粉防己碱对链脲佐菌素所致糖尿病小鼠海马的保护机制

》首页 》 医学论文 》粉防己碱对链脲佐菌素所致糖尿病小鼠海马的保护机制
作者:无, 字数:17032

  [摘要]目的 探討粉防己碱对链脲佐菌素(STZ)所致糖尿病小鼠海马的保护机制。方法 将32只10~12周龄雄性C57小鼠随机分为四组,每组各8只,分别为对照组、粉防己碱对照组、模型组、粉防己碱治疗组。造模后,水迷宫实验检测小鼠空间学习记忆能力;酶标比色法检测海马组织匀浆中超氧化物歧化酶(SOD)和丙二醛(MDA)含量;酶联免疫吸附测定(ELISA)检测海马组织匀浆中白介素-1β(IL-1β)、白介素-6(IL-6)与肿瘤坏死因子-α(TNF-α)含量;蛋白免疫印迹法检测海马组织中p-Smad2和p-Smad3蛋白表达。结果 与对照组比较,模型组小鼠空间学习记忆能力下降,水迷宫逃逸潜伏期长于对照组,差异有统计学意义(P<0.05);模型组小鼠海马组织匀浆中的SOD含量低于对照组,MDA含量高于对照组,差异有统计学意义(P<0.05);模型组小鼠的IL-1β、IL-6和TNF-α含量均高于对照组,差异有统计学意义(P<0.05);模型组小鼠的p-Smad2、p-Smad3蛋白表达量均高于对照组,差异有统计学意义(P<0.05);与模型组比较,粉防己碱治疗组小鼠空间学习记忆能力增强,水迷宫逃逸潜伏期短于模型组,差异有统计学意义(P<0.05);粉防己碱治疗组小鼠海马组织匀浆中的SOD含量高于模型组,MDA含量低于模型组,差异有统计学意义(P<0.05);粉防己碱治疗组小鼠的IL-1β、IL-6和TNF-α含量均低于模型组,差异有统计学意义(P<0.05);粉防己碱治疗组小鼠的p-Smad2、p-Smad3蛋白表达量均低于模型组,差异有统计学意义(P<0.05)。结论 粉防己碱具有拮抗STZ所致糖尿病小鼠海马损伤的作用,其机制可能与抑制其氧化应激及其下游Smad信号通路有关。
  [关键词]粉防己碱;链脲佐菌素;糖尿病模型;海马
  [中图分类号] R972          [文献标识码] A          [文章编号] 1674-4721(2020)9(a)-0004-05
  [Abstract] Objective To investigate the protective mechanism of Tetrandrine against hippocampus in Streptozocin (STZ) induced diabetic mice. Methods A total of 32 male C57 mice aged 10-12 weeks were randomly divided into four groups, 8 mice in each group, which were control group, Tetrandrine control group, model group and Tetrandrine treatment group. On the day after the establishment and administration of the diabetic model, the water maze test was used to detect the spatial learning and memory ability of the mice, the enzyme-labeled colorimetric method was used to detect the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in the homogenate of hippocampus, the enzyme linked immunosorbent assay (ELISA) was used to detect the contents of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in hippocampal tissue homogenate. Western blotting was used to detect p-Smad2 and p-Smad3 protein expression of hippocampal tissue. Results Compared with the control group, the spatial learning and memory ability of mice in the model group decreased, and the escape latency of water maze was longer than that in the control group, and the differences were statistically significant (P<0.05). The SOD content in the hippocampal tissue homogenate of the model group was lower than that of the control group, and the MDA content was higher than that of the control group, the differences were statistically significant (P<0.05). The contents of IL-1β, IL-6 and TNF-α in the model group were higher than those in the control group, and the differences were statistically significant (P<0.05). The protein expression levels of p-Smad2 and p-Smad3 in the model group were higher than those in the control group, and the differences were statistically significant (P<0.05). Compared with the model group, the spatial learning and memory ability of mice in the Tetrandrine treatment group increased, and the escape latency of the water maze was shorter than that in the model group, the differences were statistically significant (P<0.05). The SOD content in the hippocampal tissue homogenate of the Tetrandrine treatment group was higher than that of the model group, and the MDA content was lower than that of the model group, the differences were statistically significant (P<0.05). The contents of IL-1β, IL-6 and TNF-α in the Tetrandrine treatment group were lower than those in the model group, and the differences were statistically significant (P<0.05). The protein expression levels of p-Smad2 and p-Smad3 in the Tetrandrine treatment group were lower than those in the model group, and the differences were statistically significant (P<0.05). Conclusion Tetrandrine can antagonize hippocampal injury in STZ induced diabetic mice, and its mechanism may be related to inhibition of oxidative stress and its downstream Smad signaling pathway.   [Key words] Tetrandrine; Streptozocin; Diabetes model; Hippocampus
  糖尿病认知功能障碍现已成为当今国内外学者研究的重点领域。粉防己碱是从中药防己科植物粉防己根中提取的双苄基异喹啉类生物碱,为天然的钙通道阻滞剂,已被广泛应用到中枢神经系统疾病的防治中[1-4]。链脲佐菌素(Streptozocin,STZ)可诱导糖尿病认知功能障碍的发生,粉防己碱可改善海马神经干细胞的损伤,提高糖尿病小鼠学习记忆能力[1-4],但其机制尚不明确。本实验采用STZ制备糖尿病小鼠认知功能障碍模型,探讨粉防己碱改善糖尿病小鼠海马损伤的可能机制,为寻找改善糖尿病认知功能障碍的新方法提供理论与实验依据。
  1材料与方法
  1.1材料
  1.1.1实验动物及分组
  1.1.1.1实验动物  32只C57小鼠,10~12周龄,雄性,体重20~25 g,无特定病原体(SPF)级,由湖北省实验动物研究中心提供,动物合格证号:42000600033780。饲养于湖北中医药大学实验动物中心,动物许可证号:SCXK(鄂)2015-0018。动物实验流程遵循湖北中医药大学有关实验动物保护和使用指南,并经实验动物伦理委员会批准。
  1.1.1.2分组  实验动物适应性喂养1周后随机分为对照组、粉防己碱对照组、模型组以及粉防己碱治疗组共四组,每组各8只。饲养条件控制温度为20~25℃,规律照明,自由饮食。
  1.1.2仪器与试剂
  Morris水迷宫(中国医学科学院药物研究所,DMS-2);酶标仪(MD,SpectraMax M5);显影仪(伯乐,ChemiDox XRS+);高速冷冻离心机(Sigma,3K-30Z);STZ(Sigma,V900890);粉防己碱(aladdin,T107322);超氧化物歧化酶(SOD)(碧云天,S0103);丙二醛(MDA)(碧云天,S0131);白介素-1β(IL-1β)(碧云天,P301);白介素-6(IL-6)(碧云天,P1326);TNF-α(南京建成,H052);p-Smad2(abclone,A0440);p-Smad3(abclone,A11471);Smad2(abclone,A7699);Smad3(abclone,A19115);β-actin(abclone,AC026);山羊抗鼠(abclone,AS003)。
  1.2方法
  1.2.1模型的建立及给药
  模型组和粉防己碱治疗组小鼠造模期间腹腔注射给药STZ共4周,随机血糖>16.7 mmol/L并持续1周为造模成功。对照组与粉防己碱对照组小鼠造模期间腹腔注射生理盐水。造模成功后,对照组和模型组小鼠灌胃生理盐水,粉防己碱对照组和粉防己碱治疗组小鼠灌胃粉防己碱,STZ给药浓度为40 mg/kg,粉防己碱给药浓度为15 mg/kg[5-6]。
  1.2.2小鼠空间学习记忆能力测定
  获得性训练:将隐形逃逸平台固定在水迷宫中,随机抓取各组小鼠沿池壁3个定点依次头朝水池壁平稳放入,记录各组小鼠在120 s内寻找隐形平台的时间(逃逸潜伏期)及运动轨迹。若超过120 s未找到隐形平台,则将该小鼠逃逸潜伏期记为120 s,训练时间为6 d。
  探查实验:Morris水迷宫获得性训练实验结束后撤去隐形平台,将各组小鼠从第2个象限放入,记录每只小鼠在120 s内穿越隐形平台的次数[7-13]。
  1.2.3小鼠海马组织中SOD、MDA含量检测
  取各组小鼠冷冻海马组织,制备组织匀浆,3500 r/min(离心机半径为13.5 cm)离心10 min,收集上清液,按试剂盒说明书检测SOD、MDA含量[14-15]。
  1.2.4小鼠海马组织匀浆中IL-1β、IL-6和TNF-α检测
  制备小鼠海马组织匀浆上清液,将上清稀释10倍后按说明书方法操作,酶联免疫吸附测定(ELISA)法测定IL-1β、IL-6及TNF-α含量,酶标仪测定450 mm处A值[16-18]。
  1.2.5 Western bloting法检测小鼠海马组织中Smad2、Smad3、p-Smad2、p-Smad3蛋白表达量
  取各组小鼠冷冻海马组织制备蛋白样品,BCA蛋白浓度试剂盒检测各组蛋白浓度。蛋白定量后调整其浓度为相同浓度。每泳道上样20 μg,SDS-PAGE凝胶分离,PVDF膜进行转膜,5%脱脂奶粉溶液(1×TBST配制)封闭1.5 h。一抗4℃恒温孵育过夜,二抗室温孵育1.5 h,ECL发光试剂盒显色,ImageJ软件计算目的蛋白与内参蛋白比值[19]。
  1.3统计学方法
  采用SPSS 23.0统计学软件进行数据分析,计量资料用均数±标准差(x±s)表示,两组间比较采用t检验,以P<0.05为差异有统计学意义。
  2结果
  2.1小鼠一般状态观察
  对照组及粉防己碱对照组小鼠体重增长迅速,反应灵敏,毛发平伏有光泽。模型组小鼠体重增长缓慢,精神萎靡,反应迟钝,毛色无光泽,尿量显著增加并带有刺激性气味。粉防己碱治疗组小鼠一般状态优于模型组小鼠。
  2.2粉防己碱对糖尿病小鼠空间学习记忆能力的影响
  模型组小鼠寻找隐形平台的逃逸潜伏期长于对照组,穿越原始平台次数少于对照组,差异有统计学意义(P<0.05);粉防己碱治疗组小鼠逃逸潜伏期短于模型组,穿越原始平臺位置次数多于模型组,差异有统计学意义(P<0.05);粉防己碱对照组小鼠的逃逸潜伏期及穿越原始平台次数与对照组比较,差异无统计学意义(P>0.05)(图1~2)。   2.3粉防己碱对糖尿病小鼠海马组织SOD、MDA含量的影响
  模型组小鼠海马组织匀浆中的SOD含量低于对照组,MDA含量高于对照组,差异有统计学意义(P<0.05);粉防己碱治疗组小鼠海马组织匀浆中的SOD含量高于模型组,MDA含量低于模型组,差异有统计学意义(P<0.05);粉防己碱对照组与对照组的SOD、MDA含量比较,差异无统计学意义(P>0.05)(表1)。
  2.4粉防己碱对糖尿病小鼠海马区IL-1β、IL-6和TNF-α含量的影响
  模型组小鼠海马匀浆中的IL-1β、IL-6和TNF-α含量均高于对照组,差异有统计学意义(P<0.05);粉防己碱治疗组小鼠海马区的IL-1β、IL-6以及TNF-α含量均低于模型组,差异有统计学意义(P<0.05);粉防己碱对照组与对照组的IL-1β、IL-6和TNF-α含量比较,差异无统计学意义(P>0.05)(表2)。
  2.5粉防己碱对糖尿病小鼠海马区Smad2、Smad3、p-Smad2、p-Smad3蛋白表达量的影响
  四组的Smad2、Smad3蛋白表达量比较,差异无统计学意义(P>0.05);模型组小鼠海马区的p-Smad2、p-Smad3蛋白表达量均高于对照组,差异有统计学意义(P<0.05);粉防己碱治疗组小鼠海马区的p-Smad2、p-Smad3蛋白表达量均低于模型组,差异有统计学意义(P<0.05)(图3)。
  3讨论
  粉防己碱广泛应用于炎症、糖尿病、神经退行性疾病等病症的治疗中。氧化应激损伤与炎症则是认知功能障碍的主要诱因学说[20],SOD是氧化还原系统中重要的抗氧化酶,能有效抑制氧化应激;MDA水平的高低反映了机体脂质氧化程度,是评价细胞受氧化损伤程度重要指标。糖尿病认知功能障碍导致的氧化应激反应可以通过诱导炎症因子释放,从而造成细胞的损伤,其中TNF-α、IL-1β、IL-6是炎症认知功能障碍的的血清学标志之一。Smad蛋白家族可通过调控星形胶质细胞的生物学行为促进糖尿病认知功能障碍的发展,p-Smad2、p-Smad3蛋白是其中较为关键的蛋白。
  本研究结果提示,粉防己碱能够改善糖尿病小鼠海马损伤,抑制炎症因子表达,拮抗STZ诱导的糖尿病认知功能障碍,其机制可能与粉防己碱拮抗海马神经干细胞的氧化应激损伤与炎症的发生及调节Smad信号通路密切相关,由于以单一途径为靶点的实验研究结果有限,因此粉防己碱治疗糖尿病认知功能障碍的具体机制有待进一步研究。
  [参考文献]
  [1]郭敏,张如意,蔡飞.粉防己碱的药理作用研究进展[J].中国当代医药,2018,25(18):30-33.
  [2]甄攀.粉防己碱的研究进展[J].医学研究杂志,2004,33(8):79-82.
  [3]杨敬,朱安祥,胡军,等.粉防己碱的作用机制研究进展[J].国际中医中药杂志,2018,40(3):286-289.
  [4]梁晓飞,王铭,肖永红.粉防己碱对小鼠肺成纤维细胞的凋亡及Caspase-3蛋白的影响[J].中国临床药理学杂志,2016,32(6):531-533.
  [5]LI Q.Effects of tetrandrine on proliferation and activation of cardiac fibroblasts[J].Beijing Da Xue Xue Bao Yi Xue Ban,2018,50(2):331-334.
  [6]Hsu JH,Wu YC,Liou SS,et al.Mediation of Endogenous beta-endorphin by Tetrandrine to Lower Plasma Glucose in Streptozotocin-induced Diabetic Rats[J].Evid Based Complement Alternat Med,2004,1(2):193-201.
  [7]Chen WC,Hayakawa S,Yamamoto T,et al.The plasma glucose lowering action of tetrandrine in streptozotocin-induced diabetic rats[J].J Pharm Pharmacol,2004,56(5):643-648.
  [8]朱丽霞,董志,廖红,等.粉防己碱对大鼠拟痴呆模型脑保护作用研究[J].中国老年学杂志,2005,25(1):78-80.
  [9]朱丽霞.粉防己碱对痴呆大鼠模型脑保护作用的实验研究[J].中国药理学通报,2004,20(8):959-960.
  [10]陈连连,陈力学,曾照芳,等.粉防己碱对血管性痴呆大鼠学习记忆能力及神经元凋亡的影响[J].激光杂志,2011, 32(6):87-89.
  [11]He FQ,Qiu BY,Zhang XH,et al.Tetrandrine attenuates spatial memory impairment and hippocampal neuroinflammation via inhibiting NF-κB activation in a rat model of Alzheimer′s disease induced by amyloid-β(1-42)[J].Brain Res,2011,1384(2):89-96.
  [12]Chen L,Chen L,Lv Y,et al.Tetrandrine ameliorates cognitive impairment via inhibiting astrocyte-derived S100B activation in a rat model of chronic cerebral hypoperfusion[J].Neurol Res,2013,35(6):614-621.
  [13]农慧,盛庆寿,梁健,等.STZ诱导糖尿病大鼠模型的研究[J].广西医科大学学报,2010,27(1):69-72.
  [14]Wang G,Lemos JR.Tetrandrine:a new ligand to block voltage-dependent Ca2+ and Ca2+-activated K+ channels[J].Life Sci,1995,56(5):295-306.
  [15]Wu F,Xu J,Zhu Z.Protective Effect of Tetrandrine in a Rabbit Model of Osteoarthritis[J].Arch Rheumatol,2017,33(1):80-84.
  [16]Liu KC,Lin YJ,Hsiao YT,et al.Tetrandrine Induces Apoptosis in Human Nasopharyngeal Carcinoma NPC-TW 039 Cells by Endoplasmic Reticulum Stress and Ca2+/Calpain Pathways[J].Anticancer Res,2017,37(11):6107-6118.
  [17]王裕勤,曹雪滨,冯义柏.粉防己碱抑制心肌细胞磷酸化减轻缺血/再灌注损伤引发的炎症反应[J].心血管康复医学杂志,2010,19(1):57-60.
  [18]黄晏军,汤长宁,杨爽,等.汉防己碱衍生物诱导人乳腺癌MDA-MB-231细胞凋亡[J].贵州医科大学学报,2019, 44(2):125-129.
  [19]张乐之,何华美,李军,等.粉防己碱对炎症白细胞第二信使物质的作用[J].四川生理科學杂志,2001,23(3):117.
  [20]张玉琴,朱旭强,吴力新,等.表没食子儿茶素没食子酸酯对脑损伤小鼠认知和氧化应激的影响[J].中华实验外科杂志,2018,35(3):475-477.
  (收稿日期:2020-04-01)

相关

相关

相关

  • “喂”羊与“牧”羊的启示

  • 开展丰富多彩的游戏活动来提高学生的学习水平

  • “0”的困惑

  • 照耀留守儿童的阳光

  • 让生活元素在初中语文教学中遍地生花

  • 药物里的美丽误会

No comments found.

相关