An optimized recording method to characterize biophysical and pharmacological properties of acid-sensing ion channel

Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods. Methods With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASICla currents evoked by low pH external solution. Results Using cell floating method, the amplitude of hASICla currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is （21±5） ms and （270±25） ms, respectively. Inactivation time constants are （496±23） ms and （2284±120） ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC 1 a [IC50 is （3.4± 1.1 ） μmol/L and （2.4± 0.9） μmol/L, respectively]. Both recording methods have similar pH activation ECs0 （6.6±0.6, 6.6±0.7, respectively）. Conclusion ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC la current was re-confirmed and the biophysical and pharmacological properties of hASIC la channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.

Does closure of acid-sensing ion channels reduce ischemia/reperfusion injury in the rat brain?

Acidosis is a common characteristic of brain damage. Because studies have shown that permeable Ca2＋-acid-sensing ion channels can mediate the toxic effects of calcium ions, they have become new targets against pain and various intracranial diseases. However, the mechanism associated with expression of these channels remains unclear. This study sought to observe the expression characteristics of permeable Ca2＋-acid-sensing ion channels during different reperfusion inflows in rats after cerebral ischemia. The rat models were randomly divided into three groups： adaptive ischemia/reperfusion group, one-time ischemia/reperfusion group, and severe cerebral ischemic injury group. Western blot assays and immunofluorescence staining results exhibited that when compared with the one-time ischemia/reperfusion group, acid-sensing ion channel 3 and Bcl-x/I expression decreased in the adaptive ischemia/reperfusion group. Calmodulin expression was lowest in the adaptive ischemia/reperfusion group. Following adaptive reperfusion, common carotid artery flow was close to normal, and the pH value improved. Results verified that adaptive reperfusion following cerebral ischemia can suppress acid-sensing ion channel 3 expression, significantly reduce Ca2＋ influx, inhibit calcium overload, and diminish Ca2＋ toxicity. The effects of adaptive ischemia/reperfusion on suppressing cell apoptosis and relieving brain damage were better than that of one-time ischemia/reperfusion.

Identification and Function of Acid-sensing Ion Channels in RAW 264.7 Macrophage Cells

Activation of acid-sensing ion channels （ASICs） plays an important role in neuroinflammation. Macrophage recruitment to the sites of inflammation is an essential step in host defense. ASIC1 and ASIC3 have been reported to mediate the endocytosis and maturation of bone marrow derived macrophages. However, the expression and inflammation-related functions of ASICs in RAW 264.7 cells, another common macrophage, are still elusive. In the present study, we first demonstrated the presence of ASIC 1, ASIC2a and ASIC3 in RAW 264.7 macrophage cell line by using reverse transcriptase polymerase chain reaction （RT-PCR）, Western blotting and immunofluorescence experiments. The non-specific ASICs inhibitor amiloride and specific homomeric ASICla blocker PcTxl reduced the production of iNOS and COX-2 by LPS-induced activating RAW 264.7 cells. Furthermore, not only amiloride but also PcTxl inhibited the migration and LPS-induced apoptosis of RAW 264.7 cells. Taken together, our findings suggest that ASICs promote the inflammatory response and apoptosis of RAW 264.7 cells, and ASICs may serve as a potential novel target for immunological disease therapy.

Involvement of Acid-sensing Ion Channel 1a in Functions of Cultured Human Retinal Pigment Epithelial Cells

In the retina, pH fluctuations may play an important role in adapting retinal responses to different light intensities and are involved in the fine tuning of visual perception. Acidosis occurs in the subretinal space （SRS） under pathological conditions such as age-related macular degeneration （AMD）. Although it is well known that many transporters in the retinal pigment epithelium （RPE） cells can maintain pH homeostasis efficiently, other receptors in RPE may also be involved in sensing acidosis, such as acid-sensing ion channels （ASICs）. In this study, we investigated whether ASICla was ex- pressed in the RPE cells and whether it was involved in the function of these cells. Real-time RT-PCR and Western blotting were used to analyze the ASICla expression in ARPE-19 cells during oxidative stress induced by hydrogen peroxide （H202）. Furthermore, inhibition or over-expression of ASICla in RPE cells was obtained using inhibitors （amiloride and PCTxl） or by the transfection of cDNA encod- ing hASICla. Cell viability was determined by using the MTT assay. The real-time RT-PCR and West- ern blotting results showed that both the mRNA and protein of ASICla were expressed in RPE cells. In- hibition of ASICs by amiloride in normal RPE cells resulted in cell death, indicating that ASICs play an important physiological role in RPE cells. Furthermore, over-expression of ASICla in RPE cells pro- longed cell survival under oxidative stress induced by H2O2. In conclusion, ASICla is functionally expressed in RPE cells and may play an important role in the physiological function of RPE cells by pro-tecting them from oxidative stress.

Ischemic postconditioning protects against ischemic brain injury by up-regulation of acid-sensing ion channel 2a

Ischemic postconditioning renders brain tissue tolerant to brain ischemia,thereby alleviating ischemic brain injury.However,the exact mechanism of action is still unclear.In this study,a rat model of global brain ischemia was subjected to ischemic postconditioning treatment using the vessel occlusion method.After 2 hours of ischemia,the bilateral common carotid arteries were blocked immediately for 10 seconds and then perfused for 10 seconds.This procedure was repeated six times.Ischemic postconditioning was found to mitigate hippocampal CA1 neuronal damage in rats with brain ischemia,and up-regulate acid-sensing ion channel 2a expression at the m RNA and protein level.These findings suggest that ischemic postconditioning up-regulates acid-sensing ion channel 2a expression in the rat hippocampus after global brain ischemia,which promotes neuronal tolerance to ischemic brain injury.

Effect of Activation of the Ca2+-Permeable Acid-Sensing Ion Channel 1a on Acid-Induced Vascular Endothelial Cell Injury of Henoch-Schönlein Purpura Children

Acidosis in local environment plays a critical role in cell injury. One key mediator of acidosis-induced cell injury is the acid-sensing ion channels (ASICs), particularly ASIC1a. Herein, we investigated the role of ASIC1a in acid-induced vascular endothelial cell injury of Henoch-Schonlein purpura (HSP) children. Acid-induced ASIC1a, Calpain and Calcineurin expression in vascular endothelial cells pretreated with IgA1 isolated from HSP were detected by real time quantitative polymerase chain reaction and western blot methods, respectively. Cell cytotoxicity was measured by interleukin-8 and nitric oxide production with ELISA. The results showed acid-induced ASIC1a, Calpain and Calcineurin expression in cells increased, especially at PH6.5. The cytotoxicity of vascular endothelial cells was increased by extracellular acidosis. Moreover non-specific or specific blockers of ASIC1a, Amiloride and PcTX-1 could remarkably decrease these parameters. These findings show that increased [Ca2+]i, mediated via ASIC1a, might contribute to acid-induced vascular endothelial cell injury of HSP.

P2X7R过表达的巨噬细胞MSU晶体诱导痛风炎症反应过程中IL-1β、TNF-α、NLRP3表达观察

目的观察嘌呤能受体P2X配体门控离子通道7的配体(P2X7R)过表达白血病细胞诱导分化的巨噬细胞单钠尿酸盐(MSU)晶体诱导痛风炎症反应过程中NOD样受体家族3(NLRP3)蛋白、IL-1β、TNF-α表达情况。方法取人单核细胞白血病细胞系THP-1,并随机分为过表达组、空白组、模型组、对照组;过表达组和空白组分别转染P2X7R过表达质粒、空白载体质粒,转染5 d,将过表达组、空白组、模型组THP-1细胞用100 ng/mL的PMA刺激3 h后分化为巨噬细胞,另将MSU晶体用氢氧化钠溶解配制成浓度为100μg/mL的MSU乳糜状悬液加入培养液中孵育6 h;对照组正常培养。分别采用RT-PCR法和Western blot法测算巨噬细胞P2X7R mRNA、蛋白,ELISA法检测巨噬细胞上清液IL-1β、TNF-α,Western blot法测算巨噬细胞NOD样受体家族3(NLRP3)蛋白。结果与对照组比较,过表达组、空白组、模型组P2X7R mRNA和蛋白相对表达量升高,细胞上清液IL-1β、TNF-α水平升高,细胞NLRP3蛋白相对表达量升高(P均<0.05);与模型组、空白组比较,过表达组P2X7R mRNA、蛋白相对表达量升高,细胞上清液IL-1β、TNF-α水平升高,细胞NLRP3蛋白相对表达量升高(P均<0.05)。结论P2X7R过表达白血病细胞诱导分化的巨噬细胞MSU晶体诱导痛风炎症反应过程中IL-1β、TNF-α、NLRP3表达增加,IL-1β、TNF-α水平升高可能通过激活NLRP3蛋白来实现。

基于PKC-P2X3信号通路观察电针对神经病理性疼痛小鼠神经修复的作用机制

目的:基于蛋白激酶C(PKC)-配体门控型非选择性离子通道3(P2X3)信号通路观察电针对神经病理性疼痛小鼠神经修复的作用机制。方法:48只小鼠随机分成假手术组、模型组、针刺组与电针组,每组12只。除假手术组外,其余小鼠构建坐骨神经慢性限制性损伤(CCI)模型。术后第8天针刺组与电针组接受针刺与电针干预,连续7 d。于术前、术后3 d、5 d、7 d、10 d、12 d、14 d对各组进行机械缩足反射阈值(MWT)与热刺激缩足反射潜伏期(TWL)测试。术后15 d处死所有小鼠,HE染色观察坐骨神经组织形态,ELISA检测脊髓组织IL-1β、TNF-α水平,Western blot检测脊髓组织PKC、P2X3水平,免疫荧光检测脊髓神经元PKC、P2X3表达情况。结果:假手术组神经元细胞大小不一,细胞膜完整,细胞质呈细小颗粒状,细胞核大而圆,核仁居中清晰可见;模型组可见萎缩的神经元,有髓神经纤维紊乱,轴突肿胀,神经元间形成间隙;与模型组相比,针刺组、电针组萎缩神经元数量减少(P<0.05),有髓神经纤维排布情况改善,电针组较针刺组改善更为明显。与相同时间点的假手术组相比,术后3 d、5 d、7 d、10 d、12 d、14 d模型组、针刺组、电针组患侧后肢的MWT、TWL值明显下降(P<0.05),IL-1β、TNF-α、PKC、P2X3表达量、PKC、P2X3阳性细胞数、平均光密度值升高(P<0.05);与模型组相比,术后10 d、12 d、14 d针刺组、电针组MWT、TWL值明显上升(P<0.05),IL-1β、TNF-α、PKC、P2X3表达量、PKC、P2X3阳性细胞数、平均光密度值降低(P<0.05);与针刺组相比,术后10 d、12 d、14 d电针组MWT、TWL值升高(P<0.05),IL-1β、TNF-α、PKC、P2X3表达量、PKC、P2X3阳性细胞数、平均光密度值降低(P<0.05)。结论:电针可能减轻CCI模型小鼠神经炎症反应,降低PKC、P2X3表达水平,改善坐骨神经细胞形态与有髓神经纤维排布情况,缩小神经元间间隙,提升MWT、TWL值,缓解神经病理性疼痛程度。

Modulatory effects of the fruits of Tribulus terrestris L. on the function of atopic dermatitis-related calcium channels,Orai1 and TRPV3

Objective: To examine the effects of Tribulus terrestris L.(T. terrestris) extract on the modulation of calcium channels to evaluate its use in topical agents for treatment of atopic dermatitis.Methods: The 70% methanol extract of T. terrestris was prepared. Human HEK293 T cells with over-expressed calcium release-activated calcium channel protein 1(Orai1),transient receptor potential vanilloid 1, or transient receptor potential vanilloid 3(TRPV3)were treated with T. terrestris extract. Modulation of ion channels was measured using a conventional whole-cell patch-clamp technique.Results: T. terrestris extract(100 mg/m L) significantly inhibited Orai1 activity in Orai1-stromal interaction molecule 1 co-overexpressed HEK293 T cells. In addition, T. terrestris extract significantly increased the TRPV3 activity compared with 2-Aminoethyl diphenylborinate(100 mmol/L), which induces the full activation of TRPV3.Conclusions: Our results suggest that T. terrestris extract may have a therapeutic potential for recovery of abnormal skin barrier pathologies in atopic dermatitis through modulating the activities of calcium ion channels, Orai1 and TRPV3. This is the first study to report the modulatory effect of a medicinal plant on the function of ion channels in skin barrier.

天香丹抑制P2X7/NLRP3表达改善动脉粥样硬化的作用机制研究

目的:探讨天香丹对动脉粥样硬化小鼠嘌呤能配体门控离子通道7(P2X7)、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)的影响。方法:选取8周龄无特定病原体(SPF)级雄性载脂蛋白E基因敲除(ApoE-/-)小鼠30只,予以高脂饲料喂养12周,造模成功后随机分为模型组、阿托伐他汀组、天香丹组,每组10只;另选取C57BL/6J小鼠10只予普通饲料喂养,设为空白对照组。酶联免疫吸附法(ELISA)检测血清白细胞介素(IL)-18、IL-1β、腺嘌呤核苷三磷酸(ATP)的水平;免疫组化检测组织中P2X7、NLRP3的表达。结果:ELISA结果显示:与空白对照组相比,模型组IL-18、IL-1β、ATP水平升高(P<0.05);与模型组相比,阿托伐他汀组、天香丹组IL-18、IL-1β、ATP水平降低(P<0.05)。免疫组化结果显示:与空白对照组相比,模型组P2X7、NLRP3表达均明显升高(P<0.05);与模型组相比,阿托伐他汀组、天香丹组P2X7、NLRP3表达均明显降低(P<0.05)。结论:天香丹改善动脉粥样硬化与调节钾稳态抑制炎症小体的激活有关。

EFFECT OF COXSACKIEVIRUS B3 ON ION CHANNEL CURRENTS IN RAT VENTRICULAR MYOCYTES

To investigate the effects of coxsackievirus B 3(CVB 3) on ion channel currents in rat ventricular myocytes. Methods.Rat hearts were isolated with collagenase to acquire single ventricular myocytes, L type voltage dependent calcium channel(VDCC)current (I Ca ),Na + current (I Na ), outward potassium current (I out ), inwardly rectifying potassium current(I KI ) were recorded using whole cell patch clamp techniques. ResultsCVB 3 infection increased I Ca and I out , while decreased I KI ; but it had no obvious effect on I Na . Conclusion.The effects of CVB 3 on I Ca 、 I out 、 I KI may be one of the mechanisms of myocytes damage and the occurrence of abnormal electroactivities induced by CVB 3 infection.

神经营养因子3-壳聚糖载体诱导神经干细胞分化为神经元的亚型及其电生理特性

背景:前期研究已证实神经营养因子3-壳聚糖载体可支持神经干细胞的存活和增殖,同时可高效诱导神经干细胞向神经元方向分化。目的:观察神经营养因子3-壳聚糖载体对神经元发育进程、发育各阶段电生理特性及发育成熟神经元亚型的影响。方法:取第3代新生大鼠脊髓神经干细胞,分4组培养:空白对照组加入神经干细胞培养基,壳聚糖组加入含壳聚糖的神经干细胞培养基,NT3组加入含神经营养因子3的神经干细胞培养基,NT3-壳聚糖组加入含神经营养因子3-壳聚糖载体的神经干细胞培养基。利用免疫荧光染色观察神经干细胞发育各阶段标志物表达情况,借助全细胞膜片钳技术评价神经干细胞发育过程中电生理特性的变化情况,利用免疫荧光染色观察神经干细胞分化21 d后中间神经元的亚型。结果与结论:①Nestin、DCX、Tuj1及MAP2免疫荧光染色显示,神经营养因子3-壳聚糖载体维持了神经干细胞池的稳态,并且通过加速神经母细胞的发育进程来促进神经元发育成熟;②全细胞膜片钳记录发育过程中的细胞发现,营养因子神经营养因子3和神经营养因子3-壳聚糖在发育早期对神经干细胞膜功能以及细胞膜上离子通道的发育成熟具有一定的促进作用,但是仅有神经营养因子3-壳聚糖可将这一优势维持到发育中后期,即分化后7-14 d;③免疫荧光染色显示,神经干细胞分化21 d后,NT3-壳聚糖组成熟神经元可表达运动神经元特异性标记物HB9、V1类型中间神经元FOXP1、V2类型中间神经元特异性标记物LHX3,以及调控机械性痛觉感觉中间神经元的特异性标记物VGLUT3;④结果显示,神经营养因子3-壳聚糖载体促进了神经干细胞向神经母细胞的发育,在发育早期对细胞膜功能及细胞膜上的离子通道发育成熟具有一定的促进作用,可诱导发育成熟的神经元亚型多样化。

Transient Receptor Potential Ion Channels in the Etiology and Pathomechanism of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a disabling condition of unknown cause having multi-system manifestations. Our group has investigated the potential role of transient receptor potential (TRP) ion channels in the etiology and pathomechanism of this illness. Store-operated calcium entry (SOCE) signaling is the primary intracellular calcium signaling mechanism in non-excitable cells and is associated with TRP ion channels. While the sub-family (Canonical) TRPC has been traditionally associated with this important cellular mechanism, a member of the TRPM sub-family group (Melastatin), TRPM3, has also been recently identified as participating in SOCE in white matter of the central nervous system. We have identified single nucleotide polymorphisms (SNPs) in TRP genes in natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs) in CFS/ME patients. We also describe biochemical pathway changes and calcium signaling perturbations in blood cells from patients. The ubiquitous distribution of TRP ion channels and specific locations of sub-family group members such as TRPM3 suggest a contribution to systemic pathology in CFS/ME.

P2X3R对心肌梗死大鼠交感神经重构和室性心律失常的影响

目的 探究嘌呤能配体门控离子通道3受体(P2X3R)信号转导对心肌梗死(MI)后交感神经重构和结构重构以及室性心律失常易感性的影响。方法 成年雄性SD大鼠随机分为3组:(1)sham组;(2)MI组;(3)MI+A317491(MI+A)组。超声心动图评价大鼠心脏功能,酶联免疫吸附测定检测血清和左心室梗死周边区去甲肾上腺素(NE)浓度,记录各组大鼠的心电图分析心率变异性(HRV),记录电生理参数,免疫荧光法染色酪氨酸羟化酶(TH)和生长相关蛋白-43(GAP-43)阳性神经纤维分析自主神经重构,天狼星红染色观察心肌纤维化程度。蛋白质印迹法检测P2X3R、TH、GAP-43、α-平滑肌肌动蛋白、纤维连接蛋白、转化生长因子-β1等蛋白表达水平。结果 与MI组比较,A317491可显著降低MI+A组梗死周边区P2X3R蛋白表达、血清和梗死周边区NE浓度,延长心室有效不应期和动作电位持续时间,改善HRV和室性心律失常诱发率,减轻心肌纤维化,降低心室TH和GAP-43阳性神经密度以及TH和GAP-43蛋白表达,保护心脏功能。结论 A317491可能通过抑制P2X3R改善MI后的交感神经重构和结构重构,保护心脏功能,进一步降低MI后室性心律失常易感性。

髓核致炎性神经痛大鼠背根神经节ASIC3表达相关性研究

【目的】观察髓核致炎后不同时期大鼠背根神经节(DRG)3型酸敏感离子通道(ASIC3)的表达,及其与痛敏形成的关系。【方法】将SD大鼠尾部髓核0.4mg移植于左侧腰5DRG而建立炎性神经痛动物模型,或术毕DRG周围给予ASIC3抑制剂阿米洛利0.1mg,用von-Fair细丝检测机械性痛敏阈值,观察DRG的ASIC3阳性细胞数,并检测不同时间点DRG的ASIC3蛋白表达。【结果】髓核移植后大鼠机械痛阈较术前持续降低(P<0.001),DRG上ASIC3阳性细胞数增多(P<0.05),术后第7天时表达至高峰(P<0.05);ASIC阻断剂阿米洛利可抑制模型鼠痛阈降低(P<0.001)。【结论】髓核移植使大鼠DRGASIC3表达增加,抑制ASIC3表达可减轻机械性痛敏,ASIC3上调可能是椎间盘突出致炎性神经痛的重要促进因素。

酸敏感离子通道3在胃食管反流大鼠中的表达和意义

目的检测酸敏感离子通道3(ASIC3)在胃食管反流大鼠食管黏膜及背根神经节(DRG)中的表达变化,探讨其在胃食管反流病(GERD)发病中的作用。方法将Sprague-Dawley雄性大鼠随机分为实验组(G组)和对照组(S组)。实验组采用限制幽门及结扎胃底方法建立GERD大鼠模型。于造模后15 d处死两组大鼠,通过HE染色对大鼠食管黏膜进行组织病理学检测,通过蛋白质印迹法(Western blotting)和实时定量聚合酶链反应(RT-PCR)检测大鼠食管黏膜及DRG中ASIC3蛋白及mRNA表达变化。结果HE染色显示G组大鼠食管黏膜慢性炎症改变,S组大鼠食管黏膜无异常;Western blotting显示G组大鼠DRG中ASIC3蛋白表达高于S组(6.75±0.74 vs 5.07±0.72)(P<0.05),食管黏膜中ASIC3蛋白表达高于S组(8.04±0.67 vs 7.31±0.740)(P<0.05);RT-PCR同样显示,G组大鼠DRG中ASIC3 mRNA表达高于S组(0.00030±0.00003 vs 0.00013±0.00002)(P<0.05),食管黏膜中ASIC3 mRNA表达高于S组(0.01073±0.00231 vs 0.00088±0.0007)(P<0.05)。结论 ASIC3在DRG及食管黏膜上表达上调可能是导致GERD食管内脏高敏感的原因之一。

补肾方骨青颗粒对佐剂性关节炎大鼠的干预和影响软骨表达酸敏感离子通道3的实验研究

目的:观察补肾方骨青颗粒对佐剂性关节炎(adjuvant arthritis,AA)大鼠的干预作用及对软骨酸敏感离子通道3(acid-sensitive ion channel 3,ASIC3)表达的影响。方法:将大鼠随机分成空白对照组、AA模型组、阳性对照药组(阿司匹林50mg/kg)、骨青颗粒治疗剂量组(8.4 g生药/kg)、骨青颗粒高剂量组(16.8 g生药/kg)。除空白对照组外其余各组大鼠左后足趾皮内注射完全弗氏佐剂(complete Freund’s adjuvant,CFA)诱导AA,致炎后第10天起各组灌胃给予相应药物,连续给药10 d。记录大鼠体重足趾容积和关节炎评分,实验结束后,光学显微镜观察大鼠踝关节病理变化,用RT-PCR和Western blot分析大鼠膝关节软骨细胞中ASIC3 mRNA及蛋白表达。结果:经治疗后,骨青颗粒能明显抑制AA大鼠踝关节的肿胀,显著降低AA大鼠关节软骨细胞ASIC3 mRNA及蛋白的表达,效果与阳性对照药物阿司匹林无显著性差异。结论:骨青颗粒可减轻佐剂性关节炎大鼠的关节炎症,保护关节软骨,并能下调软骨ASIC3的表达。

强筋壮骨祛风合剂对髓核致炎大鼠背根神经节中3型酸敏感离子通道的影响

目的:观察强筋壮骨祛风合剂对髓核致炎大鼠背根神经节中3型酸敏感离子通道(acid-sensing ion channel 3,ASIC3)的影响。方法:选取40只清洁级成年雄性SD大鼠,随机分为空白组、假手术组、模型组、阿米洛利组和中药组,每组8只。除空白组外,其余各组大鼠均手术暴露L5背根神经节。假手术组暴露L5背根神经节后直接缝合切口;模型组和中药组取出大鼠尾部的髓核组织置于暴露的L5背根神经节上,缝合切口;阿米洛利组将取自大鼠尾部的髓核组织置于暴露的L5背根神经节上,并在暴露的L5背根神经节周围滴入含0.1 mg盐酸的阿米洛利药液1 m L,缝合切口。自造模后第1天开始,中药组按5 m L·kg-1以强筋壮骨祛风合剂灌胃,每天2次,连续30 d;空白组、假手术组、模型组不进行药物干预。分别于造模前1 d及造模后3、5、9、15、23、29 d,采用Up-Down法测定大鼠左后足50%机械刺激缩足阈值(50%paw withdrawal threshold,50%PWT)。中药组药物干预结束后,处死所有大鼠,取出L5背根神经节分成2份,一份采用免疫组织化学法计算ASIC3阳性面积,另一份采用Western Blot技术检测ASIC3蛋白表达量。结果:造模后3 d内,模型组、阿米洛利组和中药组均有部分大鼠出现左后足外翻以及足趾背伸现象。造模前后不同时间50%PWT的差异有统计学意义,即存在时间效应(F=50.132,P=0.000)。5组50%PWT总体上比较,组间差异有统计学意义,即存在分组效应(F=288.219,P=0.000)。除造模前1 d时外(F=0.332,P=0.854),造模后各时点5组大鼠50%PWT比较,组间差异均有统计学意义(F=288.015,P=0.000;F=308.588,P=0.000;F=374.945,P=0.000;F=560.713,P=0.000;F=343.043,P=0.000;F=459.914,P=0.000);造模后各时点模型组的50%PWT均低于空白组、假手术组、阿米洛利组和中药组(P=0.010,P=0.011,P=0.001,P=0.001;P=0.023,P=0.022,P=0.000,P=0.004;P=0.013,P=0.013,P=0.003,P=0.001;P=0.012,P=0.002,P=0.002,P=0.002;P=0.004,P=0.000,P=0.000,P=0.000;P=0.003,P=0.005,P=0.003,P=0.013);造模后各时点空白组和假手术组50%PWT比较,差异均无统计学意义(P=0.640,P=0.710,P=0.590,P=0.480,P=0.330,P=0.390);造模后各时点阿米洛利组50%PWT均高于中药组(P=0.021,P=0.011,P=0.013,P=0.002,P=0.001,P=0.005)。时间因素与分组因素存在交互效应(F=2.358,P=0.002)。5组大鼠背根神经节中ASIC3阳性面积和ASIC3蛋白表达量比较,组间差异均有统计学意义(F=205.890,P=0.030;F=101.740,P=0.001);空白组、假手术组、阿米洛利组和中药组背根神经节中ASIC3阳性面积和ASIC3蛋白表达量均小于模型组(P=0.004,P=0.003,P=0.001,P=0.007;P=0.000,P=0.000,P=0.000,P=0.000);阿米洛利组背根神经节中ASIC3阳性面积和ASIC3蛋白表达量均大于中药组(P=0.001;P=0.000)。结论:强筋壮骨祛风合剂可以降低髓核致炎大鼠病变的背根神经节中ASIC3的水平,提高大鼠的疼痛阈值,但其作用效果不及盐酸阿米洛利。

Tungsten oxide/nitrogen-doped carbon nanotubes composite catalysts for enhanced redox kinetics in lithium-sulfur batteries

The sluggish redox kinetics of polysulfides in lithium-sulfur(Li-S)batteries are a significant obstacle to their widespread adoption as energy storage devices.However,recent studies have shown that tungsten oxide(WO_(3))can facilitate the conversion kinetics of polysulfides in Li-S batteries.Herein,we fabricated host materials for sulfur using nitrogen-doped carbon nanotubes(N-CNTs)and WO_(3).We used low-cost components and simple procedures to overcome the poor electrical conductivity that is a disadvantage of metal oxides.The composites of WO_(3) and N-CNTs(WO_(3)/N-CNTs)create a stable framework structure,fast ion diffusion channels,and a 3D electron transport network during electrochemical reaction processes.As a result,the WO_(3)/N-CNT-Li2S6 cathode demonstrates high initial capacity(1162 mA·h·g^(-1) at 0.5℃),excellent rate performance(618 mA·h·g^(-1) at 5.5℃),and a low capacity decay rate(0.093%up to 600 cycles at 2℃).This work presents a novel approach for preparing tungsten oxide/carbon composite catalysts that facilitate the redox kinetics of polysulfide conversion.

背根神经节内代谢型谷氨酸受体2与酸敏感性离子通道3和辣椒素受体1的共存研究(英文)

代谢型谷氨酸受体(mGluR)2/3在伤害性信息从外周向脊髓传递的过程中发挥着重要作用。以往研究证明在大鼠中mGluR2参与了机械性超敏和热超敏的形成,因此本研究拟采用免疫荧光组织化学染色技术揭示背根节(DRG)中mGluR2和酸敏感性离子通道3(ASIC3),一个多觉机械性感受器,或者和热伤害性感受器辣椒素受体(TRPV1)的共存情况。结果显示:mGluR2主要存在于DRG神经元的胞浆中。计数结果显示DRG中35.85%的神经元呈mGluR2免疫阳性。在这些阳性神经元中,82.61%为小细胞(直径小于30μm);5.79%为中等细胞(直径为30～50μm);11.59%为大细胞(直径大于50μm)。进一步在免疫荧光双重标记切片上可观察到分别有42.45%和79.78%的小型mGluR2阳性神经元同时表达ASIC3或TRPV1免疫阳性。以上结果提示mGluR2主要存在于DRG中的小神经元中,这些神经元通常被认为是外周Aδ-或C-纤维传入的伤害性感觉神经元,在这类神经元中mGluR2与ASIC3或TRPV1均有大量共存,提示这些共存可能与机械性或热超敏的产生或者维持有着重要的联系。