Effect of concentrated decoction of Chinese herbal compound Buyanghuanwutang on cAMP-PKA-CREB signaling pathway in hippocampus of rats with vascular dementia

AIM: To evaluate the role of concentrated decoction of Chinese herbal compound Buyanghuanwutang (BYHWT) in cyclic adenosine monophosphate(cAMP)-protein kinase A(PKA)-cAMP response element-binding protein(CREB) signaling pathway in hippocampus of rats with vascular dementia (VD). METHODS: The rats were randomly divided into sham operation group (sham-operated rats treated with normal saline), VD model group (VD rats treated with normal saline), BYHWT treatment group (VD rats treated with BYHWT) and nimodipment treating group (VD rats treated with nimodipine). The rat model of VD was build by the method of four-vessel occlusion. The rats in all 4 groups were administered with the corresponding reagents for successive 30 days. The content of cAMP was measured by radioimmunoassay. The expression of PKA catalytic subunit (PKAc) was observed by Western blotting. The changes of DNA-binding activity of CREB in rat hippocampus were detected by electrophoretic mobility shift assay. RESULTS: The content of cAMP, the expression of PKAc and the DNA-bingding activity of CREB in the hippocampus of VD rats were lower than those in the hippocampus of sham-operated rats (P<0.01). The above indexes in both nimodipine treatment group and BYHWT treatment group were definitely higher than those in VD model group (P<0.01). CONCLUSION: BYHWT increases the content of cAMP, the expression of PKAc and the DNA-binding activity of CREB in VD rat hippocampus, thus strengthening the cAMP-PKA-CREB signaling pathway.     

Erythropoietin attenuates cerebral ischemia-induced cognitive dysfunction through stimulation of hippocampal CREB phosphorylation in C57BL/6 mice

AIM: To investigate the neuroprotective effect of erythropoietin (EPO) on cognitive dysfunction and neuronal injury in hippocampal CA1 region induced by cerebral ischemia in mice. METHODS: Male C57BL/6 green fluorescent protein-transgenic mice were randomly divided into 3 groups: sham operation group (sham), ischemia/reperfusion group (I/R) and EPO-treated group. Transient cerebral global ischemia was induced by bilateral common carotid artery occlusion (2-VO). The step-down test was used to measure the capacity of learning and memory of the animals in each group. Nissl staining was applied to examine the neuronal number in hippocampal CA1 region. The expression of phosphorylated cAMP response element-binding protein (pCREB) was determined by Western blotting. Alterations of dendritic morphology in hippocampal CA1 region were evaluated using laser scanning confocal microscopy and Neurolucida software. RESULTS: Transient cerebral ischemia caused deficits of spatial learning and memory, and delayed neuronal death and loss of dendritic spines in hippocampal CA1 region were also obvious. The EPO treatment significantly improved the cognitive function in cerebral ischemic mice, and the protein expression of pCREB was obviously increased. At the same time, neuronal death and loss of dendritic spines were reduced in hippocampal CA1 region. CONCLUSION: Erythropoietin increases the protein expression of pCREB, and reduces neuronal death and loss of dendritic spines. These processes may be responsible for erythropoietin-mediated neuroprotective effects and the improvement of cognitive function in cerebral ischemic mice.     

PI3K/Akt regulates endoplasmic reticulum stress-mediated GRP78 induction

AIM: To investigate the effect of PI3K/Akt pathway on endoplasmic reticulum (ER) stress-mediated glucose-regulated protein 78 (GRP78) induction in human embryonic kidney 293 cells (HEK293) cells.METHODS: PI3K inhibitor LY294002, dominant negative kinase-dead mutant vector for HA-Akt (K179M) and Akt1 siRNAs were used to block the PI3K/Akt pathway under ER stress. Constitutively active expression vectors for Akt (myr-HA-Akt) were used to up-regulate Akt activity under ER stress. The effects of PI3K/Akt on ER stress-mediated GRP78 induction in HEK293 cells were determined by Western blotting and RT-PCR. RESULTS: GRP78 induction was inhibited by LY294002, Akt1 (K179M) and Akt1 siRNA, but was increased by myr-Akt1 in dithiothreitol-and thapsigargin-treated HEK293 cells. However, both myr-Akt2/3 and Akt2/3 siRNA had no effect on GRP78 induction in HEK293 cells under ER stress. Furthermore, the PI3K/Akt pathway didnt regulated GRP78mRNA induction but increased GRP78 protein stability.CONCLUSION: PI3K/Akt promotes GRP78 accumulation through increasing the stability of GRP78 protein in HEK293 cells under ER stress.     

Role of translationally controlled tumor protein in PRL-3-promoted metastasis of colon cancer cells

AIM: To study the role of translationally controlled tumor protein (TCTP) in the proliferation, migration and invasion of phosphatase of regenerating liver-3(PRL-3)-promoted colon cancer cells.METHODS: The vectors pAcGFP-C3 and pAcGFP-C3-PRL-3 were constructed and transfected into the colon cancer cell line LoVo.LoVo-PRL-3 cells stably expressing PRL-3 and LoVo-control cells were established. The expression levels of PRL-3 and TCTP in both cells were detected by Western blotting and real-time PCR. The specific siRNA sequence for TCTP mRNA and control-siRNA were synthesized and transfected into the LoVo-PRL-3 cells. TCTP expression at mRNA and protein levels in LoVo-PRL-3 was detected by Western blotting and real-time PCR 24 h, 48 h and 72 h after transfection. The proliferation, migration and invasion abilities of LoVo-control cells, LoVo-PRL-3 cells, TCTP-siRNA and control-siRNA cells were detected by CCK-8 assay and the method of Transwell cell culture chambers.RESULTS: The expression of TCTP at mRNA and protein levels in LoVo cells was significantly increased after PRL-3 transfection (P<0.05). TCTP mRNA was significantly inhibited 24 h, 48 h and 72 h after transfection of TCTP-siRNA (P<0.01). TCTP protein was also significantly inhibited 48 h and 72 h after transfection (P<0.01). Compared with LoVo-control cells, the proliferation, migration and invasion abilities of LoVo-PRL-3 cells were significantly enhanced (P<0.05). However, lowering the up-regulated expression of TCTP in LoVo-PRL-3 cells inhibited the proliferation, migration and invasion abilities (P<0.05). CONCLUSION: PRL-3 promotes proliferation, migration and invasion of colon cancer cells by up-regulating the TCTP expression. siRNA targeting TCTP may be an effective method for prevention and treatment of colon cancer cell metastasis.     

Anti-Sonic hedgehog blocking antibody enhances killing effect of PBMCs on cervical carcinoma HeLa cells

AIM: To investigate the effect of anti-Sonic hedgehog(Shh) blocking antibody on the killing effect of peripheral blood mononuclear cells(PBMCs) on cervical carcinoma HeLa cells. METHODS: The expression levels of Shh and Shh signaling molecules in HeLa cells were detected by immunocytochemistry and RT-PCR. PBMCs from health peoples were isolated by the method of Ficoll density gradient centrifugation, and then co-cultured with HeLa cells in vitro. The expression of CD3, CD69 and CD71 was assayed by flew cytometry. The concentrations of IFN-γ, IL-10 and IL-4 in culture supernatants were detected by ELISA. The killing effect of PBMCs on HeLa cells was observed under microscope. RESULTS: Shh and Shh signaling molecules were expressed in HeLa cells. The level of Shh expression didn't change significantly in the 6th passage of HeLa cells. CD3⁺ cells were increased in the co-culture system. The expression of CD69 and CD 71, and the secretion of IFN-γ were increased, while the secretion of IL-10 was decreased in the co-culture system treated with anti-Shh blocking antibody. Anti-Shh blocking antibody has no effect on the secretion of IL-4. The killing effect of PBMCs on HeLa cells was strengthened by anti-Shh blocking antibody. CONCLUSION: Anti-Shh blocking antibody promotes the activation of PBMCs and enhances the killing effect of PBMCs on cervical carcinoma HeLa cells.     

Effect of growth arrest-specific protein 6 on H9c2 cell apoptosis induced by anoxia-reoxygenation via PI3K/Akt survival pathway

AIM: To investigate the effect of growth arrest-specific protein 6(Gas 6) on H9c2 cell apoptosis induced by anoxia-reoxygenation (A/R) and its possible relationship with PI3K/Akt pathway. METHODS: Cultured H9c2 cell line of cardiomyocytes was randomly divided into 4 groups: normal control group, anoxia-reoxygenation group (A/R), anoxia-reoxygenation+Gas6 group (A/R+Gas6) and anoxia/reoxygenation+Gas6+LY294002 group (A/R+Gas6+LY294002). The procedure of A/R was performed in cultured H9c2 cells by 3 h of anoxia and then 3 h of reoxygenation. The viability of the cells and the activity of caspase-3 were detected by automatic biochemistry analytic instrument. Cell apoptotic rates were evaluated by flow cytometry. The protein level of phosphorylated Akt(p-Akt) was determined by Western blotting. RESULTS: Compared with control group, the cell viability was significantly decreased, and caspase-3 activity, cell apoptotic rate and the protein level of p-Akt were increased in A/R group. Compared with A/R group, the caspase-3 activity and cell apoptotic rate reduced markedly, while the cell viability and the protein level of p-Akt were significantly increased in A/R+Gas6 group .The effect of Gas6 was inhibited by LY294002. CONCLUSION: Gas6 may protect the H9c2 cells from anoxia-reoxygenation-induced apoptosis. Its mechanism is possibly involved in the activation of PI3K/Akt survival pathway via increasing the phosphorylation of Akt protein.     

Effect of ROCK1 and ROCK2 silencing by shRNA on hypoxia-induced apoptosis of rat cardiomyocytes

AIM: To investigate the effects of Rho-associated coiled-coil protein kinase-1 (ROCK1) and ROCK2 on apoptosis induced by hypoxia in rat cardiomyocytes. METHODS: Rat cardiomyocytes were cultured primarily and identified using an antibody targeting α-actin of striated muscle. ROCK1-shRNA and ROCK2-shRNA were transiently transfected into the cells by liposome. After 48 h, these cells were subject to hypoxia for 6 h. The cells were divided into 5 groups: blank control group, hypoxia group, hypoxia+negative control shRNA group, hypoxia+ROCK1-shRNA group and hypoxia+ROCK2-shRNA group. The beating frequency and rhythm of the cardiomyocytes were assessed by microscopy. The activity of lactate dehydrogenase (LDH) in the cell culture supernatants was detected by automatic biochemical analyzer. The cell survival rate was analyzed by the method of MTT. The cell apoptotic rate was assessed by flow cytometry. Western blotting was used to determine the expression of ROCK1, ROCK2, caspase-3 and p-PI3K. RESULTS: The primary culture of the cardiomyocytes was successful. Western blotting results showed that the transfection of ROCK1-shRNA or ROCK2-shRNA decreased the expression of ROCK1 or ROCK2 in the cardiomyocytes. Hypoxia slowed down the beat frequency of the cardiomyocytes, also made the rhythm disorder. Hypoxia increased the release of LDH and decreased the cell survival rate. Flow cytometry results showed that hypoxia increased the cell apoptotic rate. Hypoxia increased the expression of caspase-3 and decreased the expression of p-PI3K. Transfection of ROCK1-shRNA and ROCK2-shRNA into the cardiomyocytes reduced all the effects of hypoxia mentioned above. CONCLUSION: Down-regulation of ROCK1 and ROCK2 expression suppresses the apoptosis of rat cardiomyocytes induced by hypoxia. The mechanism is associated with the inhibition of caspase-3 activation and the up-regulation of p-PI3K expression.     

Proliferative effect of PDGF and anti-proliferative activity of AMPK on vascular smooth muscle cells

AIM: To investigate the proliferative effect of platelet-derived growth factor (PDGF) and anti-proliferative activity of AMP-activated protein kinase (AMPK) on vascular smooth muscle cells (VSMCs). METHODS: The proliferation of VSMCs cultured with PDGF and activation of AMPK were observed. VSMCs were divided in 4 groups: control group; PDGF group; 5-aminoimidazole-4 -carboxamide-1-β-D-riboside (AICAR) group and AICAR+PDGF group. The time course of AMPK activation was determined. The protein level of mTOR was also measured. RESULTS: Compared with control group, the proliferative rate in PDGF group was significantly increased. The growth of VSMCs was inhibited in a time-dependent manner and the activity of p-mTOR was significantly decreased in AICAR group. Compared with control group, the expression of p-AMPK in PDGF group was significantly decreased, and that in AICAR group and AICAR+PDGF group was significantly increased. The expression of p-AMPK in AICAR+PDGF group was higher than that in PDGF group. The activity of p-mTOR in PDGF group was significantly higher than that in control group, while that of AICAR group and AICAR+PDGF group was significantly decreased. The expression of p-mTOR in AICAR+PDGF group was lower than that in PDGF group. CONCLUSION: Stimulation of VSMCs with PDGF promotes the cell proliferation, which can be inhibited by AICAR. The proliferation of VSMCs activated by AMPK is probably correlated with the down-regulation of mTOR expression.     

Effects of atorvastatin on expression of PAPP-A induced by TNF-α and IL-1β in rat aortic endothelial cells

AIM: To investigate the effects of atorvastatin on the expression of pregnancy-associated plasma protein A(PAPP-A)induced by TNF-α and IL-1β in endothelial cells. METHODS: The rat aortic endothelial cells were isolated from thoracic aortas and cultured by the tissue explant method. The cells in passage 3-4 were used in the experiment and were randomly divided into 4 groups: blank control group: the cells were treated without any intervention; atorvastatin concentration groups: the cells were incubated with atorvastatin at the concentrations of 0.1, 1 and 10 μmol/L for 24 h; atorvastatin time groups: the cells were incubated with atorvastatin at the concentration of 10 μmol/L for 6 h,12 h and 24 h; atorvastatin+inflammatory factors groups: the cells were pre-incubated with 60 μg/L TNF-α or 20 μg/L IL-1β for 1 h, then different concentrations of atorvastatin (0.1, 1.0, 10 μmol/L) were added for 6 h,12 h and 24 h. MTT reduction assay was used to observe the cell proliferation. The mRNA expression of PAPP-A was detected by RT-PCR. The protein level of PAPP-A in the supernatants of cultured cells was measured by ELISA. RESULTS: Compared with blank control group, no significant change of cell proliferation was observed after the intervention of atorvastatin and TNF-α/IL-1β for 3 h, 6 h, 12 h, 24 h and 48 h, indicating that the drugs had no toxic effects on the cells. No significant difference of PAPP-A expression between atorvastatin groups and blank control groups was found. Compared with TNF-α groups and IL-1β groups, PAPP-A expressions in atorvastatin intervention groups significantly decreased. The protein level of PAPP-A was gradually decreased with the raised concentration of atorvastatin and the prolonged time in a concentration- and time-dependent manner. CONCLUSION: Atorvastatin doesn't influence the PAPP-A expression, but inhibits the expression of PAPP-A activated by inflammatory factors in a concentration- and time-dependent manner in primary cultured rat aortic endothelial cells.     

Inhibitory effect of 5-fluorouracil on ultra-microstructure and insulin secretion of pancreatic β cells

AIM: To investigate the molecular mechanisms of β cell dysfunction induced by 5-fluorouracil (5-FU) in islet β cell line (NIT-1 cells). METHODS: The NIT-1 cells were treated with different concentrations of 5-FU. The content of insulin in the culture medium was determined by radioimmunoassay. Cell apoptosis was observed by flow cytometry with annexin V/PI staining. The ultra-microstructural changes of NIT-1 cells were observed under transmission electron microscope. The expression of pancreatic and duodenal homeobox protein 1(PDX-1) at mRNA and protein levels in NIT-1 cells was examined by RT-PCR and Western blotting, respectively. RESULTS: Exposed to the low glucose concentration (5.6 mmol/L), insulin secretion in NIT-1 cells was not significantly decreased following a 24 h treatment with 5.0 to 40.0 mg/L 5-FU (P>0.05). On the contrary, the high glucose (16.7 mmol/L)-stimulated insulin secretion in NIT-1 cells was inhibited by 5.0 to 40.0 mg/L of 5-FU in a dose-dependent manner after 24 h of incubation (P<0.01). The apoptosis rate of NIT-1 cells was significantly increased as compared to those in the control levels(P<0.05). The structural changes of mitochondria were the main apoptotic changes under transmission electron microscope. Significant down-regulation of PDX-1 expression at mRNA and protein levels was observed in NIT-1 cells treated with 5-FU at the concentration of 10.0 mg/L to 40.0 mg/L(P<0.05).CONCLUSION: 5-FU inhibits the insulin secretion in islet β cell induced by high glucose. A relative deficiency in insulin secretion following 5-FU treatment is related to the changes of β cell ultra-microstructure and the reduction of β cell numbers, by which an increase in apoptosis of pancreatic β cells is induced. Down-regulation of PDX-1 expression may play a pivotal role in increasing the apoptosis of pancreatic β cells induced by 5-FU in high-glucose condition.