川芎嗪对顺铂诱导豚鼠耳蜗细胞凋亡的影响

目的:探讨川芎嗪对顺铂耳蜗毒性中细胞凋亡的影响及可能的作用机制。方法:将60只健康白色红目豚鼠随机分为3组:对照组、顺铂组和中药组,各组动物均于用药前及停药后测试听觉脑干诱发电位(ABR).电镜观察螺旋神经节细胞超微结构损伤情况,利用TUNEL测定各组耳蜗细胞凋亡情况。结果:对照组ABR阈值为40.94±6.75 db,顺铂组ABR阈值为76.26±4.54 db,中药组ABR阈值为58.98±5.82 db,三组比较,差异具有显著性(P<0.01);耳蜗透射电镜显示中药组较顺铂组耳蜗组织超微结构损伤明显减轻;TUNEL检测顺铂组有大量阳性细胞,与中药组比较,差异具有显著性(P<0.01)。结论:川芎嗪可以通过抑制细胞凋亡来拮抗顺铂的耳蜗毒性。     

顺铂耳毒性大鼠耳聋模型的研究

摘要 目的：探讨顺铂对大鼠造成的听力损伤及耳蜗细胞形态学变化。方法：体内实验,运用顺铂腹腔注射的方法,连续七天注射,通过听性脑干反应检测,观察顺铂对不同日龄的大鼠听力损伤情况;测听后取耳蜗,通过基底膜铺片和冰冻切片的免疫荧光染色,观察听力损伤后对耳蜗毛细胞和螺旋神经元的影响。体外实验,耳蜗器官培养免疫荧光染色,观察顺铂对耳蜗毛细胞和螺旋神经元的影响。结果：顺铂具有耳毒性,会对大鼠听力造成损伤,高频听力损伤更加严重,而且对不同日龄的大鼠造成的听力损失不同,小日龄的大鼠对顺铂耳毒性更加敏感。体内实验,顺铂耳毒性造成听力损失,会引起大鼠耳蜗毛细胞的缺失,但未观察到明显的螺旋神经元缺失,也没有观察到明显的Cleaved caspase-3阳性螺旋神经元细胞。体外实验,可以观察到顺铂同时引起毛细胞和螺旋神经元产生明显的损伤。结论：体、内外实验,都可以建立稳定的顺铂耳毒性大鼠耳聋模型,对研究顺铂损伤耳蜗毛细胞的发生机制和保护奠定了实验基础。     

Cav1.2在顺铂诱导C57BL/6J小鼠耳蜗螺旋神经元凋亡中的作用*

目的: 探究顺铂(CDDP)诱导C57BL/6J小鼠耳蜗螺旋神经元(SGNs)凋亡过程中Cav1.2的作用及其可能的机制。方法: 动物实验:选取8周龄雄性C57BL/6J小鼠分为以下两组(10只/组):生理盐水组(Control组)和顺铂给药组(Cisplatin组)。Control组每天腹腔注射生理盐水,Cisplatin组每周期前4 d以3 mg/kg的剂量进行顺铂腹腔注射,后10 d每日注射生理盐水,重复三个周期。给药结束后,听性脑干反应(ABR) 检测小鼠听力阈值变化; 小鼠内眦采血,并断颈取耳蜗,超氧化物歧化酶(SOD)以及丙二醛(MDA)试剂盒检测血清及耳蜗组织的SOD活性和MDA含量;免疫印迹法(Western blot)检测耳蜗组织相关凋亡蛋白表达;苏木精-伊红HE染色观察小鼠耳蜗螺旋神经节形态学变化; TUNEL 染色观察小鼠耳蜗SGNs凋亡情况;免疫荧光观察耳蜗SGNs上Cav1.2的分布和表达。细胞实验:原代培养SGNs,根据CCK8选择顺铂5 μmol/L干预12 h并随机分为:对照组(Control)、溶剂组(DMSO)、Cav1.2阻断剂组(N)、顺铂组(Cisplatin)、顺铂与Cav1.2阻断剂共同孵育组(Cisplatin+N)。Western blot检测Cav1.2蛋白表达;Hoechst33342染色观察各组SGNs凋亡情况,流式细胞术检测各组SGNs凋亡率,Western blot检测相关凋亡蛋白的表达,CA²⁺探针检测细胞内钙离子浓度变化,线粒体膜电位检测试剂盒(JC-1)检测膜电位变化,线粒体超氧化物指示剂(MitoSOX^TM-red)检测线粒体释放ROS情况。结果: 动物实验:与Control组相比,Cisplatin组小鼠听力阈值升高(P<0.01), 血清及耳蜗组织MDA含量、耳蜗组织凋亡蛋白 Cleaved-caspase-3、Bax 蛋白水平和TUNEL阳性率、Cav1.2蛋白表达水平等均明显升高(P<0.05, P<0.01);血清及耳蜗组织SOD活性、耳蜗组织抗凋亡蛋白 Bcl-2 蛋白水平和SGCs密度均明显降低(P<0.05,P<0.01)。细胞实验:与Control组相比,Cisplatin组的Cav1.2表达、细胞凋亡率、Cleaved-caspase-3、Bax蛋白水平、细胞内钙离子浓度以及ROS释放均明显增加(P<0.05,P<0.01);而细胞的Bcl-2蛋白水平和线粒体膜电位则明显降低(P<0.01);Cav1.2阻断剂可部分逆转上述改变(P<0.05)。 结论: 顺铂可能通过上调Cav1.2促进钙内流,进而使线粒体ROS增多,引起SGNs氧化应激损伤从而诱导线粒体途径的细胞凋亡。     

雌激素对老年C57BL/6J小鼠耳蜗螺旋神经节细胞凋亡的影响*

目的:观察雌激素对于老年C57BL/6J小鼠耳蜗螺旋神经节细胞凋亡的影响,并探讨雌激素对老年性耳聋保护作用的可能机制。方法:将40只C57BL/6J雌鼠分为以下4组(10只/组): 3 m组(3月龄组),12 m组(12月假手术组); 12 m OVX组(12月去卵巢组),在9月龄行双侧卵巢切除术,正常饲养至12月龄;12 m OVX+E2组(雌激素干预组)在9月龄行双侧卵巢切除术,经过1月洗脱期后,给予皮下注射雌激素100 μg/(kg·d),持续2月至12月龄,其余各组小鼠正常喂养。至12 m OVX+E2组小鼠给药结束后,尾静脉采血,酶联免疫吸附( ELISA) 法检测各组小鼠血清雌激素水平;听性脑干反应(ABR)检测各组小鼠听力阈值变化;用2%戊巴比妥钠麻醉小鼠,断颈后取双侧耳蜗,石蜡包埋切片,苏木精-伊红HE染色观察各组小鼠耳蜗螺旋神经节形态学变化;TUNEL 染色观察各组小鼠耳蜗螺旋神经节神经元凋亡情况;取耳蜗螺旋神经节,应用实时荧光定量PCR(QRT-PCR)检测各组小鼠耳蜗螺旋神经节凋亡蛋白Caspase-3、Bax、Bcl-2 mRNA 表达水平。结果:12 m组与3 m组相比,小鼠听力阈值升高(P＜0.01),耳蜗螺旋神经节细胞出现缺失严重及异常凋亡(P＜0.01);12 m OVX组小鼠听力阈值高于12 m组(P＜0.01),且螺旋神经节细胞缺失加重,细胞凋亡增多(P＜0.01),凋亡蛋白 Caspase-3、Bax mRNA水平升高(P＜ 0.01),抗凋亡蛋白Bcl-2 mRNA水平降低(P＜0.01);外源性给予雌激素12 m OVX+E2组,较12 m OVX组,听力阈值降低(P＜0.01),细胞凋亡减少,凋亡蛋白 Caspase-3、Bax mRNA水平降低(P＜0.01),抗凋亡蛋白Bcl-2 mRNA水平升高(P＜0.01)。结论:雌激素可抑制老年C57BL/6J小鼠耳蜗螺旋神经节细胞凋亡,从而实现对老年性耳聋的保护作用。     

丹参注射液对顺铂致听力损伤的拮抗作用

目的:探讨丹参注射液对顺铂致小鼠耳毒性的保护作用,为顺铂耳毒性作用的防治提供实验依据.方法:应用听觉脑干反应(ABR)检测给药前后各组小鼠不同滤波click听力阈值的变化.结果:顺铂可引起小鼠体重明显下降和各滤波click听力阈值的升高.而丹参拮抗组小鼠给药前后体重和ABR听力阈值的改变明显低于单纯顺铂给药组,并呈剂量依赖关系.结论:丹参可明显减轻顺铂对小鼠的听力损伤.     

Caspase-3在老年豚鼠耳蜗螺旋神经节细胞的表达

目的检测caspase-3在老年豚鼠耳蜗的表达。方法实验分两组:实验组和对照组,实验组豚鼠年龄为33至35个月之间,对照组豚鼠年龄为2至3个月。用免疫组织化学方法检测caspase-3在两组豚鼠耳蜗的表达。结果Caspase-3在实验组耳蜗的表达呈阳性,阳性区域主要存在于耳蜗螺旋神经节细胞。在对照组耳蜗的表达呈阴性。结论Caspase-3在老年豚鼠耳蜗螺旋神经节细胞中呈阳性表达,提示caspase-3在豚鼠耳蜗老化过程中起重要作用。     

Caspase-3在豚鼠内淋巴积水耳蜗中的表达

目的观察Caspase-3在豚鼠内淋巴积水耳蜗中的表达。方法实验分正常对照组和实验组,每组10只豚鼠。用破坏并阻塞豚鼠内淋巴囊的方法造成豚鼠内淋巴积水模型。3周后处死豚鼠,取耳蜗分别用石蜡及火棉胶包埋、切片,免疫组织化学方法观察caspase-3在耳蜗的表达。结果caspase-3在豚鼠内淋巴积水耳蜗中表达呈阳性,阳性区域为耳蜗外侧壁和螺旋神经节细胞。结论caspase-3在豚鼠内淋巴积水耳蜗中呈阳性表达,提示在内淋巴积水病理过程中存在耳蜗细胞凋亡。     

四君子丸对Lewis肺癌小鼠肿瘤组织Bax和Bcl2表达的影响

本实验通过观察四君子丸对Lewis肺癌小鼠肿瘤组织生长抑制作用及凋亡相关蛋白Bax和Bcl2表达的影响,探讨培土生金法中药复方干预肺癌的作用机制。首先在小鼠右侧腋窝皮下,接种浓度为1×107个/m L的小鼠肺癌Lewis细胞悬液0.1 m L,建立肺癌小鼠模型,成瘤后,将小鼠分为3组:Lewis肺癌组、中药治疗组、顺铂组。中药治疗组每天采用四君子丸0.0936 g/10 g,灌胃一次,连续14 d;顺铂组每周一次腹腔注射顺铂0.05mg/10 g,持续2次。然后取材,采用TUNEL荧光染色法观察各组肿瘤组织细胞凋亡的变化,采用免疫组化和免疫印迹测定肿瘤组织Bax和Bcl2蛋白表达的变化。TUNEL法荧光染色显示:和Lewis肺癌组比较,中药治疗组、顺铂组细胞凋亡指数显著增加(P0.01);免疫组化和免疫印迹结果显示:和Lewis肺癌组比较,中药治疗组和顺铂组Bax的蛋白表达水平显著升高,Bcl-2的蛋白表达水平显著降低(P0.01)。实验表明四君子丸能够干预Lewis肺癌小鼠模型肿瘤组织中Bax和Bcl2的表达,从而促进肿瘤细胞凋亡。     

华蟾素联合顺铂对人骨肉瘤U2OS细胞增殖和凋亡的影响

目的:研究华蟾素联合顺铂对人骨肉瘤U2OS细胞增殖和凋亡的影响,并探寻联合治疗增敏的可能分子机制。方法:采用不同浓度的华蟾素、顺铂单药和华蟾素联合顺铂处理人骨肉瘤U2OS细胞1-3天,通过CCK-8法检测其对U2OS细胞生长的抑制作用;平板克隆实验检测其对U2OS细胞集落形成能力的影响;流式细胞仪检测其对U2OS细胞凋亡的影响;RT-PCR及Western blotting检测Bax、Bcl-2、caspase-3、caspase-9等凋亡相关分子mRNA及蛋白水平的表达。结果:单用华蟾素或顺铂均可以浓度和时间依赖的方式抑制骨肉瘤U2OS细胞的增殖并诱导其凋亡,两药联合应用具有协同效应,并可以上调促凋亡基因Bax下调抑制凋亡基因Bcl-2的表达;联合用药组凋亡相关蛋白caspase-3、caspase-9的表达较单药组明显增加。结论:华蟾素联合顺铂与单一用药相比能够显著抑制人骨肉瘤细胞U2OS细胞的增殖,促进其凋亡,两药联合应用对骨肉瘤细胞的杀伤效应具有一定的协同效应,其机制可能与激活凋亡通路有关。     

内耳免疫反应诱导Fas和FasL表达与凋亡的关系

目的研究内耳免疫反应过程中是否存在细胞凋亡,以及细胞凋亡是否与Fas和FasL信号转导有关.方法选用雌性白色豚鼠16只,随机分为实验组和对照组各8只,以钥孔虫戚血蓝蛋白(keyhole limpet hemocyanin,KLH)全身免疫后,实验组以相同抗原进行内耳免疫,对照组内耳注射等量的磷酸盐缓冲生理盐水(phosphate buffered saline,PBS),在内耳免疫5d后处死动物,取内耳免疫侧耳蜗做石蜡切片.通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling,TUNEL)检测内耳凋亡细胞,免疫组化检测内耳Fas和FasL的表达.结果实验组豚鼠内耳Corti器毛细胞,血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞,而对照组动物切片仅在支持细胞、血管纹和螺旋神经节细胞中发现极少数TUNEL染色阳性细胞.免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性,而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达,FasL蛋白表达阴性.结论内耳免疫反应可诱导细胞凋亡的发生,Fas-FasL途径是参与此过程重要的信号转导途径之一.     

5,7-Dihydroxy-4-methylcoumarin modulates the JNK/FoxO1 signaling pathway to attenuate cisplatin-induced ototoxicity by suppressing oxidative stress and apoptosis in vitro

5,7-Dihydroxy-4-methylcoumarin (D4M) is attributed to free radical scavenging effects, with wide application for anti-oxidation. This work aimed to assess D4M's impact on cisplatin-induced ototoxicity. The cell viability was estimated with CCK-8 assay. Apoptosis was detected by the Annexin V-FITC and PI assay. The reactive oxygen species (ROS) level was determined by MitoSOX-Red and CellROX-Green probes. Mitochondrial membrane potential was analyzed with TMRM staining. Immunofluorescence was utilized for hair cells and spiral ganglion neuron detection. Apoptosis-associated proteins were assessed by cleaved caspase-3 and TUNEL staining. These results showed that D4M pretreatment protected hair cells from cisplatin-induced damage, increased cell viability, and decreased apoptosis in House Ear Institute-Organ of Corti1 (HEI-OC1) cells and neonatal mouse cochlear explants. D4M significantly inhibited cisplatin-induced mitochondrial apoptosis and reduced ROS accumulation. In addition, the protective effect of D4M on cisplatin-induced ototoxicity was also confirmed in cochlear hair cells and spiral ganglion neurons in neonatal mice. Mechanistic studies showed that D4M markedly downregulated p-JNK and elevated the expression ratio of p-FoxO1/FoxO1, thereby reducing cisplatin-induced caspase-dependent apoptosis. Meanwhile, D4M-related protection of HEI-OC1 cells was significantly blunted by JNK signaling induction with anisomycin. This study supports the possibility that D4M may be used as a new compound to prevent cisplatin-related hearing loss.     

大鼠脑创伤后caspase-3的过度表达与细胞凋亡的关系

目的：探讨大鼠脑创伤后海马神经组织中casepase-3表达及其在细胞凋亡中的机制。方法：雄性Wistar大鼠72只随机分成对照组和创伤组。用Marmarou方法造成大鼠重型弥漫性颅脑创伤,采用免疫组织化学检测海马CA1区神经细胞casepase-3蛋白表达情况,原位细胞DNA断裂检测末端标记（TUNEL）法观察大鼠海马CA1区神经细胞凋亡动态变化。同时行TUNEL与caspase-3双标染色。结果：对照组海马区神经细胞casepase-3未见明显表达,创伤组海马CA1区神经细胞casepase-3表达在伤后3小时开始升高,伤后3天达高峰（P〈0．01）,伤后7天下降明显。对照组海马区未见TUNEL阳性细胞,创伤组海马区TUNEL阳性细胞伤后3小时开始增多,伤后3天达高峰（P〈0．01）,伤后7天下降。可见创伤组TUNEL染色与caspase-3免疫染色双标阳性的细胞伤后6小时细胞数量逐渐增多,于伤后3天达高峰（P〈0．01）,伤后7天双标阳性细胞数量下降。Casepase-3表达与TUNEL阳性细胞明显相关（P〈0．01）。结论：大鼠脑创伤后casepase-3的过度表达是影响大鼠脑创伤后神经细胞凋亡原因之一,抑制casepase-3活性表达对神经组织起保护作用。     

20(S)-Ginsenoside Rh1 inhibits cisplatin-induced hearing loss by inhibiting the MAPK signaling pathway and suppressing apoptosis in vitro

As an anticancer drug, cisplatin is widely used, but its clinical application is restricted due to its severe side effects of ototoxicity. Therefore, this study was dedicated to assessing the benefit of ginsenoside extract, 20(S)-Ginsenoside Rh1 (Rh1), on cisplatin-induced ototoxicity. HEI-OC1 cells and neonatal cochlear explants were cultured. Cleaved caspase-3, TUNEL, and MitoSOX Red were observed in vitro by immunofluorescence staining. CCK8 and LDH cytotoxicity assays were detected to measure cell viability and cytotoxicity. Our results showed that Rh1 significantly increased cell viability, reduced cytotoxicity, and alleviated cisplatin-induced apoptosis. In addition, Rh1 pretreatment decreased the excessive accumulation of intracellular reactive oxygen species. Mechanistic studies indicated that Rh1 pretreatment reversed the increase of apoptotic protein expression, accumulation of mitochondrial ROS, and activation of the MAPK signaling pathway. These results suggested that Rh1 can act as an antioxidant and anti-apoptotic agent against cisplatin-induced hearing loss by suppressing the excessive accumulation of mitochondrial ROS, activation of MAPK signaling pathway and apoptosis.     

Alpha-lipoic acid protects against cisplatin-induced ototoxicity via the regulation of MAPKs and proinflammatory cytokines

Cisplatin is an effective antineoplastic drug that is widely used to treat various cancers; however, it causes side effects such as ototoxicity via the induction of apoptosis of hair cells in the cochlea. Alpha-lipoic acid (ALA) has been reported to exert a protective effect against both antibiotic-induced and cisplatin-induced hearing loss. Therefore, this study was conducted to (1) elucidate the mechanism of the protective effects of ALA against cisplatin-induced ototoxicity using in vitro and ex vivo culture systems of HEI-OC1 auditory cells and rat cochlear explants and (2) to gain additional insight into the apoptotic mechanism of cisplatin-induced ototoxicity. ALA pretreatment significantly reduced apoptotic cell death of the inner and outer hair cells in cisplatin-treated organ of Corti explants and attenuated ototoxicity via marked inhibition of the increase in the expression of IL-1β and IL-6, the phosphorylation of ERK and p38, the degradation of IκBα, the increase in intracellular levels of ROS, and the activation of caspase-3 in cisplatin-treated HEI-OC1 cells. This study represents the first histological evaluation of the organ of Corti following treatment with ALA, and these results indicate that the protective effects of ALA against cisplatin-induced ototoxicity are mediated via the regulation of MAPKs and proinflammatory cytokines.     

Overexpression of XIAP inhibits cisplatin-induced hair cell loss

Cisplatin is a platinum-containing drug with ototoxicity commonly used clinically and has significant efficacy against a variety of solid tumors. One of the most important mechanisms of ototoxicity is that cisplatin induces apoptosis of hair cells. According to relevant literature, X-linked inhibitor of apoptosis protein (XIAP, anti-apoptotic protein) could inhibit the apoptotic pathway. We hypothesized that this protein might protect cochlear hair cells from cisplatin-induced injury. To figure it out, we treated cochlea of normal mice with various concentrations of cisplatin to observe the response and morphology of hair cells and determine a reasonable concentration. Next, Western Blot and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) experiments were conducted to make an investigation about the expression of XIAP protein and mRNA. In addition, we constructed and identified XIAP overexpressing mice. Finally, we treated cochlear tissues of normal and overexpressing mice with cisplatin to investigate the cyto-protection of XIAP on hair cells, respectively. It was found that 50 μmol/L cisplatin resulted in significant loss and disorganization of hair cells, while simultaneously downregulating the protein and mRNA of XIAP. In XIAP overexpressing mice, the loss and disorganization of hair cells were significantly lessened. These results showed that XIAP can lessen cisplatin-induced hair cell loss and play a role in otoprotection.     

Deafness due to degeneration of cochlear neurons in caspase-3-deficient mice

Mice that lack caspase-3, which functions in apoptosis, were generated by gene targeting and shown to undergo hearing loss. The ABR threshold of the caspase-3(-/-) mice was significantly elevated compared to that of caspase-3(+/+) mice at 15 days of age and was progressively elevated further by 30 days. Distortion product otoacoustic emissions were not detectable in caspase-3(-/-) mice at 15 days of age. Caspase-3(-/-) mice exhibited marked degeneration of spiral ganglion neurons and a loss of inner and outer hair cells in the cochlea at 30 days of age, although no such changes were apparent at 15 days. The degenerating neurons manifested features, including cytoplasmic vacuolization, distinct from those characteristic of apoptosis. Spiral ganglion neurons and cochlear hair cells thus appear to require caspase-3 for survival but not for initial development. The mapping of both the human caspase-3 gene and the locus responsible for an autosomal dominant, nonsyndromic form of hearing loss (DFNA24) to chromosome 4q35 suggests that the caspase-3(-/-) mice may represent a model of this human condition.