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.     

Effect of ixazomib on apoptosis and NF-κB signaling pathway in pancreatic cancer cells

AIM:To investigate the effects of ixazomib on the apoptosis and NF-κB signaling pathway in pancreatic cancer cells. METHODS:Human pancreatic cancer cell lines CFPAC-1 and PANC-1 were cultured, and the cells were treated with ixazomib at 0, 10, 20, 30 and 40 nmol/L for 12, 18, 24 and 48 h. The expression of NF-κB p65, IκB kinase (IKK), Bax and caspase-3 in the cells at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by CCK-8 assay. The apoptosis was analyzed by flow cytometry. RESULTS:Treatment with ixazomib at 10~40 nmol/L inhibited the viability of PANC-1 cells and CFPAC-1 cells, and the inhibitory rate was increased significantly with the increases in the concentration and time (P<0.05). Compared with the control cells, treatment with ixazomib significantly increased the apoptotic rates of PANC-1 cells and CFPAC-1 cells in a dose- dependent manner (P<0.05), and significantly decreased the mRNA expression levels of NF-κB p65 and IKK in the PANC-1 cells and CFPAC-1 cells (P<0.05), while the mRNA expression levels of apoptotic factors Bax and caspase-3 in the PANC-1 cells and CFPAC-1 cells were significantly increased (P<0.05). The results of Western blot showed that treatment with ixazomib significantly decreased the protein levels of NF-κB p65 and IKK in the PANC-1 cells and CFPAC-1 cells (P<0.05), which was consistent with the results of mRNA expression. The protein levels of apoptosis factors Bax and caspase-3 in the CFPAC-1 cells were significantly increased (P<0.05), and the protein level of caspase-3 in the PANC-1 cells was increased significantly (P<0.05). However, Bax protein did not increase significantly in 10 nmol/L ixazomib group. CONCLUSION:Ixazomib, a proteasome inhibitor, inhibits the viability of pancreatic cancer cells and promotes apoptosis by inhibiting the activation of NF-κB signaling pathway in a time- and dose-dependent manner.     

Salvianolate attenuates oxidative stress injury of human endothelial EA.hy926 cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolate on oxidative damage induced by hydrogen peroxide in human endothelial EA.hy926 cells.METHODS: EA.hy926 cells were cultured in vitro and divided into the following groups:control group, damage group, and anti-damage groups (salvianolate+damage groups). The cell viability was measured by CCK-8 assay. The migration ability of the EA.hy926 cells was detected by Transwell assay. The content of nitric oxide (NO) in the culture supernatant of the EA.hy926 cells was examined. The levels of vascular endothelial growth factor (VEGF) were detected by ELISA. The apoptosis,mitochondrial membrane potential and intracellular superoxide anion content of the EA.hy926 cells were analyzed by flow cytometry. The protein levels of caspase-3, cleaved caspase-3, Bcl-2, Bax, NF-κB and p53 were determined by Western blot. RESULTS: Compared with damage group, the viability of EA.hy926 cells pretreated with salvianolate at different concentrations was significantly increased (P<0.05). The apoptotic rate was significantly decreased (P<0.05). Savianolate enhanced the migration ability of the cells. The levels of VEGF, NO and mitochondrial transmembrane potential were increased (P<0.05), and the intracellular ROS level was significantly decreased (P<0.05). The protein levels of NF-κB, p53, Bax and cleaved caspase-3 were significantly decreased, and the protein level of Bcl-2 was markedly increased(P<0.05). CONCLUSION: Savianolate reduces the damage of EA.hy926 cells by hydrogen peroxide exposure, and its mechanism may be related to the blocking of NF-κB signaling pathway.     

Epigallocatechin gallate induces apoptosis of ovarian cancer cell line SKOV-3 via regulation of SIRT1-P53 pathways

AIM: To study the effect of epigallocatechin gallate (EGCG) on the growth and apoptosis of ova-rian cancer cell line SKOV-3 and its molecular mechanism. METHODS: SKOV-3 cells were treated with different concentrations of EGCG (0~50 μmol/L), SRT1720 (1 μmol/L) or EX527 (1 μmol/L) for 24 h. The cell activity was evaluated by CCK-8 assay. The apoptosis of SKOV-3 cells was analyzed by flow cytometry. The mRNA expression of Bcl-2 and Bax was detected by real-time PCR. SIRT1 deacetylase fluorometric assay kit was used to detect the activity of SIRT1. The protein levels of SIRT1 and acetylated P53 (Ac-P53) were determined by Western blot. RESULTS: EGCG or EX527 decreased the deacetylase activity and protein expression of SIRT1, and increased the level of Ac-P53 in a dose-dependent manner. Moreover, SRT1720 abrogated the effects of EGCG on the activity, apoptosis and SIRT1-P53 pathways in ovarian cancer cell line SKOV-3. CONCLUSION: EGCG inhibits the activity and induces apoptosis of ovarian cancer cell line SKOV-3 by regulating SIRT1-P53 pathways.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells

AIM:To explore the effect of pyrrolidine dithiocarbamate (PDTC),an NF-κB inhibitor,on the proliferation and apoptosis of human multiple myeloma U266 cells and its mechanisms.METHODS:The U266 cells were treated with PDTC at different concentrations (0,25,50,100 and 200 μmol/L)in vitro.The growth inhibitory rate of the U266 cells was detected by CCK-8 assay and cell counting.The cell cycle of the U266 cells was determined by flow cyto-metry,and the apoptosis was examined by flow cytometry with Annexin V-FITC/PI staining.The effect of PDTC on the expression of DNA methyltransferase 1(DNMT1) at mRNA and protein levels was measured by RT-qPCR and Western blot,respectively.The effects of PDTC on the protein levels of NF-κB (P65),DNMT1,Bcl-2,cyclin D1,cleaved caspase-3 and cleaved caspase-8 were determined by Western blot.RESULTS:The protein level of NF-κB (P65) was decreased after treatment with PDTC for 48 h or 72 h.PDTC inhibited the proliferation of U266 cells in both dose-and time-dependent manners.After treatment with PDTC for 48 h,the percentage of U266 cells in G₂ phase increased compared with control group (P<0.05).PDTC induced the apoptosis of U266 cells in a dose-dependent manner.The expression of DNMT1 at mRNA and protein levels decreased (P<0.05).The results of Western blot showed that the expression of Bcl-2 in PDTC groups decreased,while the protein levels of cyclin D1,cleaved caspase-3 and cleaved caspase-8 were higher than those in control group (P<0.05).CONCLUSION:The NF-κB inhibitor PDTC inhibits the proliferation of U266 cells by inducing cell apoptosis.It may be related to the down-regulated expression of DNMT1,cell cycle arrest and activation of the apoptotic pathways.     

Effect of integrin β1 on multidrug resistance in gastric cancer SGC-7901 cells

AIM: To study the effect of integrin β1 on multidrug resistance in gastric cancer and its possible mechanisms. METHODS: The expression of integrin β1 at mRNA and protein levels in the SGC-7901 cells and SGC-7901/DDP cells was determined by qPCR and Western blot. The expression of integrin β1 in the SGC-7901/DDP cells was silenced by antisense oligodeoxynucleotide. The cell viability was detected by the CCK-8 assay, the cell apoptosis were analyzed by flow cytometry, and the protein levels of integrin β1, Bcl-2/Bax, cleaved caspase-3/caspase-3, cytochrome C (Cyt-C) and p-AKT/AKT were determined by Western blot.RESULTS: The expression of integrin β1 at both mRNA and protein levels was significantly upregulated in SGC-7901/DDP cells. The expression of integrin β1 was increased in SGC-7901 cells treated with chemotherapeutic agents such as cisplatin, paclitaxel and 5-fluorouracil. Knockdown of integrin β1 induced apoptosis of SGC-7901/DDP cells with an increased sensitivity to the chemotherapeutic agents. Meanwhile, knockdown of integrin β1 downregulated the protein levels of Bcl-2/Bax, p-AKT^Ser473 and p-AKT^Thr308, while promoted the release of Cyt-C and upregulated the protein level of cleaved caspase-3. CONCLUSION: Knockdown of integrin β1 increases the sensitivity of SGC-7901/DDP cells to the chemotherapeutic agents, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of AKT pathway by inhibiting phosphorylations of AKT at Ser473 and Thr308.     

Effects of TRAF3 on inhibiting cyst formation induced by NF-κB signaling in kidney

AIM: To investigate the effects of tumor necrosis factor (TNF) receptor associated factor 3 (TRAF3) on the signaling pathway of NF-κB and the expression of Bax and Bid in renal collecting duct epithelial cells of polycystic kidney disease (PKD), and to observe the tubulogenesis of TRAF3 transgenics for exploring the protective effect of TRAF3 on the cystogenesis and development of PKD. METHODS: The signaling changes of NF-κB and expression of Bax and Bid in PKD renal collecting duct epithelial cells and TRAF3 transgenic PKD renal collecting duct epithelial cells were determined by the Western blot. The percentage of apoptotic cells was measured by flow cytometry with Annexin V staining. The caspase-3 activity was also measured. The 3D Matrigel culture was performed to examine abnormal tubulomorphogenesis in vitro. RESULTS: The over-expression of TRAF3 significantly inhibited the signaling pathway of NF-κB in PKD renal collecting duct epithelial cells and significantly downregulated the expression of Bax and Bid, and also decreased the cell apoptosis. More branch structures were observed in the TRAF3 transgenic PKD renal collecting duct epithelial cells. CONCLUSION: TRAF3 is a negative regulatory inhibitor for Bax and Bid by regulating the NF-κB signaling and may be a new target for inhibiting the cyst formation in PKD.     

Protective effect of β-asarone on primary rat hippcampal neurons against hypoxia/hypoglycemia and reperfusion injury

AIM：To investigate the protective effect of β-asarone against hypoxia/hypoglycemia and reperfusion injury in primary rat hippocampal neurons. METHODS：Cell viability, the activity of caspase-3, the protein expression of p-JNK and Bcl-2, and the mRNA expression of Bcl-2 and caspase-3 were determined by MTT assay, spectrophoto-metry, Western blotting and real-time PCR. RESULTS：Compared with normal control group, the cell viability decreased and the activity of caspase-3 increased obviously, the expression of p-JNK protein and caspase-3 mRNA increased obviously, and the expression of Bcl-2 protein decreased obviously in model group (P<0.05). Compared with model group, different doses of β-asarone inhibited the changes of the above indexes (P<0.05). CONCLUSION：β-asarone inhibits JNK-mediated chondrosome signaling pathway, thereby attenuating the process of hippocampal neuron apoptosis after hypoxia/hypoglycemia and reperfusion.     

Effect of HIPK2 gene on viability,apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation

AIM: To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation (H/R). METHODS: HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by Lipofectamine^TM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca²⁺ fluorescence intensity were analyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS: Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION: Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epithelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.     

Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells via PI3K/Akt signaling pathway

AIM:To investigate the effect of apelin-13 on nicotine-induced apoptosis of cardiomyocytes and its potential molecular mechanism. METHODS:Rat H9c2 cells were treated with nicotine (10 μmol/L) to induced apoptosis. Flow cytometry was used to detect apoptotic rate. Western blot was used to determined the expression of related proteins. RESULTS:Compared with control group, nicotine treatment significantly increased the apoptotic rate of the H9c2 cells (P<0.01), and the protein levels of apoptosis-related proteins Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with nicotine group, apelin-13+nicotine significantly decreased the apoptotic rate of the H9c2 cells (P<0.01) and the the protein levels of Bax and cleaved caspase-3, but markedly increased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with apelin-13+nicotine group, apelin-13+nicotine+PI3K/Akt inhibitor LY294002 significantly increased the apoptotic rate of the H9c2 cells (P<0.01) and the protein levels of Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt and p-PI3K (P<0.05). CONCLUSION:Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells through PI3K/Akt signaling pathway.     

Influence of Bcl-2 inhibitor on Astragalus injection-reduced expression of caspase-3 in rat hippocampal neurons with oxygen-glucose deprivation and reoxygenation

AIM: To observe the influence of Bcl-2 inhibitor on the expression of caspase-3 reduced by Astra-galus injection in rat hippocampal neurons with oxygen-glucose deprivation and reoxygenation (OGD/R). METHODS: The primary rat hippocampal neurons cultured in vitro for 8 d were chosen and randomly divided into 6 groups: normal control group, model group (OGD/R group), Astragalus injection group, Astragalus injection solvent (sterile deionized water)group, Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group. The cells in all groups were tested 24 h after they were treated with reoxygenation after deprived of oxygen and glucose for 30 min except normal control group. The cell type and rate of positive cells were observed by immunohistochemistry. The protein levels of Bcl-2 and cleaved caspase-3 in the hippocampal neurons were measured by Western blotting. The mRNA expression of caspase-3 was detected by RT-PCR. RESULTS: Compared with normal control group, the caspase-3 positive rate of the cells, the protein levels of Bcl-2 and cleaved caspase-3, and the mRNA expression of caspase-3 in model group enhanced significantly (P < 0.05). Compared with model group, the expression of Bcl-2 in Astragalus injection group obviously enhanced, while the caspase-3 positive rate of the cells, the protein level of cleaved caspase-3 and the mRNA expression of caspase-3 in the Astragalus injection group decreased significantly (P < 0.05). No significant difference in injection solvent group, Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group was observed (P > 0.05). The expression of Bcl-2 was decreased sharply in Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group. CONCLUSION: Bcl-2 inhibitor antagonizes the inhibitory effect of Astragalus injection on caspase-3 expression in rat hippocamal neurons with OGD/R, which may be one of the possible target for the inhibitory action of Astragalus injection on the apoptosis of rat hippocampal neurons induced by OGD/R.     

Effect of PARP-1 on cisplatin resistance in breast cancer MCF-7 cells

AIM: To study the effect of poly(ADP-ribose) polymerase-1 (PARP-1) on cisplatin resistance of human breast cancer MCF-7 cells and its possible mechanisms.METHODS: The expression of PARP-1 at mRNA and protein levels in MCF-7 cells and MCF-7/DDP cells was determined by real-time PCR and Western blot. The expression of PARP-1 in the MCF-7/DDP cells was blocked by PARP-1 siRNA. The cell viability and apoptosis were detected by the CCK-8 assay and flow cytometry analysis, respectively. Furthermore, the protein levels of PARP-1, Bcl-2, Bax, cleaved caspase-3, caspase-3, cytochrome C (Cyto-C), extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK) were detected by Western blot.RESULTS: The expression of PARP-1 at both mRNA and protein levels was significantly up-regulated in the MCF-7/DDP cells. The expression of PARP-1 was increased in the MCF-7 cells treated with cisplatin. Knockdown of PARP-1 induced the apoptosis of MCF-7/DDP cells with an increased sensitivity to cisplatin. Meanwhile, knockdown of PARP-1 down-regulated the protein levels of Bcl-2/Bax and p-ERK, but up-regulated the protein levels of cleaved caspase-3 and Cyto-C. After incubated with a specific ERK inhibitor U0126, the cell viability in PARP-1 siRNA group was down-regulated significantly.CONCLUSION: Knockdown of PARP-1 increases the sensitivity of MCF-7/DDP cells to cisplatin, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of ERK signaling pathway by inhibiting phosphorylation of ERK.     

Nisin induces apoptosis of human osteosarcoma MG63 cells via oxidative stress

AIM To investigate the effect of nisin on apoptosis of human osteosarcoma MG63 cells and its related oxidative stress mechanism. METHODS The MG63 cells were cultured in the medium containing different concentrations of nisin with or without antioxidant N-acetyl-L-cysteine （NAC）. The cell viability was measured by CCK-8 assay. Apoptosis was analyzed by flow cytometry with annexin-V/PI staining. The production of intracellular reactive oxygen species （ROS） was detected by redox-sensitive dye 2',7'-dichlorofluorescein diacetate （DCFH-DA）. The 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolyl carbocyanine iodide （JC-1） was used to detect the changes of mitochondrial membrane potential （MMP）. The protein levels of apoptosis-associated molecules Bax, Bcl-2 and cleaved caspase-3 were determined by Western blot. RESULTS Nisin decreased the viability of MG63 cells and promoted the apoptosis in a dose-dependent manner. It also up-regulated the protein level of cleaved caspase-3, increased the protein expression ratio of Bax/Bcl-2, triggered a large amount of intracellular ROS generation and reduced the MMP （P<0.05）. Moreover, antioxidant NAC significantly inhibited nisin-induced apoptosis of MG63 cells, down-regulated the protein level of cleaved caspase-3, decreased Bax/Bcl-2 ratio, reduced intracellular ROS level, and restored the MMP （P<0.05）. CONCLUSION Nisin may promote oxidative stress in human osteosarcoma cells, activate mitochondrial apoptosis pathway, and eventually induce apoptosis.     

Lycopene protects primary mouse cerebrocortical neurons against t-BHP-induced damage in vitro

AIM: To investigate the protective effect of lycopene on primary mouse cerebrocortical neurons exposed to tert-butyl hydroperoxide (t-BHP) and its mechanisms of in vitro.METHODS: Primary cerebrocortical neurons of newborn C57 mice were extracted and divided into normal group, t-BHP group, lycopene+t-BHP group and lycopene group. The neuronal damage was induced by t-BHP exposure for 24 h, and the cell viability was examined by MTT assay. ROS content was measured by flow cytometry, and the protein levels of Bax, Bcl-2, caspase-3, cleaved caspase-3 and cytochrome C were examined by Western blot.RESULTS: The primary mouse cortical neurons expressed MAP-2 protein. Lycopene at concentration of 4 μmol/L reversed the decrease in cell viability. Flow cytometry revealed that lycopene treatment attenuated ROS content under the condition of t-BHP exposure. In addition, the protein level of Bcl-2 was increased, and the expression of Bax, cleaved caspase-3 and cytochrome-C was suppressed in lycopene+t-BHP group.CONCLUSION: The protective effect of lycopene on cortical neurons with t-BHP-induced injury may be involved in the mechanism of neuronal antioxidative response by down-regulating caspase-3 and Bax/Bcl-2 through the mitochondrial apoptotic pathway.     

Emodin reduces hypoglycemia/hypoxia-induced injury of microglia by TLR4/NF-κB signaling pathway

AIM: To investigate the mechanism of emodin on the protection of glucose-deficient/anoxic microglia. METHODS: A microglia BV2 cell model induced by hypoglycemia/hypoxia (HH) was established. The glucose-deficient/anoxic cells treated with emodin were labeled as HH+emodin (20, 40 and 80 μmol/L) groups. The BV2 cells with TLR4 over-expression treated with emodin under hypoglycemia/hypoxia condition was labeled as HH+pcDNA-TLR4+ emodin (40 μmol/L) group. The cell viability was measured by MTT assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were detected by ELISA. The apoptosis was analyzed by flow cytometry. The protein levels of Bax, Bcl-2, TLR4, p-IκB and IκB were determined by Western blot. RESULTS: Compared with HH+DMSO group, the viability was significantly increased, the levels of LDH and TNF-α and apoptotic rate were significantly decreased, the protein levels of Bax, TLR4 and p-IκB were significantly decreased, the protein level of Bcl-2 was significantly increased in HH+emodin groups (P<0.05). Over-expression of TLR4 reversed the effect of emodin on promoting the viability and inhibiting apoptosis in the BV2 cells. CONCLUSION: Emodin has a protective effect on hypoglycemia/hypoxia induced microglia, and its mechanism may be related to the inactivation of TLR4/NF-κB signaling pathway.     

Effect of Ginsenoside Rh2 on apoptosis of human osteosarcoma cell line MG-63

AIM: To investigate the effect of Ginsenoside Rh2(Rh2) on the apoptosis of human osteosarcoma cell line MG-63.METHODS: The cell viability was determined by MTT assay. MG-63 cell apoptotic rate was examined by flow cytometry with Annexin V-PI double staining. The expression of Bcl-2, Bax, cytochrome C(Cyt C) and cleaved caspase-3 were measured by Western blot.RESULTS: Rh2 enhanced the apoptosis of MG-63 cells in a dose-dependent manner. Furthermore, after treatment with Rh2, the release of mitochondrial Cyt C and Bax expression were increased, while Bcl-2 and the ratio of Bcl-2/Bax were decreased as compared with control group(P<0.05). The protein level of cleaved caspase-3 was also increased(P<0.05).CONCLUSION: Ginsenoside Rh2 accelerates the apoptosis of MG-63 cells through mitochondria-dependent pathway, suggesting that Rh2 is a novel approach for the treatment of osteosarcoma.     

Angiotensin-(1-7)/Mas receptor axis protects cardiomyocytes against high glucose-induced injury by modulating nuclear factor-κB pathway

AIM: To study whether the angiotensin-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardiomyocytes against high glucose(HG)-induced injury by inhibiting nuclear factor-κB(NF-κB) pathway. METHODS: The cell viability was measured by CCK-8 assay. The intracellular levels of reactive oxygen species(ROS) were detected by DCFH-DA staining. The number of apoptotic cells was tested by Hoechst 33258 nuclear staining. Mitochondrial membrane potential(MMP) was examined by JC-1 staining. The levels of NF-κB p65 subunit and cleaved caspase-3 protein were determined by Western blotting. RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose(HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated(p) NF-κB p65, peaking at 60 min. Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30 μmol/L and HG for 24 h inhibited HG-induced cytotoxicity, evidenced by an increase in cell viability. On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage, indicated by decreases in the number of apoptotic cells, cleaved caspase-3 level, ROS generation and MMP loss. However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor(Mas receptor). Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG. CONCLUSION: Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway.     

Lutein suppresses t-BHP-induced apoptosis in retinal ganglion cells

AIM: To investigate the protective effects of lutein on retinal ganglion cells in vitro. METHODS: The effect of lutein on tert-butyl hydroperoxide (t-BHP)-treated retinal ganglion cells (RGC-5 cell line) was determined. The protein expression of Brn-3 and MAP-2 was examined by the method of immunocytochemistry to identify the RGC-5 cells. The RGC-5 cells were induced by a 24 h exposure of t-BHP, and the cell viability was examined by MTT assay. The apoptotic ratio of the RGC-5 cells after exposed to t-BHP or/ and lutein treatment was analyzed by flow cytometry assay with Annexin V-FITC /PI staining. The activation of caspase-3 was detected by immunocytochemistry and the protein levels of Bcl-2, Bax, cleaved caspase-3, JNK and c-Jun were determined by Western blot. RESULTS: The RGC-5 cells expressed Brn-3 and MAP-2 proteins. Lutein treatment prevented t-BHP-induced RGC-5 cell apoptosis and increased the cell activity. Compared with control group, exposure of the RGC-5 cells to t-BHP decreased the expression of anti-apoptotic protein Bcl-2, increased the Bax/Bcl-2 ratio, up-regulated the level of cleaved caspase-3, also promoted the phosphorylation of JNK and c-Jun. Lutein partly reversed the effects of t-BHP on the RGC-5 cells mentioned above. CONCLUSION: Lutein protects RGC-5 cells against t-BHP-induced apoptosis by up-regulating Bcl-2 expression and inhibiting caspase-3 activation through modulating the JNK signaling pathway.     

Ethanol extract from Cortex Albiziae attenuates mouse acute liver injury induced by carbon tetrachloride via modulation of NF-κB pathway

AIM:To investigate the protective effect of ethanol extract from Cortex Albiziae on acute liver injury, and to explore its possible mechanism. METHODS:Acute liver injury in mice was induced by single intraperitoneal injection of 25% carbon tetrachloride (olive oil solubilization). The effective parts of ethanol extract from Cortex Albizziae against acute liver injury were screened. The pathological changes of the liver tissues were examined by pathological sections with HE staining. The activity of total superoxide dismutase (T-SOD) and the content of malondialdehyde (MDA) of the liver tissues were detected, the serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were mea-sured by ELISA, and the protein expression levels of NF-κB p65, Bcl-2 and Bax in the liver cells of the mice in each group were determined by Western blot. RESULTS:Compared with model group, the serum levels of AST and ALT in low-dose n-butanol phase of ethanol extract from Cortex Albiziae (AB-L, 4 mg·kg^-1·d^-1) group and high-dose n-butanol phase of ethanol extract from Cortex Albiziae (AB-H, 8 mg·kg^-1·d^-1) group were significantly decreased. The necrosis extent and degree of the hepatocytes and infiltration of inflammatory cells were significantly lower than that in model group. Compared with model group, the serum levels of TNF-α and IL-6 in AB-H group and AB-L group were significantly decreased (P<0.05). The protein level of NF-κB p65 in the nuclei of mouse liver cells in AB-H group and AB-L group were also decreased significantly (P<0.05). Compared with model group, the protein expression of Bax was decreased, the protein expression of Bcl-2 was increased, and the Bcl-2/Bax ratio was increased in AB-L group and AB-H group. CONCLUSION:The n-butanol phase of ethanol extract from Cortex Albiziae may protect the liver by reducing the activation of NF-κB p65, inhibiting the excessive release of inflammatory cytokines IL-6 and TNF-α, and decreasing hepatocyte apoptosis via regulating Bcl-2 and Bax expression.