Endoplasmic Reticulum Stress Induces MUC5AC and MUC5B Expression in Human Nasal Airway Epithelial Cells

OBJECTIVES: Endoplasmic reticulum (ER) stress is known to be associated with inflammatory airway diseases, and three major transmembrane receptors: double-stranded RNA-activated protein kinase-like ER kinase, inositol requiring enzyme 1, and activating transcription factor 6 (ATF6) play important roles in ER stress-related proinflammatory signaling. However, the effects of ER stress and these three major signaling pathways on the regulation of the production of airway mucins in human nasal airway epithelial cells have not been elucidated. METHODS: In primary human nasal epithelial cells, the effect of tunicamycin (an ER stress inducer) and 4-phenylbutyric acid (4-PBA, ER stress inhibitor) on the expression of MUC5AC and MUC5B was investigated by reverse transcriptasepolymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and immunoblot analysis. Small interfering RNA (siRNA) transfection was used to identify the mechanisms involved. RESULTS: Tunicamycin increased the expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules, including spliced X-box binding protein 1 (XBP-1), transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP), and ATF6. In addition, 4-PBA attenuated the tunicamycin-induced expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules. Furthermore, siRNA knockdowns of XBP-1, CHOP, and ATF6 blocked the tunicamycin-induced mRNA expressions and glycoprotein productions of MUC5AC and MUC5B. CONCLUSION.: These results demonstrate that ER stress plays an important role in the regulation of MUC5AC and MUC5B via the activations of XBP-1, CHOP, and ATF6 in human nasal airway epithelial cells.

Melatonin attenuates cerebral ischemia-reperfusion injury in mice by inhibiting endoplasmic reticulum stress / 国际脑血管病杂志

Objective To investigate the effect of melatonin against cerebral ischemia reperfusion injury (CIRI) in mice and its mechanism.Methods Thirty male C57BL/6 mice were randomly divided into sham operation group,CIRI group,and melatonin treatment group (n =10 in each group).A middle cerebral artery occlusion model was induced by suture method.The degree of brain injury was evaluated by neurological function score,brain water content,and cerebral infarction volume.Western blot analysis was used to detect apoptosis-related proteins Bim,Bcl-2,and endoplasmic reticuhm stress-related molecules C/ EBP homologous protein (C/EBP) expression.Results Compared with the CIRI group,the neurological function score was significantly improved,the degree of cerebral edema was significantly reduced,and the volume of cerebral infarction was significantly reduced in the melatonin treatment group (all P ＜0.05).In addition,the expression of Bcl-2 was significantly up-regulated in the melatonin treatment group,and the expression of Bim and CHOP was significantly down-regulated (all P ＜ 0.05).Conclusion Melatonin may play an anti-CIRI role by regulating CHOP,and endoplasmic reticulum stress plays an important role in CIRI.

Effect of sevoflurane postconditioning on expression of CHOP in a rat model of hemorrhagic shock and resuscitation / 中华麻醉学杂志

Objective To evaluate the effect of sevoflurane postconditioning on the expression of CCAAT/enhancer-binding protein homologous protein (CHOP) in a rat model of hemorrhagic shock and resuscitation.Methods Thirty-six healthy adult male Sprague-Dawley rats,weighing 300-350 g,were divided into 3 groups (n=12 each) using a random number table:sham operation group (group S),hemorrhagic shock and resuscitation group (group HSR) and sevoflurane postconditioning group (group SP).Hemorrhagic shock was induced by withdrawing 40% of the total blood volume from the right carotid artery over an interval of 30 min,and 1 h later the removed blood was reinfused via the left jugular vein for resuscitation.Group SP inhaled 2.4% sevoflurane for 30 min starting from the onset of reinfusion.Mean arterial pressure was monitored and recorded at a 10 min interval.Before withdrawing blood (T0),immediately after the end of withdrawing blood(T1), at 1 h after the end of withdrawing blood(T2) and immediately after the end of reinfusion (T3),blood samples were collected from the common carotid artery for blood gas analysis.At 4 days after reinfusion,6 rats of each group were selected to detect spatial learning and memory ability by using Morris water maze test.The animals were then sacrificed,brains were removed for determination of neuronal apoptosis in hippocampal CA1 area using TUNEL.The rest 6 rats in each group were sacrificed at 72 h after reinfusion,and the hippocampus was isolated to detect the expression of CHOP by Western blot.Results Compared with group S,mean arterial pressure was significantly decreased,and lactic acid concentrations were increased at T1,2 in HSR and SP groups,and the escape latency was significantly prolonged,the percentage of time staying at the target quadrant was decreased,the number of apoptotic neurons in hippocampal CA1 area was increased,and the expression of CHOP was up-regulated in group HSR (P<0.05).Compared with group HSR,the escape latency was significantly shortened,the percentage of time staying at the target quadrant was increased,the number of apoptotic neurons in hippocampal CA1 area was decreased,and the expression of CHOP was down-regulated in group SP (P<0.05).Conclusion The mechanism by which sevoflurane postconditioning improves cognitive function is related to down-regulation of CHOP expression and inhibition of apoptosis in hippocampal neurons in a rat model of hemorrhagic shock and resuscitation.

Endoplasmic reticulum stress and renal lesion in mice with combination of high-fat diet and streptozotocin-induced diabetes

ABSTRACT PURPOSE : To investigate in the kidney the pathologic changes and expression of GRP78 and CHOP in the Kunming (KM) mice with combination of high-fat diet and streptozotocin-induced diabetes. METHODS : Sixty two male KM mice were randomly divided into a normal control (NC) group (n=20) and a high-fat diet (HFD) group (n=42). After a four-week dietary manipulation, the KM mice in the HFD group were injected intraperitoneally with streptozotocin to induce diabetes. After diabetic models were successfully established, the kidneys were excised and conserved for further test. RESULTS : No significant difference in the body weight was observed after the dietary manipulation (p=0.554). After the streptozotocin was injected, fasting blood glucose levels in the diabetes group (DM) were significantly higher than that in the NC group (p＜0.0001). Glomerular atrophy observed under light microscope in the DM group was more serious compared with the NC group. The expression of GRP78 and CHOP in the kidneys of the mice in the DM group were higher compared with the NC group. CONCLUSION : Renal lesion occurs in the diabetic Kunming mice induced by combination of high-fat diet and low-dose streptozotocin, and endoplasmic reticulum stress and CHOP may contribute to the injury process.

Effect of fluvastatin on the expressions of caspase-12, CHOP and JNK in ischemia-reperfusion brain injury in rats / 中国医师杂志

Objective To investigate the effect of fluvastatin on the expressions of caspase-12,CCAAT/enhancer-binding protein homologous protein(CHOP), and c-Jun N-terminal kinases (JNK) in ischemia-reperfusion brain injury in rats.Methods Forty two rats were randomly divided into sham operation group (6 rats), ischemia-reperfusion (I/R) group (18 rats), and fluvastatin (Flu) group (18 rats).The rats of I/R and Flu groups were molded by modified Longa intraluminal thread, then put to death at 2 h occlusion and 24 h reperfusion point.Expressions of caspase-12, CHOP, and JNK were detected with immunohistochemistry and Western blot.Results Immunohistochemistry and Western blot showed that the expressions of caspase-12, CHOP, and JNK were increased at 24 h reperfusion.Compared to I/R group, the expressions of caspase-12 and CHOP in Flu group were decreased significantly (all P ＜0.01);and the expression of JNK had no difference between I/R and Flu groups(P ＞ 0.05).Conclusions The increased expression of caspase-12, CHOP, and JNK showed that endoplasmic reticulum stress was involved in the pathological process of ischemia-reperfusion brain injury.Fluvastatin could inhibit the expression of caspase12 and CHOP, and could delete endoplasmic reticulum stress (ERS) in ischemia-reperfusion brain injury.

Change of endoplasmic reticulum stress-related molecules in the course of sciatic nerve injury of diabetic rats / 中国医师杂志

Objective To investigate the dynamic changes of endoplasmic reticulum stress-related molecules including glucose regulated protein (GRP78), C/EBP homologous protein (CHOP), and caspase-12 in sciatic nerve of diabetic rats and explore its mechanisms.Methods Rats were randomly divided into normal control group (NC) and diabetes mellitus group (DM) that were induced by intraperitoneal injection of Streptozocin after 4 weeks of high-fat chow feeding.Sciatic nerves were isolated for three times at 4 weeks, 8 weeks and 12 weeks after induction of diabetes.The expressions of GRP78, CHOP,and caspase-12 were detected with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses.The morphology of sciatic nerve was investigated with electron microscope.Results With the extension of the course, demyelinating and axonal injury appeared in sciatic nerve of diabetic rats.The expressions of GRP78 mRNA and protein in DM group were significantly higher than NC group at 4 weeks and 8 weeks after induction of diabetes(P ＜0.05, P ＜0.01).The expressions of CHOP mRNA and protein in DM group were significantly higher than NC group at 8 weeks and 12 weeks after induction of diabetes (P ＜ 0.05).The expressions of caspase-12 mRNA and protein in DM group were significantly higher than NC group at 8 weeks after induction of diabetes(P ＜ 0.05, P ＜ 0.01).Conclusions Endoplasmic reticulum stress-related molecules (GRP78, CHOP, and caspase-12) contributed to the peripheral nerve injury of diabetic rats, and displayed dynamic changes.

Preliminary mechanism of edaravone against cell apoptosis after spinal cord injury in rats / 天津医药

Objective To investigate the effects of edaravone (EDA) on cell apoptosis induced by endoplasmic reticu?lum stress (ESR) after spinal cord injury (SCI) in rats. Methods Thirty-six healthy adult SD rats were randomly divided in?to three groups (12 rats for each group):Sham group, SCI group and EDA group. The rat model of SCI was made by Allen’s method and the sham group was only received laminectomy and kept the spinal cord intact. Rats in sham group and SCI group accepted the same volume and frequency of saline injection as EDA group. The EDA group was given 10 mg/kg EDA once every 12 h intraperitoneally. Three days after injuring, the spinal cords were harvested, and the protein levels of C/EBP homologous protein (CHOP), Cleaved caspase-12 and Cleaved caspase-3 were detected by Western blot assay. Immunofluo?rescence staining was used to analyze the positive ratio of caspase-12 and CHOP in spinal cord of three groups. Meanwhile, TUNEL staining was used to identify cell apoptosis of spinal cord. Results Compared with sham group, the protein levels of CHOP, Cleaved caspase-12 and Cleaved caspase-3 were obviously higher in SCI group (P<0.01);the proportion of Cas?pase-12 and CHOP positive cells was significantly increased (P<0.01), and the apoptotic rates were also significantly in?creased in spinal cord (P<0.01). However, compared with SCI group, the protein levels of CHOP , Cleaved caspase-12 and Cleaved caspase-3 were significantly decreased in EDA group (P<0.01);the proportion of Caspase-12 and CHOP positive cells was significantly reduced (P<0.01), and the apoptotic rates were also significantly decreased in spinal cord (P<0.01). Conclusion EDA has neuroprotective potential to spinal cord injury. The mechanism of its neuroprotective effect may asso?ciate with its inhibitory effect to the cell apoptosis induced by endoplasmic reticulum stress after SCI.

Effect of Shenfu injection on the expressions of ORP150, XBP1 and CHOP mRNAs in hypoxic-ischemic neonatal rats / 国际脑血管病杂志

Objective To investigate the effect of Shenfu injection on the expressions of endoplasmic reticulum stress-related factors oxygen-regulated protein 150 (ORP150),X-box binding protein 1 (XBP1),and C/EBP homologous protein (CHOP) mRNAs in cerebral cortical neurons after hypoxic-ischemic brain damage (HIBD) in neonatal rats.Methods The 7 day-newborn Sprague-Dawley rats were randomly divided into sham operation,normal saline and Shenfu treatment (Shenfu injection,10 ml/kg per day,peritoneal injection,for 3 days) groups.They were redivided into 3 h,6 h,12 h,24 h,3 d and 7 d subgroups at different time points after modeling (n =8 in each group).A HIBD model of the neonatal rats was induced.Reverse transcription-polymerase chain reaction was used to detect the expressions of ORP150,XBP1,and CHOP mRNAs in rat cerebral cortex.Results The expressions of ORP150,XBP1 and CHOP mRNAs of the sham operation group was very week.The expressions of ORP150,XBP1 and CHOP mRNAs in the normal saline group and the Shenfu treatment group were significantly upregulated compared to those in the sham operation group at 3 h after modeling (all P ＜0.05); the expressions of XBP1 and CHOP mRNAs reached the peak at 6 and 12 h,respectively.Then they decreased mildly and closed to the level in the sham operation group at day 7.The expressions of XBP1 and CHOP mRNAs in the Shenfu treatment group at 12,24 h and day 3 after modeling were significantly lower than those in the normal saline group (all P ＜0.05).The XBP1 mRNA expression was significantly positively correlated with the CHOP mRNA expression in the normal saline group (r =0.649,P ＜0.05).Conclusions HIBD in neonatal rats induces endoplasmic reticulum stress response.ORP150,XBP1 and CHOP may be involved in the delayed neuronal damage process after HIBD in neonatal rats.Shenfu injection downregulates the expression of XBP1 and CHOP mRNAs and inhibits endoplasmic reticulum stress response.

The Effects of Taurine Postconditioning in Pancreatic Injury Following Limb Ischemia Reperfusion in Rats / 天津医药

Objective To observe changes of the pancreas after limb ischemia reperfusion, and the protective effect of taurine (Tau) postconditioning. Methods Twenty-four male SD rats were randomly divided into control group (Control), limb ischemia reperfusion (LIR) group, and taurine postconditioning (Tau) group (n=8 for each group). Plasma contents of xanthine oxidase (XOD), malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), pancreatic amylase (AMS), pancreatic lipase (LPS) and trypsin were detected in three groups. Morphological changes of the pancreas were observed using optical microscopy. The expressions of anti-C/enhancer-binding protein homologous protein (CHOP) in pancreas were analyzed by immunohistochemistry assay. TUNEL staining was performed to estimate the apoptosis of pancre-atic cells. Results Compared with the control group and Tau group, plasma contents of MDA, XOD, ROS, AMS, LPS, and trypsin were significantly increased in LIR group, but the level of SOD was significantly lower in LIR group ( P<0.05). HE staining showed that acinar structure was disrupted , pancreatic lobule gap widened, stromal vascular dilatation and conges-tion were observed in LIR group. The perivascular infiltration of inflammatory cells appeared, islet cells were damaged with irregular islet. The immunohistochemical analysis showed the increased expression of CHOP in pancreas in LIR group than that of Tau group. And the pancreatic apoptosis was enhanced in LIR group detected by TUNEL staining. Conclusion Tau-rine postconditioning can ameliorate pancreatic injury following limb ischemia reperfusion, which may be related to the inhi-bition of oxidative stress, down-regulation of CHOP expression, thereby reducing endoplasmic reticulum stress-induced apoptosis.

Ischemic preconditioning in the protection of global cerebral ischemia in rats by reducing endoplasmic reticulum stress / 国际脑血管病杂志

Objective To investigate the effect of inhibition of endoplasmic reticulum stress (ERS) in ischemic preconditioning-induced cerebral ischemia tolerance.Methods A total of 120 adult male SpragueDawley rats were randomly allocated into three groups:sham operation,global cerebral ischemic and ischemic preconditioning groups (ischemic preconditioning for 3 minutes,and global cerebral ischemia for 15 minutes after 2 days).Three time points (day 1,day 3 and day 7) were set.Sugawara method was used to observe the changes of neurological behavior in rats.TUNEL staining was used to observe the conditions of cortical neuronal apoptosis.Immunofluorescence staining and Western blot analysis were used to detect the expression levels of ERS-related protein CHOP,GRP78,and caspase-12.Results The neurological behavior score showed that the sham operation group did not have neurological deficits.Both the global cerebral ischemic group and the ischemic preconditioning group had obvious neurological deficits,and they improved gradually with the passage of time,but after modeling,the neurological scores at each time point in the global cerebral ischemic were significantly lower than those in the ischemic preconditioning group:at day 1∶11.00 ±0.63 vs.14.33 ±0.33 (t =21.74,P＝0.001); at day 3∶ 12.17±0.31vs.15.17±0.48 (t=27.93,P =0.000); at day 7:14.67±0.49 vs.16.33 ±0.33 (t =7.81,P=0.020).TUNEL staining showed that at day 7 after ischemia,the positive cell count per mm2 in the sham operation,global cerebral ischemic and ischemic preconditioning groups were 4.83 ±1.85vs.395.67± 43.43 and 146.17± 27.38 respectively (F=23.62,P=0.001).The ischemic preconditioning group was significantly lower than that in the global cerebral ischemic group (P =0.001).Immunofluorescence staining showed that at day 7 after ischemia,the numbers of positive cells of CHOP (26.50±3.89vs.82.33±4.25; P=0.000),GRP78 (15.00±2.02vs.35.67±2.99; t=0.000),and caspase-12 (22.33 ± 2.76 vs.66.50± 7.25; P=0.000) in the ischemic preconditioning group were significantly less than those in the global cerebral ischemic group.Western blotting showed that at day 7 after ischemia,the expression levels of CHOP (1.22 ± 0.38 vs.3.22 ± 0.51; t =24.50,P =0.001),GRP78 (1.78 ± 0.45 vs.3.16 ± 0.76; t =14.29,P =0.005),and caspase-12 (2.89 ± 0.53 vs.5.96 ± 0.67; t =77.73; P =0.000) in the ischemic preconditioning group were significantly lower than those in the global cerebral ischemic group.Conclusions Ischemic preconditioning demonstrated a neuroprotective effect for the second lethal ischemia,its mechanism may be associated with the relief of ERS and downregulation of ERS-related protein.

Expression of CHOP in Squamous Tumor of the Uterine Cervix

BACKGROUND: High-risk human papillomavirus (HR-HPV) infection and abnormal p53 expression are closely involved in carcinogenesis of squamous cell carcinoma (SqCC) of uterine cervix. Recent studies have suggested that virus-induced endoplasmic reticulum (ER) stress modulates various cell survival and cell death signaling pathways. The C/EBP homologous protein (CHOP) is associated with ER stress-mediated apoptosis and is also involved in carcinogenesis of several human cancers. We hypothesized that CHOP is involved in the carcinogenesis of uterine cervical cancer in association with HR-HPV and/or p53. METHODS: Immunohistochemistry was used to analyze CHOP and p53 protein expression of tissue sections from 191 patients with invasive cancer or preinvasive lesions of the uterine cervix (61 cases of SqCC, 66 cases of cervical intraepithelial neoplasia [CIN] III, and 64 cases of CIN I). RESULTS: CHOP was expressed in 59.4% of CIN I, 48.5% of CIN III, and 70.5% of SqCC cases. It was also significantly more frequent in invasive SqCC than in preinvasive lesions (p=0.042). Moreover, CHOP expression significantly correlated with HR-HPV infection and p53 expression (p=0.009 and p=0.038, respectively). CONCLUSIONS: Our results suggest that CHOP is involved in the carcinogenesis of the uterine cervix SqCC via association with HR-HPV and p53.

Effect of sevoflurane preconditioning on CHOP expression in cerebral cortex after focal cerebral ischemia-reperfusion injury in rats / 中华麻醉学杂志

Objective To investigate the effect of sevoflurane preconditioning on CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) expression in the cerebral cortex after focal cerebral ischemiareperfusion (I/R) injury in rats and the mechanism. Methods Thirty-six male SD rats weighing 250-280 g were randomly divided into 3 groups ( n = 12 each) : sham operation group (group S) , focal cerebral I/R group (group I/R) and sevoflurane preconditioning group (group Sevo-pc). The animals were anesthetized with intraperitoneal chloral hydrate 300 mg/kg. In groups I/R and Sevo-pc, focal cerebral ischemia was induced by middle cerebral artery occlusion using a nylon thread with rounded tip inserted into the right internal carotid artery and advanced cranially until resistance was met. The occlusion was maintained for 1 h followed by 24 h reperfusion. Group Sevo-pc inhaled 2.7% sevoflurane for 1 h before ischemia. Neurological deficits were assessed and scored at the end of 24 h reperfusion and then the rats were decapitated. Their brains were immediately removed. The cerebral infarct size was determined by TTC staining. The CHOP expression in the ischemic cerebral cortex was determined by immunohistochemistry. The number of apoptotic neurons was counted using TUNEL. Results The neurological deficit scores were significantly higher, the cerebral infarct size was significantly larger, and the CHOP expression and the number of apoptotic neurons were significantly higher in groups I/R and Sevo-pc than in group S ( P ＜ 0.01) . The neurological deficit scores were significantly lower, the cerebral infarct size was significantly smaller, and the CHOP expression and the number of apoptosis neurons were significantly lower in group Sevo-pc than in group I/R ( P ＜ 0.05 or 0.01) . Conclusion Sevoflurane preconditioning may protect the brain against focal cerebral I/R injury by down-regulating CHOP expression in the cerebral cortex in rats.

Involvement of GADD153 and cardiac ankyrin repeat protein in cardiac ischemia-reperfusion injury

Oxidative stress is critical for causing cardiac injuries during ischemia-reperfusion (IR), yet the molecular mechanism for this remains unclear. In the present study, we observe that hypoxia and reoxygenation, a component of ischemia, effectively induces apoptosis in the cardiac myocytes from neonatal rats and it concomitantly leads to induction of GADD153, an apoptosis-related gene. Furthermore, IR injury of rat heart showed a GADD153 overexpression in the ischemic area where the TUNEL reaction was positive. A downregulation of cardiac ankyrin repeat protein (CARP) was also observed in this ischemic area. Promoter deletion and reporter analysis revealed that hypoxia transcriptionally activates a GADD153 promoter through the AP-1 element in neonatal cardiomyocytes. Ectopic overexpression of GADD153 resulted in the downregulation of CARP expression. Accordingly, the induction of GADD153 mRNA were followed by the CARP down-regulation in an in vivo rat coronary ischemia/reperfusion injury model. These results suggest that GADD153 over-expression and the resulting downregulation of CARP may have causative roles in apoptotic cell death during cardiac IR injury.