Astragalus injection inhibits the expression of JNK3 mRNA after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of rats

AIM: To investigate the effect of astragalus injection on the expression of c-jun N terminal kinase (JNK3) mRNA interrelated with apoptosis after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of rats. METHODS: The hippocampal neurons cultured for eight days were divided into 4 groups: normal control group, original astragalus injection group, hypoxia/hypoglycemia and reoxygenation group, astragalus injection group. Hypoxia/ hypoglycemia and reoxygenation group, astragalus injection group and original astragalus injection group were treated with hypoglycemia and reoxygenation after deprived of oxygen and glucose for 30 min. Methods of in situ hybridization and RT-PCR were used respectively to measure the expression of JNK3 mRNA after hypoxia/hypoglycemia and reoxygenation 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. RESULTS: In normal control group the volume of hippocampal neuronal nucleolus was accretion, cellular tuber was distinct and cytokinesis was dyed by yellow a lot. In hypoxia/hypoglycemia and reoxygenation group the hippocampal neuronal nucleolus was crimple, cellular tuber was shrinked, large number of cytokinesis was dyed by yellow and yellow granule was observed. Compared with normal control group, the numbers of JNK3 mRNA positive neuronal cells at each time point increased obviously in the hypoxia/hypoglycemia and reoxygenation group (P<0.05). The change of neuronal configuration and the numbers of JNK3 mRNA positive neuronal cells in original astragalus injection group accorded with hypoxia/ hypoglycemia and reoxygenation group (P>0.05). In astragalus injection group the hippocampal neuronal nucleolus was crimple slightly and segmental cytokinesis was dyed by yellow.Compared to hypoxia/hypoglycemia and reoxygenation group, the numbers of JNK3 mRNA positive neuronal cells at each time point were less obviously in the astragalus injection group besides 120 h (P<0.05). Compared to normal control group, the mean optic density of expression of JNK3 mRNA in hippocampal neurons of rats at each time point increased obviously in hypoxia/ hypoglycemia and reoxygenation group (P<0.05). Compared to hypoxia/hypoglycemia and reoxygenation group, the mean optic density of JNK3 mRNA expression at each time point in original astragalus injection group had no obvious change (P>0.05), however the mean optic density of JNK3 mRNA expression in hippocampal neurons of rats at each time point decreased obviously in the astragalus injection group besides 120 h (P<0.05). CONCLUSION: Astragalus injection inhibits the expression of JNK3 mRNA after hypoxia/hypoglycemia and reoxygenation, accordingly inhibits hippocampal neuronal apoptosis.     

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.     

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.     

Astragalus injection inhibits expression of Apaf-1 in rat hippocampus after cerebral ischemia and reperfusion

AIM: To investigate the effect of Astragalus injection on the expression of apoptotic protease-activating factor 1 (Apaf-1) in the hippocampus of global cerebral ische-mia-reperfusion rats. METHODS: Male SD rats were randomly divided into 4 groups with 30 each: sham operation group, cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group, and cerebral ischemia-reperfusion+vehicle group. The global cerebral ischemia-reperfusion model of the rats was established by 4-vessel occlusion. The rats in cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group and cerebral ischemia-reperfusion+vehicle group were further divided into 7 subsets, according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. After reperfusion, the brains were removed at the corresponding time points. The protein expression of Apaf-1 in hippocampal neurons was detected by immunohistochemistry and Western blotting. The mRNA expression of Apaf-1 was observed by RT-PCR. RESULTS: Compared with sham operation group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h increased obviously in cerebral ischemia-reperfusion group (P<0.05). Compared with cerebral ischemia-reperfusion group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h decreased obviously in cerebral ischemia-reperfusion+Astragalus injection group (P<0.05). However, those in cerebral ischemia-reperfusion+vehicle group had no obvious change (P>0.05). CONCLUSION: Astragalus injection inhibits the expression of Apaf-1 at mRNA and protein levels in hippocampus of global cerebral ischemia-reperfusion rats, thus inhibiting the apoptosis of hippocampal neurons.     

Effects of Astragalus injection on neuronal apoptosis and expression of JNK3 in rat hippocampus after cerebral ischemia reperfusion

AIM:To investigate the effects of Astragalus injection on neuronal apoptosis and expression of c-Jun N-terminal kinase 3(JNK3) in the rat hippocampus after cerebral ischemia reperfusion. METHODS:The rat model of cerebral ischemia reperfusion was set up by a four-vessel occlusion method. The SD rats were randomly divided into 4 groups:sham operation group, cerebral ischemia reperfusion group(model group), cerebral ischemia reperfusion+Astragalus injection group(Astragalus injection group) and cerebral ischemia reperfusion+vehicle group(vehicle group). The rats in model group, Astragalus injection group and vehicle group after transient global cerebral ischemia(30 min) were then divided into 7 subgroups according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. The apoptosis of the neuron in the hippocampus was measured by the method of TUNEL staining. The expression of JNK3 at mRNA and protein levels was determined by real-time PCR and Western blotting,respectively. RESULTS:Compared with sham operation group, the number of apoptotic neurons increased in model group(P<0.05). Compared with model group, the number of apoptotic neurons decreased obviously in Astragalus injection group(P<0.05). Compared with sham operation group, the expression of JNK3 at mRNA and protein levels in the hippocampus increased obviously in model group at all time points except 120 h(P<0.05). Compared with model group, the expression of JNK3 at mRNA and protein levels in the hippocampus decreased obviously in Astragalus injection group at all time points except 120 h(P<0.05). CONCLUSION:Astragalus injection decreases neuronal apoptosis in rat hippocampus after cerebral ischemia reperfusion by inhibiting the expression of JNK3 at mRNA and protein levels.     

Protective role and mechanism of peroxysome proliferator activated receptor-γ in injury of cultured rat cortical neurons induced by hypoxia/ reoxygenation

AIM: To observe the role of peroxysome proliferator activated receptor-γ (PPAR-γ) and the relationship of cyclooxygenase-2 (COX-2) and PPAR-γ in injury of cultured rat cortical neurons induced by hypoxia/reoxygenation. METHODS: Primary rat cortical neurons were cultured. Experiments include control group, hypoxia/ reoxygenation group and hypoxia/ reoxygenation with PPAR-γ agonist group. Cell viability was surveyed by MTT assay. COX-2 protein expression was measured by Western blotting.RESULTS: Neuron viability raised dramatically in hypoxia/reoxygenation with PPAR-γ agonist group, compared with hypoxia/reoxygenation group (P<0.05). The COX-2 protein expression in hypoxia/ reoxygenation with PPAR-γ agonist group decreased significantly compared with hypoxia/ reoxygenation group (P<0.05). CONCLUSION: PPAR-γ agonist inhibits the expression of COX-2 and reduces obviously cortical neuron injury induced by hypoxia/ reoxygenation. It may protect cortical neurons by down-regulating the expression of COX-2.     

JNK pathway promotes apoptosis of rat hippocampal neurons after cerebral ischemia and reperfusion

AIM:To investigate the effect of c-Jun N-terminal kinase(JNK) pathway on the apoptosis of hippocampal neurons after cerebral ischemia-reperfusion(IR) in SD rats. METHODS:Ninety rats were randomly divided into 5 groups:sham group, cerebral IR group,cerebral IR+JNK inhibitor(SP600125) group,cerebral IR+JNK agonist(anisomycin) group and cerebral IR+vehicle group. The brain samples were collected 24 h after reperfusion. The protein level of caspase-3 in hippocampal neurons was measured by immunohistochemical and Western blotting techniques. The mRNA expression of caspase-3 in the hippocampus was determined by real-time fluorescence quantitative PCR. The apoptosis of hippocampal neurons was detected by TUNEL staining. RESULTS:Compared with sham group, the expression of caspase-3 at mRNA and protein levels in cerebral IR group increased obviously(P<0.05). Compared with cerebral IR group, the expression of caspase-3 at mRNA and protein levels in cerebral IR+JNK inhibitor group decreased obviously(P<0.05), and those in cerebral group increased obviously(P<0.05). However, the expression of caspase-3 at mRNA and protein levels in cerebral IR+vehicle group had no obvious change(P>0.05).The apoptosis of hippocampal neurons in each group was consistent with the changes of caspase-3 at mRNA and protein levels. CONCLUSION:Activation of JNK pathway enhances caspase-3 expression in rat hippocampal neurons after cerebral IR,thus promoting the apoptosis of the neurons.     

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.     

Role of cyclooxygenase-2 in injury induced by hypoxia/reoxygenation in cultured rat cortical neurons

AIM: To observe the role of cyclooxygenase-2 (COX-2) in injury induced by hypoxia and reoxygenation in cultured rat cortical neurons and protective effects of COX-2 specific inhibitor NS398.METHODS: Primary rat cortical neuronal cells were cultured. Experiments were divided into control group, hypoxia/reoxygenation group and hypoxia/reoxygenation with COX-2 inhibitor group. Cell viability was measured by MTT assay. COX-2 protein expression was examined by Western blotting. Apoptosis was measured by DNA agarose electrophoresis.RESULTS: The expression levels of COX-2 increased significantly after neurons were treated with hypoxia and reoxygenation, compared with control group and hypoxia/reoxygenation with COX-2 inhibitor group (P＜0.05). COX-2 specific inhibitor NS398 protected neurons from death (P＜0.05 and P＜0.01), DNA fragmentation analysis showed DNA fragmentation was inhibited significantly by NS398.CONCLUSION: COX-2 is involved in the pathogenesis of neuron apoptosis induced by hypoxia/reoxygenation. COX-2 specific inhibitor significantly protects cortical neurons against hypoxia/reoxygenation injury and inhibits apoptosis induced by hypoxia.     

Effects of Tribulus terrestris L. saponin on apoptosis and changes in cytosolic calcium induced by hypoxia/reoxygenation in rat cortical neurons

AIM: To study the effect of Tribulus terrestris L. saponin (TTLS) on apoptosis and changes in cytosolic calcium concentration induced by hypoxia/re-oxygenation in rat cortical neurons. METHODS: Rat cortical neurons in primary culture were used, and a apoptosis model was induced by hypoxia/reoxygenation. LDH releasing rate was detected by spectrophotometry. The apoptosis rate of cortical neurons was analyzed quantitatively by flow cytometry with Annexin V-FITC and PI staining. Intracellular free Ca2+(［Ca2+］i) was observed with a confocal laser-scanning microscope and determined by mean fluorescent value with Fluo-3 fluometry. RESULTS: Compared to control group, three hours of hypoxia and twelve hours of reoxygenation group induced cortical neuronal apoptosis and significantly increased the intracellular free Ca2+ concentration（P<0.01). Compared with model group, TTLS decreased the percentage of neuronal apoptosis and reduced neuronal ［Ca2+］i（P<0.01).CONCLUSION: TTLS could obviously reduce hypoxia/reoxygenation-induced apoptosis and alleviate the damage degree of rat cortical neurons.The mechanism might be related to inhibiting the calcium overload induced by hypoxia/reoxygenation in rat cortical neurons.     

Effect of Ginsenoside Rb1 on apoptosis induced by hypoxia/hypoglycemia and reoxygenation in cultured rat hippocampal neurons

AIM: The aim of this work is to investigate the protective effects of Ginsenoside Rb₁ (Rb₁) on apoptosis induced by hypoxia/hypoglycemia and reoxygenation in cultured rat hippocampal neurons. METHODS: Apoptosis were measured by flow cytometry; Morphological changes and neuronal necrosis were examined under microscope; The leakage of lactic dehydrogenase (LDH) and the product of nitric oxide (NO) were measured. RESULTS: hypoxia/hypoglycemia cultures for 5 h and reoxygenation induced neuronal apoptosis and necrosis,and significantly increased the leakage of LDH and the product of NO. The effects were enhanced with the extending time of reoxygenation. Rb₁ could significantly decrease the percentage of neuronal apoptosis and necrosis, and reduce the leakage of LDH and the product of NO. CONCLUSION: Rb₁ had an effect of anti-neuronal apoptosis. This effect might be related to the inhibition of the activity of NO synthase and NO production.     

Preliminary mechanism of senegenin against H/R-induced apoptosis of primary cortical neurons

AIM：To explore the preliminary mechanism of senegenin (Sen) on inhibiting hypoxia/reoxygenation(H/R)-induced apoptosis of primary cortical neurons. METHODS：The cultured cortical neurons were randomly divided into normal group (control group), model group (H/R group), Sen+H/R group and Sen group. Flow cytometry was used to evaluate the effect of Sen on H/R-induced cell apoptosis. The protein levels of JNK, p-JNK, c-Jun, p-c-Jun, Bcl-2 and Bax were assessed by Western blotting. RESULTS：The apoptotic rate in H/R group was obviously higher than that in control group (P<0.05), while the apoptotic rate in Sen+H/R group was obviously lower than that in H/R group (P<0.05), suggesting that the model of apoptosis was established successfully. The results of Western blotting showed that Sen increased the expression of JNK and c-Jun, inhibited the phosphorylation of JNK and c-Jun (P<0.05), increased the protein level of Bcl-2 and inhibited the protein level of Bax in H/R treated primary cortical neurons (P<0.05). CONCLUSION：Sen has a protective effect against H/R-induced neuronal apoptosis by increasing the expression of JNK and c-Jun, inhibiting the phosphorylation of JNK and c-Jun, increasing the protein level of Bcl-2 and decreasing the protein level of Bax.     

Preliminary study on pyroptosis involved in cerebral ischemia-reperfusion injury

AIM:To investigate the changes of pyroptosis in hippocampus and cortex at different time points after cerebral ischemia-reperfusion, and to explore its mechanism from NLRP3-mediated classical pyroptosis pathway, and to analyze the role of pyroptosis in different parts of cerebral injury. METHODS:SD rats were randomly divided into sham operation group (sham group) and model group (MCAO/R group). The rats in model group was further divided into cerebral ischemia-reperfusion 6 h group (MCAO/R 6 h group), 12 h group (MCAO/R 12h group)and 24 h group (MCAO/R 24 h group). The rat model was established on rats by middle cerebral artery occlusion and reperfusion (MCAO/R) induced by modified right-side thread method. Neurologic function score, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and morphological observation were used to evaluate the degree of nervous cell injury. TUNEL and caspase-1 immunofluorescence double staining were used to detect pyroptosis. The protein expression of NLRP3, cleaved caspase-1, pro-caspase-1 and interleukin-1β (IL-1β) was determined by Western blot. RESULTS:Neurological damage occurred at different times after cerebral ischemia-reperfusion. TTC staining showed that the volume of cerebral infarction gradually increased with the prolongation of reperfusion time (P<0.05). The hippocampal CA1 area and cortical area showed typical morphological features such as loose tissue structure, interstitial edema, disordered arrangement of nerve cells, deepening of nucleus staining, nuclear fragmentation and decreased cell number. Immunofluorescence double staining showed that there was a phenomenon of pyroptosis at different time after cerebral ischemia-reperfusion. The pyroptosis of hippocampal CA1 and cortical area was most obvious at 12 h and 24 h after reperfusion (P<0.05). Western blot analysis showed that the expression of NLRP3, cleaved caspase-1, pro-caspase-1 and IL-1β in NLRP3-mediated classic pyroptosis pathway was regulated in different degrees after cerebral ischemia-reperfusion. The protein expression of NLRP3 in hippocampus was significantly increased at 12 h and 24 h after reperfusion (P<0.05), and the protein expression of NLRP3 in cortex was significantly increased at 6 h after reperfusion (P<0.05). The protein expression of pro-caspase-1 in hippocampus was significantly increased at each time points of reperfusion (P<0.05), and the protein expression of pro-caspase-1 in the cortex was significantly increased at 24 h after reperfusion (P<0.05). The protein expression of cleaved caspase-1 in the hippocampus was significantly increased at 12 h after reperfusion (P<0.05), and increased in the cortex at 24 h after reperfusion (P<0.05). The protein expression of IL-1β in the hippocampus was significantly increased at 24 h after reperfusion (P<0.05), and increased in the cortex at 6 h after reperfusion (P<0.05). CONCLUSION:Pyroptosis is involved in neuronal injury after cerebral ischemia-reperfusion. The classic pyroptosis pathway plays an important regulatory role in hippocampus and cortex, especially in hippocampus, suggesting that hippocampus is the main part of secondary nerve impairment induced by pyroptosis and inflammation after cerebral ischemia-reperfusion.     

Effects of several harmful factors on expression of glycine receptor α1 subunit in neonatal rat myocardial cells

AIM: To study the expression of glycine receptor α₁ subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells. METHODS: Neonatal rat myocardial cells were cultured in vitro. The expression of glycine receptor α₁ subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell viability was measured by CCK-8 assay, and the expression of glycine receptor α₁ subunit was determined by Western blotting. RESULTS: The expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells was positively detectable by Western blotting. Compared with control group, no significant difference of the cell viability (P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed. The expression of glycine receptor α₁ subunit was increased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION: Glycine receptor α₁ subunit exists in the neonatal rat myocardial cells. A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptor α₁ subunit, but HG downregulates the expression of glycine receptor α₁ subunit in cultured neonatal rat myocardial cells.     

Effect of Danhong injection on respiratory inhibition induced by hypoxia

AIM：To investigate the effect of Danhong injection on respiratory inhibition induced by hypoxia and its related mechanism. METHODS：The electromyogram of the diaphragm was monitored to observe the respiratory responses of the rats to hypoxia. The expression of acid-sensingion channel 1a (ASIC1a) in the brainstem after hypoxia was detected by the technique of immunohistochemical staining. RESULTS：The respiratory responses of the rats to hypoxia were initiatory excitation and consequent inhibition. In contrast to pre-hypoxia, the respiration of the rats in hypoxia group was inhibited 30 min after hypoxia (P<0.05), but the respiration of the rats in hypoxia plus Danhong group was still excited (P<0.05), indicating that the respiration of the rats in hypoxia plus Danhong group was not yet depressed at the same time. The ASIC1a positive neurons were detected in the nuclei of trapezoid body and solitary tract. Compared with control group, the expression of ASIC1a was obviously enhanced after hypoxia. In contrast to hypoxia group, the expression of ASIC1a in the rats in hypoxia plus Danhong group was remarkably reduced (P<0.05). CONCLUSION：Danhong injection resists the occurrence of respiratory inhibition after hypoxia, and ASIC1a may participate in this process.     

Effect of miR-25 on apoptosis of H9c2 cells induced by hypoxia/reoxygenation

AIM:To investigate the role and mechanism of microRNA-25 (miR-25) in apoptosis of H9c2 cells induced by hypoxia/reoxygenation. METHODS:The H9c2 cells with over-expression of miR-25 were treated with hypo-xia/reoxygenation. Real-time PCR was used to detect the expression of miR-25 and high mobility group box-1 (HMGB1) mRNA. Western blot was performed to examine the protein expression levels of HMGB1, Bcl-2 and cleaved caspase-3. Flow cytometry was used to analyze the proportion of apoptotic cells and the cell cycle. Dual-luciferase reporter assay was used to confirm that HMGB1 was the target gene of miR-25 in the H9c2 cells. Moreover, the H9c2 cells transfected with HMGB1-shRNA were subjected to hypoxia/reoxygenation to verify whether HMGB1 participated in the regulation of apoptosis of H9c2 cells. RESULTS:Over-expression of miR-25 significantly reduced the protein expression levels of HMGB1 and cleaved caspase-3, and increased the expression of Bcl-2 and the entrance into S phase in H9c2 cells induced by hypoxia/reoxygenation (P<0.01). The result of dual-luciferase reporter assay showed that compared with the control group, transfection with HMGB1-3' UTR-psi-CHECK2+miR-25 mimic strongly inhibited the luciferase activity (P<0.05). After the H9c2 cells transfected with HMGB1-shRNA was treated with hypoxia/reoxygenation, the expression of Bcl-2 was up-regulated, the expression of cleaved caspase-3 was down-regulated, and the cells in S phase were increased (P<0.05). CONCLUSION:miR-25 reduces apoptosis of H9c2 cells induced by hypoxia/reoxygenation, and its mechanism may be related with the inhibition of HMGB1 expression via interacting with its 3'-UTR.     

Inhibitory effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease rats via SIRT1/NF-κB signaling pathway

AIM: To investigate the effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease (AD) rats via SIRT1/NF-κB signaling pathway and its mechanism. METHODS: AD rat model was established by intragastric administration of AlCl₃ and intraperitoneal injection of D-galactose. After treated with butylphthalide at 25 mg/kg (low dose), 50 mg/kg (medium dose) and 100 mg/kg (high dose), the effects of butylphthalide on the morphology of hippocampal neurons, apoptosis rate, and the protein levels of Bcl-2, Bax, cleaved caspase-3 and the SIRT1/NF-κB signaling pathway associated proteins were determined by HE staining, flow cytometry and Western blot, respectively. After treated with SIRT1 agonist SRT1720 and inhibitor sirtinol, the role of SIRT1/NF-κB signaling pathway in hippocampal neuronal apoptosis was observed. On the basis of giving 50 mg/kg butylphthalide, sirtinol was administered, and the effect of butylphthalide on neuronal apoptosis regulated by SIRT1/NF-κB signaling pathway was evaluated. RESULTS: The morphology of hippocampal neurons in the AD rats were improved, the apoptosis rate of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited, and the protein levels of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted by butylphthalide significantly (P<0.05). The protein expression of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted, and the apoptosis of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited by SRT1720 remarkably (P<0.05), whereas the effect of sirtinol was contrary to that of SRT1720. After sirtinol treatment, the inhibitory effect of butylphthalide on apoptosis of hippocampal neurons, the protein levels of Bax and cleaved caspase-3, and the promotion of Bcl-2 protein expression in hippocampal neurons were markedly weakened (P<0.05). CONCLUSION: Butylphthalide inhibits the apoptosis of hippocampal neurons in the AD rats by down-regulating the protein expression of Bax and cleaved caspase-3, and up-regulating the protein expression of Bcl-2 through activating SIRT1/NF-κB signaling pathway.     

Fuzi polysaccharide protects neonatal rat cardiomyocytes with hypoxia-reoxygenation by inhibiting endoplasmic reticulum stress

AIM: To investigate whether the protection mechanism of Fuzi polysaccharide (FPS) is related to inhibition of endoplasmic reticulum stress in cultured neonatal rat cardiomyocytes with hypoxia/reoxygenation (H/R). METHODS: Cultured rat myocardial cells were divided into control group, H/R group (hypoxia for 3 h and reoxygenation for 6 h) and different concentrations of FPS (0.1 g/L, 1 g/L, 10 g/L or 20 g/L) +H/R groups. The cell survival was detected by MTT assay and cell apoptosis of cardiomyocytes was measured by flow cytometry using Annexin V-FITC staining. The expression of glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-12 were determined by Western blotting. The mRNA expression of CHOP and caspase-12 was detected by quantitative PCR. RESULTS: After reoxygenation, the expression of GRP78, CHOP and caspase-12 in cardiomyocytes was increased. Compared with H/R group, the expression of GRP78, CHOP and caspase-12 in FPS+H/R groups was significantly inhibited, the survival rate of cardiomyocytes was increased and the apoptosis of cardiomyocytes was inhibited. This protective effect of FPS was in a dose-dependent manner and reached its peak at 10 g/L. CONCLUSION: Fuzi polysaccharide protects cardiomyocytes from H/R injury. The mechanism is related to inhibiting endoplasmic reticulum stress.     

Mild hypothermia protects against injury of rat hippocampal neurons induced by oxygen-glucose deprivation

AIM：To study the protective effect of mild hypothermia (31~32 °C) on rat hippocampal neurons against oxygen-glucose deprivation (OGD)-induced injury and its possible mechanisms. METHODS：An OGD experimental model of rat hippocampal neurons in vitro was established to simulate cerebral ischemic-hypoxic injury. The rat hippocampal neurons were randomly divided into 4 groups：control group, mild hypothermia group, OGD group and mild hypothermia+OGD group. The cell morphology was observed under light and electron microscopes. The neuronal apoptosis was detected by flow cytometry. The activity of caspase-3 in the cytoplasm was measured by colorimetry. RESULTS：The neuronal injury was apparent after OGD, with a great increase in apoptotic rate (P<0.01). Compared with OGD group, the morphology of neuronal injury in mild hypothermia+OGD group was attenuated, and the neuronal apoptotic rate and the activity of caspase-3 in the cytoplasm decreased. The activity of caspase-3 in the cytoplasm increased after OGD, and was positively correlated with the neuronal apoptotic rate (r=0.823, P<0.05). The activity of caspase-3 in the cytoplasm also increased after mild hypothermia and OGD, and was also positively correlated with the neuronal apoptotic rate (r=0.841, P<0.05). CONCLUSION：OGD can increase caspase-3 activity in the neuronal cytoplasm and induce neuronal apoptosis. Restraint on caspase-3 activity in the neuronal cytoplasm may be the mechanism by which mild hypothermia protects against neuronal injury induced by OGD.