Effects of human thioredoxin on pneumocyte apoptosis and Bcl-2/Bax expression in rats with lung ischemia/reperfusion injury

AIM: To explore the relationship between apoptosis in the lung tissues and lung ischemia/reperfusion injury, and to observe the effects of human thioredoxin (hTrx) on apoptosis in lung ischemia/reperfusion injury. METHODS: The single lung in situ ischemia/reperfusion animal model was used. Eighty four Wistar rats were randomly divided into control group (control), groups of ischemia for 1 h and reperfusion for different times (IR1h, IR3h, IR5h), and groups of IR+human thioredoxin treatment (IR1h +hTrx, IR3h +hTrx and IR5h +hTrx). Transmission electron microscope (TEM), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immunocytochemistry techniques were used to observe apoptosis, apoptosis signal-regulating kinase 1 (ASK1) and expression of Bcl-2 and Bax in various phases of lung ischemia/reperfusion. RESULTS: Cell apoptosis in lung tissues was significantly high, ASK1, Bcl-2 and Bax protein were up-regulated in lung tissues of lung ischemia/reperfusion injury as compared to control (all P<0.01). Compared to IR group, hTrx suppressed apoptosis as well as expression of ASK1 and Bax protein (P<0.01), Bcl-2 protein and the ratio of Bcl-2/Bax were up-regulated in lung tissues (all P<0.05 or P<0.01). There was a significant correlation between the expression of ASK1, Bax protein and cell apoptosis (r=0.775, r=0.814, respectively; all P<0.01). There was a negative correlation between cell apoptosis and Bcl-2/Bax protein (r=-0.275, P<0.05). CONCLUSION: Initiating cell apoptosis by the activation of Bcl-2/Bax system in lung tissues may contribute to the pathogenesis of lung ischemia/reperfusion injury. The protective effects of hTrx include suppressing the expression of ASK1, down-regulating the ratio of Bcl-2/Bax and blocking apoptosis in lung tissues in lung ischemia/ reperfusion injury.     

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.     

Role of sulfur dioxide in acute lung injury induced by ischemia-reperfusion of hind limbs in rats

AIM：To explore the role of endogenous and exogenous sulfur dioxide(SO2) in acute lung injury(ALI) induced by ischemia-reperfusion(IR) of limbs in rats. METHODS：The rat model of ALI was induced by ischemia and reperfusion of the hind limbs using a tourniquet. The rats(n=96) were randomly divided into 6 groups: sham, IR, sham+SO2, sham+hydroxamate(HDX), IR+SO2 and IR+HDX. The morphological changes of the lung tissues were observed under light microscope. Meanwhile, polymorphonuclear neutrophils(PMN) in alveolar septum, lung coefficient, lung levels of malondialdehyde(MDA) and intercellular adhesion molecule(ICAM)-1, serum tumor necrosis factor(TNF)-α and interleukin(IL)-1, the content of SO2 and the activity of aspartate aminotransferase(AST) in the lung tissues, and 24 h survival rate of the rats were measured. RESULTS：IR of the rat limbs resulted in the damage of the lung tissues, and the increases in PMN in alveolar septum, lung coefficient, the lung levels of MDA and ICAM-1 and the serum levels of TNF-α and IL-1 were also observed with the reductions of SO2 content and AST activity. Pretreatment with SO2 donor Na2SO3/NaHSO3 alleviated the changes of the indicators above. HDX, an inhibitor of SO2-producing enzymes, aggravated the changes above. CONCLUSION：Down-regulation of AST/SO2 pathway is involved in the pathogenesis of limb IR-induced ALI. Administration of exogenous SO2 might attenuate lung injury through anti-inflammation and anti-oxidation.     

PI3K/Akt signaling pathway regulates autophagy induced by acute kidney injury in septic rats

AIM：To investigate the autophagy induced by sepsis and acute kidney injury, and the regulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in this process. METHODS：The rats were subjected to cecal ligation and puncture (CLP) or sham operation. Histopathologic changes of the renal tissues were examined by HE staining. Blood urea nitrogen (BUN) and serum creatinine (SCr) were measured by chemical colorimetry. The protein expression of microtubule-associated protein light chain 3 I/II (LC3 I/II), beclin-1 and p-Akt at different time points after CLP was detected by Western blotting. In vitro, human proximal tubular epithelial cell line HK-2 were treated with LPS to induce autophagy. The protein expression of LC3 I/II and p-Akt in the HK-2 cells after LPS treatment at different time points and different concentrations was detected by Western blotting. These molecules in HK-2 cells and apoptosis of HK-2 cells treated with LPS plus PI3K inhibitor or Akt inhibitor were also detected. RESULTS：Compared with sham group, the severe changes of renal histopathological injuries in CLP groups were observed, the levels of BUN and SCr in CLP groups were significantly increased. LC3 I/II, beclin-1 and phosphorylation of Akt gradually increased after CLP. After treatment with LPS, the expression of p-Akt (308) in the HK-2 cells gradually increased in a dose- and time-dependent fashion. The expression of beclin-1 and p-Akt (472) reached a peak at 8 h or 10 mg/L LPS treatment. Treatment with PI3K or Akt inhibitor down-regulated the expression of LC3 and promoted the apoptosis of HK-2 cells. CONCLUSION：Autophagy in the kidney is induced by sepsis and acute kidney injury. PI3/Akt signaling pathway may be involved in this process.     

Long-term insulin treatment improves postischemic cardiac structure and function via increasing serum BNP levels

AIM：To investigate the influence of long-term insulin treatment on postischemic cardiac structural and functional changes, and to further explore the underlying mechanisms. METHODS：Adult male SD rats were randomly divided into 4 groups (8~10 rats per group): sham group, myocardial infarction (MI) + saline (1 mL·kg-1·d-1, hypodermic injection for 4 weeks) group, MI + insulin (2 U·kg-1·d-1, hypodermic injection for 4 weeks) group and MI + insulin (2 U·kg-1·d-1, hypodermic injection for 4 weeks) + wortmannin ［a phosphatidylinositol 3-kinase (PI3K) inhibitor; 15 μg·kg-1·d-1, intraperitoneal injection 15 min before each insulin treatment］ group. The rats in the latter 3 groups were subject to ligation of the left anterior descending coronary artery, while those in sham group underwent the same surgical procedures without tying the sutures. The cardiac structural and functional changes were observed by echocardiogram, heart catheterization and microscopy with HE and Masson trichrome staining. Blood glucose was determined by Roche blood glucose meter, and the serum levels of insulin and brain natriuretic peptide (BNP) were detected by ELISA. The protein expression and phosphorylation of PI3K, Akt, glycogen synthase kinase 3β (GSK3β) and p38 mitogen-activated protein kinase (p38 MAPK) in myocardial tissues were detected by Western blotting. The mRNA expression of BNP, β-myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) in myocardial tissues was determined by real-time fluorescence quantitative PCR. RESULTS：At the end of the 4th week, MI rats receiving long-term insulin treatment showed decreased ratio of heart length/heart weight, smaller systolic left ventricle cavity, thicker systolic interventricular septum, and increased cardiac ejection fraction, left ventricular development pressure and instantaneous first derivate of left ventricle pressure (P<0.05 vs MI + saline group). Moreover, insulin treatment significantly increased the phosphorylation of PI3K and Akt and the serum level of BNP, and inhibited the phosphorylation of p38 MAPK (P<0.05 vs MI + saline group), but did not change the mRNA expression of BNP in myocardial tissues. The effects of insulin on BNP were not blocked by wortmannin (P>0.05 vs MI + insulin group). CONCLUSION：Insulin improves postischemic cardiac structure and function by increasing serum BNP levels possibly independent of PI3K-Akt signaling pathway.     

Resveratrol inhibits chondrosarcoma via mitochondrial and PI3K/Akt signaling pathways

AIM：To investigate the inhibitory effects of resveratrol on chondrosarcoma and the relation with mitochondrial and PI3K/Akt pathways. METHODS：Chondrosarcoma SW1353 cells were treated with resveratrol at concentrations of 25, 50 and 100 μmol/L for the time intervals of 24 h, 48 h and 72 h. The viability and apoptosis of the SW1353 cells in the presence or absence of resveratrol were analyzed by CCK8 assay and Hoechst 33258 staining, respectively. The protein levels of Bcl-2, Bax, activated caspase-3, Akt and p-Akt were detected by Western blotting. The cell migration ability was determined by wound scratch assay. RESULTS：Exposure of the cells to resveratrol resulted in a decrease in the cell viability in a dose- and time-dependent manner (P<0.05). visible nuclei with apoptotic characteristics in resveratrol group were observed. The protein levels of activated caspase-3 and Bax were increased, and Bcl-2 and p-Akt were decreased compared with control group. The total Akt were not significantly changed. Resveratrol also significantly reduced the migration of tumor cells. CONCLUSION：Resveratrol induces apoptosis of chondrosarcoma, which plays a role of part through mitochondrial and PI3K/Akt signaling pathways.     

Effect of ischemic post-conditioning on Egr-1 and IL-1β expression in ischemia-reperfusion injured lung in rats

AIM: To investigate the protective effects of ischemic post-conditioning on the expression of early growth response factor 1 (Egr-1) and interleukin-1β(IL-1β) in ischemia-reperfusion injured lung in rats. METHODS: The model of lung ischemia-reperfusion injury was established in 24 rats and the rats were randomly allocated to 3 different groups (n=8 in each group): (1) sham group: only sham operation (thoracotomy) and no ischemia for 3 h; (2)ischemia-reperfusion group (I/R group): interruption of pulmonary perfusion and ventilation for 1 h followed by reperfusion for 2 h; (3) ischemic post-conditioning group (IPostC group): ischemic post-conditioning (5 min of reperfusion and 5 min of ischemia for 3 times) between the end of ischemia and the beginning of the reperfusion followed by reperfusion for 1.5 h. The lung tissues (prepared to small pieces of about 20 mg) were collected and homogenized at the end of the experiment. The concentration of myeloperoxidase (MPO) in the homogenate was determined. The wet to dry weight ratio (W/D) of the lung tissues was also measured at the end of reperfusion. The pathological changes of the lung tissues were observed under light microscope after reperfusion. The mRNA expression of Egr-1 and IL-1β in the lung tissues was detected by RT-PCR. RESULTS: Compared with sham group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D were significantly increased in I/R group (P<0.05). The inflammatory responses of the lungs in I/R group were significantly severer than those in sham group. Compared with I/R group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D in IPostC group were significantly decreased (P<0.05). The inflammatory responses of the lungs in IPostC group were also significantly attenuated. CONCLUSION: Ischemic post-conditioning significantly reduces ischemic reperfusion injury of the lung by inhibiting the expression of Egr-1 and IL-1β.     

Effects of panax notoginseng saponins on pneumocyte apoptosis and Fas/FasL expression in rabbits with lung ischemia/reperfusion injury

AIM: AIM: To explore the relationship between apoptosis in the lung tissues and lung ischemia/reperfusion injury, and observe effects of panax notoginseng saponins (PNS) on apoptosis in lung ischemia/reperfusion injury. METHODS: Single lung in situ ischemia/reperfusion animal model was used. Eighty four Japanese white rabbits were randomly divided into control group (control), ischemia/reperfusion 1 h group (IR1h), IR3h, IR5h, Panax Notoginseng Saponins 1 h group (PNS1h), PNS3h and PNS5h. TUNEL, immunocytochemistry and in situ hybridization techniques were used to observe apoptosis and Fas/FasL expression in various phases of lung ischemia/reperfusion. RESULTS: Cell apoptosis in lung tissues were significantly high, Fas/FasL mRNA and its protein were up-regulated in lung tissues of lung ischemia/reperfusion injury compared with control (all of P<0.01). The PNS suppressed apoptosis as well as expression of Fas/FasL mRNA and its protein (P<0.05 or P<0.01, respectively). There was a significant correlation between expression of Fas/FasL protein, Fas/FasL mRNA and cell apoptosis (r=0.540，0.658，0.668，0.686；all P<0.01). CONCLUSIONS: Activation of Fas/FasL system and its initiating cell apoptosis of lung tissues may contribute to the pathogenesis of lung ischemia/reperfusion injury. The protective effects of PNS include suppressing the activation of Fas/FasL system and blocking apoptosis in lung tissues in lung ischemia/reperfusion injury.     

ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway, thus promoting the apoptosis of cardiomyocytes.     

Protective effect of rapid ischemic preconditioning against spinal cord ischemic injury in rabbits

AIM: To evaluate the protective effect of rapid phase of ischemic preconditioning against spinal cord ischemic injury in rabbits. METHODS: Thirty six male New Zealands white rabbits were randomly assigned to 3 groups (12 in each group): ischemia and reperfusion injury group (IR group), ischemic preconditioning + IR group (IPC+IR group) and sham operation group (sham). In IR group, spinal cord ischemia was induced by an infrarenal aorta clamping for 20 min; The rabbits in IPC+IR group underwent a 6 min ischemic preconditioning followed by 30 min of reperfusion before the 20 min clamping; The rabbits in sham group underwent the same procedures as the IR group except for infrarental aortic unclamping. Neurologic status was scored at 8, 12, 24 and 48 h after reperfusion. All animals were sacrificed at 48 h after reperfusion and the spinal cords (L_5-7) were removed for histopathologic study and determination of the activity of Na⁺, K⁺-ATPase. RESULTS: The neurologic function scores in sham group and IPC+IR group at each observation interval were higher than those in IR group (P＜0.01). Compared to IR group, there were more normal neurons in anterior horn of spinal cord in sham group and IPC+IR group (P＜0.01); the activity of Na⁺, K⁺-ATPase in sham group and IPC+IR group were higher than those in IR group (P＜0.01). CONCLUSION: The rapid phase of ischemic preconditioning has a protective effect against spinal cord ischemic injury in rabbits, and this neuroprotection may be related to the maintenance of Na⁺, K⁺-ATPase activity.     

X-ray irradiation increases sensitivity of myocardium to ischemia/reperfusion injury in rats

AIM：To observe the sensitivity of myocardium to ischemia/reperfusion (I/R) injury in the rats with chest radiotherapy. METHODS：The radiation-induced heart disease model was established by local 20 Gy of X-ray irradiation in the chest. Male Wistar rats (n=42) were randomly divided into 6 groups:sham trauma group, trauma group, sham trauma+sham operation group, sham trauma +I/R group, trauma+sham operation group and trauma+I/R group. The rats were subjected to 30 min of ischemia and 1 h of reperfusion 2 week after trauma. The left ventricular developed pressure (LVDP) and ±dp/dtmax were recorded by BL-410 biological signal recording and analysis system. The serum cardiac troponin I (cTnI) and creatine kinase isoenzyme (CK-MB) were measured by ELISA. The myocardial infarct size was determined by nitroblue tetrazolium(NBT) staining method and BI2000 image analysis software. RESULTS：Compared with sham trauma+I/R group, significant decreases in LVDP and ±dp/dtmax were observed in trauma+I/R group (P<0.01) with significant increases in the infarct size and the concentrations of cTnI and CK-MB (P<0.01). CONCLUSION:Chest X-ray irradiation increases the sensitivity of myocardium to I/R injury in rats.     

Effect of intervention for mast cell function before reperfusion on intestinal ischemia-reperfusion-induced early liver injury in rats

AIM：To explore the effect of intervention for mast cell function before reperfusion on intestinal ischemia-reperfusion (IR)-induced early liver injury. METHODS：Adult SD rats (n=35) were randomized into 5 groups with 7 rats each: sham operation group (S group), IR group, cromolyn sodium treatment group (IR+C group, 25 mg/kg), ketotifen treatment group (IR+K group, 1 mg/kg), compound 48/80 treatment group (IR+CP group, 0.75 mg/kg). IR was induced by superior mesenteric artery occlusion for 75 min followed by 4 h of reperfusion. The agents were intravenously administered 5 min before reperfusion. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and histamine, and the liver levels of lactate dehydrogenase (LDH), tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), malondialdehyde (MDA) and superoxide dismutase (SOD) were assessed. The liver histopathologic changes were also evaluated. RESULTS：IR resulted in severe liver injury as demonstrated by great increases in injury scores, concomitant significant increases in serum levels of AST, ALT and histamine, and liver levels of LDH, TNF-α, IL-8, and MDA, accompanied by reduced SOD activity (all P<0.05 vs S group). Treatment with cromolyn sodium or ketotifen markedly alleviated IR-mediated liver injury as confirmed by significant reduction of the above biomedical changes, whereas compound 48/80 further aggravated liver injury by dramatically enhancing the biomedical changes (all P<0.05 vs IR group). CONCLUSION：Inhibition of mast cell function before reperfusion may reduce early liver injury induced by intestinal ischemia reperfusion. Histamine, oxidative stress and inflammatory response may provide promising effects on it.     

Minocycline suppresses sodium nitroprusside-induced apoptosis in PC12 cells by regulating PI3K/Akt signaling

AIM：To explore the role of PI3K/Akt signaling in the anti-apoptotic effect of minocyline (MC) on the apoptosis of PC12 cells induced by sodium nitroprusside (SNP). METHODS：PC12 cells were divided into 4 groups: blank control group, SNP (500 μmol/L) group, MC (10 μmol/L)+SNP group and LY294002+MC+SNP group. The cell viability was observed by MTT assay. The expression of Akt and p-Akt was determined by Western blotting. RESULTS： The viability of the PC12 cells decreased after exposed to 500 μmol/L SNP for 24 h. Meanwhile, MC at concentration of 10 μmol/L significantly blocked the effect of SNP, such as decreasing the cell viability. Pretreatment with LY294002 for 60 min prior to exposure of the PC12 cells to MC and SNP down-regulated the expression of p-Akt induced by SNP. CONCLUSION：Minocycline regulates PI3K/Akt signaling pathway to restrain the apoptosis of PC12 cells induced by SNP.     

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.     

Effect of down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.     

Adipolin/CTRP12 protects against LPS-induced ARDS by up-regulating alveolar epithelial sodium channel in mice

AIM: To investigate the effect of adipolin/CTRP12 in LPS-induced acute respiratory distress syndrome(ARDS) and its potential regulation on alveolar epithelial sodium channel(ENaC) in mice. METHODS: C57BL/6J mice(n=40) were randomly divided into control group, LPS group, adipolin group and wortmannin(PI3K inhibitor) group with 10 mice in each group using random number table. The pathological changes of the lung tissues were evaluated by HE staining. The alveolar fluid clearance(AFC) was measured by Evans blue-marked albumin, and the concentrations of total protein in bronchoalveolar lavage fluid(BALF) were assessed by bicinchoninic acid(BCA) method. In BALF, the levels of IL-1β and TNF-α were determined by ELISA, and the activity of myeloperoxidase(MPO) was detected by an MPO assay kit. The total cell counts and polymorphonuclear neutrophil(PMN) counts in the BALF were analyzed by Giemsa staining. The mRNA levels of α-ENaC were assessed by qPCR, while the protein levels of α-ENaC and p-Akt were determined by Western blot. RESULTS: Compared with control group, the classic ARDS pathological changes were observed in the mice in LPS group, manifesting by severe pathological lung injury(P<0.05), increases in W/D weight ratio, total protein levels, cell counts, MPO activitiy, and IL-1β and TNF-α levels in the BALF, and decrease in AFC(P<0.05), accompanied by down-regulated levels of α-ENaC and p-Akt in the lung tissues(P<0.05). The deteriorating effects triggered by LPS were significantly reversed by administration of adipolin. However, PI3K inhibitor wortmannin canceled the beneficial effects of adipolin on LPS-induced ARDS, as evidenced by aggravated lung injury, increased levels of W/D weight ratio, protein levels, cell counts, MPO activity, and IL-1β and TNF-α levels in the BALF(P<0.05), and decreased levels of AFC, α-ENaC and p-Akt in the lung tissues. CONCLUSION: Adipolin protects against LPS-induced ARDS in the mice by up-regulating α-ENaC and enhancing AFC via PI3K/Akt signal pathway.     

Pretreatment of hypertonic saline attenuates the hepatic ischemia reperfusion injury induced by neutrophils

AIM: To explore the effect of the pretreatment of hypertonic saline (HTS) in hepatic ischemia reperfusion (I/R) injury.METHODS: The rats were divided into sham group (sham group), ischemia reperfusion group (IR group) and pretreatment of hypertonic saline group (HTS group). Partial hepatic ischemia reperfusion model was used. The rats were sacrificed at the time of 1 h, 3 h, 6 h, 12 h and 24 h after reperfusion in each group, respectively. Blood samples were obtained to examine ALT. The expression of the CD11b/CD18 (Mac-1) on the neutrophils was analyzed by flow cytometry. RT-PCR and Western blotting were used to examine the expression of intercellular adhesion molecule-1 (ICAM-1) in livers and chromatometry was performed to detect the activity of myeloperoxidase (MPO) in livers. The morphology of hepatocytes and the structure of sinusoid were observed by histological examinations. RESULTS: ① HTS pretreatment decreased the level of ALT at the time points of 3 h, 6 h and 12 h after reperfusion (P<0.05). ② Mac-1 expression in HTS group was lower at 6 h and 12 h after reperfusion compared with IR group (P<0.05). ③ MPO activity in HTS group was lower at 6 h, 12 h and 24 h compared with IR group (P<0.05). ④ RT-PCR and Western blotting analysis indicated that the pretreatment of HTS inhibited the expression of ICAM-1 in livers after reperfusion. ⑤ Moderate hepatocyte swelling and few neutrophil infiltration were observed in HTS group.CONCLUSION: Pretreatment with HTS has the effect on hepatic ischemia reperfusion injury by inhibiting the expression of Mac-1 on circulating neutrophils and the expression of ICAM-1 in the liver.