PQS attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibiting endoplasmic reticulum stress

AIM:To study the effect of Panax quinquefoliumsaponin (PQS) on cardiomyocyte apoptosis induced by thapsigargin (TG). METHODS:Primary cultured cardiomyocytes from neonatal SD rats were divided into control group, TG group, PQS (40 mg/L, 80 mg/L and 160 mg/L)+TG group, si-PERK+TG group, and mock+TG group. The cells were treated with 1 μmol/L TG for 24 h to induce apoptosis. The PERKgene in the cardiomyocytes was knocked down by RNAi. The cell viability was detected by CCK-8 assay. Apoptosis was analyzed by flow cytometry. Wes-tern blotting was used to determine the expression of ERS molecules GRP78, CRT, ATF4 and CHOP, anti-apoptosis protein Bcl-2 and pro-apoptosis protein Bax. RESULTS:Compared with control group, TG significantly and the apoptosis, reduced the cell viability (P<0.05), increased the phosphorylation of PERK and eIF2α, increased the expression of GRP78, CRT, ATF4, CHOP and pro-apoptosis protein Bax, and decreased the expression of anti-apoptosis protein Bcl-2 (P<0.05). Compared with TG group, PQS treatment (160 mg/L) significantly reduced the apoptosis and increased the cell viability (P<0.05). All the 3 different concentrations of PQS significantly increased the expression of anti-apoptosis protein Bcl-2 and reduced the expression of pro-apoptosis protein Bax (P<0.05) in a dose-dependent manner. PQS pretreatment and knockdown of PERK both reduced the protein levels of GRP78, CRT, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, CHOP and pro-apoptosis protein Bax, and increased the expression of anti-apoptosis protein Bcl-2 (P<0.05). CONCLUSION: PQS at concentration of 160 mg/L attenuated cardiomyocyte apoptosis induced by TG. PQS had the similar effect as PERKknockdown on cardiomyocyte apoptosis. The mechanism may be associated with inhibiting PERK-eIF2α-ATF4-CHOP pathway of ERS-related apoptosis.     

Effect of CHOP expression knock-down on apoptosis of renal tubular epithelial cells

AIM:To study the effect of C/EBP homologous protein (CHOP) on the apoptosis of renal tubular epithelial HK2 cells. METHODS:The serum mRNA levels of CHOP in the patients with acute kidney injury and healthy controls were detected by qPCR. In vitro, renal tubular epithelial HK2 cells were divided into control group, negative group (transfected with negative control siRNA), si-CHOP group (transfected with CHOP siRNA), and induced by transforming growth factor-β1 (TGF-β1). The viability of the cells was measured by MTT assay, and the apoptotic rate was analyzed by flow cytometry. The protein levels of nuclear antigen Ki-67, proliferating cell nuclear antigen (PCNA), caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with the healthy controls, the serum mRNA levels of CHOP in the patients with acute kidney injury were increased significantly (P<0.05). Transfection with CHOP siRNA significantly decreased the expression of CHOP in the renal tubular epithelial HK2 cells (P<0.05). Knock-down of CHOP expression by siRNA significantly increased the viability of renal tubular epithelial HK2 cells (P<0.05), decreased the apoptotic rate (P<0.05), increased the expression of Ki-67 and PCNA (P<0.05), and down-regulated the protein level of cleaved caspase-3 (P<0.05). CONCLUSION:The serum mRNA levels of CHOP were increased in the patients with acute kidney injury. Knock-down of CHOP expression inhibits the apoptosis of renal tubular epithelial cells by regulating the expression of proliferation-and apoptosis-related proteins.     

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）~–51.9 kal ? 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miR396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miR396家族成员的表达,尤其是在不定根诱导期和根系生长期更为显著。     

Expression of endoplasmic reticulum-associated apoptosis protein CHOP in lung tissues of COPD model rats

AIM: To investigate whether cigarette smoke (CS) promotes the expression of endoplasmic reticulum-associated apoptosis protein CCAAT/enhancer-binding protein homologous protein (CHOP) in rat lung tissues.METHODS: Adult male Wistar rats (n=40) were randomly divided into 4 groups with 10 rats in each group:control group, CS-2 group (exposed to CS for 2 months), CS-4 group (exposed to CS for 4 months) and ex-smoking (Ex-S) group (exposed to CS for 4 months and then quit smoking for 1 month). The percentage of forced expiratory volume in 0.3 second to forced vital capacity (FEV_0.3/FVC) and peak expiratory flow (PEF) were measured. TUNEL assay was used to detect the apoptotic cells. In situ hybridization and RT-PCR were used to determine the mRNA expression of CHOP. The methods of immunohistochemistry and Western blot were used to determine the protein expression of CHOP. Western blot was also used to determine the protein levels of protein kinase R-like endoplasmic reticulum kinase (PERK), p-PERK, eukaryotic initiation factor (eIF) 2α and p-eIF2α.RESULTS: The pulmonary function greatly decreased in the rats exposed to CS for 2 months in comparison with control group (P<0.05), markedly decreased in the rats exposed to CS for 4 months as compared with the rats after exposure to CS for 2 months (P<0.05), and was improved little in ex-smoking rats (P>0.05). The structural destruction of the lung was observed in the rats exposed to CS for 2 months, and more obvious changes were found in the rats exposed to CS for 4 months. However, the structural destruction of the lung remained obvious in ex-smoking rats. The apoptotic cells were markedly increased in the rats exposed to CS for 2 months and were even more in the rats exposed to CS for 4 months. The apoptotic cells were alveolar epithelial cell I (ACE I), ACE Ⅱ, vascular endothelial cells and bronchial epithelial cells. The protein levels of p-PERK, p-eIF2α and CHOP were remarkably increased in the rats after exposure to CS for 2 months compared with the control rats (P<0.05), significantly elevated in the rats exposed to CS for 4 months compared with the rats exposed to CS for 2 months (P<0.05), and slightly decreased in ex-smoking rats in comparison with the rats after exposure to CS for 4 months (P>0.05). The total protein levels of PERK and eIF2α did not change between the control rats and those exposed to CS.CONCLUSION: CS promotes the development of chronic obstructive pulmonary disease (COPD) by inducing the expression of endoplasmic reticulum-associated apoptosis protein CHOP via PERK/eIF2α/CHOP signaling pathway.     

Protective effect of morinda officinalis oligosaccharides monomer HexB on hypoxia/reoxygenation-induced injury in HUVECs

AIM:To explore the protective effect of morinda officinalis oligosaccharides monomer HexB on hypoxia/reoxygenation (H/R)-induced injury in human umbilical vein endothelia cells (HUVECs). METHODS:HUVECs were treated with HexB, 4-phenylbutyric acid (4-PBA) and thapsigargin (TG), respectively. The cells were divided into control group, HexB group, H/R group, HexB+H/R group, 4-PBA+H/R group, TG group and HexB+TG group. The cell viability was measured by CCK-8 assay. The apoptotic rate was detected by flow cytometry. Western blot was used to determine the protein levels of endoplasmic reticulum stress (ERS) related molecules chaperone protein glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), apoptosis-related protein caspase-12 and phosphorylated c-Jun NH₂-terminal kinase (p-JNK). RESULTS:The viability of HUVECs was reduced in H/R group and TG group (P<0.05), increased in HexB+H/R, 4-PBA+H/R and HexB+TG group (P<0.05). The apoptotic rate, the protein levels of GRP78, CHOP, caspase-12 and p-JNK were increased in H/R group and TG group (P<0.05), weakened in the HexB+H/R group (P<0.05), 4-PBA+H/R group and HexB+TG group (P<0.05). No significant change in the apoptotic rate, cell viability, protein levels of GRP78, CHOP, caspase-12, p-JNK between HexB+H/R group and 4-PBA+H/R group was observed. CONCLUSION:HexB attenuates HUVECs injury caused by H/R through suppressing ERS and apoptosis. The possible mechanism may be involved in the apoptotic pathways related to GRP78, CHOP, caspase-12 and p-JNK.     

Effects of miR-130a on viability and apoptosis of rat basilar arterial smooth muscle cells

AIM: To investigate the regulatory effects of microRNA (miR)-130a on the biological characteristics of rat basilar arterial smooth muscle cells (BASMCs) and its underlying mechanisms. METHODS: The expression of miR-130a in rat BASMCs was measured by real-time PCR. After knockdown of miR-130a with inhibitor in the BASMCs, the cell viability, cell cycle distribution and apoptosis were detected by CCK-8 assay and flow cytometry. The expression of cell cycle-and apoptosis-related molecules, such as cyclin D1, cyclin-dependent kinase 2 (CDK2), p21, Bcl-2 and cleaved caspase-3/caspase-3 at protein levels was determined by Western blot. The growth arrest-specific homeobox protein (Gax) expression at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: AngiotensionⅡ (AngⅡ) up-regulated the expression of miR-130a and down-regulated the expression of Gax (P<0.05). Transfection with miR-130a inhibitor partly reversed the increase in AngⅡ-induced cell viability and promoted the Gax expression. Furthermore, the early cell apoptotic rate was significantly increased after down-regulation of miR-130a (P<0.05), and the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb were significantly decreased, accompanied with the up-regulation of p21 and cleaved caspase-3 (P<0.05). CONCLUSION: Down-regulation of miR-130a restrains the viability and promotes the apoptosis of BASMCs by promoting Gax expression and regulating cell cycle-and apoptosis-related molecules, suggesting that miR-130a may be a potential therapeutic target of brain vascular remodeling during hypertension.     

Downregulation of DEK induces cell apoptosis via inhibition of NF-κB signaling pathway in gastric carcinoma SGC-7901 cells

AIM: To investigate the effect of DEK downregulation on the apoptosis of gastric carcinoma SGC-7901 cells, and to explore its associations with NF-κB signaling pathway and apoptosis related proteins. METHODS: SGC-7901 cells with different treatments were divided into 3 groups including untreated group, control siRNA group and DEK siRNA group. The expression of DEK at mRNA and protein levels in the SGC-7901 cells was detected by real-time PCR and Western blot. The cell apoptosis was examined by flow cytometry. Furthermore, the activities of caspase-3 and caspase-9 in the SGC-7901 cells were investigated by Caspase-Glo^®-3/9 kit. Finally, the expression of key regulatory protein p65 of NF-κB signaling pathway and apoptosis-related proteins Bcl-2 and Bax in the SGC-7901 cells was investigated by Western blot. RESULTS: Compared with untreated group and control siRNA group, the expression of DEK at mRNA and protein levels was significantly downregulated in DEK siRNA group (P<0.05). In addition, the ratios of early phase apoptosis and total apoptosis in DEK siRNA group were markedly higher than those in untreated group and control siRNA group (P<0.05). Most notably, the decrease in p65 and Bcl-2 proteins, increase in Bax protein and the increases of caspase-3 and caspase-9 activities were observed in DEK siRNA group. CONCLUSION: Downregulation of DEK mediates cell apoptosis of gastric carcinoma may be tightly associated with NF-κB signaling pathway.     

miR-155-specific siRNA enhances chemosensitivity of Burkitt lymphoma Raji cells to cytosine arabinoside by inducing apoptosis

AIM：To investigate the effect of miR-155-specific siRNA alone or in combination with cytosine arabinoside (Ara-C) on the growth and apoptosis of Burkitt lymphoma Raji cells. METHODS：miR-155-specific siRNA and/or Ara-C were used to treat the cells. Quantitative real-time polymerase chain reaction was used to detect the expression of miR-155. The growth of the cells was analyzed by CKK-8 assay. The cell apoptosis was determined by flow cytometry. RESULTS：The miR-155 expression level of the cells transfected with miR-155 siRNA was significantly lower than that in the 2 control groups. Ara-C or miR-155 siRNA alone inhibited the growth of Raji cells in a dose-depend manner. miR-155 siRNA combined with Ara-C produced more inhibition of cell proliferation (P<0.05). After treatment for 48 h, the apoptotic rate of Raji cells in miR-155 siRNA+Ara-C group ［(38.4±1.4)%］ was higher than that in Ara-C group ［(16.5±0.3)%］ and miR-155 siRNA group ［(14.6±0.3)%］, with statistically significant difference (P<0.05). The expression of caspase-3 in Ara-C+miR-155 siRNA group was increased significantly as compared with Ara-C group and miR-155 siRNA group. CONCLUSION：miR-155-specific siRNA enhances the chemosensitivity of Raji cells to Ara-C by inducing apoptosis through the caspase-3 pathway.     

Over-expression of P21 attenuates cisplatin-induced HK-2 cells injury

AIM:To investigate the effect of P21 on cisplatin-induced renal tubular epithelial cells injury.METHODS:The expression of P21 at mRNA and protein levels in cisplatin treated human renal tubular epithelial cells (HK-2) cells was determined by RT-qPCR and Western blot. Over-expression of P21in the HK-2 cells was induced by the transfection of pcDNA3-P21. The cell viability and cell apoptosis were detected by CCK-8 assay and flow cytometry, respectively. Furthermore, the protein expression of kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), caspase-3, glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP), and phosphorylation level of eucaryotic translation initiation factor 2α (eIF2α) and protein kinase R-like endoplasmic reticulum kinase (PERK) were detected by Western blot.RESULTS:Cisplatin increased the mRNA and protein levels of P21 in a time-and concentration-dependent manner in the HK-2 cells. Over-expression of P21 inhibited cisplatin-induced cell apoptosis, and down-regulated the expression of KIM-1 and NGAL. Furthermore, Over-expression of P21 decreased the protein levels of GRP78, p-PERK, p-eIF2α, CHOP and cleaved caspase-3.CONCLUSION:Over-expression of P21 attenuates cisplatin-induced HK-2 cells injury, and the mechanism may be related to the modulation of endoplasmic reticulum stress pathway and inhibition of cell apoptosis.     

Liraglutide ameliorates liver injury of type 2 diabetic mice via micro-RNA-33-AMPK pathway

AIM: To observe the effects of liraglutide on the level of microRNA-33 (miR-33) and the expression of AMP-activated protein kinase (AMPK) and apoptosis-related proteins in mice with type 2 diabetes mellitus (T2DM), and to explore its possible mechanism. METHODS: High-fat diet and intraperitoneal injection of streptozocin were used to establish the type 2 diabetic model in C57BL/6 mice. The mice were randomly divided into 4 groups (n=15):in control group, the normal mice were subcutaneously injected with equivalent volume of saline; in model group, the T2DM mice were subcutaneously injected with equivalent volume of saline; in low-and high-dose liraglutide treatment groups, the T2DM mice were subcutaneously injected with 100 and 200 μg·kg^-1·d^-1, respectively. After 4 weeks of administration, the levels of FBG, TG, TC, HDL-C, LDL-C, ALT and AST were determined. HE staining was used to observe the pathological changes of the liver tissues. The protein level of cleaved caspase-3 in the liver tissue was detected by the technique of immunofluorescence. The protein levels of p-AMPK/AMPK and apoptosis-related proteins were detected by Western blot. The expression of miR-33 in the liver tissues was detected by real-time PCR. RESULTS: Compared with model group, the contents of FBG, TG, TC, LDL-C, ALT and AST were decreased significantly, while the content of HDL-C was increased significantly in low-dose liraglutide group and high-dose liraglutide group (P<0.05). The protein levels of phosphorylated AMPK and Bcl-2 were up-regulated significantly, and the expression of cleaved caspase-3 was down-regulated significantly (P<0.05). The level of miR-33 was decreased significantly (P<0.01). CONCLUSION: Liraglutide alleviates liver injury in type 2 diabetic mice, and the mechanism may be associated with reducing the level of miR-33 and increasing the phosphorylation of AMPK in the liver tissues, thereby inhibiting hepatocyte apoptosis.     

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.     

Model construction of rat coronary artery smooth muscle cell endoplasmic reticulum stress induced by thapsigargin

AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress (ERS) model in CASMCs of SD rats. METHODS: CASMCs were cultured by tissue explant method. The morphological characteristics were observed under optical microscope. The marker proteins of CASMCs, including α-SMA and SM-MHC, were identified by immunofluorescence technique. The protein expression levels of BiP and CHOP, the marker molecules of ERS, were determined by Western blot. RESULTS: The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical "hill and valley" growth pattern of CASMCs at 9~10 d. The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed. The results of Western blot showed that the protein expression of BiP and CHOP in TG (1 and 2 μmol/L) treatment groups was increased compared with control group. Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION: CASMCs can be successfully cultured by tissue explant method. ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.     

Cepharanthine promotes apoptosis of RL-952 cells by regulating eIF4E-related miR-215

AIM:To investigate the effect of cepharanthine (CEP) on the viability and apoptosis of human endometrial carcinoma RL-952 cells and its mechanisms. METHODS:RL-952 cells were treated with cepharanthine at different concentrations. The cell viability and apoptosis were measured by MTT assay and flow cytometry, respectively. The optimal drug concentration was also screened. The miR-215 expression induced by cepharanthine was detected by real-time PCR. The effects of cepharanthine on the protein le-vels of eukaryotic initiation factor 4E (eIF4E) and p-eIF4E were determined by Western blot. The downstream target genes were predicted by bioinformatic software. Finally, after miR-215 was transfected into RL-952 cells, the change of GFP expression was evaluated by flow cytometry and the protein levels of eIF4E and p-eIF4E were determined by Western blot. RESULTS:With the increasing concentrations of cepharanthine, the inhibitory rate of the viability in RL-952 cells was increased gradually and the rate of apoptosis was increased. The most effective concentration was 30 μmol/L. After treatment with cepharanthine at this concentration, the expression level of miR-215 was significantly higher than that in control group (P<0.01). The bioinformatic software prediction indicated that there were binding sites of miR-215 in the 3'UTR of eIF4E. The protein levels of eIF4E and p-eIF4E in the cepharanthine group were lower than those in control group (P<0.05). After the miR-215 and pcDNA-GFP-eIF4E-3'UTR were cotransfected into the RL-952 cells, the expression of GFP declined (P<0.05). After transfection with miR-215, the protein le-vels of eIF4E and p-eIF4E were decreased (P<0.05). CONCLUSION:Cepharanthine effectively inhibits the viability and promotes the apoptosis of RL-952 cells. The mechanism may be related to up-regulating the expression of eIF4E-related miR-215 and then suppressing the protein levels of eIF4E and its active form p-eIF4E.     

4-PBA attenuates damage of primary hippocampal neurons via inhibiting endoplasmic reticulum stress

AIM:To investigate the inhibitory effect of 4-phenylbutyric acid (4-PBA), an aromatic short-chain fatty acid with the functions of endoplasmic reticulum (ER) molecule chaperon, on ER stress-induced decreases in both dendritic spine density and synaptic protein expression. METHODS:Primary hippocampal neurons were cultured from neonatal rats. The primary hippocampal neurons were transfected with enhanced green fluorescent protein plasmids at DIV (day in vitro) 5~7. At DIV 20, the neurons were divided into DMSO group, tunicamycin group and tunicamycin+4-PBA (treated with 4-PBA 1 h before tunicamycin) group. The expression levels of ER stress marker protein BiP and synaptic proteins were detected by Western blot. After immunofluorescence staining, the neurons were morphologically observed under confocal laser scanning microscope, and the density of dendritic spines was analyzed. At last, the cell viability was measured by MTT assay. RESULTS:Tunicamycin induced ER stress in the primary hippocampal neurons, characterized by significantly increased level of BiP in tunicamycin group, which was reduced in tunicamycin+4-PBA group (P<0.05). 4-PBA inhibited tunicamycin-induced decreases in both dendritic spine density and synaptic protein expression in the primary hippocampal neurons. 4-PBA attenuated tunicamycin-induced decrease in the cell viability. CONCLUSION:4-PBA not only attenuates ER stress of primary hippocampal neurons but also inhibits the decreases in both dendritic spine density and synaptic protein expression induced by tunicamycin.