Magnesium Ascorbyl Phosphate Promotes Bone Formation Via CaMKII Signaling.

Dysregulation of bone homeostasis is closely related to the pathogenesis of osteoporosis. Suppressing bone resorption by osteoclasts to attenuate bone loss has been widely investigated, but far less effort has been poured toward promoting bone formation by osteoblasts. Here, we aimed to explore magnesium ascorbyl phosphate (MAP), a hydrophilic and stable ascorbic acid derivative, as a potential treatment option for bone loss disorder by boosting osteoblastogenesis and bone formation. We found that MAP could promote the proliferation and osteoblastic differentiation of human skeletal stem and progenitor cells (SSPCs) in vitro. Moreover, MAP supplementation by gavage could alleviate bone loss and accelerate bone defect healing through promoting bone formation. Mechanistically, we identified calcium/calmodulin-dependent serine/threonine kinase IIα (CaMKIIα) as the target of MAP, which was found to be directly bound and activated by MAP, then with a concomitant activation in the phosphorylation of ERK1/2 (extracellular regulated kinase 1/2) and CREB (cAMP-response element binding protein) as well as an elevation of C-FOS expression. Further, blocking CaMKII signaling notably abolished these effects of MAP on SSPCs and bone remodeling. Taken together, our data indicated that MAP played an important role in enhancing bone formation through the activation of CaMKII/ERK1/2/CREB/C-FOS signaling pathway and may be used as a novel therapeutic option for bone loss disorders such as osteoporosis. © 2023 American Society for Bone and Mineral Research (ASBMR).

CaMKII and CaV3.2 T-type calcium channel mediate Connexin-43-dependent inflammation by activating astrocytes in vincristine-induced neuropathic pain.

Vincristine (VCR), an alkaloid isolated from vinca, is a commonly used chemotherapeutic drug. However, VCR therapy can lead to dose-dependent peripheral neurotoxicity, mainly manifesting as neuropathic pain, which is one of the dominant reasons for limiting its utility. Experimentally, we discovered that VCR-induced neuropathic pain (VINP) was accompanied by astrocyte activation; the upregulation of phospho-CaMKII (p-CaMKII), CaV3.2, and Connexin-43 (Cx43) expression; and the production and release of inflammatory cytokines and chemokines in the spinal cord. Similar situations were also observed in astrocyte cultures. Interestingly, these alterations were all reversed by intrathecal injection of KN-93 (a CaMKII inhibitor) or L-Ascorbic acid (a CaV3.2 inhibitor). In addition, KN-93 and L-Ascorbic acid inhibited the increase in [Ca2+]i associated with astrocyte activation. We also verified that knocking down or inhibiting Cx43 level via intrathecal injection of Cx43 siRNA or Gap27 (a Cx43 mimetic peptide) relieved pain hypersensitivity and reduced the release of inflammatory factors; however, they did not affect astrocyte activation or p-CaMKII and CaV3.2 expression. Besides, the overexpression of Cx43 through the transfection of the Cx43 plasmid did not affect p-CaMKII and CaV3.2 expressions in vitro. Therefore, CaMKII and CaV3.2 may activate astrocytes by increasing [Ca2+]i, thereby mediating Cx43-dependent inflammation in VINP. Moreover, we demonstrated that the CaMKII signalling pathway was involved in VCR-induced inflammation, apoptosis, and mitochondrial damage. Collectively, our findings show a novel mechanism by which CaMKII and CaV3.2 mediate Cx43-dependent inflammation by activating astrocytes in neuropathic pain induced by VCR.

Total alkaloids from the seed embryo of Nelumbo nucifera Gaertn. improve cognitive impairment in APP/PS1 mice and protect Aß-damaged PC12 cells.

The seed embryo of Nelumbo nucifera Gaertn. is a famous traditional Chinese medicine and food which is considered conducive to the prevention of Alzheimer's disease (AD). In this study, the effect and mechanism of TASENN (total alkaloids from the seed embryo of Nelumbo nucifera Gaertn.) on AD mice and amyloid-ß (Aß) injured PC12 cells were evaluated. HPLC-UV analysis showed that the extracted TASENN (purity = 95.6%) mainly contains Liensinine, Isoliensinine, and Neferine (purity was 23.01, 28.02, and 44.57%, respectively). In vivo, oral treatment with TASENN (50 mg/kg/day for 28 days) improved the learning and memory functions of APP/PS1 transgenic mice, ameliorated the histopathological changes of cortical and hippocampal neurons, and inhibited neuronal apoptosis. We found that TASENN reduced the phosphorylation of Tau and the formation of neurofibrillary tangles (NFTs) in APP/PS1 mouse brain. Moreover, TASENN down-regulated the expression of APP and BACE1, ameliorated Aß deposition, and inhibited microglial proliferation and aggregation. The elevated protein expression of CaM and p-CaMKII in APP/PS1 mouse brain was also reduced by TASENN. In vitro, TASENN inhibited the apoptosis of PC12 cells injured by Aß25-35 and increased the cell viability. Aß25-35-induced increase of cytosolic free Ca2+ level and high expression of CaM, p-CaMKII, and p-Tau were decreased by TASENN. Our findings indicate that TASENN has a potential therapeutic effect on AD mice and a protective effect on PC12 cells. The anti-AD activity of TASENN may be closely related to its negative regulation of the CaM pathway.

An in vivo and in vitro model on the protective effect of cilnidipine on contrast-induced nephropathy via regulation of apoptosis and CaMKâ ¡/mPTP pathway.

Contrast-induced nephropathy (CIN) is an acute kidney injury (AKI) observed after the administration of contrast media. Calcium channel blockers (CCBs) have been reported to exert a renal protective effect. This study aims to investigate the role of cilnidipine, a novel CCBs, on CIN by regulating the calcium/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)/mitochondrial permeability transition pore (mPTP) pathway. Here, iohexol, a representative contrast media, was used to establish CIN model. KN-93 (CaMKⅡ inhibitor) and atractyloside (mPTP opener) were administered in rats, and CaMKⅡ overexpression was used in Human proximal tubular epithelial cells. Markers of renal injury (serum creatinine, blood urea nitrogen, and urinary NAGL), hematoxylin-eosin stain, oxidative stress (ROS, superoxide dismutase [SOD], and malondialdehyde [MDA] levels), cell death (MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]), mitochondrial function (mPTP, mitochondrial membrane potential [MMP], and ATP) were assessed. Western blots were used to measure the expression levels of Bax/Bcl-2, caspase-3, CaMKⅡ/mPTP signaling pathways. Results showed that cilnidipine markedly improved kidney function, and alleviated tubular cell apoptosis, oxidative stress and mitochondrial damage induced by iohexol in vitro and in vivo. The underlying mechanism may be that cilnidipine relieved CaMKⅡ activation and mPTP opening induced by iohexol. All of these protective effects of cilnidipine were attenuated by CaMKⅡ overexpression and atractyloside (mPTP opener) pretreatment. Moreover, KN-93 (CaMKⅡ inhibitor) treatment showed a similar renal protective effect with cilnidipine, while the protective effect of cilnidipine on kidney in CIN rats was not further suppressed by KN-93 cotreatment. These in vitro and in vivo results point toward the fact that cilnidipine might be a novel therapeutic drug against contrast-induced nephrotoxicity in a CaMKⅡ-dependent manner.

MicroRNA-145 Protects against Myocardial Ischemia Reperfusion Injury via CaMKII-Mediated Antiapoptotic and Anti-Inflammatory Pathways.

MicroRNA-145 (miR-145) has been shown to play an important role in cardiovascular system disorders; however, the underlying mechanism is not completely understood. The purpose of this study was aimed at elucidating the cardioprotective effects of miR-145 against myocardial ischemia/reperfusion (I/R) injury. We established a rat myocardial I/R model with 45 min left anterior descending coronary artery (LAD) occlusion and 2 h reperfusion. The levels of myocardial enzymes, apoptotic, inflammatory, and oxidative indices were determined. The arrhythmia score was assessed by programmed electrical stimulation (PES). Quantitative real-time PCR and western blot were applied to evaluate the expression levels of miR-145 and related target proteins, respectively. I/R injury decreased the expression of miR-145; however, upregulated miR-145 markedly reduced the elevation of ST segment, decreased corrected QT (QTc) intervals, and attenuated I/R-induced electrophysiological instability. Furthermore, miR-145 suppressed myocardium apoptotic, inflammatory, and oxidative response as well as the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), ryanodine receptor2 (RyR2 Ser2814), apoptosis signal-regulating kinase 1 (ASK1), c-Jun NH2-terminal kinases (JNK), and nuclear translocation of nuclear factor kappa-B (NF-κB) p65. In summary, overexpression of miR-145 alleviates I/R-induced myocardial electrophysiological instability and apoptotic and inflammatory response via inhibition of the CaMKII-mediated ASK1 antiapoptotic pathway and NF-κB p65 anti-inflammatory pathways.