Xianjie Wen

Cardioprotection of sevoflurane postconditioning by activating extracellular signal-regulated kinase 1/2 in isolated rat hearts

AbstractAim:The activation of extracellular signal-regulated kinase (ERK)1/2 protects against ischemic–reperfusion injury. Whether ERK1/2 mediates the cardioprotection of sevoflurane postconditioning is unknown. We tested whether sevoflurane postconditioning produces cardioprotection via an ERK1/2-dependent mechanism.Methods:In protocol 1, Langendorff-perfused Sprague–Dawley rat hearts (n=84, 12 per group), with the exception of the Sham group, were subjected to 30 min ischemia followed by 90 min reperfusion and were assigned to the untreated (control) group, followed by 4 cycles of ischemic postconditioning (25 s of each), 3% (v/v) sevoflurane postconditioning (for 5 min and 10 min of washout), and the PD98059 solvent DMSO (<0.2%), ERK1/2 inhibitor PD98059 (20 μmol/L), and Sevo+PD administration. Left ventricular hemodynamics and coronary flow at 30 min of equilibrium were recorded at 30, 60, and 90 min of reperfusion, respectively. Acute infarct size was measured by triphenyltetrazolium chloride staining. The configuration of mitochondria was observed by an electron microscope. Western blot analysis was used to determine the contents of cytosolic and mitochondrial cytochrome c at the end of reperfusion. In protocol 2, after 15 min of reperfusion, the expression of total and phosphorylated forms of ERK1/2 and its downstream target p70S6K was determined by Western blotting.Results:No differences in baseline hemodynamics were observed among the experimental groups (P>0.05). After reperfusion, compared with the control group, sevoflurane postconditioning and ischemic postconditioning significantly (P<0.05) improved functional recovery and largely (P<0.05) decreased myocardial infarct size (22.9%±4.6% and 21.2%±3.8%, vs 39.4%±5.7%, both P<0.05). Sevoflurane-mediated protection was abolished by PD98059.Conclusion:Anesthetic postconditioning by sevoflurane effectively protects against reperfusion damage by activating ERK1/2 in vitro.

Intrathecal administration of Cav3.2 and Cav3.3 antisense oligonucleotide reverses tactile allodynia and thermal hyperalgesia in rats following chronic compression of dorsal root of ganglion

AbstractAim:The present study aimed to elucidate the role of T-subtype calcium channels (Cav3.1, Cav3.2, and Cav3.3) in the pathogenesis of neuropathic pain at spinal level.Methods:The chronic compression of the dorsal root ganglion (CCD) rat model was adopted. The antisense oligonucleotide of Cav3.1, Cav3.2, and Cav3.3 or normal saline (NS) were intrathecally administered twice per day from the first day to the fourth day after operation. Paw mechanical withdrawal threshold and paw thermal withdrawal latency were measured to evaluate the tactile allodynia and thermal hyperalgesia, respectively.Results:CCD rats developed reliable tactile allodynia and thermal hyperalgesia after operation. Intrathecal administration of antisense oligonucleotide of Cav3.2 and Cav3.3 significantly relieved tactile allodynia and thermal hyperalgesia in CCD rats, but not Cav3.1.Conclusion:Cav3.2 and Cav3.3 subtype calcium channels in the spinal cord may play an important role in the pathogenesis of neuropathic pain, which may contribute to the management of the neuropathic pain.

Neurotoxicity Induced by Bupivacaine via T-Type Calcium Channels in SH-SY5Y Cells

There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca2+ ([Ca2+]i), cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation.

SEVOFLURANE POST‐CONDITIONING PROTECTS AGAINST MYOCARDIAL REPERFUSION INJURY BY ACTIVATION OF PHOSPHATIDYLINOSITOL‐3‐KINASE SIGNAL TRANSDUCTION

1 The mechanisms underlying myocardial protection by sevoflurane post‐conditioning are unclear. In the present study, we tested two hypotheses: (i) that sevoflurane post‐conditioning produces cardioprotection via a phosphatidylinositol‐3‐kinase (PI3‐K)‐dependent pathway; and (ii) combining sevoflurane and ischaemic post‐conditioning offers an additional benefit against reperfusion injury. 2 Rat isolated perfused hearts were exposed to 25 min ischaemia followed by 90 min reperfusion. Sevoflurane post‐conditioning was induced by administration of sevoflurane (3.0 vol%) for 15 min from the onset of reperfusion. In some groups, 15 µmol/L LY294002, a selective PI3‐K inhibitor, was coadministrated with sevoflurane. Other groups of hearts were exposed to ischaemic post‐conditioning or combined sevoflurane plus ischaemic post‐conditioning in the presence and absence of LY294002. After 15 min reperfusion, phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β) was determined by Western blot analysis. Infarct size was determined by 2,3,5‐triphenyltetrazolium chloride staining and subsarcolemmal mitochondrial lesions were assessed by electron microscopy after 90 min reperfusion. 3 Sevoflurane post‐conditioning significantly decreased infarct size compared with control hearts (31 ± 2 vs 42 ± 3%, respectively; P < 0.05), diminished mitochondrial lesions and increased phosphorylation of Akt and GSK3β, as did ischaemic post‐conditioning. However, combined sevoflurane plus ischaemic post‐conditioning did not further improve the cardiaoprotective effects compared with either intervention alone. Sevoflurane‐mediated cardioprotection was abolished or inhibited by 15 µmol/L LY294002. 4 In conclusion, sevoflurane acts during early reperfusion after ischaemia to salvage the myocardium by activating PI3‐K. The combination of sevoflurane plus ischaemic post‐conditioning does not offer any additional benefit over either intervention alone.

Sevoflurane inhibits invasion and migration of lung cancer cells by inactivating the p38 MAPK signaling pathway.

PurposeSevoflurane is used widely during lung cancer surgery. However, the effect of sevoflurane on the invasion and migration of lung carcinoma cells remains unclear. The aims of this study were to explore the role of matrix metalloproteinase (MMP)-2 and MMP-9 in the effect of sevofluane on the invasion and the role of fascin and ezrin on the effect of sevofluane on the migration of human lung adenocarcinoma A549 cells. We also investigated whether sevoflurane regulates the expression of these molecules through the p38 mitogen-activated protein kinase (MAPK) signaling pathway.MethodsThe invasion of cells was evaluated using the Transwell invasion assay, and the migration of cells was determined using the wound healing assay. The expression of MMP-2, MMP-9, ezrin, fascin, and phospho-p38 MAPK in cells was determined by western blotting.ResultsA significant inhibition of cell invasion and migration was found in A549 cells which had been treated with sevoflurane. The data also revealed that sevoflurane could decrease the phosphorylation level of p38 MAPK, which is involved in the downregulation of MMP-2, MMP-9, fascin, and ezrin expression, accompanied by a concomitant inhibition of the invasion and migration of A549 cells. SB203580, a p38 MAPK inhibitor, augmented the downregulation of the expression of these proteins.ConclusionThe anti-invasion effect of sevoflurane on A549 cells was associated with a downregulation of both MMP-2 and MMP-9 expression, while the anti-migration effect was associated with a downregulation of both fascin and ezrin expression. These effects could occur partly as a result of inactivation of the p38 MAPK signaling pathway.

Neuroprotective effect of ginkgolide B on bupivacaine-induced apoptosis in SH-SY5Y cells.

Local anesthetics are used routinely and effectively. However, many are also known to activate neurotoxic pathways. We tested the neuroprotective efficacy of ginkgolide B (GB), an active component of Ginkgo biloba, against ROS-mediated neurotoxicity caused by the local anesthetic bupivacaine. SH-SY5Y cells were treated with different concentrations of bupivacaine alone or following preincubation with GB. Pretreatment with GB increased SH-SY5Y cell viability and attenuated intracellular ROS accumulation, apoptosis, mitochondrial dysfunction, and ER stress. GB suppressed bupivacaine-induced mitochondrial depolarization and mitochondria complex I and III inhibition and increased cleaved caspase-3 and Htra2 expression, which was strongly indicative of activation of mitochondria-dependent apoptosis with concomitantly enhanced expressions of Grp78, caspase-12 mRNA, protein, and ER stress. GB also improved ultrastructural changes indicative of mitochondrial and ER damage induced by bupivacaine. These results implicate bupivacaine-induced ROS-dependent mitochondria, ER dysfunction, and apoptosis, which can be attenuated by GB through its antioxidant property.

Interleukin-33 signaling contributes to renal fibrosis following ischemia reperfusion

&NA; Acute kidney injury caused by ischemia‐reperfusion injury (IRI) is a major risk factor for chronic kidney disease, which is characterized by renal interstitial fibrosis. However, the molecular mechanisms underlying renal fibrosis induced by IRI are not fully understood. Our results showed that interleukin (IL)−33 was induced markedly after IRI insult, and the kidneys of mice following IRI plus IL‐33 treatment presented more severe renal fibrosis compared with mice treated with IRI alone. Therefore, we investigated whether inhibition of IL‐33 protects against IRI‐induced renal fibrosis. Mice were administrated with soluble ST2 (sST2), a decoy receptor that neutralizes IL‐33 activity, or vehicle by intraperitoneal injection for 14 days after IRI challenge. We revealed that mice treated with sST2 exhibited less severe renal dysfunction and fibrosis in response to IRI compared with vehicle‐treated mice. Inhibition of IL‐33 suppressed bone marrow–derived fibroblast accumulation and myofibroblast formation in the kidneys after IRI stress, which was associated with less expression of extracellular matrix proteins. Furthermore, inhibition of IL‐33 also showed a significant reduction of F4/80+ macrophages and CD3+ T cells in the kidneys of mice after IRI treatment. Finally, Treatment with IL‐33 inhibitor reduced proinflammatory cytokine and chemokine levels in the kidneys of mice following IRI insult. Taken together, our findings indicate that IL‐33 signaling plays a critical role in the pathogenesis of IRI–induced renal fibrosis through regulating myeloid fibroblast accumulation, inflammation cell infiltration, and the expression of proinflammatory cytokines and chemokines.

Inhibitory gene expression of the Cav3.1 T-type calcium channel to improve neuronal injury induced by lidocaine hydrochloride.

Cav3.1 is a low-voltage-activated (LVA) calcium channel that plays a key role in regulating intracellular calcium ion levels. In this study, we observed the effects of lidocaine hydrochloride on the pshRNA-CACNA1G-SH-SY5Y cells that silenced Cav3.1 mRNA by RNA interference, and investigated the roles of p38 MAPK in these effects. We constructed the pNC-puro-CACNA1G-SH-SY5Y cells and pshRNA-CACNA1G -SH-SY5Y cells by the RNA interference. All the cells were cultured with or without 10mM lidocaine hydrochloride for 24 h. The cell morphology, cell viability, Cav3.1 and p38 protein expression, cell apoptosis rate and intracellular calcium ion concentration were detected. We found that all cells treated with 10mM lidocaine hydrochloride for 24 h showed cellular rounding, axonal regression, and cellular floating. Compared with the cells in SH-SY5Y+Lido group and NC+Lido group, those in the RNAi+Lido group showed similar changes, but of smaller magnitude. Additionally, following lidocaine hydrochloride all cells displayed increased Cav3.1 and p38 MAPK protein, apoptosis rate, and intracellular calcium ion levels; however,these changes in the RNAi+Lido group were less pronounced than in the SH-SY5Y+Lido and NC+Lido groups. The cell viability decreased following lidocaine hydrochloride treatment, but viability of the cells in the RNAi+Lido group was higher than in the SH-SY5Y+Lido and NC+Lido groups. The results showed that Cav3.1 may be involved in neuronal injury induced by lidocaine hydrochloride and that p38 MAPK phosphorylation was reduced upon Cav3.1 gene silencing.

CaMK II γ down regulation protects dorsal root ganglion neurons from ropivacaine hydrochloride neurotoxicity

T-type calcium channels are intimately involved in the local anesthetics neurotoxicity. Does CaMKIIγ regulate T-type calcium currents in local anesthetics neurotoxicity? This study generated pAd-CaMKIIγ and pAd-shRNA adenovirus vectors to up- and down-regulate CaMKIIγ mRNA expression in dorsal root ganglion neurons (DRG). Normal DRG (Normal group), empty vector DRG (Empty vector group), pAd-CaMKIIγ DRG (pAd-CaMKIIγ group) and pAd-shRNA DRG (pAd-shRNA group) were treated or untreated with 3 mM ropivacaine hydrochloride for 4 h. Cell viability, apoptosis rate, CaMKIIγ, pCaMKIIγ, Cav3.2, and Cav3.3 expression were detected. Ultrastructural changes in DRG were observed under a transmission electron microscope. The results demonstrated that the cell viability of DRG treated with ropivacaine hydrochloride decreased markedly, the apoptosis rate, CaMKIIγ, pCaMKIIγ, Cav3.2, Cav3.3 expression increased significantly. CaMKIIγ up-regulation aggravated ropivacaine hydrochloride-induced cell damage and increased Cav3.2 and Cav3.3 expression. In conclusion, CaMKIIγ regulated Cav3.2 and Cav3.3 expression in DRG, which was involved with ropivacaine hydrochloride-induced cell injury.

One cell model establishment to inhibit CaMKIIγ mRNA expression in the dorsal root ganglion neuron by RNA interfere

Abstract CaMKIIγ in dorsal root ganglion neurons is closely related to the neuropathic pain, neuron injury induced by local anesthetics. To get great insight into the function of CaMKIIγ in dorsal root ganglion neurons, we need one cell model to specially inhibit the CaMKIIγ mRNA expression. The present study was aimed to establish one cell model to specially inhibit the CaMKIIγ mRNA expression. We designed the CaMKIIγ shRNA sequence and connected with pYr-1.1 plasmid. The ligation product of the CaMKIIγshRNA and pYr-1.1 plasmid was recombined with pAd/PL-DEST vector into pAD-CaMKIIγ-shRNA. adenovirus vector. pAD-CaMKIIγ-shRNA. adenovirus vector infected the dorsal root ganglion neuron to inhibit the CaMKIIγ mRNA expression in vitro. The pAD-CaMKIIγ-shRNA adenovirus vector was verified to be correct by the digestion, sequence. And pAD-CaMKIIγ-shRNA. adenovirus vector can infect the DRG cells to inhibit the CaMKIIγ mRNA or protein expression by the real-time polymerase chain reaction (PCR) or western blotting. Those results showed that we successfully constructed one adenovirus vector that can infect the dorsal root ganglion neuron to inhibit the CaMKIIγ mRNA and protein expression. That will supply with one cell model for the CaMKIIγ function study.