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.     

Morphology of endoplasmic reticulum and expression of GRP78 in rat fibrotic liver

AIM: To observe the changes of endoplasmic reticulum and the biomarker of endoplasmic retidum stress (ERS), glucose-regulated protein 78(GRP78),in the process of liver fibrosis induced by carbon tetrachloride (CCl₄). METHODS: Male Wistar rats weighing 180 g to 220 g were divided into control group and liver fibrosis group. The rats in liver fibrosis group were induced by hypodermic injection of CCl₄ for 4 weeks or 8 weeks. The liver index and the serumactivity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed. The liver fibrosis and the morphological changes of endoplasmic reticulum were observed under light and electronic microscopes, respectively. Additionally, the expression of GRP78 at mRNA and protein levels was determined by real-time PCR and the method of immunohistochemistry, respectively. RESULTS: The liver index, serum ALT and AST activity in liver fibrosis group were obviously higher than those in control group. Swelling and reduced number of endoplasmic reticulum were observed in the hepatocytes of fibrotic rats compare to the controls. The levels of GRP78 protein and GRP78 mRNA in the liver of hepatic fibrotic rats were obviously higher than those in the control rats. CONCLUSION: In the process of liver fibrosis induced by CCl₄, the obvious morphological changes of endoplasmic reticulum and increased expression of ERS protein indicate that ERS plays an important role in the liver fibrosis.     

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.     

Changes of portal hemodynamics in the development of experimental liver cirrhosis

AIM: To understand the formation of portal hypertension through the change of portal hemodynamics on experiment cirrhosis. METHODS: Carbon tetrachloride was subcutaneously injected in the rat. The changes of the portal hemodynamics in the pathological process of liver tissue were observed. RESULTS: The liver underwent degeneration, necrosis of hepatocytes, and the normal architecture of the liver lobules was replaced by pseudolobule, which consist of regenerative hepatocytes and fibrous septa. The diameter, the blood flow velocity and the blood flow quantity of portal were significantly higher than that in former group (P＜0.05 or P＜0.01) two weeks after the injection of carbon tetrachloride. In the fifteenth week, these parameters were lower than that before owing to the forming of portacaval collateral circulation (P＜0.01). The congest index of the portal in second week, fifth week and fifteenth week were statistically higher than its predecessor (P＜0.05 or P＜0.01), except that in tenth week, which had no statistical significance (P>0.05). CONCLUSION: The changes in hemodynamics of the portal are in accordance with the changes in pathology of liver in the formation of liver cirrhosis.     

Changes of endoplasmic reticulum stress-related molecules CHOP and TRB3 in rat fibrotic liver

AIM: To observe the changes of endoplasmic reticulum stress-related molecules CCAAT/enhancer-binding protein homologous protein(CHOP) and Tribbles homolog 3（TRB3） in the process of liver fibrosis induced by carbon tetrachloride (CCl4). METHODS: Male Wistar rats weighing 180 g to 200 g were divided into 4-week normal control group, 8-week normal control group, 4-week liver fibrosis group and 8-week liver fibrosis group. The rats in liver fibrosis groups were induced by subcutaneous injection of 40% CCl4 for 4 weeks or 8 weeks. The pathological changes of the liver were observed under light microscope. The protein level of ATF6 was determined by Western blotting. The protein and mRNA levels of CHOP and TRB3 in the liver were analyzed by immunohistochemistry, Western blotting and real-time PCR, respectively. The apoptosis of hepatocytes was measured by TUNEL assay. RESULTS: Pseudolobuli formed in the liver tissue of hepatic fibrotic rats. Compared with the control rats, the protein level of p90ATF6 was obviously decreased, the protein level of p50ATF6 was obviously increased, and the protein and mRNA levels of CHOP and TRB3 were obviously higher in the hepatocytes of hepatic fibrotic rats. The apoptosis of hepatocytes was also increased in 4-week and 8-week fibrosis groups. CONCLUSION: In the process of liver fibrosis induced by 40% CCl4, the obviously increased expression of endoplasmic reticulum stress-related molecules CHOP and TRB3 at protein and mRNA levels indicates that endoplasmic reticulum stress may play an important role in the liver fibrosis by promoting the apoptosis of hepatocytes.     

Roles of four liver cells in regulation of PA-plasmin system during liver fibrogenesis in rats

AIM: To investigate the roles of hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs) and endothelial cells (ECs) in the regulation of PA-plasmin system during liver fibrogenesis in rats. METHODS: Experimental liver fibrosis was induced in rats by injection of carbon tetrachloride (CCl₄) twice a week for 12 weeks. Four kinds of liver cells were separated from the normal and fibrotic livers of the rats. The expression levels of urokinase plasminogen activator (uPA), uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) in liver cells were determined by Northern and Western blotting. RESULTS: The expression of PAI-1 and uPAR was markedly increased in HSCs during liver fibrosis in rats as compared to those in the ECs. CONCLUSION: HSCs and ECs may play very important roles in the regulation of PA-plasmin system during liver fibrogenesis in rats. The activated HSCs are main cells to secrete PAI-1 and uPAR.     

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 sorafenib on liver regeneration after partial hepatoectomy in liver cirrhotic rats

AIM: To study the effect of sorafenib on the liver regeneration after partial hepatectomy (PH) in cirrhotic rats. METHODS: Thirty Wistar rats with liver cirrhosis induced successfully with diethylnitrosamine (DEN) underwent 30% PH and then were randomly divided into 2 groups (n=15). The rats in experimental group were fed with sorafenib at dose of 30 mg·kg^-1·d^-1 from the 1st day to the 10th day after PH, while those in control group were fed with vehicle by gavage. The blood and liver tissues of the rats were collected after PH and at the end of the experiment. Liver regeneration rate (LRR) and proliferating cell nuclear antigen (PCNA) expression were assessed for determining the hepatocyte proliferation. The content of alanine transaminase (ALT), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), angiogenesis related factors including vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet-derived growth factor receptor β (PDGFR-β) and micro-vessel density (MVD) were measured in both groups. RESULTS: LRRs on day 10 after PH were 45.43%±3.36% and 44.21%±2.77% in experimental group and control group, respectively (P>0.05), and the expression of PCNA in hepatic tissues of the rats was not found by the method of immunohistochemistry in both groups. Liver function index had no significant difference between the 2 groups (P>0.05). However, other than VEGF, sorafenib resulted in inhibition of VEGFR-2 and PDGFR-β expression and reduction of MVD in experiment group, and significant difference between the 2 groups was observed (P<0.01). CONCLUSION: Sorafenib does not influence live regeneration after PH in liver cirrhotic rats.     

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 calories restriction on ER stress in the liver of high fat diet rats

AIM: To study the effect of calories restriction on endoplasmic reticulum(ER) chaperone protein 78-kD glucose regulated protein (GRP78) mRNA expression in the liver of high fat diet rats, in order to explore the mechanism of how calories restriction improves insulin resistance. METHODS: Wistar rats (n=24) were randomly divided into 3 groups: normal chow (NC) group, was fed free normal chow (18.94% of calories as fat) for 12 weeks; high fat group (HF) was fed high fat diet (50.55% of calories as fat) for 12 weeks; calories restriction group (CR) was fed high fat diet for 8 weeks at first, then given 50% of diet consumed by the same age NC group. Changes of body weight, height, and food intake were recorded. At the end of experiment, HOMAIR, the rate of visceral fat (including perirenal fat and epididymal fat) vs weight, plasma protein, blood lipid (including total cholesterol and triglyceride), hepatic GRP78 mRNA and hepatic histological changes (including light microscopic studies and electron microscopic studies) were detected. RESULTS: (1) Animals in HF group had an obviously elevation of fasting insulin (27.51±3.51) mU/L vs (15.46±2.25) mU/L, triglyceride (1.35±0.25) mmol/L vs (0.67±0.10) mmol/L, total cholesterol (2.59±0.34) mmol/L vs (1.41±0.28) mmol/L and insulin resistance index HOMAIR (5.85±0.23 vs 2.85±0.60) compared with NC group, and also had obviously lipid accumulations in the liver. (2) After calories restriction, all the abnormal elevated biochemical indicators were decreased to normal levels, the hepatic lipid accumulations were also improved. (3) The changes of liver ultrastructure in HF group showed rough endoplasmic reticulum enlargement, fragmentation, taking off grain, and with glycogen solution. The changes in CR group were nearly the same as those in NC group. (4) High fat diet induced the expression of GRP78 mRNA, calories restriction might reverse it. CONCLUSION: Reasonable food calories restriction is a good method to improve insulin resistance, partly due to improvement of endoplasmic reticulum stress in liver.     

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.     

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...     

Low density lipoprotein from patients of diabetes mellitus induces apoptosis of murine macrophages via activating caspase-12 pathway

AIM:To explore the effect of low density lipoprotein from the patients of diabetes mellitus (DM-LDL) on the activation of caspase-12 an important molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, in the murine macrophages, and to clarify the underlying molecular mechanisms of apoptosis. METHODS:Murine macrophage RAW264.7 was exposed to DM-LDL (25, 50 and 100 mg/L), normal low density lipoprotein (n-LDL, 50 mg/L), or tunicamycin (TM, 4 mg/L) for 24 h. Additionally, RAW264.7 macrophages were precultured with 4-phenylbutyric acid (PBA, 5 mmol/L) for 1 h and then exposed to DM-LDL (100 mg/L) for 24 h. The cell viability and apoptosis were detected by MTT assay and flow cytometry with Annexin V-FITC/propidium iodide staining, respectively. Lactate dehydrogenase (LDH) activity in the media was measured by assay kit. The protein level of caspase-12 was determined by Western blot. RESULTS:Similar to TM (an ERS inducer), treatment with DM-LDL caused significantly decrease in the viability and increase in LDH activity in the media and apoptotic rate of the RAW264.7 macrophages (P<0.05). Additionally, DM-LDL induced activation of caspase-12 especially at the dose of 50 and 100 mg/L (P<0.01). However, the ERS inhibitor PBA protected RAW264.7 macrophages from DM-LDL-induced decrease in viability and increase in LDH activity and apoptosis (P<0.05). Furthermore, PBA attenuated DM-LDL-induced activation of caspase-12 (P<0.05). CONCLUSION:DM-LDL may induce apoptosis in RAW264.7 macrophages, and the mechanism may be related to the activation of caspase-12.     

Changes of serum levels of TXA2 and PGl2 in cirrhosis patients during liver transplantation

AIM：To study the changes of serum levels of thromboxane A₂ (TXA₂) and prostacyclin (PGI₂) in cirrhosis patients during liver transplantation.METHODS：Samples were obtained from 24 cirrhosis patients in end at five time points during liver transplantation.TXA₂ and PGI₂ level were measured by radioimmunoassay.Arterial and mixed venous blood samples used for blood gas analysis were taken at the same time.Intrapulmonary shunt (Qs/Qt) was calculated according to the standard formula.The hemodynamics parameters including continuous cardiac output index (CI),HR,mean artery blood pressure (MABP),MPAP,CVP,PAWP,SVRI,PVRI were measured during liver transplantation.RESULTS：(1) MABP decreased significantly in the early stage of anhepatic period and neohepatic period.(2) CVP,MPAP and PAWP decreased significantly during anhepatic period.They increased significantly after graft reperfusion and remain the high level.(3) CI declined significantly during anhepatic period and increased at 10 min postreperfusion of new liver.(4) SVRI and PVRI increased during anhepatic period and were higher than baseline level at 15 min after reperfusion.SVRI was lower than baseline level at 30 min after reperfusion.(5) Compared with the baseline level,6-keto-PGF1α and TXB₂ increased significantly.Compared with the level before vascular cross-clamping,6-keto-PGF1α decreased during neohepatic period and it had significant difference in statistics at the end of operation.CONCLUSION：Serum levels of TXA₂ and PGI₂ significantly change during liver transplantation and may affect the system and pulmonary circulation to some extent.     

Autophagy protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting C/EBP homologous protein expression

AIM: To investigate the protective effect of autophagy on oxidized low density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: The RAW264.7 macrophages were pretreated with 3 mmol/L 3-methyladenine (3-MA), 1 μmol/L rapamycin (Rap) or 4 mmol/L 4-phenylbutyric acid (PBA) respectively for 1 h and then treated with ox-LDL (100 mg/L) for 12 h. The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The activities of lactate dehydrogenase (LDH) in the medium and caspase-3 in the cells were determined by detection kits. The protein levels of beclin-1 (a molecular marker of autophagy), glucose-regulated protein 78 (GRP78, an endoplasmic reticulum stress marker) and C/EBP homologous protein (CHOP, a key-signaling component of endoplasmic reticulum stress-induced apoptosis) were examined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope.RESULTS: Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability, and dramatic elevation in LDH leakage, cell apoptosis and caspase-3 activity, which were promoted by 3-MA (an autophagy inhibitor) and inhibited by Rap (an autophagy inducer). ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3, which were inhibited by 3-MA and promoted by Rap. Interestingly, 3-MA enhanced, while Rap blocked, the CHOP upregulation induced by ox-LDL. Moreover, PBA (endoplasmic reticulum stress inhibitor) significantly inhibited ox-LDL-induced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granulation of LC3. CONCLUSION: Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages, and moderates activation of autophagy may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression.     

Effect of capsaicin on liver fibrogenesis in rats

AIM：To investigate the effects of capsaicin on rat hepatic stellate cells (HSCs) in vitro and on the liver fibrogenesis in vivo. METHODS：HSCs were cultured with different concentrations of capsaicin. The levels of reactive oxygen species (ROS) were tested with a DCFH-DA kit. The proliferation of HSCs was detected by CCK-8 assay. The expression of α-smooth muscle actin in HSCs was evaluated by Western blotting. The expression of fibrosis-related genes was detected by RT-PCR. The apoptosis of HSCs was measured by flow cytometry. The rat model of liver fibrosis was established by intraperitoneal injection of carbon tetrachloride. Capsaicin at different concentrations was given by gavage. The pathologic changes of the liver sections were observed under microscope with HE staining. Hydroxyproline content in the liver tissues and the levels of collagen Ⅲ and hyaluronic acid in the serum were also measured. RESULTS：Capsaicin inhibited the generation of ROS in a concentration-dependent manner. Compared with control, the proliferation and activation of HSCs were inhibited (P<0.05) and the apoptosis of HSCs was promoted by capsaicin (P<0.05). Capsaicin down-regulated the expression of tissue inhibitor of metalloproteinase 1 and transforming growth factor β 1 in activated HSCs (P<0.05). Capsaicin decreased the levels of hydroxyproline, collagen III and hyaluronic acid in the rats (P<0.05). CONCLUSION：Capsaicin inhibits the proliferation and activation, and promotes the apoptosis of hepatic stellate cells, thus down-regulating the fibrogenesis level of the liver in rats．     

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.     

Changes of histone modification levels in rats with liver fibrosis

AIM: To observe the changes of histone modifications in the liver of rats with hepatic fibrosis and its possible role in the development of hepatic fibrosis. METHODS: Male Wistar rats (n=20) were randomly divided into liver fibrosis group and normal control group. The liver fibrosis model was established by hypodermic injection of CCl₄, and the rats in normal control group were injected with vegetable oils. At the end of the 8th week, all rats were killed. Liver function indexes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and liver fibrosis indexes including haluronic acid (HA), laminin (LN), collagen type IV (Col Ⅳ) and procollagen type Ⅲ (PCⅢ) were determined by biochemical and RIA methods. The liver index was analyzed, and the liver fibrosis degree and the morphological change of the liver were detected by HE and Masson staining. The levels of α-smooth muscle actin (α-SMA), collagen type Ⅰ (ColⅠ), H3K4me2, H3K9me2, acH3K9 and acH4K12 were detected by Western blot. RESULTS: After hypodermic injection of CCl₄ for 8 weeks, the liver index, ALT, AST, HA, LN, Col Ⅳ and PCⅢ of the rats in liver fibrosis group were higher than those in control group (P<0.05). Compared with control group, the level of acH4K12 was decreased (P<0.05), while acH3K9, H3K9me2, α-SMA and ColⅠ were increased (P<0.05), but H3K4me2 had no significant change.CONCLUSION: The levels of acH4K12, acH3K9 and H3K9me2 may play essential roles in the pathogenesis of liver fibrosis, and these histone modifications may regulate gene expression associated with extracellular matrix metabolism.     

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.