Clinical value of fluorescence in situ hybridization with MDM2 and DDIT3 probe in diagnosis of liposarcoma / 北京大学学报(医学版)

OBJECTIVE@#To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma.@*METHODS@#In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features.@*RESULTS@#The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis.@*CONCLUSION@#Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.

Protective effect of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress / 中国中药杂志

To explore the protective effect and mechanism of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress. Tunicamycin was used to establish the damage model in L02 cells. Methyl thiazolyl tetrazolium(MTT) colorimetric assay was used to investigate the survival rate of ethyl acetate extract from B. bipinnata in L02 cells injury induced by endoplasmic reticulum stress; the protein expressions of endoplasmic reticulum stress-related molecule glucose regulated protein 78(GRP78), PKR-like ER kinase(PERK), eukaryotic initiation factor-2(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(CHOP), B-cell CLL/lymphoma 2(Bcl-2), Bal-2 associated X apoptosis regulator(Bax) were examined by Wes-tern blot. The expressions of the above proteins were also detected after endoplasmic reticulum stress inhibitor(4-phenyl butyric acid) and CHOP shRNA-mediated knockdowns were added. The expressions of GRP78, PERK, CHOP in L02 cells were observed by immunofluorescence method. The results showed that ethyl acetate extract from B. bipinnata could significantly increase the survival rate of L02 cell injury caused by endoplasmic reticulum stress in a dose and time-dependent manner(P<0.05 or P<0.01). The expression levels of GRP78, PERK, eIF2α, ATF4, CHOP and Bax in the drug treatment groups were significantly down-regulated(P<0.05 or P<0.01), while Bcl-2 was significantly up-regulated(P<0.01). After endoplasmic reticulum stress inhibitor and CHOP shRNA-mediated knockdowns were added, the expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax in the drug treatment groups were significantly down-regulated(P<0.01), whereas Bcl-2 was significantly up-regulated(P<0.01). Immunofluorescence results showed that the expressions of GRP78, PERK, CHOP were consistent with the Western blot method. In conclusion, ethyl acetate extract from B. bipinnata has a significant protective effect on the damage of L02 cells caused by endoplasmic reticulum stress. The mechanism may be related to the inhibition of endoplasmic reticulum stress and the down-regulation of apoptosis in cells through the PERK/eIF2α/ATF4/CHOP signaling pathway.

Glutamine protects intestine against ischemia-reperfusion injury by alleviating endoplasmic reticulum stress induced apoptosis in rats

Abstract Purpose Glutamine, as an essential part of enteral nutrition and parenteral nutrition agent, has been widely recognized to be a kind of important intestinal mucosa protectant in clinical practice and experimental research. However, the mechanisms of its protective effects are still not fully understand. Consequently, this study aimed to explore the potential mechanism of glutamine on ischemia-reperfusion (I/R) injury induced endoplasmic reticulum (ER) stress in intestine. Methods An experimental model of intestinal I/R in rats was established by 1 hour occlusion of the superior mesenteric artery followed by 3 hours of reperfusion. Morphologic changes of intestinal mucosa, apoptosis of epithelial cells, and expression of intestinal Grp78, Gadd153, Caspase-12, ATF4, PERK phosphorylation (P-PERK) and elF2αphosphorylation(P-elF2α) were determined. Results After I/R, the apoptotic index of intestinal mucosa epithelial cells observably increased with notable necrosis of intestinal mucosa, and the expressions of Grp78, Gadd153, Caspase-12, ATF4, P-PERK and P-elF2αall were increased. However, treatment with glutamine could significantly relieve intestinal I/R injury and apoptosis index. Moreover, glutamine could clearly up-regulate the expression of Grp78, restrain P-PERK and P-elF2α, and reduce ATF4, Gadd153 and Caspase-12 expressions. Conclusion Glutamine may be involved in alleviating ER stress induced intestinal mucosa cells apoptosis.

Fluorofenidone inhibits apoptosis of renal tubular epithelial cells in rats with renal interstitial fibrosis

This study aimed to investigate the mechanism of fluorofenidone (AKF-PD) in treating renal interstitial fibrosis in rats with unilateral urinary obstruction (UUO). Thirty-two male Sprague-Dawley rats were randomly divided into sham, UUO, UUO + enalapril, and UUO + AKF-PD groups. All rats, except sham, underwent left urethral obstruction surgery to establish the animal model. Rats were sacrificed 14 days after surgery, and serum was collected for renal function examination. Kidneys were collected to observe pathological changes. Immunohistochemistry was performed to assess collagen I (Col I) protein expression, and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining to observe the apoptosis of renal tubular epithelial cells. The expression of Fas-associated death domain (FADD), apoptotic protease activating factor-1 (Apaf-1), and C/EBP homologous protein (CHOP) proteins was evaluated by immunohistochemistry and western blot analysis. AKF-PD showed no significant effect on renal function in UUO rats. The pathological changes were alleviated significantly after enalapril or AKF-PD treatment, but with no significant differences between the two groups. Col I protein was overexpressed in the UUO group, which was inhibited by both enalapril and AKF-PD. The number of apoptotic renal tubular epithelial cells was much higher in the UUO group, and AKF-PD significantly inhibited epithelial cells apoptosis. The expression of FADD, Apaf-1, and CHOP proteins was significantly upregulated in the UUO group and downregulated by enalapril and AKF-PD. In conclusion, AKF-PD improved renal interstitial fibrosis by inhibiting apoptosis of renal tubular epithelial cells in rats with UUO.

G protein-coupled estrogen receptor alleviates cerebral ischemia-reperfusion injury through inhibiting endoplasmic reticulum stress / 生理学报

The aim of this study was to investigate whether G protein-coupled estrogen receptor (GPER) could alleviate hippocampal neuron injury under cerebral ischemia-reperfusion injury (CIRI) by acting on endoplasmic reticulum stress (ERS). The CIRI animal model was established by middle cerebral artery occlusion (MCAO). Female ovariectomized (OVX) Sprague-Dawley (SD) female rats were randomly divided into 4 groups: control, ischemia-reperfusion injury (MCAO), vehicle (MCAO+DMSO), and GPER-specific agonist G1 (MCAO+G1) groups. The neurobehavioral score was assessed by the Longa score method, the morphological changes of the neurons were observed by the Nissl staining, the cerebral infarction was detected by the TTC staining, and the neural apoptosis in the hippocampal CA1 region was detected by TUNEL staining. The distribution and expression of GRP78 (78 kDa glucose-regulated protein 78) in the hippocampal CA1 region were observed by immunofluorescent staining. The protein expression levels of GRP78, Caspase-12, CHOP and Caspase-3 were detected by Western blot, and the mRNA expression levels of GRP78, Caspase-12, and CHOP were detected by the real-time PCR. The results showed that the neurobehavioral score, cerebral infarct volume, cellular apoptosis index, as well as GRP78, Caspase-12 and CHOP protein and mRNA expression levels in the MCAO group were significantly higher than those of control group. And G1 reversed the above-mentioned changes in the MCAO+G1 group. These results suggest that the activation of GPER can decrease the apoptosis of hippocampal neurons and relieve CIRI, and its mechanism may involve the inhibition of ERS.

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.

Effect of naringin on oxidative stress and endoplasmic reticulum stress in diabetic cardiomyopathy / 中国中药杂志

To explore the protective effect of naringin(Nar) on the injury of myocardium tissues induced by streptozotocin(STZ) in diabetic rats and the relationship with oxidative stress and endoplasmic reticulum stress(ERS)， the male SD rats were intraperitoneally injected with streptozotocin(STZ， 60 mg·kg⁻¹) to establish the diabetic rat model and then randomly divided into the type 1 diabetic rat group(T1DR)， the low-dose Nar group(Nar25)， the middle-dose Nar group(Nar50) and the high-dose Nar group(Nar100). The normal rats were designed as control group(Con). Nar25， Nar50， Nar100 groups were orally administered with Nar at the doses of 25.0， 50.0， 100.0 mg·kg⁻¹ per day， respectively， while the normal group and the T1DR group were orally administered with saline. At the 8th week after treatment， fasting plasma glucose and heart mass index were measured. The pathological changes in myocardial tissues were observed by microscope. The cardiac malondialdehyde(MDA) level and superoxide dismutase(SOD) activities were measured. The gene and protein expressions of glucose-regulated protein 78(GRP78)， C/EBP homologous protein(CHOP)， cysteinyl aspartate-specific proteinase 12(caspase 12) were detected by qRT-PCR and Western blot. According to the results， compared with control group， the myocardial structure was damaged， the content of MDA was increased， while the activities of SOD were decreased(<0.05) in T1DR group. GRP78， CHOP and caspase 12 mRNA and protein expressions were increased significantly in T1DR group(<0.05， <0.01). Compared with T1DR group， myocardial structure damage was alleviated in Nar treatment group. The content of MDA was decreased， while the activities of SOD were increased significantly. The mRNA and protein expressions of GRP78， CHOP and caspase 12 were increased， especially in middle and high-dose groups(<0.05， <0.01). After treatment with Nar for 8 weeks， myocardial structure damage was obviously alleviated in Nar treatment groups. The content of MDA was decreased， while the activities of SOD were increased significantly in myocardial tissues. The mRNA and protein expressions of GRP78， CHOP and caspase 12 were increased， especially in middle and high-dose groups(<0.05， <0.01). The findings suggest that Nar may protect myocardium in diabetic rats by reducing mitochondrial oxidative stress injuries and inhibiting the ERS-mediated cell apoptosis pathway.

Effects of excessive endoplasmic reticulum stress on lung ischemia/reperfusion induced myocardial injury in mice / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of excessive endoplasmic reticulum stress on lung ischemia/reperfusion (I/R) induced myocardial injury in mice.@*METHODS@#Forty healthy SPF male C57BL/6J mice were divided into 4 groups randomly (=10):sham operation group (Sham group), lung I/R group (I/R group), endoplasmic reticulum stress (ERS) pathway agonist Tunicamycin group (TM) and ERS inhibitor 4-phenyl butyric acid group (4-PBA). The model of lung I/R injury was established by clamping the left hilum of lung for 30 min followed by 180 min of reperfusion. In sham group, only sternotomy was performed, the hilum of lung was not clamped, and the mice were mechanically ventilated for 210 min. In TM and 4-PBA groups, TM 1mg/kg and 4-PBA 400 mg/kg were injected intraperitoneally, respectively, at 30 min before establishment of the model. At 180 min of reperfusion, blood samples were collected from the orbit for determination of myocardial enzyme. The animals were then sacrificed, and hearts were removed for determination of light microscope, TUNEL, Caspase 3 enzymatic activity, real-time polymerase chain reaction and Western blot.@*RESULTS@#Compared with sham group, the cardiomyocytes had obvious damage under light microscope, and the serum creatine kinase-MB (CK-MB) and lactic dehydrogenase (LDH) activities, apoptosis index and Caspase 3 enzymatic activity were increased significantly, the expressions of p-Jun N-terminalkinase(p-JNK), Caspase 12, CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose regulated protein 78(GRP78) protein and mRNA were up-regulated in I/R, TM and 4-PBA groups (<0.01). Compared with I/R group, the cardiomyocytes damage was obvious under light microscope, and the serum CK-MB and LDH activities, apoptosis index and Caspase 3 enzymatic activity were increased significantly, the expressions of p-JNK, Caspase 12, CHOP and GRP78 protein and mRNA were up-regulated in group TM; while all above changes were relieved in group 4-PBA (<0.01). Compared with TM group, the cardiomyocytes damage was relieved under light microscope, and the serum CK-MB and LDH activities, apoptosis index and Caspase 3 enzymatic activity were decreased significantly, the expressions of p-JNK, Caspase 12,CHOP and GRP78 protein and mRNA were down-regulated in group 4-PBA.@*CONCLUSIONS@#The excessive endoplasmic reticulum stress participates in myocardial injury induced by lung ischemia/reperfusion (I/R) and inhibit excessive endoplasmic reticulum stress response can relieved myocardial injury.

Effect of dexmedetomidine on apoptosis and CHOP in hypoxia/reoxygenation injury A549 cell / 中国应用生理学杂志

OBJECTIVES@#To investigate the effects of dexmedetomidine (Dex) on injury of A549 cells induced by hypoxia/reoxygenation(H/R)and the influence of C/EBP homologous protein (CHOP) expression.@*METHODS@#Logarithmic growth phase A549 cells(it originated from alveolar type Ⅱ epithelial cell line) were randomly divided into 4 groups (=10):normoxic control group (N), Dex group (D), hypoxia/reoxygenation group (H), hypoxia/reoxygenation + Dex group(HD). At the beginning of modeling, 1 nmol/L Dex was puted into D and HD groups. N and D groups were cultured in the normoxic incubator for 30 h. H and HD group were incubated in the anoxic cultivation for 6 h, fo llowed by normoxic culture for 24 h. Then A549 cells were observed under the inverted microscope to observe the morphological changes. Cell activity was detected by cell counting Kit-8(CCK-8) and the apoptosis index(AI) was detected by in situ end labeling (TUNEL) method. The expression of CHOP、glucose-regulated protein of molecular weight 78 kDa (Grp78)、cysteinyl aspirate-specificprotease-3 (caspase-3) protein and CHOP、Grp78 mRNA were detected by Western blot and RT-PCR.@*RESULTS@#Compared with N group, the number of adherent cells in H group decreased significantly, and cell morphology changed. The absorbance value in H group decreased obviously (<0. 01). The AI value and expression of CHOP, Grp78, caspase-3 proteins and CHOP, Grp78 mRNA were significantly increased (<0.01). Compared with H group, the cell damage in HD group was decreased, the absorbance value increased (<0.01), the number of apoptosis cells decreased relatively (<0.01), the expression of CHOP, caspase-3 protein and CHOP mRNA decreased (<0. 01).@*CONCLUSIONS@#Dex has notable effects against H/R injury, which may be related to effective inhibition of apoptosis mediated by the CHOP's signal path.

Protective effect of prostaglandin E1 against brain injury induced by hyperoxia in neonatal rats / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the protective effect of prostaglandin E1 (PGE-1) against brain injury induced by hyperoxia in neonatal rats and observe the changes in the expression of glucose-regulated protein 78 (GRP78) and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), and to provide a theoretical basis for the clinical application of PGE-1 in the treatment of neonatal brain injury induced by hyperoxia.</p><p><b>METHODS</b>Sixty neonatal Wistar rats were randomly divided into air control group, hyperoxic brain injury model group, and hyperoxic brain injury+PGE-1 group. All rats except those in the air control group were treated to establish a hyperoxic brain injury model. From the first day of modeling, the rats in the hyperoxia brain injury+PGE-1 group were intraperitoneally injected with PGE-1 2 μg/kg daily for 7 consecutive days, while the other two groups were treated with normal saline instead. The water content of brain tissue was measured; the pathological changes of brain tissue were evaluated by hematoxylin-eosin staining; the apoptosis of brain cells was assessed by nuclear staining combined with TUNEL staining; the protein expression of GRP78 and CHOP in brain tissue was measured by Western blot.</p><p><b>RESULTS</b>The water content of brain tissue in the hyperoxic brain injury model group was significantly higher than that in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the water content of brain tissue in the hyperoxic brain injury+PGE-1 group was significantly higher than that in the air control group (P<0.05). The pathological section of brain tissue showed inflammatory cell infiltration and mild cerebrovascular edema in the brain parenchyma in the hyperoxic brain injury model group; the periparenchymal inflammation and edema in the hyperoxic brain injury+PGE-1 group were milder than those in the hyperoxic brain injury model group. The apoptosis index of brain tissue in the hyperoxic brain injury model group was significantly higher than that in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the apoptosis index of brain tissue in the hyperoxic brain injury+PGE-1 group was significantly higher than that in the air control group (P<0.05). The protein expression of GRP78 and CHOP in brain tissue was significantly higher in the hyperoxic brain injury model group than in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the protein expression of GRP78 and CHOP was significantly higher in the hyperoxic brain injury+PGE-1 group than in the air control group (P<0.05).</p><p><b>CONCLUSIONS</b>PGE-1 has a protective effect against hyperoxia-induced brain injury in neonatal rats, which may be related to the inhibition of cell apoptosis by down-regulating the expression of GRP78 and CHOP.</p>

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.

Ellagic acid inhibits proliferation and induces apoptosis in human glioblastoma cells

PURPOSE: To investigate the anticancer activity of ellagic acid (EA) in U251 human glioblastoma cells and its possible molecular mechanism. METHODS: The cells were treated with EA at various concentrations for different time periods. Cell viability and cell proliferation were detected by cell counting kit-8(CCK-8) assay and live/dead assay respectively. Cell apoptosis were measured with Annexin V-FITC/PI double staining method by flow cytometry and Mitochondrial membrane potential assay separately. Cell cycle was measured with PI staining method by flow cytometry. The expressions of Bcl-2, Survivin, XIAP, Caspase-3, Bax, JNK, p-JNK, ERK1/2, p-ERK1/2, p38, p-p38, DR4, DR5, CHOP and GRP78-related proteins were detected by western blot after EA treatment. RESULTS: Cell viability and proliferation of glioblastoma cells treated with EA were significantly lower than the control group. EA caused robust apoptosis of the glioblastoma cells compared to the control group. EA significantly decreased the proportion at G0/G1 phases of cell cycling accompanied by increased populations at S phase in U251 cell lines. And the expressions of anti-apoptotic proteins were dramatically down-regulated. CONCLUSION: Ellagic acid potentially up-regulated DR4, DR5 and MAP kinases (JNK, ERK1/2 and p38). EA also caused significant increase in the expressions of CHOP and GRP78. Our findings suggest that EA would be beneficial for the treatment of glioblastoma.

Effect of hydrogen sulfide on oxidative stress and endoplasmic reticulum stress in diabetic cardiomyopathy / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of hydrogen sulfide (H₂S) on oxidative stress and endoplasmic reticulum stress (ERS) in a rat model of diabetic cardiomyopathy (DCM).</p><p><b>METHODS</b>Thirty male SD rats were randomly divided into control group, diabetes group and treatment group( n = 10). Intraperitoneal injection of streptozotocin was utilized to establish a rat model of DCM. The rats with DCM in treatment group were intraperitoneally injected with NaHS solution. After treated for 12 weeks, the hearts isolated from rats were perfused on a langendorff apparatus. The ventricular hemodynamic parameters were measured. The ultrastructures of myocardium were observed using electron microscopy. The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in myocardial tissue were determined by spectrophotometry. The expressions of C/EBP homologous protein( CHOP), glucose-regulated protein 78 (GRP78) and Caspase 12 at mRNA level in myocardium were detected using RT-PCR.</p><p><b>RESULTS</b>Compared with control group, the cardiac function and myocardial ultrastructure were damaged obviously in diabetic rats. In myocardial tissue, the content of MDA was increased, while the activities of SOD and GSH-Px were decreased. CHOP, GRP78 and Caspase 12 mRNA expressions were increased significantly. Compared with diabetes group, cardiac function and myocardial ultrastructure damage were improved in treatment group. The content of MDA was decreased, while the activities of SOD and GSH-Px were increased significantly. The mRNA levels of CHOP, GRP78 and Caspase 12 were increased.</p><p><b>CONCLUSION</b>H2S can protect myocardium in diabetic rats, maybe it is related to reduce oxidative stress damage and inhibition of the ERS-induced apoptosis pathway.</p>

Effect of Tongxinluo on Apoptosis of Rat Cardiac Microvascular Endothelial Cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the protective effects of Tongxinluo (TXL) on apoptosis of rat cardiac microvascular endothelial cells (RCMECs) resulting from homocysteine (Hcy) induced endoplasmic reticulum stress (ERS), and to determine the signaling pathway behind its protection.</p><p><b>METHODS</b>Primary cultured RCMECs were isolated from neonatal rats using tissue explant method. The morphology of RCMECs was observed using inverted microscope, identified and differentiated by CD31 immunofluorescence method. Selected were well growing 2nd-4th generations of RCMECs. The optimal action time was determined by detecting the expression of glucose regulated protein 78 (GRP78) using immunofluorescence method. In the next experiment RCMECs were divided into 5 groups, i.e., the blank control group, the Hcy induced group (Hcy 10 mmol/L, 10 h), the Hcy + TXL group (Hcy 10 mmol/L + TXL 400 µg/mL), the Hcy +LY294002 group (Hcy 10 mmol/L + LY294002 5 µmol/L, LY294002 as the inhibitor of PI3K), the Hcy + LY294002 + TXL group (Hcy 10 mmol/L + LY294002 5 µmol/L + TXL 400 µg/mL). The apoptosis rate of RCMECs was detected by flow cytometry. mRNA and protein expressions of GRP78, C/ EBP homologous protein (CHOP), and cysteinyl aspartate specific proteinase-12 (caspase12) were detected by real-time reverse transcription PCR (RT-PCR) and Western blot respectively. Expression levels of phosphorylation of phosphatidylinositol 3-kinase (P-PI3K), total phosphatidylinositol 3-kinase (T- P13K) , phosphorylation of kinase B (P-Akt) , and total kinase B (T-Akt) were detected by Western blot.</p><p><b>RESULTS</b>Ten hours Hcy action time was determined. Compared with the blank control group, the apoptosis rate was increased (22.77%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, protein expressions of P-PI3K and P-Akt,ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy induced group (P < 0.05, P < 0.01). Compared with the Hcy induced group, the apoptosis rate was decreased (10.17%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were decreased, and expression levels of P-PI3K, P-Akt, P-PI3K/T-PI3K, and P-Akt/T-Akt were increased in the Hcy + TXL group (P < 0.05, P < 0.01). Compared with the Hcy + TXL group, the apoptosis rate was increased (17.9%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, expression levels of P-PI3K and P-Akt, ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy + TXL + LY294002 group (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>TXL could inhibit the apoptosis of RCMECs resulting from Hcy-induced ERS and its mechanism might be associated with activating PI3K/Akt signaling pathway.</p>

Role of PERK/eIF2α/CHOP Endoplasmic Reticulum Stress Pathway in Oxidized Low-density Lipoprotein Mediated Induction of Endothelial Apoptosis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>PERK/eIF2α/CHOP is a major signaling pathway mediating endoplasmic reticulum (ER) stress related with atherosclerosis. Oxidized LDL (ox-LDL) also induces endothelial apoptosis and plays a vital role in the initiation and progression of atherosclerosis. The present study was conducted to explore the regulatory effect of ox-LDL on PERK/eIF2α/CHOP signaling pathway in vascular endothelial cells.</p><p><b>METHODS</b>The effects of ox-LDL on PERK and p-eIF2α protein expression of primary human umbilical vein endothelial cells (HUVECs) were investigated by Western blot analysis. PERK gene silencing and selective eIF2α phosphatase inhibitor, salubrinal were used to inhibit the process of ox-LDL induced endothelial cell apoptosis, caspase-3 activity, and CHOP mRNA level.</p><p><b>RESULTS</b>Ox-LDL treatment significantly increased the expression of PERK, PERK-mediated inactivation of eIF2α phosphorylation, and the expression of CHOP, as well as the caspase-3 activity and apoptosis. The effects of ox-LDL were markedly decreased by knocking down PERK with stable transduction of lentiviral shRNA or by selective eIF2α phosphatase inhibitor, salubrinal.</p><p><b>CONCLUSION</b>This study provides the first evidence that ox-LDL induces apoptosis in vascular endothelial cells mediated largely via the PERK/eIF2α/CHOP ER-stress pathway. It adds new insights into the molecular mechanisms underlying the pathogenesis and progression of atherosclerosis.</p>

Expressions of CCAAT/enhancer-binding Protein Homologous Protein and Calnexin in the Hippocampus of a Mouse Model of Mesial Temporal Lobe Epilepsy / 中国医学科学院学报

Objective To explore the temporal and spatial distribution of CCAAT/enhancer-binding protein homologous protein (CHOP) and calnexin (CNX) in the dentate gyrus of mesial temporal lobe epilepsy (mTLE) mouse model. Methods We used kainic acid (KA) to induce acute phase (12 h and 24 h) mTLE mouse models and performed Western blotting and immunofluorescence to detect the different expressions and distribution pattern of CHOP and CNX in CA3 of the hippocampus. Results Compared with the controls,the expressions of CHOP(F=1.136,P=0.4069) and CNX (F=2.378,P=0.2087) did not increase in CA3 of hippocampus 12 h following KA injection in the acute phase of mTLE mouse models,whereas the expressions in CA1 and CA3 of hippocampus 24 h after injection were significantly higher (F=8.510,P=0.0362;F=6.968,P=0.0497,respectively). As shown by immunofluorescence analysis,CHOP was expressed mainly in CA3 of hippocampus 12 h after KA injection,and increased in CA1 and CA3 24 h after KA administration. Compared with the controls,the expressions of CHOP(F=24.480,P=0.0057) and CNX (F=7.149,P=0.0478) were significantly higher 24 h after KA injection.Conclusions The expression of CHOP increases along with the progression of seizures,indicating the increased level of endoplasmic reticulum stress. An increasing number of CNX,which serves as molecular chaperone,may be needed to facilitate the unfolded protein to complete the folding process.

Association between endoplasmic reticulum stress pathway mediated by inositol-requiring kinase 1 and AECII apoptosis in preterm rats induced by hyperoxia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the association between endoplasmic reticulum stress (ERS) pathway mediated by inositol-requiring kinase 1 (IRE1) and the apoptosis of type II alveolar epithelial cells (AECIIs) exposed to hyperoxia.</p><p><b>METHODS</b>The primarily cultured AECIIs from preterm rats were devided into an air group and a hyperoxia group. The model of hyperoxia-induced cell injury was established. The cells were harvested at 24, 48, and 72 hours after hyperoxia exposure. An inverted phase-contrast microscope was used to observe morphological changes of the cells. Annexin V/PI double staining flow cytometry was performed to measure cell apoptosis. RT-PCR and Western blot were used to measure the mRNA and protein expression of glucose-regulated protein 78 (GRP78), IRE1, X-box binding protein-1 (XBP-1), and C/EBP homologous protein (CHOP). An immunofluorescence assay was performed to measure the expression of CHOP.</p><p><b>RESULTS</b>Over the time of hyperoxia exposure, the hyperoxia group showed irregular spreading and vacuolization of AECIIs. Compared with the air group, the hyperoxia group showed a significantly increased apoptosis rate of AECIIs and significantly increased mRNA and protein expression of GRP78, IRE1, XBP1, and CHOP compared at all time points (P<0.05). The hyperoxia group had significantly greater fluorescence intensity of CHOP than the air group at all time points. In the hyperoxia group, the protein expression of CHOP was positively correlated with the apoptosis rate of AECIIs and the protein expression of IRE1 and XBP1 (r=0.97, 0.85, and 0.88 respectively; P<0.05).</p><p><b>CONCLUSIONS</b>Hyperoxia induces apoptosis of AECIIs possibly through activating the IRE1-XBP1-CHOP pathway.</p>

Endoplasmic Reticulum Stress Induces the Early Appearance of Pro-apoptotic and Anti-apoptotic Proteins in Neurons of Five Familial Alzheimer's Disease Mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Amyloid β (Aβ) deposits and the endoplasmic reticulum stress (ERS) are both well established in the development and progression of Alzheimer's disease (AD). However, the mechanism and role of Aβ-induced ERS in AD-associated pathological progression remain to be elucidated.</p><p><b>METHODS</b>The five familial AD (5×FAD) mice and wild-type (WT) mice aged 2, 7, and 12 months were used in the present study. Morris water maze test was used to evaluate their cognitive performance. Immunofluorescence and Western blot analyses were used to examine the dynamic changes of pro-apoptotic (CCAAT/enhancer-binding protein homologous protein [CHOP] and cleaved caspase-12) and anti-apoptotic factors (chaperone glucose-regulated protein [GRP] 78 and endoplasmic reticulum-associated protein degradation-associated ubiquitin ligase synovial apoptosis inhibitor 1 [SYVN1]) in the ERS-associated unfolded protein response (UPR) pathway.</p><p><b>RESULTS</b>Compared with age-matched WT mice, 5×FAD mice showed higher cleaved caspase-3, lower neuron-positive staining at the age of 12 months, but earlier cognitive deficit at the age of 7 months (all P < 0.05). Interestingly, for 2-month-old 5×FAD mice, the related proteins involved in the ERS-associated UPR pathway, including CHOP, cleaved caspase-12, GRP 78, and SYVN1, were significantly increased when compared with those in age-matched WT mice (all P < 0.05). Moreover, ERS occurred mainly in neurons, not in astrocytes.</p><p><b>CONCLUSIONS</b>These findings suggest that compared with those of age-matched WT mice, ERS-associated pro-apoptotic and anti-apoptotic proteins are upregulated in 2-month-old 5×FAD mice, consistent with intracellular Aβ aggregation in neurons.</p>

The role of cell apoptosis mediated by endoplasmic reticulum stress (ERS) of deep tissue injury of pressure ulcer of rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the the expression of endoplasmic reticulum stress (ERS) related factors in deep tissue injury (DTI) at pressure ulcer rat and to investigate the ERS mechanism of DTI in muscle tissue and protective effect of 4-phenylbutyric acid (4-PBA) in local tissue.</p><p><b>METHODS</b>Fifty male SD rats were randomly devided into control group, model group, experimental group NS group and PBA group, the experimental groups were divided into 4 d, 7 d, 14 d and 21 d group according to the observation time (n = 5). Rats in the PBA group were administrated with gastric perfusion of 4-PBA after the modeling; the NS group was given normal saline of the same quantity. Using HE staining to observe morphologic character. The expression of glucose regulated protein 78 (GRP78), CHOP, Caspase 12 were detected by immunohistochernical staining. Cell apoptosis was detected by TUNEL assay.</p><p><b>RESULTS</b>HE staining results showed that each group demonstrated compression injury compared with control group: cellular swelling, ompaction of nuclear, and apoptosis in muscle tissue. The new muscle fiber in 4-PBA group fused faster than those in NS group. The number of TUNEL positive cells peaked at 4 day after compression, then got decreased on day 7 in muscle tissue, apoptosis positive cells were diminished after 4-PBA treatment. The immunohistochemical staining results showed that the expression of protein GRP78, CHOP, Caspase 12 peakd 4 d after modeling and decreased gradually. The GRP78, CHOP, Caspase 12 protein expression were significantly higher than those of PBA group at all time points (P < 0.05).</p><p><b>CONCLUSION</b>Cell apoptosis induced by endoplasmic reticulum stress took part in deep tissue injury resulting of pressure ulcer, which mechanism might be related to reducing apoptosis mediated by CHOP, Caspase 12.</p>

Ethanol promotes saturated fatty acid-induced hepatoxicity through endoplasmic reticulum (ER) stress response / 中国天然药物

Serum palmitic acid (PA), a type of saturated fatty acid, causes lipid accumulation and induces toxicity in hepatocytes. Ethanol (EtOH) is metabolized by the liver and induces hepatic injury and inflammation. Herein, we analyzed the effects of EtOH on PA-induced lipotoxicity in the liver. Our results indicated that EtOH aggravated PA-induced apoptosis and lipid accumulation in primary rat hepatocytes in dose-dependent manner. EtOH intensified PA-caused endoplasmic reticulum (ER) stress response in vitro and in vivo, and the expressions of CHOP, ATF4, and XBP-1 in nucleus were significantly increased. EtOH also increased PA-caused cleaved caspase-3 in cytoplasm. In wild type and CHOP(-/-) mice treated with EtOH and high fat diet (HFD), EtOH worsened the HFD-induced liver injury and dyslipidemia, while CHOP knockout blocked toxic effects of EtOH and PA. Our study suggested that targeting UPR-signaling pathways is a promising, novel approach to reducing EtOH and saturated fatty acid-induced metabolic complications.