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Objective:To evaluate the effect of pre-injection of young rat plasma on cognitive dysfunction after cerebral ischemia-reperfusion (I/R) in aged rats and the role of phosphatidylinositol 3-kinase/serine threonine protein kinase (PI3K/Akt) signaling pathway.Methods:Seventy-two SPF-grade healthy male Sprague-Dawley rats, aged 18 months, weighing 600-650 g, were divided into 4 groups ( n=18 each) by the random number table method: control group (group C), cerebral I/R group (group IR), pre-injection of young rat plasma group (group P) and PI3K inhibitor LY294002 group (group LY). In group P and group LY, young rat plasma 100 μl/time was injected via the tail vein. In group C and group IR, the equal volume of normal saline was injected via the the tail vein, 2 times a week for 4 weeks. Then the model of cerebral I/R injury was developed under sevoflurane anesthesia in IR, P and LY groups. LY294002 0.3 mg/kg was injected through the tail vein at 1 h before anesthesia in LY group. The neurological deficit score (Longa score) was performed at 24 h after reperfusion, and then 6 rats were randomly sacrificed, and brain tissues were obtained to determine the cerebral infarct volume. Spontaneous mobility and anxiety-like behavior were assessed by the open field test at day 29 of reperfusion, and cognitive function was assessed by the novel object recognition test at day 30 of reperfusion. At the end of the behavioral test, rats were sacrificed, hippocampal tissues were isolated for determination of the expression of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt), postsynaptic dense protein-95 (PSD-95) and synaptic vesicle protein (SYN) (by Western blot), and the dendritic length and dendritic spine density of neurons in the hippocampal CA1 region. Results:There was no significant difference in motor speed, distance traveled, and time of staying at the center of the open field among the four groups ( P>0.05). Compared with group C, the Longa score and cerebral infarct volume were significantly increased, the percentage of novel object exploration and discrimination index were decreased, the expression of p-PI3K, p-Akt, PSD-95 and SYN in hippocampal tissues was down-regulated, and the dendritic length and dendritic spine density of hippocampal neurons were decreased in IR, P and LY groups ( P<0.05). Compared with group IR, Longa score and cerebral infarct volume were significantly decreased, the percentage of novel object exploration and discrimination index were increased, the expression of p-PI3K, p-Akt, PSD-95 and SYN in hippocampal tissues was up-regulated, and the dendritic length and dendritic spine density of hippocampal neurons were increased in group P ( P<0.05), and no significant change was found in the parameters mentioned above in group LY ( P>0.05). Compared with group P, Longa score and cerebral infarct volume were significantly increased, the percentage of novel object exploration and discrimination index were decreased, the expression of p-PI3K, p-Akt, PSD-95 and SYN in hippocampal tissues was down-regulated, and the dendritic length and dendritic spine density of hippocampal neurons were decreased in group LY ( P<0.05). Conclusions:Pre-injection of young rat plasma can attenuate cognitive dysfunction after cerebral I/R in aged rats, and the mechanism is related to activation of hippocampal PI3K/Akt signaling pathway and improvement in synaptic plasticity.
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Objective:To evaluate the role of adenosine monophosphate-dependent protein kinase/p38 mitogen-activated protein kinase/nuclear factor E2-associated factor 2 (AMPK/p38 MAPK/Nrf2) pathway in myocardial ischemia-reperfusion (I/R) injury in diabetic rats.Methods:Clean-grade healthy Sprague-Dawley male rats, aged 2-3 months, weighing 220-280 g, were fed with a high fat diet, and 1% streptozotocin 50 mg/kg was intraperitoneally injected for 4 consecutive days to develop the model of diabetes mellitus.Thirty diabetic rats were divided into 3 groups ( n=10 each) using the random number table method: sham operation group (sham group), myocardial I/R group (I/R group), and AMPK inhibitor compound C+ myocardial I/R group (C+ I/R group). The model of myocardial I/R injury was developed by ligation of the left anterior descending coronary artery for 30 min followed by 120 min reperfusion.Compound C 0.5 mg/kg was injected via the caudal vein at 30 min before ischemia in C+ I/R group, while the equal volume of normal saline was given instead in Sham group and I/R group.At 120 min of reperfusion, the percentage of myocardial infarct size was calculated, the serum concentrations of creatine kinase isoenzymes (CK-MB) and lactic dehydrogenase (LDH) were determined by enzyme-linked immunosorbent assay, the levels of glutathione (GSH), superoxide dismutase (SOD) and reactive oxygen species (ROS) in myocardial tissues were measured by enzyme-linked immunosorbent assay, and the expression of AMPK, phosphorylated AMPK (p-AMPK), phosphorylated p38 MAPK (p-p38 MAPK), Nrf2 and heme oxygenase-1 (HO-1) in myocardium was determined by Western blot. Results:Compared with Sham group, the percentage of myocardial infarct size and serum CK-MB and LDH levels were significantly increased, the levels of GSH and SOD in myocardial tissues were decreased, ROS level was increased, and the expression of AMPK, p-AMPK, p-p38 MAPK, Nrf2 and HO-1 was up-regulated in I/R group ( P<0.05). Compared with I/R group, the percentage of myocardial infarct size and serum CK-MB and LDH levels were significantly increased, the levels of GSH and SOD in myocardial tissues were decreased, ROS level was increased, and the expression of AMPK, Nrf2 and HO-1 was down-regulated in C+ I/R group ( P<0.05). Conclusions:AMPK/p38 MAPK/Nrf2 signaling pathway is involved in the mechanism of endogenous antioxidant stress during myocardial I/R in diabetic rats.
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Objective:To investigate the role of PI3K/Akt signaling pathway in hydromorphone postconditioning on alleviating myocardial ischemia/reperfusion (I/R)-induced apoptosis in rats.Methods:Forty healthy male SD rats were randomly(random number) divided into five groups, with 8 rats in each group:①sham group;②I/R group;③I/R+hydromorphone group (I/R+H group);④I/R+PI3K inhibitor group (I/R+W group); and⑤I/R+hydromorphone+PI3K inhibitor group (I/R+H+W group). The myocardial ischemia/reperfusion injury model was established by ligating the left anterior descending coronary artery for 30 min and reperfusion for 120 min. After the experiment, the area of myocardial infarction was measured by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining. The amount of serum lactate dehydrogenase (LDH) leakage was estimated by colorimetry . The cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay. The protein expressions of p-Akt, Bcl-2 and Bax were detected by Western blot. Comparisons among groups were carried out by analysis of variance (ANOVA).Results:Compared with the sham group, the area of myocardial infarction, serum LDH leakage and cardiomyocyte apoptosis were significantly increased, p-Akt and Bax expression were upregulated, Bcl-2 expression was downregulated in the I/R group ( P<0.05). Compared with the I/R group, the area of myocardial infarction, serum LDH leakage and cardiomyocyte apoptosis were markedly decreased, p-Akt and Bcl-2 expression were upregulated and Bax expression was downregulated in the I/R+H group ( P<0.05). Compared with the I/R+H group, the area of myocardial infarction, serum LDH leakage and cardiomyocyte apoptosis were significantly increased, p-Akt and Bcl-2 expression were downregulated, and Bax expression was upregulated in the I/R+H+W group ( P<0.05). Conclusions:Hydromorphone postconditioning can alleviate cardiomyocyte apoptosis induced by myocardial ischemia/reperfusion, and its protection mechanism may be related to the activation of PI3K/Akt signaling pathway.
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Objective:To evaluate the effect of pre-infusion of young rat plasma on postoperative cognitive function in aged rats and role of phosphatidylinositol 3 kinase/serine-threonine protein kinase (PI3K/Akt) signaling pathway.Methods:Sixty SPF healthy male Sprague-Dawley rats, aged 18 months, weighing 550-650 g, were divided into 4 groups ( n=15 each) using a random number table method: control group (group C), operation group (group O), young rat plasma group (group P) and PI3K inhibitor LY294002 group (group LY). The young rat plasma 100 μl/time was injected via the caudal vein twice a week for 4 consecutive weeks in group P and group LY, while the equal volume of normal saline was given instead in group C and group O. Rats received internal fixation for unilateral tibial fracture under sevoflurane anesthesia in O, P and LY groups.Rats received no treatment in group C. PI3K inhibitor LY294002 0.3 mg/kg was injected through the caudal vein before anesthesia in group LY.The ability of spontaneous activity was evaluated by open field test at 3 days after surgery, and then the cognitive function was assessed by Morris water maze test.The rats were sacrificed after the end of behavioral testing, and the hippocampal tissues were isolated for determination of the expression of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt), synapsin, synaptophysin I and synaptic vesicle protein (by Western blot) and for microscopic examination of the ultrastructure of hippocampal neurons (with a transmission electron microscope). The number of synapses was recorded. Results:There was no significant difference in the movement speed and length and time spent in the central zone among the four groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-PI3K, p-Akt, synapsin, synaptophysin I and synaptic vesicle protein was down-regulated, and the number of synapses was reduced in O and LY groups ( P<0.05), and no significant change was found in the parameters mentioned above in group P ( P>0.05). Compared with group O, the escape latency was significantly shortened, the number of crossing the original platform was increased, the expression of p-PI3K, p-Akt, synapsin, synaptophysin I and synaptic vesicle protein was up-regulated, and the number of synapses was increased in group P ( P<0.05), and no significant change was found in the parameters mentioned above in group LY ( P>0.05). Compared with group P, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-PI3K, p-Akt, synapsin, synaptophysin I and synaptic vesicle protein was down-regulated, and the number of synapses was reduced in group LY ( P<0.05). Conclusion:Pre-infusion of young rat plasma can improve postoperative cognitive function in aged rats, and the mechanism is related to activation of PI3K/Akt pathway and improvement of synaptic plasticity.
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Objective To explore the time and dose effects of AKT (a kind of protein serine/threonine kinase) inhibitor GSK2141795 on the apoptosis of human hepatocellular cell line Huh7. Methods Huh7 cells were treated with GSK2141795 at the concentrations of 0, 0.3, 1, 3, 10 and 30 µmol/L for 24 h. A concentration of 10 µmol/L GSK2141795 was selected to treat Huh7 cells for 0, 2, 6, 12, 24 and 48 h. The protein expression levels of AKT and phosphorylated AKTS473 (p-AKTS473) were determined by Western blotting and cell apoptosis was detected by flow cytometry. The expression levels of apoptosis-related proteins (Bad, Bcl-2 and Caspase-9) were measured by qPCR and Western blotting. Results With the increase of GSK2141795 concentration, AKT protein level in Huh7 cells was gradually decreased and the p-AKTS473 protein level was gradually increased within the range of 0-10 µmol/L. With the prolongation of GSK2141795 treatment time, the AKT protein level was gradually decreased and the p-AKTS473 protein level was gradually increased within the range of 0-24 h. At 48 h of treatment, the AKT protein and p-AKTS473 protein expression levels were increased compared with 0 h. With the increase of GSK2141795 concentration and treatment time, the proportion of apoptotic cells was gradually increased, the expression levels of apoptotic molecules Bad and Caspase-9 were gradually increased, and the expression level of apoptotic antagonist Bcl-2 was gradually decreased. Conclusion AKT inhibitor GSK2141795 can effectively inhibit AKT protein expression, and induce apoptosis of Huh7 cells through Bad-Bcl-2 pathway in a dose-and time-dependent manner. In addition, the expression level of AKT protein in Huh7 cells can increase again after long-term stimulation by GSK2141795, suggesting the existence of a negative feedback signal loop.
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Objective To investigate the expression level of receptor-interacting protein serine threonine kinase (RIP) 3 in macrophages/monocytes activation in autoimmune hepatitis (AIH) and its regulation on inflammatory cytokines.Methods The degree of macrophage infiltration and the expression of RIP3 in liver tissues from patients with AIH or hepatic cysts by double-immunofluorescence.After 24 hours treated with different concentrations (0,1,3,6,10 μg/mL) of lipopolysaccharide (LPS),Necrostatin-1,the specific inhibitor of RIP3 signaling pathway,and 6-thioguanine,the active metabolite of azathioprine,the expression levels of RIP1 (RIP3 upstream signal molecule),RIP3 and mixed-lineage kinase domain-like protein (MLKL,RIP3 downstream substrate) in RAW264.7 macrophages were detected by Western blotting.The expression of macrophage-associated cytokine at mRNA level of each treatment group was determined by real time quantitative polymerase chain reaction (PCR).Student's t test and sum rank test were performed for statistical analysis.Spearman analysis was performed for the correlation analysis.Results Compared with hepatic cysts adjacent liver tissues,the infiltration of CD68 positive macrophages in liver tissue of AIH patients was significantly increased (4.75 ± 0.96 vs 28.86 ± 6.23),and the difference was statistically significant (t =7.80,P＜0.05),and the expression level of RIP3 also was significantly increased (15,11 to 22 vs 0,0 to 1),and the difference was statistically significant (Z=-2.66,P＜0.05).In vitro,compared with those of control group,the expression levels of RIP1,RIP3 and MLKL stimulated by LPS at 0,1,3,6,10 μg/mL were significantly increased,and the differences were statistically significant (t=4.00,4.90,6.40,10.30;3.80,9.30,9.80,9.00;4.90,9.90,9.30 and 7.70;all P＜0.05),and were dose-dependent (r=0.91,0.86 and 0.79,all P＜0.05).Furthermore,compared with those of LPS-stimulated group,the expressions of RIP1,RIP3,MLKL of LPS+Necrostatin-1 group were significantly decreased (0.73±0.11 vs 0.47±0.13,0.60±0.07 vs 0.37 ± 0.05,0.65 ± 0.22 vs 0.38 ± 0.04,respectively),and the differences were statistically significant (t=2.60,4.50 and 2.10,all P＜0.05).And the expressions of interleukin (IL)-1β,IL-6and IL-10 at mRNA levels were also decreased (810.3±200.8 vs 463.7±118.1,1 504.4±482.7 vs 290.4±106.9,1 358.6 ± 559.2 vs 677.8 ± 297.6,respectively),and the differences were statistically significant (t=5.40,12.52,5.70,all P＜0.05).However,the expressions of IL-4 and TGF-β at mRNA levels up-regulated (0.3±0.2 vs 0.6±0.3,0.4±0.1 vs 0.9±0.4,respectively),and the differences were statistically significant (t=4.60 and 6.10,both P＜0.05).Compared with those of the LPS-stimulated group,the expressions of IL-1β,IL-6 and IL-10 at mRNA levels of LPS and 6-thiopurine stimulated group significantly down-regulated (810.3±200.8 vs 283.4±65.5,1 504.4±482.7 vs 354.4±73.8,1 358.6± 559.2 vs 625.6±336.3),and the differences were statistically significant (t=4.30,10.60 and 3.50,all P＜0.05);however,the expressions of IL-4 and TGF-β at mRNA levels significantly up-regulated (0.3±0.2 vs 0.6±0.1 and 0.4±0.1 vs 0.5±0.1),and the differences were statistically significant (t=5.20and 12.50,P＜0.05).Conclusions The regulation effects of 6-thiopurine on RIP3 signaling pathway and related cytokines are similar to those of Necrostatin-1.And the expression of RIP3 signaling protein increasing in activated macrophages of liver tissues from AIH patients is closely related to the regulation of IL-6.The RIP3 mediated inflammatory signaling pathway in macrophage may be involved in the genesis and development of AIH and may be a potential therapeutic target.
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Objective To investigate the mechanism underlying sufentanil postconditioning-induced reduction of myocardial ischemia-reperfusion (I/R) injury in rats through evaluating the relationship between phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/Akt) signaling pathway and mitochondrial permeability transition pore (mPTP).Methods Forty-eight pathogen-free healthy male SpragueDawley rats,aged 3 months,weighing 250-300 g,were divided into 4 groups (n=12 each) using a random number table:sham operation group (group S),group I/R,sufentanil postconditioning group (group SP) and snfentanil postconditioning plus PI3K inhibitor wortmannin group (group SP +W).The rats were anesthetized with 20％ urethane 5 ml/kg.Myocardial I/R was induced by ligation of the anterior descending branch of left coronary artery for 30 min,followed by 120 min reperfusion.Sufentanil 1.0 μg/kg was injected via the sublingual vein at 5 min before reperfusion in SP and SP+W groups.Wortmannin 15 pμg/kg was injected via the sublingual vein at 5 min before reperfusion,and then sufentanil 1.0 μg/kg was given in group SP+W.Blood samples were taken from the abdominal aorta at the end of reperfusion for detection of serum cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB) concentrations.The rats were then sacrificed and hearts were removed for determination of cell apoptosis (by TUNEL),nicotinamide adenine dinueleotide (NAD+) content (by speetrophotometry),and expression of phosphorylated Akt (p-Akt) in myocardial tissues (by Western blot).Apoptosis index (AI) was calculated.The myocardial mitochondria and cytoplasm were isolated for detection of the expression of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) using Western blot.Results Compared with group S,the serum cTnI and CK-MB concentrations and AI were significantly increased,the content of NAD+ was decreased,the expression of p-Akt was up-regulated,the expression of Cyt e and AIF in mitochondria was down-regulated,and the expression of Cyt c and AIF in cytoplasm was up-regulated in I/R,SP and SP+W groups (P＜0.05).Compared with group I/R,the serum cTnI and CK-MB concentrations and AI were significantly decreased,the content of NAD+ was increased,the expression of p-Akt was up-regulated,the expression of Cyt c and AIF in mitochondria was up-regulated,and the expression of Cyt c and AIF in cytoplasm was down-regulated in group SP (P＜0.05).Compared with group SP,the serum cTnI and CK-MB concentrations and AI were significantly increased,the content of NAD+ was decreased,the expression of p-Akt was down-regulated,the expression of Cyt c and AIF in mitochondria was down-regulated,and the expression of Cyt c and AIF in cytoplasm was up-regulated in group SP+W (P＜0.05).Conclusion Sufentanil postconditioning can activate PI3K/Akt signaling pathway,inhibit mPTP opening,mitigate mitochondrial injury and inhibit apoptosis in cardiomyocytes,thus attenuating myocardial I/R injury in rats.
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Objective To explore the effect and the possible pathway of different concentrations of QLT0267,which was the inhibitor of the integrin-linked kinase (ILK),on the process of high glucose-induced tubularepithelial-myofibroblast transdifferentiation (TEMT) in human renal tubular epithelial cells (HK-2).Methods HK-2 cells were exposed to 30 mmol/L GS,and TEMT model was established.After excluding the effect of high osmotic in TEMT,HK-2 cells were divided into 6 groups by different concentrations of GS and QLT0267 for 48 hours.The rate of the cell proliferation was calculated by MTT.The expression of ILK and α-smooth muscle actin (α-SMA) were determined by immunofluorescence and Western blot,and the expression of protein kinase B (AKT),phosphorylated protein kinase B (p-AKT),and E-cadherin were determined by Western blot.Results (1) The expression of ILK,p-AKT,and α-SMA in HK-2 cells were unregulated and the expression of E-cadherin was downregulated for 48 hours with glucose treating vs control (P ＜ 0.05);(2) The proliferation rate in high glucose group was higher than the group which concentration of QLT0267 was greater than 5 μmol/L (P ＜ 0.05);(3) With the concentrations of QLT0267 increased,the expression of p-AKT,α-SMA was gradually decreased (all P ＜ 0.05),and the expression of E-cadherin was gradually increased (all P ＜ 0.05).Conclusions 30 μmol/L of GS can lead to TEMT in HK-2 cell.The QLT0267 with concentration greater than 5 μmol/L may prevent the activation of ILK downstream proteins,then partially inhibits cell proliferation and TEMT in HK-2 cell.
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Objective To explore the expression of phosphoinositide 3 kinase (PI3K)and Akt in gastric carcinoma tissues and the distal normal tissues,and to analyze their correlation with the clinical pathologic characteristics of gastric cancer.Methods The S-P immunohistochemistry and RT-PCR were used to detect the expression of PI3K and Akt protein and mRNA in 60 cases of gastric carcinoma tissues and 25 cases of normal gastric mucosal tissues.Results The expression of PI3K and Akt in the gastric carcinoma tissues was significantly higher than that in the distal normal gastric tissues with statistical difference (P <0.05)and their expression in gastric carcinoma tissues was significantly related with the pathologic differentiation degree,TNM stage,invasion depth and lymph node metastasis (P <0.05).Conclusion PI3k/Akt protein and mRNA participate in the occurrence,development, infiltration and metastasis process of gastric cancer,and play an important role in the canceration of gastric carcinoma.
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Objective To explore the role of receptor‐interacting protein kinase 3 (RIP3)‐mediated necroptosis in ulcerative colitis (UC ) . Methods The colonic mucosa tissues of eighty‐five patients diagnosed with UC were collected .Disease staging and pathological classification of UC patients were evaluated according to Mayo standard and Truelove standard ,respectively .The expressions of RIP3 and tumor necrosis factor (TNF)‐α were detected by immunohistochemistry .The cell inflammation model in HT‐29 cells was induced by interferon (IFN)‐γ.The expression of RIP3 mRNA and interleukin (IL)‐1βmRNA was determined by real time polymerase chain reaction (RT‐PCR) .Twenty healthy individuals were studied as controls .A single factor analysis of variance was performed in the comparison among groups ,and Pearson correlation analysis was used for correlation analysis .Results Among 85 patients with UC , 27 cases were mild ,30 cases were moderate and 28 cases were severe;for pathological classification ,26 were gradeⅠ ,31 were gradeⅡ and 28 were grade Ⅲ .The Mayo score was positively correlated with pathological grade (r=0 .997 , P<0 .05) .The expression of RIP3 and TNF‐α in colonic mucosa tissues of UC group increased along with the pathological classification grade .The expression of RIP3 of gradeⅠto Ⅲ was 1 .81 ± 0 .98 ,2 .77 ± 1 .26 and 4 .86 ± 2 .77 ,respectively ,and the differences were statistically significant (F=26 .10 ,P<0 .05) .The expression of TNF‐αof gradeⅠto Ⅲ was 1 .35 ± 0 .69 ,2 .61 ± 1 .41 and 4 .43 ± 2 .17 ,respectively ,and the differences were statistically significant (F=31 .80 ,P<0 .05) .The expression of IL‐1β mRNA of IFN‐γ group ,IFN‐γ+ z‐VAD group and IFN‐γ+z‐VAD+ TNF‐αgroup was 0 .68 ± 0 .27 ,1 .47 ± 0 .12 and 1 .86 ± 0 .16 ,respectively ,and the differences were statistically significant (F=38 .45 , P<0 .05) .The expression of RIP3 mRNA of IFN‐γgroup ,IFN‐γ+z‐VAD group and IFN‐γ+z‐VAD+TNF‐αgroup was 0 .46 ± 0 .13 ,1 .21 ± 0 .29 and 2 .06 ± 0 .20 ,respectively , and the differences were statistically significant (F= 55 .15 , P< 0 .05) .Conclusions TNF‐α/RIP3 signal pathway is up‐regulated in UC .RIP3‐mediated necroptosis promotes the death of colonic epithelial cells and exacerbates inflammatory response .RIP3‐mediated necroptosis may be an important mechanism in UC .
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Objective To evaluate the effect of sevoflurane preconditioning on autophagy during ischemiareperfusion (I/R) injury to isolated rat hearts and the role of PI3K/Akt signaling pathway.Methods Healthy adult male Wistar rats,aged 6-8 weeks,weighing 250-280 g,were anesthetized.Their hearts were excised and perfused in a Langendorff apparatus.Eighty-four isolated rat hearts with I/R injury were randomly divided into 7 groups (n =12 each):normal control group (NC group),I/R group,sevoflurane preconditioning group (S + I/R group),normal control plus wortmannin group (NC + W group),I/R plus wortmannin group (I/R + W group),sevoflurane preconditioning plus wortmannin group (S + I/R + W group),and solvent group (S + I/R + D group).In NC group,the hearts were continuously perfused with K-H solution for 180 min.In I/R group,the hearts were perfused with K-H solution for 30 min,and then subjected to ischemia for 30 min followed by 120 min of reperfusion.In S+ I/R group,the hearts were perfused with K-H solution for 10 min,and then with K-H solution saturated with 2.5％ sevoflurane for 15 min,followed by 5 min washout with K-H solution before ischemia.In NC + W group,wortmannin (PI3K inhibitor) 15 μg/kg was injected intraperitoneally at 30 min before chest opening,and the other procedures were similar to those previously described in group NC.In I/R + W group,wortmarnin 15 μg/kg was injected intraperitoneally at 30 min before chest opening,and the other procedures were similar to those previously described in group I/R.In S + I/R + W group,wortmannin 15 μg/kg was injected intraperitoneally at 30 min before chest opening,and the other procedures were similar to those previously described in S + I/R group.In S + I/R + D group,dimethyl sulfoxide 1.5 ml/kg was injected intraperitoneally at 30 min before chest opening,and the other procedures were similar to those previously described in group S + I/R.The HR,± dp/dtmax,left ventricular developed pressure (LVDP) and left ventricular end-diastolic pressure (LVEDP) were recorded at the end of equilibration and reperfusion.At the end of reperfusion,coronary effluent was collected to detect lactate dehydrogenase (LDH) activity,and myocardial specimens were obtained to calculate the percentage of myocardial infract size.The expression of autophagy marker LC3-Ⅱ and Akt,phosphor-Akt (p-Akt),mammalian target of rapamycin (mTOR),and phosphor-mTOR (p-mTOR) was determined by Western blot.Results Compared with NC group,no significant change was found in the parameters of hemodynamics in NC + W group,and HR,± dp/dtmax and LVDP were significantly decreased,LVEDP,myocardial infract size,and LDH activity were increased,LC3-Ⅱ expression was up-regulated,and the expression of p-Akt and p-mTOR was down-regulated in the other groups.Compared with group I/R,HR,± dp/dtmax,and LVDP were significantly increased,LVEDP,myocardial infract size,and LDH activity were decreased,LC3-Ⅱ expression was downregulated,and the expression of p-Akt and p-mTOR was up-regulated in S + I/R and S + I/R + D groups,and no significant change was found in each parameter in S+ I/R+ W group.Compared with S + I/R group,HR,± dp/ dtmax and LVDP were significantly decreased,LVEDP,myocardial infract size and LDH activity were increased,LC3-Ⅱ expression was up-regulated,and the expression of p-Akt and p-mTOR was down-regulated in S + I/R + W group,and no significant change was found in each parameter in S + I/R + D group.Conclusion Sevoflurane preconditioning can decrease autophagy of myocardial cells during I/R through activating PI3K/Akt signaling pathway and enhancing mTOR activity in the downstream,thus protecting isolated rat hearts against I/R injury.
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Objective To evaluate the regulatory role of acetylcholine receptor in muramyl dipeptide (MDP)-induced activation of Nod-like receptor 2/receptor-interacting protein 2 (2NLR2/RIP2) pathway in macrophages of mice.Methods RAW264.7 cells at the logarithmic growth phase were seeded in 12-well plates (density 1 × 106 cells/ml,2 ml/well),a total of 108 wells.The cells were randomly divided into 3 groups (n =36 each) using a random number table:control group (group C),MDP group (group M),and GTS-21 (a7nAChR specific agonist) group (group G).The cells were routinely cultured in group C.MDP with the final concentration of 10 μg/ml was added to the culture medium in group M.MDP with the final concentration of 10μg/ml and GTS21 with the final concentration of 50 μg/ml were added to the culture medium in group G.The cells were incubated for 24 h.At 1,6 and 24 h of incubation with MDP,12 wells were chosen and the cell suspension was obtained for measurement of NLR2 mRNA expression (by real-time fluorescent quantitative PCR),RIP2 expression (by Western blot),and concentrations of tumor necrosis factor-alpha (TNF-α) and high mobility group box-1 (HMGB1) in the culture media (by ELISA).Results Compared with group C,the levels of NLR2 mRNA,RIP2,TNFα and HMGB1 were significantly increased at each time point in group M (P ＜ 0.05).Compared with group M,the levels of NLR2 mRNA,RIP2,TNF-α and HMGB1 were significantly decreased at each time point in group G (P ＜ 0.05).Conclusion Acetylcholine receptor can suppress MDP-induced transduction of NLR2/RIP2 pathway in macrophages of mice.
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CONTEXT: CHEK2 encodes a cell cycle checkpoint kinase that plays an important role in the DNA damage repair pathway, activated mainly by ATM (Ataxia Telangiectasia Mutated) in response to double-stranded DNA breaks. A germline mutation in CHEK2, 1100delC, has been described as a low penetrance allele in a significant number of families with breast and colorectal cancer in certain countries and is also associated with increased risk of contralateral breast cancer in women previously affected by the disease. About 5%-10% of all breast and colorectal cancers are associated with hereditary predisposition and its recognition is of great importance for genetic counseling and cancer risk management. OBJECTIVES: Here, we have assessed the frequency of the CHEK2 1100delC mutation in the germline of 59 unrelated Brazilian individuals with clinical criteria for the hereditary breast and colorectal cancer syndrome. METHODS: A long-range PCR strategy followed by gene sequencing was used. RESULTS: The 1100delC mutation was encountered in the germline of one (1.7%) individual in this high risk cohort. This indicates that the CHEK2 1100delC is not commonly encountered in Brazilian families with multiple diagnoses of breast and colorectal cancer. CONCLUSION: These results should be confirmed in a larger series of families and further testing should be undertaken to investigate the molecular mechanisms underlying the hereditary breast and colorectal cancer phenotype.
INTRODUÇÃO: CHEK2 codifica uma proteína quinase envolvida em um ponto de checagem do ciclo celular que desempenha um papel importante na via de reparação do DNA, danos ativados principalmente por ATM (Ataxia Telangiectasia Mutado) em resposta a danos na dupla hélice do DNA. A mutação germinativa 1100delC no gene CHEK2 tem sido descrita como um alelo de baixa penetrância em um número significativo de famílias com câncer de mama e cólon em certos países e também está associada com risco aumentado de câncer de mama contralateral em mulheres previamente afetadas pela doença. Cerca de 5%-10% de todos os cânceres de mama e colorretais estão associados a predisposição hereditária e o seu reconhecimento é de grande importância para o aconselhamento genético e gestão do risco de câncer. OBJETIVOS: Neste estudo foi avaliada a frequência da mutação germinativa 1100delC no gene CHEK2 em 59 diferentes indivíduos brasileiros com critérios clínicos para a síndrome de câncer de mama e cólon hereditários. MÉTODO: Utilizamos como estratégia a realização do PCR de longo alcance seguido de sequenciamento. RESULTADOS: A mutação 1100delC foi encontrada em um indivíduo (1,7%), indicando que esta mutação germinativa não é comumente encontrada em famílias brasileiras com múltiplos diagnósticos de câncer de mama e câncer colorretal. CONCLUSÃO: Estes resultados devem ser confirmados em uma série maior de famílias, e estudos adicionais devem ser realizados para investigar a patologia molecular do fenótipo HBCC.
Subject(s)
Female , Humans , Male , Middle Aged , Breast Neoplasms/genetics , Colonic Neoplasms/genetics , Germ-Line Mutation/genetics , Protein Serine-Threonine Kinases/genetics , Brazil , Genetic Predisposition to Disease , Genotype , Pedigree , Phenotype , Polymerase Chain ReactionABSTRACT
Objective To explore the mechanism of apeptosis in rheumatoid arthritis synoviocyte induced by dihydroartemisinin. Methods Synovial tissues were cut from rheumatoid arthritis patients when who was under knee prosthesis. Apoptosis was detected with flow cytometry. Western blot was performed to assess ser473-phosphorylated Akt. EMSA (electrophoretic mobility shift assay) was used to ana-lyze NF-κB activation. Results Dihydroartemisin can induce apoptosis in rheumatoid arthritis synoviocyte in a dose-dependent manor from 2.5μmol/L to 10μmol/L. Rheumatoid Arthritis synoviocyte cultured with dihydroartemisinin in 5μmol/L or 10μmoL/L can significantly in-hibit serine 473 phosphorylation in Akt and activation of NF-κB. Conclusion Dihydroartemisinin can induce apoptosis in rheumatoid arthri-tis synoviocyte through Akt signal pathway.
ABSTRACT
Objective To investigate the clinical features and LRRK2 gene mutation in patients with autosomal dominant familial Parkinson's disease (PD). Methods The clinical features of 16 autosomal dominant familial PD probands were analyzed in terms of age at onset, onset symptoms, UPDRS scores, response to the levodopa treatment and drug-induced dyskinesia. The LRRK2 gene exons 5,13,31,32,35,37,41 and 48 of 16 probands were sequenced after polymerase chain reaction. The novel mutation was further screened in 24D sporadic PD patients and 214 controls using PCR-RFLP for the genotypo frequency analysis. Results Clinically, most of 16 probands had late-onset age. Resting tremor (9patients, 56. 25%,t=0.558,P=0.679)and bradykinesia (9 patients,56.25%,t=0.369,P=0.454)were common onset symptoms followed by rigidity(6 patients,37.50%,t=1.324,P=0.735)and postural instability(5 patients,31.25%,t=2.369,P=0.956).Majority of them had good response to levedopa treatment and rare occurrence of drug-induced dyskinesia. Among the 16 autosomal dominant familial PD probands,6 variants were identified:c.457 T＞C(Leu153Leu),c.1432 G＞T(Asp478Tyr),c.5457 T＞C(Gly1819Gly),c.7153 G＞A(Gly2385Arg),IVS31+28 T＞G and IVS37+162 T＞C. The c.1432G＞T(Asp478Tyr)variant was a novel mutation and it was not detected in 240 sporadic PD patients and 214 controls. The reported mutations associated with the PD, such as Arg1441 Cys/Gly/His, Arg1514Gln, Tyr1699Cys, Ile2012Thr, Gly2019Ser and Ile2020Thr,were not found in our study. Conclusions The autosomal dominant familial PD patients present with classical symptoms of PD and bear the LRRK2 variantsAsp478Tyr and Gly2385Arg.
ABSTRACT
Objective To investigate the dynamical changes of Rho kinase in the cerebrospinal fluid (CSF) and its relationship with cerebral vascular spasm CVS. Methods CSF were collected on the ist, 3rd, 7th, 10th and 14th day after subarachnoid hemorrhage. The expression of Rho-kinase mRNA in CSF was determined by RT-PCR. The expression of endothelin-1 in CSF was determined by radioimmuno-assay. TCD was used to measure the velocity of the cerebral artery. Results The levels of ET-1 and Rho-kinase mRNA in CSF were re-markably increased on the 3rd day, and reached at the peak on the 7th day after subarachnoid hemorrhage, which were significantly higher than those without CVS. Conclusion There is a positive correlation between the level of Rho-kinase mRNA and ET-1 in CSF. Rho-kinase may participate in the development of CVS.