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.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Effects of adenovirus-mediated shRNA targeting PTEN on proliferation and apoptosis of activated hepatic stellate cells in vitro

AIM:To investigate the down-regulation of phosphatase and tensin homolog deleted on chromosome 10(PTEN) gene by adenovirus-mediated short hairpin RNA(shRNA) on proliferation and apoptosis of activated hepatic stellate cells(HSCs) in vitro and the related signaling transduction pathways. METHODS:The activated HSCs were cultured in vitro and transfected with recombinant adenovirus expressing shRNA targeting PTEN. The proliferation of HSCs was measured by MTT assay and the apoptosis was assessed by TUNEL and flow cytometry. Western blotting was used to detect the protein levels of PTEN, Bax, Bcl-2, Akt, p-Akt, ERK1/2 and p-ERK1/2 in HSCs, and real-time fluorescent quantitative PCR was applied to detect the mRNA expression of Akt and ERK1. RESULTS:The recombinant adenovirus expressing shRNA targeting PTEN was successfully transfected into activated HSCs in vitro, and significantly promoted the proliferation of HSCs in a time-dependent manner within a certain extent. The apoptotic rate of HSCs was significantly decreased 72 h after transfection(P<0.05). Meanwhile, reduced expression of Bax and elevated expression of Bcl-2 were induced 72 h after transfection(P<0.05). Furthermore, the expression of p-Akt and p-ERK1/2 were increased significantly(P<0.05), while no significant difference in the expression of Akt and ERK1 at mRNA and protein levels was observed(P>0.05). CONCLUSION:Down-regulation of PTEN by adenovirus-mediated shRNA dramatically promotes the proliferation of activated HSCs, and inhibits the apoptosis through Bcl-2/Bax pathway. In addition, the phosphorylation of Akt and ERK1/2 is increased, indicating that PI3K/Akt and ERK1/2 signal transduction pathways may play an important role in the regulation of proliferation and apoptosis of HSCs.     

Role of miR-486-5p in apoptosis of human bone marrow mesenchymal stem cells induced by hydrogen peroxide

AIM: To investigate the role of microRNA-486-5p (miR-486-5p) in the apoptosis of human bone marrow mesenchymal stem cells (hMSCs) induced by hydrogen peroxide (H₂O₂). METHODS: The hMSCs were cultured in vitro and exposed to serum-free medium and H₂O₂ (10 mmol/L). The changes of miR-486-5p expression in oxidative stress-related apoptosis of hMSCs were measured by real-time PCR. The hMSCs were transfected with miR-486-5p mimic or inhibitor at concentration of 30 nmol/L by Lipofectamine RNAiMAX. The effect of miR-486-5p on H₂O₂-induced decrease in cell viability was evaluated by MTT assay. Hoechst 33342 staining and flow cytometry were applied to determine the role of miR-486-5p in the apoptosis of hMSCs. The protein expression was evaluated by Western blotting. Caspase-3 activity was determined using a caspase-3 activity kit. RESULTS: Compared with control group, the expression of miR-486-5p significantly decreased after treated with H₂O₂ (P<0.05). In addition, over-expression of miR-486-5p in the hMSCs reduced the cell viability, accelerated apoptosis, down-regulated Bcl-2/Bax ratio, caspase-3 enzyme precursor content and phosphorylation of Akt, and activated caspase-3 activity. Conversely, down-regulation of miR-486-5p significantly inhibited H₂O₂-induced cell apoptosis and the caspase-3 activity, increased cell viability and up-regulated Bcl-2/Bax ratio and phosphorylation level of Akt. CONCLUSION: Over-expression of miR-486-5p promotes H₂O₂-induced hMSCs apoptosis, and repression of miR-486-5p protects hMSCs from H₂O₂-induced cellular apoptosis, which may be mediated by regulating Akt signaling pathway.     

Effect of sulodexide on apoptosis of human dermal microvascular endothelial cells exposed to hypoxia

AIM: To investigate the effect of sulodexide (SDX) on the apoptosis of human dermal microvascular endothelial cells (HDMECs) exposed to hypoxia and its underlying mechanism. METHODS: The HDMECs were cultured and divided into normoxia control group cultured under normoxic condition; hypoxia control group cultured in a humid incubator maintained at 37℃ with 5% CO₂ and 1% O₂ for 24 h; treatment groups treated with SDX at 0.25, 0.5 and 1 LSU/mL for 24 h under hypoxic condition. The cell viability was measured by CCK-8 assay. The apoptotic rate of the HDMECs was analyzed by flow cytometry. The activity of caspase-3 in HDMECs was examined by caspase-3 activity assay kit. The expression of pro-apoptotic factor P53, caspase-3, Bax and anti-apoptotic factor Bcl-2 at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: SDX increased the viability of HDMECs exposed to hypoxia, but also decreased the apoptosis. Furthermore, SDX down-regulated the expression of pro-apoptotic factor P53, Bax and caspase-3 at mRNA and protein levels as well as the activity of caspase-3, while the expression of anti-apoptotic factor Bcl-2 was up-regulated. CONCLUSION: SDX significantly increases the viability and decreases the apoptosis of HDMECs exposed to hypoxia. Inhibition of the mitochondrial apoptosis pathway may be involved in the underlying mechanism.     

Effects of CARHSP1 gene expression on viability,apoptosis and immune factors of vascular endothe-lial cells induced by hypoxia in atherosclerosis

AIM: To investigate the effect of calcium-regulated heat stable protein 1 (CARHSP1) gene expression on the viability, apoptosis and expression of interleukin-6 (IL-6) and C-reactive protein (CRP) in vascular endothe-lial cells induced by hypoxia.METHODS: The protein expression of CARHSP1 was detected by Western blot in atherosclerotic plaques. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia, and the cells were divided into normal culture group, hypoxia group, hypoxia+CARHSP1-siRNA group and hypoxia+pcDNA3.1-CARHSP1 group. The viability and apoptotic rate of the HUVECs were measured by CCK-8 assay and flow cytometry, respectively. The mRNA expression of IL-6 and CRP was detected by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot.RESULTS: The protein expression of CARHSP1 in atherosclerotic plaques was significantly higher than that in control group (P<0.05). Hypoxia significantly increased the expression of CARHSP1. The cell viability and the protein expression of Bcl-2 were significantly lower in hypoxia group than those in normal culture group (P<0.05). The apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were significantly higher than those in normal culture group (P<0.05). Compared with hypoxia group, the cell viability and protein expression of Bcl-2 were significantly increased in hypoxia+CARHSP1-siRNA group, while the apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were decreased significantly (P<0.05). The cell viability and protein expression of Bcl-2 were decreased significantly in hypoxia+pcDNA3.1-CARHSP1 group, while the apoptotic rate and the protein le-vels of IL-6, CRP, cleaved caspase-3 and Bax were increased significantly (P<0.05).CONCLUSION: The expression of CARHSP1 is increased in atherosclerotic plaques, and inhibition of CARHSP1 expression improves the viability, reduces the apoptosis, and down-regulates the expression of IL-6 and CRP in the HUVECs. Over-expression of CARHSP1 exerts the opposite effect.     

C-reactive protein induces apoptosis in human renal tubular epithelial cells

ATM: To explore whether the C-reactive protein (CRP) level in microinflammation state induces the apoptosis of renal tubular epithelial cells. METHODS: HK-2 cells were stimulated with recombinant human CRP. Annexin-FITC-PI staining and flow cytometry were used to detect the percentage of apoptotic cells. Morphology observation of apoptosis was assessed by Hoechst 33258 staining. Caspase-3 activity was measured by a colorimetric assay. The expression of apoptotic gene bax and anti-apoptotic gene bcl-2 at mRNA levels was determined by real-time PCR. RESULTS: CRP induced apoptosis of HK-2 cells in a time- and dose-dependent manner. The maximal apoptotic effect of CRP concentration was 10 mg/L CRP at concentration of 20 mg/L. CRP treatment was associated with the characteristic morphological features of apoptosis such as condensation, fragmentation or margination of nuclear chromatin. CRP exposure increased caspase-3 activity, up-regulated the mRNA expression of Bax and down-regulated the mRNA expression of Bcl-2. CONCLUSION: Slightly increased CRP level has the potential to induce apoptosis of renal tubular cells.     

Effect of advanced glycosylation end products on function of human adipose tissue-derived stem cells in vitro

AIM：To explore the effect of advanced glycosylation end products (AGEs) on the function of human adipose-derived stem cells (hADSCs) in promoting wound healing. METHODS：hADSCs were isolated by conventional method in vitro and divided into control bovine serum albumin (BSA) group, low-dose AGE-BSA group and high-dose AGE-BSA group. The proliferation and migration of hADSCs with different treatments were determined by WST-8 assay and Transwell assay, respectively. In addition, the expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1(IGF-1) at mRNA and protein levels was determined by real-time PCR and ELISA analysis. RESULTS：Compared with control group, the proliferation and migration abilities were significantly inhibited in the hADSCs of AGE-BSA group. The mRNA expression of VEGF, HGF and IGF-1 in AGE-BSA group was obviously lower than that in control group. The contents of VEGF, HGF and IGF-1 in hADSCs-conditioned me-dium in AGE-BSA group were also obviously lower than those in control group. CONCLUSION：AGEs alter the intrinsic properties of hADSCs and impair their functions in promoting wound healing, thus affecting the therapeutic potential of hADSCs in the treatment of diabetic ulcers.     

Effect of 27nt-miRNA on apoptosis of human umbilical vein endothelial cells induced by oxidized low-density lipoprotein

AIM To investigate the effect of 27nt-miRNA （27nt-miR） on apoptosis of human umbilical vein endothelial cells （HUVECs） induced by oxidized low-density lipoprotein （Ox-LDL） and its underlying mechanism. METHODS HUVECs were cultured in vitro and grouped as below： normal control group， Ox-LDL group， 27nt-miR+Ox-LDL group， anti-27nt-miR+Ox-LDL group and negative control+Ox-LDL group. The cells in Ox-LDL group were treated with Ox-LDL at 40 mg/L for 48 h， while those in normal control group were untreated but cultured normally. The cells in 27nt-miR+Ox-LDL group， anti-27nt-miR+Ox-LDL group and negative control+Ox-LDL group were transfected with their corresponding lentiviral vectors under the same procedure， followed by treatment with Ox-LDL at 40 mg/L for 48 h to induce apoptosis. The cell viability was measured by CCK-8 assay. The migration capacity was detected by scratch assay. The caspase-3 activity was measured by caspase-3 activity assay kit. The apoptotic rate was analyzed by Hoechst 33258 and flow cytometry. The mRNA and protein expression levels of Bcl-2， Bax and caspase-3 were determined by RT-qPCR and Western blot. RESUITS： Compared with negative control+Ox-LDL group， the cell viability and migration ability were significantly decreased by over-expression of 27nt-miR in the HUVECs （P<0.05）， while the activity of caspase-3 and apoptosis induced by Ox-LDL were significantly increased （P<0.05）. Furthermore， the mRNA and protein expression levels of Bax and caspase-3 were significantly up-regulated （P<0.05）， and the mRNA and protein expression level of Bcl-2 was down-regulated in 27nt-miR+Ox-LDL group （P<0.05）. Meanwhile， all the above indexes showed an opposite tendency in anti-27nt-miR+Ox-LDL group. CONCLUSION 27nt-miR promotes Ox-LDL-induced apoptosis and inhibits the viability and migration of HUVECs in vitro， possibly through regulating the expression of apoptotic/anti-apoptotic proteins such as Bax，caspase-3 and Bcl-2.     

Bone marrow-derived mesenchymal stem cells protect against hypoxia-induced injury and apoptosis of PC12 cells via up-regulation of erythropoietin expression

AIM： To investigate the effects of bone marrow-derived mesenchymal stem cells (BM-MSCs) on cobalt chloride (CoCl2)-induced injury and apoptosis of PC12 cells. METHODS：PC12 cells were divided into control group, CoCl2 group, BM-MSCs-siCTL+CoCl2 group and BM-MSCs-siEPO+CoCl2 group. The viability of the PC12 cells was measured by MTT assay. Flow cytometry and Hoechst 33258 staining were used to determine the apoptotic rate and the changes of chromatin distribution in PC12 cells. The expression of erythropoietin (EPO) in BM-MSCs was measured by RT-PCR and Western blotting. The mRNA expression of Bcl-2 and Bax in PC12 cells was detected by RT-PCR. Caspase-9 and caspase-3 assay kits were used to detect the activity of caspase-9 and caspase-3. RESULTS：The viability of PC12 cells treated with CoCl2 for 24 h and 48 h decreased to （43.0±6.4）% and （33.8±5.7）%, respectively, while 1∶15 ratio of BM-MSCs co-culture increased the cell viability to （77.9±3.8）% and （75.2±9.7）%,respectively. The expression of EPO in BM-MSCs was up-regulated after treated with 0.6 mmol/L CoCl2 for 24 h and 48 h, while EPO siRNA significantly abrogated the EPO expression in BM-MSCs. BM-MSCs-siCTL co-culture significantly inhibited the apoptosis of PC12 cells induced by CoCl2. However, EPO siRNA the protective effect of BM-MSCs. Compared with CoCl2 treatment group, BM-MSCs co-culture induced remarkable increase in the expression of Bcl-2 and decrease in the expression of Bax in PC12 cells, which was reversed by EPO siRNA. BM-MSCs-siCTL co-culture remarkably abrogated the CoCl2 induced up-regulation of caspase-9 and -3, while BM-MSCs-siEPO co-culture significantly reversed the down-regulation of caspase-9 and -3 induced by BM-MSCs-siCTL co-culture. CONCLUSION：BM-MSCs protect PC12 cells from apoptosis induced by CoCl2. The protective effect of BM-MSCs might be executed by up-regulating the expression of EPO.     

Effects of shRNA-mediated hWAPL silencing on proliferation and apoptosis of human cervical cancer CaSki cells

AIM：To investigate the role of human wings apart-like (hWAPL) protein in proliferation and apoptosis of human cervical cancer CaSki cells through hWAPL gene silencing by specific short hairpin RNA (shRNA) duplexes. METHODS：The relative hWAPL mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. Cell proliferation was detected by MTT assay, and the apoptosis was determined by Annexin V-PE and Hoechst 33258 staining. Western blotting was used to analyze the expression of cleaved caspase-3, p21 and p27. The effect of hWAPL gene silencing on the in vivo tumorigenic capacity of CaSki cells was investigated in a tumor-bearing nude mouse model. RESULTS：Real-time fluorescence quantitative PCR and Western blotting showed that hWAPL mRNA and protein expression in CaSki cells was efficiently inhibited by hWAPL shRNA. The shRNA-mediated hWAPL silencing inhibited the proliferation and induced the apoptosis of CaSki cells. Additionally, the expression levels of cleaved caspase-3, p21 and p27 were up-regulated in hWAPL knockdown cells. Knockdown of hWAPL also inhibited the in vivo tumorigenic capacity of CaSki cells. CONCLUSION：hWAPL is involved in the regulation of the proliferation and apoptosis of CaSki cells in vitro and in vivo, and might serve as a therapeutic target in cancer treatment.     

Effects of quercetin on apoptosis of alveolar polymorphonuclear neutrophils in severe acute pancreatitis rats with lung injury

AIM：To explore the effects of quercetin (Que) on the apoptosis of alveolar polymorphonuclear neutrophils (PMN) isolated from severe acute pancreatitis (SAP) rats with lung injury. METHODS：Forty-eight SD rats were randomly divided into 4 groups：sham group, SAP group, low-dose (50 mg/kg) Que group and high-dose (100 mg/kg) Que group. SAP was induced by retrograde administration of 5% sodium taurocholate into the biliary pancreatic duct. The SAP rats in Que groups were given quercetin, while the rats in sham group and SAP group received an infusion of physiological saline. Alveolar PMN were harvested by the collection of bronchoalveolar lavage fluid. The cell morphological changes were observed under fluorescent microscope. The cell apoptotic index was analyzed by flow cytometry. The protein levels of Bax and Bcl-2 were examined by Western blotting. Caspase-3 activity was measured by fluorescence spectrophotometry. RESULTS：Cell shrinkage and condensation of chromosomes were observed in alveolar PMN from SAP rats. Compared with sham group, the apoptotic index of alveolar PMN reduced in SAP group. The protein expression of Bax was significantly reduced, that of Bcl-2 was significantly enhanced, and caspase-3 activity was attenuated. After Que pretreatment, the apoptotic index of alveolar PMN increased, the protein expression of Bax was significantly enhanced, that of Bcl-2 was significantly reduced, and caspase-3 activity increased. The effects of Que presented a concentration-dependent manner, indicating that Que alleviated SAP-induced lung injury. CONCLUSION：The apoptosis of alveoar PMN is delayed in SAP rats. Quercetin induces apoptosis of alveolar PMN by up-regulating the expression of Bax and down-regulating the expression of Bcl-2.     

Roles of microRNA-29a in expression of Bcl-2 and Mcl-1 in rat cardiomyocytes

AIM: To investigate the regulatory mechanisms of microRNA-29a (miR-29a) on the expression of Bcl-2 and Mcl-1 in rat cardiomyocytes (CM cells). METHODS: The CM cells were isolated from the hearts of newborn rats and transfected with miR-29a mimic (100 nmol/L) by Lipofectamine RNAiMAX. The expression of Bcl-2 and Mcl-1 at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and Western blotting. The luciferase assay was performed in HEK293T cells and CM cells, which were co-transfected with plasmid DNA and miRNA using Lipofectamine 2000. RESULTS: Transfection of miR-29a mimics significantly reduced the expression levels of Bcl-2 and Mcl-1 in CM cells as compared with the control cells (P<0.05). In addition, HEK293T cells co-transfected with miR-29a mimic and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid significantly reduced the luciferase activity as compared with control group (P<0.05). While CM cells transfected with miR-29a inhibitor and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid in succession, the luciferase activity was increased inversely (P<0.05). CONCLUSION: miR-29a may regulate apoptosis by targeting the bcl-2 and mcl-1 genes.     

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.     

The mechanism of apoptosis induced by homoharringtonine in HL-60 cells

AIM: To study the signal transduction pathway of apoptosis initiation induced by homoharringtonine in HL-60 cells. METHODS: After establishing the model of apoptosis initiation induced by homoharringtonine in HL-60 cells, at the point of apoptosis initiation, molecular caspase-3, Bcl-2, Bax and Fas/FasL were measured with flow cytometry and transmission electron microscope. ERK2 and P38 expression in HL-60 cells were detected by using immunohistochemistry. RESULTS: The model of apoptosis initiation induced by homoharringtonine was established in HL-60 cells. At the point of apoptosis initiation, upregulation of caspase-3 and decrease in Bcl-2/Bax were observed. However, the expression of Fas/FasL did not significantly change. ERK2 expression decreased and P38 expression increased. CONCLUSIONS: Caspase-3, Bcl-2, Bax and mitogen activated protein kinase pathways were involved in signal transduction of apoptosis initiation induced by homoharringtonine in HL-60 cells.     

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 CREB-shRNA on mitochondrial morphology and cell apoptosis in OGD/R-induced cortical neurons

AIM: To construct recombinant lentiviral vector with short hairpin RNA (shRNA) of CREB gene, and to investigate the effect of CREB gene silencing on mitochondrial morphology and cell apoptosis in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cortical neurons. METHODS: Three lentiviral vectors pLentiLox3.7 (PLL) inserted shRNA fragments targeting CREB gene were co-transfected with the packaging plasmids psPAX2 and pMD2.G to the 293T cells, and the virus particles, which was infected with the primary cortical neurons, was encapsulated. The protein expression of CREB was detected by Western blot. The mitochondrial morphology, cell apoptosis and the expression of Bcl-2 and Bax were evaluated by the methods of MitoTracker red, TUNEL and Western blot in OGD/R induced cortical neurons after CREB gene silencing. RESULTS: The pLL-CREB-shRNA1 was the most effective shRNA, which inhibited 80% CREB gene expression in the cortical neurons. The mitochondrial was appeared dot and fragment morphology in OGD/R induced cortical neurons with transfected pLL-CREB-shRNA1 plasmid. In addition, the expression of Bcl-2 was decreased, the expression of Bax, and the apoptosis of the neurons were increased by tranfected with pLL-CREB-shRNA1. CONCLUSION: CREB shRNA recombinant lentiviral vector specifically inhibits the expression of CREB gene. CREB gene silencing promotes the cell apoptosis and mitochondrial morphological changes in the cortical neurons induced by OGD/R.