CN107184596B - 硫氢化钠在制备治疗脊髓小脑性共济失调药物中的应用 - Google Patents
硫氢化钠在制备治疗脊髓小脑性共济失调药物中的应用 Download PDFInfo
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Abstract
本发明属于一种硫氢化钠在制备治疗脊髓小脑性共济失调药物中的应用。本发明测出SCA3小鼠的记忆能力明显低于WT小鼠,SCA3小鼠海马内硫化氢的量明显低于WT小鼠。海马中的H2S与SCA3小鼠生长过程中的空间学习和记忆有更多的关系。硫氢化钠治疗SCA3小鼠的机制可能是:硫氢化钠通过上调海马内硫化氢的含量能够改善其线粒体的功能,进而有可能使海马有髓神经纤维通路MBP含量增加来减轻其髓鞘损伤,或许就可以改善SCA3小鼠的记忆障碍。
Description
技术领域
本发明属于医药技术领域,具体涉及硫氢化钠在制备治疗脊髓小脑性共济失调药物中的应用。
背景技术
SCA(脊髓小脑性共济失调)发病率约占神经遗传病的10%左右,其中SCA3发病率最高,神经遗传病的致病蛋白存在突变,进而导致了结构的不正常折益,这也导致大量的分子伴侣被招募到致病蛋白上,进而可能影响细胞内功能正常的蛋白质的正常折叠的进行。
另一SCA主要的病理生理学机制为线粒体功能障碍,线粒体DNA突变导致SCA延迟显性及表型高度异质性。已有研究表明,线粒体损伤在SCA3发病机制中起着重要作用,线粒体的主要功能是合成ATP,其损伤直接影响合成ATP。ATP生成的减少也能造成细胞水肿和内质网扩张,若进一步加重则将导致细胞变性坏死和细胞功能的丧失。
髓鞘的损伤和脱落会影响到神经元的功能。有研究表明:记忆、认知功能降低与中枢神经***有髓神经纤维髓鞘结构的改变存在着密切的联系。在中枢神经***中,组成髓鞘的主要成分之一是髓鞘碱性蛋白(MBP),约占30-40%。MBP起到维持CNS髓鞘结构和功能的稳定、维持髓鞘结构和脑发育的作用,是判断有无髓鞘脱失的特异性生化指标。有学者证明,MBP有促进轴突再生的作用。髓鞘内MBP含量的正常依赖于线粒体的正常能量供应。
发明内容
本发明解决其技术问题采用的技术方案是,硫氢化钠作为活性成分在制备治疗脊髓小脑性共济失调药物中的应用,尤其是在治疗脊髓小脑性共济失调3型药物中的应用。
本发明所述药物为单一成分的硫氢化钠或者为硫氢化钠和其它化合物组成的复方药物。
本发明所述药物包括硫氢化钠和药学上可接受的载体。
本发明所述药物的剂型为任意一种临床可接受的口服给药剂型、注射给药剂型或外用给药剂型。
本发明所述药物的剂型为片剂、胶囊剂、颗粒剂、丸剂、注射剂、煎膏剂、悬浮剂、分散剂、糖浆剂、栓剂、凝胶剂、气雾剂或贴剂等。
本发明所述药物中硫氢化钠的活性剂量为0.06-0.62mg/kg/d。
进一步,本发明所述药物中硫氢化钠的活性剂量为0.31mg/kg/d,上述剂量可以一个剂量单位或分成几个剂量单位给药。本发明给药剂量取决于多种因素,如患者或动物的性别、年龄、体重、所患疾病的严重程度、自身的肝、肾功能状态及给药途径等。因此,本发明的剂量可以有一定范围的变化。
本发明检测SCA3小鼠、WT小鼠海马内硫化氢的含量,同时也通过免疫组化的方法测出MBP含量在WT小鼠和SCA3小鼠大脑海马有髓神经纤维通路的差异。
本发明通过Morris水迷宫筛选建立痴呆小鼠模型,经过小鼠定位航行试验和空间探索试验,在学习记忆能力、生化指标上进行科学的评估,证明了海马中的H2S与小鼠生长过程中的空间学习和记忆有更多的关系,通过改变海马硫化氢的含量能够改善其线粒体的功能,进而有可能使海马有髓神经纤维通路MBP含量增加来减轻其髓鞘损伤,或许就可以改善SCA3小鼠的记忆障碍。可见,本发明为今后治疗脊髓小脑性共济失调应用于临床提供参考依据。
附图说明
图1为不同小鼠组在Morris水迷宫平均行驶距离;
图2为不同小鼠组在Morris水迷宫平均平台潜伏期;
图3为不同小鼠组在Morris水迷宫平均游泳速度;
图4为不同小鼠给与NaHS后海马组织H2S含量;
图5为空白对照组小鼠海马有髓神经纤维的丝状着色图;
图6为SCA3模型组小鼠海马有髓神经纤维的丝状着色图;
图7为SCA3低剂量小鼠组小鼠海马有髓神经纤维的丝状着色图;
图8为SCA3中剂量小鼠组海马有髓神经纤维的丝状着色图;
图9为SCA3高剂量小鼠组海马有髓神经纤维的丝状着色图。
具体实施方式
下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
1.1模型建立
雄性SCA3基因小鼠和雄性野生型(WT)小鼠均来源于郑州大学实验动物中心,体重20-23g,年龄12个月,在所有实验中使用。PCR鉴定,从中随机选出SCA3模型组和空白对照组。给药,每天新鲜配制100mmol/LNaHS母液(用无菌生理盐水配制),NaHS:10μmol/kg组以100份生理盐水稀释、50μmol/kg组用20份生理盐水稀释、1000μmol/kg组用10份生理盐水稀释,每日腹腔注射一次。每周监测小鼠的体重和H2S含量,根据小鼠体重的改变相应调整给药量,连续4周。WT小鼠组每日等量腹腔注射生理盐水做为空白组,SCA3小鼠分为低剂量组(n=12)NaHS10μmol/kg,中剂量组(n=12)NaHS50μmol/kg,高剂量组(n=12)NaHS100μmol/kg,大约28天,期间进行称重,H2S含量检测。
1.2学习和记忆能力评价
1.2.1水迷宫测试
1.2.1.1实验前的准备工作
实验开始前,水温调整至22-24℃。向水池内加入无毒的白色涂料污染至水不透明,水面高度调节至使水没过平台0.5cm。实验前先将所有实验动物进行Morris水迷宫初筛,以筛选出先天性痴呆及其他条件不符合实验条件的小鼠。
1.2.1.2定位航行实验
实验时长4d,按逆时针方向每天改变小鼠游泳的开始象限,每只小鼠每天训练4次,每个象限1次,两次训练的时间间隔约为25min。用摄像头记录小鼠的游泳轨迹,通过视频信号采集卡进行视频信号的采集和数字转换,用行为学记录软件EthoVisionXT8(Noldus公司,德国)进行信号的记录与分析。每次游泳训练小鼠找到水下平台后,让其停留在平台上30s,以巩固其记忆。小鼠每次游泳训练的最大时长为120s,如果小鼠在120s内没有自行找到水下平台,则将其引导至水下平台上并让其在平台上停留30s。
1.2.1.3空间探索实验
定位航行实验结束后24h开始进行训练空间探索实验,水下平台即被移除,小鼠的游泳时间依旧为120s。用行为学记录软件EthoVisionXT8记录这段时间内小鼠在迷宫内游泳轨迹。
1.2.1.4统计学处理
数据以均值(Mean)±标准误(SEM)表示,组间进行方差分析(ANOVA)和t检验和线性回归分析,P<0.05被认为有统计学意义。
1.2.2标本的固定、取材和制作
将各组小鼠用10%水合氯醛(3mL/kg)腹腔注射完全麻醉后,在大脑海马上按照“快、轻、小、准”的四字原则,选用锋利的刀片快速取材(冰上操作),操作完毕置液氮中保存,为方便电镜和硫化氢测定的使用次日将其移入-80℃冰箱保存。各组其余的小鼠经过全身麻醉后,开胸暴露心脏,在右心耳处切一小口,将针头经左心室插人升主动脉内,向内注人4℃冰生理盐水至右心耳流出液澄清后,再注人4%多聚甲醛250ml,速度先快后慢地持续约1h左右,断头取脑。将脑组织置于4%多聚甲醛中在4℃冰箱内固定6h,经脱水后用石蜡包埋制成切片。
1.3测定H2S含量
分光光度法间接测定脑组织中H2S含量。开颅取脑,分离出双侧海马组织、前脑皮质和纹状体放入液氮保存备用,用0~4℃的50mmol/L磷酸钾缓冲液(pH8.0)匀浆(12%质量体积比),匀浆液离心(20000r,15min,4℃),上清液(75μL)移至另一离心管中,在室温下加入0.25mL1%醋酸锌及0.45mL双蒸水孵育10min,然后加入10%三氯乙酸0.25mL,再次离心(14000r,10min,4℃),收集清澈的上清液,加入20mmol/LN,N-二甲基苯二胺·7.2mol/LHCl缓冲液133μL及30mmol/LFeCl3·1.2mol/LHCl缓冲液133μL,充分混匀,20min后,用全自动酶标仪在波长670nm测定吸光度,根据H2S标准曲线计算溶液中的H2S含量,脑组织中硫化氢含量以单位重量的组织硫化氢的量(nmol/g)表示。
1.4测定MBP的表达情况
每组均随机选取4只小鼠,全部用作免疫组化检测,各组所取小鼠经4%多聚甲醛(温度:4℃)灌注,断头取脑,进行固定、脱水、透明、浸蜡后制作蜡块,石蜡切片经脱蜡复水后,进行抗原修复和免疫组化,DAB显色,常规脱水、透明、中性树胶封片,显微镜观察并摄片。在高倍显微镜下每张切片上随机摄取双侧海马各4个视野的图片,用image-pro-plus6.0图像处理软件对MBP的阳性表达情况进行分析,以其平均光密度值作为统计数据,统计后取其平均值作为判断MBP的阳性表达情况。
1.5统计学处理
采用成组设计随机对照研究,每组样本计量结果用p表示,组间比较采用t检验,p<0.05为差异具有显著性,p<0.01差异具有极显著性。
结果
SCA3小鼠,WT小鼠和SCA3NaHS低、中、高剂量组小鼠在Morris水迷宫实验中空间学习和记忆的差异。
2.1.1距离
不同小鼠组在Morris水迷宫平均行驶距离如图1所示,在Morris水迷宫定位航行训练中,12月龄的WT小鼠组(n=12),SCA3小鼠组(n=12),SCA3NaHS低剂量小鼠组(n=12),SCA3NaHS中剂量小鼠组(n=12),SCA3NaHS高剂量小鼠组(n=12)游泳距离。**p<0.01或***p<0.001。
4天的训练试验的数据说明,在Morris水迷宫平均行驶距离中12月龄的SCA3小鼠组比同龄WT小鼠组明显延长(967.4±71.4cmvs653.3±64.5cm,p<0.01);同样12月龄SCA3NaHS小鼠组比SCA3模型小鼠组平均行驶距离明显缩短(高剂量组:713.2±68.6cm;中剂量组:823.4±64.5cm;低剂量组:881.7±77.2cm;SCA3模型组:967.4±71.4cm,p<0.01或<0.001)。
2.1.2平台潜伏期
不同小鼠组在Morris水迷宫平均平台潜伏期如图2所示,在Morris水迷宫定位航行训练中,12月龄的WT小鼠组(n=12),SCA3小鼠组(n=12),SCA3NaHS低剂量小鼠组(n=12),SCA3NaHS中剂量小鼠组(n=12),SCA3NaHS高剂量小鼠组(n=12)的平均平台潜伏期。**p<0.01或***p<0.001。
4天的训练试验的数据说明,在Morris水迷宫平均平台潜伏期中12月龄的SCA3小鼠组比同龄WT小鼠组明显延长(43.8±3.4svs31.4±3.2s,p<0.01);同样12月龄SCA3NaHS小鼠组比SCA3模型小鼠组平均平台潜伏期明显缩短(高剂量组:32.5±3.0s;中剂量组:35.3±2.4s;低剂量组:38.2±3.5s;SCA3模型组:43.8±3.4s,p<0.01)。
2.1.3游泳速度
不同小鼠组在Morris水迷宫平均游泳速度如图3所示,在Morris水迷宫定位航行训练中,12月龄的WT小鼠组(n=12),SCA3小鼠组(n=12),SCA3NaHS低剂量小鼠组(n=12),SCA3NaHS中剂量小鼠组(n=12),SCA3NaHS高剂量小鼠组(n=12)的游泳速度。
4天的训练试验的数据说明,在Morris水迷宫游泳速度中12月龄WT小鼠组和SCA3小鼠组间没有统计学差异(p>0.05),同样12月龄SCA3NaHS小鼠组(高、中、低剂量)比SCA3模型小鼠组间也没有统计学差异(p>0.05)。
2.2SCA3小鼠海马H2S含量
免疫组化结果显示,12月龄SCA3小鼠海马H2S含量下降,给予NaHS后海马H2S含量增加。如表1所示,SCA3模型小鼠组(M)与正常对照组(NC)相比,SCA3模型小鼠组(M)海马组织H2S含量明显降低(p<0.01);给予NaHS干预后,SCA3低剂量小鼠组(NL)与SCA3模型小鼠组(M)比较,海马组织H2S浓度无统计学差异(p>0.05);SCA3NaHS中剂量小鼠组(NM)和SCA3高剂量小鼠组(NH)与SCA3模型小鼠组(M)相比,海马组织H2S含量非常显著增加(p均<0.01)。
表1SCA3小鼠给予NaHS后海马组织H2S含量
注:NC组:正常对照组;M组:SCA3模型组,与NC组比较P=0.000000014(P<0.01);NL组:SCA3小鼠NaHS低剂量组,与NC组比较P=0.000000167(P<0.01);NM组:SCA3小鼠NaHS中剂量组;与NC组比较P=0.000007620(P<0.01),与M组比较P=0.000000058(P<0.01)。NH组:SCA3小鼠高剂量给予NaHS组,与NC组比较P=0.000120234(P<0.01),与M组比较P=0.0000000099(P<0.01)。▲与正常对照组(NC)比较p<0.01;★与SCA3模型组(M)比较p<0.01。
2.312月龄SCA3小鼠与WT小鼠海马有髓神经纤维MBP阳性表达
SCA3模型小鼠组(M)与正常对照组(NC)相比,期海马有髓神经纤维MBP平均光密度明显降低,丝状着色减少(p<0.01);给予NaHS干预后,SCA3中、高剂量小鼠组(NM和NH)与SCA3模型组(M)小鼠相比,海马有髓神经纤维MBP平均光密度均升高,丝状着色增加(p<0.01)。海马有髓神经纤维MBP免疫组化平均光密度值如表2所示。不同组小鼠的海马有髓神经纤维的丝状着色图如图5-9所示。
表2海马有髓神经纤维MBP免疫组化平均光密度值
注:NC组:正常对照组;M组:SCA3模型组;NL组:SCA3小鼠低剂量给予NaHS组;NM组:SCA3小鼠中剂量给予NaHS组;NH组:SCA3小鼠高剂量给予NaHS组。▲p<0.01与正常对照组(NC)比较;★p<0.01与SCA3模型组(M)比较。
本实验已测出SCA3小鼠的记忆能力明显低于WT小鼠,SCA3小鼠海马内硫化氢的量明显低于WT小鼠。海马中的H2S与SCA3小鼠生长过程中的空间学习和记忆有更多的关系。并已通过免疫组化的方法测出MBP含量在WT小鼠和SCA3小鼠大脑海马内的差异,SCA3小鼠的大脑海马内有髓神经纤维通路的MBP含量明显低于WT小鼠。
因此,我们有理由认为,SCA3小鼠记忆功能障碍可能是由于其海马内有髓神经纤维通路髓鞘碱性蛋白含量的降低引起的。我们推测硫氢化钠治疗SCA3小鼠的机制可能是:硫氢化钠通过上调海马内硫化氢的含量能够改善其线粒体的功能,进而有可能使海马有髓神经纤维通路MBP含量增加来减轻其髓鞘损伤,或许就可以改善SCA3小鼠的记忆障碍。
Claims (6)
1.硫氢化钠作为唯一有效成分在制备治疗脊髓小脑性共济失调 3型引起的记忆障碍的药物中的应用,其特征在于:所述药物中硫氢化钠的活性剂量为0.06-0.62 mg/kg/d,硫氢化钠通过上调海马内硫化氢的含量能够改善其线粒体的功能,进而使海马有髓神经纤维通路MBP含量增加来减轻其髓鞘损伤,改善记忆障碍。
2.根据权利要求1所述的应用,其特征在于,所述药物中硫氢化钠的活性剂量为0.31mg/Kg/d。
3.根据权利要求1 所述的应用,其特征在于所述药物为单一成分的硫氢化钠或者为硫氢化钠和其它化合物组成的复方药物。
4.根据权利要求1所述的应用,其特征在于,所述药物包括硫氢化钠和药学上可接受的载体。
5.根据权利要求3或4所述的应用,其特征在于,所述药物的剂型为任意一种临床可接受的口服给药剂型、注射给药剂型或外用给药剂型。
6.根据权利要求1至5中任一项所述的应用,其特征在于,所述药物的剂型为片剂、胶囊剂、颗粒剂、丸剂、注射剂、煎膏剂、悬浮剂、分散剂、糖浆剂、栓剂、凝胶剂、气雾剂或贴剂。
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