PERK signal pathway is involved in hypoxia-induced endoplasmic reti-culum stress and apoptosis in cultured cardiac myocytes

AIM:To investigate the role of endoplasmic reticulum(ER) stress in the process of hypoxia-induced neonatal rat myocardial injury through PERK signal pathway. METHODS:Neonatal rat cardiac myocytes were randomly divided into control group and hypoxia 1 h, 4 h, 8 h, 12 h and 24 h groups. Cell viability was evaluated by determining the intracellular content of ATP. Apoptosis was measured by high-content analysis(HCA) cell imaging system. The protein levels of GRP78, calreticulin, p-PERK, p-eIF2α, ATF4 and CHOP were detected by Western blotting at different time points. The primary cultured neonatal rat cardiac myocytes were treated with an agonist of PERK pathway salubrinal and the cell apoptosis was observed under hypoxia. RESULTS:In the early phase, hypoxia induced an increase in the expression of calreticulin and GPR78. In the middle phase of hypoxia, the levels of p-PERK, p-eIF2α and ATF4 were increased. In the later phase of hypoxia, increased CHOP level was also observed. Salubrinal effectively protected the cardiac myocytes from hypoxic injury. CONCLUSION:Hypoxia activates ER stress in cardiac myocytes and also activates PERK signal pathway. PERK signaling protects cardiac myocytes from hypoxic damage in the early stage and triggers apoptosis of the cells in the later phase.     

Effect of acteoside on behavioral changes and endoplasmic reticulum stress in prefrontal cortex of depressive rats

AIM: To explore the effect of acteoside on behavioral changes and endoplasmic reticulum stress (ERS) in prefrontal cortex of depressive rats. METHODS: Sprague-Dawley (SD) rats (n=108) were randomly divided into 6 groups:control group, model group, fluoxetine (20 mg/kg) group, low-dose (30 mg/kg) acteoside group, medium-dose (60 mg/kg) acteoside group and high-dose (120 mg/kg) acteoside group, with 18 rats in each group. The depressive-like rat model was established by chronic unpredictable mild stress (CUMS) combined with solitary way for 28 d. The rats in fluoxetine group and acteoside groups were treated with fluoxetine (20 mg/kg) or acteoside (30 mg/kg, 60 mg/kg and 120 mg/kg) once daily by intragastric administration for 3 weeks. The rats in control group and model group were both given equal volume of saline by intragastric administration for 3 weeks. The behavioral changes were detected by the open-field test and sugar preference experiment. The protein expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) was assessed by immunofluorescence and Western blot. The caspase-3 activity was measured by spectrophotometer. RESULTS: Compared with control group, the total distance, time spent in the center and sugar intake were all decreased, the expression of GRP78 and CHOP was increased, and the activity of caspase-3 was increased in model group, fluoxetine group and acteoside groups (P<0.05). Compared with model group, the total distance, time spent in the center and sugar intake were increased, the expression of GRP78 and CHOP was reduced, and the activity of caspase-3 was decreased (P<0.05) in fluoxetine group and acteoside groups. CONCLUSION: Acteoside improves depressive-like behaviors in depressive rats, which may be related to the inhibition of ERS and neuronal apoptosis in prefrontal cortex.     

Effect of endoplasmic reticulum stress on brain injury following chronic intermittent hypoxia in growing rats

AIM: To explore the role of endoplasmic reticulum stress (ERS) in brain injury following chronic intermittent hypoxia in growing rats and the protective effect of treatment with salubrinal. METHODS: Healthy male SD rats (3~4-week-old, 100~120 g, n=64) were randomly assigned to 8 groups (8 rats in each group):the groups of intermittent hypoxia for 2 and 4 weeks (2IH and 4IH), the groups of control (C) for 2 and 4 weeks (2C and 4C), the groups of dimethylsulfoxide (DMSO) for 2 and 4 weeks (2DMSO and 4DMSO) and the groups of salubrinal for 2 and 4 weeks (2SAL and 4SAL). The 8-arm radial maze was used to assess the working memory error (WME), reference memory error (RME) and total error (TE) of the rats. The changes of neuronal apoptosis in the hippocampus were observed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The activity of superoxide dismutase (SOD), and the protein levels of endoplasmic reticulum stress marker compounds, C/EBP homologous protein (CHOP), phosphorylated eukaryotic translation initiation factor 2 alpha (p-eIF2α) and phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), were analyzed. RESULTS: Chronic intermittent hypoxia (CIH) significantly increased RME, WME, TE and neuronal apoptotic index (AI) (P<0.01), and decreased the activity of SOD in the hippocampus and serum (P<0.01). The protein levels of p-PERK and CHOP progressively increased in hippocampus in IH groups (P<0.01), and p-eIF2α was downregulated (P<0.05). Treatment with salubrinal significantly decreased RME (P<0.05), WME (P<0.05), TE (P<0.01) and AI (P<0.01), and increased the activity of SOD (P<0.01). Salubrinal induced the phosphorylation of eIF2α significantly after CIH in hippocampus and downregulated the level of CHOP (P<0.01). CONCLUSION: Chronic intermittent hypoxia upregulates the protein levels of p-PERK and CHOP in the hippocampus, and decreases p-eIF2α protein and the activity of SOD. Salubrinal, a selective inhibitor of eIF-2α dephosphorylation, increases the activity of SOD and prevents CHOP protein activation throughout CIH exposure. Our findings suggest ERS-mediated cell apoptosis is one of the underlying mechanisms of cognitive dysfunction in OSAHS children. Further, a specific ERS inhibitor salubrinal should be tested for neuroprotection against CIH-induced brain injury.     

苹果miR396家族鉴定及在不定根发育过程中的表达分析

分析了苹果miR396家族进化特性及其在苹果不定根发育过程中的表达模式。结果表明:苹果miR396家族有4条成熟体和7条前体序列(pre-miRNA)。Mfold预测显示Pre-miR396家族7个成员序列均可形成典型稳定的茎环二级结构,最小折叠自由能介于–62.9 kal·mol^-1(pre-miR396b)～–51.9kal·mol^-1(pre-miR396g)之间。系统发育进化树分析显示,pre-miR396家族亲缘关系可分为3个亚组(G1、G2、G3),每个亚组内基因数量不同,分别含有11、9、19个。靶基因预测显示,苹果miR396靶基因包括MdGRF1、MdGRF2和MdGRF5等,降解组测序进一步验证了mi R396对其候选靶基因MdGRF1、MdGRF2和MdGRF5的剪切关系。苹果miR396家族成员在侧根和果实中的表达量显著高于其他组织,其候选靶基因表达量则在花芽和腋芽中显著高于其他组织;不定根发育过程中,miR396家族不同成员表达模式存在显著差异,整体上呈上调表达趋势,其候选靶基因呈下调表达趋势;外源IBA处理显著诱导...     

IL-1β induces human mesangial cell injury via exacerbating lipid-induced endoplasmic reticulum stress

AIM：To investigate the molecular mechanism that interleukin-1 beta (IL-1β) exacerbates lipid-induced endoplasmic reticulum stress (ERS) and the injury of human mesangial cells (HMCs). METHODS：HMCs were cultured and divided into control group, low-density lipoprotein (LDL) group, IL-1β+LDL group and 4-phenyl butyric acid (4-PBA)+IL-1β+LDL group. Oil red O staining was used to evaluate the accumulation of lipid droplet in the cells. The mRNA levels of glucose-regulated protein 78(GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK) and α-smooth muscle actin (α-SMA) were examined by real-time PCR. Immunocytochemistry was used to observe GRP78 expression. The protein level of NF-κB p65 was measured by Western blotting. The releases of IL-6 and TGF-β1 in the culture supernatants of HMCs were detected by ELISA. RESULTS：Compared with LDL group, the intracellular lipid accumulation, the mRNA levels of GRP78 and PERK, the protein expression of GRP78 and NF-κB p65, and the release of IL-6 were significantly increased in IL-1β+LDL group. Dramatically reduced intracellular lipid accumulation, down-regulated GRP78 and PERK mRNA expression, decreased protein levels of GRP78 and NF-κB p65, and suppressed IL-6 release were observed in 4-PBA+IL-1β+LDL group as compared with IL-1β+LDL group. The mRNA level of α-SMA was higher in IL-1β+LDL group than that in LDL group, and that in 4-PBA+IL-1β+LDL group was significantly depressed. CONCLUSION：IL-1β exacerbates lipid-induced ERS, thus promoting the injury of HMCs.     

Effect of endoplasmic reticulum stress protein BiP on type 2 diabetic neuropathic pain in rats treated with curcumin

AIM: To investigate the effects of immunoglobulin heavy chain-binding protein (BiP),an endoplasmic reticulum stress protein, on mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), spinal dorsal horn and dorsal root ganglion (DRG) in type Ⅱ diabetic neuropathic pain rats treated with curcumin. METHODS: The rats were fed with a high-fat and high-fructose diet for 8 weeks to induce insulin resistance, and then were intraperitoneally injected with streptozotocin (STZ, 35 mg/kg). Eighty-one rats were selected into experimental design as their blood glucose ≥ 16.7 mmol/L 3 d after STZ injection and their MWT and TWL were decreased to 85% of the baseline values 14 d after STZ injection. The rats were divided into 3 groups (n=27 each): DNP group: type 2 diabetic neuropathic pain; DCur group: type 2 diabetic neuropathic pain and intraperitonal injection of curcumin at a dose of 100 mg·kg^-1·d^-1; DSC group: type 2 diabetic neuropathic pain and intraperitonal injection of corn oil at a dose of 4 mL/kg. Another 27 normal SD male rats fed with normal forage were adopted as control group (C group). MWT and TWL were measured at the time points of 3 d, 7 d and 14 d after curcumin injection. The lumbar segment 4~6 of the spinal cord and the corresponding DRG were removed at the same time. The expression of BiP was determined by immunohistochemical staining and Western blotting. RESULTS: Compared with C group, the rats in DNP group developed hyperglycemia and a decrease in MWT and TWL, as well as an increase in the activity of BiP in spinal dorsal horn and DRG (P<0.05). Compared with DNP group, the rats in DCur group at the time point of 7 d significantly attenuated mechanical allodynia and thermal hyperalgesia, and these effects were correlated with the inhibition of BiP hyper-activation at the time point of 14 d after treatment with curcumin (P<0.05). No significant difference of MWT, TWL and the expression of BiP between DNP group and SC group was observed. CONCLUSION: BiP participates in the pathogenesis of type Ⅱ diabetic neuropathic pain. Curcumin attenuates the MWT and TWL in type 2 diabetic neuropathic pain rats. The mechanism may be involved in the inhibition of BiP expression by curcumin.     

Effects of bisoprolol plus peridopril on myocardial endoplasmic reticulum stress in rats with doxorubicin-induced heart failure

ATM: To investigate the effects of bisoprolol (Bis) plus peridopril (Per) on myocardial endoplasmic reticulum stress (ERS) in rats with heart failure.METHODS: Male SD rats were randomly divided into normal control group, sham group, doxorubicin (DOX) group, bisoprolol treatment group (DOX+Bis group), peridopril treatment group (DOX+Per group) and bisoprolol plus peridopril treatment group (DOX+Bis+Per group). A rat model of heart failure was induced by intraperitoneal injection of DOX. Distilled water, bisoprolol, peridopril, and bisoprolol plus peridopril were administrated by gastric gavage for 35 d, respectively. The indexes of cardiac functions and plasma levels of brain natriuretic peptide (BNP) were measured, myocardial apoptosis was analyzed by TUNEL assay and myocardial protein expression of GRP78, CHOP, JNK, caspase-12 and SERCA2a was detected by Western blot.RESULTS: Compared with normal control group and sham group, cardiac output (CO), left ventricular fractional shortening (FS), and left ventricular ejection fraction (EF) of the rats in DOX group decreased significantly (P<0.01), the cardiomyocyte apoptotic index increased significantly (P<0.01), the myocardial protein levels of SERCA2a decreased significantly, and GRP78, CHOP, JNK and caspase-12 increased significantly (P<0.01). Compared with DOX group, CO, FS and EF of the rats in DOX+Bis group, DOX+Per group and DOX+Bis+Per group increased significantly (P<0.01), cardiomyocytes apoptotic indexes in DOX+Bis group, DOX+Per group and DOX+Bis+Per group decreased significantly (P<0.01), myocardial protein levels of SERCA2a in DOX+Bis group, DOX+Per group and DOX+Bis+Per group increased significantly, while GRP78, CHOP, SERCA2a, JNK and caspase-12 decreased significantly (P<0.05). Indicators except JNK in DOX+Bis+Per group were changed more significantly than those in DOX+Bis group or DOX+Per group (P<0.05).CONCLUSION: Bisoprolol plus peridopril therapy improves cardiac functions in a rat model of doxorubicin-induced heart failure with more significant effectiveness than using bisoprolol or peridopril alone, which may be related to inhibition of myocardial ERS and apoptosis.     

Effect of homocysteine-mediated endoplasmic reticulum stress on lipid metabolism in hepatocytes

AIM: To explore the effect of homocysteine (Hcy)-mediated endoplasmic reticulum stress on lipid metabolism in hepatocytes.METHODS: HepG2 cells used in the study were treated with 5 mmol/L Hcy.The concentrations of Hcy,triglycerides (TGE) and cholesterol (CHO) in the cells were measured.In high methionine diet-induced hyperhomocysteinemia C57BL/6 mice,the concentrations of TGE and CHO in hepatocytes were analyzed.The mRNAs and proteins expressions of glucose-regulated protein 78 (GRP-78) and sterol regulatory element binding protein (SREBP-1) were also assessed.RESULTS: The concentrations of Hcy and lipids (TGE,CHO) in HepG2 cells at different time point were elevated after treated with 5 mmol/L Hcy (P<0.05,vs 0 hour).The levels of plasma Hcy and TGE or CHO in hepatocytes of high methionine diet mice at different time point were significantly increased (P<0.05,vs 0 week).However,the TGE and CHO levels in plasma had no change (P>0.05,vs 0 week).The mRNAs and proteins expressions of GRP-78 and SREBP-1 in mice at different time point after high methionine diet were higher than that at 0 week (P<0.05).CONCLUSION: It suggests that Hcy-induced endoplasmic reticulum stress causes dysregulation of the endogenous sterol response pathway,also increases hepatic biosynthesis and uptake of TGE and CHO.     

PERK-mediated endoplasmic reticulum stress is involved in angiotensin II-induced myocardial hypertrophy

AIM: To investigate the role of protein kinase R-like endoplasmic reticulum kinase (PERK)-mediated endoplasmic reticulum stress (ERS) in angiotensin II (AngⅡ) -induced myocardial hypertrophy. METHODS: In the hypertrophy model of AngⅡ-induced cardiomyocytes isolated from neonatal Sprague-Dawley rats, the methods of morphological observation, [³H]-leucine incorporation and surface area measurement were employed to assess the cardiomyocyte hypertrophy. Real-time PCR, RT-PCR and Western blotting were used to detected the expression of glucose-regulated protein 78 (GRP78), calreticulin (CRT), PERK, eukaryotic initiation factor 2α (eIF2α) and C/EBP homologous protein (CHOP) at mRNA and protein levels. RESULTS: Compared with control group, Ang II-treated cardiomyocytes showed that the mRNA and protein expression of CRT increased by 146.4% and 125.3%, respectively (P<0.05). The mRNA and protein expression of GRP78 increased by 84.0% and 77.6%, respectively (P<0.05). The mRNA and protein expression of PERK increased by 165.4% and 132.1%, respectively (P<0.05).The mRNA and protein expression of eIF2α was increased by 110.9% and 46.5%, respectively (P<0.05). The mRNA and protein expression of CHOP also increased by 117.7% and 63.3%, respectively (P<0.05). CONCLUSION: PERK-mediated ERS response is involved in AngⅡ-induced cardiomyocyte hypertrophy.     

Prostaglandin E1 protects heart after myocardial infarction by inhibiting endoplasmic reticulum stress in rats

AIM To investigate the effect of prostaglandin E1 (PGE1) on heart after myocardial infarction (MI) in rats and its related molecular mechanism. METHODS Fifty male SD rats were divided into sham group, model group and model+PGE1 group. The MI rat model was established by ligation of left anterior descending coronary artery. Cardiac function in the rats was detected by echocardiogaphy. The myocardial histomorphologic changes were evaluated by HE and Masson staining. The MI area was measured by TTC staining. The cardiomyocyte death was detected by TUNEL staining. The protein levels of endoplasmic reticulum stress (ERS)-related factors glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and caspase-12, and apoptosis-related factors Bcl-2, Bax and cleaved caspase-3 were determined by immunofluorescence staining and Western blot. RESULTS Compared with sham group, the cardiac function in model group was decreased, with significant myocardial pathological changes. The MI area was enlarged, and the death of cardiomyocytes was promoted. The protein levels of GRP78, CHOP, caspase-12, Bax and cleaved caspase-3 in the myocardial tissues were significantly increased, while Bcl-2 was decreased (P<0.01). Compared with model group, the cardiac function in model+PGE1 group was significantly improved, and the myocardial pathological damage was significantlty attenuated. The MI area and myocardial cell death were significantly reduced. The protein levels of GRP78, CHOP, caspase-12, Bax and cleaved caspase-3 in the myocardial tissues were significantly decreased, while Bcl-2 was increased (P<0.01). CONCLUSION PGE1 reduces collagen deposition and inflammation, and improves cardiac function by reducing ERS level, thus protecting cardiomyocytes from MI damage.     

Effect of SET7/9-mediated endoplasmic reticulum stress on arsenic-induced hepatocyte apoptosis

AIM:To investigate the effect of SET7/9 (SET domain containing 7/9)-mediated endoplasmic reticulum stress (ERS) on protein kinase R-like endoplasmic reticulum kinase (PERK) signaling pathway, and to explore the mechanisms of arsenic-induced hepatocyte apoptosis. METHODS:Human liver LO2 cells were divided into control group, arsenic poisoning model group, negative transfection group and SET7/9 siRNA transfection group. The apoptosis of the LO2 cells in each group was analyzed by flow cytometry. The protein levels of SET7/9, glucose-regulated protein 78 (GRP78), PERK and p-PERK in the LO2 cells of each group were observed by Western blot. RESULTS:Inhibition of SET7/9 expression reduced the apoptotic rate of arsenic-induced LO2 cells. Arsenic exposure increased the expression of SET7/9 in the LO2 cells. Arsenic exposure increased the protein levels of GRP78 and p-PERK in the LO2 cells, but decreased the protein levels of GRP78 and p-PERK after transfection with SET7/9 siRNA (P<0.05). CONCLUSION:Arsenic exposure induces hepatocyte apoptosis by increasing SET7/9 to activate ERS by PERK signaling pathway.     

Role of FAT/CD36 in high-fat diet-induced adipose tissue inflammation

AIM: To investigate the role of fatty acid translocase/CD36 (FAT/CD36) in adipose tissue inflammation induced by a high-fat diet. METHODS: C57BL/6J mice were fed with a normal-chow diet (NCD) or a high-fat diet (HFD) for 14 weeks. The content of free fatty acid (FFA) in the serum was measured by ELISA. The expression of CD36, cytokines and chemokines at mRNA and protein levels in the adipose tissues was determined by real-time polymerase chain reaction and Western blotting. Immunohistochemical staining was used to examine the macrophages infiltration in the adipose tissues. The inflammatory responses in CD36 knockout mice and wild type mice with high-fat diet were analyzed. RESULTS: The levels of FAT/CD36 were higher in HFD group than that in NCD group. HFD feeding enhanced the mRNA and protein expression of IL-1β, IL-6, TNF-α, MCP-1 and MIP-1, as well as promoted macrophage infiltration in the adipose tissues. Interestingly, as fed with HFD, the expression of cytokines/chemokines and macrophage infiltration were significantly reduced in adipose tissues of the CD36 knockout mice, compared with the wild type mice. CONCLUSION: High-fat diet promotes adipose tissue inflammation in the mice in a FAT/CD36-dependent manner.     

Excessive endoplasmic reticulum stress induces apoptotic cell death in chronic cyclosporine A nephrotoxicity

AIM：To investigate the impact of excessive endoplasmic reticulum stress on apoptotic cell death in a rat model of chronic cyclosporine A (CsA) nephrotoxicity. METHODS：Male Sprague-Dawley rats on a low-salt diet were subcutaneously injected with vehicle (olive oil, 1 mL·kg-1·d-1) or CsA (15 mg/kg) daily for 1 or 4 weeks. Tubulointerstitial fibrosis and apoptotic cell death were estimated by trichrome staining and TUNEL staining. In addition, immunohistochemistry and immunoblotting were used to evaluate the expression of immunoglobulin-binding protein (BiP), eukaryotic initiation factor 2α (eIF2α), growth arrest and DNA damage-inducible protein 153 (GADD153), caspase-12 and caspase-3. RESULTS：The rats treated with CsA for 1 week did not develop tubulointerstitial fibrosis and TUNEL-positive cells, whereas 4-week treatment with CsA induced typical tubulointerstitial fibrosis and increased TUNEL-positive cells. CsA induced a significant increase in BiP and caspase-12 expression peaked at 1 week, and then returned to normal levels at 4 weeks. In contrast, the expression of eIF2α, GADD153 and caspase-3 in CsA-treated rat kidneys were significantly increased in a time-dependent manner. CONCLUSION：Excessive endoplasmic reticulum stress causes apoptotic cell death by depleting molecular chaperones and stimulating the proapoptotic pathway in chronic CsA nephrotoxicity.     

Role of endoplasmic reticulum stress in Bim-mediated cardiomyocyte apoptosis induced by hypoxia

AIM:To investigate the role of endoplasmic reticulum stress (ERS) in the process of Bim-mediated cardiomyocyte apoptosis induced by hypoxia. METHODS:Cardiomyocytes were isolated from neonatal Sprague-Dawley rats aged 1~3 days, and primarily cultured in vitro. The antibody targeting α-striated muscle actin was used to identify the cardiomyocytes. The siRNAs targeting bim were transfected into cardiomyocytes with liposome, followed by detecting the expression of Bim by Western blotting. Cardiomyocytes were divided into five groups: blank control group, hypoxia group, hypoxia+liposome group, hypoxia+negative control siRNA group and hypoxia+Bim-siRNA group. The cell viability was determined by MTT assay, and the cell apoptotic rate and the intracellular calcium concentration were measured by flow cytometry. The protein expression of caspase-12 and inositol 1,4,5-triphosphate (IP3) was detected by Western blotting. RESULTS:Immunohistochemical identification confirmed that rat cardiomyocytes were successfully cultured. Green fluorescence was observed in the cells transfected with negative control siRNA under fluorescence microscope. The expression of Bim was obviously inhibited after transfected with Bim-siRNAs and the silencing efficiency of Bim-siRNA-2 was the highest (86.73%). Compared with blank control group, the viability of cardiomyocytes in hypoxia group was significantly reduced (P<005). Compared with hypoxia+negative control siRNA group, the viability of cardiomyocytes in hypoxia+Bim-siRNA group was significantly increased (P<005). The apoptotic rate and the intracellular calcium concentration of cardiomyocytes were obviously increased in hypoxia group (P<0.01), and were both decreased after bim silencing (P<005 or P<0.01). The expression of caspase-12 and IP3 was up-regulated in hypoxia group (P<005), and was down-regulated after bim silencing (P<005 or P<0.01). CONCLUSION: Cardiomyocyte apoptosis induced by hypoxia can be inhibited by silencing the expression of bim gene. Caspase-12 and IP3, as markers of ERS, may participate in the process of Bim-mediated cardiomyocyte apoptosis induced by hypoxia.     

Effect of berberine on endoplasmic reticulum stress-autophagy pathway in human ovarian cancer SKOV3 cells

AIM: To investigate the regulatory effect of berberine on the endoplasmic reticulum stress-auto-phagy pathway in human ovarian cancer SKOV3 cells. METHODS: Human ovarian cancer SKOV3 cells were cultured in vitro, and berberine at doses of 12.5, 25, 50, 100, 200 and 400 μmol/L were added. After exposure for 12 h, 24 h and 48 h, the viability of the SKOV3 cells was measured by MTT assay. The cells were divided into control group, berberine (50 μmol/L) group, berberine (100 μmol/L) group, and berberine (200 μmol/L) group. After treatment with berberine for 24 h, the effects of berberine on the morphological changes of SKOV3 cells were observed under inverted phase-contrast microscope. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) and ubiquitin-binding protein p62 was observed by indirect immunofluorescence method under laser confocal microscope. The protein expression of beclin-1,LC3,p62, CCAAT/lenhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) was determined by Western blot. RESULTS: Berberine at 12.5, 25, 50, 100, 200 and 400 μmol/L significantly decreased the viability of SKOV3 cells at 12 h, 24 h and 48 h, and the IC₅₀ values of 12 h, 24 h and 48 h were (764.7±0.3) μmol/L, (231.6±0.1) μmol/L and (96.2±0.1) μmol/L, respectively. Laser confocal microscopy showed that the LC3 and p62 proteins were scattered and the fluorescence intensity was increased, while the point-like aggregation was also observed. Berberine at 200 μmol/L obviously enhanced the co-localization of LC3 and p62 proteins. Compared with control group, the expression of endoplasmic reticulum stress-related proteins GRP78 and CHOP, and autophagy-related proteins beclin-1, LC3 and p62 in berberine (200 μmol/L) group was increased significantly (P<0.05). CONCLUSION: Berberine may promote endoplasmic reticulum stress in SKOV3 cells by regulating autophagy.     

Panax quinquefolium saponins protect rat myocardium from ischemia/reperfusion injury through attenuating excessive endoplasmic reticulum stress

AIM: To investigate whether excessive endoplasmic reticulum stress (ERS) is involved in the protective mechanism of Panax quinquefolium saponins (PQS) against ischemia/reperfusion (I/R) injury in rat myocardium. METHODS: The model of myocardial I/R injury in vivo was made by ligating the left anterior descending artery for 45 min followed by 24 h of reperfusion in SD rats. The hemodynamics and serum content of cardiac troponin T (cTnT) were measured. The myocardial infarct size was measured by Evans blue and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Cardiomyocyte apoptosis was detected using in situ TDT-mediated dUTP nick end labeling (TUNEL). The protein levels of glucose-regulated protein 78 (GRP78), calreticulin (CRT), C/EBP homologous protein (CHOP), caspase-12, apoptosis-associated proteins Bax and Bcl-2 were determined by Western blotting. RESULTS: Compared with I/R group, the mean arterial pressure in PQR+IR group was decreased by 32.0%, and left ventricular±dp/dtmax was increased by 64.0% and 35.0%, respectively.The serum content of cTnT was decreased by 53.3%, the percentage of area of necrosis (AN)/area at risk (AAR) was reduced by 65.5% and the apoptosis rate was decreased by 54.9%.The myocardial pathological changes were improved. Bcl-2 protein expression was increased by 110.0% and that of Bax was decreased by 47.8%. CRT protein expression was decreased by 43.4 %, CHOP protein expression and the protein level of cleaved caspase-12 were decreased by 38.6% and 23.7% in PQS+I/R group. CONCLUSION: PQS alleviates I/R injury in myocardium by inhibition of excessive ERS.     

Role of endoplasmic reticulum stress in trimethylamine N-oxide-induced oxidative stress in human umbilical vein endothelial cells

AIM: To investigate whether endoplasmic reticulum stress is involved in trimethylamine N-oxide (TMAO)-mediated oxidative stress in human umbilical vein endothelial cells (HUVECs). METHODS: The cell viability was examined by CCK-8 assay. The cells were stained by DCFH-DA, and the intracellular level of reactive oxygen species (ROS) was observed by phase-contrast microscopy and detected by flow cytometric analysis. The protein levels of phospho-IRE-1α, IRE-1α and GRP78/BiP were detected by Western blot. RESULTS: TMAO exerted no significant effect on the viability of HUVECs. For a long period (>24 h), even a low concentration (10 μmol/L) of TMAO increased the oxidative stress level in the HUVECs (P<0.05). TMAO increased the phosphorylation level of IRE-1α and significantly up-regulated the protein level of GRP78/BiP in HUVECs (P<0.01). Pretreatment with STF-083010, an inhibitor of IRE1α, for 1 h reduced TMAO-induced oxidative stress in HUVECs (P<0.05). CONCLUSION: Endoplasmic reticulum stress is involved in TMAO-induced oxidative stress in HUVECs.     

Effects of fructose sodium diphosphate on expression of CHOP and JNK in endoplasmic reticulum stress and islet apoptosis in rats with type 2 diabetes

AIM: To study the effects of fructose sodium diphosphate (FDP) on the expression of CHOP and c-Jun N-terminal binase(JNK) in endoplasmic reticulum stress and islet apoptosis in the rats with type 2 diabetes mellitus (T2DM). METHODS: T2DM model was established in male Wistar rats by feeding of high lipid diet and injection of streptozotocin. The rats were divided into 4 groups (n=8):normal control group, T2DM model group, T2DM+low-dose FDP (2 mL穔g^-1穌^-1, ip) group and T2DM+high-dose FDP (5 mL穔g^-1穌^-1, ip) group. The rats in the treatment groups received FDP for 8 weeks. The levels of fasting blood glucose (FBG), fasting serum insulin (FINS) and insulin sensitivity index (ISI) were measured. TUNEL was used to detect the islet apoptosis. The protein levels of CHOP and JNK were determined by the method of immunohistochemistry. RESULTS: (1) Compared with normal control group, FBG, FINS, the expression of CHOP and JNK, and apoptosis in T2DM model group were significantly increased (P<0.01). The level of ISI was significantly decreased. (2) Compared with T2DM model group, the levels of FBG and FINS, the expression of CHOP and JNK, and apoptosis in high-dose FDP group were significantly decreased. The level of ISI was significantly increased (P<0.01). However, the level of FBG, the expression of CHOP and JNK, and apoptosis in low-dose FDP group were significantly decreased. Compared with low-dose FDP group, the levels of FBG and FINS, the expression of CHOP and JNK, and apoptosis in high-dose FDP group were significantly decreased. The level of ISI was significantly increased (P<0.01 or P<0.05). CONCLUSION: FDP may prevent islet cells from apoptosis in T2DM rats by decreasing the expression of CHOP and JNK.