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AIM: To investigate the effect of inhibiting Ca2+/calmodulin-dependent protein kinase Ⅱ(CAMKⅡ)expression in adult retinal pigment epithelial cell line-19(ARPE-19)cells on the migration, invasion, and tube formation of human umbilical vein endothelial cells(HUVECs)in a non-contact co-culture system.METHODS: RNA sequencing was performed on ARPE-19 cells overexpressing CAMKⅡ-δ, and bioinformatics was used to analyze the biological functions of the differentially expressed genes. Transwell inserts was used to construct a non-contact co-culture system of ARPE-19 and HUVECs. The experimental groups included: blank group: only HUVECs were inoculated without ARPE-19 cells; control group: ARPE-19 and HUVECs cells were co-cultured with complete medium; AIP group(CAMKⅡ inhibition group): ARPE-19 cells in AIP(160 nmol/L)were co-cultured with HUVECs in complete medium. The migration, invasion and tube formation abilities of HUVECs were detected. The protein expression levels of CAMKⅡ/AMPK/mTOR/VEGFA were detected by Western blotting.RESULTS:Bioinformatics analysis found that the differentially expressed genes could affect biological processes such as cell growth and death and cell movement. The scratch test and transwell migration test showed that the relative mobility of HUVECs in the AIP group was significantly lower than that in the control group(all P<0.05). However, the invasion and tube formation assay showed that the relative invasion rate and tube formation rate of the AIP group were not significantly different from those of the control group(both P>0.05). Western blotting results showed that the expression levels of CAMKⅡ, P-mTOR, and VEGFA proteins in the AIP group were significantly lower than those in the control group, while the expression level of the P-AMPK protein was significantly higher than that in the control group(all P<0.05).CONCLUSION:In the non-contact co-culture system, inhibition of CAMKⅡ expression in ARPE-19 cells significantly reduced the migration ability of HUVECs, but it cannot change the invasion and tube formation ability, which may be achieved by AMPK/mTOR/VEGFA.
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@#Objective To investigate the effect of microparticles(MPs)derived from bone marrow mesenchymal stem cells(BMSCs) on myocardial hypertrophy and its mechanism.Methods The osteogenic differentiation and adipogenic differentiation of mesenchymal stem cells(MSCs) were induced. After isolation and purification,the morphological characteristics were observed by transmission electron microscope,and the MPs surface antigen was identified by flow cytometry. Myocardial hypertrophy model was induced by using isoprenaline(ISO)in rats,which were measured for the cardiac structure and function by echocardiography,and then detected for various indexes of the heart and isolated left ventricle. Single ventricular myocytes of rats were acutely isolated and divided into control group(Control group),cardiomyocyte hypertrophy group(ISO group),MPs group(MPs group),and MPs supernatant group(Supernatant group). The mRNA expressions of atrial natriuretic peptide(ANP)and B-type natriuretic peptide(BNP)were detected by qRTPCR. The expression levels of calmodulin-dependent protein kinaseⅡ（CaMKⅡ）and phosphorylated calmodulin-dependent protein kinaseⅡ（p-CaMKⅡ）were detected by ELISA. The L-type calcium current(LCa-L)in single ventricular myocyte of various groups was recorded by whole-cell patch clamp.Results The bone nodules of MSCs osteogenic differentiation turned red after alizarin red staining,and lipid droplets of adipogenic differentiation turned red after oil red O staining;Under transmission electron microscope,MPs membrane had a complete structure,a clear outline and a diameter of about200 nm;The positive rates of CD29 and CD90 on the surface of MPs were(98. 24 ± 0. 82)% and(97. 69 ± 1. 83)%,respectively. Compared with Control group,the left ventricular end diastolic dimension(LVEDD)reduced signifi-cantly(t =5. 065,P < 0. 05),while the interventricular septum end-diastolic dimension(IVSd),left ventricular posterior wall dimension(LVPWd),heart weight to body weight ratio(HW/BW),and heart weight to tibial length ratio(HW/Tibia)significantly increased in ISO group(t = 4. 013,2. 368,4. 392,5. 043 and 6. 120,respectively,each P < 0. 05),indicating that the hypertrophic model was successfully established. The expression levels of ANP and BNP mRNA in hypertrophic cardiomyocytes of rats in ISO group were significantly higher than those in Control group(t = 25. 120 and18. 261,respectively,each P < 0. 01);While the expression levels of ANP and BNP mRNA in MPs group significantly reduced after incubation with 48 μg/mL MPs for 48 h compared with ISO group(t = 12. 110 and 3. 526,respectively,each P < 0. 05);The expression levels of CaMK Ⅱand p-CaMKⅡ in ISO group were significantly higher than those in Control group(t = 3. 278 and 4. 181,respectively,each P < 0. 05),while the expression of p-CaMK Ⅱ in MPs group decreased significantly(t = 5. 420,P < 0. 05);The calcium current density in ISO group was significantly higher than that in Control group(t = 15. 261,P < 0. 01),while that in MPs group was significantly lower than that in ISO group(t =6. 216,P < 0. 05).Conclusion MSC-MPs can significantly inhibit ISO-induced cardiomyocyte hypertrophy in rats,which is related to its down-regulation of cardiomyocyte CaMKⅡ and inhibition of L-type calcium channel.
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Recent studies have found that in the development of epilepsy, cyclic adenosine monophosphate response element binding protein (CREB) may cause recurrent epilepsy by inhibiting the expression of γ-aminobutyric acid, resulting in neuron damage and weakened effect of antiepileptic drug targets. Antiepileptic drugs can not control the extent or frequency of seizures, and then the patients are in a persistent state, hence the development of drug-resistant epilepsy. Therefore, the mechanism of CREB leading to drug-resistant epilepsy was reviewed in this paper, hoping to provide ideas for the treatment of drug-resistant epilepsy patients.
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Myocardial infarction(MI), an acute coronary syndrome that poses a serious risk to human health, involves multiple pathophysiological processes, including calcium overload. Existing therapeutic approaches and preventive measures have limitations and cannot effectively repair myocardial cells with poor regenerative potential. Exploring multiple programmed modes of cardiomyocyte death could help find potential targets for the treatment of myocardial infarction, and the potential role of ferroptosis as a novel mode of cell death in myocardial infarction has attracted great attention. The aim of this study was to investigate whether Ca
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AIM: To investigate t h e impacts of theaflavins (TFs) on neuronal apoptosis and blood-brain barrier (BBB) by regulating the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)/5 '-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. METHODS: Ninety rats were randomly separated into sham operation group, model group, low-dose TFs group (20 mg/kg TFs), high-dose TFs group (40 mg/kg TFs), and high-dose TFs + STO-609 group (40 mg/kg TFs + 10 ΜL CaMKK2 inhibitor-STO-609), positive control group (2 mg/kg nimodipine injection), with 15 rats in each group. A rat model of intracerebral hemorrhage was induced by collagenase type VII. The behavior of rats and the water content of brain tissue were detected; the serum of rats was isolated, and the levels of inflammatory factors-vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule-1 (ICAM-1) were detected; brain tissue around the hematoma was collected to detect neuronal apoptosis, BBB permeability parameter-EB level, and expressions of p-CaMKK2/CaMKK2, p-AMPK/AMPK and apoptosis-related protein Bax. RESULTS: Compared with the sham operation group, the mNSS score, ICAM-1, TNF-α, VCAM-1, brain tissue water content, apoptosis rate, EB level and Bax protein expression in the model group were all increased, both pCaMKK2/CaMKK2 and p-AMPK/AMPK were decreased (P < 0.05); compared with the model group, the mNSS score, ICAM-1, TNF-α, VCAM-1, brain water content, apoptosis rate, EB level and Bax expression in the low- and high-dose TFs groups and the positive control group were all lower than those in the model group, both pCaMKK2/CaMKK2 and p-AMPK/AMPK were increased (P < 0.05); compared with the high-dose TFs group, the mNSS score, ICAM-1, TNF-α, VCAM-1, brain tissue water content, apoptosis rate, EB level and Bax expression were all increased in the high dose TFs + STO-609 group, both p-CaMKK2/CaMKK2 and p-AMPK/AMPK were decreased (P < 0.05). CONCLUSION: TFs can reduce neuronal apoptosis, inflammatory response, BBB permeability, and play a protective role in rats with cerebral hemorrhage injury. Its mechanism is related to the activation of CaMKK2/AMPK signaling pathway.
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OBJECTIVE@#To observe the effect of acupoint thread-embedding on tight junction of intestinal mucosal epithelial barrier in rats with ulcerative colitis (UC) under the state of "deficiency and stasis", and to explore its mechanism.@*METHODS@#Sixty male SD rats were randomly divided into a control group (@*RESULTS@#Compared with the control group, in the model group the body weight was decreased (@*CONCLUSION@#The thread-embedding could repair the tight junction of intestinal mucosa epithelium and reduce the permeability of intestinal mucosa epithelium, which may be related to the decrease of the expression of CaMKⅡ, MLCK and other protein kinases.
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Animals , Male , Rats , Acupuncture Points , Colitis, Ulcerative/therapy , Epithelium , Intestinal Mucosa , Rats, Sprague-Dawley , Tight JunctionsABSTRACT
Z-VAD-FMK was combined with hypoxia-reoxygenation (H/R) injury to establish a necroptosis model of H9c2 cells to mimic the pathological changes of myocardial ischemia reperfusion injury (MIRI) in vitro and to study the effect and mechanism of tilianin against myocardial ischemia-reperfusion injury. A cell counting kit-8 (CCK-8) was used to detect cell viability, and commercial kits were used to detect lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the cell culture supernatant. Hoechst 33342/PI immunofluorescence staining was used to detect cell death. DCFH-DA, BBcellProbeTMM61, and JC-1 probes were used to detect reactive oxygen species (ROS), mitochondrial permeability transition pore (mPTP), and mitochondrial membrane potential (MMP), respectively. An enzyme-linked immunosorbent assay (ELISA) method was used to detect the release of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). The results show that the cell viability, SOD activity, and MMP of the model group induced by H/R injury decreased, as compared with control group, but the necroptosis rate, LDH level, and ROS release increased significantly. Furthermore, mPTP of the model group cells opened, and TNF-α, IL-1β, and IL-6 levels were significantly higher. Molecular docking modeling showed that tilianin can bind to calmodulin-dependent protein kinase II (CaMKII), and Western blot results showed that compared with control group, the expression levels of p-CaMKII and phospho-mixed lineage kinase domain-like protein increased in the model group, and tilianin could decrease the expression level of these proteins. The above results indicate that tilianin can protect H9c2 cells by inhibiting the phosphorylation of CaMKⅡ at threonine 287, protecting mitochondrial function, and inhibiting the opening of mPTP to prevent necroptosis. This study has value for research on new methods to treat H/R injury.
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Objective:To observe the effect of electroacupuncture at the Baihui acupoint on learning and memory ability and on the calmodulin kinase (CaMK)Ⅱ-Tau protein signal pathway in rats exposed to infrasound, and to explore its mechanism.Methods:Forty-eight Sprague-Dawley rats were randomly divided into a blank group, an infrasound group, a Baihui group and a non-acupoint group, each of 12. The rats in the blank group were placed in an infrasound chamber without infrasound for 2 hours daily. Those in the other 3 groups were exposed to 8Hz, 130dB infrasound in the chamber for 2 hours daily for 7 consecutive days. The rats in the Baihui and non-acupoint groups were given electroacupuncture within 2 hours after the infrasound exposure at the Baihui acupoint or elsewhere respectively. The rats in the blank and infrasound groups were given the same grasping and fixation, but no electroacupuncture. On the 6th and 7th day of intervention, Morris water maze positioning and navigation experiments and spatial exploration experiments were used to quantify the rats′ spatial learning and memory ability. Nissl staining was used to observe any changes in the morphology of the neurons in the hippocampus of 6 rats in each group. The expression of phosphorylated calmodulin-dependent protein kinase II (P-CaMKⅡ) and phosphorylated Tau protein (P-Tau) in the hippocampus was also documented using western blotting.Results:After 6 or 7 days the average escape latency of the rats in the infrasound group was significantly longer than the blank group′s average. Platform quadrant time and distance ratios and the number of times crossing the platform area were also significantly lower. Compared with the infrasound group, the average escape latency of the Baihui group was significantly shorter, with the platform quadrant time and distance ratios and the number of times crossing the platform area significantly higher. After 7 days, the damage to hippocampal neurons among the rats in the infrasound group was significantly aggravated and the number of neurons was reduced significantly compared with the blank group. Compared with the infrasound group, significantly fewer neurons in the hippocampus were damaged in the Baihui group and the number of neurons had increased significantly. After the intervention the levels of P-CaMKⅡand P-Tau protein in the infrasound group had increased significantly compared with the blank group, but those levels in the Baihui group were significantly lower, on average.Conclusion:Electroacupuncture at the Baihui acupoint can improve the learning and memory ability of rats exposed to infrasound, and has some protective effect against infrasound brain damage. That may be due to its inhibiting Tau protein hyperphosphorylation in the hippocampus by reducing CaMKⅡ activity.
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Objective To clarify the protective effect of allopregnanolone (APα) on cell line SH-SY5Y damaged by 6-hydroxydopamine (6-OHDA) and its possible molecular mechanism. Methods 6-OHDA, APα, γ-aminobutyric acid A receptor (GABAAR) antagonist, voltage-gated L-type Ca2
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Redox-altered plasticity refers to redox-dependent reversible changes in synaptic plasticity
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Alzheimer's disease（AD） is an incipient aging neurodegenerative disease， which increases rapidly along with the development trend of social aging and seriously threatens the health of the people. In the absence of effective preventive measures， it will have an enormous impact on the socio-economic and healthcare system. The study found that abnormal cell signal transduction is a key link in many diseases. Cell signal transduction theory has been widely used to clarify the essence of traditional Chinese medicine visceral image and the mechanism of traditional Chinese medicine. 'Correlation of Liver and Kidney' is one of the core plates of the theory of 'Correlation of Five Organs'， which is suitable for explaining the pathogenesis of complex diseases and the correlation of multiple syndromes， and guiding the prescription of clinical syndrome. Hei Xiaoyaosan， as the first choice compound for the prevention and treatment of AD based on the theory of "Correlation of Liver and Kidney' in our team， can play the effects of prevention and treatment by soothing liver and nourishing blood， strengthening spleen and tonifying kidney， and promoting brain collaterals and dredging viscerab spirit. Based on the theory of 'Correlation of Liver and Kidney'， this paper expounds the pathogenesis of AD from the perspective of traditional Chinese medicine， and puts forward the methods and ideas of the preventing and treating of AD from Ca2+-calcium/calmodulin dependent protein （CaM）/calcium/calmodulin dependent protein kinaseⅡ（CaMKⅡ）-cyclic adenosine phosphate reactive element binding protein （CREB） cell signal transduction pathway by consulting literatures and previous studies.
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Objective To investigate the effects of therapeutic hypothermia (TH) on myocardial Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) and cell autophagy after cardiopulmonary resuscitation (CPR) in swine.Methods Twenty healthy male domestic swine weighing 33-40 kg were randomly (random number) divided into 3 groups:sham group (n=4),CPR group (n=8) and TH group (n=8).Sham animals only underwent general preparation without experiencing cardiac arrest and resuscitation.The animal model was established by 8 min of electrically induced ventricular fibrillation and then 5 min CPR in the CPR and TH groups.Successful resuscitation was regarded as an organized rhythm with a mean arterial pressure of greater than 50 mmHg for 5 min or more.After successful resuscitation,body temperature was decreased to 33 ℃ by a cooling blanket and then maintained until 24 h post-resuscitation,and followed by a rewarming at a rate of 1 ℃/h for 5 h in the TH group.A normal temperature was maintained by the blanket throughout the experiment in the sham and CPR groups.At 6,12,24 and 30 h after resuscitation,the values of stroke volume (SV) and global ejection fraction (GEF) were measured by PiCCO,and meanwhile the serum concentrations of cardiac troponin Ⅰ (cTnI) were measured by ELISA assay and the serum activities of creatine kinase-MB (CK-MB) were evaluated by an automatic biochemical analyzer.At 30 h after resuscitation,the animals were sacrificed and left ventricular myocardium was obtained for the determination ofCaMK Ⅱ,microtubule-associated protein light chain 3 Ⅱ (LC3 Ⅱ) and p62 expressions by Western blot.The variables were compared with One way analysis of variance and then the Bonferroni test among the three groups.Results Compared with the sham group,myocardial dysfunction and injury after resuscitation were observed in the CPR and TH groups,which were indicated by decreased SV and GEF and also increased cTnI concentration and CK-MB activity in serum (all P＜0.05).Compared with the CPR group,the values of SV and GEF were significantly increased at 6 h after resuscitation,and serum cTnI concentration and CK-MB activity were significantly decreased starting 12 h after resuscitation in the TH group [SV (mL):25.0±6.9 vs 31.9±3.3 at 6 h,26.7±5.1 vs 34.6±3.7 at 12 h,28.8±3.3 vs 35.7±3.2 at 24 h,29.2±5.2 vs 36.7±3.3 at 30 h;GEF (％):17.1±2.7 vs 19.9±1.8 at 6 h,18.7±1.9 vs 21.6±1.8 at 12 h,19.3±2.3 vs 23.0±2.4 at 24 h,21.0±1.7 vs 23.7±1.7 at 30 h;cTnI (pg/mL):564±51 vs 466±56 at 12 h,534±38 vs 427±60 at 24 h,476±55 vs 375±46 at 30 h;CK-MB (U/L):803±164 vs 652±76 at 12 h,693±96 vs 557±54 at 24 h,633±91 vs 480±77 at 30 h,all P＜0.05].Tissue detection indicated that the expression of CaMK Ⅱ and LC3 Ⅱ were increased while the expression of p62 was decreased in post-resuscitation myocardium in the CPR and TH groups compared with the sham group (all P＜0.05).However,the expression of CaMK Ⅱ and LC3 Ⅱ were decreased and the expression of p62 was increased in postresuscitation myocardium in the TH group compared to the CPR group (CaMK Ⅱ:0.73±0.06 vs 0.58±0.05;LC3 Ⅱ:0.69±0.09 vs 0.50±0.07;p62:0.40±0.07 vs 0.68±0.14,all P＜0.05).Conclusion The mechanism of TH alleviating post-resuscitation myocardial dysfunction and injury may be related to the inhibition of CaMK Ⅱ expression and cell autophagy.
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Objective::To study the effect of Hei Xiaoyaosan on the expression of calcium calmodulin-dependent protein kinase Ⅱ alpha(CaMKⅡα) and its phosphorylation in hippocampus and cortex of mice with Alzheimer's disease. Method::After weighing, 30 APP/PSI transgenic male mice were divided into model group, donepezil hydrochloride group and Hei Xiaoyaosan group according to random principle and 10 in each group.At the same age, wild-type C57BL/6 10 mice of the same species were treated as blank group. Donepezil hydrochloride group (6 g·kg-1) and Hei Xiaoyaosan group (3.25 mg·kg-1) were administered for 90 days, then the behavior of all the mice were detected by Morris water maze, the expression of CaMKⅡα, p-CaMKⅡα proteins in hippocampus and cortex by immunohistochemical technique and Western blot. Result::After intervention 3 months, compared with blank group, the average escaping latency periods prolonged significantly and the number of cross-platform and effective areas were decreased distinctly in model group mice(P<0.01), CaMKⅡα protein relative expression decreased significantly(P<0.01), p-CaMKⅡα protein relative expression increased significantly(P<0.01). Compared with the model group, the escape latency of donepezil hydrochloride and Hei Xiaoyaosan group were significantly shortened, and the number of crossing platforms and effective areas was significantly increased (P<0.05, P<0.01), the expression of CaMKⅡα protein in the hippocampus and cortex of drug groups was significantly increased (P<0.01), p-CaMKⅡα protein in the hippocampus and cortex of drug groups was significantly decreased (P<0.05, P<0.01). Conclusion::Hei Xiaoyaosan can improve the learning and memory ability of AD mice by regulating the expression of CaMKⅡα and its phosphorylation, which are key proteins involved in the mechanism of cell memory formation in different brain regions of AD mice.
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Calmodulin-binding transcription activators (CAMTAs) are a family of transcription factors that play an important role in plants’ response to the various biotic and abiotic stresses. The common bean (Phaseolus vulgaris L.) is one of the most important crops in the world and plays a pivotal role in sustainable agriculture. To date, the composition of CAMTA genes in genomes of Phaseolus species and their role in resistance to drought stress are not known. In this study, five PhavuCAMTA genes were characterized in common bean genome through bioinformatics analysis, the morphological and biochemical response of 23 Ph.vulgaris genotypes to different levels of drought stress were evaluated and the expression patterns of PhCAMTA1 in the leaf tissues of sensitive and tolerant genotypes were analysed. Gene structure, protein domain organization and phylogenetic analyses showed that the CAMTAs of Phaseolus were structurally similar and clustered into three groups as other plant CAMTAs. Genotypes showeda differential response to drought stress. Thus, the plant height, number of nodes and flower, total chlorophyll and total protein content, and activity of antioxidant enzymes (ascorbate peroxidase and catalase) in plants significantly influenced by genotype and drought stress interaction. Moreover, the resistant and susceptible genotypes were identified according to three-dimensional plots and the expression patterns of PhavuCAMTA1 gene were studied using real-time quantitative polymerase chain reaction. The results of the present study serve as the basis for future functional studies on the Phaseolus CAMTA family.
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Objective The aim of this study was to investigate the effect of Ca2+ /calmodulin - dependent kinase II (CaMKII)γ RNA interference on the expression of nuclear factor of activated T-cells cytoplasmic 1(NFATc1),tyrosine kinase(c-Src)and tartrate resistant acid phosphatase(TRAP)genes,and its role and molecular mechanism in osteoclast differentiation. Methods The CaMKII γ RNA interference vector was constructed by lentivirus and transfected into RAW264. 7 cells. The experiment was di-vided into three groups:A,B and C,which were the control group,negative vector group and interference vector group. After transfec-tion for 12 hours,osteoclasts induced by 50 ng/mL RANKL and the cells were harvested after induction for 5 days. Real-time quanti-tative PCR,Western blot and immunofluorescence were used to detect the expression of NFATc1,TRAP and c-Src genes in three groups. Results The mRNA levels of NFATc1,TRAP and c-Src in the group C decreased by 49. 86% ,43. 65% and 53. 57% ,re-spectively(P<0. 001),and the protein levels decreased by 54. 22% ,46. 75% and 45. 86% ,respectively(P<0. 001). There was no significant difference between the A and the B groups(P>0. 05). The fluorescence intensity of the above genes in the group C was significantly weaker than that in the A and B groups,and the formation of osteoclasts was significantly less than that in the A and B groups. Conclusion CaMKIIγ RNA interference significantly inhibited the expression of NFATc1,TRAP and c-Src genes,sugges-ting that CaMKIIγ plays a key regulatory role in osteoclast differentiation.
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Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when coexpressed with CaM and CaMΔN. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ΔEF-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.
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Binding Sites , Calcium , Calcium Signaling , Calmodulin , Ion Channels , Ions , Patch-Clamp Techniques , Polycystic Kidney Diseases , Transient Receptor Potential ChannelsABSTRACT
Objective Mild hypothermia (MHT) can effectively protect the brain in traumatic brain injury (TBI). This study was to investigate the effects of MHT on the calmodulin (CAM) expression and brain edema in the rat model of TBI. Methods Ninety adult SD rats were randomly divided into a sham operation, a normal temperature and an MHT group of equal number. Immediately after TBI, the rats of the MHT group maintained at a rectal temperature of (32 ± 0.5) °C for 6 hours. Modified neurological severity scores (mNSS) were obtained from 6 rats in each group at 1, 3, 5 and 7 days after modeling, and the rest of the animals subjected to brain MRI at 6, 12, 24 and 48 hours and then killed for determination of the CAM gene transcription and protein expression in the brain tissue by real-time PCR, immunohistochemistry and Western blot. Results The mNSSs were significantly higher in the MHT and normal temperature groups than in the sham operation control (P < 0.05) at all time points, neurological severity markedly decreased in the MHT group compared with the normal temperature group (P < 0.05). At 6, 12, 24 and 48 hours, the expression of CAM mRNA was remarkably down-regulated in the MHT group (1.83 ± 0.19, 1.72 ± 0.12, 1.59 ± 0.06 and 1.60 ± 0.07) in comparison with the normal temperature group (2.76 ± 0.25, 2.49 ± 0.18, 2.04 ± 0.14 and 1.65 ± 0.09) (P < 0.05), even lower in the MHT than in the normal temperature group (P < 0.05), but higher in both of the two groups than in the sham operation group (P < 0.05). At 6, 12, 24 and 48 hours, the volume of brain edema was significantly reduced in the MHT group ([32.14 ± 4.52], [36.52 ± 4.10], [42.10 ± 4.38] and [46.25 ± 5.02] mm3) as compared with the normal temperature group ([48.56 ± 5.35], [53.13 ± 6.31], [59.23 ± 6.82] and [62.35 ± 7.25] mm3) (P < 0.05). Conclusion Mild hypothermia can improve the neurological function and reduce the CAM expression and brain edema in the brain tissue of rats with traumatic brain injury, which may be related to the neuroprotective effect of mild hypothermia.
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Objective To evaluate the effects of hypothermia on Ca2+∕calmodulin-dependent pro-tein kinase Ⅱ ( CaMKⅡ) and cell autophagy in brain tissues after cardiac arrest and cardiopulmonary re-suscitation ( CA-CPR) in swine. Methods Twenty-one healthy male white swine, weighing 33-40 kg, were divided into 3 groups using a random number table method: sham operation group ( group S, n=5) , CA-CPR group ( n=8) and hypothermia group ( group H, n=8) . The experimental model of CA-CPR was established in CA-CPR and H groups. The Swan-Ganz catheters were placed in the right femoral artery and vein to monitor the pressure of thoracic aorta and right atrium and body temperature and to collect blood sam-ples. A pacing catheter was advanced from the right external jugular vein into the right ventricle. Ventricu-lar fibrillation was induced by using a 1 mA alternating current through the pacing catheter. Once ventricular fibrillation was successfully induced, mechanical ventilation was discontinued for 8 min, and then CPR was initiated. Epinephrine 20 μg∕kg was intravenously injected at 2. 5 min of CPR, followed by repetition once every 3 min. Defibrillation was delivered at 5 min of CPR, and then spontaneous circulation was evaluated. If the spontaneous circulation was not restored, CPR was immediately resumed for 2 min, and then defibril-lation was delivered again. Mechanical ventilation was continued for 30 h after successful CPR. At 5 min af-ter successful resuscitation, body temperature was decreased to 33 ℃ by using a cooling blanket, then maintained at 33 ℃ until 24 h after resuscitation, and finally increased at a rate of 1℃∕h for 5 h in group H. The temperature was maintained at a normal level of 37. 5-38. 5 ℃ with the aid of a cooling blanket in S and CA-CPR groups. At 1, 6, 12, 24 and 30 h after resuscitation (T1-5), blood samples were collected from the femoral vein for measurement of the concentration of neuron specific enolase ( NSE) and S100βprotein in serum by enzyme-linked immunosorbent assay. Five animals in each group were then sacrificed, and brains were removed to determine the expression of CaMKⅡ, microtubule-associated protein 1 light chain 3 Ⅱ( LC3Ⅱ) and p62 in cerebral cortex by Western blot. Neurological deficit score was evaluated in the remaining three swine at 48, 72 and 96 h after resuscitation (T6-8) in CA-CPR and H groups. Results Compared with group S, the concentrations of NSE and S100β protein in serum were significantly in-creased at T1-5 , the expression of CaMKⅡand LC3Ⅱin cerebral cortex was up-regulated, and the expres-sion of p62 in cerebral cortex was down-regulated in CA-CPR and H groups (P<0. 05). Compared with group CA-CPR, the concentrations of NSE and S100βprotein in serum were significantly decreased at T3-5, the neurological deficit score was decreased at T6-8 , the expression of CaMKⅡand LC3Ⅱin cerebral cortex was down-regulated, and the expression of p62 in cerebral cortex was up-regulated in group H ( P<0. 05) . Conclusion The mechanism by which hypothermia alleviates brain injury after CA-CPR may be related to inhibiting CaMKⅡ activation and reducing cell autophagy in brain tissues of swine.
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Objective To explore the protective effect and mechanism of swimming rehabilitation training on learning and memory impairment of cerebral ischemia reperfusion gerbil. Methods Forty adult healthy male gerbils were randomly divided into sham group,sham+swimming group (Sham+S group),cere-bral ischemia / reperfusion group ( I/R group), cerebral ischemia/reperfusion+swimming group ( I/R+S group),with 10 rats in each group. The gerbil models of cerebral ischemia/reperfusion in I/R group and I/R+S group were established by blocking bilateral common carotid artery,while for gerbils in Sham group and Sham+S group, only bilateral common carotid arteries of gerbils were exposed, but no arteries were clamped. Morris water maze was used to detect the changes of learning and memory function in rats. Oxida- tive stress injury in hippocampal neurons was detected by detection kit analysis. And the expression of Bax, Bcl-2 and CaMK Ⅱ protein in hippocampal tissue was detected by Western blot. Results Compared with Sham group,the gerbils in I/R group had longer positioning cruise time and less shuttle times ( both P<0. 01). Compared with I/R group,the positioning cruise time and shuttle times in I/R+S group were signifi-cantly shortened and increased respectively (both P<0. 01). Compared with sham group( SOD:(123. 13± 7. 50)U/mg,GSH:(42. 10±2. 17) μg/g,GSH-Px:(61. 37±2. 51) μg/g,MDA:( 2. 91± 0. 23) nmol/mg), the activities of SOD,GSH,GSH-Px in I/R group decreased significantly,while the content of MDA increased significantly(SOD:(75. 50±6. 96)U/mg,GSH:(22. 50±1. 64) μg/g,GSH-Px:(33. 15±2. 04)μg/g,MDA:(5. 96±0. 32)nmol/mg;all P<0. 01). Furthermore,compared with I/R group,the above indexes in I/R+S group were significantly reversed(SOD:(110. 30±5. 90)U/mg,GSH:(34. 31±1. 73)μg/g,GSH-Px:(50. 13 ±2. 31)μg/g,MDA:(3. 57±0. 29) nmol/mg;all P<0. 01). Compared with Sham group,the expression of Bax protein in hippocampus of gerbils in I/R group was increased,while the expression of Bcl-2 protein and p-CaMK Ⅱ protein was decreased (all P<0. 05). Compared with I/R group,the expression of Bax protein in hippocampus of gerbils in I/R+S group was decreased,while the expression of Bcl-2 protein and p-CaMK Ⅱprotein was increased (all P<0. 05). Conclusion Swimming rehabilitation training can improve learning and memory impairment of gerbils after ischemia-reperfusion through anti-oxidative stress and anti-apoptosis, which may be related to CaMK Ⅱ signaling system.
ABSTRACT
Calcium (Ca2+) has long been recognized as a crucial intracellular messenger attaining stimuli specific cellular outcomes via localized signaling. Ca2; binding proteins, such as calmodulin (Ca.M) and its target proteins are key to Ca2;-dependent signaling events. Calcium/calmodulin-dependent protein kinase type II (CaMK II) is a highly abundant polymer enzyme comprising a significant fraction of total protein in mammalian forebrain and forming a major component of the postsynaptic density. In recent years, studies have shown that CaMK D contains four subtypes of a, (3, y and 8, in which CaMK II a and p are mainly expressed in nerve tissues and 7 and 6 are expressed in the whole body. They are essential for certain synaptic plasticity and memory consolidation processes, both in the central nervous system and in the excitability of the nervous system and in some neurological diseases. CaMK II may play an important role in the pathogenesis of some nervous system diseases. Previous studies have also shown that CaMK II 8 plays an important role in promoting neuronal survival. The structure of CaMK H and its role in nervous system and its relationship with related nervous system diseases will be reviewed.