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Objective To evaluate the effect of propofol on subunit 2B-containing N-methyl-D-aspartate receptor (NR2B)/calcium/calmodulin-dependent protein kinase Ⅱ alpha (CaMKⅡα) signaling pathway in brain injury induced by hepatic ischemia-reperfusion (I/R) in preadolescent mice.Methods Sixty-four healthy clean-grade C57BL/6 mice,aged 2 weeks,weighing 4-6 g,were randomized into 4 groups (n=16 each) using a random number table method:sham operation group (S group),hepatic I/R group (HI/R group),propofol control group (P group),and propofol plus hepatic I/R group (P+ HI/R group).The model of 70％ liver I/R injury was established by clamping the left and middle lobe vascular trunk in anesthetized mice.Propofol 20 mg/kg was intraperitoneally injected before operation at 30 min before establishing the model in P and P+HI/R groups.The equal volume of normal saline was given instead in P and P + HI/R groups.Eight mice of each group were sacrificed at 6h of reperfusion,hippocampal tissues were obtained for examination of pathological changes of hippocampal tissues and for determination of neuronal apoptosis (by TUNEL) and expression of NR2B,phosphorylated NR2B (p-NR2B),CaMKⅡα and phosphorylated CaMKⅡα (p-CaMKⅡα) (by Western blot).The remaining 8 mice in each group were used for Morris water maze test at 1 month after establishing the model.Apoptosis index was calculated.Results Compared with group S,the escape latency was significantly prolonged,the percentage of the time of staying at the original platform quadrant was decreased,the expression of p-NR2B and p-CaMKⅡα was up-regulated,and apoptosis index was increased in HI/R and P+HI/R groups (P＜0.05),and no significant change was found in the parameters mentioned above in group P (P＞0.05).Compared with group HI/R,the escape latency was significantly shortened,the percentage of the time of staying at the original platform quadrant was increased,the expression of p-NR2B and p-CaMKⅡα was down-regulated,and apoptosis index was decreased (P＜0.05),and the pathological changes of hippocampal tissues were significantly attenuated in group P+HI/R.Conclusion The mechanism by which propofol reduces brain injury induced by hepatic I/R may be related to inhibiting NR2B/CaMKⅡα signaling pathway in preadolescent mice.
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Objective To evaluate the effect of propofol on the expression of programmed death-ligand-1 (PD-L1) in pancreatic cancer cells and the relationship with NMDA/Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ)/hypoxia-inducible factor-1α (HIF-1α) pathway.Methods Human pancreatic cancer cells were divided into 5 groups (n=16 each) by a simple random sampling method:control group (group C),propofol group (group P),KN93 (CaMK Ⅱ inhibitor) group,MK801 (NMDA receptor antagonist) group and propofol plus rapastinel (NMDA receptor agonist) group (group PR).Cells were cultured in DMEM supplemented with 10％ fetal bovine serum in group C.Cells were incubated for 8 h with 50 μmol/L propofol in group P.Cells were incubated for 8 h with 10 μmol/L KN93 in group KN93.Cells were incubated for 8 h with 500 μmol/L MK801 in group MK801.Cells were incubated for 8 h with 50 μmol/L propofol and 20 μmol/L rapastinel in group PR.After the end of treatment in each group,the cell viability was measured using CCK8 assay,the expression of PD-L1,HIF-1α,CaMK Ⅱ and phosphorylated CaMK Ⅱ (p-CaMK Ⅱ) was detected by Western blot,and intracellular calcium concentrations were determined by Fluo3/AM probe.Results Compared with group C,the cell viability was significantly decreased,the expression of PD-L1,HIF-1α and p-CaMK Ⅱ was down-regulated,and intracellular calcium concentrations were decreased in P,KN93 and MK801 groups (P＜0.05),and no significant change was found in group PR (P＞0.05).Compared with group P,the cell viability was significantly enhanced,the expression of PD-L1,HIF-1α and p-CaMK Ⅱ was up-regulated,and intracellular calcium concentrations were increased in group PR (P＜0.05).Conclusion The mechanism by which propofol inhibits the malignant potential of pancreatic cancer cells may be related to inhibiting NMDA/CaMK Ⅱ/HIF-1α pathway and down-regulating PD-L1 expression.
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Objective@#To investigate the effect of zoledronate on protein interaction between Ca2+/calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and calmodulin and protein expression of nuclear factor of activation of T cells-1 (NFATc1) and tartrate resistant acid phosphatase (TRAP) during osteoclast differentiation. @*Methods@#Mouse RAW264.7 cells were divided into group A and B and were cultured. Group A was induced with 50 mg/L receptor activator of NF-κB ligand (RANKL) for osteoclastogenesis, and group B was treated with 1×10-6 zoledronate for two days from day 2. Co-immunoprcipitation (Co-IP) and reverse Co-IP were used to detect the protein-binding between CaMKⅡ and calmodulin. Western-blotting and immunofluorescent cytochemistry were also used to detect the protein level of NFATc1 and TRAP in both groups. Osteoclast formation was also analyzed. @*Results@#In group B, the number of osteoclasts, number and size of dentin resorption lacunaes were 11.3±1.5, 8.7±2.1 and (5 034.4±775.4) μm2 respevtively, which were significantly lower than those (37.7±5.7, 23.0±4.0 and [15 042.7±1 906.0] μm2) in group A (P<0.01). Co-IP and reverse Co-IP examination indicated that protein-binding between CaMKⅡ and calmodulin significantly decreased by 59.8% and 50.9% in group B compared with group A (P<0.01). The protein level of calmodulin and CaMKⅡ in total cellular proteins also significantly decreased by 52.1% and 51.5% in group B compared with group A (P<0.01). NFATc1 and TRAP protein decreased by 52.4% and 38.9% in group B than in group A (P<0.01), respectively. @*Conclusions@#Zoledronate could significantly inhibit protein-binding between CaMKⅡ and calmodulin and down-regulate protein level of NFATc1 and TRAP.
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Objective To evaluate the effect of sevoflurane on hippocampal calcium/calmodulindependent protein kinase Ⅱ (CaMK Ⅱ)/cyclic adenosine monophosphate response element-binding protein (CREB) signaling pathway in aged rats.Methods Sixty pathogen-free healthy male Sprague-Dawley rats,aged 18 months,weighing 600-750 g,were divided into 2 groups (n=30 each) using a random number table:control group (group C) and sevoflurane group (group Sev).Group Sev inhaled 2％ sevoflurane in the mixture of 50％ air and oxygen (2 L/min) 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 6 days before anesthesia and 1 day after anesthesia.The escape latency,swimming distance,frequency of crossing the original platform and time of staying at the platform quadrant Ⅱ were recorded.On 1,3 and 7 days after anesthesia,the rats were sacrificed,and the hippocampus was obtained for determination of the expression of CaMK Ⅱ,phosphorylated CaMK Ⅱ,CREB and phosphorylated CREB by Western blot.Results Compared with group C,the escape latency and swimming distance were significantly prolonged,the frequency of crossing the original platform was decreased,and the time of staying at the platform quadrant Ⅱ was reduced on 5th day of training and 1 day after anesthesia,and the expression of CaMK Ⅱ,phosphorylated CaMK Ⅱ,CREB and phosphorylated CREB was down-regulated after anesthesia in group Sev (P＜ 0.05).Conclusion Sevoflurane leads to cognitive decline through inhibiting hippocampal CaMK Ⅱ/CREB signaling pathway in aged rats.
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Objective To evaluate the role of spinal CX3C chemokine receptor 1 (CX3CR1) in inflammatory pain and the relationship with calmodulin (CaM)-calmodulin-dependent protein kinaseⅡ(CaMKⅡ) signaling pathways in mice.Methods Ninety-six pathogen-free healthy male C57BL6 mice,weighing 25-27 g,were divided into 3 groups using a random number table:control group (group C,n=30),inflammatory pain group (group IP,n=36) and CX3CR1 antagonist group (group CA,n=30).Inflammatory pain was induced by injecting complete Freund′s adjuvant (CFA) 50 μl into the plantar surface of right hind paws in IP and CA groups,while the equal volume of normal saline was given instead in group C.In group CA,CX3CR1 antagonist (diluted to 1 μg/5 μl in phosphate buffer solution) was intrathecally injected at 1 h before CFA injection.The thermal paw withdrawal latency (TWL) was measured at 30 min before CFA injection (T0) and 30 min,1 h,2 h and 4 h after CFA injection (T2-4).The animals were then sacrificed,and the spinal cord was removed for determination of the expression of phosphorylated CaMKⅡ (p-CaMKⅡ),phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB) and c-fos (by Western blot) and expression of CaMKⅡ,CREB and c-fos mRNA (using real-time polymerase chain reaction).Immunofluorescence was used to determine that p-CAMKⅡ was expressed in microglia.Results Compared with group C,the TWL was significantly shortened at T2-4,and the expression of p-CaMKⅡ,p-CREB and c-fos protein and mRNA was up-regulated at T1-4 in IP and CA groups (P<0.05).Compared with group IP,the TWL was significantly prolonged at T2-4,and the expression of p-CaMKⅡ,p-CREB and c-fos protein and mRNA was down-regulated at T1-4 in group CA (P<0.05).p-CaMKⅡ was co-expressed with the microglial specific biomarker.Conclusion CX3CR1 is involved in the development and maintenance of inflammatory pain through activating CaM-CaMKⅡsignaling pathways in mice.
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Objective To investigate the influence of dexmedetomidine on expressions of protein kinase c (PKC)γ, cal?cium/calmodulin-dependent protein kinase (CaMK)Ⅱαand pCaMKⅡαin spinal cord in rats with incisional pain (IP) and remifentanil-induced hyperalgesia. Methods Forty male Sprague-Dawley rats, aged 2-3 months, weighing 240-260 g, were randomly divided into 5 groups (n=8 each):blank control group (group C), remifentanil+incisional pain group (group R+I), dexmedetomidine + remifentanil + incisional pain group (group D+R+I), dexmedetomidine + remifentanil + incisional pain+phorbol myristate acetate+DMSO group (group D+R+I+P+DMSO) and dexmedetomidine+remifentanil+incisional pain+DMSO group (group D+R+I+DMSO). The incisional pain rat model was established by a plantar incision in left hind paw. Remifentanil was infused at a rate of 1.2μg·kg-1·min-1 for 90 min via the caudal vein. Dexmedetomidine was adminis?tered subcutaneously at a dose of 50μg/kg at 30 min before plantar incision. Phorbol myristate acetate and DMSO were intra?thecally injected at a dose of 10 μL. Paw withdrawal latency (PWL) to thermal stimulation and paw withdrawal threshold (PWT) to von Frey hair stimulation were measured 24 h before remifentanil infusion (T0) and at 2, 6, 24 and 48 h (T1-4) after intraveonus remifentanil injection. The rats were sacrificed after the last behavioral test and the L 4-6 segment of spinal cord was removed to determine the expressions of PKCγ, CaMKⅡαand pCaMKⅡαin spinal cord by Western blot analysis. Re? sults Compared with group C, the value of PWL was significantly shortened and PWT was significantly decreased except T0, and the expressions of PKCγ, CaMKⅡαand pCaMKⅡαwere up-regulated in other groups. Compared with group R+I, the value of PWL was significantly prolonged and PWT was significantly increased, the expressions of PKCγ, CaMKⅡαand pCaMKⅡαwere down-regulated in group D+R+I and group D+R+I+DMSO. Compared with group D+R+I, the value of PWL was significantly shortened and PWT was significantly decreased, the expressions of PKCγ, CaMKⅡαand pCaMKⅡαwere up-regulated in group D+R+I+P+DMSO. Compared with group D+R+I+P+DMSO, the value of PWL was significantly prolonged and PWT was significantly increased, the expressions of PKCγ, CaMKⅡαand pCaMKⅡαwere down-regulated in group D+R+I+DMSO. Conclusion Dexmedetomidine can reduce the expressions of PKCγ, CaMKⅡαand pCaMKⅡαin spinal cord in rats with IP and hyperalgesia induced by remifentanil.
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Objective To evaluate the effect of intrathecal resveratrol on activation of Ca2+/ calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in spinal dorsal horn neurons of rats with bone cancer pain.Methods Thirty-two adult female Sprague-Dawley rats,weighing 180-220 g,were equally randomized into 4 groups using a random number table:sham operation group (group S),bone cancer pain group (B group),and bone cancer pain + solvent control group (BD group).Walker 256 mammary gland cancer cell suspension (4× 105 cells/ml) 5 μl was injected into the medullary cavity of the right tibia in B,BR and BD groups.Normal saline 5 μl was given in group S.On 12,13 and 14 days after injection of mammary gland cancer cells,resveratrol 200 μg/10 μl was injected intrathecally once a day in group BR,and 1％ dimethyl sulfoxide 10 μl was intrathecally injected once a day in group BD.Before injection of mammary gland cancer cells (T0) and on 3,5,7,10,12 and 14 days after injection of mammary gland cancer cells (T1-6),mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.The rats were then sacrificed and L4,5 segments of the spinal cord were removed for confirmation of the location of phosphorylated CaMK Ⅱ (p-CaMK Ⅱ) in spinal dorsal horn neurons (by immunofluorescence) and for detection of p-CaMK lⅡ expression (using Western blot).Results Compared with group S,MWT and TWL were significantly decreased at T2-6,and p-CaMK Ⅱ expression was upregulated at T6,and p-CaMK Ⅱ was mainly co-expressed with neurons in B,BR and BD groups.Compared with group B,MWT and TWL were significantly increased at T5,6,and p-CaMK Ⅱ expression was down-regulated at T6 in group BR.There was no significant difference in MWT,TWL,and p-CaMK Ⅱ expression at each time point between group B and group BD.Conclusion Resveratrol can alleviate hyperalgesia in rats with bone cancer pain and inhibited activation of CaMK Ⅱ in spinal dorsal horn neurons may be involved in the mechanism.
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Calcium/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) has multiple functions, which made it play a central role in cardiovascular disease. Especially it activates numerous downstream targets in various signaling pathways that promotes vascular disease, heart failure, myocardial hypertrophy and arrhythmias. CaMKⅡcan impact calcium balance and increase calcium leak in myocardial cell via phosphorylating L type calcium channel, Ryanodine receptor (RyR 2) and phos?pholamban (PLN), and regulate ATP sensitive potassium current (IKATP) and late sodium current by affecting sodium channels and potassium channels. In addition, It can directly regulate transcription via activating the silk crack the original activated protein kinases (MAPKs) and acetylation enzyme (HDAC). These mechanisms have important roles in myocardial hypertro?phy, heart failure and arrhythmia. So we focus to demonstrating the structure and action mechanism of CaMKⅡto improve a new therapy of cardiovascular disease.
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Objective To evaluate the role of calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in cognitive dysfunction caused by chronic pain in rats.Methods The experiment was performed in 2 parts.In experiment Ⅰ,24 pathogen-free male Sprague-Dawley rats,weighing 180-220 g,were randomly divided into 4 groups (n =6 each) using a random number table:sham operation group (group S),m-AIP injected before sham operation group (group M-S),chronic sciatic nerve injury group (group N-C),and m-AIP injected before chronic constriction injury (CCI) group (group M-C).The sciatic nerve was only exposed but not ligated.Chronic pain was induced by CCI in N-C and M-C groups.The animals were anesthetized with intraperitoneal 1％ pentobarbital sodium.The sciatic nerve was exposed and 4 ligatures were placed on the sciatic nerve at 1 mm intervals.Normal saline 20 μ1 and m-AIP 20 μ/ were injected intrathecally at 15 min before sham operation in S and M-S groups,respectively,and at 15 min before CCI in N-C and M-C groups,respectively.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before CCI and on 4,7,10,14,17,21 and 28 days after CCI.Step-through latency (STL) was measured before CCI and on 7,14,21 and 28 days after CCI.In experiment Ⅱ,18 pathogen-free male Sprague-Dawley rats,weighing 180-220 g,were randomly divided into 3 groups (n =6 each) using a random number table:m-AIP injected after sham operation group (group C-N),control after CCI group (group C-N) and m-AIP injected after CCI group (group C-M).Group S-M received intrathecal injection of m-AIP 20 μl at 7 days after sham operation.Normal saline 20 μl and m-AIP 20 μ/ were injected intrathecally at 7 days after CCI in C-N and C-M groups,respectively.MWT,TWL and STL were measured before administration and at 2,4 and 8 h after administration.Results In experiment Ⅰ,compared with group S,MWT was significantly decreased at each time point after CCI,TWL was shortened at each time point after CCI and STL was shortened on 7,14 and 21 days after CCI in N-C group,and MWT was significandy decreased at each time point,TWL was shortened at each time point,and STL was shortened on 14 and 21 days after CCI in group M-C.Compared with group N-C,MWT was significantly increased on 4,7 and 10 days after CCI,TWL was prolonged on 4 and 7 days after CCI,and STL was prolonged on 7 days after CCI in group M-C.In experiment Ⅱ,compared with group S-M,MWT was significantly decreased,and TWL and STL were shortened at each time point after administration in C-N group,and TWL at 8 h after administration and STL at each time point after administration were shortened,MWT was decreased at 8 h after administration,and no significant change was found in MWT and TWL at 2 and 4 h after administration in group C-M Compared with group C-N,MWT was significantly increased,and TWL was prolonged at 2 and 4 h after administration,and no significant change was found in STL at each time point after administration in group C-M.Conclusion CaMK Ⅱ is involved in the development of cognitive dysfunction caused by chronic pain in rats.
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Objective To evaluate the relationship between calmodulin protein kinase Ⅱ (CaMK Ⅱ) and levosimendan against arrhythmias induced by myocardial ischemia-reperfusion (I/R) in rats in vitro.Methods Thirty male Wistar rats,weighing 250-300 g,were randomly divided into 3 groups (n =10 each) using a random number table:control group (group C),I/R group and levosimendan group (group L).Their hearts were rapidly excised and perfused in a langendorff apparatus with K-H solution saturated with 95％ O2-5％ CO2 at 36.5-37.5 ℃.At 20 min of equilibration,the hearts were perfused with K-H solution for 60 min in group C.The hearts were subjected to 30 min of ischemia followed by 30 min reperfusion with K-H solution in group I/R.The hearts were subjected to 30 min of ischemia followed by 30 min reperfusion with K-H solution containing 300 nmol/L levosimendan in group L.Left ventricle developed pressure (LVDP),left ventricle end-diastolic pressure (LVEDP),+ dP/dt-dP/dtmax and heart rate (HR) were recorded immediately before ischemia and at 15 and 30 min of reperfusion.Arrhythmia was recorded during reperfusion and scored.Specimens were obtained from the apex of heart at 30 min of reperfusion for determination of the intracellular calcium concentration ([Ca2 +] i).Myocardial specimens were obtained from the left ventricle at 30 min of reperfusion to detect CaMK Ⅱ activity.Results Compared with group C,arrhythmia score,[Ca2+]i and CaMK [Ⅱ activity were significantly increased,and LVDP,+ dP/dtmax,-dP/dtmax and HR were decreased,and LVEDP was increased at 15 and 30 min of reperfusion in group I/R.Compared with group I/R,the number of ventricular premature beat,arrhythmia score,[Ca2+] i and CaMK Ⅱ activity were significantly decreased,and LVDP,+ dP/dtmax,-dP/dtmax and HR were increased,and LVEDP was decreased at 15 and 30 min of reperfusion in group L.Conclusion Inhibition of CaMK Ⅱ activity is involved in the mechanism by which levosimendan decreases the development of arrhythmias induced by myocardial I/R in rats.
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Arrhythmias can develop in various cardiac diseases, such as ischemic heart disease, cardiomyopathy and congenital heart disease. It can also contribute to the aggravation of heart failure and sudden cardiac death. Redox stress and Ca2+ overload are thought to be the important triggering factors in the generation of arrhythmias in failing myocardium. From recent studies, it appears evident that Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a central role in the arrhythmogenic processes in heart failure by sensing intracellular Ca2+ and redox stress, affecting individual ion channels and thereby leading to electrical instability in the heart. CaMKII, a multifunctional serine/threonine kinase, is an abundant molecule in the neuron and the heart. It has a specific property as "a memory molecule" such that the binding of calcified calmodulin (Ca2+/CaM) to the regulatory domain on CaMKII initially activates this enzyme. Further, it allows autophosphorylation of T287 or oxidation of M281/282 in the regulatory domain, resulting in sustained activation of CaMKII even after the dissociation of Ca2+/CaM. This review provides the understanding of both the structural and functional properties of CaMKII, the experimental findings of the interactions between CaMKII, redox stress and individual ion channels, and the evidences proving the potential participation of CaMKII and oxidative stress in the diverse arrhythmogenic processes in a diseased heart.
Subject(s)
Arrhythmias, Cardiac , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calmodulin , Cardiomyopathies , Death, Sudden, Cardiac , Heart , Heart Diseases , Heart Failure , Ion Channels , Memory , Myocardial Ischemia , Myocardium , Neurons , Oxidation-Reduction , Oxidative Stress , Protein KinasesABSTRACT
Objective To evaluate the role of calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in the neuronal damage induced by lidocaine.Methods SH-SY5Y cells were seeded in 96-well plates (100 μl/hole) with a density of 5 × 105/ml and randomly divided into 4 groups (n =63 each):normal culture group (C group),CaMK Ⅱ inhibitor KN93 (K group),lidocaine group (L group) and KN93 + lidocaine group (KL group).KN93 (final concentration 1 μmol/L) was added to the culture medium and the cells were then cultured for 24 h in group K.Lidocaine (final concentration 10 mmol/L) was added to the culture medium and the cells were then cultured for 24 h in group L.KN93 (final concentration 1 μmol/L) and lidocaine (final concentration 10 mmol/L) were added to the culture medium and the cells were then cultured for 24 h in group KL.The cell morphology was examined with microscope after 24 h of incubation.The viability of cells was measured by MTT assay before incubation and at 1,6,12 and 24 h of incubation.The apoptosis in the cells was assessed by flow cytometry.The apoptotic rate was calculated.Results Compared with C and K groups,the cell viability was significantly decreased and the apoptotic rate was increased in L and KL groups (P ＜ 0.05).The cell viability was significantly higher and the apoptotic rate was lower in group KL than in group L (P ＜ 0.05).There was no significant difference in the cell viability and apoptotic rate between C group and K group (P ＞ 0.05).The pathological changes were obviousin group L and significantly reduced in group KL.Conclusion CaMK Ⅱ is involved in the neuronal damage induced by lidocaine.
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Objective To evaluate the role of calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in the up-regulation of expression of Cav3.2 T-type calcium channels in spinal cord in a rat model of neuropathic pain (NP).Methods Forty-eight male Sprague-Dawley rats,aged 3 months,weighing 220-250 g,were randomly divided into 6 groups (n =8 each)∶ sham operation group (group S),group NP,dimethyl sulfoxide group (group D) and different concentrations of a specific CaMK Ⅱ inhibitor KN93 groups (groups K1-3).NP was produced by chronic compression of dorsal root ganglion.The rats in groups D and K1-3 received a single intrathecal injection of dimethyl sulfoxide and KN93 15,30,60 nmol/L (10 μl),respectively,on 5th day after NP.Paw withdrawal threshold to von Frey filament stimulation (MWT) and paw withdrawal latency to thermal nociceptive stimulus (TWL) were measured before NP,before intrathecal injection on 5th day after NP,and at 30 and 60 min and 3,6 and 8 h after intrathecal injection on 5th day after NP (T1-7).The rats were sacrificed after the measurement of pain threshold at T7 and their lumbar enlargements were removed to detect the expression of Cav3.2 mRNA and protein using Western blot and RT-PCR.Results Compared with group S,MWT was significantly decreased,TWL was shortened and the expression of Cav3.2 mRNA and protein was up-regulated in NP,D and K1-3 groups (P ＜ 0.05).Compared with NP group,MWT was significantly increased,TWL was prolonged and the expression of Cav3.2 mRNA and protein was down-regulated in a concentration-dependent manner in K1-3 groups (P ＜ 0.05),and no significant change was found in the parameters mentioned above in group D (P ＞ 0.05).Conclusion CaMK Ⅱ is involved in the development and maintenance of chronic NP by up-regulating the expression of Cav3.2 T-type calcium channels in rat spinal cord.
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Objective To investigate the regulation and the mechanism of Melatonin in calcium overload of pancreatic acinar cell in acute necrotizing pancreatitis (ANP). Methods Fifty-four Sprague-Dawley (SD) rats were equally divided into three groups:sham-operation group (SO group),ANP group (created with retrograde cholangiopancreatography injection of sodium taurocholate) and MT group (ANP model made after intra-peritoneal injection MT for 30 mins).Rats were sacrificed at 1,4 and 8hours after operation and pancreas tissues were underwent pathological examination.The free calcium concentration of pancreas tissues was determined by fluorescence minitoring method; and the expression of CaMK Ⅱ in pancreas tissues at mRNA and protein level was tested by real-time PCR and Western Blot.Results Pancreatic pathological injury in ANP groups was progressively severe as time extended,which was obviously ameliorated in MT group compared with ANP group (the t value of compared pathological score at 1,4 and 8 hour was:-7.95,-9.72 and -7.69,all P=0.00).Compared at same time point,the free Ca2+ concentration of pancreas tissues in ANP group was significantly higher than that of SO group (the t value of 1,4 and 8 hour was 13.09,18.58 and 16.56,all P=0.00).It was a little bit higher in MT group compared with that of SO group,however was significantly lower than that of ANP group (the t value of 1,4 and 8 hour was -10.03,-11.75 and -11.02,all P =0.00).Compared with SO group,the expression of CaMK Ⅱ at mRNA and protein level significantly increased in ANP group; MT significantly inhibited its expression.Conclusions The expression of CaMK Ⅱ may be inhibited by MT interfere,and then lower the calcium overload in pancreatic acinar cell,which play a role in pancreas tissue protection.
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ObjectiveTo investigate the effect of propofol repeated anesthesia on the expression of CaMK Ⅱ α in the hippocampus in neonatal rats.MethodsThirty-two SD rats aged 7 days weighing 12-16 g were randomly divided into 2 groups (n =16 each): group C received intraperitoneal 0.9％ normal saline 7.5 ml/kg once a day for 7 days and group P received propofol 75 mg/kg once a day for 7 days.Learning and memory function were assessed using Morris warier maze at 28 days old of rats.The animals were sacrificed at 24 h after the tests and brain tissues were removed.The expression of CaMK Ⅱ α and phosphorylated CaMK Ⅱ α (pCaMK Ⅱ α) in hippocampal CAI region were determined by immunochemistry and Western bolt.ResultsCompared with group C,the escape latency was significantly prolonged,space exploration time shortened and expression of CaMK Ⅱ α and pCaMK Ⅱ α down-rugulated in group P than in group C( P ＜ 0.01 ).ConclusionPropofol repeated anesthesia decreases congnitive function through down-regulating the expression and inhibiting the activity of CaMK Ⅱ α in hippocampus in neonatal rats.
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ObjectiveTo investigate the effects of propofol on calcium/calmodulin-dependent protein kinase Ⅱ α ( CaMK Ⅱ α) activity in hippocampus in mentally depressed rats after electroconvulsive therapy (ECT).MethodsHealthy adult male SD rats aged 2-3 months weighing 180-220 g were used in this study.Mentally depressed model was induced by chronic unpredictable mild stress.Forty mentally depressed rats were randomly divided into 4 groups (n =10 each): mental depression group (group D),propofol group (group P),ECT group (group E),propofol + ECT group (group DPE).Groups D and P received intraperitoneal normal saline 8 ml/kg or propofol 80 mg/kg once a day for 7 consecutive days respectively.Group E received ECT once a day for 7 consecutive days.Group DPE received propofol 80 mg/kg + ECT once a day for 7 consecutive days.Sucrose preference test was performed at 1 d before and 1 d after treatment,and Morris water maze test was performed at 1 d before and 3 d after treatment.The rats were sacrificed after Morris water maze test,and hippocampi were removed for determination of CaMK Ⅱ α and phosphorylated CaMK Ⅱ α(pCaMK Ⅱ α )expression,and pCaMK Ⅱ α/CaMK Ⅱ α ratio was caculated.ResultsCompared with group D,the sucrose preference percentage was significantly increased in groups E and DPE,the escape latency prolonged and space exploration time shortened,and the expression of CaMK Ⅱ α and pCaMK Ⅱ α down-regulated,pCaMK Ⅱ α/CaMK Ⅱ α ratio decreased in group E,the escape latency was significantly shortened and space exploration time prolonged,and the expression of pCaMK Ⅱ α up-regulated in group DPE ( P ＜ 0.05).Compared with group E,the escape latency was significantly shortened,space exploration time prolonged,and the expression of CaMK Ⅱ α and pCaMK Ⅱ α up-regulated,and pCaMK Ⅱ α/CaMK Ⅱ α ratio increased in group DPE ( P ＜ 0.05).ConclusionPropofol can reduce the cognition impairment induced by ECT in mentally depressed rats through enhancing CaMK Ⅱ α activity in hippocampus.
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Objective To investigate the effect of calcium/calmodulin-dependent protein kinaseⅡ (CaMK[KG-*6]Ⅱ) inhibitor,KN93 on calcium overloading of atrial muscle cells in neonatal rats and detect the expression of CaMK[KG-*6]Ⅱ. Methods The atrial muscle cells from neonatal rats were primarily cultured for 96 h and then divided into 6 group,control,calcium overloading group,KN93 group (0.5 ?mol/L),low-,moderate-and high-dose of KN93+ calcium overloading group. A model of calcium overloading for atrial muscle cells was established by using calcium ionophore (ionomycin,1.0 ?mol/L). For the later 3 groups,KN93 at doses of 0.25,0.5 and 1.0 ?mol/L was added into the culture medium for 30 min followed by 1.0 ?mol/L ionomycin treatment for another 30 min. The identification of ?-actin was performed by immunofluorescence staining. In the present of Fluo-3/AM (an indicator of calcium),intracellular calcium and the expression of CaMK[KG-*6]Ⅱ were detected under the intervention of KN93 with laser cofocal microscopy and Western blotting respectively. Results More than 90% of cultured cells were positive to ?-actin antibody. Compared with the control group,the fluorescence intensity of intracellular Ca2+ was increased significantly by ionomycin (660.16?108.47 vs 376.12?57.57,P