Effect of Changpu Yujin Decoction on Synaptosome SNARE Protein Complex in Tourette Syndrome Model Rats
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摘要: 目的 研究菖蒲郁金汤对抽动秽语综合征(Tourette syndrome, TS)模型大鼠突触体中SNARE蛋白复合体的调控作用, 揭示菖蒲郁金汤治疗TS的药效机制。方法 选择3周龄SD大鼠240只, 依据随机数字表将SD大鼠分为空白组(60只)和造模组(180只), 采用3, 3'-亚氨基二丙腈(IDPN)腹腔注射法制备TS动物模型。造模成功后, 再次随机将造模组大鼠分为模型组、硫必利组及菖蒲郁金汤组，每组60只。从造模完成后次日(即d0)开始灌胃给药, 空白组与模型组给予生理盐水, 其余各组给予相应药物, 每日1次, 连续给药4周。各组大鼠分别于第0、7、14、21、28天(即d0，d7，d14，d21，d28), 随机抽取12只大鼠, 麻醉后取纹状体, 采用Percoll密度梯度离心法制备纹状体中的突触体, 并在透射电镜下进行形态学鉴定。ELISA法检测纹状体多巴胺(Dopamine, DA)含量，qPCR和Western blot法检测突触体中SNAP-25、Syntaxin-1a和VAMP-2 mRNA及蛋白的表达, 免疫组织化学法检测纹状体上述指标的表达。结果 与空白组相比, 模型组各观察点纹状体中DA含量降低(P < 0.01), 突触体/纹状体中SNAP-25、Syntaxin-1a、VAMP-2 mRNA及蛋白的表达量显著降低(P < 0.05, P < 0.01);与模型组相比较, 硫必利组及菖蒲郁金汤组在干预期间的各观察点(d7，d14，d21，d28)纹状体中DA含量以及突触体/纹状体内SNAP-25、Syntaxin-1a、VAMP-2 mRNA及蛋白的表达量呈逐渐升高趋势。在治疗结束后(d28), 菖蒲郁金汤组突触体中SNAP-25、Syntaxin-1a、VAMP-2 mRNA, Syntaxin-1a蛋白及纹状体中SNAP-25、VAMP-2蛋白的表达高于硫必利组(P < 0.05, P < 0.01)。结论 菖蒲郁金汤通过调控SNAP-25、Syntaxin-1a、VAMP-2的表达促进DA的释放, 进而发挥其抗抽动的作用。
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关键词:
- 菖蒲郁金汤 /
- 抽动秽语综合征 /
- 突触体 /
- SNARE蛋白复合体
Abstract: OBJECTIVE To study the regulatory effect of Changpu Yujin Decoction on the SNARE protein complex in the synaptosomes of Tourette syndrome (TS) model rats, and to reveal the pharmacodynamic mechanism of Changpu Yujin Decoction in the treatment of TS.METHODS 240 SPF SD male rats (3 weeks) were randomly divided into blank group (n=60) and model group (n=180). Model group received intraperitoneal injection of 3, 3'-iminodipropionitrile to establish TS model. After the TS model was successfully established, the model rats were randomly divided into model group, tiapride group, and Changpu Yujin Decoction group, each with 60 rats. The intragastric administration was started from the next day after the TS model was made (d0). The blank group and the model group were given normal saline, and the other groups were given corresponding drugs, once a day, for 4 consecutive weeks. At d0, d7, d14, d21 and d28, 12 rats were randomly selected from each group. After anesthesia, the striatum was taken out, and the synaptosomes in the striatum were prepared by Percoll density gradient centrifugation. Morphological identification was performed under transmission electron microscope; The level of dopamine (DA) in the striatum was examined by ELISA; Expression of SNAP-25, Syntaxin-1a and VAMP-2 in the synaptosomes/striatumwere measured by qPCR, Western blot analysis and immunohistochemical staining.RESULTS Compared with the blank group, the DA content in the striatum in the model group was reduced (P < 0.01), and the expressions of SNAP-25, Syntaxin-1a, VAMP-2 mRNA and protein in the synaptosome/striatum significantly reduced (P < 0.05, P < 0.01); Compared with the model group, the DA content in striatum and the mRNA and protein expression levels of SNAP-25, Syntaxin-1a, VAMP-2 in the tiapride group and Changpu Yujin group showed a gradual increase trend during the intervention period (d7, d14, d21, d28); At d28 (after the end of treatment), the expressions of SNAP-25, Syntaxin-1a, VAMP-2 mRNA, Syntaxin-1a protein in synaptosome and SNAP-25 and VAMP-2 protein in the striatum were higher than those of tiapride group (P<0.05, P < 0.01).CONCLUSION Changpu Yujin Decoction promotes the release of DA by regulating the expression of SNAP-25, Syntaxin-1a, and VAMP-2, thereby exerting its anti-tic effect.-
Key words:
- Changpu Yujin Decoction /
- Tourette syndrome /
- synaptosome /
- SNARE protein complex
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图 2 各组大鼠突触体中SNAP-25、Syntaxin-1a、VAMP-2 mRNA的表达情况
注: 与空白组比较, **P < 0.01;与模型组比较, #P < 0.05, ##P < 0.01;与硫必利组比较, ▲P < 0.05；与d0比较, ★P < 0.05, ★★P < 0.01。x±s, n=3。
图 3 不同时间点各组大鼠突触体中目标蛋白的表达情况
注: 与空白组比较, *P < 0.05, * *P < 0.01;与模型组比较, #P < 0.05, ##P < 0.01;与硫必利组比较, ▲P < 0.05。x±s, n=3。
图 4 各组大鼠纹状体SNAP-25蛋白的表达
注: 与空白组比较, * *P < 0.01;与模型组比较, ##P < 0.01;与硫必利组比较, ▲P < 0.05；与d0比较, ★P < 0.05, ★★P < 0.01。x±s, n=3。
图 5 各组大鼠纹状体Syntaxin-1a蛋白的表达
注: 与空白组比较, * *P < 0.01;与模型组比较, #P < 0.05, ##P < 0.01;与d0比较, ★P < 0.05, ★★P < 0.01。x±s, n=3。
图 6 各组大鼠纹状体VAMP-2蛋白的表达
注: 与空白组比较, * *P < 0.01;与模型组比较, #P < 0.05, ##P < 0.01;与硫必利组比较, ▲▲P < 0.01；与d0比较, ★P < 0.05, ★★P < 0.01。x±s, n=3。
表 1 行为学评分表
程度评分 刻板行为 运动行为 0 无刻板运动 安静或正常活动 1 旋转行为(顺时针或逆时针的旋转运动) 过度兴奋 2 头颈部垂直运动过多 探究行为增加 3 头颈部垂直运动过多加旋转行为 不停跑动 4 头向侧摆, 合并头颈部垂直运动过多 不停跑动加惊跳 
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表 2 目的基因序列
引物 引物序列(5'→3') 长度/bp VAMP-2 上游: CAGCTGGTGTGTAAGTGTCTTGGAG 160 下游: GCAGCAGATCAGGCAGATGG SNAP-25 上游: CGGCATCATCGGAAACCTC 135 下游: GCACGTTGGTTGGCTTCATC Syntaxin-1α 上游: GGCCGTCAAGTACCAGAGCAA 78 下游: ATGATGATGCCCAGAATCACACA
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表 3 各组大鼠纹状体DA含量(x±s, pg·mL-1, n=3)
组别 d0 d7 d14 d21 d28 空白组 104.34±3.76 103.88±4.78 105.02±5.95 104.38±4.45 103.62±4.52 模型组 38.62±3.33** 38.63±3.58** 37.71±3.43** 41.76±2.87** 40.30±2.92** 硫必利组 40.70±1.56** 72.45±4.28**##★★ 84.00±6.13**##★★ 87.55±6.20**##★★ 96.91±5.54**##★★ 菖蒲郁金汤组 39.93±1.96** 61.69±5.75**##▲▲★★ 73.48±7.61**##▲▲★★ 80.36±4.87**##★★ 94.95±2.82**##★★ 注: 与空白组比较, **P < 0.01;与模型组比较, ##P < 0.01;与硫必利组比较, ▲▲P < 0.01；与d0比较, ★★P < 0.01。
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