ABSTRACT
Aim To study the role of Cx43 hemichannel in rats with acute incision pain. Methods Adult male rats were randomly divided into normal saline group(C group), incision pain group(I group), Gap19 group. Western blot was used to determine the expression of Cx43 and GFAP at 6 h, 3 d and 7 d after paw incision surgery of rats. The rats were intrathecal injected with Gap19 30 min before incision surgery, followed by the measurement of the paw withdrawal threshold to mechanical stimuli at several time points. Immunofluorescence was used to detect the expression of Cx43 and GFAP. ELISA was performed to detect the different inflammatory cytokine levels in rats after surgery. Results Compared with group C, the expression of Cx43 and GFAP of rats increased significantly 6 h after incision surgery in group I, while their expression at postoperative 3 d and 7 d showed no obvious alteration. Compared with the preoperative baseline, the mechanical paw withdrawal threshold in I group and Gap19 group rats was significantly reduced at 2 h, 6 h, 24 h and 3 d post-incision(P<0.01), but it had no obvious change on postoperative 7 d. Compared with group I, the mechanical paw withdrawal threshold in rats of Gap19 group increased 2 h, 6 h, 24 h post-incision, while it showed no statistical difference on 3 d and 7 d post-incision. Compared with group C, immunofluorescence results showed that the expression of GFAP and Cx43 in spinal cord dorsal horn astrocytes also increased significantly in Group I 6 h after incision surgery. Compared with group I, GFAP and the expression of Cx43 were markedly reduced in Gap19 group. There was no statistical difference on 7 d post-incision. Compared with those in group C, the inflammation factors including IL-1β, IL-6 and TNF-αobviously increased in group I. However, in contrary to the rats in group I, the rats pretreated with gap19 showed increased expression of Cx43 and GFAP in spinal cord dorsal horn. Conclusions Specific inhibition of Cx43 hemichannel significantly suppresses astrocyte activation and alters the inflammatory microenvironment in the spinal cord dorsal horn and reduces postoperative hyperalgesia in rats with acute incision pain.
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Objective: To investigate the effect of silver nanoparticles (AgNPs) on hemichannel activity in human skin keratinocytes (HaCaT) and to explore the role of hemichannel in AgNP-induced anti-proliferative effect on HaCaT cells.Methods: HaCaT cells were exposed to 0, 0.1, 0.2, 1.0, 5.0, 10.0, 15.0 and 20.0 μg/cm2 of AgNPs for 24 h and cell viability was assessed by propidium iodide (PI) staining with flow cytometry.Hemichannel activity was examined by ethidium bromide (EB) uptake experiments in cells exposed to AgNPs with and without hemichannel inhibitor carbenoxolone (CBX).Afterward, HaCaT cells were seeded at a low density of 1×104cell /cm2 or a normal density of 4×104cell /cm2 and cultured for 24 h.Cell proliferation was measured by cell counting kit-8 (cck-8) in low-or normal-density cultured cells exposed to AgNPs for 24 h with and without 100 μmol/L CBX.Results: Cell viability showed no significant differences between the control and AgNP-exposed groups with the concentration less than 10 μg/cm2.Exposure to AgNPs increased EB uptake in a time-and dose-dependent manner in HaCaT cells and EB fluoresce density was increased to 116.67%, 124.85% and 139.53% of the control after exposure to 10 μg/cm2 AgNPs for 2 h, 12 h and 24 h.After being treated with 25, 50 and 100 μmol/L CBX, 10 μg/cm2 AgNP-induced increase in EB uptake was significantly reduced in HaCaT cells (P<0.01).The cellular proliferation rate was increased inlow-and normal-density cultured cells after AgNPs exposure with 100 μmol/L CBX.After being treated with 100 μmol/L CBX, 10 μg/cm2 AgNP-induced anti-proliferation in low-density cultured cells was significantly restrained (P<0.01).Conclusion: AgNPs could enhance hemichannel activity of HaCaT cells.Hemichannel activation was involved in AgNP-induced anti-proliferative effect.
ABSTRACT
Connexins (Cx) are membrane proteins and monomers for forming gap junction (GJ) channels. Cx46 and Cx50 are also known to function as conductive hemichannels. As part of an ongoing effort to find GJ-specific blocker(s), endocrine disruptors were used to examine their effect on Cx46 hemichannels expressed in Xenopus oocytes. Voltage-dependent gating of Cx46 hemichannels was characterized by slowly activating outward currents and relatively fast inward tail currents. Bisphenol A (BPA, 10 nM) reduced outward currents of Cx46 hemichannels up to ~18% of control, and its effect was reversible (n=5). 4-tert-Octylphenol (OP, 1 microM) reversibly reduced outward hemichannel currents up to ~28% (n=4). However, overall shapes of Cx46 hemichannel current traces (outward and inward currents) were not changed by these drugs. These results suggest that BPA and OP are likely to occupy the pore of Cx46 hemichannels and thus obstruct the ionic fluxes. This finding provides that BPA and OP are potential candidates for GJ channel blockers.
Subject(s)
Connexins , Endocrine Disruptors , Gap Junctions , Membrane Proteins , Oocytes , XenopusABSTRACT
Objective To observe ultrastructure of the morphological relationship between neuron and astrocytes in hippocampi of pentylenetetrazol (PTZ)-kindled epileptic rats,and to investigate the communicative ways between them.Methods Epilepsy models of 10 kindled rats established by intraperitoneal injection of PTZ[i.p.,35 mg/(kg · d)],assigned as kindled group.Five rats received 9 g/L saline as control group.Three days after being kindled,the ultrastructural relationship between neurons and the astrocytes was observed with transmission electron microscope and Cx43 labelling immuno-electron microscopy.Results 1.Synapses increased in hippocampi of PTZ-kindled epileptic rats.2.Gap junctions were observed between astrocytes and neurons.3.Astrocytic process extended into the synaptic cleft between pre-synaptic and post-synaptic membranes which formed the synaptic complex.4.The Cx43-hemichannels existed between astrocytes and neurons.Conclusions In hippocampi of PTZ-kindled epileptic rats,ultrastructure of morphological relationship between neurons and astrocytes includes synaptic complex,gap junctions and hemichannels,which might be communicative forms between neurons and astrocytes.