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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 relationship between mechanical ventilation-induced apoptosis in hippocampal neurons and mammalian taget of rapamycin ( mTOR) signaling pathway in mice. Methods Fifty healthy male C57BL∕6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 2 groups ( n=25 each) using a random number table method: control group ( group C ) and mechanical ventilation group ( group V) . The mice breathed spontaneously for 6 h in group C, and the mice were mechanically ventilated for 6 h in group V. Open field test and contextual fear conditioning test were conducted at 1 and 3 days after the end of ventilation. Hippocampal tissues were obtained at 1 day after the end of ventilation for determina-tion of the expression of mTOR, phosphorylated mTOR (p-mTOR), microtubule-associated protein 1 light chain 3Ⅱ( by Western blot) and apoptosis in hippocampal neurons ( by TUNEL) . The p-mTOR∕mTOR ratio and apoptosis index were calculated. Results Compared with group C, the time animals spent in the central square was significantly prolonged, the number of crossing the grid was reduced, the percentage of freezing time was decreased, the expression of microtubule-associated protein 1 light chain 3Ⅱwas up-regulated, and the p-mTOR∕mTOR ratio and apoptosis index were increased in group V ( P<0. 05) . Conclusion The mech-anism by which mechanical ventilation induces apoptosis in hippocampal neurons may be related to activation of mTOR signaling pathway in mice.
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Objective To evaluate the role of mammalian target of rapamycin (mTOR) in the synaptic plasticity of entorhinal area-hippocampal formation in rats with inflammatory pain.Methods Twenty-four healthy adult male Sprague-Dawley rats,weighing 180-240 g,were divided into 4 groups (n =6 each) by using a random number table method:control group (group C),inflammatory pain group (group IP),dimethyl sulfoxide (DMSO) group and mTOR inhibitor rapamycin group (group R).Inflammatory pain model was established by subcutaneous injection of 50 μl bee venom into the plantar surface of the left hindpaw.The equal volume of normal saline was subcutaneously injected into the plantar surface of the left hindpaw in group C.In group DMSO,2％ DMSO was administered by intragastric gavage for 3 days,1 ml per day,and the inflammatory pain model was established at 1 h after administration on 3rd day.In group R,rapamycin was administered by intragastric gavage for 3 days,1 ml per day,and the inflammatory pain model was established at 1 h after administration on 3rd day.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 2 h after establishing the model.The rats were sacrificed after measurement of the pain threshold,and hippocampi were removed to prepare hippocampal slices.Hippocampal CA1 region and dentate gyrus (DG region) were located with an inverted microscope.Planar microelectrode array technique was used to record the number of channels and the standardized amplitude of evoked effective field excitatory postsynaptic potentials (fEPSPs) (fEPSPs amplitude＞20％ of the baseline value) at different stimulus intensities.Results Compared with group C,MWT was significantly decreased,TWL was shortened,the number of effective fEPSP channels at different stimulus intensities was increased,and the amplitude of standardized fEPSPs in hippocampal DG and CA1 regions was increased in group IP (P＜0.05 or 0.01),and no significant change was found in the parameters mentioned above in group R (P＞0.05).Compared with group IP,MWT was significantly increased,TWL was prolonged,the number of effective fEPSP channels at different stimulus intensities was decreased,and the amplitude of standardized fEPSPs in hippocampal DG and CA1 regions was decreased in group R (P＜0.05 or 0.01),and no significant change was found in the parameters mentioned above in group DMSO (P＞0.05).Conclusion mTOR is involved in the changes in the synaptic plasticity of entorhinal areahippocampal formation in rats with inflammatory pain.
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Objective To evaluate the role of classⅠhistone deacetylase (HDAC) in myocardial ischemia-reperfusion (I∕R) injury in diabetic rats and the relationship with adenosine monophosphate-acti- vated protein kinase (AMPK)∕mammalian target of rapamycin (mTOR) signaling pathway. Methods SPF healthy adult male Sprague-Dawley rats, weighing 210-220 g, were used in this study. Type I diabetes mellitus was induced by single intraperitoneal injection of streptozotocin dissolved in citrate buffer 60 mg∕kg, and 8 weeks later the rats with type I diabetes mellitus were used for experiment. Forty-eight diabetic rats were divided into 4 groups (n＝12 each) by using a random number table method: sham operation group (group S), myocardial I∕R group (group I∕R), myocardial I∕R plus class I HDAC inhibitor MS-275 group (group I∕R+MS) and myocardial I∕R plus MS-275 plus AMPK inhibitor Compound C group ( group I∕R+MS+CC). Myocardial I∕R was induced by ligation of the left anterior descending branch of the coronary ar-tery for 45 min followed by 180 min of reperfusion in anesthetized rats. In group I∕R+MS, MS-275 10 mg∕kg was intraperitoneally injected once a day for 7 consecutive days, and myocardial I∕R was produced after the end of administration. AMPK inhibitor Compound C 0. 5 mg∕kg was intravenously injected at 30 min before ischemia in group I∕R+MS+CC. Six rats were sacrificed at the end of reperfusion for determina-tion of myocardial infarct size. Another 6 rats were selected at the end of reperfusion and sacrificed for deter-mination of the level of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in serum (by en-zyme-linked immunosorbent assay), expression of AMPK, phosphorylated AMPK ( p-AMPK), mTOR, phosphorylated mTOR (p-mTOR), ubiquitin-binding protein P62 (P62), microtubule-associated protein 1 light chain 3 Ⅰ(LC3 Ⅰ) and LC3Ⅱ in myocardial tissues (by Western blot). The ratios of p-AMPK∕AMPK, p-mTOR∕mTOR and LC3Ⅱ∕Ⅰwere calculated. Results Compared with group S, the myocardial infarct size and levels of serum CK-MB and LDH were significantly increased in I∕R, I∕R+MS and I∕R+M+CC groups, the ratios of p-AMPK∕AMPK and LC3Ⅱ∕Ⅰwere significantly increased, p-mTOR∕mTOR ratio was decreased, and P62 expression was down-regulated in group I∕R+MS (P＜0. 05), and no significant change was found in p-AMPK∕AMPK ratio, p-mTOR∕mTOR ratio, LC3Ⅱ∕Ⅰ ratio or P62 expression in I∕R and I∕R+M+CC groups (P＞0. 05). Compared with group I∕R, the myocardial infarct size and levels of serum CK-MB and LDH were significantly decreased, the ratios of p-AMPK∕AMPK and LC3Ⅱ∕Ⅰwere in-creased, p-mTOR∕mTOR ratio was decreased, and P62 expression was down-regulated in group I∕R+MS (P＜0. 05), and no significant change was found in the parameters mentioned above in group I∕R+M+CC (P＞0. 05). Compared with group I∕R+MS, the myocardial infarct size and levels of serum CK-MB and LDH were significantly increased, the ratios of p-AMPK∕AMPK and LC3Ⅱ∕Ⅰ were decreased, p-mTOR∕mTOR ratio was increased, and P62 expression was up-regulated in group I∕R+M+CC ( P＜0. 05). Con-clusion Class Ⅰ HDAC is involved in myocardial I∕R injury through enhancing AMPK∕mTOR signaling pathway-regulated level of autophagy in diabetic rats.
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Objective To evaluate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on mammalian target of rapamycin (mTOR) signaling pathways in lung tissues of rats with acute lung injury (ALI).Methods Healthy pathogen-free adult male Sprague-Dawley rats were selected,and the BMSCs were obtained and cultured in vitro.One hundred and five healthy clean adult male SpragueDawley rats,weighing 170-190 g,were divided into 5 groups (n=21 each) using a random number table:control group (group C),PBS group,group ALI,ALI plus BMSC group (group ALI+BMSCs),and ALI plus phosphate buffer solution (PBS) group (group ALI+PBS).Group C received no treatment.PBS 0.5 ml was injected via the tail vein in group PBS.Lipopolysaccharide (LPS,0.5 ml) 5 mg/kg was intraperitoneally injected to establish the model of ALI in group ALI.BMSCs (0.5 ml) 1×104 cells/ml were injected via the tail vein after intraperitoneal injection of LPS in group ALI+ BMSCs.PBS 0.5 ml was injected via the tail vein after intraperitoneal injection of LPS in group ALI+PBS.Arterial blood samples were collected for blood gas analysis at 6,24 and 48 h after injection of BMSCs.Lungs were then removed for determination of wet/dry weight ratio (W/D ratio) and expression of mTOR,nuclear factor kappa B (NF-κB) and tumor necrosis factor-alpha (TNF-o) in lung tissues (by Western blot) and for examination of the pathologic changes of lungs tissues (using haematoxylin and eosin staining).Results Compared with group C,pH value and PaO2 were significantly decreased,PaCO2 and W/D ratio were increased,and the expression of mTOR,NF-κB and TNF-α was up-regulated at each time point in ALI,ALI+BMSCs and ALI+PBS groups (P＜0.05).Compared with group ALI,pH value and PaO2 were significantly increased,PaCO2 and W/D ratio were decreased,the expression of mTOR,NF-κB and TNF-α was down-regulated at each time point (P＜0.05),and the pathologic changes of lungs tissues were significantly attenuated in group ALI+BMSCs.Conclusion The mechanism by which BMSCs reduce ALI may be associated with inhibiting mTOR signaling pathways in lung tissues of rats.
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Objective To evaluate the effect of dexmedetomidine on the mammalian target of rapamycin(mTOR)/tau protein signaling pathway in the hippocampus of aged rats after splenectomy.Methods One hundred and fifty pathogen-free healthy male Sprague-Dawley rats,aged 18 months,weighing 400-540 g,were divided into 5 groups(n=30 each)using a random number table:control group(group C),sham operation group(group S),operation group(group O),normal saline group(group NS)and dexmedetomidine group(group D).Group C received no treatment.Ten percent chloral hydrate 0.3 ml/100 g was injected intraperitoneally in group S.Group O underwent splenectomy.Dexmedetomidine 50 μg/kg was injected intraperitoneally at 5 min before splenectomy in group D.The equal volume of normal saline was injected intraperitoneally at 5 min before splenectomy in group NS.Morris water maze test was performed at day 7 after surgery.At days 1,3 and 7 after surgery,the rats were sacrificed,and the hippocampi were removed for examination of the pathological changes in the hippocampal CA3 region and for determination of the expression of mTOR protein and mRNA,tau protein mRNA and phosphor-tau protein(pS396 tau protein)(by real-time polymerase chain reaction or Western blot).Results Compared with group C,the escape latency and swimming distance were significantly prolonged,and the expression of mTOR protein and mRNA,tau protein mRNA and pS396 tau protein was up-regulated in O,D and NS groups(P0.05).Compared with group O,the escape latency and swimming distance were significantly shortened,and the expression of mTOR protein and mRNA,tau protein mRNA and pS396 tau protein was down-regulated in group D(P0.05).The pathological changes in the hippocampal CA3 region were significantly attenuated in group D as compared with group O.Conclusion The mechanism by which dexmedetomidine improves postoperative cognitive function may be associated with inhibited activation of mTOR/tau protein signaling pathway in the hippocampus of aged rats.
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Objective To investigate the effects of hydrogen-rich saline on hippocampal mammalian target of rapamycin (mTOR)/tau pathway after operation in aged rats.Methods One hundred fifty healthy male Sprague Dawley rats, aged 18 months, weighing 400-540 g, were randomly divided into 5 groups (n=30 each) using a random number table: control group (group C);sham operation group (group S);operation group (group O);hydrogen-rich saline treatment group (group HS);normal saline group (group NS).Splenectomy was performed in group O.Hydrogen-rich saline 1 ml/100 g was injected intraperitoneally at 5 min before splenectomy in group HS.Normal saline 1 ml/100g was injected intraperitoneally at 5 min before splenectomy in group NS.Morris water maze test was performed at 1, 3 and 7 days after surgery, and the escape latency and swimming distance were recorded.After the end of the test, the rats were sacrificed, and the brains were removed for examination of the pathological changes in the hippocampal CA3 region (under light microscope), and for determination of the expression of mTOR mRNA and tau mRNA (using reverse transcriptase polymerase chain reaction) and mTOR and pS396 tau (by Western blot).Results Compared with C and S groups, the escape latency and swimming distance were significantly prolonged, and the expression of mTOR protein and mRNA, tau mRNA and pS396 tau was up-regulated in O, HS and NS groups (P＜0.05).Compared with O and NS groups, the escape latency and swimming distance were significantly shortened, and the expression of mTOR protein and mRNA, tau mRNA and pS396 tau was down-regulated in group HS (P＜0.05).The pathological changes of the hippocampal tissues were mitigated in group HS when compared with group O.Conclusion The mechanism by which hydrogen-rich saline mitigates postoperative cognitive dysfunction may be associated with inhibited activation of hippocampal mTOR/tau pathway in aged rats.
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Objective To evaluate the role of mammalian target of rapamycin (mTOR) signaling pathway in dexmedetomidine-induced reduction of renal ischemia-reperfusion (I/R) injury in rats and the relationship with hypoxia-inducible factor 1 (HIF-1α).Methods Seventy-two male Sprague-Dawley rats, aged 10-12 weeks, weighing 220-260 g, were randomly divided into 4 groups (n=18 each) using a random number table: sham operation group (group S), group I/R, dexmedetomidine group (group Dex) ,and rapamicyn + dexmedetomidine group (group Rpm+Dex).Renal I/R was produced by occlusion of bilateral renal pedicles for 35 min follow by reperfusion in anesthetized rats in I/R, Dex and Rpm+Dex groups.Bilateral renal pedicles were only exposed, and then the abdominal cavity was closed in group S.Dexdetomidine 50 μg/kg was injected intraperitoneally at 30 min before I/R in group Dex.In group Rpm+Dex, rapamicyn 1.5 mg/kg and dexdetomidine 50 μg/kg were injected intraperitoneally, and renal I/R model was established 30 min later.Immediately after onset of reperfusion, and at 4 and 24 h of reperfusion (T1-3) , blood samples were collected from the caudal vein for measurement of serum creatinine and blood urea nitrogen (BUN) concentrations.After blood sampling at T1-3, the rats were sacrificed, and the renal specimens were obtained for detection of HIF-1αt, erythropoietin (EPO) and mTOR expression by Western blot.Their kidneys were removed at T3, and cut into sections which were stained with haematoxylin and eosin and examined under microscope.Acute renal tubular necrosis was scored.The cell apoptosis in renal tissues was detected by TUNEL assay, and apoptosis index (AI) was calculated.Results Compared with group S,the concentrations of serum creatinine and BUN, expression of HIF-1α, EPO and mTOR at T2,3 , AI at T3 and acute renal tubular necrosis score were significantly increased in the other three groups (P＜ 0.05).Compared with group I/R, the concentrations of serum creatinine and BUN were significantly decreased, and the expression of HIF-1α, EPO and mTOR was up-regulated at T2,3 , and AI and acute renal tubular necrosis score were decreased in group Dex (P＜0.05) , and no significant change was found in the parameters mentioned above in group Rpm + Dex (P ＞ 0.05).Conclusion The mTOR signaling pathway is involved in dexmedetomidine-induced reduction of renal I/R injury, which may be related to dexmedetomidine-produced up-regulation of HIF-1α expression in renal tissues of rats.
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Objective To evaluate the role of hippocampal mammalian target of rapamycin (mTOR) signaling pathways in the cognitive dysfunction after splenectomy in aged rats.Methods Sixtythree male Sprague-Dawley rats,aged 18 months,weighing 400-540 g,were randomly divided into 3 groups (n=21 each) using a random number table:control group (group C),operation group (group O) and mTOR inhibitor rapamycin group (group M).Morris water maze test was performed to evaluate cognitive function before operation and at 1,3 and 7 days after operation.After the end of Morris water maze test carried out at 1,3 and 7 days after operation,7 rats selected randomly in each group were sacrificed,and the brains were removed for detection of the expression of mTOR and phosphorylated tau protein at Ser-396 site (pS396 tau) in hippocampal tissues by using Western blot.Results Compared with group C,the escape latency was significantly prolonged,the ratio of time spending in the target quadrant was decreased,and the expression of pS396 tau was up-regulated at 1 and 3 days after operation,and the expression of mTOR was up-regulated at each time points after operation in group O,and the escape latency was significantly prolonged,the ratio of time spending in the target quadrant was decreased,and the expression of mTOR and pS396 tau was up-regulated at 1 and 3 days after operation in group M.Compared with group O,the escape latency was significantly shorten,the ratio of time spending in the target quadrant was increased,and the expression of pS96 tau was down-regulated at 1 and 3 days after operation,and the expression of mTOR was down-regulated at each time point after operation in group M.Conclusion The hippocampal mTOR signaling pathways are involved in the development of cognitive dysfunction after splenectomy in aged rats.
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Objective To evaluate the role of hippocampal mammalian target of rapamycin (mTOR) signaling pathway in inflammatory pain in rats.Methods Sixty adult male Sprague-Dawley rats,weighing 180-240 g,were randomly divided into 4 groups (n=6 each) by using a random number table:control group (group C),inflammatory pain group (group IP),dimethyl sulfoxide (DMSO) group and rapamycin (inhibitor of mTOR) group (group R).Inflammatory pain was produced by injection of honey bee venom 50 μl into the plantar surface of the left hindpaw.While the equal volume of normal saline was given instead in group C.In group D,2％ DMSO was injected through a gastric tube into stomach 1 ml per day lasting for 3 days,and inflammatory pain was produced at 1 h after the last injection on 3rd day.In group R,rapamycin 10 mg/kg (in 2％ DMSO) was injected through a gastric tube into stomach 1 ml per day lasting for 3 days,and inflammatory pain was produced at 1 h after the last injection on 3rd day.At 2 h after the model was established,the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.Rats were sacrificed after measurement of pain threshold,and hippocampal tissues were obtained for detection of the expression of mTOR,phosphorylated mTOR (p-mTOR),ribosomal S6 kinase (S6K) and phosphorylated S6K (p-S6K).Results Compared to group C,the MWT was significantly decreased,TWL was shortened,the expression of p-mTOR and p-S6K was up-regulated,and no significant change was found in the expression of mTOR and S6K in IP and DMSO groups,and no significant change was found in group R in the MWT,TWL and expression of p-mTOR,mTOR,p-S6K and S6K.Compared to group IP,no significant change was found in group DMSO in the MWT,TWL and expression of p-mTOR,mTOR,p-S6K and S6K,and the MWT was significantly increased,TWL was prolonged,the expression of p-mTOR and p-S6K was down-regulated,and no significant change was found in the expression of mTOR and S6K in group R.Conclusion Hippocampal mTOR signaling pathways are involved in the development of inflammatory pain in rats.
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Objective: To observe the effects of low concentration of CGP57380, an inhibitor of mitogen-activated protein kinase-interacting kinase 1 (MNK1) on the proliferation and apoptosis of human lung adenocarcinoma A549 cells. Methods: A549 cells were treated with low concentrations (1-4 μmol/L) CGP57380 for 24-72 hours, then the cell proliferation was assessed by MTT assay, and the apoptosis and cell cycle distribution were detected by flow cytometry. The expression levels of phosphorylated MNK1 (p-MNK1) and phosphorylated human eukaryotic translation initiation factor 4E (p-eIF4E) proteins in A549 cells treated with 1 μmol/L CGP57380 for 48 hours were examined by Western blotting. Results: Comparing to the control, the proliferation of A549 cells was inhibited after treatment with different concentrations of CGP57380 for 48 hours (all P < 0.05). A549 cells treated with 2-4 μmol/L CGP57380 for 72 hours were induced G2/M cell cycle arrest (both P < 0.05). CGP57380 at different concentrations could induce dose-dependent apoptosis in A549 cells. The expression levels of p-MNK1 and p-eIF4E were significantly lower in A549 cells treated with 1 μmol/L CGP57380 than those in the control (P < 0.05). Conclusion: Low concentration of CGP57380 can inhibit the proliferation of human lung adenocarcinoma A549 cells and induce their apoptosis, implying that MNK1 gene may be a candidate target for the treatment of lung cancer. Copyright © 2013 by TUMOR.
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Objective To evaluate the role of mTOR in spinal cord in the development of diabetic neuropathic pain in rats.Methods Sixty adult male Sprague-Dawley rats,aged 2 months,weighing 180-220 g,were used in the study.Forty-five rats among them were chosen randomly and diabetes mellitus was induced by intraperitoneal streptozotocin (STZ) 60 mg/kg and confirmed by blood glucose ＞ 16.7 mmol/L on day 3 after STZ injection.The left 15 rats received intraperitoneal injection of the equal volume of citric acid-sodium citrate buffer and served as normal control group (group C).Paw withdrawal threshold to von Frey filament stimulation was measured in the right hind paw before STZ injection and on 3,6,9,12,15,18,and 21 days after STZ injection.The diabetic rats with mechanical pain threshold decreasing by more than 50％ of the baseline were allocated to diabetic neuropathic pain group (group DP),and by less than 25 ％ of the baseline were allocated to diabetic non-neuropathic pain group (group NP).The rats were sacrificed at 21 days after STZ injection,and their lumbar enlargements of the spinal cord were removed for determination of the expression of mTOR and phosphorylated mTOR (p-mTOR) by Western blot.Results The expression of mTOR was significantly up-regulated in DP and NP groups when compared with group C (P ＜ 0.05),the expression of p-mTOR was up-regulated in DP group,and no significant change was found in the expression of p-mTOR in group NP (P ＞ 0.05).Compared with group NP,the expression of p-mTOR was significantly up-regulated (P ＜ 0.05),and no significant change was found in the expression of mTOR in group DP (P ＞ 0.05).Conclusion Activation of mTOR in the spinal cord is involved in the development of diabetic neuropathic pain in rats.