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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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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
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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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 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.
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Objective: To study the protective effect of Huayu Qutan decoction on vascular dementia (VD) gerbils and to explore whether its mechanism is related to Calcium ion-calmodulin-dependent protein kinase Ⅱ (CaMKⅡ)/cyclic adenosine effect element binding protein (CREB)/brain-derived neurotrophic factor (BDNF) signaling pathway. Method: Forty healthy gerbils were randomly divided into sham operation group, model group, low, medium and high dose groups (5.35, 10.7, 21.4 g·kg-1) of removing blood stasis and expelling phlegm. Eight gerbils in each group were divided into model group and removing blood stasis and expelling phlegm group. Gerbils were given corresponding drugs twice a day after operation. Water maze experiment was conducted 21 days later to investigate the spatial learning and memory ability of gerbils. The expression of p-CaMKⅡ/CaMKⅡ, p-CREB/CREB and BDNF in the hippocampus of gerbils were detected by Western blot and immunohistochemistry. Result: Compared with sham operation group, the incubation period and the number of platform trips of gerbil in the model group were significantly reduced, p-CaMKⅡ/CaMKⅡ, p-CREB/CREB, and BDNF protein expression were significantly reduced (PPPConclusion: Huayu Qutan decoction improves the learning and memory abilities of gerbils with vascular dementia, and its mechanism may be related to the activation of CaMKⅡ/CREB/BDNF signaling pathway.
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Objective:To investigate the role of Na+/Ca2+ exchanger (NCX) in myocardial ischemiareperfusion injury and the underlying mechanisms.Methods:Forty Sprague-Dawley rats were divided into 4 groups randomly:a control group,a KBR7943 group,an ischemia-reperfusion group (IR group),and an IR plus KB-R7943 group (KB-R7943+IR group).Isolated Sprague Dawley male rat hearts underwent Langendorffperfusion.The ratio of left ventricular developed pressure (LVDP),left ventricular end-diastolic pressure (LVEDP),the infarct size of myocardium,and the lactate dehydrogenase (LDH) activity in the coronary flow was determined.HE staining was used to assess the change of myocardial morphology.Western blot was used to determine the levels of cleaved caspase-3,cytochrome c and the phosphorylation of Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) and the Thr17 site ofphospholamban.Results:Compared with the control group,IR group significantly induced an enlarged infarct size,reduction of the ratio of LVDP,up-regulation of cytochrome c,cleaved caspase-3,p-CaMKⅡ and p-phospholamban,and increased in the activity of LDH,the level of LVEDP (P＜0.01) and the disordered myocardial morphology.These effects were significantly attenuated in the presence of KB-R7943 treatment (10 μmol/L).Conclusion:NCX mediates myocardial ischemia-reperfusion-induced cell apoptosis and necrosis through activation of CaMKⅡ.
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Atrial structural remodeling and electrical remodeling are the core of atrial fibrillation.Oxidative stress directly activates calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ), and induces electrical remodeling and atrial structural remodeling characterized by reduced atrial effective refractory period, which becomes the pathological basis of atrial fibrillation.Therefore, the study of the relationship between the oxidative CaMKⅡ and atrial remodeling will help to elucidate the pathogenesis of atrial fibrillation and to prevent or reverse atrial remodeling by lowering CaMKⅡ phosphorylation to reduce the incidence of atrial fibrillation.
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Heart failure is mainly characterized by myocardial systolic dysfunction,which is based on excitement-contraction coupling on the cellular level,and calcium (Ca2+) signaling plays a very important role in this process.The ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is the major Ca2+ source of required for cardiac muscle excitation contraction coupling.RyR2 phosphorylation is the basis of SR calcium release,and RyR2 phosphorylation is mainly controled by protein kinase A (PKA) and calcium/calmodulin dependent protein kinase Ⅱ (CaMK Ⅱ).Although widely research in this area,excessive activation of RyR2 phosphorylation involved in the pathogenesis of heart failure are still controversial,which is discussed in this review.
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Objective To elucidate the relationship between Ca2+/calmodulin-dependent protein kinaseⅡ(CaMKⅡ)and autophagy during the development of cardiac hypertrophy. Methods Rat embryonic cardiac cell line H9c2 cells was treated by angiotensin Ⅱ to establish cardiomyocyte hypertrophy model in vitro and using antagonists and gene function gain and loss to analyze AMPK-LC3Ⅱ autophagy signaling pathway. Results The phosphorylation of CaMKⅡ and autophagy related signaling-AMPK and autophagy marker LC3Ⅱ were rapidly increased by angiotensinⅡtreatment at early stage. However ,the above changes were highly blocked by CaMKⅡinhibitor and HDAC4 inhibition. Conclusion CaMKⅡ is the center factor of regulating cardiac hypertrophy ,it mediates autophagy through directly regulating AMPK or indirectly regulating HDAC4 during the development of cardiac hypertrophy.
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Calcium/cahnodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) is a multifunctional serine/threonine protein kinase.It plays a vital role in the regulation of neurotransmitters,cell metabolism,and synaptic plasticity,as well as in many diseases of the nervous system.As CaMK Ⅱ alpha is specifically located at excitatory neurons,it has long been regarded as crucial for the treatment of epilepsy.CaMK Ⅱ shows decreased activity after seizures in different in vivo and in vitro models of epilepsy.This article provides a new theoretical basis for further research on the pathological process of epilepsy and its treatment,by exploring several recent experimental studies on CaMK Ⅱ and epilepsy.
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AIM:To investigate the change of late sodium current (INaL) and the effect of Ca2+/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) inhibitor KN-93 on INaLin the cardiomyocytes after isoproterenol-induced heart fai-lure (HF) in rabbits. METHODS:The rabbit model of HF was induced by injecting isoproterenol (300 μg·kg-1· d-1) for 15 d. One month later, all rabbits received by echocardiography and HE staining to observe the morphological changes of myocardium for evaluating the HF model. The protein expression of NaV1.5, CaMKⅡδ and phosphorylated CaMKⅡδ was determined by Western blot. The ventricular myocytes were isolated from the rabbits of normal saline(NS) group and HF group by Langendorff perfusion, and the whole-cell patch-clamp technique was used to record INaL. RE-SULTS:Compared with NS group,the heart rate in HF group was increased (P<0.01), the ventricular cavity was en-larged (P<0.05),and the cardiac function was decreased(P<0.01). Compared with NS group,the cardiomyocytes in HF group arranged in disorder, vacuolar degeneration and myocardial interstitial edema were observed, and fibrous tissue increased. The protein levels of NaV1.5,CaMKⅡδ and phosphorylated CaMKⅡδ in HF group were higher than those in NS group(P<0.01). INaLin HF group significantly increased compared with NS group (P<0.01). After adding sea anemone toxin Ⅱ (ATXⅡ), the density of INaLin HF group and NS group was significantly increased, but that in HF group increased more obviously than that in NS group (P<0.01). After ATXⅡ had induced stable current, we added KN-93 into NS group and HF group,and we found that the ATXⅡ-increased INaLin NS group and HF group was signifi-cantly decreased(P<0.05).CONCLUSION:CaMKⅡinhibitor KN-93 inhibits the increase in INaLin HF rabbits,which may be related to the activity of CaMKⅡδ and the regulation of CaMKⅡ δ on INaL.
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OBJECTIVE To determine the functional role of hydrogen sulfide (H2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca2 +/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) using wild type and CSE knockout mouse models. METHODS Continuous subcutaneous injection isoprenaline (7.5 mg·kg-1 per day), once a day for 4 weeks to induce heart failure in male C57BL/6 (6-8 weeks old) mice and CSE-/- mice. 150 μmol·L-1 H2O2 was used to induce oxidative stress in H9c2 cells. Echocardiograph was used to detect cardiac parameters. H&E stain and Masson stain was to observation histopathological changes. Western blot was used to detect protein expression and activity. The siRNA was used to silence protein expression. HPLC was used to detect H2S level. Biotin assay was used to detect the level of S-sulfhydration protein. RESULTS Treatment with S-propyl-L-cysteine (SPRC) or sodium hydrosulfide (NaHS), modulators of blood H2S levels, attenuated the development of heart failure in animals, reduced lipid peroxidation, and preserved mitochondrial function. The inhibition CaMKⅡ phosphorylation by SPRC and NaHS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds. Interestingly, CaMKⅡ activity was found to be elevated in CSE-/- mice as compared to wild type animals and the phosphorylation status of CaMK Ⅱ appeared to relate to the severity of heart failure. Importantly, in wild type mice SPRC was found to promote S-sulfhydration of CaMKⅡ leading to reduced activity of this protein however, in CSE-/- mice S-sulfhydration was abolished following SPRC treatment. CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of CaMKⅡ is presented. SPRC mediated S-sulfhydration of CaMKⅡ was found to inhibit CaMKⅡ activity and to preserve cardiovascular homeostasis.
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OBJECTIVE To determine the functional role of hydrogen sulfide (H2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca2 +/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) using wild type and CSE knockout mouse models. METHODS Continuous subcutaneous injection isoprenaline (7.5 mg·kg-1·d-1), once a day for 4 weeks to induce heart failure in Male C57BL/6 (6-8 weeks old) mice and CSE-/- mice. 150 μmol·L-1 H2O2 was used to induce oxidative stress in H9c2 cells. Echocardiograph was used to detect cardiac parameters. H&E stain and Masson stain was to observation histopathological changes. Western blot was used to detect protein expression and activity. The siRNA was used to silence protein expression. HPLC was used to detect H2S level. Biotin assay was used to detect the level of S- sulfhydration protein. RESULTS Treatment with S-propyl-L-cysteine (SPRC) or sodium hydrosulfide (NaHS), modulators of blood H2S levels, attenuated the development of heart failure in animals, reduced lipid peroxidation, and preserved mitochondrial function. The inhibition CaMKⅡ phosphorylation by SPRC and NaHS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds. Interestingly, CaMKⅡ activity was found to be elevated in CSE-/- mice as compared to wild type animals and the phosphorylation status of CaMKⅡ appeared to relate to the severity of heart failure. Importantly, in wild type mice SPRC was found to promote S-sulfhydration of CaMKII leading to reduced activity of this protein however, in CSE-/- mice S-sulfhydration was abolished following SPRC treatment. CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of CaMKⅡ is presented. SPRC mediated S-sulfhydration of CaMKII was found to inhibit CaMKⅡ activity and to preserve cardiovascular homeostasis.
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Objective To investigate the effects of N-butylphthalide (NBP) on cognitive function in acute severe carbon monoxide (CO) poisoning rats and its mechanism. Methods 120 health Sprague-Dawley (SD) rats were randomly divided into three groups (n = 40): normal control group (NC group), CO poisoning group (CO group) and NBP treatment group (NBP group). The acute severe CO poisoning model was established in a hyperbaric oxygen chamber by intoxicated with 1 000 ×10-6CO for 40 minutes, followed with 3 000 ×10-6CO for another 20 minutes, and then received hyperbaric oxygen therapy 1.5 hours once a day until sacrificed. Rats in NBP group were administered orally NBP 60 mg/kg for 2 times daily until death. NC group and CO group were treated with equal amount of pure olive oil. Four rats in each group were taken from 1, 3, 7, 14, 30 days after model setup, respectively. The cognitive function score was assessed by Morris water maze test. The changes in ultrastructure of hippocampus were observed under transmission electron microscope. The expressions of calpain 1 and Ca2+/calmodulin dependent protein kinase Ⅱ(CaMK Ⅱ) in hippocampus of brain tissue were detected by immunofluorescence staining, and the localization of the two target proteins in neurons was observed by immunofluorescence double staining. Results Compared with NC group, the escape latency at 1 day after poisoning in CO group was significantly prolonged (s: 55.6±3.2 vs. 44.5±3.5, P < 0.05), and the times of the platform crossing was significantly decreased (times: 1.3±0.8 vs. 6.6±1.2, P < 0.05);the ultrastructure of hippocampus was obviously injured; the protein expressions of calpain 1 and CaMK Ⅱ in brain tissue were significantly increased at 1 day after CO poisoning [calpain 1 (A value): 41.24±5.21 vs. 6.44±1.13, CaMK Ⅱ (A value): 56.19±5.04 vs. 9.84±1.53, both P < 0.05], and the protein expression of calpain 1 reached the peak at 3 days (A value: 59.34±6.11), the protein expression of CaMK Ⅱ reached the peak at 1 day (A value:56.19±5.04). Compared with CO group, the cognitive function was significantly improved in NBP group in the late stage of poisoning [7-30 days, escape latency (s): 40.3±1.9 vs. 49.1±3.1 at 7 days, 30.1±2.9 vs. 39.4±3.1 at 30 days;times of the platform crossing (times): 2.8±1.0 vs. 1.0±0.9 at 14 days, 3.2±0.8 vs. 1.0±0.9 at 30 days, all P < 0.05];the degree of injury of hippocampal neuron was relatively slight; the protein expression of calpain 1 in brain tissue was significantly decreased from 3 days after CO poisoning (A value: 39.63±3.03 vs. 59.34±6.11, P < 0.05), and the protein expression of CaMK Ⅱ was significantly decreased from 1 day after CO poisoning (A value: 42.22±3.84 vs. 56.19±5.04, P < 0.05). Immunofluorescence double staining suggested that calpain 1 and CaMK Ⅱ protein could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed that the expression of calpain 1 and CaMK Ⅱ was positively correlated (R 2= 0.852, P = 0.002). Conclusions NBP treatment could maintain ultrastructure integrity of hippocampus, balance the expression levels of calpain 1 and CaMK Ⅱproteins, and significantly improve cognitive impairment induced by CO poisoning, thus play a protective role against hippocampus damage in rats with acute severe CO poisoning.
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OBJECTIVE: To observe the changes of intracellular calcium ([Ca2＋]i) concentration and expression of calcium/calmodulin dependent protein kinaseⅡ (CaMKⅡ) in spinal dorsal horn neurons of spared nerve injury (SNI) rats, so as to explore its mechanisms underlying improvement of neuropathic pain. METHODS: One hundred and ten SD rats were randomly divided into 5 groups: sham control, model, EA, AP-5 and L-NAME groups. The sham group underwent only a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right "Weizhong" (BL 40) and "Huantiao" (GB 30) for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg·kg－1·d－1) and L-NAME (a non-selective antagonist for nitric oxide synthase [NOS], 60 mg·kg－1·d－1) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, and the calcium fluorescence intensity (shown by Fluo-3/AM calcium fluorescence indicator) of the superficial layer of the lumbar spinal cord (L 4－L 6) was measured by immunohistochemical staining and the expression of spinal cord (L 4－L 6) CaMK Ⅱ protein was detected by Western blot (WB). RESULTS: After modeling, the mechanical pain threshold was significantly decreased on day 10 and 16 after operation in comparison with the sham operation group and baseline data of pre-operation in each group (P<0.01), and remarkably increased in the EA, AP-5 and L-NAME groups relevant to the model group on day 16 (P<0.01, P<0.05), while the effect of EA was significantly superior to that of AP-5 and L-NAME groups (P<0.05), suggesting a reduction of EA analgesia after administration of AP-5 and L-NAME. The concentration of intracellular [Ca2＋]i was significantly higher in the model group than in the sham group, and considerably lower in the EA, AP-5 and L-NAME groups than in the model group (P<0.01, P<0.05). Moreover, the expression level of CaMKⅡ shown by WB and immunohistochemical staining was significantly higher in the model group than in the sham group (P<0.05) and obviously lower in the EA group (not the AP-5 and L-NAME groups) than in the model group on day 16 after the intervention (P<0.05). It suggests an involvement of glutamate NMDA receptor and NMDAR-NOS/NO signaling in the analgesic effect and CaMKⅡ expression down-regulation of EA. CONCLUSIONS: EA can ease pain in rats with neuropathic pain, which is closely related to its effect in reducing the calcium concentration and the expression of CaMKⅡ in the lumbar spinal cord, possibly mediated by glutamate NMDA receptor and NMDAR-NOS/NO signaling.
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Objective To explore the effects of prenatal taurine supplementation on the expression of protein kinase A(PKA) and c-fos,Ca2+/calmodulin-dependent protein kinase Ⅱ (GaMK Ⅱ) in fetal rat brains with intrauterine growth restriction and its significance.Methods Fifteen pregnant Sprague-Dawley rats were randomly divided into 3 groups:the control group,the intrauterine growth restriction (IUGR) group and the IUGR with prenatal taurine supplementation group(the taurine group),with 5 fetal rats in each group.All the fifteen fetal rat brains were detected as following:the expression of PKA,c-fos,CaMK Ⅱ mRNA in fetal rat brains was detected by way of real-time polymerase chain reaction(real-time PCR),while the expression changes of PKA,CaMK Ⅱ,and c-fos protein in fetal rat brains were detected by using Western blot,and the number of PKA,CaMK Ⅱ,c-fos positive cells in fetal rat brains was detected by using immunohistochemistry.Results The control group,the IUGR group and the taurine group:Comparison of the expression of PKA,CaMK Ⅱ,c-fos mRNA among 3 groups were of significant differences(F =7.934,P =0.021 ; F =5.568,P =0.043 ;F =7.332,P =0.024).Comparison of the expression of PKA,CaMK Ⅱ,c-fos protein among the 3 groups were of significant differences(F =57.743,P =0.000 ; F =163.405,P =0.000 ; F =160.136,P =0.000).Comparison of the number of PKA,CaMK Ⅱ,c-fos positive cells among the 3 groups were of significant differences (F =42.903,P =0.000 ;F =329.123,P =0.000 ; F =43.674,P =0.000).Compared with the control group,the expression of mRNA,protein,positive cells of three indicators in IUGR group was less,and the difference was statistically significant (P ＜ 0.05).The expression of mRNA,protein,positive cells of three indicators in fetal rat brains of the taurine group were not different from the control group.The expression of mRNA,protein,positive cells of three indicators in fetal rat brains of the taurine group were significantly more than the IUGR group(P ＜ 0.05).Conclusions Prenatal taurine supplementation can improve PKA-CaMK Ⅱ,c-fos mRNA and protein levels in fetal rat brain tissue with IUGR,and increase its number of positive cells and may enhance the regenerative capacity of the central nervous system,so as to reduce the IUGR brain injury and promote its role in brain development.