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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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Redox-altered plasticity refers to redox-dependent reversible changes in synaptic plasticity
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@#Objective To study the effect of infrasound on expression of calmodulin-dependent protein kinase II (CaMKII) and tau pro-tein in hippocampus of rats. Methods Fifty-six male Sprague-Dawley rats were randomized into control group (n=8), 1-day group (n=8), 7-day group (n=8) and 14-day group (n=32), and the 14-day group was subgrouped as 1-hour, 6-hour, 24-hour, 48-hour subgroups, naming after the time after infrasound exposure, 8 in each subgroup. All the test groups were put in an infrasound field with 8 Hz, 130 dB for 2 hours daily, while the control group was put in the infrasound instrument without infrasound exposure for 2 hours daily. The expression of pT286-CaMKII and tau protein in hippocampus was detected with immunohistochemisty, Western blotting and enzyme-linked immunoabsor-bent assay. Results The expression of pT286-CaMKII was the most in 14-day group (F>14.912, P<0.001), as well as the expression of tau pro-tein (F>36.229, P<0.001), and secondary in 7-day group (P<0.05). For 14-day group, the expression of tau protein was the most in 1-hour and 6-hour subgroups, and dropped down in 24-hour subgroup, although more than that in the control group (P<0.05). Conclusion Exposure of 8 Hz, 130 dB infrasound may induce phosphorylation of CaMKII and tau protein, and the expression of tau protein in hippocampal cells in rat, which may disturb their learning and memory function.
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[ ABSTRACT] AIM:To study the expression profiles and the role of Ca 2+/calmodulin-dependent kinase II delta ( CaMKIIδ) during osteoclast differentiation .METHODS:Mouse RAW264.7 cells were induced by receptor activator of nuclear factor κB ligand ( RANKL) at 50μg/L for osteoclastogenesis .Tartrate-resistant acid phosphatase ( TRAP) staining and bone resorption lacunae examination were performed to verify osteoclast formation .The expression of CaMKIIδat mR-NA and protein levels was also determined by immunofluorescent cytochemistry , RT-qPCR and Western blot at days 0, 1, 3 and 5.RESULTS:TRAP positive multinuclear cells with bone resorption function were formed after 5 d of induction. The mRNA levels of CaMKIIδdetected by RT-qPCR were 1.028 ±0.041, 2.478 ±0.087, 10.524 ±1.284 and 42.914 ± 2.667 at days 0, 1, 3 and 5, respectively, while the protein levels of CaMKIIδ detected by Western blot were 0.762, 0.963, 1.802 and 3.136, respectively.The changes of protein level were also verified by immunofluorescence cytochemis -try, in which the fluorescence intensity increased in a time-dependent manner (P<0.05).CONCLUSION:The expres-sion of CaMKIIδincreases with the differentiation of osteoclasts .CaMKIIδmay play a key role in the osteoclastogenesis .
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Objective To observe the effect of tanshinone IIA on the myocardial calmodulin (CaM) and calmodulin dependent protein kinase II (CaMK) mRNA expression and heart function of acute myocardial infarction rats. Methods A total of 100 rats were randomly divided into the sham operation group, the model group, and the tanshinone ⅡA group. The model of acute myocardial infarction was established by the ligation of left anterior descending coronary artery ligation with 45 min, and reperfusion with 45 min, and the above process was repeated 3 times. Tanshinone IIA group was treated with intravenous injection of 10 mg/kg sodium tanshinone ⅡA, while the sham operation group and the model group with 10 mg/kg saline, and all treatment last 7 days. The mRNA expressions of CaM and CaMKII were determined. TTC staining was used to observe areas of myocardial infarction. The left ventricular maximum rise/fall rate (±LVdp/dtmax), myocardial contraction tension, heart rate and heart function index were detected pre-and post-treatment by the Langendorff device perfusion of isolated heart. Results Compared with the model group, the mRNA expression of and CaM mRNA (1.29 ± 0.19 vs. 2.31 ± 0.21) and CaMK II (1.10 ± 0.07 vs. 2.13 ± 0.18) in the tanshinone IIA group were significantly lower (P<0.05), and the areas of myocardial infarction (25.12%± 0.43%vs. 35.15%± 0.64%) significantly decreased (P<0.05), and the IT (2.03 ± 0.14 g vs. 1.06 ± 0.12 g), the ±LV dp/dtmax (4 701.2 ± 135.3 mmHg/s vs. 3 214.7 ± 110.2 mmHg/s, 2 518.7 ± 65.4 mmHg/s vs.1 960.3 ± 62.5 mmHg/s) significantly increased (P<0.05). Conclusion Tanshinone IIA may protect myocardial ischemia by regulating the mRNA expression of CaM and CaMKⅡ.
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Objective: To explore the changes of protein expression and activity of calcium/calmodulin-dependent protein kinase-II (CaMK-II) in myocardium nucleus and sarcoplasmic reticulum in experimental rabbits with heart failure (HF). Methods: A total of 16 rabbits were divided into 2 groups: Sham group and HF group, the HF model was established by volume overload plus pressure overload.n=8 in each group and all animals were treated for 7 weeks. Left ventricular structure, hemodynamic parameters and protein expression and activity of CaMK-II in myocardium nucleus and sarcoplasmic reticulum were examined and compared between 2 groups. Results: Compared with Sham group, HF group presented increased left ventricular mass index (LVMI) (1.32 ± 0.06) g/kg vs (3.61 ± 0.09) g/kg, LVEDP (-1.50 ± 0.50) mmHg vs (23.00 ± 2.37) mmHg, allP Conclusion: Increased protein expression and activity of CaMK-II in myocardium nucleus and sarcoplasmic reticulum might be one of the mechanisms for HF occurrence in experimental rabbits.
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Objective: To explore the role of calcium/calmodulin-dependent protein kinase-II δ (CaMK-II δ) in doxorubicin (DOX) induced cardio-toxicity in experimental rats. Methods:①The rat’s cardiomyocytes were treated by DOX and the cell proliferation, protein expression and activity of CaMK-II δ were examined.②CaMK-II δ gene was knocked out by CRISPR method, the changes of DOX induced cell apoptosis and NF-κb activity and miR-146a expression were detected. Results: DOX could inhibit cardiomyocyte proliferation, the protein expression level of CaMK-II δ was similar and the activity was increased. CRISPR method may effectively knock out CaMK-II δ gene. Compared with normal cells, the cells from CaMK-II δ knocked out rats had decreased sensitivity to DOX induction, suppressed NF-κb activation and miR-146a up-regulation. Conclusion: CaMK-II δ participated in DOX induced cardio-toxicity in experimental rats and NF-κb and miR-146a were involved in this process.
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Objective: To investigate the effect of endocannabinoid system on cardiac hypertrophy in experimental rats with chronic intermittent hypoxia and to study the impact of endocannabinoid antagonist, rimonabant in such pathological processing. Methods: A total of 48 male Wistar rats were divided into 6 groups. 4 and 6 weeks of Normal control group, 4 and 6 weeks of Hypoxia group, 4 and 6 weeks of Hypoxia with rimonabant intervention group. n=8 in each group. The rats were sacrificed to measure left ventricular mass index (LVMI), the myocardial cell morphological changes were observed by optical microscope, the expression of cardiac calcium/calmodulin-dependent protein kinase II (CaMKII) and cardiotrophin-1 (CT-1) were detected by immunohistochemistry at 4 and 6 weeks respectively. Results: Compared with 4 and 6 weeks of Normal control group, the LVMI, cardiac hypertrophy condition, CaMKII and CT-1 were increased in 4 and 6 weeks of Hypoxia group, all P Conclusion: The Chronic intermittent hypoxia could induce myocardial hypertrophy via endocannabinoid system disorders, such pathological processing could be reduced by rimonabant intervention.
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Tamoxifen, an antiestrogen, has previously been shown to induce apoptosis in HepG2 human hepatoblastoma cells through activation of the pathways independent of estrogen receptors, i.e., intracellular Ca2+ increase and generation of reactive oxygen species (ROS). However, the mechanism of tamoxifen to link increased intracellular Ca2+ to ROS generation is currently unknown. Thus, in this study we investigated the possible involvement of calmodulin, a Ca2+ activated protein, and Ca2+/ calmodulin-dependent protein kinase II in the above tamoxifen-induced events. Treatment with calmodulin antagonists (calmidazolium and trifluoroperazine) or specific inhibitors of Ca2+/calmodulin-dependent protein kinase II (KN-93 and KN-62) inhibited the tamoxifen-induced apoptosis in a dose-dependent manner. In addition, these agents blocked the tamoxifen-induced ROS generation in a concentration-dependent fashion, which was completely suppressed by intracellular Ca2+ chelation. These results demonstrate for the first time that, despite of its well-known direct calmodulin-inhibitory activity, tamoxifen may generate ROS and induce apoptosis through indirect activation of calmodulin and Ca2+/calmodulin-dependent protein kinase II in HepG2 cells.
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Humans , Apoptosis , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calmodulin , Carcinoma, Hepatocellular , Estrogen Receptor Modulators , Hep G2 Cells , Hepatoblastoma , Protein Kinases , Reactive Oxygen Species , Receptors, Estrogen , TamoxifenABSTRACT
AIM: To observe the change of Ca2+ / calmodulin dependent protein in kinase II (CaMK II) signal pathway in opioids dependent NG108-15 cells and the relationships between Ca2+ / calmodulin dependent protein kinase II (CaMK II) signal pathway and cAMP accumulation. METHODS: NG108-15 cells were used as an in vitro model system. Competitive protein binding assay and radioimmunoassay were used to examine the intracellular cAMP accumulation. Calmodulin activity was assayed by PDE method. CaMK H activity was assayed by τ-32P incorporation of syntide-2. mRNA expression of mDOR was measured by RT-PCR. RESULTS DPDPE long-term treatment also increased cAMP accumulation, and resulted in opioids dependence in NG108-15 cells; DPDPE long-term treatment increased calmodulin activity and CaMK II activity in NG108-15 cells. Specific calmodulin antagonist W-7 was found to inhibit significantly the elevation of calmodulin and CaMK II activity which resulted from DPDPE long-term treatment, and CaMK II inhibitor KN-62 also inhibited elevation of CaMK II activity by DPDPE long-term treatment. DPDPE long-term treatment increased cAMP accumulation, when W-7 or KN-62 was added at the same time, cAMP accumulation decreased. When naloxone was added to NG108-15 cells which were long-term treated by DPDPE, calmodulin and CaMK II activity increased more. mRNA level of mDOR did not change significantly in opioids dependent NG108-15 cells. CONCLUSION: Ca2+/CaMK II signal pathway was involved in the mechanisms of opioids dependence through regulating cAMP level when DPDPE is long-term administered to NG108-15 cells.
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AIM:To study the effects of different duration of sleep deprivation (SD) on neurogranin (Ng),protein kinase C (PKC) and Ca2+-calmodulin dependent protein kinase II (CaMK II) mRNA expressions in hippocampus and forebrain of rats. METHODS:Male Wistar rats were divided into three groups:24 h,48 h and 72 h experimental groups. Each group was divided into sleep deprivation group (SD group) and control group (C group). SD model was established by sleep deprivation box. One step methods by Trizol was used to extract hippocampus and forebrain neuronal total RNA. The changes of Ng,PKC and CaMK II mRNA expressions were detected by SYBRA green I RT-PCR. RESULTS:(1) The expression of Ng mRNA in hippocampus of SD group was significantly lower than that in C group after 24 h,48 h and 72 h SD (P