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.     

Reversal effect of shikonin on cisplatin resistance of ovarian cancer SKOV3/DDP cells

AIM:To explore the reversal effect of shikonin on cisplatin resistance of ovarian cancer SKOV3/DDP cells and its potential mechanism. METHODS:The proper conditions of treatment with shikonin and cisplatin were determined by CCK-8 assay. The cell cycle and apoptotic rate were analyzed by flow cytometry. The protein levels of cell cycle-and apoptotic-related molecules, such as cyclin D1, cyclin-dependent kinases 2 (CDK2), P18, p-Rb, Bcl-2, Bax and cleaved caspase-3, were determined by Western blot. RESULTS:The results of CCK-8 assay showed that compared with cisplatin group, combined treatment with shikonin and cisplatin had a better inhibitory effect on the growth of cisplatin-resistant SKOV3/DDP cells. The cell cycle G₁/S transition was inhibited, while early apoptotic rate was increased after combined use of shikonin and cisplatin. The results of Western blot showed that compared with cisplatin group, the cells in combination group had lower protein levels of cyclin D1, CDK2, p-Rb and Bcl-2, accompanied with higher protein levels of P18, Bax and cleaved caspase-3. CONCLUSION:Shikonin reverses the cisplatin resistance of ovarian cancer SKOV3/DDP cells. The mechanism may be related to the regulation of cell cycle-and apoptotic-related molecules, and further inhibition of cell viability and promotion of cell apoptosis.     

Schisandra total lignin attenuates apoptosis of endoplasmic reticulum pathway to delay mouse brain aging

AIM:To investigate the role of schisandra total lignin (SCL) in anti-aging of the mouse brain. METHODS:A D-galactose induced mouse aging model was established. The following groups in this study were set up: control group, model group, SCL low dose group ［SCL (L)］, SCL moderate dose group ［SCL (M)］ and SCL high dose group ［SCL (H)］. Learning and memory abilities were measured by mouse jumping experiments. The expression of ubiquitin (Ub), glucose-regulated protein 78 （GRP78）, protein disulfide isomerase (PDI), CCAAT /enhancer-binding protein homologous protein (CHOP), Bcl-2 and Bax proteins was detected by Western blotting. In addition, the protein expression of Bcl-2 and Bax in the aging cerebral cortex was also observed by immunohistochemistry. RESULTS:In learning test, compared with control group, the number of errors within 5 min increased in model group (P<0.05). Compared with model group, the number of errors within 5 min decreased in SCL (L) group, SCL(M) group and SCL(H) group (P<0.05). In memory test, compared with control group, incubation period of the first jumping off the platform was shorter and the number of errors within 5 min increased in model grou(P<0.05). Compared with model group, the incubation period of the first jumping off the platform was longer and the number of errors within 5 min decreased in SCL (L) group , SCL(M) group and SCL(H) group (P<0.05). Compared with control group, the protein expression of Ub, GRP78, PDI, CHOP and Bax was increased (P<0.05), while Bcl-2 protein level and Bcl-2/Bax ratio were decreased in model group(P<0.05). Compared with model group, the protein expression of Ub, GRP78, PDI, CHOP and Bax was decreased in SCL (L) group, SCL(M) group and SCL(H) group (P<0.05), while Bcl-2 protein level and Bcl-2/Bax ratio were increased (P<0.05). In control group, neuronal morphology was normal, the protein expression of Bcl-2 was positive and Bax was negative in the cytoplasm. In model group, the neurons were degeneration, the protein expression of Bcl-2 was negative and Bax was positive in the cytoplasm. In SCL (L) group, SCL (M) group and SCL (H) group, the number of degenerative neurons decreased, the protein expression of Bcl-2 was positive and Bax was negative in the cytoplasm. CONCLUSION: SCL inhibit D-galactose-induced brain aging in mice by attenuating apoptosis of endoplasmic reticulum pathway.     

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.     

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...     

Role of endoplasmic reticulum stress and autophagy mediated by endocannabinoid N-arachidonic acid aminoethanol in ovarian cancer cells

AIM To study the effect of endocannabinoid N-arachidonic acid aminoethanol （AEA） on ovarian cancer and its mechanism. METHODS The serum levels of AEA in healthy control group and ovarian cancer group were analyzed by ELISA, and the diagnostic value of AEA in ovarian cancer patients was evaluated by ROC curve. The effects of AEA on the viability, migration and invasion abilities of the ovarian cancer cells were detected by CCK-8 assay, Transwell cell invasion test and Scratch test. The effect of AEA on ovarian cancer was further verified by the measurement of tumor volume, tumor weight and visual map of tumor tissue. Meanwhile, flow cytometry and Western blot were used to determine the effect of AEA on the apoptosis of ovarian cancer cells, so as to explore the mechanism of AEA promoting the apoptosis of ovarian cancer cells through detecting the endoplasmic reticulum stress and autophagy related proteins by Western blot. RESULTS The serum levels of N-arachidonic aminoethanol in the patients with ovarian cancer were significantly decreased. ROC results suggested that AEA was a sensitive biological marker to distinguish the patients with ovarian cancer from healthy dedividuals. In addition, AEA inhibited the cell viability, migration and invasion abilities of ovarian cancer cells, and inhibited the growth of ovarian cancer tissues. CONCLUSION By promoting endoplasmic reticulum stress and affecting autophagy of ovarian cancer cells, AEA promotes apoptosis of ovarian cancer cells.     

Effects of calpain-2 and autophagy-related protein 5 on hepatocyte apoptosis induced by endoplasmic reticulum stress

AIMTo investigate the effects of calpain-2 and autophagy-related protein 5 (Atg5) on apoptosis of BRL-3A rat normal liver cells during endoplasmic reticulum stress (ERS) induced by dithiothreitol (DTT). METH?ODS: BRL-3A cells were treated with DTT at 2.0 mmol/L for 0, 6, 12 and 24 h to induce ERS. Real-time cell analysis (RTCA) was used to measure the effect of DTT on BRL-3A cell proliferation. Apoptosis and cell cycle distribution were analyzed by flow cytometry. The mRNA expression of calpain-2 and Atg5 was detected by real-time PCR. The protein levels of calpain-2, Atg5, Atg7, Atg12 and microtubule-associated protein 1 light chain 3 (LC3) were determined by Western blot. The interaction between calpain-2 and Atg5 was investigated by co-immunoprecipitation (Co-IP). RESULTSThe proliferation of BRL-3A cells treated with DTT was significantly inhibited. The apoptosis of BRL-3A cells was significantly increased after DTT treatment for 6, 12 and 24 h as compared with 0 h group (P<0.05). The cell cycle was arrested in G₁ phase after DTT treatment (P<0.05). After DTT treatment for 6, 12 and 24 h, the mRNA expression of calpain-2 and Atg5 in the BRL-3A cells was significantly increased as compared with 0 h group (P<0.05). The protein levels of calpain-2, Atg12 and Atg7 in the cells treated with DTT for 6, 12 and 24 h were significantly higher than those in 0 h group, and the ratio of LC3-II/LC3-I was also significantly higher than that in 0 h group, while Atg5 expression was significantly lower than that in 0 h group (P<0.05). The results of Co-IP found that the anti-calpain-2 antibody precipitated Atg5 protein from the cell lysates, and the anti-Atg5 antibody also precipitated calpain-2 from the cell lysates, which confirmed the interaction between calpain-2 and Atg5. CONCLUSION Calpain-2 may participate in ERS-induced hepatocyte apoptosis by interacting with Atg5.     

Role of juglone in regulating oxidative stress and apoptosis in ovarian cancer SKOV3 cells

AIM: To study the cytotoxicity of juglone on ovarian cancer SKOV3 cells. METHODS: The activity of SKOV3 cells was detected by MTT assay. The cells apoptosis was examined by flow cytometry. The level of reactive oxygen species (ROS) was measured by DCF-DA staining. The protein levels of cytochrome C (Cyt C) and activated caspase-3 were evaluated by Western blot. RESULTS: MTT assay showed that juglone significantly inhibited the growth of SKOV3 cells in dose- and time-dependent manners (P<0.05). The early apoptotic rate and late apoptotic rate of SKOV3 cells in 50 μmol/L juglone group at 24 and 48 h were higher than those in control group (P<0.01).Moreover, juglone induced ROS accumulation, and increased the protein levels of Cyt C and activated caspase-3.CONCLUSION: Juglone inhibits the cell growth and induces the apoptosis of SKOV3 cells by ROS accumulation.     

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.     

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.     

Effects of capsaicin on cognitive impairment and mitochondria-associated endoplasmic reticulum membranes in rats with chronic cerebral hypoperfusion

AIM:To observe the effects of capsaicin on cognitive impairment and mitochondria-associated endoplasmic reticulum membranes (MAMs) of hippocampal CA1 area in the rats with chronic cerebral hypoperfusion (CCH), and to investigate the underlying molecule mechanism of cognitive defects induced by ischemia. METHODS:Healthy male Sprague-Dawley (SD) rats(n=48) were randomly divided into sham operation (sham) group,CCH model (CCH) group, capsaicin group,and solvent group, 12 rats in each group. Capsaicin at 2.5 mg/kg was intraperitoneally injected twice a week for 4 weeks, starting on the 7th day after surgery. The rats in solvent group were given the same amount of solvent at the same time and under the same conditions. Morris water maze, object recognition test and open field test were conducted to analyze the cognitive related behavior performance on the 4th week after surgery. The changes of MAMs in the hippocampal CA1 region were observed under transmission electron microscope, the co-localization of the MAMs was observed by immunofluorescence double-labeling, and the expression of mitofusin 2 (Mfn2) in the hippocampal tissue was determined by Western blot.RESULTS:Four weeks after the operation, the behavior tests showed that the cognitive function of CCH rats was impaired compared with sham operation group. Compared with solvent group, spatial learning and memory in capsaicin group was improved significantly. The results of transmission electron microscope and confocal microscope showed that the distance of MAMs in the hippocampal CA1 area of CCH rats was increased compared with sham operation group, and the co-localization of the contacts was decreased (P<0.05). Compared with solvent group, the correlation between the mitochondria and ER in capsaicin group was increased (P<0.05). The protein level of Mfn2 in CCH group was significantly lower than that in sham group (P<0.05). Compared with solvent group, the protein level of Mfn2 in capsaicin group was higher (P<0.05). CONCLUSION:CCH rats showed decreased cognitive function and loosen MAMs. Capsaicin improves the cognitive behavior of CCH rats by up-regulation of MAMs.     

Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in cardiomyocytes via inducing autophagy

AIM: This study aims to explore the effect of abietic acid (AA) on advanced glycosylation end products (AGEs)-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes. METHODS: H9c2 cells were divided into 5 groups. The cells in control group were treated with saline for 24 h. The cells in AGEs treatment group were treated with AGEs (100 mg/L) for 24 h. The cells in AGEs+AA (10, 25 and 50 μmol/L) groups were simulta-neously treated with AGEs (100 mg/L) and AA (10, 25 and 50 μmol/L) for 24 h. The cell viability was measured by MTT assay. The protein levels of myoglobin (Mb), creatine kinase MB isoenzyme (CK-MB), cardiac troponin I (cTnI), C/EBP homologous protein (CHOP), cleaved caspase-12, GADD34, BiP, LC3, P62 and beclin 1 were determined by Western blot. The levels of lactate dehydrogenase (LDH) were measured by ELASA. The apoptosis was analyzed by flow cytometry. RESULTS: The low concentration (<50 μmol/L) of abietic acid had no obvious effect on the viability of H9c2 cells. The high concentration (>50 μmol/L) of abietic acid decreased the viability of H9c2 cells. The levels of Mb, CK-MB, cTnI and LDH in AGEs group were higher than those in control group (P<0.05). Compared with AGEs group, the levels of Mb, CK-MB, cTnI and LDH in AGEs+AA (10, 25 and 50 μmol/L) groups were obviously reduced (P<0.05). Abietic acid at concentrations of 10, 25 and 50 μmol/L inhibited AGEs-induced apoptosis, elevated the protein levels of CHOP and cleaved caspase-12, and attenuated expression of GADD34 and BiP (P<0.05). Moreover, abietic acid at concentrations of 10, 25 and 50 μmol/L suppressed AGEs-induced decreased ratio of LC3-Ⅱ/LC3-Ⅰ and expression of beclin 1, and enhanced the expression of P62 (P<0.05). 3-Methyladenine, an inhibitor of autophagy, reversed the effect of abietic acid on the protein levels of LC3, Mb, cleaved caspase-12 and BiP (P<0.05). CONCLUSION: Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes via inducing autophagy.     

Suberoylanilide hydroxamic acid induces apoptosis of HepG2 cells by endoplasmic reticulum stress apoptotic pathway

AIM: To investigate the effect of suberoylanilide hydroxamic acid (SAHA) on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells and to explore its possible mechanism. METHODS: HepG2 cells were treated with SAHA at different concentrations for 48 h. The proliferation of HepG2 cells was detected by real-time cellular analysis. The protein levels of acetylated histones H3K9 and H3K27, glucose-regulated protein 78 (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK) and p-PERK were determined by Western blot. The cell apoptosis was analyzed by flow cytometry. RESULTS: Compared with control group, treatment with SAHA at 0.1 μmol/L and 1 μmol/L for 48 h showed no significant inhibitory effect on the proliferation of HepG2 cells, while SAHA at 6 μmol/L and 12 μmol/L significantly inhibited the proliferation of HepG2 cells (P<0.05). The results of Western blot showed that the protein levels of acH3K9, acH3K27, GRP78 and p-PERK increased significantly after treated with SAHA at diffe-rent concentrations for 48 h, while the protein level of PERK was decreased significantly (P<0.05). The results of flow cytometry analysis showed that the apoptotic rates of the HepG2 cells increased with the increase in SAHA concentration. CONCLUSION: SAHA up-regulates the acetylation of H3K9 and H3K27 in the HepG2 cells and induces apoptosis of HepG2 cells by activating the endoplasmic reticulum stress-related apoptosis pathway.     

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.     

NAC-1 -specific siRNA enhances paelitaxel-induced apoptosis of ovarian cancer cell line HO8910

AIM: To observe the effect of NAC-1 -specific siRNA alone, or in combination with paelitaxel on proliferation and apoptosis of human ovarian cancer cell line HO8910. METHODS: Ovarian cancer cells were treated with NAC-1 siRNA alone or in combination with paelitaxel. The level of NAC-1 mRNA was assessed by real-time quantitative PCR. Western blotting analysis was used to detect NAC-1 protein and the activation of epidermal growth factor receptor(EGFR) downstream signals,Akt and ERK. The cell proliferation rate was measured by MTT assay, and the cell cycle and apoptosis were determined by flow cytometry. RESULTS: After treated with NAC-1 -specific siRNA for 48 h, the expression of NAC-1 at mRNA and protein levels in HO8910 cells decreased by 71.1% and 80.5%, respectively. The cells in G₁ phase increased. The protein levels of p-Akt and p-ERK were decreased by 43.7% and 49.8%, respectively. After treated with NAC-1 -specific siRNA for 72 h, the proliferation inhibitory rate of the cells was increased to 45.6% as compared with the cells treated with negative siRNA. Apoptotic rate of the cells treated with NAC-1 siRNA (0.5 μmol/L combined with 2 μmol/L of paelitaxel) for 72 h was (30.93±4.57)%,higher than that of the cells treated with paelitaxel alone[(23.85±3.65)%]. CONCLUSION: NAC-1 siRNA suppresses NAC-1 gene expression and EGFR downstream signaling activation, inhibits cell proliferation and enhances the responsiveness of ovarian cancer cells to paelitaxel. The combination treatment produces synergistic inhibition.     

Dexmedetomidine reduces renal injury induced by lung ischemia/reperfusion in mice through inhibiting endoplasmic reticulum stress response

AIM:To investigate the effect of dexmedetomidine (DEX) on renal injury induced by lung ischemia/reperfusion (I/R) in mice and its relationship with endoplasmic reticulum stress response.METHODS:Healthy SPF male C57BL/6J mice,weighing 20~24 g,aged 8~10 weeks,were randomly divided into 5 groups (n=10 each):sham operation group (sham group),I/R group,atipamezole (Atip) group,DEX group,and DEX+Atip group.In vivo lung I/R model was established by occlusion of the left pulmonary artery for 30 min followed by 180 min of reperfusion in the mice.The Atip (250 μg/kg),DEX (20 μg/kg) and DEX+Atip were intraperitoneally infused into the mice before left pulmonary hilus was blocked in Atip group,DEX group and DEX+Atip group,and other operations were the same as I/R group.After experiment,the mice were killed,and the renal tissues were harvested to observe the morphological changes.The enzymatic activity of caspase-3,serum creatinine and blood urea nitrogen,and cell apoptotic index of the renal cells were also analyzed.The expression of c-Jun N-terminal kinase (JNK),caspase-12,CCAAT/enhancer-binding protein homdogous protein (CHOP) and glucose-regulated protein 78(GRP78) at mRNA and protein levels in the renal tissues was determined by RT-PCR and Western blot.RESULTS:Compared with sham group,the enzymatic activity of caspase-3,serum creatinine and blood urea nitrogen,renal cell apoptotic index,and the mRNA and protein levels of JNK,caspase-12,CHOP and GRP78 in I/R group were significantly increased (P<0.01),and the renal tissues had obvious damage under light microscope.Compared with I/R group,Atip group and DEX+Atip group,the enzymatic activity of caspase-3,serum creatinine and blood urea nitrogen,renal cell apoptotic index,and the mRNA and protein levels of JNK,caspase-12 and CHOP in DEX group were significantly decreased,and the expression level of GRP78 significantly increased (P<0.01).Furthermore,the renal tissue damage was obvious reduced.CONCLUSION:DEX effectively relieves the renal injury induced by lung I/R in mice,which may be associated with exciting α₂-adrenergic receptor and inhibiting endoplasmic reticulum stress response.     

Resveratrol induces apoptosis in human ovarian cancer SKOV3 cells via Sirt3-SOD2-ROS pathway

AIM: To investigate whetier resveratrol induces apoptosis of human ovarian cancer SKOV3 cells through Sirt3-SOD2-ROS pathway. METHODS: SKOV3 cells were cultured in vitro and treated with resveratrol at 0, 2.5, 5, 10, 20, 40 and 80 mg/L for 24 h. The inhibitory effect of resveratrol on the viability of SKOV3 cells was measured by MTT assay. SKOV3 cells were randomly divided into blank control group, 10 mg/L resveratrol group, 20 mg/L resveratrol group and 40 mg/L resveratrol group. After 24 h of treatment, Hoechst 33342 staining and confocal microscopy were used to observe the nuclear changes. The protein levels of silent mating type information regulation 2 homolog 3 (Sirt3), superoxide dismutase 2 (SOD2), Bcl-2, Bax and cleaved caspase-3 were determined by Western blot. RESULTS: Treatment with resveratrol at 2.5, 5, 10, 20, 40 and 80 mg/L for 24 h significantly reduced the viability of SKOV3 cells. The observation by confocal microscopy showed that the nucleus of SKOV3 cells was markedly condensed and heavily stained with the increase in the concentration of resveratrol. Compared with blank control group, the red fluorescence intensity of ROS in different concentrations of resveratrol groups was significantly reduced. The results of Western blot showed that the protein levels of Sirt3, SOD2, Bax and cleaved caspase-3 in resveratrol groups were significantly higher than those in control group, while the protein expression of Bcl-2 was significantly lower than that in control group (P<0.05). CONCLUSION: Resveratrol induces apoptosis of SKOV3 cells by regulating Sirt3-SOD2-ROS pathway.     

Panax quinquefolium saponin attenuates ventricular remodeling after acute myocardial infarction in rats by inhibiting endoplasmic reticulum stress-related apoptosis

AIM: To investigate the effect of Panax quinquefolium saponin (PQS) on ventricular remodeling after acute myocardial infarction (AMI) in rats and its mechanism. METHODS: Ninety healthy male SD rats were randomly divided into sham group, AMI group, taurine 300 mg·kg-1·d-1 group, PQS 50 mg·kg-1·d-1 group, PQS 100 mg·kg-1·d-1 group and PQS 200 mg·kg-1·d-1 group. AMI models were produced by ligating the left coronary arteries in SD rats. The rats in each treatment group were gavaged with drugs dissolved in water (10 mL·kg-1·d-1), and the rats in sham group and AMI group received equal volume of water. Four weeks after MI, the left ventricle fractional shortening, ejection fraction and structure were evaluated by echocardiography. Myocardial infarct size was measured by 2,3,5-triphenyltetrazolium chloride staining. The hydroxyproline level was measured by colorimetric method. Apoptosis of the cardiomyocytes was detected by TUNEL. In addition, the expression of endoplasmic reticulum stress-related molecules in the noninfarcted myocardium was determined by Western blotting. RESULTS: Compared with AMI group, the left ventricular end-systolic dimension in PQS 50 mg·kg-1·d-1 group, PQS 100 mg·kg-1·d-1 group and PQS 200 mg·kg-1·d-1 group decreased by 17.2%, 20.3% and 38.8% respectively,and the left ventricular end-diastolic dimension decreased by 8.91%, 8.95% and 17.20%, respectively.The left ventricular end-systolic volume decreased by 31.4%, 38.5% and 67.0%, respectively, and the left ventricular end-diastolic volume decreased by 18.2%, 18.8% and 34.2%, respectively.The left ventricular ejection fraction increased by 44.9%, 60.1% and 118.0%, respectively,and the fractional shortening increased by 55.4%, 71.0% and 148.0%, respectively.The infarction size decreased by 4.6%, 39.5% and 55.8%, respectively,and the hydroxyproline level in noninfarcted myocardium decreased by 34.5%, 35.9% and 48.7%, respectively. Compared with AMI group, the myocardial apoptotic index in PQS 200 mg·kg-1·d-1 group decreased by 27.3%, the protein expression of Bcl-2 increased by 114.0%, and that of Bax, GRP78, CRT and CHOP decreased by 53.1%, 79.9%, 80.8% and 42.5%, respectively. The above mentioned protective effects in PQS 200 mg·kg-1·d-1 group and taurine group were similar. The Spearman correlation analysis revealed that CHOP expression had significant positive correlation with apoptotic index (r=0.797, P<0.01). CONCLUSION: PQS attenuates ventricular remodeling in rats. The underlying mechanism may be associated with the inhibition of CHOP-mediated endoplasmic reticulum stress-related cardiomyocyte apoptosis.