Effects of Senegenin against hypoxia/reoxygenation-induced injury in PC12 cells.

OBJECTIVE: To investigate the effect and the potential mechanism of Senegenin (Sen) against injury induced by hypoxia/reoxygenation (H/R) in highly differentiated PC12 cells. METHODS: The cultured PC12 cells were treated with H/R in the presence or absence of Sen (60 µmol/L). Four groups were included in the experiment: control group, H/R group, H/R+Sen group and Sen group. Cell viability of each group and the level of lactate dehydrogenase (LDH) in culture medium were detected for the pharmacological effect of Sen. Hoechst 33258 staining and annexin V/propidium iodide double staining were used to analyze the apoptosis rate. Moreover, mitochondrial membrane potential (△Ψm), reactive oxygen species (ROS) and intracellular free calcium ([Ca(2+)]i) were measured by fluorescent staining and flow cytometry. Cleaved caspase-3 and activity of NADPH oxidase (NOX) were determined by colorimetric protease assay and enzyme linked immunosorbent assay, respectively. RESULTS: Sen significantly elevated cell viability (P<0.05), decreased the leakage of LDH (P<0.05) and apoptosis rate (P<0.05) in H/R-injured PC12 cells. Sen maintained the value of △Ψm (P<0.05) and suppressed the activity of caspase-3 (P<0.05). Moreover, Sen reduced ROS accumulation P<0.05) and [Ca(2+)]i increment (P<0.05) by inhibiting the activity of NOX (P<0.05). CONCLUSION: Sen may exert cytoprotection against H/R injury by decreasing the levels of intracellular ROS and [Ca(2+)]i, thereby suppressing the mitochondrial pathway of cellular apoptosis.

Effect of hypobaric hypoxia on the P2X receptors of pyramidal cells in the immature rat hippocampus CA1 sub-field.

PRIMARY OBJECTIVE: This study was designed to evaluate the effect of hypobaric hypoxia (HH) on the function and expression of P2X receptors in rat hippocampus CA1 pyramidal cells. RESEARCH DESIGN: The functional changes of P2X receptors were investigated through the cell HH model and the expressional alterations of P2X receptors were observed through the animal HH model. METHODS AND PROCEDURE: P2X receptors mediated currents were recorded from the freshly dissociated CA1 pyramidal cells of 7-day-old SD rats by whole cell patch clamp recording. The expression and distribution of P2X receptors were observed through immunohistochemistry and western blot at HH 3-day and 7-day. MAIN OUTCOMES AND RESULTS: In acute HH conditions, the amplitudes of ATP evoked peak currents were decreased compared to control. The immunohistochemistry and western blot results reflected there was no change in P2X receptors expression after 3 days HH injury, while P2X receptors expression was up-regulated in response to 7 days HH injury. CONCLUSIONS: These findings supported the possibility that the function of P2X receptors was sensitive to HH damage and long-term function decrease should result in the expression increase of P2X receptors.

Effect of lappaconitine on neuropathic pain mediated by P2X3 receptor in rat dorsal root ganglion.

ATP facilitates initiation and transmission of the neuropathic pain at the dorsal root ganglion (DRG) level via the P2X receptors, especially the subtype P2X(3). Lappaconitine (LA) is an active principle isolated from Chinese herbal medicine and possesses analgesic effect. The aim of this study was to investigate the effect of LA on chronic constriction injury (CCI)-induced neuropathic pain mediated by P2X(3) receptor in the DRG neurons. In the presence of CCI and/or LA, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and P2X(3) receptor expression in the DRG neurons was evaluated by immunohistochemistry and Western blotting. Following intrathecal administration of P2X(3) receptor oligonucleotide, the effect of LA on pain thresholds was assessed. Furthermore, the effect of LA on the P2X(3) receptor agonists ATP- and α,ß-meATP-induced inward currents (I(ATP) and I(α,ß-meATP)) in the acutely dissociated rat DRG neurons was investigated by whole cell patch-clamp. The results included: (1) There showed reduction of pain thresholds, enhancement of I(ATP) and I(α,ß-meATP) and up-regulation of P2X(3) receptor expression in rat DRG neurons when neuropathic pain occurred. (2) In the presence of LA, the decreased pain thresholds, the up-regulated P2X(3) receptor expression and the enhanced I(ATP) and I(α,ß-meATP) were reversible in the CCI rats. (3) The down-regulated P2X(3) receptor expression with pretreatment of P2X(3) receptor antisense oligonucleotide significantly attenuated the analgesic effect of LA. These results indicate that the analgesic effect of LA involves decrease of expression and sensitization of the P2X(3) receptors of the rat DRG neurons following CCI.

Role of midbrain periaqueductal gray P2X3 receptors in electroacupuncture-mediated endogenous pain modulatory systems.

Extracellular ATP facilitates pain transmission at peripheral and spinal sites via the P2X receptors and the P2X3 subtype is an important candidate for this effect. Electroacupuncture (EA) has been clinically utilized to manage chronic pain. In this study, with neuropathic pain model of Sprague-Dawley (SD) rats, the P2X3 receptor protein level and expression location in the midbrain periaqueductal gray (PAG), a crucial site in endogenous pain modulatory system, were evaluated by Western blotting and immunohistochemistry. The results showed (1) pain thresholds were decreased while P2X3 receptor expression was up-regulated in the lateral PAG (lPAG) when neuropathic pain occurred. When the lPAG was pretreated with P2X3 receptors, antagonist A-317491 attenuated the antinociceptive effect produced by intra-lPAG injection of alpha,beta-methylene-ATP (alpha, beta-meATP), an agonist for P2X3 receptor. (2) Multiple EA treatments begot enhanced pain thresholds and increased P2X3 receptor immunoreactivity in the lPAG in neuropathic pain rats. Conversely, the down-regulated P2X3 receptor expression in the lPAG with antisense oligodeoxynucleotide (ODN) for P2X3 gene significantly attenuated the antinociceptive effect of EA treatment. These results suggest that P2X3 receptors in the lPAG play an inhibitory role in pain modulation and EA exerts a marked therapeutic effect in relieving neuropathic pain in CCI rats, which may be related to its regulative effect on the expression of P2X3 receptors in the lPAG. In conclusion, P2X3 receptors in the lPAG are involved in the supraspinal antiociception effect of EA treatment.