Protective effect of Huangqi injection combined with puerarin injection on myocardium of type 2 diabetes mice

AIM:To investigate the effect of Huangqi injection combined with puerarin injection on the myocardium of the mice with type 2 diabetes. METHODS:Diabetic KKAy mice were randomly divided into model group and treatment group (Huangqi injection combined with puerarin injection). The male KKAy mice of the same age were used as control group. All mice were sacrificed at 21, 24 and 28 weeks. Morphological changes of the myocardium were observed by HE staining. Apoptosis of the cardiomyocytes was measured by TUNEL staining. The mRNA levels of glucose-regulated protein 78(GRP78), C/EBP hoinologous protein (CHOP) and p53 up-regulated modulator of apoptosis (PUMA) were detected by real-time PCR, and the protein levels of GRP78, CHOP, PUMA, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, poly(ADP-ribose) polymerase (PARP) and cleaved PARP were determined by Western blot. RESULTS:Cardiomyocyte hypertrophy, partly dissolved sarcoplasm and necrosis were observed in model group, and these lesion were alleviated in treatment group. Obvious increased apoptosis in model group and significantly decreased apoptosis of cardiomyocytes in treatment group was observed (P<0.05). At 21, 24 and 28 weeks, the mRNA and protein levels of GRP78, CHOP and PUMA and the protein levels of cleaved caspase-3, cleaved caspase-9 and cleaved PARP in model group were increased significantly as compared with control group (P<0.01), and these in treated group were decreased compared with model group. CONCLUSION:Huangqi injection combined with puerarin injection has cardioprotective effects on type 2 diabetes mice and its mechanism of the action was implemented via inhibiting the activation of endoplasmic reticulum stress and caspase pathway, thus resulting in suppressed apoptosis.     

Poly (I∶C) promotes iodine excess-induced chronic lymphocytic thyroiditis in NOD mouse

AIM: To investigate the effects of poly (I∶C) as virus mimics on iodine excess-induced chronic lymphocytic thyroiditis in NOD mouse. METHODS: Female, 32 NOD mice were randomly divided into 4 groups: (1) control; (2) high iodine; (3) poly (I∶C); (4) high iodine+poly (I∶C). Nine weeks after administration, mice were sacrificed. The following parameters were determined: body weight, thyroid weight and anatomic form. Thyroid hormone (T4) in serum was measured by radioimmunoassay, the thyroid morphology was observed through HE staining, apoptosis was detected by TUNEL, the mRNA expression levels of TRAIL, TRAIL-sR1, ICAM-1 and CXCL10 were determined by the method of real time RT-PCR. RESULTS: Compared to control group and poly (I∶C) group, the thyroid absolute weight and relative weight in high iodine group were increased (P<0.01), the level of total T4 in serum was decreased (P<0.05), inflammation and apoptosis were obviously observed, the mRNA expressions of TRAIL, TRAIL-sR1, CXCL10 and ICAM-1 were upregulated (P<0.05). Compared to high iodine group, thyroid absolute weight and relative weight in high iodine+poly (I∶C) group were further increased, the level of total T4 in serum was further decreased (P<0.05), the ratio of inflammatory degree Ⅳ increased to 50.0%, the numbers of apoptosis cells were further enhanced, the mRNA expressions of TRAIL, TRAIL-sR1, ICAM-1 and CXCL10 were further upregulated (P<0.05). Otherwise, the tendency of all parameters in poly (I∶C) group was similar to that in control group (P>0.05). CONCLUSION: Poly (I∶C) aggravates chronic lymphocytic thyroiditis induced by excess of iodine associated with increase in infiltration of lymphocytes and induction of apoptosis.     

Effects of diabetes on ischemic/reperfusion injury and cell apoptosis in rats myocardium via alterations in peroxynitrite

AIM: To determine the effects of different-term streptozotocin(STZ)-induced diabetes on ischemia/reperfusion (I/R) injury and cell apoptosis in rats myocardial via alterations in myocardial peroxynitrite.METHODS: The models of I/R injury were induced by occlusion and reperfusion of the left descending coronary artery (LDCA) in rats.I/R-induced infarct size was determined using triphenyltetrazolium chloride (TTC) staining.Quantified caspase-3 expression was used to represent apoptosis by Western blotting analysis.Peroxynitrite formation as indicated by nitrotyrosine level was measured by morphometric analysis.RESULTS: Two weeks after STZ treatment,infarct size (35.00%±3.00%) was smaller in 2 weeks diabetic hearts (2WKD) as compared with time-matched control group (2WKC) (51.00%±3.30%),whereas after 16 weeks of diabetes (16WKD),the infarct size (61.00%±3.00%) was bigger in the diabetic hearts as compared with the 16WKC group (50.00%±2.00%,P<0.05).After I/R,caspase-3 expression was lower in 2WKD+I/R group (A value：481±77) than that in 2WKC+I/R group (A value：1033±46),while caspase-3 was higher in the 16WKD+I/R group (A value：1206±78) than that in 16WKC+I/R group (A value：940±72,P<0.05).Nitrotyrosine was lower in 2WKD hearts (A value：211±13) than that in controls (A value：409±12),but was higher in 16WKD group (A value：506±37) compared with controls (A value：378±46,P<0.05).CONCLUSION: Short- and long-term STZ induced diabetes exerts opposite influences on myocardial I/R injury and cell apoptosis,and these contradictory influences may depend on different alterations in myocardial peroxynitrite.     

Anti-apoptosis effect of liraglutide on islet via regulating microRNA-375

AIM: To observe the anti-apoptosis effect of liraglutide on the islet through microRNA-375 (miR-375) for providing additional pharmacodynamic evidence for its clinical application. METHODS: For in vitro study, C57BL/KsJ-db/m mice aged 8 weeks served as normal control group. A total of 40 male genetically diabetic C57BL/KsJ-db/db mice at the same age were randomly divided into diabetic control group （the db/db mice were injected subcutaneously with equivalent amount of saline) and liraglutide group (the db/db mice were injected subcutaneously with liraglutide at dose of 300 μg·kg^-1·d^-1). After 8 weeks of administration, body weight (BW) was measured and blood was collected for detection of fasting blood glucose (FBG), fasting blood insulin (FINS), triglyceride (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C). Before sacrifice, intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were conducted. The histopathological features in the islet tissue were examined with HE staining. The apoptosis in the islet tissue was detected by TUNEL staining. The protein levels of caspase-3, Bcl-2 and Bax were determined by Western blot. The level of miR-375 in the islet tissue was detected by qPCR. Forin vitro study, the MIN-6 cells were cultured and divided into control group (incubated with equivalent amount of solvent), miR-375 mimic group and miR-375 mimic+ liraglutide group. The cell viability was examined by MTT assay. The protein levels of caspase-3, Bcl-2 and Bax were detected by Western blot. RESULTS: In thein vitro study, compared with control group, the levels of BW, FBG, FINS, TC, TG and LDL-C were decreased significantly in liraglutide group. The islet apoptosis was reduced by the administration of liraglutide. The expression of Bcl-2 was up-regulated significantly, while the protein levels of caspase-3 and Bax were down-regulated significantly in liraglutide group. The level of miR-375 was decreased significantly. In the in vitro study, the cell viability was decreased in miR-375 mimic group and increased in miR-375 mimic+liraglutide group. Moreover, the expression of Bcl-2 was decreased and the protein levels of caspase-3 and Bax were increased with the incubation of miR-375 mimic, while the expression of Bcl-2 was increased and the protein levels of caspase-3 and Bax were decreased with the co-incubation of miR-375 mimic and liraglutide. CONCLUSION: Liraglutide attenuates islet apotosis, and the mechanism may be associated with its effects of reducing the elevated level of miR-375 in islet tissues.     

Effects of NOD8 on H2O2-induced apoptosis in human hepatocytes

AIM: To observe the effects of NOD8 on H2O2-induced apoptosis in human L02 hepatocytes. METHODS: pEGFP-C2 and pEGFP-NOD8 plasmids were transfected into L02 cells by JetPRIME, respectively. The apoptosis of these transfected cells was induced by H2O2. The cells were divided into pEGFP-C2 group, pEGFP-C2+H2O2 group and pEGFP-NOD8+H2O2 group. MTT assay was used to detect the cell viability. NOD8 protein expression was determined by Western blotting. The cell apoptosis was observed by Hoechst 33342 staining and apoptotic rate was evaluated by flow cytometry. The caspase-3 activity was analyzed by a colorimetric method. RESULTS: L02 cells were stimulated by H2O2 at concentrations of 0.2～2 mmol/L for 6 h, and H2O2 at concentration of 1 mmol/L was chosen to induce apoptosis determined by MTT assay. The protein expression of NOD8 significantly increased in the cells transfected with pEGFP-NOD8 plasmid. More cellular nucleus with strong blue fluorescence by Hoechst 33342 staining in pEGFP-C2+ H2O2 group were observed, indicating that apoptosis was increased, while the apoptosis in pEGFP-NOD8+H2O2 group significantly reduced. The apoptotic rate in pEGFP-C2+H2O2 group was obviously increased, whereas that in pEGFP-NOD8+H2O2 group was significantly decreased. The caspase-3 activity in pEGFP-C2+H2O2 group was remarkably increased. By contrast, the activity of caspase-3 was significantly reduced in pEGFP-NOD8+H2O2 group.CONCLUSION: NOD8 inhibits H2O2-induced apoptosis in L02 cells and the mechanism may be related to inhibition of caspase-3 activity.     

Protective effect of pentoxifylline against spinal cord ischemia/reperfusion injury in rabbits

AIM: To investigate the protection of pentoxifylline against spinal cord ischemia/reperfusion injury.METHODS: Rabbits sustained spinal cord ischemia with 45 min cross-clamping of the infrarenal aorta. Groups were as follows: sham operation (n=8); ischemic control (n=20), receiving only vehicle; PTX A (n=20), receiving PTX before clamping and PTX B (n=20), receiving PTX at the onset of reperfusion. Rabbits were evaluated for hind-limb motor function with the Tarlov scoring system at 48 h. Serum was assayed with ELISA for TNF-α and spinal cords were harvest for MPO activity, histopathologic analysis and TUNEL staining. Immunohistochemistry was used for PECAM-1 and caspase-3 detection, and the numbers of necrosic and apoptotic neuron were counted at 12 h, 24 h, 48 h and 72 h of reperfusion. The necrotic and apoptotic neurons were also observed with transmission electron microscope.RESULTS: Improved Tarlov scores were observed in PTX-treated rabbits as compared with ischemic control rabbits at 48 h. The significant reductions of TNF-α in serum, activity of MPO, immunoreactivity of the PECAM-1 and caspase-3 were found in PTX-treated rabbits. The numbers of necrosic and apoptotic neuron were higher in PTX-treated rabbits than that in the ischemic control rabbits (P<0.05). No necrosic and apoptotic neuron were found in the sham operation group. CONCLUSION: PTX induces protection against ischemia/reperfusion injury in the spinal cord, thereby preventing both necrosis and apoptosis.     

Effects of Le Er Mai on apoptosis of hippocampus neuronal cells in anaphase of cerebral ischemic reperfusion injury in rats

AIM: To investigate the effects and mechanism of Le Er Mai (LEM) on the apoptosis of hippocampus neuronal cells in the anaphase of cerebral ischemic reperfusion injury in rats.METHODS: A rat model of middle cerebral artery occlusion reperfusion (MCAO) was produced with the intraluminal filament. During reperfusion for 30 d after 2 h of ischemia, the TUNEL staining methods were used to detect apoptosis of hippocampus neuronal cells, and immunohistochemical technique were employed to examine the protein expression of Fas, Bax, caspase-3 and caspase-9 in the hippocampial. The gene expressions of fas, bax, caspase-3 and caspase-9 in hippocampial were examined by RT-PCR. RESULTS: After 2 h ischemia and 30 d reperfusion, compared with sham-operated group, TUNEL-positive staining cells and expression levels of Fas, Bax as well as caspase-3 and caspase-9 obviously increased, and the mRNA expressions of fas, bax, caspase-3 and caspase-9 in hippocampial markedly up-regulated in model group. Compared with model group, LEM at dose of 2.00 g/kg or 0.87 g/kg, and flunarizinum significantly reduced apoptosis and decreased the protein expressions of Fas, Bax, caspase-3 and caspase-9 in hippocampial, and down-regulated the mRNA expressions of fas, bax, caspase-3 and caspase-9 (P<0.05), those action of LEM in 0.87 g/kg dosage group was lower than those in 2.00 g/kg dosage group.CONCLUSION: LEM obviously lower the injury of hippocampial in the anaphase of cerebral ischemia reperfusion through inhibiting the apoptosis of hippocampus neuronal cells. The mechanism of LEM may be related to regulate the expression of signal transduction pathway correlated gene of apoptosis in neuronal cells.     

Protective effect of paeoniflorin on nerve cells in APP/PS1 mice and its mechanism

AIM: To investigate the therapeutic and preventive effects of paeoniflorin (PF) on APP/PS1 mice, and to explore the possible mechanism. METHODS: Fifteen male 5-month-old APP/PS1 non-dominant mice were chosen as normal control group, 15 male 5-month-old APP/PS1 double transgenic mice were used as model group, and 15 male 5-month-old APP/PS1 double transgenic mice treated with 5 mg/kg PF by intraperitoneal injection were allocated in administation group. The learning and memory ability of the mice in each group was detected by Morris water maze. The apoptosis was assessed by TUNEL fluorescence staining. The protein expression of PI3K, Akt, p-PI3K, p-Akt, caspase-3, caspase-9, Bcl-2 and Bax in cerebral cortex and hippocampus was detected by Western Blot. The protein expression levels and distribution of caspase-3 and caspase-9 were detected by immunohistochemistry. RESULTS: (1) Compared with normal control group, the learning and memory ability declined in APP/PS1 model group. Compared with APP/PS1 model group, PF obviously improve the ability of learning and memory in mice. (2) Compared with normal control group, the apoptosis of nerve cells in APP/PS1 model group significantly increased and distributed in wider areas, while that in PF group was reduced (P<0.05). (3) Compared with APP/PS1 model group, PF could significantly lower pro-apoptotic factors, caspase-3, caspase-9 and Bax (P<0.05), and increase the expression of anti-apoptotic factors, p-PI3K, p-Akt and Bcl-2 (P<0.05). CONCLUSION: PF can up-regulate the expression of Bcl-2 and down-regulate the expression levels of caspase-9, caspase-3 and Bax via the activation of PI3K/Akt pathway, thereby inhibiting the nerve cell apoptosis and protecting the nerve cells, so as to treat neurodegenerative diseases.     

Mechanisms of histone acetylation modification in paternal alcohol consumption-induced heart development abnormalities of offspring

AIM:To investigate the role of histone acetylation modification in paternal alcohol consumption-induced heart development abnormalities of offspring. METHODS:Male C57 mice were exposed to 40% alcohol for 6 weeks at a dose of 10 mL·kg^-1·d^-1. Normal saline was used as negative control and non-treatment was for blank control. Heart samples of the newborn offspring mice were collected. Birth weight was measured. Cardiac ultrastructure was observed by transmission electron microscope (TEM). The apoptosis of cardiomyocytes was measured by TUNEL method. The mRNA levels of caspase-3 and Bcl-2 were detected by qPCR. The protein levels of active caspase-3 and Bcl-2 were determined by Western blot. ChIP-qPCR was employed to analyze the levels of acH3K9 and acH3K27 on the promoters of caspase-3 and Bcl-2. RESULTS:Low birth weight was found in the mice of alcohol exposure group. TEM observation showed broad existence of sarcomere dissolution, enlargement of sarcoplasmic reticulum and disorder of sarcomere arrangement. The results of TUNEL displayed increased cardiac apoptosis in alcohol group compared with the controls. The mRNA levels of caspase-3 and the protein levels of active caspase-3 were increased compared with control group (P<0.05). The protein level of active caspase-3 was decreased compared with the controls (P<0.05). Both mRNA and protein levels of Bcl-2 were decreased compared with the controls (P<0.05). The acH3K9 on the promoter of caspase-3 went up significantly (P<0.05), and acH3K27 on the promoter of Bcl-2 was decreased (P<0.05). CONCLUSION:Paternal alcohol exposure causes heart development abnormalities in offspring mice. Histone acetylation may play an important role in the apoptosis of cardiomyocytes.     

Effects of salinomycin combined with L-asparaginase on proliferation and apoptosis of human acute T-cell leukemia Jurkat cells

AIM:To investigate the effects of salinomycin alone or in combination with L-asparaginase on the growth and apoptosis of human acute T-cell leukemia Jurkat cells, and the possible mechanism. METHODS:The growth of Jurkat cells was tested by Cell Counting Kit-8 in vitro. The levels of cytochrome C, Bcl-2, caspase-3, caspase-8 and caspase-9 were measured by Western blotting. Flow cytometry was used to assay cell apoptosis. RESULTS:Salinomycin or L-asparaginase alone inhibited the growth of Jurkat cells in a dose-dependent manner. The IC50 value of L-asparaginase was 8.12 IU/L, while that of salinomycin was 0.75 μmol/L. Salinomycin combined with L-asparaginase induced more significant inhibition of cell proliferation (P<0.05). Western blotting showed that the expression of Bcl-2 protein in combination group was significantly reduced, and the expression of caspase-3, caspase-8, caspase-9 and cytochrome C was significantly increased (P<0.05). Flow cytometry showed that the apoptotic rates of Jurkat cells incubated with salinomycin (0.5 μmol/L), L-asparaginase (2.5 IU/L) and both drugs for 48 h were (7.11±0.23)%, (25.43±0.47)% and (39.12±1.97)%, respectively, and significantly higher than that in control group ［(6.67±0.13)%, P<0.05］.CONCLUSION: Salinomycin synergizes with L-asparaginase-induced cytotoxicity in vitro, and the combined treatment with salinomycin and L-asparaginase induces the apoptosis of Jurkat cells.     

Influence of amylin on apoptosis of human pancreatic islet β-cells and its molecular mechanism

AIM: To investigate the molecular mechanism of amylin in inducing apoptosis of human pancreatic islet β-cells. METHODS: Human pancreatic islet cells were isolated and cultured. The cells were treated with amylin or amylin and aminoguanidine (AG group) for 24 h, respectively. Apoptosis of pancreatic islet β-cells was studied by in situ TUNEL method combined with double staining for insulin and ELISA. The levels of insulin, NO2-/NO3- and glutathione (GSH), p53 mRNA and bcl-2 mRNA were also detected. RESULTS: (1) The enrichment factor and the apoptosis rate of pancreatic islet β-cells in amylin group were markedly higher than that in control group and AG group (P<001). (2) The insulin level in amylin group was significantly lower than that in control group and AG group (P<005). (3) The levels of NO2-/NO3- and p53 mRNA in amylin group were significantly higher than that in control group and AG group (P<001), while the levels of GSH and bcl-2 mRNA were markedly decreased as compared with control group and AG group. CONCLUSIONS: Amylin increases apoptosis of human pancreatic islets β cells, resulting in decrease in insulin secretion. This may be due to increased expression of p53 and decreased expression of bcl-2 during the of oxidative stress induced by amylin.     

siRNA targeting TRIM29 gene induces apoptosis of nasopharyngeal carcinoma cells by inhibiting PI3K/AKT signaling pathway

AIM:To investigate the effect of TRIM29 gene expression silencing on the apoptosis and PI3K/AKT signaling pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were divided into blank group, negative control (NC) group (transfected negative control siRNA) and si-TRIM29 group (transfected TRIM29 specific siRNA). The viability of the 5-8F cells transfected with si-TRIM29 for 0~96 h was measured by CCK-8 assay. The apoptotic rate and the protein levels of TRIM29, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, t-AKT and p-AKT in the 5-8F cells transfected with si-TRIM29 for 48 h were determined by flow cytometry and Western blot, respectively. PI3K/AKT signal specific inhibitor LY294002 at 10 μmol/L and si-TRIM29 alone or in combination were treated with the 5-8F cells, and the cells were divided into blank group, LY294002 group and LY294002+si-TRIM29 group. The apoptotic rates in the 3 groups were detected by flow cytometry. RESULTS:The protein expression of TRIM29 in the 5-8F cells transfected with TRIM29 siRNA was significantly lower than that in blank group (P<0.05). Compared with blank group, the cell viability was significantly decreased, the apoptotic rate was significantly increased, the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax were significantly increased, and the protein levels of Bcl-2 and p-AKT were significantly decreased in si-TRIM29 group (P<0.05). The apoptotic rate in LY294002 group was higher than that in blank group, while that in LY294002+si-TRIM29 group was even higher than that in LY294002 group (P<0.05). CONCLUSION:Silencing of TRIM29 gene expression induces apoptosis of nasopharyngeal carcinoma 5-8F cells by inhibiting PI3K/AKT signaling pathway.     

Ultrastructural impairment of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice

AIM: To investigate the ultrastructural changes of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice. METHODS: BALB/c mice were randomly divided into diabetic group and control group. The expression of insulin and platelet-endothelial cell adhesion molecule-1 (CD31) in islet microvessels was detected by immunohistochemical staining. The ultrastructural changes of islet β cells and islet microvessels were observed under transmission electron microscope. RESULTS: Compared with control group, the number of islet β cells, ratio of β cells/α cells, average number of secretory granules in β cells and insulin expression area per islet in diabetic group were significantly decreased (P<0.01). Besides, diabetic group had fewer microvessels with lower expression of CD31 (P<0.01). Mitochondria in islet microvascular endothelial cells and pericytes in diabetic group were swelling. The basement membrane of islet microvessels became thicker in diabetic group (P<0.01). CONCLUSION: Islet microvascular endothelial cells were impaired in type 1 diabetic mice.     

Effects of extract of Ginkgo biloba on cognitive function and apoptosis of hippocampus neuronal cells in type 1 diabetic rats

AIM: To investigate the protective mechanism of extract of Ginkgo biloba (EGB) on apoptosis of hippocampus neuronal cells in type 1 diabetic encephalopathic rats. METHODS: Thirty-six male Sprague-Dauley rats were divided into 3 groups: control group, diabetic group and EGB-treated group. Streptozotocin was injected intraperitoneally to the animals in later two groups to induce diabetes. The rats in EGB-treated group were injected intraperitoneally with EGB, and the same volume of normal saline was injected to the rats in other groups. At the end of the 12th week, the spatial learning and memory abilities of rats in each group were examined by Morris water maze test. Blood glucose and serum insulin concentration were measured. The neuron densities in hippocampus were measured by Image-Pro Plus 6.0 software. The expressions of Bax, Bcl-2, caspase-3 were assayed by Western blotting and immunohistochemistry. RESULTS: Compared to control group, the level of blood glucose (P<0.01), the protein expression of Bax (P<0.01) and caspase-3 (P<0.01) in hippocampus neuronal cells, and the ratio of Bax/Bcl-2 (P<0.01) and the escape latency (P<0.01) in diabetic group, were significantly increased, while the serum insulin concentration (P<0.01), the neuronal density (P<0.05) in CA1,CA2 hippocampal regions and the platform searching score (P<0.01) were significantly deceased. After treated with EGB, the serum insulin concentration (P<0.05), the neuronal density (P<0.05) in CA1,CA2 hippocampal regions and the platform searching score (P<0.01) were significantly increased, while the level of blood glucose (P<0.01), the protein expression of Bax (P<0.05), caspase-3 (P<0.05) in hippocampus neuronal cells, the ratio of Bax/Bcl-2 (P<0.01) and the escape latency (P<0.05) were significantly deceased than those in diabetic group. The protein expression of Bcl-2 in hippocampus neuronal cells did not alter in any experimental rats. CONCLUSION: EGB improves the spatial learning and memory capacity in diabetic rats by decreasing the expression of Bax, Bax/Bcl-2 ratio and down-regulating caspase-3 to reduce neurocyte apoptosis and increase the neuron density in CA1, CA2 hippocampal regions, suggesting that effective regulation of neuron apoptosis associated genes may be one of the mechanisms for EGB to treat diabetic encephalopathy.     

NOD8 inhibits autophagy in human pancreatic cancer cells and effect of apoptosis on autophagy regulatd by NOD8

AIM: To explore whether NOD8 inhibits autophagy in human pancreatic cancer cells and its underlying mechanisms, and to investigate the effect of apoptosis on the autophagy regulated by NOD8. METHODS: The empty plasmid pEGFP-C2 and recombinant plasmid pEGFP-NOD8 were transfected into the Panc-1 cells using JetPRIME reagent.The untransfected cells served as control group. The protein levels of NOD8, autophagy-related proteins beclin-1 and LC3-II, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway-related proteins Akt, p-Akt, mTOR and p-mTOR were determined by Western blot 48 h after transfection. Meanwhile, the number of LC3 spots was quantified by immunofluorescence staining. Furthermore, after a broad caspase inhibitor Z-VAD-FMK was applied to NOD8-over-expressing cells, the protein expression levels of beclin-1 and LC3-II were detected by Western blot and the number of LC3 spots was observed by immunofluorescence staining. RESULTS: The protein level of NOD8 in pEGFP-NOD8 group was significantly higher than that in control group and pEGFP-C2 group (P<0.01). The protein expression of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8 group were significantly decreased as compared with control group and pEGFP-C2 group. Moreover, the protein levels of p-AKT and p-mTOR in pEGFP-NOD8 group were higher than those in control group and pEGFP-C2 group, while no significant difference of mTOR and AKT protein expression was found among these 3 groups. Furthermore, the protein levels of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8+Z-VAD-FMK group were significantly increased compared with pEGFP-NOD8 group. CONCLUSION: NOD8 inhibits autophagy in the Panc-1 cells and its mechanism may be related to the activation of PI3K/Akt/mTOR pathways. Apoptosis enhances the inhibitory effect of NOD8 on autophagy.     

Effect of biological clock gene Timeless silencing on apoptosis and invasion of ovarian cancer SKOV3 cells

AIM:To evaluate the effect of biological clock gene Timeless (TIM) silencing on the apoptosis and invasion ability of human ovarian cancer SKOV3 cells. METHODS:The protein expression of TIM in the ovarian cancer tissues and normal ovarian tissues was detected by immunohistochemistry, and the correlation between the protein expression of TIM in ovarian cancer tissues and the pathological features was analyzed. The ovarian cancer SKOV3 cells were transfected with PBS (blank control group), control siRNA (siRNA control group) or TIM siRNA (TIM siRNA group). The protein expression of TIM, Bcl-2, Bax, MMP-2, MMP-9, caspase-3 and caspase-9 was determined by Western blot. The apoptosis was detected by flow cytometry. The invasion ability was measured by Transwell chamber test. RESULTS:The positive expression rate of TIM in the ovarian cancer tissues (84.0%) was significantly higher than that in the normal ovarian tissues (10.0%; P<0.01). TIM expression was associated with ovarian cancer differentiation, depth of invasion, lymph node metastasis and TNM stage (P<0.05), but was not associated with age and pathological type (P>0.05). The protein expression levels of TIM, MMP-2, MMP-9 and Bcl-2 in TIM siRNA group were significantly decreased as compared with control group and siRNA control group (P<0.01), and the protein expression of Bax, caspase-3 and caspase-9 in TIM siRNA group was significantly increased as compared with blank control group and siRNA control group (P<0.01). No significant difference of the protein expression of TIM, MMP-2, MMP-9, Bcl-2, Bax, caspase-3 and caspase-9 between blank control group and siRNA control group was observed (P>0.05). The apoptotic rate in TIM siRNA group was significantly higher than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). The penetrated cell number in TIM siRNA group was significantly less than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). CONCLUSION:Silencing of TIM gene in ovarian cancer SKOV3 cells by siRNA promotes apoptosis, and inhibits cell invasion.     

Injury pathways of hypoxia/reoxygenation in AC16 cardiomyocyte

AIM:To investigate the effects of hypoxia/reoxygenation (H/R) for different reoxygenation times on cardiomyocyte injury. METHODS:Human cardiomyocyte AC16 was cultured in glucose-free and serum-free DMEM with 1% O₂ for 24 h, 10% fetal bovine serum and low glucose DMEM combined with 21% O₂ were used to establish reoxygenation for 2 h, 6 h and 12 h, respectively. The cell viability was measured by CCK-8 assay. The protein levels of different cell injury pathway related molecules, such as LC3-Ⅱ/-I (autophagy), caspase-1 and gasdermin D (pyroptosis) and caspase-3 and Bax/Bcl2 (apoptosis), were determined by Western blot. RESULTS:Compared with blank control group, the cell viability in each H/R group was continuously decreased with the extension of reoxygenation time (P<0.05). The expression of LC3-Ⅱ/-I was up-regulated in hypoxia group and H/R group compared with blank control group (P<0.05). In addition, the protein levels of cleaved caspase-1 and cleaved gasdermin D were increased in H/R groups for 6 h and 12 h, respectively (P<0.05). Cleaved caspase-3 and Bax/Bcl2 were increased after reoxygenation for 12 h (P<0.05). CONCLUSION:Autophagy in hypoxia-induced AC16 cells is up-regulated, and then decreased by reoxygenation. The cell pyroptosis is activated earlier than the apoptosis during reoxygenation.