplasias Hepáticas/genética , Necrosis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Línea Celular Tumoral , ARN Mensajero/metabolismo , AutofagiaRESUMEN
Objective:To observe the intervention mechanism of phlegm-stasis co-treatment for the JNK signaling pathway in the myocardium of diabetes rats.Methods:Totally 50 male SD rats of SPF grade were selected. Diabetes model was established by single intraperitoneal injection of 55 mg/kg streptozotocin (STZ) solution. After continued feeding for 3 weeks, the rats were divided into normal group, model group, alachloramine group, blood stasis removing group, phlegm removing group and phlegm-blood stasis co-treatment group according to random number table method, with 6 rats in each group. Xiaoxianxiong Decoction (4.05 g/kg), Xuefu Zhuyu Decoction (7.02 g/kg), Didang Xianxiong Decoction (8.10 g/kg) were administered to the stomach respectively in the phlegm removing group, the blood stasis removing group and the phlegm-blood stasis co-treatment group. Alachloramine (3 mg/kg) was administered to the stomach by gavage in the alachloramine group. After 8 weeks, HE staining was used to observe the morphological changes of myocardial tissue in diabetic rats. Masson staining was used to observe the deposition of collagen fibers in the myocardial interstitium in rats. The expression of JNK1 protein was determined by immunohistochemistry. JNK1 mRNA, IRS1 mRNA and NLRP3 expression levels were detected by Real-time PCR. Western blot was used to detect the protein expressions of IRS-1, p-Akt and NLRP3.Results:The myocardial cells in the model group were disorganized, with hypertrophy, blurred texture, inflammatory infiltration of interstitium, increased collagen fibers, and focal necrosis. All treatment groups could improve fibrosis, inflammatory infiltration and reduce myocardial collagen deposition in different degrees. Compared with the model group, the mRNA and protein expressions of JNK1 and NLRP3 bodies decreased ( P<0.01), the IRS-1 mRNA and protein increased ( P<0.01), and p-Akt protein expression increased ( P<0.01). Conclusions:The phlegm and stasis co-treatment can effectively improve the cardiomyopathy of diabetes rats, and the effect is better than the phlegm-resolving method or the stasis resolving method alone. The mechanism may be related to the inhibition of JNK signaling pathway activation, reduce the expressions of JNK1 and NLRP3, and increase the IRS-1 and Akt.
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Aim To investigate the effect of resveratrol (RSV)on apoptosis of PC 12 cells induced by advanced glycation end products(AGEs) and its possible molecular mechanism. Methods MTS assay was used to detect the effects of AGEs (0, 50, 100, 200, 400 g • L"1) and RSV (0, 5, 10, 20, 40 jxmol • L"1) on cell viability. PC12 cells were treated with AGEs (400 g • L-1) and RSV (10 |xmol • L"1). TUNEL staining was used to detect apoptosis; flow cytometry was used to detect apoptosis and reactive oxygen species (ROS) ; Western blot was used to detect protein expression of p-JNK, JNK, PUMA, Bcl-2, Bax and Caspase-3. Apoptosis was observed by flow cytometry after pretreat- ment with JNK specific phosphorylation inhibitor (sp600125). Results Compared with normal control group, the cell viability of AGEs group decreased, the apoptotic rate and ROS levels increased, the expressions of p-JNK, PUMA, Bax and Caspase-3 protein in-creased, the expression of Bcl-2 protein decreased; Compared with AGEs group, the cell viability of the AGEs + RSV group increased, the levels of apoptotic rate and ROS were reduced, the expressions of p-JNK, PUMA, Bax and caspase-3 protein decreased, the expression of Bcl-2 protein increased. Sp600125 could partially reverse the effect of AGEs on PC)2 cell apopto-sis. Conclusions RSV can significantly inhibit the apoptosis of PC 12 cells induced by AGEs, which may be related to the activation of ROS-JNK pathway.
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A stable hepatoma cell line(Hep G2 cell) insulin resistance model was established and used to analyze the effect of effective components of Mori Folium in alleviating insulin resistance,and preliminary explore the mechanism for alleviating insulin resistance. The Hep G2 insulin action concentration and the duration of action were investigated using the glucose oxidase method(GOD-POD method) to establish a stable Hep G2 insulin resistance model. Normal control group,model group,Mori Folium polysaccharide group,Mori Folium flavonoid group and rosiglitazone group were divided to determine the glucose consumption. The effect of Mori Folium effective components on Hep G2 insulin resistance was analyzed. The mRNA expressions of JNK,IRS-1 and PDX-1 in each group were detected by Real-time quantitative PCR(qRT-PCR). The protein expressions of p-JNK,IRS-1 and PDX-1 were detected by Western blot. And the mechanism of effective components of Mori Folium in alleviating insulin resistance was investigated. The results showed that the glucose consumption was significantly decreased in the insulin resistance cells after incubation with 25. 0 mg·L-1 insulin for 36 h(P<0. 01),and the model was relatively stable within 36 h. Mori Folium polysaccharides and flavonoids all alleviated insulin resistance,among which Mori Folium flavonoids had better effect in alleviating Hep G2 insulin resistance(P<0. 05). The qRT-PCR analysis showed that Mori Folium polysaccharides and flavonoids could inhibit JNK and IRS-1 mRNA expressions,while enhancing PDX-1 mRNA expression. Western blot analysis displayed that Mori Folium polysaccharides and flavonoids could inhibit p-JNK and IRS-1 protein expressions,while enhancing PDX-1 protein expression. Mori Folium polysaccharides and flavonoids can alleviate insulin resistance in Hep G2 cells,and its mechanism may be the alleviation of insulin resistance by inhibiting JNK signaling pathway.
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Humanos , Medicamentos Herbarios Chinos , Farmacología , Glucosa , Células Hep G2 , Proteínas de Homeodominio , Metabolismo , Insulina , Proteínas Sustrato del Receptor de Insulina , Metabolismo , Resistencia a la Insulina , MAP Quinasa Quinasa 4 , Metabolismo , Sistema de Señalización de MAP Quinasas , Morus , Química , Hojas de la Planta , Química , Transactivadores , MetabolismoRESUMEN
Objective: To investigate the effects of Hedyotis diffusa Willd (HDW) on the proliferation, cell cycle, apoptosis, migration and invasion of human renal carcinoma cells, and to explore the possible mechanisms. Methods: After human renal adenocarcinoma ACHN cells and human renal proximal tubule HK-2 cells were treated with HDW for 24 h, the cell proliferation was detected by CCK-8 assay, and the cell cycle and apoptosis were detected by FCM assay. The phenotypes of ACHN and HK-2 cells treated with HDW were observed by light microscopy and rhodamine staining. The expressions of mesenchymal-epithelial transition (MET)-related proteins were detected by immunofluorescence staining and Western blotting, respectively. The effects of HDW on the migration and invasion of ACHN and HK-2 cells were detected by wound-healing and Transwell chamber assay. RNAsequencing was used to detect the effect of HDW on the transcriptome in ACHN cells. The expression levels of RAP1 pathway-related genes [RAP1 GTPase activating protein (RAP1GAP), RAS guanyl releasing protein 2 (RASGRP2), RAP guanine nucleotide exchange factor 3 (RAPGEF3), membrane-associated guanylate kinase, WW and PDZ domain containing 1 (MAGI1) and G protein subunit alpha i1 (GNAI1)] were detected by real-time fluorescent quantitative PCR. The expression levels of mitogen-activate protein kinase (MAPK) pathway-related proteins [c-Jun N -terminal kinase (JNK), phospho-JNK (p-JNK), protein kinase B (PKB, Akt), phospho-Akt (p-Akt), extracellular signalregulated kinase (ERK) and phospho-ERK (p-ERK)] were detected by Western blotting. Results: HDW selectively inhibited the proliferation of ACHN cells (P < 0.01), blocked cell cycle at S-phase (P < 0.01), and induced apoptosis (P < 0.01). After treatment with HDW, the morphology of ACHN cells significantly changed. HDW promoted the expressions of MET-related proteins (E-cadherin 1 and β-catenin) in ACHN cells, and inhibited the expressions of epithelial-mesenchymal transition (EMT)-related proteins (Vimentin and Snail1) (all P < 0.01). HDW inhibited the migration and invasion of ACHN and HK-2 cells (all P < 0.01), and there was no significant difference between ACHN and HK-2 cells. HDW affected the expressions of cell cycle-related genes and transcription factor E2F and Myc target genes, and activated the p53 signaling pathway. HDW significantly downregulated the expression levels of RAP1GAP, RASGRP2, RAPGEF3, MAGI1 and GNAI1 (all P < 0.000 1) and the phosphorylation level of JNK in ACHN cells. Conclusion: HDW may selectively inhibit cell proliferation, and promote MET, cell cycle arrest and apoptosis of ACHN cells by inhibiting RAP1-JNK signaling pathway.
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Objective To study the effects of acetated ringer's solution resuscitation in hemorrhagic shock rats on inflammatory mediators on lung tissue and their JNK (c-Jun N-terminal kinase) signaling pathways. Methods Thirty-two SD rats were randomly(random number) divided into four groups: shock without resuscitation group (CR, n=8), saline group (NR, n=8), lactated ringer's solution group (LR, n=8) and acetated ringer's solution group (AR,n=8). The rats of NR group, LR group and AR group were prepared into shock models (mean arterial blood pressure maintained at 40-45 mmHg),The rats of NR group, LR group and AR group were in the shock for 60 min and then the corresponding kinds of liquid were administered for 30 min and observation was carried out for 4 hours. The rats of CR group without liquid resuscitation were observed for 4 hours after shock. After that, the lung tissues of rats were taken from NR group, LR group and AR group as well as from CR group 4 hours after shock (if the rats died, the lung tissues were immediately taken). The levels of TNF-α, IL-4 and IL-10 mRNA in lung were measured by real-time polymerase chain reaction (RT-PCR),and Western blot was used to measure the levels of JNK phosphorylation and MKP-1 acetylation. The one-way ANOVA was used for comparison among groups. Between the two groups, the comparison was analyzed by using LSD-t test. Results The IL-4 mRNA expression of lung tissue in AR group was higher than that in CR group, NR group and LR group (CR group:0.42±0.34; NR group:2.60±0.66; LR group:6.24±2.95; AR group: 11.08±4.24; P＜0.05).The IL-10 mRNA expression of lung tissue in AR group was significantly higher than that in CR group, NR group and LR group (CR group:0.25±0.25; NR group:2.79±1.62; LR group:3.51±1.66; AR group:9.35±2.86;P＜0.01).The TNF-a mRNA expression in AR group was significantly lower than that in CR group, NR group and LR group (CR group:4.98±1.26; NR group:2.50±0.76; LR group:3.87±3.00; AR group:0.19±0.09; P＜0.01). The level of JNK phosphorylation in lung tissue of rats in AR group was significantly lower than that in CR group, NR group and LR group (CR group:0.52±0.12; NR group:0.42±0.08; LR group:0.30±0.08; AR group:0.17±0.06;P＜0.01). The level of MKP-1 acetylation in lung tissue of rats in AR group was significantly higher than that in CR group, NR group and LR group (CR group:0.14±0.07; NR group:0.30±0.07; LR group:0.37±0.02; AR group:0.48±0.06;P＜0.01). Compared with normal saline and lactated ringer's solution, acetated ringer's solution used in hemorrhagic shock rats could promote MKP-1 acetylation, inhibit the phosphorylation of JNK, significantly inhibit the lung tissue TNF-a released, promote the release of anti-inflammatory factors, IL-4 and IL-10. Conclusions The acetated ringer's solution for resuscitation of hemorrhagic shock in rats could reduce inflammation of lung tissue in a certain extent, probably by enhanced the acetylation of MKP-1 to inhibited JNK signaling pathway and reduced lung tissue inflammation.
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Objective: To explore the anti-inflammatory immune mechanism in moxibustion treatment of Crohn.s disease (CD) from the perspective of c-Jun N-terminal kinase (JNK) signaling pathway, through observing the regulatory effect ofmoxibustion on colonic JNK, c-Jun, monocyte chemoattractant protein 1 (MCP-1) and cyclooxygenase 2 (COX2) in CDmodel rats. Method: Male Sprague-Dawley rats of clean grade were randomized into a normal group, a model group, amoxibustion group and a sham moxibustion group. CD model was developed by the mixture of 2, 4, 6 Trinitro-benzene-sulfonic acid (TNBS) and ethanol via enema. Hematoxylin-eosin (HE) staining was used to observe the morphologicalchanges in rat.s colon tissues for pathological scoring; enzyme-linked immunosorbent assay (ELISA) was used to detectthe contents of MCP-1, COX2, JNK, and c-Jun in colon tissues; real-time fluorescence quantitative PCR was adopted toexamine the mRNA expressions of JNK and c-Jun in rat.s colon. Result: Compared with the normal group, the modelgroup showed more significant colonic damage and thus had a higher colonic damage score (P ＜ 0.01), manifested astopical inflammation which involved the submucosa, fissuring ulcers and granuloma; the model group also showedincreased contents of protein MCP-1 and COX2, and elevated contents of JNK protein and mRNA in colon (all P ＜ 0.05), while the change in the content of c-Jun was insignificant (all P＞ 0.05) . Compared with the model group and shammoxibustion group, the colonic damage score was lower in the moxibustion group (P ＜ 0.01, P ＜ 0.05), with improvementin colonic structure and inflammation; the contents of MCP-1 and COX2 in colon tissues declined, so did the proteincontent and mRNA expression of JNK (all P ＜ 0.05), while the change in the content of c-Jun was insignificant (all P＞0.05) . There were no significant differences between the model group and sham moxibustion group comparing all theindexes (all P＞ 0.05) . Conclusion: Moxibustion down-regulates the expressions of JNK protein and mRNA in CD rat.scolon, as well as the contents of MCP-1 and COX2 in colon tissues, which is possibly one significant mechanism formoxibustion to ease intestinal inflammation and promote the repair of colon tissues in CD.
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AIM:To investigate the effect of dihydroartemisinin ( DHA) adjuvant treatment on enhancing the antitumor effect of 5-fluorouracil (5-FU) against gastric cancer .METHODS:The gastric cancer BGC-823 cells were di-vided into control group , DHA group, 5-FU group, 5-FU+DHA group and 5-FU+DHA+SIRT1 plasmid group.The via-bility of BGC-823 cells treated with DHA and 5-FU was measured by MTT assay .The expression of SIRT1 and NADPH ox-idase, activation of caspase-9 and caspase-3, and phosphorylation of ASK1 and JNK in the BGC-823 cells treated with DHA and 5-FU were determined by Western blot .The production of ROS and the apoptosis of the BGC-823 cells treated with DHA and 5-FU were analyzed by flow cytometry .RESULTS:Dihydroartemisinin significantly inhibited the expression of SIRT1 and increased NADPH oxidase protein level (P<0.05).DHA increased the sensitivity of BGC-823 cells to 5-FU, thus decreasing the IC50 of 5-FU to the gastric cancer cells.However, transfection with SIRT1 plasmid decreased the cytotoxicity of DHA and 5-FU co-treatment to the BGC-823 cells.DHA promoted the production of ROS and phosphoryla-tion of ASK1 and JNK induced by 5-FU in the BGC-823 cells ( P<0.05 ) .However , ROS scavenger N-acetylcysteine ( NAC) or JNK specific inhibitor SP600125 inhibited the cell death and activation of caspase-9 and caspase-3 induced by DHA and 5-FU co-treatment (P<0.05).In addition, NAC significantly inhibited the phosphorylation of JNK in the BGC-823 cells co-treated with DHA and 5-FU.However, treatment with SP600125 did not influence the ROS production in the BGC-823 cells, indicating that JNK was the downstream target of ROS pathway .CONCLUSION: Combination of DHA with 5-FU induces caspase-dependent apoptosis in gastric cancer cells through the SIRT 1/NADPH oxidase/ROS/JNK sig-naling pathway .
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Objective To investigate the correlation between JNK signal and the apoptosis of VSMC as well as the expression of Cathepsin B and to explore the role of JNK signal in the development of cerebral aneurysm. Methods Rat models of cerebral aneurysm were established and histopathologic changes of cerebral aneurysm and the apoptosis of VSMC were analyzed. Rat models were respectively subject to subcutaneous injection of Cathepsin B siRNA and JNK inhibitor SP600125. Western blot technique was used to detect the expression of proteins like Cathepsin B, Caspase-3, and p-JNK. Spearman's rho was used to examine the correlation between p-JNK and Cathepsin B, as well as the expression of relevant proteins. Results The success rate of modeling rats with cerebral aneurysm was 88.75%. After the respective injection of Cathepsin B siRNA, SP600125 and their combination, the cell densities of VSMC of rats with cerebral aneurysm all increased significantly (P < 0.05 or P < 0.01), but the apoptosis rate of VSMC decreased significantly (P < 0.01). Compared with normal rats, the expression of Cathepsin B, Caspase-3 and p-JNK in Cerebral aneurysm models increased significantly. Effectively intervening Cathepsin B genes with Cathepsin B siRNA could significantly inhibit the expression of Cathepsin B and Caspase-3, but hardly influence the expression of p-JNK. JNK inhibitor SP600125 had no influence on the expression of Cathepsin B and Caspase-3, but effectively inhibited the expression of p-JNK. In cerebral aneurysm tissues, positive correlation was observed between the expression of p-JNK and Cathepsin B, the correlation coefficient was r = 0.640. Conclusion After the attack of cerebral aneurysm, proteins like Cathepsin B, Caspase-3 and p-JNK are all involved in the apoptosis of VSMCs. This process may be realized by Cathepsin B which activates the apoptosis mechanism of Caspase-3 and mediate the apoptosis of VSMC through the JNK signaling pathway. Therefore, silencing Cathepsin B gene or inhibiting the conduction through JNK signaling pathway can mitigate the apoptosis of vascular smooth muscle cells in cerebral aneurysm.
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OBJECTIVE@#To investigate the correlation between JNK signal and the apoptosis of VSMC as well as the expression of Cathepsin B and to explore the role of JNK signal in the development of cerebral aneurysm.@*METHODS@#Rat models of cerebral aneurysm were established and histopathologic changes of cerebral aneurysm and the apoptosis of VSMC were analyzed. Rat models were respectively subject to subcutaneous injection of Cathepsin B siRNA and JNK inhibitor SP600125. Western blot technique was used to detect the expression of proteins like Cathepsin B, Caspase-3, and p-JNK. Spearman's rho was used to examine the correlation between p-JNK and Cathepsin B, as well as the expression of relevant proteins.@*RESULTS@#The success rate of modeling rats with cerebral aneurysm was 88.75%. After the respective injection of Cathepsin B siRNA, SP600125 and their combination, the cell densities of VSMC of rats with cerebral aneurysm all increased significantly (P < 0.05 or P < 0.01), but the apoptosis rate of VSMC decreased significantly (P < 0.01). Compared with normal rats, the expression of Cathepsin B, Caspase-3 and p-JNK in Cerebral aneurysm models increased significantly. Effectively intervening Cathepsin B genes with Cathepsin B siRNA could significantly inhibit the expression of Cathepsin B and Caspase-3, but hardly influence the expression of p-JNK. JNK inhibitor SP600125 had no influence on the expression of Cathepsin B and Caspase-3, but effectively inhibited the expression of p-JNK. In cerebral aneurysm tissues, positive correlation was observed between the expression of p-JNK and Cathepsin B, the correlation coefficient was r = 0.640.@*CONCLUSION@#After the attack of cerebral aneurysm, proteins like Cathepsin B, Caspase-3 and p-JNK are all involved in the apoptosis of VSMCs. This process may be realized by Cathepsin B which activates the apoptosis mechanism of Caspase-3 and mediate the apoptosis of VSMC through the JNK signaling pathway. Therefore, silencing Cathepsin B gene or inhibiting the conduction through JNK signaling pathway can mitigate the apoptosis of vascular smooth muscle cells in cerebral aneurysm.
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Objective To investigate the effects of omega-3 polyunsaturated fatty acids (ω-3 PUFA) supplementation on neuron apoptosis,brain edema,activation of microglia,inflammatory response and neural function after traumatic brain injury (TBI) in rats,so as to understand the protection of ω-3 PUFA in rats following TBI and its mechanism.Methods TBI model was established using Feeney's method.Ninety SD rats were randomly divided into 5 groups:sham operation group (sham group),TBI group,TBI + selective activator of c-Jun N-terminal kinase (JNK) anisomycin group (TBI + Aniso group),TBI + ω-3 PUFA supplementation group (TBI + ω-3 group),and TBI + ω-3 PUFA supplementation + JNK activation group (TBI + ω-3 + Aniso group).We measured rat behavioral outcomes by modified neurological severity score (mNSS) on day 1,3,and 7 after TBI.Brain water content was measured with wet-dry weight method.The neuron apoptosis and microglial activation (identified by specific marker IBA-1) in TBI cerebral cortex were determined by TUNEL staining and immunofluorescence.Inflammatory cytokines [tumor necrosis factor-α (TNF-α),interleukin (IL)-1α,IL-1β,and IL-6] and the JNK signaling pathway (JNK,pJNK) were tested with reverse transcription-polymerase chain reaction and Western blot,respectively.Results Compared with the sham group,the levels of brain cell apoptosis,brain edema,neuron apoptosis,and inflammatory-relatived factors (TNF-α,IL-1 α,IL-1β,and IL-6) were significantly increased in the other four groups (P ＜ 0.05).Compared with the TBl group,ω-3 PUFA supplementation reduced brain water content following TBI,especially on day 3 after TBI [(78.14 ± 0.57) ％ vs.(82.31 ± 0.81) ％,P ＜ 0.01],and improved neurological function score (P ＜ 0.05).Meanwhile,ω-3 PUFA supplementation suppressed neuron apoptosis,the activation of microglia,and the mRNA and protein expressions of inflammatory cytokines (TNF-α,IL-1α,IL-1 β,IL-6).The activation of JNK signaling pathway was also inhibited by ω-3 PUFA.Conclusion ω-3 PUFA supplementation may markedly reduce brain edema,suppress neuron apoptosis,and improve neurological outcomes after TBI in rats,possibly mediated by inhibiting JNK signaling pathway and microglial activation,reducing microglia-induced cerebral inflammatory responses,demonstrated as down-regulated expression of TNF-α,IL-1α,IL-1β,and IL-6.
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Objective To investigate the effect and mechanisms of factor fibroblast growth factor inducible 14(Fn14)in the high glucose induced-cardiomyocyte hypertrophy. Method To observe the expression of collagenⅠ, connective tissue growth factor ( CTGF ) , transforming growth factor-β1 ( TGF-β1 ) , and Fn14 in high glucose induced-cardiomyocyte hypertrophy. Fn14 expressions was down-regulated by siRNA interference technique, and then the expressions of collagen Ⅰ, CTGF, and TGF-β1 were observed, and the mechanism was also explored. Results The expression of collagen I, CTGF and TGF-β1 was significantly up-regulated after high glucose induced-cardiomyocyte hypertrophy for 72 h. At the same time, the expression of Fn14 was increased after 72 h-treatment, and reached the peak at concentration of 30 mmol/L high glucose. High glucose could not up-regulated the expression of collagenⅠ, CTGF, and TGF-β1 after siFn14 interference, while the same result was observed in the expression of p-JNK. Conclusion The expressions of collagenⅠ, CTGF, TGF-β1, and Fn14 in cardiomyocyte of neonatal rats were induced by high glucose. While Fn14 expression was inhibited, the expressions of collagenⅠ, CTGF, and TGF-β1 were down-regulated, which seems to be involved with p-JNK signaling pathway.
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Objective To observe the influence of exterior-interior meridian acupoint selection on hippocampal cell apoptosis and c-Jun N-terminal kinase ( JNK) in ischemia-reperfusion injury rats, and to explore the possible protective mechanism for ischemia-reperfusion injury. Methods One hundred and twenty SD rats were randomly divided into sham-operation group (SH), model group (I/R), electroacupuncture (EA) group and JNK inhibitor SP600125 ( SP) group. The ischemia-reperfusion model was established by occlusion of middle cerebral artery with intraluminal thread. EA group was given EA on acupoints of Zusanli ( ST 36) , Sanyinjiao ( SP 6) , Chize ( LU 5) , Hegu ( LI 4) , once a day, based on the method of exterior-interior meridian acupoint selection. We observed the neurological behavioral changes by Longa neurological function standard, detected the activation of phosphorylated JNK ( p-JNK) by immunohistochemical method and examined the hippocampal apoptotic index by TUNEL method. Results The neurologic impairment scores of EA group and SP group were lower than I/R group (P<0.05) . Apoptotic index of I/R group was higher than SH group (P<0.01) and that of EA group and SP group was lower than I/R group (P <0.05) . The level of p-JNK in I/R group was higher than SH group ( P <0.01) and that of EA group and SP group was lower than I/R group ( P <0.05). Conclusion Exterior-interior meridian acupoint selection therapy has certain effect on alleviating neurologic impairment and reducing apoptosis in ischemia- reperfusion injury rats, and the mechanism might be associated with the inhibition of the activation of JNK signaling pathway.
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Objective To observe the autophagy of 87-MG and U251 glioma cells induced by ceramide and explore the possible mecha-nism. Methods The viability and apoptosis of 87-MG and U251 cells were detected by MTT assay and flow cytometry, respectively. Autoph-agic-related protein expressions of LC3B/LC3A and Beclin-1 were determined by Western blotting. The activation of JNK/c-Jun signaling pathway induced by ceramide with or without the treatment of JNK specific inhibitor SP600125 was also measured. Results 24 hours after treatment of ceramide, the growth of 87-MG and U251 cells was significantly inhibited time-dependently (P<0.05);and the number of au-tophagic cells increased dose-dependently (P<0.05). The levels of LC3B/LC3A and Beclin-1 significantly increased after ceramide treat-ment (P<0.05). JNK signaling pathway was activated in the 87-MG and U251 cells and the phosphorylation of c-Jun also increased after ce-ramide treatment. This activation of autophagy could be reversed by the pre-treatment of SP600125. Conclusion Ceramide may induce au-tophagy in 87-MG and U251 glioma cells and the mechanism may be related to the activation of JNK/c-Jun signaling pathway.
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@#Objective To observe the autophagy of 87-MG and U251 glioma cells induced by ceramide and explore the possible mechanism. Methods The viability and apoptosis of 87-MG and U251 cells were detected by MTT assay and flow cytometry, respectively. Autophagic- related protein expressions of LC3B /LC3A and Beclin-1 were determined by Western blotting. The activation of JNK/c-Jun signaling pathway induced by ceramide with or without the treatment of JNK specific inhibitor SP600125 was also measured. Results 24 hours after treatment of ceramide, the growth of 87-MG and U251 cells was significantly inhibited time-dependently (P<0.05); and the number of autophagic cells increased dose-dependently (P<0.05). The levels of LC3B/LC3A and Beclin-1 significantly increased after ceramide treatment (P<0.05). JNK signaling pathway was activated in the 87-MG and U251 cells and the phosphorylation of c-Jun also increased after ceramide treatment. This activation of autophagy could be reversed by the pre-treatment of SP600125. Conclusion Ceramide may induce autophagy in 87-MG and U251 glioma cells and the mechanism may be related to the activation of JNK/c-Jun signaling pathway.
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This study was aimed to explore the function of c-Jun N-terminal kinase (JNK) signaling pathway in the induction of brain ischemic tolerance, and observe the function of Shu-Xue Tong-Mai (SXTM) capsule pretreatment. Ischemic preconditioning was performed for 3 min on rats to induce cerebral ischemic tolerance. Rat model of cere-bral ischemia reperfusion (the ischemia pretreatment group, I/R group) was established 24 h later. Western blot was used to detect the protein expression of JNK and phosphorylation of c-Jun N-terminal kinase (P-JNK), comparing to the expression with the sham operation group, I/R group and SXTM capsule group. Tunel method was applied to de-tect the apoptosis of neurons. Relationship between expression of JNK, P-JNK and apoptosis of neurons was also studied. The results showed that compared with the model group, expressions of P-JNK in ischemia preconditioning group and SXTM group were declined significantly (P < 0.05); and the apoptosis of neurons quantity was also de-clined (P< 0.05). It was concluded that ischemia preconditioning can decrease the apoptosis of neurons in cerebral ischemia reperfusion, and improve neurologic function. Its mechanism related to the inhibition of JNK signaling path-way. SXTM capsule pretreatment can protect the cerebral by inhibiting the JNK signaling pathway.