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Objective:To evaluate the role of protein kinase A (PKA)-cyclic adenosine monophosphate response element binding protein (CREB) signaling pathway in sevoflurane-induced reduction of cardiopulmonary bypass (CPB)-induced cognitive impairment in rats.Methods:Forty healthy male Sprague-Dawley rats, aged 4 months, weighing 300-350 g, were divided into 4 groups ( n=10 each) using a random number table method: control group (group C), group CPB, CPB+ sevoflurane group (group CS) and CPB+ sevoflurane+ PKA inhibitor H89 group (group CSH). After H89 5 μl was injected into the lateral ventricle in group CSH, the rats in group CS and group CSH were exposed to 2.4% sevoflurane for 1 h, and then the CPB model of beating heart without blood priming for 60 min was developed in CPB, CS and CSH groups.The autonomic movement ability was evaluated using the open field test at 2nd day after CPB.Morris water maze test was used to assess the cognitive function at 3rd day after CPB.The rats were sacrificed after the Morris water maze test, the brain was removed and the hippocampal tissues were isolated for determination of the apoptosis rate of hippocampal neurons (by flow cytometry) and expression of PKA, phosphorylated CREB (p-CREB) and brain-derived neurotrophic factor (BDNF) (by Western blot). Results:There was no significant difference in movement speed, distance and time of staying at the central region 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 time of staying at the original platform quadrant was shortened, the apoptosis rate of hippocampal neurons was increased, and the expression of PKA, p-CREB and BDNF was down-regulated in the other three groups ( P<0.05). Compared with group CPB, the escape latency was significantly shortened, the number of crossing the original platform was increased, the time of staying at the original platform quadrant was prolonged, the apoptosis rate of hippocampal neurons was decreased, and the expression of PKA, p-CREB and BDNF was up-regulated in group CS ( P<0.05), and no significant change was found in the indexes mentioned above in group CSH ( P>0.05). Compared with group CS, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, and the time of staying at the original platform quadrant was shortened, the apoptosis rate of hippocampal neurons was increased, and the expression of PKA, p-CREB and BDNF was down-regulated in group CSH ( P<0.05). Conclusions:Sevoflurane can reduce the apoptosis in hippocampal neurons by activating PKA-CREB signaling pathway, and thus reducing the cognitive impairment induced by CPB in rats.
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Objective To evaluate the effect of sevoflurane on hippocampal cyclic AMP (cAMP)-protein kinase A (PKA)-cAMP response element-binding protein (CREB) signaling pathway in aged rats.Methods Sixty healthy aged male Sprague-Dawley rats,aged 18 months,weighing 600-750 g,were divided into control group (C group,n =30) and sevoflurane group (group Sev,n =30) using a random number table method.Group Sev inhaled 2％ sevoflurane for 4 h.Group C inhaled the mixture of 50％ air and oxygen (2 L/min) for 4 h.Morris water maze test was performed on 1-6 days before anesthesia and at 1 day after the end of anesthesia (at the corresponding time points in group C).The animals were sacrificed and hippocampi were removed at 1,3 and 7 days after anesthesia (at the corresponding time points in group C) for determination of the hippocampal cAMP content (using enzyme-linked immunosorbent assay) and expression of hippocampal CREB,phosphorylated CREB (p-CREB) and PKA (using Western blot),and the p-CREB/CREB ratio was calculated.Results Compared with group C,the escape latency and swimming distance were significantly prolonged,the frequency of crossing the original platform was decreased,the time of staying at the original platform quadrant was shortened at 1 day after the end of anesthesia,and the expression of hippocampal cAMP and PKA was down-regulated at 1,3 and 7 days after the end of anesthesia,and the expression of CREB and p-CREB was down-regulated and p-CREB/CREB ratio was decreased at 1 day after the end of anesthesia in group Sev (P＜ 0.05).Conclusion The mechanism of sevoflurane-induced postoperative cognitive dysfunction may be related to inhibiting hippocampal cAMP-PKA-CREB signaling pathway in aged rats.
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Objective To evaluate the role of cyclic adenosine monophosphate-protein kinase A-cAMP response element-binding protein (cAMP-PKA-CREB) signaling pathway in hypoxic preconditioninginduced reduction of propofol-induced central neurotoxicity in the developing rats.Methods A total of 70 SPF male Sprague-Dawley rats,aged 7 days,weighing 10-15 g,were divided into 7 groups (n=10 each) using a random number table method:normal saline group (N group),propofol group (P group),hypoxic preconditioning plus propofol group (HP group),hypoxic preconditioning plus propofol plus PKA inhibitor H89 group (HPH group),propofol plus PKA agonist SP-CAMP group (PS group),normal saline injected via the lateral cerebral ventricle group (NI group),and 5％ dimethyl sulfoxide (DMSO) injected via the lateral cerebral ventricle group (DI group).In P group,propofol 50 mg/kg was intraperitoneally injected,and an increment of propofol 50 mg/kg was given after recovery of righting reflex.The equal volume of normal saline was given instead in N group.In HP group,hypoxic preconditioning (rats were subjected to 5 cycles of 10-min hypoxia of 8％ O2 and 1O-min normoxia of 21％ O2) was performed,and propofol was intraperitoneally injected at 2 h after the end of hypoxic preconditioning and the method was similar to those previously described in P group.In HPH group,H89 5 μmol/5 μl was injected via the lateral cerebral ventricle,and 30 min later the other treatment was similar to those previously described in HP group.In PS group,SP-CAMP 20 nmol/5 μl was injected via the lateral cerebral ventricle,and 30 min later propofol was injected using the method previously described in P group.In NI and DI groups,5 μl normal saline and 5％ DMSO were injected via the lateral cerebral ventricle,respectively.Rats were immediately sacrificed after the righting reflex was recovered,brains were removed and hippocampi were isolated and cut into sections which were stained with haematoxylin and eosin for determination of PKAc and p-CREB positive cells (by i mmuno-histochemistry) and expression of cleaved caspase-3,Bcl-2,Bax,PKAc and posphorylated (p-CREB) protein (by Western blot).Results Compared with N group,the expression of cleaved caspase-3 and Bax was significantly up-regulated,the expression of Bcl-2,PKAc and p-CREB was downregulated,and the percentage of PKAc and p-CREB positive cells was decreased (P＜0.05),hippocampal cells had irregular arrangement,and cells was atrophied in P group.Compared with P group,the expression of cleaved caspase-3 was significantly down-regulated,the expression of Bcl-2,PKAc and p-CREB was up-regulated,and the percentage of PKAc and p-CREB positive cells was increased in HP and PS groups,and the expression of Bax was down-regulated (P＜0.05),the hippocampal cells were arranged neatly,the cytoplasm was abundant,and the nuclei were visible in HP group.Compared with HP group,the expression of cleaved caspase-3 was significantly up-regulated,the expression of Bcl-2,PKAc and p-CREB protein was down-regulated and the percentage of PKAc and p-CREB positive cells was decreased (P＜0.05),the cells had irregular arrangement and shrinked,and nuclear condensation was found in cells in HPH group.Conclusion The mechanism by which hypoxic preconditioning reduces propofol-induced central neurotoxicity may be related to activating cAMP-PKA-CREB signaling pathway in the developing rats.
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Objective To evaluate the effect of electroacupuncture (EA) preconditioning on activity of AMP-activated protein kinase (AMPK) in hippocampal neurons during cerebral ischemiareperfusion (I/R) in mice.Methods A total of 60 male C57BL6 mice,aged 7 weeks,weighing 20-22 g,were randomly divided into 5 groups (n=12 each) using a random number table:control group (C group),sham operation group (S group),I/R group,acupuncture at acupoint Baihui preconditioning group (EA + I/R group),and acupuncture at non-acupoint preconditioning group (NEA + I/R group).Baihui acupoints were stimulated with electric stimulator (frequency 2 Hz/15 Hz,intensity 1 mA) for 30 min once a day for 5 consecutive days.At 24 h after the last stimulation,the model of cerebral I/R injury was established.Bilateral common carotid arteries were occluded by clipping for 15 min followed by reperfusion.Neurological deficit score (NDS) was assessed at 3 days after operation.Then the mice were sacrificed and the brains were immediately harvested for microscopic examination and for determination of apoptosis in hippocampal neurons (using TUNEL) and expression of phosphor-AMPKα (pAMPKα) and caspase-3 (by Western blot).Results Compared with group C,NDS and the number of apoptotic neurons in hippocampal CA1 region were significantly increased,and the expression of pAMPKα and caspase-3 was up-regulated in I/R and EA+I/R groups,and no significant change was found in the parameters mentioned above in group S.Compared with group I/R,NDS and the number of apoptotic neurons in hippocampal CA1 region were significantly increased,the expression of pAMPKα was up-regulated,and the expression of caspase-3 was down-regulated in group EA +I/R,and no significant change was found in the parameters mentioned above in group NEA+I/R.The pathological changes in hippocampal CA1 region were significantly attenuated in group EA + I/R as compared with group I/R.Conclusion The mechanism by which EA preconditioning mitigates apoptosis in hippocampal neurons during cerebral I/R is related to promotion of AMPK activation in mice.
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Objective To investigate the production and mechanism of chemokine (C-C motif) ligand 5 (CCL5) by macrophages in U14 cervical cancer-bearing mice during infection. Methods The U14 cervical cancer cells were injected in C57BL/6 mice to induce tumor-bearing condition. Lipopolysaccharide (LPS) was injected into C57BL/6 mice to induce infection. The protein expression of CCL5 in the serum and the CCL5 mRNA expression in inflammatory cells were measured by ELISA and fluorescence quantitative-PCR in four groups. Macrophages were induced in the tumor conditioned medium (TCM) which extracted from mice serum. The protein expression levels of CCL5, prostaglandin E2 (PGE2) and cyclic adenosine monophosphate (cAMP) in the medium and CCL5, PGE2 and cAMP mRNA expression in the macrophages were detected in different groups. In order to determine whether the inhibition was related to PGE2, selective cyclooxygenase 2(COX-2) inhibitor NS398 was used to reverse this phenomenon and protein kinase A (PKA) inhibitor H89 demonstrated the mechanism through blocking cAMP/PKA signaling pathway. Results (1) The protein and mRNA level of CCL5 in tumor-bearing mice were respectively (151±35) pg/ml and 1.0, which were lower than those in the tumor-free mice (691 ± 85) pg/ml and 4.5 ± 0.8, there were significant difference between them (all P<0.05). The protein and mRNA level of PGE2 in tumor-bearing mice were (1 198±83) pg/ml and 5.8±0.8, which were higher than those in the tumor-free mice (187±25) pg/ml and 1.0, the difference were significant (all P<0.05). The protein and mRNA level of CCL5 in tumor-free+LPS mice were (4 049±141) pg/ml and 31.5±2.0, which were higher than those in the tumor-bearing+LPS mice (1 951±71) pg/ml and 12.1±2.8, the difference were also significant (P<0.05). The protein and mRNA level of PGE2 in tumor-free+LPS mice were (676±70) pg/ml and 3.4±0.4, which were lower than those in tumor-bearing+LPS mice (2 550±382) pg/ml and 11.6±0.9, the difference were also significant (all P<0.05). (2) Macrophages were cultured in vitro using TCM derived from mice. The protein and mRNA level of CCL5 in tumor-bearing mice TCM were respectively (1 626 ± 177) pg/ml and 28.6 ± 1.2, which were higher than those in the tumor-free mice TCM [(27 ± 3) pg/ml and 1.0], there were significant difference (P<0.05). The protein and mRNA level of PGE2 in tumor-bearing mice TCM were (790 ± 156) pg/ml and 1.7 ± 0.3, which were higher than those in the tumor-free mice TCM [(448 ± 115) pg/ml, 1.0], the difference were significant (all P<0.05). The protein and mRNA level of cAMP in tumor-bearing mice TCM were (164 ± 30) pg/ml and 1.6 ± 0.3, which weres higher than those in the tumor-free mice TCM [(118 ± 25) pg/ml,1.0], the difference were significant (all P<0.05). The protein and mRNA level of CCL5 in tumor-free + LPS mice TCM were (10 475 ± 742) pg/ml and 212.0 ± 5.7, which were higher than those in the tumor-bearing+LPS mice TCM [(6 375±530) pg/ml, 142.3±2.5], the difference were significant (all P<0.05). The protein and mRNA level of PGE2 in tumor-free+LPS mice TCM were (2 438±95) pg/ml and 4.3±0.7, which weres lower than those in the tumor-bearing + LPS mice TCM [(3 441 ± 163) pg/ml, 5.9 ± 0.3], the difference were significant (all P<0.05). The protein and mRNA level of cAMP in tumor-free+LPS mice TCM were (340 ± 13) pg/ml and 4.1 ± 0.4, which were lower than those in the tumor-bearing + LPS mice TCM [(542 ± 42) pg/ml, 5.4 ± 0.5], the difference were significant (all P<0.05). (3) Using COX-2 inhibitor NS398 in the tumor-bearing+LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (7 691±269) pg/ml and 159.0±8.9, (2 820±152) pg/ml and 4.9 ± 0.3, (465 ± 8) pg/ml and 4.3 ± 0.4, respectively, and there were significant difference (all P<0.05), compared to before treatment. Using PKA inhibitor H89 in the tumor-bearing+LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (8 375±520) pg/ml and 177.0±8.8, (2 650±35) pg/ml and 4.7 ± 0.4, (368 ± 13) pg/ml and 3.1 ± 0.7, respectively, and there were significant difference (all P<0.05), compared to before treatment. Conclusion TCM of U14 cells activated macrophages to release PGE2 could inhibit the expression of CCL5 levels by cAMP/PKA signaling pathway.
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Objective To investigate the expression of aquaporins-3 (AQP3) in amniotic epithelial cells regulated by cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal pathway and to explore the mechanisms of its expression.Methods The amniotic epithelial cells were collected from 30 patients who underwent elective caesarean sections at term with normal amniotic fluid volume and primarily cultured.The cultured cells were treated with (1) forskolin groups: different concentration (0,2.5,5,50 or 100 μmol/L) of forskolin treated cells for 2 hours,and the optimal concentration of forskolin treated cells with different time (0,1,2,10 or 20 hours) ; (2)SP-cAMP groups: different concentration (0,2.5,5,50 or 100 μmol/L) of SP-cAMP treated cells for 2 hours,and the optimal concentration of SP-cAMP treated cells with different time (0,1,2,10 or 20 hours); (3)H-89 groups: different concentration (0,5,10,50 or 100 μmol/L) of H-89 treated cells for 2 hours,and the optimal concentration of H-89 treated cells with different time (0,1,2,10 or 20 hours).The level of intracellular cAMP and activity of PKA were detected by using ELISA,and immunohistochemistry was used to detect the localization of AQP3,the protein expression of total cAMP-response element binding protein (CREB) and phospho-CREB (p-CREB) and AQP3 were assessed by western blot analysis.Cell proliferation was assessed by cell counting kit-8 (CCK-8)assay.Results (1) The brown staining of AQP3 was detected in both cell membrane and cytoplasm in each group.(2) There was no significant change of the cell proliferation rate among groups with different concentration of forskolin,SP-cAMP and H-89 treatment (P ＞ 0.05).(3) After different concentration of forskolin treated 2 hours,the expression of total CREB had no significant difference among them(P ＞ 0.05).While the expression of cAMP level,PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 2.5 μmol/L,5 μmol/L,50 μmol/L forskolin group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 5 μmol/L forskolin group were higher than that in 2.5 μmol/L and 50 μmol/L (P ＜ 0.05).The optimal forskolin concentration was 5 μmol/L.(4) After different concentration of SP-cAMP treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 5 μμmol/L,50 μmol/L SP-cAMP group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 50 μmol/L SP-cAMP group were higher than that in 5 μmol/L (P ＜0.05).The optimal SP-cAMP concentration was 50 μmol/L (5) After different concentration of H-89 treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were lower in 10 μmol/L,50 μmol/L and 100 μmol/L H-89 group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 10 μmol/L H-89 group were lower than that in 50 μmol/L,100 μmol/L (P ＜ 0.05).The optimal H-89 concentration was 10 μmol/L.(6) p-CREB and AQP3 protein expression were significantly lower in 5 μmol/L forskolin combined 10 μmol/L H-89 incubating 2 hours group when compared with 5 μmol/L forskolin,but higher than that in 10 μmol/L H-89 treated group (P ＜ 0.05).Total CREB was no significant difference among the three groups (P ＞ 0.05).Conclusion cAMP-PKA signal transduction pathway may regulate AQP3 protein expression in human amniotic epithelial cells.
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Objective To evaluate the role of cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal transduction pathway in lidocaine-induced up-regulation of the expression of surfactant protein-A (SP-A) in rat alveolar epithelial type Ⅱ cells (AEC Ⅱ).Methods Healthy male Sprague-Dawley rats were sacrificed and AEC Ⅱ were isolated,purified and incubated in 24-well culture plates (100μd/hole) with density of 1 × 106/ml.After being incubated for 2 h,the culture medium was replaced with serum-free medium DMEM.The cells were randomly divided into 4 groups (n =48 each):control group (group C),forskolin (adenylate cyclase agonist) group (group F),lidocaine 200 μg/ml group (group L),and PKA inhibitor H89 + L group (group P+ L).Forskolin 10 μmol/ml was added to DMEM in group F.Lidocaine 200 μg/ml was added to DMEM in group L.H89 10μnol/ ml was added to DMEM and AEC Ⅱ were incubated for 10 min,and then lidocaine 200 μg/ml was added in group P + L.At 6,12 and 24 h of incubation (T1-3),cAMP content and PKA activity (using ELISA),and expression of SP-A mRNA (by real-time fluorescent quantitative PCR) and SP-A (by Western blot) were measured.Results Compared with group C,the expression of SP-A mRNA and SP-A was significantly upregulated,and cAMP level and PKA activity were increased at T1-3 in group F and at T2,3 in group L.Compared with group L,the expression of SP-A and SP-A mRNA was down-regulated,PKA activity was decreased,and no significant change was found in cAMP level at T1-3 in group P + L.Conclusion Lidocaine can up-regulate the expression of SP-A in AEC Ⅱ of rats through activating cAMP-PKA signal transduction pathway.
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Objective To investigate the mechanism of cyclic adenosine monophosphate / protein kinase A signal transduction pathway in severe acute pancreatitis(SAP)-associated lung injury.Methods Seventy-two male healthy SD rats were completely randomized into three groups:sham operation (SO) group(n =8),SAP group and SAP plus H89 (cAMP inhibitor) group,then the latter two groups were divided into four sub-groups with eight rats in each sub-group according to the sampling time of 3,6,12 and 24 h,and the total numbers of groups were nine.The content change of TNF-α and IL-1β in serum,protein levels of cAMP-dependent protein kinase catalytic subunit (PKA C) and phosphorylated vasodilator-stimulated phosphoprotein(p-VASP) and the expression of VSAP mRNA in lung tissue were detected by enzyme-linked immunosorbent assay (ELISA),immunohistochemistry and quantitative real time PCR,respectively.Pathological changes of the pancreas and lung tissues were also observed.Results Compared with the SO group,the serum levels of TNF-α and IL-1β in the SAP group were obviously increased at different time points(P ＜0.05).Pathological changes of the pancreas and lung tissues were aggravated significantly.The protein levels of PKA C,p-VASP and the expression of VSAP mRNA in lung tissue were increased significantly (P ＜0.05)which peaked at 12 h in the SAP group [TNF-α was (266.07 ± 17.14) pg/mL,IL-1β(169.17 ±25.92) pg/mL,PKA C(210.69 ±6.32) × 103,p-VASP (56.62 ±0.57) × 103,VASP mRNA(2.06 ±0.21)],which had positive correlation with the serum level of TNF-α and IL-1β.Compared with the SAP group,pathological changes of the pancreas and lung tissues were alleviated significantly,the protein levels of PKA C,p-VASP and the expression of VSAP mRNA in lung tissue were decreased significantly in the SAP plus H89 group at different time points(P ＜ 0.05).Conclusion The cyclic adenosine monophosphate / protein kinase A signal transduction pathway is found to participate in the pathological process of SAP-associated lung injury through the up-regulations of TNF-α,IL-1 β and phospho-VASP.
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Objective To evaluate the changes in 5'-adenosine monophosphate-activated protein kinase (AMPK) signal transduction pathway in hippocampal neurons of aged rats during transient global cerebral ischemia/reperfusion (I/R).Methods Ninety-six aged male Sprague-Dawley rats aged 18-22 months,weighing 450-600 g,were randomly allocated to one of two groups (n=48 each):sham operation group (group OS) and transient global cerebral I/R group (group OTIR).Ninety-six yong male Sprague-Dawley rats aged 3 months,weighing 200-250 g,were randomly divided into 2 groups (n=48 each):sham operation group (group AS) and transient global cerebral I/R group (group ATIR).The global cerebral I/R was produced by 3 min four-vessel occlusion followed by reperfusion according to Pulsinelli.On 3,5 and 7 days of reperfusion,12 rats in each group were chosen and sacrificed.Their brains were removed and hippocampal CA1 region was dissected for detection of neuronal apoptosis (by TUNEL) and expression of phosphorylated AMPKα (p-AMPKα) (by Western blot).The apoptotic rate (AR) was calculated.Results Compared with OS group,the AR was significantly increased and the expression of p-AMPKα was up-regulated at each time point in OTIR group,and the AR was significantly decreased and the expression of p-AMPKα was down-regulated at each time point in AS group (P ＜ 0.05).Compared with AS groupthe AR was significantly increased at each time point and the expression of p-AMPKα was up-regulated on day 3 and 5 of reperfusion in ATIR group (P ＜ 0.05).The AR was significantly lower at each time point and the expression of p-AMPKα was down-regulated on day 5 and 7 of reperfusion in ATIR group than in OTIR group (P ＜ 0.05).Conclusion Transient global cerebral I/R can activate AMPK signal transduction pathway in hippocampus of aged rats.The activation of AMPK signal transduction pathway is stronger and the cerebral I/R injury is more severe in aged rats than in young rats.
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Objective To evaluate the effects of propofol anesthesia on hippocampal protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling pathway in neonatal rats.Methods One hundred and seventy-five male Sprague-Dawley rats,aged 7 days,weighing 8-15 g,were randomly divided into 5 groups (n =35 each) using a random number table:control group (C group) and propofol 25,50,100 and 200 mg/kg groups (P~ groups).Groups P1 and P2 received intraperitoneal propofol 25 and 50 mg/kg,respectively.Groups P3 and P4 received intraperitoneal propofol 100 and 200 mg/kg,respectively,and after righting reflex completely recovered,an increment of propofol 50 mg/kg was given until the total amount was finished.Five animals in each group were chosen and arterial blood samples were obtained immediately after the animals were fully awake for blood gas analysis.Five animals in each group were chosen at 2 h after fully awake and the age of 9 weeks,the rats were sacrificed and their brains were removed for microscopic examination of the ultrastructure of hippocampal neurons and for determination of PKA mRNA,CREB mRNA,PKA protein and pCREB protein in hippocampus (using RT-PCR and Western blot analysis).Results There was no significant difference in the indexes of blood gas analysis anong the five groups (P ＞ 0.05).Nuclear swelling and fragmentation,chromatin condensation,apoptotic bodies,decreased number of synapses and widened synaptic space were observed in P2,P3 and P4 groups.Compared with group C,the expression of PKA mRNA,CREB mRNA,PKA protein and pCREB protein was significantly down-regulated at 2 h after fully awake and the age of 9 weeks in P1,P2,P3 and P4 groups (P ＜ 0.05).Conclusion The mechanism by which propofol anesthesia induces neurotoxicity in neonatal rats may be related to inhibition of the activity of PKA-CREB signaling pathway.
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Objective To evaluate the role of c-AMP-protein kinase A (cAMP-PKA) on the up-regulation of heme oxygenase-1 (HO-1) expression during lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats.Methods Forty-eight healthy male Sprague-Dawley rats,weighing 180-220 g,aged 2.5-3.0 months,were randomly divided into 4 groups (n =12 each)∶ normal control group (group C),ALI group (group ALI),H89 +ALI group (group H + ALI) and H89 group (group H).In group C,normal saline (solvent for LPS) 0.5 ml was injected via the femoral vein and normal saline (solvent for H89) 0.5 ml was injected subcutaneously 2 h later.In group ALI,10 mg/kg LPS 0.5 ml was injected via the femoral vein and normal saline 0.5 ml was injected subcutaneously 2 h later.In group H +ALI,10 mg/kg LPS 0.5 ml was injected via the femoral vein and 5 mg/kg H89 0.5ml was injected subcutaneously 2 h later.In group H,normal saline 0.5 ml was injected via the femoral vein and 5 mg/kg H89 0.5 ml was injected subcutaneously 2 h later.The rats were then sacrificed at 6 h after iv injection of LPS and the lungs were removed for microscopic examination and lung water content.The pathological changes of the lung were scored.The expression of HO-1 and PKA (by Western blot) and HO-1 mRNA (by RT-PCR) was detected.Results Compared with group C,the pathological score and lung water content were significantly increased,and the expression of HO-1,PKA and HO-1 mRNA was up-regulated in groups ALI and H +ALI (P ＜0.05),and no significant change was found in the parameters mentioned above in group H (P ＞ 0.05).The pathological score and lung water content were significantly higher,and the expression of HO-1,AP-1 and HO-1 mRNA was significantly lower in group H + ALI than in group ALI (P ＜ 0.05).Conclusion Activation of signaling pathway c-AMP-PKA is involved in the up-regulation of HO-1 expression during LPS-induced ALI in rats.
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It is not yet understood how the enhanced expression of pancreatic adenocarcinoma up-regulated factor (PAUF; a novel oncogene identified in our recent studies), contributes to the oncogenesis of pancreatic cells. We herein report that PAUF up-regulates the expression and transcriptional activity of beta-catenin while the suppression of PAUF by shRNA down-regulates beta-catenin. The induction of beta-catenin by PAUF is mediated by the activities of Akt and GSK-3beta, but inhibition of downstream ERK does not reduce beta-catenin expression. To test whether PAUF emulates either the Wnt3a-mediated or the protein kinase A-mediated signaling pathway for the stabilization of beta-catenin, we examined the phosphorylation status of beta-catenin in the presence of PAUF compared with that of beta-catenin during treatment with Wnt3a or dibutyryl cAMP, a cell permeable cyclic AMP analogue. PAUF expression induces phosphorylation at Ser-33/37/Thr-41 and Ser-675 of beta-catenin but no phosphorylation at Ser-45, indicating that a unique phosphorylation pattern of beta-catenin is caused by PAUF. Finally, the expression of PAUF up-regulates both cyclin-D1 and c-Jun, target genes of beta-catenin, leading to a rapid proliferation of pancreatic cells; conversely decreased PAUF expression (by shRNA) results in the reduced proliferation of pancreatic cells. Treatment with hexachlorophene (an inhibitor of beta-catenin) reduces the proliferation of pancreatic cells despite the presence of PAUF. Taken together, we propose that PAUF can up-regulate and stabilize beta-catenin via a novel pattern of phosphorylation, thereby contributing to the rapid proliferation of pancreatic cancer cells.
Subject(s)
Humans , Adenocarcinoma/metabolism , Cell Line, Tumor , Cell Proliferation , Cyclin D1/metabolism , Gene Expression Regulation, Neoplastic , Glycogen Synthase Kinase 3/metabolism , HEK293 Cells , Lectins/genetics , Pancreatic Neoplasms/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction , Up-Regulation , beta Catenin/geneticsABSTRACT
Objective To investigate the effect of isoflurane on the levels of protein kinase A (PKA) and protein kinase C (PKC) in hippocampus in rats. Methods Thirty-six 3-month-old male SD rats weighing 180-220 g were randomly divided into 3 groups ( n = 12 each): group Ⅰ underwent the cognitive function test without being pretreated with isoflurane inhalation (group C); group Ⅱ and Ⅲ inhaled 1.2% isoflurane for 4 h and underwent the cognitive function test 2 days and 2 weeks later respectively (group Ⅰso1,Iso2). Morris water maze was used to assess the cognitive function and the escape latency was recorded. The animals were killed immediately after the test.The hippocampus was isolated for determination of the expression and activities of PKA and PKC.Results The escape latency was significantly longer in group Ⅲ than in group Ⅰ.The expression of PKA and PKC was significantly down-regulated and the activities of PKA and PKC were significantly decreased in group Ⅱand Ⅲ as compared with group Ⅰ . There was no significant difference in the expression and activities of PKA and PKC between group Ⅱ and Ⅲ . Conclusion Four hour 1.2% isoflurane inhalation can decrease cognitive function by inhibiting the levels of PKA and PKC in hippocampus.
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Objective To investigate the changes in cerebral cAMP and PKA levels during development of acute opioid tolerance induced by remifentanil and to determine whether post-receptor cAMP/PKA signaling pathway is involved in the process. Methods Fifty-six male Kunming mice weighing 25-35 g were randomly divided into 5 groups: group Ⅰ control (C) (n=8); group Ⅱ received morphine infused intraperitoneally (IP) at 0.6 μg'kg-1·min-1 for 120min(M) (n=8); group Ⅲ,Ⅳ,Ⅴ received remifentnil infused IP at 0.4, 0.8 and 1.6 μg·kg-1·min-1 for 120 min(R1=8, R2n=8; R3 n=24).Control group received IP infusion of normal saline. Tail-flick test was performed td measure the response of animals to a thermal nociceptive stimulus before IP infusion, at 30, 60, 90 and 120 min after beginning of IP infusion and at 15, 30, 45 and 60 min after termination of IP infusion. Eight animals were decapitated at 60 min after termination of IP infusion in all 5 groups and the other 16 animals in group R3 were decapitated at 30 and 45 min after termination of IP infusion (n=8 each) for determination of intracellular contents of cAMP and activities of PKA in cerebral cortex and inferior colliculus-striatum by ELISA or radioactive isotope [32p,] ATP-catalyzing assay. Results The tail-flick latency was significantly prolonged during IP infusion as compared with the baseline before infusion in group M, R1 , R2 and R3 but became significantly shorter at 30 and 45 min after infusion than the baseline values in group R1, R2 and R3indicating hyperalgesia after remifentauil infusion. The cerebral contents of cAMP and PKA activities at 60 min after termination of infusion were comparable or decreased in group M, R1, R2 and R3 as compared with group C. There was no significant difference in cerebral cAMP contents and PKA activities at 30, 60 and 45 min after IP remifentanil infusion in group R3. Conclusion Remifentanil can induce acute hyperalgesic effect on mice, and there is no up-regulation of post-receptor cAMP/PKA signaling pathway in the acute opioid tolerance, which is not similar to that chronic opioid tolerance.
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Objective To investigate the role of cAMP-PKA signal transduction pathway in the ischemie pleeonditioning-induced attenuation of ischemia.1eperfusion(ITR)injury in isohted rat hearts.Methods Fifty healthy adult SD rats weighing 300-350 g were anesthetized with intraperitoneal(IP)pentobarbital 50 meg/kg.Their he.arts were excised and perfused in a Langendorff apparatus with 37℃ oxygenated(95%O2-5%CO2)modified K-H solution at a constant pressure of 70 cm H2O,and randomly divided into 5 groups(n=10 each):group Ⅰ I/R;group Ⅱ ischemic preconditioning(IPC);groupⅢH89(PKA inhibitor);group Ⅳ PDTC(NF-κB inhibitor)and group Ⅴ db-cAMP.The experiment started after 10 min stabilization.The isolated hearts were tinct perfuzed for 30 min.followed by 60 min ischemia and 30 min leperfusion in group I/R(Ⅰ).Group IPC(Ⅱ)w88 subjected to 3 episodes of 5 min ischemia at 5 min intervals before I/R.Group Ⅲ and Ⅳ received 5 min perfnsion withH89 10 μmol/L and PDTC 100 μmol/L 3 times at 5 min intervals respectively before I/R.Group Ⅴ was peffnsed with db-cAMP 200 μmol/L for 30 min before I/R.Left ventricular developed pressure(LVDP)and ± dp/dtu were measured at stabilization period and 10, 20, 30 rain of reperfusion. Coronary flow (CF) was measured at stabilization period and 30 min of reperfusion and activities of LDH and creafinc kinase (CK) in the coronary effluent were determined. The myocardial specimens were obtained at 30 rain of reperfusion for determination of NF-κB-DNA binding activity (by EMSA) and expression of TNF-κ mBNA (by RT-PCR ) and p-CBEB (Ser133) (by Western blot). Results Compared with 1/R group, NF-κB-DNA binding activity and TNF-α mRNA expression were significandy decreased, ± dp/dt and CF were significandy increased, CK and LHD activities in the coronary effluent were significantly decreased in group IPC and db-cAMP (group Ⅱ , Ⅴ ) and p-CREB (Ser133) expression was significantly increased in group IPC, PDTC and db-cAMP (group Ⅱ , Ⅳ,Ⅴ ). Compared with IPC group, NF-κB-DNA binding activity and TNF-α mBNA expression were significantly increased, ± dp/dtmax, LVDP and CF were significantly decreased, CK and LDH activities were significantly increased in group H89 and PDTC (group Ⅲ, Ⅳ ) and p-CREB (Ser133) expression was significantly decreased in group H89(group Ⅲ ). Conclusion lschemic preconditioning can attenuate I/R injury in isolated hearts by inhibition of NF-κB-DNA binding activity via cAMP-PKA signal transduction pathway which reduces gene transcription of inflammatory cytokine.
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Previously we demonstrated that ATP released from LPS-activated microglia induced IL-10 expression in a process involving P2 receptors, in an autocrine fashion. Therefore, in the present study we sought to determine which subtype of P2 receptor was responsible for the modulation of IL-10 expression in ATP-stimulated microglia. We found that the patterns of IL-10 production were dose-dependent (1, 10, 100, 1,000 micrometer) and bell-shaped. The concentrations of ATP, ATP-gammaS, ADP, and ADP-beta S that showed maximal IL-10 release were 100, 10, 100, and 100 micrometer respectively. The rank order of agonist potency for IL-10 production was 2'-3'-O-(4-benzoyl)-benzoyl ATP (BzATP) = dATP > 2-methylthio-ADP (2-meSADP). On the other hand, 2-methylthio-ATP (2-meSATP), alpha,beta-methylene ATP (alpha,beta-meATP), UTP, and UDP did not induce the release of IL-10 from microglia. Further, we obtained evidence of crosstalk between P2 receptors, in a situation where intracellular Ca2+ release and/or cAMP-activated PKA were the main contributors to extracellular ATP-(or ADP)-mediated IL-10 expression, and IL-10 production was down- regulated by either MRS2179 (a P2Y1 antagonist) or 5'-AMPS (a P2Y11 antagonist), indicating that both the P2Y1 and P2Y11 receptors are major receptors involved in IL-10 expression. In addition, we found that inhibition of IL-10 production by high concentrations of ATP-gammaS (100 micrometer) was restored by TNP-ATP (an antagonist of the P2X1, P2X3, and P2X4 receptors), and that IL-10 production by 2-meSADP was restored by 2meSAMP (a P2Y12 receptor antagonist) or pertusis toxin (PTX; a Gi protein inhibitor), indicating that the P2X1, P2X3, P2X4 receptor group, or the P2Y12 receptor, negatively modulate the P2Y11 receptor or the P2Y1 receptor, respectively.
Subject(s)
Animals , Rats , Adenosine Diphosphate/analogs & derivatives , Adenosine Triphosphate/analogs & derivatives , Adenylyl Cyclases/antagonists & inhibitors , Calcium/metabolism , Chelating Agents/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Extracellular Space/drug effects , Gene Expression Regulation/drug effects , Interleukin-10/biosynthesis , Microglia/drug effects , RNA, Messenger/genetics , Rats, Sprague-Dawley , Receptor Cross-Talk/drug effects , Receptors, Purinergic P2/agonists , Thionucleotides/pharmacologyABSTRACT
Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ia. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.
Subject(s)
Male , Humans , Time Factors , Protein Kinase Inhibitors/pharmacology , Phosphorylation , Lysophospholipids/pharmacology , Luciferases/genetics , Gene Expression/drug effects , Fibroblasts/cytology , Diploidy , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP/metabolism , Cells, Cultured , Cellular Senescence/physiology , Catalytic Domain/geneticsABSTRACT
Here we determined which radiation-responsive genes were altered in radioresistant CEM/IR and FM3A/IR variants, which showed higher resistance to irradiation than parental human leukemia CEM and mouse mammary carcinoma FM3A cells, respectively and studied if radioresistance observed after radiotherapy could be restored by inhibition of protein kinase A. The expressions of DNA-PKcs, Ku70/80, Rad51 and Rad54 genes that related to DNA damage repair, and Bcl-2 and NF-kappaB genes that related to antiapoptosis, were up-regulated, but the expression of proapototic Bax gene was down-regulated in the radioresistant cells as compared to each parental counterpart. We also revealed that the combined treatment of radiation and the inhibitor of protein kinase A (PKA) to these radioresistant cells resulted in synergistic inhibition of DNA-PK, Rad51 and Bcl-2 expressions of the cells, and consequently restored radiosensitivity of the cells. Our results propose that combined treatment with radiotherapy and PKA inhibitor can be a novel therapeutic strategy to radioresistant cancers.
Subject(s)
Animals , Humans , Mice , Apoptosis/drug effects , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , DNA Damage/drug effects , DNA Repair/drug effects , Gamma Rays , Gene Expression Regulation, Neoplastic/radiation effects , Genes, bcl-2 , Neoplasm Proteins/genetics , Neoplasms/enzymology , Radiation Tolerance/geneticsABSTRACT
AIM: To investigate the inhibitory effects of cholecystokinin octapeptide(CCK-8) on nuclear factor-?B(NF-?B) activities stimulated by lipopolysaccharide(LPS) by using forskolin,the activator of adenylate cyclase,and PKA inhibitor H-89 in rat pulmonary interstitial macrophages(PIMs).METHODS: PIMs were isolated and purified.EMDA was applied to detect NF-?B activities and Western blotting was used to analyze the I?B-? protein level in rat PIMs.RESULTS: The NF-?B activity was not detected in normal control rat PIMs.The NF-?B activity in LPS-treated rat PIMs was obviously higher than that in control group(P0.05).The NF-?B activity in CCK+LPS group and LPS+Fsk group were obviously lower than that in LPS group(P
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Objective To study the molecular biological mechanism of the protective effect of angelica on myocardium during ischemia reperfusion Methods The myocardial ischemia/reperfusion was induced with the 40 min cross clamp/120 min declamping of anterior decending coronary artery Forty SD rats were randomly divided into 3 groups: group A without myocardial ischemic reperfusion , group B with intravenous adminisrtration of normal saline 0 8ml/100g before ischemia reperfusion , and group C with intravenous adminisrtration of 25% angelica 0 8ml/100g before ischemia reperfusion The content of protein kinase C (PKC) in cardiac myocyte was measured with immunohistochemical method, and the PKC activity with the isotope lable method Results Compared with those in group B, the myocardial infarct size reduced significantly in group C (P0 05) but increased obviously in group C (P0 05) The PKC activity was significantly higher in group C than that in group A(P