Hyperbaric oxygen therapy activates CREB/BDNF signaling pathway to reduce synaptic damage in hippocampal neurons of Alzheimer disease mice

AIM To investigate the effect of hyperbaric oxygen (HBO) on synaptic damage of hippocampal neurons in APP/PS1 transgenic (TG) mice and its possible mechanism. METHODS The 6-month-old male APP/PS1 TG mice were randomly divided into TG group, HBO group and cAMP response element binding protein (CREB) inhibitor H89 group, with 10 mice in each group. Ten male wild-type (WT) C57BL/6 mice of the same age were used as negative control group (WT group). The mice in HBO and H89 groups were treated with HBO for 6 cycles, while the mice in WT group and TG group were not treated. The learning and memory abilities were observed by Morris water maze. The nesting ability of the mice was detected by nesting test. The Nissl bodies in hippocampal neurons were observed by Nissl staining. The mRNA expression of CREB and brain-derived neurotrophic factor (BDNF) in hippocampus was detected by real-time PCR. The protein levels of synapsin (SYN), postsynaptic density protein 95 (PSD95), growth-associated protein 43 (GAP43), CREB, phosphorylated CREB (p-CREB) and BDNF in the hippocampus were determined by Western blot. RESULTS Compared with WT group, the learning and memory abilities of the mice in TG group were signilficantly reduced (P<0.05). In addition, the nesting score, the number of Nissl bodies in the hippocampal neurons, the mRNA expression of CREB and BDNF, and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also decreased significantly (P<0.05). Compared with TG group, the learning and memory abilities of the mice in HBO group were improved (P<0.05). Meanwhile, the nesting scores of the mice were significantly increased (P<0.05), the neurons in the hippocampus were arranged neatly, and the number of Nissl bodies, the relative mRNA expression of CREB and BDNF,and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also increased significantly (P<0.05). Compared with HBO group, the mice in H89 group had poor learning and memory abilities, lowered nesting scores and decreased number of Nissl bodies. Futhermore, the relative mRNA expression of CREB and BDNF, and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also decreased significantly (P<0.05). CONCLUSION HBO improves the learning and memory abilities of APP/PS1 TG mice, and its mechanism may be related to activating the CREB/BDNF signaling pathway to reduce synaptic damage of hippocampal neurons in mice.     

Fluorocitrate inhibits regulatory effect of ischemic postconditioning on HIF-1α/iNOS signaling pathway after cerebral ischemia in tree shrews with brain injury

AIM: To investigate the regulatory effect of HIF-1α/iNOS signaling pathway on the neuroprotection of ischemic postconditioning (PC) in tree shrews, and to explore the mechanisms of deteriorated cerebral injury after inhibiting astrocyte (AS) metabolism. METHODS: Thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews. Fluorocitrate (FC) was used to inhibit AS metabolism and the ischemic PC was established at 4 h after ischemia followed by clipped ipsilateral common carotid artery on the ischemia side for 3 times, 5 min/time. A total of 67 male tree shrews were randomly divided into 7 groups:control (n=9), ischemia (4 h and 24 h, n=9 for each group), ischemia with PC (4 h and 24 h, n=9 for each group), and FC pretreatment (4 h and 24 h, n=11 for each group). The cerebral infarction size was detected by TTC staining, and the histological changes of hippocampal neurons were observed under light microscope. The regional cerebral blood flow (rCBF) in ischemic cortex was monitored by laser Doppler brain flowmetry. The protein expression of iNOS in hippocampus was detected both by immunohistochemistry and Western blot. The production of NO detected by spectrophotometer. The level of HIF-1α in hippocampus analyzed by ELISA. RESULTS: The cerebral infarct volume was increased with prolonged duration of ischemia, and the changes of ischemia at 24 h were significant (P<0.05). The cortical rCBF was progressively decreased, and it was decreased at 4 h and 24 h after ischemia (P<0.05). The expression of HIF-1α and iNOS in hippocampus was enhanced, and the production of NO was increased significantly (P<0.05). Ischemic PC restored the cortical rCBF (P<0.05), reduced cerebral infarction volume (P<0.05), down-regulated iNOS expression and reduced NO production in the hippocampus (P<0.05). However, the cortical rCBF in FC pretreatment group was significantly lower than that in ischemic group (P<0.05), the neuronal damage was aggravated, and the infarction volume was increased after pretreatment with FC (P<0.05). CONCLUSION: Ischemic PC may reduce cerebral injury by regulating the expression of HIF-1α and iNOS. Inhibition of AS function may attenuate the protective effect mediated by ischemic PC and aggravate brain injury.     

Xinshuaikang inhibits myocardial autophagy and MAPK/ERK1/2 signaling pathway in rats with chronic heart failure

AIM:To explore the effect of Xinshuaikang on myocardial autophagy in the rats with chronic heart failure and its relationship with the MAPK/ERK1/2 signaling pathway. METHODS:The rats were divided into sham group, model group (rat model of chronic heart failure was established by ligation of anterior descending branch of left coronary artery), low-, middle-, and high-dose Xinshuaikang treatment (TL, TM and TH) groups and captopril group (treated with captopril as positive control), with 12 in each group. Doppler echocardiography was used to evaluate the cardiac function. The morphological changes of the myocardium were observed by HE staining. TUNEL staining was used to detect cardiomyocyte apoptosis. The expression of microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) in the myocardium was detected by immunofluorescence labeling. The protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ, beclin-1 and p62 in the myocardium were determined by Western blot. RESULTS:Compared with sham group, left ventricular end-diastolic dia-meter (LVEDD) and left ventricular end-systolic diameter (LVESD) in model group were increased, while left ventricular posterior wall thickness at end-diastole (LVPWTd), left ventricular posterior wall thickness at end-systole (LVPWTs), left ventricular ejection fraction (LVEF), cardiac output (CO), left ventricular diastolic pressure (LVDP), left ventricular systolic pressure (LVSP) and maximum rate of rise/decrease of left ventricular pressure (+dp/dt_max/-dp/dt_max) were decreased (P<0.05). The myocardial cells were deformed and necrotic, and the myocardial fibers were broken, with inflammatory cell infiltration. The apoptotic rate, the positive rate of LC3-Ⅱ, and the protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ/LC3-I and beclin-1 were increased, and the protein expression of p62 was decreased (P<0.05). Compared with model group, the levels of LVEDD and LVESD were decreased, LVPWTd, LVPWTs, LVEF, CO, LVSP, LVDP, +dp/dt_max and -dp/dt_max were increased in Xinshuaikang groups and captopril group (P<0.05). The morphological changes of myocardial cells were gradually returned to normal, and inflammatory cell infiltration, the apoptotic rate and the positive rate of LC3-Ⅱ were decreased. The protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ/LC3-I and beclin-1 were decreased, and the protein expression of p62 was increased (P<0.05). CONCLUSION:Xinshuaikang inhibits myocardial auto-phagy to play a role of cardiac protection in the rats with chronic heart failure, and its mechanism may be related to inhibition of MAPK/ERK1/2 signaling pathway.     

Effect of glucagon-like peptide 1 on Sesn2/AMPK/mTOR signaling pathway in liver of obese rats induced by high-fat diet

AIM: To explored the effect of glucagon-like peptide 1 receptor agonist liraglutide on Sesn2/AMPK/mTOR signaling pathway in the liver of obese rats.METHODS: Male SD rats were divided into normal chow (NC) group (n=12) and high-fat diet (HF) group (n=33). After 12 weeks, 5 rats of each group were used to assess establishment of obese rat model. The rats in HF group were divided into 4 subgroups, HF group, low dose of liraglutide (LG) group, middle dose of liraglutide (MG) group, and high dose of liraglutide (HG) group, and treated with various doses of liraglutide (0, 50, 100 and 200 μg/kg) via hypodermic injection twice a day for 4 weeks. The body weight and epididymal fat index of the rats at the 16th week were measured. The liver tissue fatty degeneration was observed. The protein levels of Sesn2, AMPK, p-AMPK, mTOR and p-mTOR were determined by Western blot.RESULTS: The body weight of rats in HF group was obviously higher than that in NC group (P<0.01). Compared with NC group, the levels of Sesn2 and p-AMPK/AMPK were significantly decreased in HF group (P<0.01), while the level of p-mTOR/mTOR was not changed. After treatment with liraglutide for 4-week, the body weight of the rats in LG, MG and HG groups was obviously lower than that in HF group (P<0.01), and epididymal fat index of the rats in MG and HG groups was obviously lower than that in HF group (P<0.01). The protein level of Sesn2 in HG group was obviously higher than that in HF group (P<0.01). The level of p-AMPK/AMPK was significantly increased in MG and HG groups (P<0.01). The level of p-mTOR/mTOR was significantly increased decreased in LG, MG and HG groups (P<0.01).CONCLUSION: Glucagon-like peptide 1 receptor agonist liraglutide affects energy metabolism and improves the state of obesity through Sesn2/AMPK/mTOR signaling pathway.     

Roles of PI3K/Akt and JAK2/STAT3 signaling pathways in protection of SO2 against limb ischemia/reperfusion-induced acute lung injury in rats

AIM: To investigate the role of PI3K/Akt and JAK2/STAT3 pathways in the protection of sulfur dioxide (SO₂) against limb ischemia/reperfusion (I/R)-induced acute lung injury (ALI) in rats. METHODS: ALI was induced by limb I/R in the SD rats. Na₂SO₃(0.54 mmol/kg, ip)/NaHSO₃ (0.18 mmol/kg, ip) as SO₂ donor was injected at 20 min before reperfusion. The inhibitors of JAK2/STAT3 and PI3K/Akt pathways, Stattic (3 mg/kg, iv) and LY294002(40 mg/kg, iv), respectively, were injected at 1 h before reperfusion. Peripheral blood and lung tissues were collected for determining the contents of the cytokines, the protein levels of the molecules related to the signaling pathways, apoptosis and histopathologic changes by ELISA, TUNEL and Western blot. RESULTS: Compared with control group, the content of MDA, the activity of MPO, lung coefficient, apoptotic index, cytokine expression, and the protein levels of p-Akt and p-STAT3 in I/R group all increased significantly, and administration of Na₂SO₃/NaHSO₃ attenuated the damage in the lung. Besides, the results of Western blot showed that the rat lung tissues expressed p-STAT3 protein and p-Akt protein. After I/R, the protein levels of p-STAT3 and p-Akt were increased. After using Na₂SO₃/NaHSO₃, p-Akt was increased, but p-STAT3 was decreased (P<0.05). CONCLUSION: Both JAK2/STAT3 and PI3K/Akt pathways are likely involved in the protective effect of SO₂ against limb I/R-induced ALI in rats. The activation of JAK2/STAT3 signaling pathway increases I/R injury. Reversely, the activation of PI3K/Akt signaling pathway reduces I/R injury. Besides, JAK2/STAT3 and PI3K/Akt signaling pathways may have crosstalk during I/R-induced ALI and JAK2/STAT3 pathway may have an impact on the P13K/Akt pathway.     

Sphingosine-1-phosphate receptor 2 attenuates influenza A virus-induced viral pneumonia through PI3K/AKT/eNOS pathway

AIM: To investigate the effects of sphingosine-1-phosphate receptor 2 (S1PR2) on influenza A virus-induced viral pneumonia.METHODS: The animal model of influenza A virus pneumonia was established by infecting wild-type C57BL/6 mice and S1pr2^-/- mice with influenza virus subtype FM1 mouse lung adaptable strain through nose drops. The pathological changes of the lung tissues of wild-type mice (model group), JTE-013 (S1PR2 effective antagonist)-challenged mice and S1pr2^-/- mice were observed, and the protein concentration, total cell number, and interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) levels were determined in the bronchoalveolar lavage fluid (BALF) at 4 d and 6 d after virus infection. The phosphorylation levels of AKT and eNOS in the lung tissues were determined by Western blot. RESULTS: Compared with the wild-type mice of control group, the influenza A virus pneumonia in JTE treatment group and S1pr2^-/- mice were more serious, and the protein concentration, total cell number and inflammatory cytokines in the BALF were remarkably increased. Moreover, the phosphorylation levels of AKT and eNOS, the downstream targets of PI3K, were significantly increased (P<0.01). CONCLUSION: S1PR2 mediates PI3K/AKT/eNOS signaling transduction pathway to regulate NO generation, and inhibit vascular permeability and inflammatory cytokine release, thus attenuating the viral pneumonia induced by influenza A virus.     

Sphingosine kinase 1 enhances invasion and migration of non-small-cell lung cancer cells by ERK1/2 signaling pathway

AIM To explore the effects of sphingosine kinase 1 (SphK1) on the migration and invasion of non-small-cell lung cancer (NSCLC) cells and its mechanism. METHODS Thirty-one tumor specimens, which were surgically resected and routinely histologically confirmed as NSCLC, and matched adjacent lung tissues were selected. Immunohistochemical staining and RT-qPCR were used to detect the expression of SphK1. The pcDNA3.1-SphK1 vector (SphK1 group), empty pcDNA3.1 vector control (NC group), SphK1 siRNA (siSphK1 group) or control siRNA (siNC group) was transfected into human lung adenocarcinoma A549 cells, and the protein levels of SphK1, E-cadherin, fibronectin and p-ERK1/2 were determined by Western blot. The effects of over-expression of SphK1 and inhibition of ERK1/2 on migration and invasion of A549 cells were evaluated by Transwell assays. RESULTS SphK1 was highly expressed in the NSCLC tissues and was associated with tumor stage. SphK1 over-expression significantly promoted the migration and invasion of A549 cells, increased the protein levels of p-ERK1/2 and fibronectin, and decreased the protein expression of E-cadherin (P<0.05), but the opposite result was observed after SphK1 interference. The ERK1/2 inhibitor U0126 significantly inhibited the up-regulation of p-ERK1/2 and fibronectin levels and the down-regulation of E-cadherin expression induced by SphK1 over-expression, and also inhibited the invasion and migration of A549 cells promoted by SphK1 over-expression (P<0.05). CONCLUSION SphK1 may reduce E-cadherin protein levels, increase fibronectin protein levels, and promote the invasion and migration of NSCLC cells through ERK1/2 signaling pathway.     

Naringenin attenuates myocardial injury by regulating AMPK/Nrf2/HO-1 signaling pathways in diabetic mice

AIM: To investigate the effects of naringenin (NAR) on the myocardium as well as its effects on adenosine monophosphate-activated protein kinase (AMPK)/nuclear factor (NF)-E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling pathways in diabetic mice. METHODS: C57BL/6 mice (n=50) were randomly divided into normal group (N group) and model group. The mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin (STZ), then the mice were divided into diabetes group (D group), diabetes+low dose of NAR intervention group (D+L-NAR group), diabetes+middle dose of NAR intervention group (D+M-NAR group) and diabetes+high dose of NAR intervention group (D+H-NAR group). The mice in intervention groups were received NAR at low, middle and high doses respectively by gavage, and the mice in N group and D group were received equal volume of normal saline. After 6 weeks, the mice were sacrificed to observe the effects of NAR at different doses on the body weight and blood glucose. The histopathological changes of the cardiac tissues were observed with HE staining. The myocardial collagen volume fraction (CVF) was calculated by Masson staining. Immumohistochemical staining was used to test the protein levels of interleukin-6 (IL-6) and IL-10, and the TUNEL was used to observe the apoptosis of myocardial tissues. The production of reactive oxygen species (ROS) in the myocardial cells was analyzed by fluorescence probe of DHE, and superoxide dismutase (SOD) activity and malondiodehyde (MDA) content in the myocardial cells were measured by SOD and MDA kits. Western blot was applied to determine the protein levels of p-AMPKα, AMPKα, Nrf2, HO-1, NAD(P)H:quinone oxidoreductase 1 (NQO1) and cleaved caspase-3 in the myocardial tissues. RESULTS: Compared with N group, the blood glucose of the mice in D groups was increased and the body weight was decreased significantly. Compared with D group, the blood glucose of the mice in NAR intervention groups was decreased and the body weight was increased. Compared with N group, the CVF, apoptotic rate and the protein levels of IL-6, cleaved caspase-3 were increased, while the protein levels of IL-10, p-AMPKα, Nrf2, HO-1, NQO1 and SOD activity were decreased, the ROS production rate and MDA content was increased significantly in D group (P < 0.05). Compared with D group, the CVF, apoptotic rate and the protein levels of IL-6, cleaved caspase-3 were relatively decreased, conversely the protein levels of IL-10, p-AMPKα, Nrf2, HO-1, NQO1 were increased in NAR intervention groups(P < 0.05). No significantly difference of the ROS production rate, SOD activity and MDA content between D group and D+L-NAR group was observed. However, the ROS production rate and MDA content was decreased,SOD activity were increased in D+M-NAR group and D+H-NAR group as compared with D group. CONCLUSIONS: NAR attenuates myocardial injury in diabetic mice, and its mechanism may be related to regulation of AMPK/Nrf2/HO-1 signaling pathway, enhancement of the antioxidant reaction, reduction of myocardial fibrosis, apoptosis and inflammation.     

Mechanism of JAK2-STAT3 signaling pathway in ischemic postconditio-ning neuroprotection after cerebral ischemia in tree shrews

AIM: To investigate the regulatory effect of JAK2-STAT3 signaling pathway on the neuroprotection of ischemic postconditioning (IPoC) in tree shrews, and to explore the mechanisms of cerebral injury deterioration after inhibiting the JAK2-STAT3 pathway. METHODS: The model of thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews and the IPoC was established at 4 h after ischemia followed by clipping ipsilateral common carotid artery on the ischemia side for 5 min (3 times). After IPoC and intracerebroventricular injection of AG490 (JAK2 inhibitor), the changes of cerebral infarction area were detected by TTC staining, and the histological and ultrastructural changes of cortical neurons were observed under light and electron microscopes, respectively. The protein levels of t-STAT3 and p-STAT3 in the cortical tissue were determined by Western blot. RESULTS: The neuronal pycnosis, mitochondrial swelling and vanish of the mitochondrial cristae were found in cortical cortex, and the infarction area was (24.78±3.30)% at 24 h after cerebral ischemia. Meanwhile, the phosphorylation level of STAT3 protein in the cortical tissue was significantly increased (P<0.01). The cortical neuronal damage and mitochondrial swelling were decreased after IPoC, the area of cerebral infarction was significantly reduced to (17.67±1.83)% (P<0.01), and the phosphorylation level of STAT3 protein was further increased (P<0.01). However, the neuronal damage was aggravated, the infarction area was expanded to (23.85±2.77)%(P<0.05) after treatment with AG490, and the phosphorylation level of STAT3 protein was also significantly reduced (P<0.05). CONCLUSION: IPoC may reduce cerebral injury by regulating the phosphorylation of STAT3 protein, and inhibition of JAK2-STAT3 signaling pathway may counteract the cerebral protective effect of IPoC and aggravate brain injury.     

Effects of ginsenoside Rg1 of Panax notoginseng on brain tissue injury and Nrf2/HO-1 signal pathway after cerebral ischemia/reperfusion in mice

AIM:To observe the effects of ginsenoside Rg1 of Panax notoginseng on brain tissue injury after mouse cerebral ischemia/reperfusion(I/R), and to explore the mechanisms involving nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signal pathway. METHODS:C57BL/6 mice were randomly divided into sham group, cerebral I/R group, ginsenoside Rg1+cerebral I/R group and edaravone+cerebral I/R group. Ginsenoside Rg1 was successively administered for 3 d. One hour after final administration, bilateral common carotid arteries were ligated to induce brain tissue injury for 20 min, and then reperfusion for 24 h. Edaravone, a drug for anti-oxidative stress injury in the treatment of ischemic cerebro-vascular disease, was used as a positive control. The brain tissues were obtained to determine the neural cellular pathology in hippocampal CA1 region. The mRNA expression of Nrf2 and HO-1 was detected by RT-PCR. The protein levels of Nrf2 in the nucleus and cytoplasm and HO-1 in the whole cells in the brain tissues were measured by Western blotting. RESULTS:After ischemia/reperfusion for 24 h, the pathological injury in the neural cells was obvious, and the cell survival rate decreased. Ginsenoside Rg1 and edaravone attenuated the neural cell injury, and significantly increased the cell survival rate. After ischemia/reperfusion for 24 h, the mRNA expression of Nrf2 and HO-1 significantly increased in the brain tissues. The protein levels of Nrf2 in the nucleus and cytoplasm in the brain tissues were increased, the nuclear translocaition rate and the protein expression of HO-1 also increased. Ginsenoside Rg1 and edaravone both decreased the protein levels of Nrf2 in the cytoplasm of the brain tissues, increased that in the nucleus, and also increased Nrf2 nuclear translocation rate and the protein expression of HO-1. The effect of edaravone was higher than that of ginsenoside Rg1, but they had no significant effect on the mRNA expression of Nrf2 in the brain tissues. CONCLUSION: Ginsenoside Rg1 has the effect of anti-brain tissue injury on cerebral ischemia/reperfusion. The mechanisms may be related to activating the Nrf2/HO-1 signal pathway, promoting Nrf2 synthesis and nuclear translocation, thus promoting the expression of downstream antioxidant protein HO-1.     

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.     

Role of ghrelin in cell protection by up-regulating HSP70 through ERK1/2 signaling pathway in PC12 cells

AIM:To study the role of ghrelin in cell protection by up-regulating heat shock protein 70 (HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway in the PC12 cells. METHODS:Sodium nitoprusside (SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group (incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group (ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group (quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group (ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group (added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS:Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased (P<0.05). Compared with SNP-injured group, ghrelin (100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells (P<0.05), and significantly up-regulated the protein expression of HSP70 (P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin (P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression (P<0.05). CONCLUSION:Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.     

Notch1 promotes renal tubulointerstitial fibrosis in renal tissues of diabetic mice by inhibiting autophagy through Akt/mTOR signaling pathway

AIM: To observe the changes of Notch1 expression and autophagy in the renal tissues of diabetic mice, and to explore the regulatory effect of Notch1 on tubulointerstitial fibrosis by inhibiting autophagy in diabetic nephro-pathy. METHODS: The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice), with 8 rats in each group. After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. The protein expression of Notch1 in the renal tubular epithelial cells was observed by immunohistochemical staining. The protein levels of Notch1, PTEN, p-Akt (Thr308), Akt, p-mTOR (Ser2448), mTOR, LC3, P62, collagen type Ⅰ (Col-Ⅰ) and collagen type Ⅲ (Col-Ⅲ) were determined by Western blot. RESULTS: Compared with the db/m mice, the blood glucose, glycosylated hemoglobin, serum creatinine, triglyceride and total cholesterol were increased in the db/db mice (P<0.01). Renal tubular epithelial cell vacuolar degeneration, renal tubular expansion and interstitial inflammatory cell infiltration in db/db mouse renal tissues with HE staining were observed. The images of Masson staining showed collagenous fiber-like substance deposition in the glomerular capillaries and renal interstitium, and disarrangement of tubular structure in the renal tissues of db/db mice. The protein expression levels of PTEN and LC3-Ⅱ were decreased (P<0.01 or P<0.05), while the protein levels of Notch1, P62, p-mTOR (Ser2448), p-Akt (Thr308), Col-I and Col-III were increased in the db/db mice as compared with the db/m mice (P<0.01). However, no significant change of total mTOR and Akt proteins between the 2 groups was found. CONCLUSION: Notch1 protein expression was increased, PTEN expression was significantly reduced, Akt/mTOR pathway was activated, autophagy was inhibited, and fibrosis was aggravated in the renal tissues of the diabetic mice.     

Calcitonin gene-related peptide triggers proliferation in HaCaT keratinocytes by ERK1/2 signaling pathway

AIM: To investigate the effect of calcitonin gene-related peptide (CGRP) on the proliferative potential of HaCaT keratinocytes and whether CGRPR and ERK1/2 pathway is involved in this progress.METHODS: ［³H］^-TdR test was used to estimate the CGRP-induced proliferative potential of HaCaT keratinocytes and the influence of CGRP8-37 (CGRP receptor 1 antagonist) and PD98059 (ERK1/2 inhibitor) on this effect.Western blotting was used to test the activation of ERK1/2 pathway.RESULTS: Exposure of HaCaT keratinocytes to CGRP induced proliferation through the CGRP receptor and ERK1/2 pathway.CGRP 8-37 and PD98059 inhibited CGRP-induced proliferation of HaCaT keratinocytes.Phosphorylation of ERK1/2 was activated by CGRP in a time-dependent manner,which was inhibited by CGRP 8-37 and PD98059.CONCLUSION: This study indicates that CGRP triggers the proliferation of HaCaT keratinocytes by CGRP receptor and ERK1/2 signaling pathway.     

Shear stress regulates NO production in vascular endothelial cells through Pim1/eNOS signaling pathway

AIM: To determine whether laminar shear stress regulates nitric oxide (NO) production in vascular endothelial cells through Pim1/endothelial nitric oxide synthase (eNOS) signaling pathway. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to laminar shear stress using a parallel-plate flow system. NO production is evaluated by NO assay kit. Pim1 protein expression and eNOS phosphorylation were determined by Western blot. A specific small interfering RNA was used to knock down Pim1 gene expression, and then the changes of above indicators were detected. RESULTS: After 15-min exposure of HUVECs to laminar shear stress (15 dyn/cm²), rapid increases in Pim1 protein expression and NO production were observed (P < 0.05). Shear stress also caused time-dependent stimulation of eNOS phosphorylation (P < 0.05). The shear-induced Pim1 expression and NO production were abrogated in the HUVECs transfected with siPim1 (P < 0.05). Pim1 silencing also prevented shear-induced rise of eNOS-Ser1177 phosphorylation (P < 0.05). CONCLUSION: Pim1 may account for shear-induced NO production in endothelial cells due to phosphorylation activation of eNOS.     

Electroacupuncture improves spatial learning and memory ability of rats with chronic cerebral hypoperfusion and signal regulation of hippocampal IL-6/JAK2/STAT3

AIM:To observe the effect of electroacupuncture (EA) on the inflammatory response and hippocampal JAK2/STAT3 signaling pathway in the rats with chronic cerebral hypoperfusion (CCH), and to explore the mechanism of EA attenuating the spatial learning and memory impairment induced by CCH. METHODS:Adult male Sprague-Dawley rats were randomly divided into sham group, model group and EA group (n=10). Modified permanent bilateral common carotid artery occlusion was used to establish animal model. The rats in EA group were stimulated at "Baihui" and "Dazhui" acupoints by 2/15 Hz frequency (30 min/d for 4 weeks), while the rats in the other 2 groups received balanced treatment. The spatial learning and memory ability and regional cerebral blood flow (rCBF) were detected by the methods of Morris water maze and laser Doppler flowmetry. The concentrations of interleukin (IL)-6 and IL-1β, the mRNA expression of JAK2 and STAT3, and the phosphorylated JAK2 and STAT3 protein levels in the hippocampus were determined by ELISA, RT-PCR and Western blot. The pathological changes of the hippocampus were observed with HE staining. RESULTS:In EA group, the rCBF, the average escape latency at every time point, and the original platform quadrant residence time were better than those in model group (P<0.01 or P<0.05). The level of IL-1β in EA group was significantly lower than that in model group (P<0.05), and the level of IL-6 was significantly increased (P<0.05). The mRNA expression of JAK2 and STAT3, and the protein levels of p-JAK2 and p-STAT3 in EA group were significantly higher than those in model group (P<0.05). The impairment of nerve cells in the hippocampal CA1 region was reduced. CONCLUSION:Electroacupuncture inhibits inflammatory response, and alleviates the hippocampal damage and the cognitive disorder by regulating IL-6/JAK2/STAT3 signaling pathways.     

Cordycepin inhibits proliferation and migration of gallbladder cancer cell SNU-308 by ERK1/2, Ezrin and Akt signaling pathways

AIM: To investigate the effects of cordycepin on the proliferation and migration abilities of gallbladder cancer cell line SNU-308 and its molecular mechanism. METHODS: The viability of SNU-308 cells treated with cordycepin at different concentrations was measured by MTT assay and the colony formation ability was also detected. The effect of cordycepin on apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The protein levels of apoptosis and autophagy markers, and the phosphorylation level of Akt, ERK1/2 and Ezrin were evaluated by Western blot. Immunofluorescence staining was also used to analyze the expression level of LC3 after cordycepin treatment. Wound healing assay and Transwell assay were performed to evaluate the migration ability of the SNU-308 cells after cordycepin treatment. Wound healing assay was also used to evaluate the effects of Akt inhibitor, ERK1/2 inhibitor and Ezrin knockdown on the changes of migration ability. RESULTS: Cordycepin significantly inhibited the viability and the ability of colony formation of gallbladder cancer cells (P<0.05). Induction of apoptosis by cordycepin were revealed by flow cytometry (P<0.05). The protein expression of Bcl-2 was down-regulated, while the protein levels of Bax, cytochrome C (Cyto C), Fas, FasL and cleaved caspase-3 were increased and the autophagy marker beclin 1 and the ratio of LC3-Ⅱ/I were upregulated by Western blot analysis (P<0.05). LC3 accumulation in the cytoplasm after cordycepin treatment was demonstrated by immunofluorescence staining. Cordycepin treatment resulted in the inhibition of cell migration were detected by Transwell assay and wound healing assay (P<0.05). The protein levels of p-Akt, p-ERK1/2 and p-Ezrin were down-regulated after cordycepin treatment (P<0.05). Besides, Ezrin knockdown, Akti-1/2 and GDC-0994 all resulted in the inhibition of migration ability (P<0.05). CONCLUSION: Cordycepin induces apoptosis and autophagy to inhibit gallbladder can-cer cell proliferation and migration by regulating ERK1/2, Ezrin and Akt signaling pathways.     

Glucagon-like peptide-1 protects against cardiac microvascular injury in diabetic rats via Keap1-Nrf2 signaling pathway

AIM: To investigate the protective effects of glucagan-like peptide-1 (GLP-1) on cardiac microvascular injury in diabetic rats and the underlying mechanism. METHODS: Diabetic rats were induced by intraperitoneal injection of streptozocin, and then randomized to 3 months of vehicle or exenatide (a GLP-1 analogue) treatment. Before and after the treatment, body weight, blood glucose and blood pressure were measured. Cardiac microvascular permeability was detected by transmission electron microscopy. Cardiac microvascular endothelial cells (CMECs) were isolated and cultured in normal glucose (5.5 mmol/L), high glucose (25 mmol/L), and high glucose plus GLP-1 (10^-8 mol/L). The production of reactive oxygen species (ROS) was examined by superoxide assay kit and dihydroethidium staining. The protein expression of GLP-1 receptor (GLP-1R), Kelch-like epichlorohydrin-associated protein 1 (Keap1), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was determined by Western blot. RESULTS: Exenatide treatment for 3 months improved the cardiac microvascular permeability in the diabetic rats. The GLP-1R was expressed in CMECs. GLP-1 inhibited high glucose-induced ROS production (P<0.05). Compared with high glucose group, the protein expression of Keap1 was decreased, and Nrf2 and HO-1 were increased significantly (P<0.05). CONCLUSION: GLP-1 inhibits oxidative stress in high glucose-induced CMECs, and improves the cardiac microvascular permeability in diabetic rats. The protective effects of GLP-1 may be related to Keap1-Nrf2 signaling pathway.     

Role of SIRT1/eNOS/NO signaling pathway in protective effect of ginsenoside Rb1 against replicative senescence of endothelial cells

AIM: To explore the effect of ginsenoside Rb1 on replicative senescence of endothelial cells and the role of SIRT1/eNOS/NO signaling pathway in this process. METHODS: The replicative senescence model of primary human umbilical vein endothelial cells (HUVECs) was established. The morphological change of the cells, the proportion of senescence-associated β-galactosidase (SA-β-Gal) positive cells and the plasminogen activator inhibitor 1 (PAI-1) expression were detected to assess the senescence model. The expression of eNOS and PAI-1 at mRNA and protein levels in the aging cells was determined by real-time PCR and Western blot before and after silencing of SIRT1 was performed. The NO concentration in the cell culture supernatant was measured by nitrate reductase assay. RESULTS: HUVECs with cumulative population-doubling level (CPDL) at 16 were chosen as the replicative senescence model in this research. Ginsenoside Rb1 at 80 μmol/L significantly reduced the expression of PAI-1 at mRNA and protein levels. Furthermore, ginsenoside Rb1 increased the expression of SIRT1 and eNOS at mRNA and protein levels, and increased the NO content. SIRT1 silencing inhibited the expression of eNOS at mRNA and protein levels and reduced NO generation, leading to an increase in the expression of PAI-1 at mRNA and protein levels. Upon intervention of ginsenoside Rb1, the eNOS and PAI-1 expression and the level of NO were not reversed. CONCLUSION: Ginsenoside Rb1 modulates SIRT1/eNOS/NO signaling pathway to prevent the replicative senescence of HUVECs.