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Objective:To investigate the expression and significance of endoplasmic reticulum stress apoptosis pathway related proteins in renal cortex of rats with chronic fluorosis.Methods:Twenty four healthy SD rats were divided into 4 groups (6 rats/group, half male and half female) according to their body mass (100 - 120 g) by random number table method, rats in control group drank tap water (fluoride content < 0.5 mg/L), and in low, medium and high fluoride groups drank tap water with fluoride content (sodium fluoride) of 10, 50 and 100 mg/L, respectively. After 180 days of feeding, dental fluorosis was examined, 24-hour urine sample was collected and the content of fluoride in urine was detected by fluoride ion selective electrode method. Renal tissue was taken after anesthesia, and the pathological changes of renal cortex were observed by hematoxylin-eosin (HE) staining. The expressions of endoplasmic reticulum stress apoptosis pathway related proteins [inositol-requiring enzyme 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), C-Jun N-terminal kinase (JNK) and phosphorylated JNK (P-JNK)] were determined by immunohistochemical staining in rat renal cortex.Results:No dental fluorosis was found in control group. The incidence of dental fluorosis in low, medium and high fluoride groups were 2/6, 5/6 and 6/6, respectively. Compared with control group [(5.707 ± 1.190) mg/L], the urinary fluoride in low, medium and high fluoride groups [(17.028 ± 3.006), (34.378 ± 12.045), (94.759 ± 31.773) mg/L] was significantly higher ( P < 0.05), and the urinary fluoride in high fluoride group was higher than that in low and medium fluoride groups ( P < 0.05). HE staining showed that, compared with control group, the cell volume of renal tubules and glomeruli in medium and high fluoride groups increased, the cells arranged closely, and the eosinophilia of the cytoplasm increased. The immunohistochemical staining results showed that there was no significant difference in the expression of JNK protein in rat renal cortex between control group and low, medium and high fluoride groups ( F = 0.07, P > 0.05). The expressions of IRE1α, ASK1 and P-JNK proteins in rat renal cortex in high fluoride group were higher than those of control, low and medium fluoride groups ( P < 0.05), and the expressions of IRE1α and ASK1 proteins in medium fluoride group were significantly higher than those in control and low fluoride groups ( P < 0.05). Conclusion:Long-term excessive fluoride intake can lead to renal cortex injury in rats, and the mechanism of injury may be related to the activation of IRE1α-ASK1-JNK endoplasmic reticulum stress apoptosis pathway.
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Objective To study the effect of small interfering ribonucleic acid (siRNA) silencing apoptosis signal-regulating kinase 1 (ASK1) on inflammatory response of lipopolysaccharide-induced alveolar epithelial A549 cells and its mechanism.Method Cell inflammation model of A549 cells was induced by lipopolysaccharide.The expression of ASK 1 in A549 cells was silenced by liposome transfection of siRNA.The mRNA and expression levels of ASK1,interleukin 6 (IL-6),interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) in A549 cells were detected by immunoblotting,real-time fluorescence quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.Result The expression of IL-6,IL-8 and TNF-α in the experimental group was significantly higher than that in the control group (P＜0.001),which indicated that the inflammatory model of A549 cells was successfully constructed.The mRNA level and expression of ASK1 in the interference group was significantly lower than that in the negative control group and the blank control group (P＜0.01),indicating that silencing ASK1 was also successful.The expressions of IL-6,IL-8 and TNF-α in the interference group (0.37±0.04,0.32±0.04,0.48 ±0.13) were significantly lower than those in the negative control group (1.04±0.11,1.22±0.19,0.93±0.14) and the blank control group (1.01±0.14,1.01 ±0.23,1.02±0.25).The expression of IL-6,IL-8 and TNF-α protein in the interference group (pg/ml) (122.6± 11.0,537.2±42.4,159.2± 19.6) were also significantly lower than those in the negative control group (267.4±20.4,1 289.8±55.3,327.0±26.3) and blank control group (246.6±18.7,1 300.3±35.6,325.2± 18.3),with significant difference (P＜0.05).There was no significant difference in each value between negative control group and blank control group (P＞0.05).Conclusion Silencing ASK1 by siRNA can down-regulate the expression of IL-6,IL-8 and TNF-α in A549 cells,suggesting that ASK 1 may be involved in the regulation of lipopolysaccharide-induced inflammation in A549 cells.
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Objective To explore the role of apoptosis signal regulating kinase 1(ASK1) in inflammatory mediated secondary injury after spinal cord injury(SCI) in rats.Methods The rat contusion SCI model was used.Forty-eight rats were randomly divided into the sham operation group(Sham),normal saline(Saline group) and inflammatory factors group (Cytokine group) respectively.The expressions of ASK1 and phosphorylated ASK1(pASK1) were detected by using Western blot.The Basso Beattie Bresnahan (BBB) scores and Grid Walking method were performed to assess the behavior changes of injured rat hindlimbs.Somatosensory evoked potential(SEP) and motor evoked potential(MEP) were used to examine the electrophysiological change.Results The expression levels of ASK1 mRNA and protein had no obvious change at 1 week after SCI;the pASK1 expression level in the Cytokine group was significantly up-regulated compared with the Saline group(P=0.002);the BBB scores at 3 or 4 weeks after SCI in the Cytokine group was significantly decreased compared with the Saline group (P =0.000,P =0.000);the hindlimbs missed step rate at 4 weeks following SCI in the Cytokine group was increased compared with the Saline group (P =0.032);the latent period of SEP and MEP in the Cytokine group was prolonged(P =0.043,P =0.045),while the wave peak value had no obvious changed (P =0.889,P=0.434).Conclusion Inflammatory cytokines may lead the hindlimbs movement dysfunction to be aggravated after SCI in rat,its mechanism may be related with the phosphorylation elevation of ASK1.
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Objective To investigate the alteration of apoptosis signal-regulating kinase 1(ASK1)phos-phorylation and matrix metalloproteinase-9(MMP-9)expression after temporal lobe epilepsy(TLE),and to explore the role and mechanism of ASK1-MMP-9 signal in TLE. Methods The lithium-pilocarpine epilepsy model was established. The ASK1 phosphorylation and MMP-9 expression were detected by western blot and immunofluo-rescence histochemistry. Using ASK1 inhibitors,the regulating effects of ASK1 on MMP-9 expression were deter-mined. And the role of ASK1 and MMP-9 in epileptic seizure was explored by inhibition of ASK1 and MMP-9. Results The ASK1 phosphorylation and MMP-9 expression increased in hippocampus in epilepsy model ,exhibiting a temporal correlation with epileptic seizure. Inhibition of ASK1 activation reduced the expression of MMP-9. And the level of epileptic seizure was significantly reduced by the inhibition of ASK1 and MMP-9. Conclusions The hippocampal ASK1 phosphorylation and MMP-9 expression distinctly increase in TLE model ,and the MMP-9 expression is induced by ASK1 activation. The ASK1-MMP-9 signal plays an important role in TLE.
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Objective To study the roles of miR-20a in lipopolysaccharide induced inflammation of A549 cells and the possible mechanisms.Method The miR-20a mimic/inhibitor were transfected into A549 cells, and the cells were stimulated using lipopolysaccharide for 24 h.Interleukin-6 ( IL-6) and IL-8 were detected at mRNA level and protein level using real-time PCR and ELISA method , respectively.Protein expression of apoptosis signal regulating kinase 1 (ASK1)、P38、P-P38、JNK and P-JNK were detected using Western blot. Result Compared to mimic negative control group , the levels of mRNA and protein expression of IL-6 and IL-8 in the mimic group were all significantly decreased ( P<0.05).Compared to inhibitor negative control group , the levels of mRNA and protein expression of IL-6 and IL-8 in the inhibitor group were all significantly increased (P<0.05).The levels of ASK1, P-P38 and P-JNK protein in the mimic group were significantly lower than the mimic negative control group (P<0.05);the level of protein expression of ASK1, P-P38 and P-JNK in the inhibitor group were all higher than the inhibitor negative control group (P<0.05).Conclusion The regulation of ASK1 by miR-20a may play an important role in the inflammation process of acute respiratory distress syndrome .
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Objective To observe the influence of Simvastatin on immature rabbit model of chronic heart failure(CHF),and to explore the possible protective mechanism of Simvastatin for the rabbits with CHF.Methods Thirty immature male rabbits were divided into 3 groups randomly:control group,heart failure group (HF group)and Simvastatin group(SIM group),10 rabbits in each group.The models of CHF were established by injecting Adrinmycin via the auricular vein of rabbits (1.5 mg/kg,once 1 week,for 12 weeks).The control group were injected the same amount of 9 g/L saline.SIM group were given both injection of Adrinmycin and Simvastatin [1.5 mg/(kg · d)for 12 weeks].The immature rabbit's cardiac function and myocardial morphology changes were evaluated.The expressions of chromogranin A (CgA) and apoptosis signal-regulating kinase 1 (ASK1) were evaluated.Results (1) In control group,the immature rabbits were all alive;in the other 2 groups,most immature rabbits were depressed and sluggish.The survival rate of HF group was 60％,while in SIM group the survival rate was 80％.(2)Compared with the control group,the left ventricular ejection fraction in HF group and SIM group decreased significantly [(40.05 ± 6.74)％,(50.18 ± 5.73) ％ vs.(65.93 ± 5.65) ％,all P ＜ 0.01],while in SIM group was higher than that of HF group,and the difference was significant (P ＜ 0.05);compared with HF group,the left ventricular end diastolic diameter and left ventricular end systolic diameter decreased in SIM group [(13.48 ± 1.24) mm vs.(16.23 ± 2.82) mm;(9.87 ± 0.85) mm vs.(11.13 ± 1.21) mm],and the differences were significant (all P ＜ 0.05).(3) Compared with the control group,the expression of CgA (mean optical density 142.24 ± 17.14,127.93 ± 12.12 vs.78.65 ± 6.78,P ＜ 0.05;integrated optical density 1 422.41 ± 167.34,1 279.37 ± 118.15 vs.786.54 ± 75.84,P ＜ 0.05) and ASK1 (mean optical density 140.32 ± 18.65,115.48 ± 12.30 vs.69.85 ± 6.54,P ＜ 0.05;integrated optical density 1 403.23 ± 165.67,1 158.79 ± 137.81 vs.698.58 ± 64.51,P ＜ 0.05) increased in the HF group and SIM group.While the expression in the SIM group decreased significantly compared with that of the HF group,and the difference was significant (P ＜ 0.05).Conclusions Simvastatin can improve cardiac function of immature rabbits with CHF.The mechanism of SIM may depress the expressions of CgA and ASK1.
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Objective To explore the changes in inositol requiring enzyme 1 (IRE1),apoptosis signal regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) mRNA levels in peripheral blood CD4+ T cells of children with acute paraquat (PQ) poisoning.Methods Blood samples of 30 cases of acute PQ poisoning (PQ group),who visited Tianjin Children's Hospital from June 2014 to June 2016,with 18 male and 12 female,aged from 2 to 14 years old,were collected,and the clinical and laboratory data were documented.Peripheral venous blood samples were collected after paraquat was taken.Thirty healthy children at the same age and of the same sex were selected as a healthy control group,18 male and 12 female,aged from 2 to 14 years old.CD4+ T cells in the peripheral blood were separated,and IRE1,ASK1 and JNK mRNA levels in peripheral blood CD4+ T cells were measured by real time polymerase chain reaction (Real-time PCR) method.Specificity of PCR products was validated through agarose gel electrophoresis.The data were statistically analyzed by SPSS 13.0 software.Results All of the 30 children had mucosal lesions,nausea,vomiting and abdomen pain,19 cases with oliguria and anuria,16 cases with alimentary tract bleeding,12 cases with headache and dizziness,11 cases with short of breath,dyspnea and difficult breathing,8 cases with convulsion,5 cases with jaundice.The IRE1,ASK1 and JNK mRNA levels in PQ group were significantly higher than those in healthy control group (1.70 ± 0.16 vs.1.02 ± 0.18,3.56 ± 0.85 vs.1.05 ± 0.31,5.22 ± 0.87 vs.1.01 ± 0.33,t =15.26,15.21,24.78,all P ＜ 0.01).Conclusions PQ increased the expressions of IRE1,ASK1 and JNK in peripheral blood CD4+ T cells,which may be related to PQ-induced oxidative stress and immune activation and lead to a complex cytokine network via endoplasmic reticulum stress and CD4+ T cell apoptosis and then results in the occurrence and development of multiple organ failure.
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OBJECTIVE@#To explore the protective effect and its molecular mechanism of apoptosis signal-regulating kinase 1 (ASK1) inhibitor (GS-459679) on acetaminophen-induced liver injury in mice.@*METHODS@#The model of liver injury was established by administration of acetaminophen (APAP) (300 mg/kg, i.p.) on C57BL/6 mice. Forty-eight male C57BL/6 mice were randomly divided into four groups, consisting of control group, GS group (GS-459679, 30 mg/kg, i.p.), APAP-induced group, and GS combined with APAP-induced group. For GS combined with APAP-induced group, mice were treated with GS 30 min prior to administration of APAP. After mice were euthanized at 6 h or 12 h, respectively, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed, and mRNA levels of TNF-α, IL-6 and IL-1β were tested. The activity of glutathione (GSH), oxidized GSH (GSSG) and malondialdehyde were quantified. In addition, ASK1, P-ASK1, JNK and P-JNK protein levels were tested in all groups.@*RESULTS@#The ASK1 and P-ASK1 levels were up-regulated in APAP-induced group. Compared to the control group, serum levels of ALT and AST, and mRNA levels of TNF-α, IL-6 and IL-1β were increased in APAP-induced group. Meanwhile, the levels of MAD and GSSG, and the ratio of GSSG/GSH were higher and the JNK was activatedin APAP-induced group compared with that in control group. However, compared to APAP-induced group, GS combined with APAP-induced group displayed a decrease of protein expression levels of ASK1, P-ASK1 and P-JNK, a reduction of serum levels of ALT and AST, a decrease in TNF-α, IL-6 and IL-1β mRNA levels, and a low ration of GSSG/GSH.@*CONCLUSIONS@#GS-459679 treatment effectively down-regulates ASK1 and P-ASK1 expression. Addition of GS-459679 decreases the generation of liver metabolites and inflammatory factors, reduces oxidative stress reaction, inhibits JNK activation, and then protects the responsiveness to APAP-induced liver injury.
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Objective: To explore the protective effect and its molecular mechanism of apoptosis signal-regulating kinase 1 (ASK1) inhibitor (GS-459679) on acetaminophen-induced liver injury in mice. Methods: The model of liver injury was established by administration of acetaminophen (APAP) (300 mg/kg, i.p.) on C57BL/6 mice. Forty-eight male C57BL/6 mice were randomly divided into four groups, consisting of control group, GS group (GS-459679, 30 mg/kg, i.p.), APAP-induced group, and GS combined with APAP-induced group. For GS combined with APAP-induced group, mice were treated with GS 30 min prior to administration of APAP. After mice were euthanized at 6 h or 12 h, respectively, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed, and mRNA levels of TNF-α, IL-6 and IL-1β were tested. The activity of glutathione (GSH), oxidized GSH (GSSG) and malondialdehyde were quantified. In addition, ASK1, P-ASK1, JNK and P-JNK protein levels were tested in all groups. Results: The ASK1 and P-ASK1 levels were up-regulated in APAP-induced group. Compared to the control group, serum levels of ALT and AST, and mRNA levels of TNF-α, IL-6 and IL-1β were increased in APAP-induced group. Meanwhile, the levels of MAD and GSSG, and the ratio of GSSG/GSH were higher and the JNK was activatedin APAP-induced group compared with that in control group. However, compared to APAP-induced group, GS combined with APAP-induced group displayed a decrease of protein expression levels of ASK1, P-ASK1 and P-JNK, a reduction of serum levels of ALT and AST, a decrease in TNF-α, IL-6 and IL-1β mRNA levels, and a low ration of GSSG/GSH. Conclusions: GS-459679 treatment effectively down-regulates ASK1 and P-ASK1 expression. Addition of GS-459679 decreases the generation of liver metabolites and inflammatory factors, reduces oxidative stress reaction, inhibits JNK activation, and then protects the responsiveness to APAP-induced liver injury.
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Objective To observe the changes of apoptosis signal-regulating kinase 1 (ASK1) expression in left ventricular myocardium of the dilated cardiomyopathy (DCM) rats induced by adriamycin and its correlation with left ventricular ejection fraction (LVEF).Methods One hundred and twenty SPF Wistar rats were divided into 2 groups as follows:control group (n =15),model group(n =105).Adriamycin was administered in the model group by intraperitoneal injection for 3 times in a week and repeated with a two-week interval for 6 times,while 9 g/L saline were administered in the control group in the same pattern,thus DCM rats model were constructed by the end of the 8th week.Both the model group and control group rats were drawn out and their LVEF were tested by the end of the 8th week and 12th week.Apoptosis index (AI) and the ASK1 in myocardium were tested respectively by apoptotic cells situ labeling,semi-quantitative analysis and Western blot.Results The AI of the control group,the 8th weekend model group and the 12th weekend model group were 0.53 ±0.27,16.13 ± 1.72,19.54 ±2.24.The AI of the 8th and the 12th weekend model groups were obviously higher than that in the control group.Comparing the differences among the 3 groups were statistically significant (F =18.98,P ＜ 0.05).The relative ASK1 expressions in ventricular myocardium of the control group,the 8th weekend model group and the 12th weekend model group were 0.169 ± 0.010,0.649 ± 0.071,0.781 ±0.077.Comparing the differences among the 3 groups were statistically significant (F =27.72,P ＜ 0.01).The LVEF (％) results of the control group,the 8th weekend model group and the 12th weekend model group were 75.41 ± 2.02,53.25 ±3.74,39.21 ±6.13.Comparing the differences among the 3 groups were statistically significant(F =154.87,P ＜0.01).There was a negative correlation between the ASK1 expression and LVFE in model group at the end of 12weeks(r =-0.86,P ＜ 0.01).Conclusions The ASK1 expression in myocardium of the DCM group increases obviously,thus apoptosis signal-regulating probably participates in the pathological process of DCM induced by ASK1.
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Neural stem cells (NSCs) have been suggested as a groundbreaking solution for stroke patients because they have the potential for self-renewal and differentiation into neurons. The differentiation of NSCs into neurons is integral for increasing the therapeutic efficiency of NSCs during inflammation. Apoptosis signal-regulating kinase 1 (ASK1) is preferentially activated by oxidative stress and inflammation, which is the fundamental pathology of brain damage in stroke. ASK1 may be involved in the early inflammation response after stroke and may be related to the differentiation of NSCs because of the relationship between ASK1 and the p38 mitogen-activated protein kinase pathway. Therefore, we investigated whether ASK1 is linked to the differentiation of NSCs under the context of inflammation. On the basis of the results of a microarray analysis, we performed the following experiments: western blot analysis to confirm ASK1, DCX, MAP2, phospho-p38 expression; fluorescence-activated cell sorting assay to estimate cell death; and immunocytochemistry to visualize and confirm the differentiation of cells in brain tissue. Neurosphere size and cell survival were highly maintained in ASK1-suppressed, lipopolysaccharide (LPS)-treated brains compared with only LPS-treated brains. The number of positive cells for MAP2, a neuronal marker, was lower in the ASK1-suppressed group than in the control group. According to our microarray data, phospho-p38 expression was inversely linked to ASK1 suppression, and our immunohistochemistry data showed that slight upregulation of ASK1 by LPS promoted the differentiation of endogenous, neuronal stem cells into neurons, but highly increased ASK1 levels after cerebral ischemic damage led to high levels of cell death. We conclude that ASK1 is regulated in response to the early inflammation phase and regulates the differentiation of NSCs after inflammatory-inducing events, such as ischemic stroke.
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Animals , Male , Mice , Cell Death , Infarction, Middle Cerebral Artery/metabolism , Lipopolysaccharides/pharmacology , MAP Kinase Kinase Kinase 5/genetics , Mice, Inbred C57BL , Microtubule-Associated Proteins/genetics , Neural Stem Cells/cytology , Neurogenesis , Neuropeptides/genetics , p38 Mitogen-Activated Protein Kinases/geneticsABSTRACT
Aim To investigate the effects of antioxidant probucol on vascular smooth muscle cells(VSMCs) apoptosis induced by H2O2.Methods H2O2 (1 mmol?L-1) was used to induce VSMCs apoptosis.The VSMCs were treated with probucol(100,10,1 ?mol?L-1) for 6 hours.For the evaluation of apoptosis,Annexin V-FITC staining,Hoechest33258 staining and the TUNEL assay were used.The expressions of ASK-1 and Trx-1 were detected by Western blot analysis.Results H2O2 could promote the apoptosis of VSMCs,increase the expression of ASK-1 and decrease the expression of Trx-1.Probucol could attenuate the apoptosis induced by H2O2 in a dose-dependent,down-regulate ASK-1 expression and increase Trx-1 expression.Conclusion Probucol can antagonize the apoptosis of VSMCs induced by H2O2.The mechanism may be correlated with a decreased expression of ASK-1 and an increased expression of Trx-1.
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Objective To study the effect of apoptosis signal regulating kinase 1(ASK1) protein expression in hypertrophic myocardium in rat models of hypertension.Methods The rat model of hypertension was established by abdominal aortic constriction.Six weeks later,the reactive oxygen species(ROS) level and ASK1 protein expression in myocardium of left ventricle were detected respectively by color matching and Western blotting.Results The left ventricular mass index,ROS level and ASK1 protein expression all increased significantly in hypertensive rats as compared with those underwent sham operation.Conclusion Under chronic pressure overload,the activated ASK1 with the increasing ROS may play an important role in signal transduction pathway of myocardial hypertrophy.
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AIM:To explore the relationship between apoptosis in the lung tissues and lung ischemia/reperfusion injury,and to observe the effects of human thioredoxin (hTrx) on apoptosis in lung ischemia/reperfusion injury. METHODS:The single lung in situ ischemia/reperfusion animal model was used. Eighty four Wistar rats were randomly divided into control group (control),groups of ischemia for 1 h and reperfusion for different times (IR1h,IR3h,IR5h),and groups of IR + human thioredoxin treatment (IR1h + hTrx,IR3h + hTrx and IR5h + hTrx). Transmission electron microscope (TEM),terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immunocytochemistry techniques were used to observe apoptosis,apoptosis signal-regulating kinase 1 (ASK1) and expression of Bcl-2 and Bax in various phases of lung ischemia/reperfusion. RESULTS:Cell apoptosis in lung tissues was significantly high,ASK1,Bcl -2 and Bax protein were upregulated in lung tissues of lung ischemia/reperfusion injury as compared to control (all P