RESUMO
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.
RESUMO
@#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.
RESUMO
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
RESUMO
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.
RESUMO
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.
RESUMO
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
RESUMO
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.
RESUMO
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.
Assuntos
Animais , Masculino , Ratos , Pontos de Acupuntura , Colite Ulcerativa/terapia , Epitélio , Mucosa Intestinal , Ratos Sprague-Dawley , Junções ÍntimasRESUMO
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.
RESUMO
Redox-altered plasticity refers to redox-dependent reversible changes in synaptic plasticity
RESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
According to the data of Pinellia ternate transcriptome,two calmodulin genes were cloned and named as Pt Ca M1 and PtCa M2 respectively. The results of bioinformatics analysis showed that Pt Ca Ms genes contained a 450 bp open reading frame,encoding149 amino acids.The identity of the coding sequences was 80%,and the identity of amino acids sequence was 91%. Pt Ca Ms genes contained EF-hand structure domain,belonging to the Ca M families. The Real-time PCR analysed the expression patterns of Pt Ca Ms in different tissues and different treatments. RESULTS:: showed that Pt Ca M1 and Pt Ca M2 gene were the highest expression level in tuber. Under Ca Cl2 treatment,the expressions of Pt Ca Ms were significantly higher than the control. Under EGTA,La Cl3 and TFP treatments,the expression level of Pt Ca Ms decreased gradually. In this study,the Pt Ca Ms gene were successfully cloned from P. ternate,which laid a foundation for the functional characteristic of Pt Ca Ms gene and the synthesis of alkaloids from P. ternata for further study.
Assuntos
Calmodulina , Genética , Clonagem Molecular , Genes de Plantas , Pinellia , Genética , Tubérculos , GenéticaRESUMO
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.
Assuntos
Sítios de Ligação , Cálcio , Sinalização do Cálcio , Calmodulina , Canais Iônicos , Íons , Técnicas de Patch-Clamp , Doenças Renais Policísticas , Canais de Potencial de Receptor TransitórioRESUMO
The aim of this study was to observe the effect of baicalin on the growth state of attention deficit hyperactivity disorder animal model and its regulation on Ca MKⅡand ERK1/2.In the present study,a total of 40 SHR rats were randomly divided into model group,methylphenidate hydrochloride group,and low,medium,and high dose baicalin groups,with 8 rats in each group.Eight WKYrats were selected as a normal control group.The methylphenidate hydrochloride group(0.07 g·L~(-1))and the low(3.33 g·L~(-1)),medium(6.67 g·L~(-1)),and high dose(10 g·L~(-1))baicalin groups received corresponding drugs by gavage administration according to the body weight(0.015 m L·g~(-1)),while the normal group and the model group received the same volume of normal saline by gavage.Thegavage administration lasted for 4 weeks,twice a day.The body weight of the rats and the amount of remaining feed were weighed daily,and the growth state of the rats was statistically evaluated weekly.Percoll density gradient centrifugation was used to prepare brain synaptosomes and an electron microscope was used to observe their structures.The Ca MKⅡand ERK1/2 protein and mRNA expression levels were detected with Western blot and Real-time PCR methods,respectively.RESULTS: showed that baicalin did not affect the normal eating and weight gain of rats,and the weight gain of rats was even more significant than that in the normal group(P<0.05).In the study of its effects on Ca MKⅡand ERK1/2 protein expression in rat synaptosomes,the expression of both proteins in each drug-administered group was higher than that in the model group(P<0.05);besides,the expression levels of Ca MKⅡand ERK1/2 protein were significantly increased in both baicalin high dose group and the methylphenidate hydrochloride group(P<0.05).The relative expression of Ca MKⅡand ERK1/2 mRNA in synaptosome was detected by PCR.The results showed that medium and high doses of baicalin and methylphenidate hydrochloride significantly increased the relative expression of Ca MKⅡand ERK1/2 mRNA in synaptosomes of SHR rats(P<0.05).In conclusion,baicalin does not affect the normal growth and development of SHR rats,so it is safe for administration.Both baicalin and methylphenidate hydrochloride could up-regulate the relative expression of Ca MKⅡand ERK1/2 in mRNA and protein,and the pharmacodynamic stability of baicalin is in a dose-dependent manner to certain extent.
Assuntos
Animais , Ratos , Transtorno do Deficit de Atenção com Hiperatividade , Modelos Animais de Doenças , Flavonoides , Peptídeos e Proteínas de Sinalização Intracelular , Sistema de Sinalização das MAP Quinases , Proteínas Serina-Treonina Quinases , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
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.
RESUMO
A redução da reatividade vascular à fenilefrina (PE) em aorta de ratas espontaneamente hipertensas (SHR) ao final da prenhez é dependente de maior produção e/ou maior biodisponibilidade de óxido nítrico (NO), consequente do aumento da fosforilação da enzima óxido nítrico sintase endotelial (eNOS) via PI3K/Akt. A glicosilação do tipo N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-traducional que compete com a fosforilação pelos mesmos sítios de ligação nas proteínas. A O-GlcNAcilação da eNOS em serina1177 leva a redução da sua atividade enquanto a fosforilação leva a sua ativação. Além destes mecanismos, a interação da eNOS com outras proteínas é capaz de regular positiva ou negativamente a sua atividade. O objetivo deste trabalho foi analisar possíveis alterações nos mecanismos de modificação pós-traducional que controlam a ativação da eNOS os quais poderiam contribuir para maior ativação e maior biodisponibilidade de NO observada em artérias de ratas prenhes. Foram avaliados o conteúdo proteico O-GlcNAc e também expressão das enzimas que participam desta modificação, O-GlcNAc transferase (OGT) e O-GlcNAcase (OGA) por Western Blotting e a atividade da OGA por ensaio bioquímico em aorta e em artéria mesentérica (2º ou 3º ramo) de ratas não prenhes (NP) e prenhes (P), normotensas (Wistar) e SHR. Ensaios de Western Blotting foram realizados também para análise da expressão das seguintes proteínas: Cav-1, p-Cav-1, CaM e Hsp90. Realizamos a contagem do número de cavéolas endoteliais da aorta e da artéria mesentérica na presença ou ausência da metil-ß-ciclodextrina (dextrina, 10 mmol/L) por microscopia eletrônica. Em estudos funcionais, avaliamos a participação da enzima OGA, pela inibição com PugNAc (100 µmol/L) e das cavéolas, utilizando um desorganizador de cavéolas, a dextrina (10 ou 20 mmol/L), na menor reatividade vascular à PE observada em aortas de ratas P. Observamos que o conteúdo de proteínas O-GlcNAciladas estava diminuído em aorta e em leito mesentérico de ratas Wistar P e SHR P. Apesar da expressão da OGT e da OGA não estar alterada, a atividade da OGA foi aumentada em aorta e leito mesentérico de ratas Wistar P, mas, encontra-se diminuída em aorta e aumentada em leito mesentérico de SHP P. A incubação com PugNAc reverteu a reduzida reatividade à PE em aorta e artéria mesentérica de ratas Wistar P mas este efeito não foi observado em vasos SHR P, demonstrando que a OGA parece ter um papel importante na redução da O-GlcNAcilação de proteínas vasculares em Wistar P. Em vasos incubados com PugNAc, a remoção do endotélio ou a incubação com L-NAME, não alterou significativamente a reatividade à PE. Juntos estes resultados sugerem que a maior atividade da eNOS observada em vasos de Wistar P, fica prejudicada na presença do PugNAc, e depende da atividade da OGA. Como não houve alteração da resposta contrátil à PE em vasos de SHR P incubados com PugNAc, possivelmente um mecanismo diferente, envolvendo a menor atividade da OGT, ocorre nestas artérias para a redução da O-GlcNAcilação da eNOS. A desorganização das cavéolas por meio da dextrina causou aumento de contração à PE e redução de potência da ACh em aortas de Wistar NP e SHR NP, porém não houve alteração em aortas de ratas Wistar P e SHR P. A dextrina não alterou o número de cavéolas em artérias de Wistar P e SHR P quando comparado com ratas NP. SHR NP apresentam um reduzido número de cavéolas das aortas em relação a Wistar NP bem como expressão reduzida de Cav-1, p-Cav-1 e CaM. A prenhez não foi capaz de alterar a expressão da Cav-1, CaM e Hsp90 em aorta e leito mesentérico de ratas normotensas e hipertensas. Estes resultados sugerem que a prenhez não altera a expressão das proteínas Cav-1, CaM e Hsp90 e possivelmente a interação com a eNOS em aorta e artérias mesentéricas de ratas normotensas e hipertensas. Em conclusão, entre os mecanismos estudados de modificação pós-traducional da eNOS, a redução da O-GlcNAcilação da eNOS, por mecanismos que envolvem a atividade da OGA e possivelmente da OGT, favoreceria a fosforilação da eNOS e consequente maior biodisponibilidade de NO, contribuindo desta forma para modulação da resposta contrátil da PE nas artérias de ratas P(AU)
Reduction of vascular reactivity to phenylephrine (PE) in aorta of spontaneously hypertensive rats (SHR) at the end of pregnancy is dependent on higher production and/or higer bioavailability of nitric oxide (NO), as a consequence of increased endothelial nitric oxide synthase enzyme (eNOS) phosphorylation, by PI3K/Akt. Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification that competes with phosphorylation by the same binding sites in proteins. O-GlcNAcylation of eNOS on serine site leads to a reduction in its activity while eNOS phosphorylation leads to its activation. In addition to these mechanisms, the interaction of eNOS with other proteins is able to regulate positively or negatively its activity. The objective of this study was to analyze possible changes in the mechanisms of post-translational modification that control the eNOS activation, which could contribute to its the greater activation and greater bioavailability of NO observed in arteries of pregnant rats. The O-GlcNAc-protein content and also the enzymes expression that participate in this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) was assessed by Western Blotting, and OGA activity were evaluated by biochemical assay in the aorta and in the artery mesenteric (2nd or 3rd branch) of non-pregnant (NP) and pregnant (P), normotensive rats (Wistar) and SHR. Western Blotting assays were also performed for expression analysis of the following proteins: Cav-1, p-Cav-1, CaM and Hsp90. We performed the counting of the number of endothelial caveolae in the aorta and the mesenteric artery in the presence or absence of methyl-ß-cyclodextrin (dextrin, 10 mmol/L) by electronic microscopy. In functional studies, we evaluated the participation of the OGA enzyme, by inhibition with PugNAc (100 µmol/L) and of the caveolae, using a caveolae disassembler, dextrin (10 or 20 mmol/L), in the reduced vascular reactivity observed in aortas or mesenteric arteries of P rats. We observed that the content of O-GlcNAcylated proteins was decreased in the aorta and in the mesenteric bed of Wistar P and SHR P rats. Although OGT and OGA expression is not altered, OGA activity was increased in the aorta and mesenteric bed of Wistar P rats but was decreased in the aorta and increased in the mesenteric bed of SHP P. Incubation with PugNAc reversed the reduced reactivity to PE in the aorta and mesenteric artery of Wistar P but this effect was not observed in SHR P arteries, demonstrating that OGA appears to play an important role in reducing O-GlcNAcylation of vascular proteins in Wistar P. In arteries incubated with PugNAc, endothelial removal or incubation with L-NAME did not significantly alter reactivity to PE. Together, these results suggest that the greater eNOS activity observed in Wistar P vessels was impaired in the presence of PugNAc, and it depends on OGA activity. As there was no change in the contractile response to PE in SHR P arteries incubated with PugNAc, possibly a different mechanism, involving the lower activity of OGT, occurs in these vessels for the reduction of O-GlcNAcylation of eNOS. Dextrin caused increased contraction of PE and decreased ACh potency in Wistar NP and SHR NP aortas, but there was no change in aortas of Wistar P and SHR P. Dextrin did not alter the number of cavelae in Wistar P and SHR P arteries compared to NP rats. SHR NP showed a lower number of caveolae than to NP Wistar as well reduced expression of Cav-1 and CaM. Pregnancy was not able to alter the expression of Cav-1, CaM and Hsp90 in the aorta and mesenteric bed of normotensive and hypertensive rats. These results suggest that pregnancy does not alter the expression of Cav-1, CaM and Hsp90 proteins and possibly interaction with eNOS in the aorta and mesenteric arteries of normotensive and hypertensive rats. In conclusion, among the studied mechanisms of post-translational modification of eNOS, the reduction of O-GlcNAcylation of eNOS, by mechanisms that involve OGA activity and possibly OGT, would favor eNOS phosphorylation and consequent greater NO bioavailability, contributing in this way for modulation of the contractile response to PE in the arteries of P rats(AU)
Assuntos
Animais , Feminino , Gravidez , Aorta , Óxido Nítrico Sintase , Hipertensão , Glicosilação , Calmodulina , Ratos Wistar , Proteínas de Choque Térmico HSP90 , Caveolina 1 , Artérias MesentéricasRESUMO
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.