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Objective:To evaluate the effect of metformin preconditioning on adenosine monophosphate-activated protein kinase(AMPK)/PTEN-induced putative protein kinase(PINK1) signaling pathway during ischemia-reperfusion (I/R) injury in diabetic rats.Methods:Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 6 weeks, weighing 120-160 g, were divided into 3 groups ( n=12 each) by the random number table method: diabetic sham operation group (DS group), diabetic myocardial I/R group (DI/R group) and diabetic myocardial I/R+ metformin preconditioning group(DI/R+ Met group). After 4 weeks of feeding a high-fat and high-glucose diet, the model of type 2 diabetes mellitus was induced by a single intraperitoneal injection of 1% streptozotocin 40 mg/kg. The myocardial I/R injury was induced by blocking the anterior descending branch of the left coronary artery for 30 min followed by 120-min reperfusion in anesthetized animals. In DI/R+ Met group, metformin 200 mg/kg was given by intragastric gavage once a day within 1 week before myocardial ischemia. Blood samples from the femoral vein were collected at 120 min of reperfusion for determination of the serum creatine kinase isoenzymes (CK-MB) and cardiac troponin I (cTnI) concentrations by enzyme-linked immunosorbent assay. Then the rats were sacrificed and myocardial tissues were obtained for examination of the pathological changes(by HE staining) and for determination of the percentage of myocardial infarct size (by the double staining of Ewan blue and TTC) and expression of myocardial autophagy-related protein Beclin-1, PTEN-induced putative kinase 1 (PINK1), phosphorylated 5′-adenosine monophosphate-activating protein kinase (p-AMPK), and ratio of microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ (LC3Ⅱ/Ⅰ) (by Western blot). Results:Compared with DS group, the percentage of myocardial infarct size and serum CK-MB and cTnI concentrations were significantly increased, the expression of Beclin-1, p-AMPK and PINK1 in myocardial tissues was up-regulated, the ratio of LC3II/I was increased( P<0.05), and the pathological changes were aggravated in DI/R group and DI/R+ Met group. Compared with DI/R group, the percentage of myocardial infarct size and serum CK-MB and cTnI concentrations were significantly decreased, the expression of Beclin-1, p-AMPK and PINK1 in myocardial tissues was up-regulated, the ratio of LC3Ⅱ/Ⅰ was increased ( P<0.05), and the pathological changes were significantly reduced in DI/R+ Met group. Conclusions:The mechanism by which metformin preconditioning reduces myocardial I/R injury is related to activation of AMPK/PINK1 signaling pathway and up-regulation of mitochondrial autophagy in diabetic rats.
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Objective:To evaluate the role of phosphatidylinositol 3-kinase (PI3K)/serine threonine protein kinase (Akt)/mammalian rapamycin target protein (mTOR) signaling pathway in edaravone-induced reduction of postoperative cognitive dysfunction in aged rats.Methods:Sixty healthy male Sprague-Dawley rats, aged 20 months, weighing 600-700 g, were divided into 4 groups ( n=15 each) using a random number table method: control group (group C), operation group (group O), edaravone group (group E) and PI3K inhibitor LY294002 group (group LY). The rats received laparotomy under 3% sevoflurane anesthesia in O, E and LY groups. Edaravone 3 mg/kg was intraperitoneally injected at 30 min before operation in E and LY groups, and LY294002 0.3 mg/kg was simultaneously injected via the tail vein in group LY. Open field test was performed at 3 days after surgery to evaluate the spontaneous activity of rats, then Morris water maze test was performed to evaluate the cognitive function of rats. The rats were sacrificed after the end of behavioral experiment to isolate hippocampal tissues for determination of the expression of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt), phosphorylated mTOR (p-mTOR), synaptophysin (SYP) and postsynaptic density protein 95 (PSD 95) (by Western blot ) and dendrite length in hippocampal CA1 area (using Golgi staining). The density of dendrites was calculated. Results:There were no statistically significant differences in exercise speed, distance, and time of staying at the center between 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, p-mTOR, SYP and PSD-95 was down-regulated, the dendritic length of neurons in hippocampal CA1 region was shortened, and the density of neurons in hippocampal CA1 region was decreased in group O ( 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, p-mTOR, SYP and PSD-95 was up-regulated, the dendritic length of neurons in hippocampal CA1 region was prolonged, and the density of neurons in hippocampal CA1 region was increased in group E ( P<0.05). Compared with group E, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-PI3K, p-Akt, p-mTOR, SYP and PSD-95 was down-regulated, and the dendritic length of neurons in hippocampal CA1 region was shortened, and the density of neurons in hippocampal CA1 region was decreased in group LY ( P<0.05). Conclusions:The mechanism by which edaravone reduces postoperative cognitive dysfunction is related to activating PI3K/Akt/mTOR signaling pathway and improving synaptic plasticity in aged rats.
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Objective:To evaluate the effect of esketamine on long-term cognitive dysfunction induced by propofol anesthesia in the developing rats and the role of phosphatidylinositol-3-kinase (PI3K)/serine-threonine protein kinase (Akt) signaling pathway.Methods:Forty-eight clean-grade healthy Sprague-Dawley rats of either sex, aged 7 days, weighing 10-15 g, were divided into 4 groups ( n=12 each) using a random number table method: fat emulsion group (C group), propofol group (P group), esketamine + propofol group (EP group), and PI3K inhibitor LY294002 + esketamine + propofol group (LYEP group). Medium/long-chain fat emulsion injection 100 mg/kg was intraperitoneally injected in C group. Propofol was intraperitoneally injected at a dose of 50 mg/kg, followed by an additional dose of 50 mg/kg after the righting reflex was restored (40-60 min later) in P group. In group EP, esketamine 10 mg/kg was intraperitoneally injected, followed by propofol administration using the same method as previously described in P group. In LYEP group, LY294002 25 μg was injected via the lateral ventricle, 30 min later ketamine 10 mg/kg was intraperitoneally injected, and then propofol was given using the same method as previously described in P group. Six rats in each group were randomly sacrificed at 2 h after emergence for microscopic examination of pathological changes of hippocampal neurons and for determination of Akt, phosphorylated Akt (p-Akt), Bax, and cleaved caspase-3 in the hippocampal tissues (using Western blot). The remaining 6 rats in each group were subjected to Y-maze test to evaluate their learning and memory abilities at 30 days after birth. The p-Akt/Akt ratio was calculated. Results:Compared with C group, the p-Akt/Akt ratio in the hippocampal tissues was significantly decreased, the expression of Bax and cleaved caspase-3 was up-regulated, the number of training sessions required for learning was increased, the correct response rate was decreased ( P<0.05), and the pathological damage to neurons in hippocampal CA1 region was found in P, EP and LYEP groups. Compared with P group, the p-Akt/Akt ratio in the hippocampal tissues was significantly increased, the expression of Bax and cleaved caspase-3 was down-regulated, the number of training sessions required for learning was decreased, the correct response rate was increased ( P<0.05), and the pathological damage to neurons in hippocampal CA1 region was significantly attenuated in EP and LYEP groups. Compared with EP group, the p-Akt/Akt ratio in the hippocampal tissue was significantly decreased, and the expression of Bax and cleaved caspase-3 was up-regulated, the number of training sessions required for learning was increased, the correct response rate was decreased ( P<0.05), and the pathological damage to neurons in hippocampal CA1 region was aggravated in LYEP group. Conclusions:Esketamine can alleviate long-term cognitive impairment caused by propofol anesthesia in the developing rats, and the mechanism may be related to activation of the PI3K/Akt signaling pathway and inhibition of apoptosis in neurons.
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Objective:To evaluate the effect of propofol on proliferation of neural stem cells (NSCs) in mice and the role of specificity protein-1 (Sp-1)-epidermal growth factor receptor (EGFR)-protein kinase B (Akt) signaling pathway.Methods:Primary NSCs harvested from both the cortices and hippocampus of C57BL/6 mouse embryos were identified by immunofluorescent staining of Nestin.NSCs at passages 3-6 were divided into 3 groups ( n=21 each) using a random number table method: normal saline control group (C group), propofol group (P group) and propofol plus Sp1 inhibitor plicamycin group (PP group). Propofol at a final concentration of 10 μmol/L was added in group P. Propofol at a final concentration of 10 μmol/L and plicamycin at a final concentration of 100 nmol/L were added in group PP.The equal volume of normal saline was added in group C. The medium was replaced after 6 h of incubation and the cells were continuously incubated.The proliferation of NSCs was assessed by direct cell counting at 24, 36, 48, 60 and 72 h after the end of treatment with drugs.At 6 h after the end of treatment with drugs, the expression of Sp1 and EGFR mRNA was detected by real-time fluorescent quantitative polymerase chain reaction, and the expression of Sp1, Akt and phosphorylated Akt (p-Akt) by Western blot. Results:Compared with group C, the count of NSCs was significantly increased at 48, 60 and 72 h after treatment with drugs, and the expression of EGFR mRNA, Sp1 protein and mRNA and p-Akt was up-regulated in group P ( P<0.05 or 0.01), and no significant change was found in each parameter in group PP ( P>0.05). Compared with group P, the count of NSCs was significantly decreased at 48 and 60 h after treatment with drugs, and the expression of EGFR protein and mRNA and p-Akt was down-regulated in group PP ( P<0.05 or 0.01). Conclusions:Propofol can promote the proliferation of NSCs, and the mechanism may be related to activation of Sp1-EGFR-Akt signaling pathway in mice.
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Objective:To evaluate the role of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway in reduction of acute lung injury (ALI) by bone marrow mesenchymal stem cells (MSCs)-conditioned medium in rats.Methods:Thirty-two healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-250 g, were divided into 4 groups ( n=8 each) according to the random number table method: control group (group C), group ALI, ALI + bone marrow MSCs-conditioned medium group (group M), and ALI + bone marrow MSCs-conditioned medium + LY294002 group (group L). A rat model of ALI was developed by endotracheal inhalation of aerosolized lipopolysaccharide (LPS) 5 mg/kg in anesthetized animals in group ALI, group M and group L. At 1 h after LPS administration, serum-free DMEM medium 1 ml was intravenously injected in group C and group ALI, bone marrow MSCs-conditioned medium 1 ml was intravenously injected in group M, PI3K inhibitor LY294002 30 mg/kg was intraperitoneally injected at 2 h before the model was developed, and bone marrow MSCs-conditioned medium 1 ml was intravenously injected at 1 h after the model was developed in group L. The blood samples were taken from the heart at 24 h after LPS administration, and serum was isolated for determination of the serum levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) (by enzyme-linked immunosorbent assay). Then the rats were sacrificed and lung tissues were obtained for determination of the lung wet/dry weight ratio (W/D ratio), expression of Akt and phosphorylated Akt (p-Akt) (by Western blot) and for microscopic examination of the pathological changes which were scored.The p-Akt/Akt ratio was calculated. Results:Compared with group C, the W/D ratio, lung injury score and serum levels of TNF-α and IL-6 were significantly increased, the expression of p-Akt in lung tissues was down-regulated, the ratio of p-Akt/Akt was decreased ( P<0.05), and pathological changes were found in lung tissues in group ALI.Compared with group ALI, the W/D ratio, lung injury score and serum levels of TNF-α and IL-6 were significantly decreased, the expression of p-Akt in lung tissues was up-regulated, the ratio of p-Akt/Akt was increased ( P<0.05), and pathological changes were significantly attenuated in group M, and no significant change was found in the parameters mentioned above in group L ( P>0.05). Compared with group M, the W/D ratio, lung injury score and serum levels of TNF-α and IL-6 were significantly increased, the expression of p-Akt in lung tissues was down-regulated, the ratio of p-Akt/Akt was decreased ( P<0.05), and pathological changes were accentuated in group L. There were no significant differences in the expression of Akt in lung tissues between the four groups ( P>0.05). Conclusions:PI3K/Akt signaling pathway is involved in reduction of ALI by bone marrow MSCs-conditioned medium in rats.
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Objective:To investigate the role of receptor-interacting protein kinse3 (RIPK3)-mediated necroptosis in diabetic mellitus-caused abolition of cardioprotection induced by sevoflurane postconditioning in rats.Methods:Eighty rats with diabetes mellitus, aged 4-5 weeks, weighing 90-100 g, were divided into 4 groups ( n=20 each) using a random number table method: sham operation group (group Sham), myocardial ischemia-reperfusion (I/R) group (group I/R), sevoflurane postconditioning group (group SP) and sevoflurane postconditiong plus RIPK3 inhibitor GSK-872 group (group GSK). Myocardial I/R was induced by 40 min occlusion of the left anterior descending branch of the coronary artery followed by 120 min reperfusion.In group SP, 2.4% sevoflurane was inhaled for 15 min at the beginning of reperfusion.In group GSK, GSK-872 3.3 mg/kg (dissolved in normal saline) was intraperitoneally injected at 24 and 2 h before surgery, and the other treatments were similar to those previously described in group SP.After 120 min of reperfusion, blood samples from the abdominal aorta were collected for determination of concentrations of serum lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB). Myocardial tissues were taken for determination of percentage of myocardial infarct size (by TTC staining) and expression of RIPK3, phospho-Ca 2+ -calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) and phospho-mixed lineage kinase domain-like protein (p-MLKL) (by Western blot), and the ultrastructure of myocardium was observed by transmission electron microscopy. Results:Compared with group Sham, the serum LDH and CK-MB concentrations and percentage of myocardial infarct size were significantly increased, the expression of RIPK3, p-MLKL and p-CaMKⅡ in myocardial tissues was up-regulated ( P<0.05), and the damage to cardiomyocytes was severe in group I/R.Compared with group I/R, no significant change was found in the parameters mentioned above in group SP ( P>0.05). Compared with group SP, the serum LDH and CK-MB concentrations and percentage of myocardial infarct size were significantly decreased, the expression of RIPK3, p-MLKL and p-CaMKⅡ in myocardial tissues was down-regulated ( P<0.05), and the damage to cardiomyocytes was reduced in group GSK. Conclusion:The mechanism of diabetic mellitus-caused abolition of cardioprotection induced by sevoflurane postconditioning is related to excessive activation of RIPK3-mediated necroptosis in rats.
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Objective:To evaluate the effect of hydrogen-rich saline on serine threonine protein kinase (Akt) /nuclear factor E2-related factor 2 (Nrf2) signaling pathway during hypoxia/reoxygenation (H/R) injury to human renal tubular epithelial cells.Methods:Human renal tubular epithelial cell line were seeded in 96-well plates at a density of 1.5×10 4 cells/ml (200 μl/well) or in 6-well plates at a density of 2×10 5 cells/ml (2 ml/well) were divided into 5 groups( n=30 each) using a random number table method: control group (group C), hydrogen-rich group (group H), group H/R, H/R plus hydrogen-rich saline group (group H/R+ H) and H/R plus hydrogen-rich saline plus Akt inhibitor uprosertib group (group H/R+ H+ U) .In group C, the cells were incubated for 28 h in an incubator filled with normoxia at 37 ℃ (5%CO 2-21%O 2-74%N 2). In group H, cells were added to the medium containing 0.6 mmol/L hydrogen-rich saline, and then incubated for 28 h in an incubator filled with normoxia at 37 ℃.In group H/R, the cells were incubated in an anaerobic chamber (37 ℃, 5%CO 2-1%O 2-94 %N 2) for 24 h, and then incubated for 4 h in an incubator filled with normoxia at 37 ℃.In group H/R+ H, the cells were incubated in an anaerobic chamber for 24 h, and then incubated for 4 h in an incubator containing 0.6 mmol/L filled with normoxia at 37 ℃.In group H/R+ H+ U, the cells were incubated for 1 h in the culture medium containing uprosertib 10 μmol/L (final concentration) and the other treatments were similar to those previously described in group H/R+ H. After treatment in each group, the cell viability was measured by MTT assay, cell apoptosis was measured using flow cytometry, superoxide dismutase (SOD) activity was measured using xanthine oxidase method), malondialdehyde (MDA) content was detected by thiobarbituric acid method, the expression of Akt, phosphorylated Akt (p-Akt), total Nrf2, nuclear Nrf2 and activated caspase-3 was detected by Western blot, and the expression of Nrf2 mRNA was detected by Real-time PCR. Results:Compared with group C, the cell viability and activity of SOD were significantly decreased, the apoptosis rate and content of MDA were increased, and the expression of p-Akt, nuclear Nrf2, total Nrf2, activated caspase-3 protein and Nrf2 mRNA was up-regulated in group H/R and group H/R+ H ( P<0.05). Compared with group H/R, the cell viability and activity of SOD were significantly increased, the apoptosis rate and content of MDA were decreased, the expression of p-Akt, nuclear Nrf2, total Nrf2 and Nrf2 mRNA was up-regulated and expression of activated caspase-3 protein was down-regulated in group H/R+ H ( P<0.05). Compared with group H/R+ H, the cell viability and activity of SOD was significantly decreased, the apoptosis rate and content of MDA were increased, the expression of p-Akt, nuclear Nrf2, total Nrf2 protein and Nrf2m RNA was down-regulated, and the expression of activated caspase-3 protein was up-regulated in group H/R+ H+ U ( P<0.05). Conclusion:The mechanism by which hydrogen-rich saline attenuates H/R injury to human renal tubular epithelial cells is related to improving activation of Akt/Nrf2 signaling pathway, decreasing oxidative stress response and inhibiting cell apoptosis.
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Objective:To evaluate the effect of activating adenosine A2B receptors on autophagy during myocardial ischemia-reperfusion (I/R) and the role of phosphatidylinositol 3-kinase/serine/threonine protein kinase (PI3K/Akt) signaling pathway in rats.Methods:Forty-eight clean-grade healthy male Sprague-Dawley rats, weighing 220-280 g, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (group Sham), myocardial I/R group (group I/R), adenosine A2B receptor agonist BAY 60-6583 group (group BAY) and BAY 60-6583+ PI3K inhibitor LY 294002 group (group BAY+ LY). Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 120-min reperfusion.BAY 60-6583 1 mg/kg was intraperitoneally injected at 5 min before reperfusion in group BAY.BAY 60-6583 1 mg/kg was intraperitoneally injected at 5 min before reperfusion and LY 294002 10 mg/kg was intraperitoneally injected at 10 min before reperfusion in group BAY+ LY.Blood samples were obtained at the end of reperfusion for determination of concentrations of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in serum (by enzyme-linked immunosorbent assay). The animals were sacrificed, and myocardial tissues were obtained for measurement of the percentage of myocardial infarct size (by Evan Blue and TTC double-staining) and for determination of the expression of microtubule-associated protein 1 light chain 3 (LC3Ⅰ), LC3Ⅱ, Beclin-1 and phosphorylated Akt (p-Akt) (by Western blot). The ratio of LC3Ⅱ/LC3Ⅰ was calculated. Results:Compared with group Sham, the serum LDH and CK-MB concentrations and percentage of myocardial infarct size were significantly increased, the expression of p-Akt was down-regulated, the expression of Beclin-1 and LC3Ⅱ was up-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group I/R ( P<0.05). Compared with group I/R, the concentrations of serum LDH, CK-MB and percentage of myocardial infarct size were significantly decreased, the expression of p-Akt was up-regulated, the expression of Beclin-1 and LC3Ⅱ was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased in the group BAY ( P<0.05), and no significant change was found in the parameters mentioned above in group BAY+ LY ( P>0.05). Compared with group BAY, the concentrations of serum LDH, CK-MB and percentage of myocardial infarct size were significantly increased, the expression of p-Akt was down-regulated, the expression of Beclin-1 and LC3Ⅱ was up-regulated and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group BAY+ LY ( P<0.05). Conclusion:Activating adenosine A2B receptors can decrease autophagy of myocardial cells during myocardial I/R injury, and the mechanism may be related to activating PI3K/Akt signaling pathway in rats.
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Objective:To evaluate the relationship between the mechanism of protective effect of hydromorphone postconditioning on myocardium and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway-mediated autophagy in rats.Methods:Forty healthy male Sprague-Dawley rats, weighing 220-250 g, were divided into 5 groups ( n=8 each) using a random number table method: sham operation group (Sham group), ischemia-reperfusion (I/R) group (group IR), hydromorphone postconditioning group (group HP), PI3K inhibitor group (group W) and hydromorphone postconditioning+ PI3K inhibitor group (group HP+ W). Myocardial ischemia was induced by 30 min occlusion of left anterior descending branch of coronary artery followed by 120 min reperfusion.In group HP, hydromorphone 0.1 mg/kg was injected via femoral vein at 5 min before reperfusion in group HP.In group HP+ W, hydromorphone 0.1 mg/kg and wortmannin (PI3K inhibitor) 15 μg/kg were injected via femoral vein at 5 min before reperfusion.In group W, wortmannin 15 μg/kg was injected via femoral vein at 5 min before reperfusion.At the end of reperfusion, the myocardial infarct size (IS) was determined by TTC staining, the activities of serum lactate dehydrogenase (LDH) was detected by colorimetry, myocardial specimens were collected for microscopic examination of the ultrastructure (with a electron microscope), the expression of phosphorylated Akt (p-Akt) and microtubule-associated protein 1 light chain 3 (LC3) was determined by Western blot and the ratio of LC3-Ⅱ/Ⅰwas calculated. Results:Compared with Sham group, IS and the activities of serum of LDH were significantly increased, p-Akt expression in myocardial tissues was up-regulated, the ratio of LC3-Ⅱ/Ⅰwas increased ( P<0.05), autophagic vacuoles were increased and the damage of ultrastructure of cardiomyocytes was obvious in group IR.Compared with group IR, IS and the activities of serum of LDH were significantly decreased, p-Akt expression in myocardial tissues was up-regulated, the ratio of LC3-Ⅱ/Ⅰwas decreased ( P<0.05), autophagic vacuoles were decreased and the damage of ultrastructure of cardiomyocytes was attenuated in group HR.Compared with group HR, IS and the activities of serum of LDH were significantly increased, p-Akt expression in myocardial tissues was down-regulated, the ratio of LC3-Ⅱ/Ⅰwas increased ( P<0.05), autophagic vacuoles were increased and the damage of ultrastructure of cardiomyocytes was aggravated in group HR+ W. Conclusion:The mechanism of protective effect of hydromorphone postconditioning on myocardium is related to activation of PI3K/Akt signaling pathway and inhibition of autophagy in rats.
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Objective:To investigate the effect of atorvastatin preconditioning on intestinal ischemia-reperfusion (I/R) injury in mice and the relationship with phosphatidylinositol 3-kinase (PI3K)/serine-threonine kinase (Akt) signaling pathway.Methods:Twenty-four healthy male C57BL/6 mice, aged 6-8 weeks, weighing 18-22 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (S group), I/R group, atorvastatin preconditioning group (A group), atorvastatin plus PI3K inhibitor LY294002 group (AL group). Atorvastatin 10 mg/kg was given by intragastric gavage for 3 consecutive days in A and AL groups, and in addition LY294002 0.3 mg/kg was intraperitoneally injected at 30 min before the last administration of atorvastatin in AL group.Intestinal I/R was produced by occlusion of superior mesenteric artery (SMA) for 45 min followed by 2 h reperfusion in anesthetized mice.The superior mesenteric artery was only isolated but not clamped in S group.The mice were sacrificed at the end of reperfusion, and small intestinal tissues were taken for determination of the pathological changes with a light microscope after HE staining and for determination of wet to dry weight ratio(W/D ratio) and expression of PI3K, phosphorylated Akt (p-Akt), autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3Ⅰ (LC3Ⅰ) and LC3Ⅱ.The intestinal damage was assessed and scored according to Chiu.The ratio of LC3Ⅱ expression to LC3Ⅰ expression (LC3Ⅱ/LC3Ⅰ) was calculated. Results:Compared with S group, Chiu′s scores and W/D ratio were significantly increased, the expression of PI3K and p-Akt was down-regulated, the expression of Beclin-1 was up-regulated, and LC3Ⅱ/LC3Ⅰ ratio was increased in I/R, A and AL groups ( P<0.05). Compared with I/R group, Chiu′s scores and W/D ratio were significantly decreased, the expression of PI3K and p-Akt was up-regulated, the expression of Beclin-1 was down-regulated, and LC3Ⅱ/LC3Ⅰ ratio was decreased in A group ( P<0.05). Compared with A group, Chiu′s scores and W/D ratio were significantly increased, the expression of PI3K and p-Akt was down-regulated, the expression of Beclin-1 was up-regulated, and LC3Ⅱ/LC3Ⅰ ratio was increased in AL group ( P<0.05). Conclusion:Atorvastatin preconditioning can mitigate intestinal I/R injury in mice, and the mechanism is related to activating PI3K/Akt signaling pathway and inhibiting the level of autophagy.
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OBJECTIVE@#To investigate the expression and clinical significance of receptor interacting protein serine-threonine kinases 1 (RIPK1) in the nucleus pulposus of patients with lumbar disc herniation (LDH).@*METHODS@#Nucleus pulposus tissue specimens of 40 patients with LDH patients underwent surgical treatment from January 2016 to January 2018 as the case group, and nucleus pulposus tissue specimens of 30 patients with lumbar spine fracture underwent surgical treatment at the same time as the control group. The expression of RIPK1 mRNA and protein of receptor interaction were detected by polymerase chain reaction (PCR) and Western blot, respectively. The expression of RIPK1 protein in the nucleus pulposus were detected by immunohistochemical staining. The concentrations of RIPK1 and tumor necrosis factor-α (TNF-α) in nucleus pulposus were detected by ELISA method. The relationship between the concentrations of RIPK1, TNF-α in nucleus pulposus and the Pearce grade of LDH patients was analyzed by one-way ANOVA. The correlation between RIPK1 and TNF-α was analyzed by Pearson.@*RESULTS@#RIPK1 was weakly positively expressed in nucleus pulposus of control group, and RIPK1 protein was positively or strongly positively expressed in case group. The expression of RIPK1 mRNA in nucleus pulposus of case group was higher than that of control group (@*CONCLUSION@#The expression levels of RIPK1 mRNA and protein in the intervertebral disc tissues of LDH patients are higher than those of normal intervertebral disc tissues, and increased with the increase of Pearce grade, which may be an important factor involved in LDH inflammatory disease.
Subject(s)
Humans , Intervertebral Disc/metabolism , Intervertebral Disc Degeneration , Intervertebral Disc Displacement/genetics , Nucleus Pulposus , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
ObjectiveTo investigate the expression and clinical significance of protein kinase CK2β in hepatocellular carcinoma (HCC) tissue and the association of CK2β expression with patient prognosis. MethodsHCC tissue and adjacent tissue samples were collected from 127 HCC patients Who were diagnosed in Department of Hepatopancreatobiliary Surgery, The first Affiliated Hospital of Zhengzhou University from Januany 2012 to June 2013, and immunohistochemistry was used to measure the expression of CK2β. The association of CK2β expression with clinical features of HCC patients was analyzed. A total of 20 HCC tissue samples, 20 adjacent tissue samples, 20 cirrhotic tissue samples, and 20 normal liver tissue samples were collected from March to June 2018 in our hospital, and Western blot and real-time PCR were used to measure the protein and mRNA expression of CK2β in these tissue samples. A one-way analysis of variance was used for comparison of means between multiple groups, and Bonferroni test was used for further comparison between two groups; the chi-square test was used for comparison of categorical data between groups. The Mann-Whitney U test or the Kruskal-Wallis H test was used to investigate the association of CK2β expression with clinical features. The Kaplan-Meier method was used for survival analysis, and the log-rank test was used for comparison between groups. ResultsWestern blot and real-time PCR showed that the expression rate of CK2β in HCC tissue was significantly higher than that in adjacent tissue, cirrhotic tissue, and normal liver tissue (P＜0.05); there was no significant difference in the expression of CK2β between adjacent tissue and cirrhotic tissue (P＞0.05), and the expression of CK2β in adjacent tissue and cirrhotic tissue was significantly higher than that in normal liver tissue (P＜0.05). Immunohistochemical staining showed that there was a significant difference in the positive rate of CK2β between HCC tissue and adjacent tissue in the 127 HCC patients (85.8% vs 63.0%, P＜0.001). There was a significant difference in CK2β expression distribution between HCC patients with different ages (Z=-2.277, P=0.023), presence or absence of liver cirrhosis (Z=-2.144, P=0.032), different tumor sizes (Z=-2.289, P=0.004), or different Edmondson-Steiner pathological grades (χ2=8.210, P=0.016). The Kaplan-Meier survival curve analysis showed that the strongly positive CK2β expression group had a significantly shorter postoperative survival time than the moderately positive, weakly positive, and negative CK2β expression groups (all P＜0.001). ConclusionCK2β may be involved in the development and progression of HCC, and its positive expression is associated with the prognosis of HCC patients.
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ObjectiveTo investigate the expression and clinical significance of protein kinase CK2β in hepatocellular carcinoma (HCC) tissue and the association of CK2β expression with patient prognosis. MethodsHCC tissue and adjacent tissue samples were collected from 127 HCC patients Who were diagnosed in Department of Hepatopancreatobiliary Surgery, The first Affiliated Hospital of Zhengzhou University from Januany 2012 to June 2013, and immunohistochemistry was used to measure the expression of CK2β. The association of CK2β expression with clinical features of HCC patients was analyzed. A total of 20 HCC tissue samples, 20 adjacent tissue samples, 20 cirrhotic tissue samples, and 20 normal liver tissue samples were collected from March to June 2018 in our hospital, and Western blot and real-time PCR were used to measure the protein and mRNA expression of CK2β in these tissue samples. A one-way analysis of variance was used for comparison of means between multiple groups, and Bonferroni test was used for further comparison between two groups; the chi-square test was used for comparison of categorical data between groups. The Mann-Whitney U test or the Kruskal-Wallis H test was used to investigate the association of CK2β expression with clinical features. The Kaplan-Meier method was used for survival analysis, and the log-rank test was used for comparison between groups. ResultsWestern blot and real-time PCR showed that the expression rate of CK2β in HCC tissue was significantly higher than that in adjacent tissue, cirrhotic tissue, and normal liver tissue (P＜0.05); there was no significant difference in the expression of CK2β between adjacent tissue and cirrhotic tissue (P＞0.05), and the expression of CK2β in adjacent tissue and cirrhotic tissue was significantly higher than that in normal liver tissue (P＜0.05). Immunohistochemical staining showed that there was a significant difference in the positive rate of CK2β between HCC tissue and adjacent tissue in the 127 HCC patients (85.8% vs 63.0%, P＜0.001). There was a significant difference in CK2β expression distribution between HCC patients with different ages (Z=-2.277, P=0.023), presence or absence of liver cirrhosis (Z=-2.144, P=0.032), different tumor sizes (Z=-2.289, P=0.004), or different Edmondson-Steiner pathological grades (χ2=8.210, P=0.016). The Kaplan-Meier survival curve analysis showed that the strongly positive CK2β expression group had a significantly shorter postoperative survival time than the moderately positive, weakly positive, and negative CK2β expression groups (all P＜0.001). ConclusionCK2β may be involved in the development and progression of HCC, and its positive expression is associated with the prognosis of HCC patients.
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BACKGROUND: Cancer is characterized by uncontrolled cellular proliferation, and Polo-like kinase 1 (PLK1), a key regulator of the cell cycle, is overexpressed in many cancers, including acute leukemia and lymphoma. However, the dynamics of PLK1 transcription in myelodysplastic syndromes (MDS) are unknown. This study aimed to investigate the transcript dynamics of PLK1 and determine its role in the pathophysiology of MDS. METHODS: PLK1 mRNA obtained from the bone marrow samples of 67 patients with MDS, 16 patients with secondary acute myeloid leukemia (sAML), and 10 healthy controls were analyzed using quantitative real-time PCR and compared according to various clinical parameters. RESULTS: The median PLK1 expression levels differed slightly, but not significantly, between MDS and sAML patients [661.21 (range, 29.38–8,987.31) vs. 1,462.05 (32.22–5,734.09), respectively], but were significantly higher (P<0.001) than the levels in the healthy controls [19.0 (1.60–49.90)]. Further analyses of PLK1 levels according to the WHO classification of MDS, prognostic risk groups, karyotype risk groups, marrow blast percentage, and depth of cytopenia did not reveal any significant associations. In patients progressing to sAML, PLK1 expression levels differed significantly according to the presence or absence of resistance to hypomethylation treatment (2,470.58 vs. 415.98, P=0.03). CONCLUSION: PLK1 is upregulated in MDS patients; however, its role in the pathophysiology of MDS is unclear. Gene upregulation in cases with pharmacotherapeutic resistance warrants further investigation.
Subject(s)
Humans , Bone Marrow , Cell Cycle , Cell Proliferation , Classification , DNA Methylation , Gene Expression , Karyotype , Leukemia , Leukemia, Myeloid, Acute , Lymphoma , Myelodysplastic Syndromes , Phosphotransferases , Protein Serine-Threonine Kinases , Real-Time Polymerase Chain Reaction , RNA, Messenger , Up-RegulationABSTRACT
Objective@#To evaluate the effect of dexmedetomidine on renal fibrosis in a mouse model of renal ischemia-reperfusion (I/R) and the role of serine-threonine kinase (Akt).@*Methods@#Sixty male C57BL/6 mice, aged 8 weeks, weighing 20-25 g, were divided into 5 groups (n=12 each) using a random number table method: sham operation group (S group), renal I/R group (I/R group), renal I/R plus dexmedetomidine group (I/R + D group), renal I/R plus dexmedetomidine plus Akt agonist SC79 group (I/R + D + SC group), and renal I/R plus dexmedetomidine plus normal saline group (I/R+ D+ NS group). Renal I/R injury model was established by clamping the bilateral renal pedicle for 30 min followed by reperfusion.Dexmedetomidine was intraperitoneally injected at 30 min before surgery in I/R+ D, I/R+ D+ SC and I/R+ D+ NS groups.SC79 was intraperitoneally injected as a bolus of 0.04 mg/kg at 1 min of reperfusion, followed by an intraperitoneal injection of the same dose every 24 h until day 7.The serum blood urea nitrogen (BUN) and Scr concentrations were detected at 24 h of reperfusion.Renal tissues were taken, and the damage to the renal tubules was scored.Renal tissues were removed at 14 days of reperfusion to detect the degree of renal fibrosis and expression of collagen 1 (COL1), fibronectin (FN), and α-smooth actin (α-SMA) (by immunofluorescence and Western blot). The expression of phosphorylated Akt (p-Akt) in renal tissues was determined by Western blot at 24 h and 14 day of reperfusion.@*Results@#Compared with group S, the serum BUN and Scr concentrations, renal tubule damage score and degree of renal fibrosis were significantly increased, and the expression of COL1, FN, α-SMA and p-Akt was up-regulated in group I/R (P<0.05). Compared with I/R group, the serum BUN and Scr concentrations, renal tubular damage score and degree of renal fibrosis were significantly decreased, and the expression of COL1, FN, α-SMA and p-Akt was down-regulated in I/R+ D and I/R+ D+ NS groups (P<0.05). Compared with I/R+ D group, the serum BUN and Scr concentrations, renal tubule damage score and degree of renal fibrosis were significantly increased , and the expression of COL1, FN, α-SMA and p-Akt was up-regulated in I/R+ D+ SC group (P<0.05).@*Conclusion@#Dexmedetomidine can reduce the degree of renal fibrosis in a mouse model of renal I/R and the mechanism is related to inhibiting activation of Akt.
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Objective@#To evaluate the role of phosphatidylinositol-3-kinase/protein kinase B/nuclear factor erythroid 2-related factor 2 (PI3K/Akt/Nrf2) signaling pathway in resveratrol preconditioning-induced cardioprotection in diabetic rats.@*Methods@#Clean-grade healthy male Sprague-Dawley rats, aged 2-3 months, weighing 240-280 g, were used in the study.The diabetes model was established by intraperitoneal injection of streptozotocin 30 mg/kg once a day for 7 consecutive days.Forty diabetic rats were selected and divided into 4 groups (n=10 each) according to the random number table method: sham operation group (S group), myocardial ischemia-reperfusion (I/R) group (I/R group), resveratrol plus myocardial I/R group (Res+ I/R group), and PI3K inhibitor LY294002 plus resveratrol plus myocardial I/R group (LY+ Res+ I/R group). The myocardial I/R injury model was established by ligating the left anterior descending coronary artery for 30 min followed by 120-min reperfusion in anesthetized rats.Resveratrol 20 mg/kg was intraperitoneally injected once a day for 7 consecutive days at one week before surgery in Res+ I/R and LY+ Res+ I/R groups.LY294002 1.5 mg/kg was intraperitoneally injected at 30 min before operation in LY+ Res+ I/R group.After 120 min of reperfusion, blood samples were taken for determination of serum creatine kinase-MB (CK-MB) and lactic dehydrogenase (LDH) concentrations (by enzyme-linked immunosorbent assay), and myocardial tissues at the ischemic area were obtained for determination of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA)levels (by enzyme-linked immunosorbent assay) and expression of Akt, phosphorylated Akt (p-Akt), glycogen synthase kinase 3β (GSK3β), phosphorylated GSK3β (p-GSK3β) and Nrf2 (by Western blot).@*Results@#Compared with group S, serum CK-MB and LDH concentrations were significantly increased, the SOD and GSH levels were decreased, the MDA level was increased, and the expression of p-Akt and p-GSK3β was down-regulated in I/R group (P<0.05). Compared with I/R group, serum CK-MB and LDH concentrations were significantly decreased, the SOD and GSH levels were increased, the MDA level was decreased, and the expression of p-Akt, p-GSK3β and Nrf2 was up-regulated in Res+ I/R group (P<0.05). Compared with Res+ I/R group, serum CK-MB and LDH concentrations were significantly increased, the SOD and GSH levels were decreased, the MDA level was increased, and the expression of p-Akt, p-GSK3β and Nrf2 was down-regulated in LY+ Res+ I/R group (P<0.05).@*Conclusion@#The mechanism of resveratrol preconditioning-induced cardioprotection is related to activating PI3K/Akt/Nrf2 signaling pathway and inhibiting oxidative stress responses in diabetic rats.
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Objective To evaluate the role of phosphatidylinositol-3-kinase/protein kinase B/nucle-ar factor erythroid 2-related factor 2(PI3K/Akt/Nrf2)signaling pathway in resveratrol preconditioning-in-duced cardioprotection in diabetic rats.Methods Clean-grade healthy male Sprague-Dawley rats,aged 2-3 months,weighing 240-280 g,were used in the study.The diabetes model was established by intraper-itoneal injection of streptozotocin 30 mg/kg once a day for 7 consecutive days.Forty diabetic rats were se-lected and divided into 4 groups(n＝10 each)according to the random number table method: sham opera-tion group(S group),myocardial ischemia-reperfusion(I/R)group(I/R group),resveratrol plus myo-cardial I/R group(Res+I/R group),and PI3K inhibitor LY294002 plus resveratrol plus myocardial I/R group(LY+Res+I/R group).The myocardial I/R injury model was established by ligating the left anterior descending coronary artery for 30 min followed by 120-min reperfusion in anesthetized rats.Resveratrol 20 mg/kg was intraperitoneally injected once a day for 7 consecutive days at one week before surgery in Res+I/R and LY+Res+I/R groups.LY294002 1.5 mg/kg was intraperitoneally injected at 30 min before operation in LY+Res+I/R group.After 120 min of reperfusion,blood samples were taken for determination of serum creatine kinase-MB(CK-MB)and lactic dehydrogenase(LDH)concentrations(by enzyme-linked immu-nosorbent assay),and myocardial tissues at the ischemic area were obtained for determination of superoxide dismutase(SOD),glutathione(GSH)and malondialdehyde(MDA)levels(by enzyme-linked immu-nosorbent assay)and expression of Akt,phosphorylated Akt(p-Akt),glycogen synthase kinase 3β(GSK3β),phosphorylated GSK3β(p-GSK3β)and Nrf2(by Western blot).Results Compared with group S,serum CK-MB and LDH concentrations were significantly increased,the SOD and GSH levels were decreased,the MDA level was increased,and the expression of p-Akt and p-GSK3β was down-regu-lated in I/R group(P＜0.05).Compared with I/R group,serum CK-MB and LDH concentrations were sig-nificantly decreased,the SOD and GSH levels were increased,the MDA level was decreased,and the ex-pression of p-Akt,p-GSK3β and Nrf2 was up-regulated in Res+I/R group(P＜0.05).Compared with Res+I/R group,serum CK-MB and LDH concentrations were significantly increased,the SOD and GSH levels were decreased,the MDA level was increased,and the expression of p-Akt,p-GSK3β and Nrf2 was down-regulated in LY+Res+I/R group(P＜0.05).Conclusion The mechanism of resveratrol precondi-tioning-induced cardioprotection is related to activating PI3K/Akt/Nrf2 signaling pathway and inhibiting oxi-dative stress responses in diabetic rats.
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Objective To evaluate the effect of dexmedetomidine on renal fibrosis in a mouse model of renal ischemia-reperfusion (I/R) and the role of serine-threonine kinase (Akt).Methods Sixty male C57BL/6 mice,aged 8 weeks,weighing 20-25 g,were divided into 5 groups (n=12 each) using a random number table method:sham operation group (S group),renal I/R group (I/R group),renal I/R plus dexmedetomidine group (I/R + D group),renal I/R plus dexmedetomidine plus Akt agonist SC79 group (I/R + D + SC group),and renal I/R plus dexmedetomidine plus normal saline group (I/R+D+NS group).Renal I/R injury model was established by clamping the bilateral renal pedicle for 30 min followed by reperfusion.Dexmedetomidine was intraperitoneally injected at 30 rmin before surgery in I/R+D,I/R+D+SC and I/R+D+NS groups.SC79 was intraperitoneally injected as a bolus of 0.04 mg/kg at 1 min of reperfusion,followed by an intraperitoneal injection of the same dose every 24 h until day 7.The serum blood urea nitrogen (BUN) and Scr concentrations were detected at 24 h of reperfusion.Renal tissues were taken,and the damage to the renal tubules was scored.Renal tissues were removed at 14 days of reperfusion to detect the degree of renal fibrosis and expression of collagen 1 (COL1),fibronectin (FN),and α-smooth actin (α-SMA) (by immunofluorescence and Western blot).The expression of phosphorylated Akt (p-Akt) in renal tissues was determined by Western blot at 24 h and 14 day of reperfusion.Results Compared with group S,the serum BUN and Scr concentrations,renal tubule damage score and degree of renal fibrosis were significantly increased,and the expression of COL1,FN,α-SMA and p-Akt was up-regulated in group I/R (P＜0.05).Compared with I/R group,the serum BUN and Scr concentrations,renal tubular damage score and degree of renal fibrosis were significantly decreased,and the expression of COL1,FN,α-SMA and pAkt was down-regulated in I/R+D and I/R+D+NS groups (P＜0.05).Compared with I/R+D group,the serum BUN and Scr concentrations,renal tubule damage score and degree of renal fibrosis were significantly increased,and the expression of COL1,FN,α-SMA and p-Akt was up-regulated in I/R+D+SC group (P＜0.05).Conclusion Dexmedetomidine can reduce the degree of renal fibrosis in a mouse model of renal I/R and the mechanism is related to inhibiting activation of Akt.
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Objective To evaluate the effect of activating adenylate-activated protein kinase (AMPK) on sepsis in aged mice and the relationship with autophagy.Methods Experiment [Twentyeight SPF female C57BL/6 mice,aged 16-19 months,weighing 25-35 g,were divided into sepsis group (group S,n=14) and sepsis plus AMPK agonist AICAR group (S+A group,n=14) by using a random number table method.In group S+A,AICAR 0.5 ml (dissolved in 5％ dimethyl sulfoxide) was administered by intragastric gavage.In group S,5％ dimethyl sulfoxide 0.5 ml was injected by intragastric gavage once a day for 7 consecutive days.The body weight of each mouse before and after administration was recorded.Sepsis was induced by intraperitoneal injection with cecal slurry 200 μl at the end of administration.Nine mice were selected in each group and observed for 7 days after establishing the model,and the survival rates were recorded.At 24 h after establishing the model,5 mice were sacrificed in each group,and spleen tissues were obtained for determination of the expression of AMPK,phosphorylated AMPK (p-AMPK),microtubule-associated protein 1 light chain 3 Ⅰ (LC3 Ⅰ) and LC3 Ⅱ (by Western blot).The ratios of p-AMPK/AMPK and LC3 Ⅱ/LC3 Ⅰ were calculated.Experiment Ⅱ Ten SPF female C57BL/6 mice,aged 16-19 months,weighing 25-35 g,were studied.The peritoneal macrophages obtained from 5 mice were extracted,cultured primarily and then randomized into control group (group C,n =5) and EG-FP E.Coli group (group E,n=5) using a random number table method.AICAR 0.5 ml was injected by intragastric gavage once a day for 7 consecutive days in the other 5 mice.The peritoneal macrophages were extracted after the end of intragastric administration,cultured primarily and then divided into 2 groups (n=5 each) using a random number table method:AICAR plus EGFP E.Coli group (group A+E) and AICAR plus autophagy inhibitor 3-methyladenine plus EGFP E.Coli group (group A+M+E).In group A+M+E,5 mmol/L 3-methyladenine 100 μl was added,and the cells were incubated for 1 h.EGFP expressingE.Coli was then added,and the cells were incubated for 1 h in E,A+E and A+E+M groups.Flow cytometry was used to detect the phagocytic ability of macrophages.Results Experiment Ⅰ The body weight was significantly lower after the end of administration than before administration in group S+A (P＜0.05).Compared with group S,the body weight was significantly decreased at the end of administration,the survival rate was increased at 7 days after establishing the model,the expression of LC3 Ⅱ in spleen tissues was up-regulated and the ratios of p-AMPK/AMPK and LC3 Ⅱ/LC3 Ⅰ were increased in group S+A (P＜0.05).Experiment Ⅱ Compared with group C,the phagocytic ability of macrophages was significantly enhanced in the other three groups (P＜0.05).Compared with group E,the phagocytic ability of macrophages was significantly enhanced in group A+E (P＜0.05),and no significant change was found in the phagocytic ability of macrophages in group A+M+E (P＞0.05).Compared with group A+E,the phagocytic ability of macrophages was significantly weakened in group A+M +E (P＜0.05).Conclusion Activating AMPK can increase the survival rate of aged mice with sepsis,and the mechanism is associated with enhancing autophagy of macrophages.
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Objective To evaluate the effect of clemastine fumarate on Toll-like receptor 4/phosphatidylinositol-3-kinase/serine-threonine kinase (TLR4/PI3K/Akt) signaling pathway during hypoxia-reoxygenation (H/R) in rat cardiomyocytes.Methods H9C2 cells of rats cultured in vitro were seeded in culture wells or dishes at a density of 1×105 cells/ml and divided into 3 groups (n=11 each) by using a random number table method:control group (group C),H/R group and clemastine fumarate group (CF group).Cardiomyocytes were exposed to 5％ CO2-95％ N2in a low-glucose DMEM medium at 37℃ for 4 h followed by 4 h reoxygenation.At 4 h of reoxygenation,the cell viability was detected by CCK-8 assay,the ultrastructure was observed with a transmission electron microscope,the expression of TLR4,PI3K,phosphorylated Akt (p-Akt) and caspase-3 was detected by Western blot,and the expression of TLR4,PI3K and caspase-3 was detected by immunofluorescence.Results Compared with group C,the cell viability was significantly decreased,the expression of TLR4 and caspase-3 was up-regulated,and the expression of PI3K and p-Akt was down-regulated in group H/R (P＜0.05).Compared with group H/R,the cell viability was significantly increased,the expression of TLR4 and caspase-3 was down-regulated,the expression of PI3K and p-Akt was up-regulated (P＜0.05),and the mitochondrial damage was significantly attenuated in group CF.Conclusion The mechanism by which clemastine fumarate alleviates H/R injury to rat cardiomyocytes may be related to inhibiting TLR4 expression and activating PI3K/Akt signaling pathway.