Osteopontin-induced ER stress results in insulin resistance in C2C12 myocytes

AIM: To investigate the potential role of osteopontin (OPN) in insulin resistance in C2C12 myocytes and its underlying mechanism. METHODS: C2C12 myoblasts were induced by low concentration of serum and insulin treatment to differentiate into myocytes. Western blot was performed to detect the protein abundance and phosphorylation using specific antibodies. Plasma membrane was isolated by centrifugation. Glucose uptake was measured by glucose uptake assay. RESULTS: OPN treatment suppressed insulin-stimulated protein kinase B (Akt) phosphorylation in a dose-and time-dependent manner, accompanied with decreased membrane translocation of glucose transporter type 4 (Glut4) and reduced glucose uptake. Neutralization of OPN specific receptor in skeletal muscle with CD44 antibody mitigated OPN-induced inhibitory impact on insulin action. Furthermore, OPN treatment resulted in endoplasmic reticulum (ER) stress and phosphorylation of c-Jun N-terminal kinase (JNK). Administration of ER stress inhibitor 4-phenylbutyrate (4-PBA) diminished the detrimental effects of OPN on JNK phosphorylation, Glut4 membrane translocation and glucose uptake. CONCLUSION: ER stress mediated OPN-induced insulin resistance in C2C12 myocytes.     

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.     

Effect of GLP-1 on insulin resistance and PKCε in rats with nonalcoholic fatty liver disease induced by high fat diet

AIM: To observe the therapeutic effect of glucagon-like peptide 1 (GLP-1) analog on nonalcoholic fatty liver disease of rats and to investigate the underlying mechanism.METHODS: SD rats (n=21) were used to establish a nonalcoholic fatty liver disease model by feeding a high fat diet for 12 weeks, and other 11 rats were fed with a normal diet for 16 weeks. The model rats were randomly divided into 2 equal groups:one group was treated with glucagon-like peptide 1 analog (0.6 mg·kg^-1·d^-1) by intraperitoneal injection for 4 weeks, the other group using saline as a control. After treatment, fasting blood glucose, serum insulin, blood lipids, liver function and the pathological changes of the hepatic tissues were evaluated and the expression of PKCε at mRNA and protein levels in the liver tissues was detected by real-time PCR and Western blot, respectively.RESULTS: Compared with model group, the intervention of GLP-1 significantly reduced insulin resistance index (HOMA-IR), improved the liver function (P<0.05), decreased the liver index and blood lipids (P<0.05). HE staining showed obvious pathological changes of the hepatic tissues in model group, and the intervention of GLP-1 significantly reduced lipid droplets in the hepatocytes and improved the structural damage of the liver. The expression of hepatic protein kinase Cε (PKCε) at mRNA and protein levels significantly decreased which were reversed by treating with GLP-1.CONCLUSION: GLP-1 shows good therapeutic effect on nonalcoholic fatty liver disease of rats, possibly by controlling lipid metabolism and reducing insulin resistance, which may be related to PKCε expression.     

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 electroacupuncture in different intensities on endoplasmic reti-culum stress in adipose tissues from high-fat diet-induced obese rats

AIM: To investigate the effect of electroacupuncture (EA) in different intensities on unfolded protein response (UPR) signaling pathway in adipose tissues from high-fat diet-induced obese rats. METHODS: Sixty SD rats were randomly divided into 2 groups: common diet group (n=10) and high-fat diet group (n=50). Thirty obese rats in high-fat diet group were chosen and divided into high-fat diet group, 5 mA EA group and 1 mA EA group (n=10 each). EA at frequency of 20 Hz and intensities of 5 mA or 1 mA was applied to ST 36 and SP 6 for 15 min once daily for 2 weeks. The body weight was detected once a week. The expression of p-PERK and CHOP/GADD153 in epididymal adipose tissues was determined by Western blotting. The content of Bcl-2 and caspase-12 in the adipose tissues was measured by ELISA. RESULTS: After EA, the body weight and the expression of p-PERK and CHOP/GADD153 in obese rats were significantly decreased (P<0.01 or P<0.05). The rats in 5 mA EA group exhibited a better improvement on the protein expression than the rats in 1 mA EA group.CONCLUSION: EA has a beneficial regulatory effect on the rats with simple obesity. Moreover, the EA density of 5 mA is superior to 1 mA.     

High-fat diet affects plasma membrane GLUT4 content in skeletal muscle from Sprague-Dawley rats

AIM: To explore the change of the amount of GLUT4 protein at the plasma membrane of the rat skeletal muscle after high-fat feeding. METHODS: The animals were divided into three groups (ten for each): group I: control; group II: high-fat feeding; group III: high-fat feeding + dietary treatment. The rat model of insulin resistance (IR) was made by feeding high-fat diet for eight weeks. And then insulin-resistant rats were fed with chow diet for 4 weeks. Fasting plasma glucose and fasting serum insulin levels were measured before and after dietary treatment, respectively. Insulin treatment was achieved by intraperitoneal injection of insulin (10 unit insulin per kg body weight) 15 minutes before killing the animals. The right hindlimb skeletal muscle was rapidly dissected. Then the expression of GLUT4 protein at the plasma membrane in all the animals was assessed with Western bloting. RESULTS: The GLUT4 content at the plasma membrane in high-fat-fed rat skeletal muscle was significantly lower (about 31%) than that in controls (P<0.01). Dietary treatment partly corrected fasting blood glucose [from(6.20±0.39)mmol/L to(5.78±0.74)mmol/L]and fasting serum insulin levels [from(17.19±1.93)mU/L to(11.68±1.28)mU/L] and increased the GLUT4 content at the plasma membrane by 1.14-fold in insulin-resistant rat skeletal muscle. CONCLUSION: High-fat feeding induces IR in Sprague-Dawley rats. The mechanism may be involved in decreased cell-surface level of GLUT4 through affecting intracellular insulin signaling and then decreasing GLUT4 trafficking.     

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.     

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.     

Radix Pseudostellariae polysaccharide attenuates high fat diet induced hepatic insulin resistance in mice

AIM: To study the role of Radix Pseudostellariae polysaccharide (RPP) in hepatic insulin resistance.METHODS: Six-week-old C57BL/6J mice were randomly divided into low-fat diet (LFD) control group and high-fat diet (HFD) model group. After 16 weeks, intraperitoneal pyruvate tolerance test (IPPTT) was performed to determine the establishment of the HFD-induced hepatic insulin resistance model. HFD containing RPP (500 mg/kg) was given for 4 consecutive weeks. IPPTT, liver malondialdehyde (MDA) level and liver mitochondrial MDA level were measured. The protein levels of p-AKT (Ser473/Thr308), p-AMPK, nuclear factor E2-related factor 2 (Nrf2), NQO1 and IκBα in the liver tissues were measured by Western blot.RESULTS: After administration of RPP, a significant reduction in the levels of blood glucose and hepatic mitochondrial MDA was observed. The levels of p-AKT (Ser473/Thr308) and p-AMPK were significantly elevated in the liver tissues. The hepatic IκBα levels were up-regulated. RPP also enhanced the expression of Nrf2 system-regulated proteins NQO1 and HO-1 in the liver tissues.CONCLUSION: Radix Pseudostellariae polysaccharides effectively reduce HFD-induced hepatic insulin resistance in C57BL/6J mice and improves liver glucose metabolism by ameliorating HFD-impaired hepatic transduction of insulin signaling, activating Nrf2-associated signaling and inhibiting the expression of inflammatory signaling proteins.     

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...     

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.     

Effects of macrophage polarization during development of liver cirrhosis in rats

AIM: To explore the state of macrophage polarization and its relation with intestinal endotoxemia-endoplasmic reticulum stress in the development of liver cirrhosis induced by multiple pathogenic factors in rats. METHODS: The male SD rats (n=36) were randomly divided into normal control group and liver cirrhosis model group, and sacrificed at the end of the 4th, 6th and 8th weeks. The rat model of liver cirrhosis was induced by multiple pathogenic factors. The levels of alanine aminotransferase (ALT), endotoxin, homocysteine (Hcy) in the plasma, and inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), arginase-1 (Arg-1) and interleukin-10 (IL-10) in the liver tissues were detected by ELISA. Histopathological change of the liver was observed under microscope with the staining of hematoxylin and eosin (HE) and van Gieson (VG). The expression of glucose-regulated protein 78 (Grp78), nuclear factor-kappa B (NF-κB), interferon-regulatory factor 5 (IRF5), CD86, CD206 and transforming growth factor-β1 (TGF-β1) at mRNA levels in the liver tissues were detected by the method of real-time fluorescence quantitative PCR.RESULTS: Compared with the corresponding normal control group, the levels of ALT, endotoxin, Hcy in the plasma and Grp78 mRNA in the liver tissues in liver cirrhosis model group were significantly and gradually increased (P<0.05). The mRNA expression of NF-κB, IRF5 and CD86, and the protein levels of iNOS, TNF-α and IL-6 in the liver tissues were significantly increased (P<0.05), and they successively increased from the 4th week to the 6th week and decreased reversely at the 8th week. The mRNA expression of CD206, TGF-β1, Arg-1 and IL-10 in the liver tissues were significantly increased from the 6th week to the 8th week (P<0.05), and no significant difference at the 4th week was observed. The level of endotoxin in the plasma was correlated with the mRNA expression of Grp78 in the liver tissues (P<0.01). Both endotoxin in the plasma and Grp78 mRNA in the liver tissues were correlated with the mRNA expression of CD86 and CD206 in the liver tissues (P<0.01).CONCLUSION: The pathway of liver damage-intestinal endotoxemia-endoplasmic reticulum stress-macrophage polarization may be critical in the pathogenesis of liver cirrhosis induced by multiple pathogenic factors.     

Protective effect of quercetin on ER stress-related apoptosis induced by thapsigargin in macrophages

AIM: To investigate the effects and possible mechanisms of quercetin (Que) on endoplasmic reti-culum stress (ERS)-related apoptosis induced by thapsigargin (TG) in RAW264.7 cells. METHODS: ER stress of RAW264.7 cells were induced by TG at concentration of 1 μmol/L for 24 h. After treated with different concentrations of Que (80, 120 and 160 μmol/L), the cell viability was determined by MTT assay.The apoptotic rate and the changes of intracellular Ca²⁺ concentration ([Ca²⁺]_i) were determined by flow cytometry, and the cell apoptotic morphology was observed under laser scanning confocal microscope.The protein levels of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were detected by Western blotting. The effect of Que on GRP78 and CHOP induced by TG with phosphatidylinositol 3-kinase (PI3K) inihibitor LY294002 at concentration of 15 nmol/L was measured by Western blotting. RESULTS: Que suppressed ER stress-related injury induced by TG in RAW264.7 cells. Compared with TG group, the cell viability increased (P<0.05), apoptotic rate and [Ca²⁺]_i decreased (P<0.05) and the changes of apoptotic morphology were alleviated. The increase in GRP78 and CHOP induced by TG as an ER stress marker was suppressed by Que (P<0.05). The suppressive effect of Que on GRP78 and CHOP was reproduced by LY294002 (P<0.05), but they failed to exhibit additive suppression. CONCLUSION: Que suppresses the ER stress induced by TG in RAW264.7 cells. The protective effect may be related to its suppression on PI3K signaling pathway.     

Glucose-regulated protein 78 promote cardiomyocyte apoptosis in liver cirrhotic rats

AIM: To explore the cardiomyocyte apoptosis induced by glucose-regulated protein 78/immunoglobulin heavy chain-binding protein (GRP78/BiP) in liver cirrhotic rats and its mechanism. METHODS: The liver cirrhotic rat model was established with multiple pathogenic factors, and sampled at the time points of 4 weeks, 6 weeks and 8 weeks. In experiment 1, the heart was collected and weighed, the thickness of the left ventricular wall was measured, and the ratios of the left ventricular wall thickness to the heart weight, and the heart weight to the body weight were calculated. In experiment 2, TUNEL was used to detect the apoptotic cardiomyocytes,and the protein levels of GRP78/BiP, CCAAT/enhancer-binding protein homologous protein/growth arrest and DNA damage-inducible protein 153 (CHOP/GADD153), caspase-12, nuclear factor-κB p65 (NF-κB p65) and B-cell lymphoma/leukemia-2 (Bcl-2) in the myocar-dium were detected by the method of immunohistochemistry. RESULTS: Compared with the normal myocardium, significant larger ratios of the left ventricular wall thickness to the heart weight and the heart weight to the body weight, a significant increase in the cardiomyocyte apoptosis, and a significant larger positive expression index of GRP78/BiP in the hearts 8 weeks after modeling were observed. The protein levels of CHOP/GADD153 and caspase-12 were gradually increased during the development of liver cirrhosis and were significantly increased at 8 weeks. The positive expression of NF-κB p65 and Bcl-2 showed consistent changes, and were markedly higher at 4 weeks than those at the other time points. The positive expression index of GRP78/BiP was positively correlated with the apoptotic index, and the levels of CHOP/GADD153 and caspase-12. The positive expression index of CHOP/GADD153 was negatively correlated with NF-κB p65 and Bcl-2. CONCLUSION: Elevated expression of GRP78/BiP may play a crucial role in the pathogenesis of liver cirrhotic cardio-myopathy mediated by endoplasmic reticulum stress.     

Role of 78 kD glucose-regulated protein in the development of liver cirrhosis promoted by intestinal endotoxemia in rats

AIM: To explore the role of 78 kD glucose-regulated protein (GRP78) in the development of liver cirrhosis in rats promoted by intestinal endotoxemia (IETM). METHODS: Fifty-one male Wistar rats were randomly divided into liver cirrhosis groups of 4th-week, 6th-week and 8th-week, and normal control group at the corresponding time points. The rat model of hepatic cirrhosis was induced by employing multiple pathogenic factors to the animals. The liver injury and hepatic fibrosis were observed with the staining of HE and VG, respectively. The expression of GRP78 at the mRNA and protein levels was measured by the methods of RT-PCR and immnunohistochemistry, respectively. The concentrations of alanine aminotransferase(ALT), endotoxin, TNF-α and homocystine (HCY) in plasma, and the content of TNF-α, malondialdehyde(MDA) and PⅢP in liver tissues were detected. RESULTS: As liver cirrhosis developed, the levels of ALT, endotoxin, TNF-α and HCY in plasma, the expression of GRP78 at mRNA and protein, the content of TNF-α, MDA and PⅢP in liver tissues, and the index of liver fibrosis were gradually increased and were significantly higher than those in normal control group (P<0.05). Elevated endotoxin in plasma was correlated positively with the protein expression of GRP78, the content of MDA and HCY in plasma and the index of liver fibrosis (P<0.01). Elevated protein expression of GRP78 was correlated positively with the content of MDA and HCY in plasma and the index of liver fibrosis (P<0.01). CONCLUSION: GRP78 plays an important role in the development of liver cirrhosis. Endoplasmic reticulum stress is a possible mechanism in the development of liver cirrhosis promoted by IETM.     

Endoplasmic reticulum stress mediates the neurotoxic effect of misfolded proteins in neurodegenerative diseases

Endoplasmic reticulum (ER) stress can be caused by disturbances in the function of the ER with the accumulation of misfolded proteins. The ER response is characterized by unfolded protein response (UPR) causing translational attenuation, induction of ER chaperones and degradation of misfolded proteins. In case of prolonged or aggravated ER stress, cellular signals leading to apoptosis are activated. ER stress has been suggested to be involved in human neurodegenerative diseases, such as Alzheimers disease, Parkinsons disease, and amyotrophic lateral sclerosis, as well as other disorders. Here we will discuss the neurotoxic effect of ER stress in these three major neurodegenerative diseases, and highlight current knowledge in this field that may reveal novel insight into disease mechanisms and help to design better therapies for these disorders.     

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 insulin resistance on fatty liver in high-fat diet-fed mice

AIM: To study the influence of insulin resistance on fatty liver in the mice fed with high-fat diet (HFD).METHODS: Male 8-week-old C57BL/6J mice were randomly divided into HFD group (with 60% calories by high saturated fatty acid) and control group (with chow diet).The mice in both groups were fed for 12 weeks. The body weight, liver weight, serum triglyceride (TG) and total cholesterol (TC), and blood glucose and insulin levels were measured. Hyperinsulinemic euglycemic clamp experiment was applied to reflect insulin sensitivity. The lipid deposition in the liver was analyzed by HE staining, Sudan IV staining and measurement of liver fat content. The phosphorylation levels of IRS1 and Akt, and the protein levels of SREBP-1 and FAS were determined by Western blot to reflect the activities of insulin signaling and lipid synthesis.RESULTS: Compared with control group, the body weight and liver weight were significantly increased in HFD group. TG and TC contents in serum and liver tissues were remarkably increased in HFD group. High-fat diet induced insulin resistance, as evidenced by increased serum insulin levels, reduced glucose infusion rate and decreases in IRS1 and Akt phosphorylation levels. In livers of HFD group, HE staining showed that the cytoplasm of hepatocytes was filled with vacuoles. Sudan IV staining also displayed that many different sizes of red lipid drops existed in the hepatocytes, and the protein levels of SREBP-1 and FAS were significantly increased. In primary normal hepatocytes with exogenous oleic acid intervention for 48 h, the phosphorylation levels of IRS1 and Akt were reduced, and the protein expression of SREBP-1 and FAS was significantly increased in a dose-dependent manner.CONCLUSION: Feeding with HFD leads to insulin resistance, resulting in activation of lipid synthesis and accumulation of lipid deposition in the liver, thus inducing fatty liver.