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吴吉, 裴方妤, 黄鹏珏, 印泥娅, 欧阳磊, 戴明轩, 朱沁泉, 张涤.孤独症谱系障碍肝肾不足证病证结合大鼠模型的构建与评价[J].湖南中医药大学学报,2025,45(7):1221-1230[点击复制] |
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| 孤独症谱系障碍肝肾不足证病证结合大鼠模型的构建与评价 |
| 吴吉,裴方妤,黄鹏珏,印泥娅,欧阳磊,戴明轩,朱沁泉,张涤 |
| (湖南中医药大学, 湖南 长沙 410208;广东省深圳市龙岗区人民医院, 广东 深圳 518000;永州职业技术学院, 湖南 永州 425100;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 目的 构建孤独症谱系障碍（ASD）肝肾不足证病证结合大鼠模型，并进行模型评价。方法 将13只SD孕鼠随机分为丙戊酸钠（VPA）组（10只）、对照组（3只）。VPA组在第13天时腹腔注射VPA溶液（600 mg·kg-1），对照组注射等体积生理盐水。第7、14、21天，测仔鼠体质量、身长、尾长，评估生长发育；第21天对两组雄性仔鼠开展行为学检测，筛选出符合ASD表现的仔鼠32只，随机分为ASD组（等体积蒸馏水）、ASD肝肾不足组（等体积蒸馏水）、六味地黄丸组（1.5 g·kg-1）、龙胆泻肝丸组（2.33 g·kg-1），另将正常雄性仔鼠设为空白组（等体积蒸馏水），每组8只。每天上午进行灌胃。每天下午，ASD肝肾不足组、六味地黄丸组、龙胆泻肝丸组予以激素（左甲状腺素钠150 μg·kg-1）结合激怒法，共14 d。干预后开展行为学检测，HE、尼氏染色观察前额叶病理改变；HE染色观察股骨病理改变；ELISA法测血清环磷酸腺苷（cAMP）、环磷酸鸟苷（cGMP）、雌二醇（E2）、睾酮（T）、游离三碘甲状腺原氨酸（FT3）、游离甲状腺素（FT4）、促甲状腺激素释放激素（TRH）、促甲状腺激素（TSH）、肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、促红细胞生成素（EPO）、1,25-二羟基维生素D3（VD3）水平；称重测胸腺指数。结果 与空白组比较，ASD组、ASD肝肾不足组存在发育迟缓、发育畸形，不同程度行为学表现异常和前额叶病理损伤，胸腺指数下降（P＜0.05，P＜0.01），ASD肝肾不足组可见股骨病理损伤；与ASD组、ASD肝肾不足组比较，各治疗组仔鼠行为学异常情况及前额叶、股骨损伤情况均得到不同程度的改善，其中六味地黄丸组改善最为明显。ELISA结果显示，与空白组比较，ASD组TSH下降（P＜0.05），TNF-α、IL-6升高（P＜0.01），VD3下降（P＜0.05）；ASD肝肾不足组血清cAMP、cAMP/cGMP、E2、E2/T升高（P＜0.05，P＜0.01），cGMP、T、FT3、FT4、TRH、TSH下降（P＜0.05，P＜0.01），TNF-α、IL-6升高（P＜0.01），VD3、EPO下降（P＜0.05，P＜0.01）。与ASD肝肾不足组比较，六味地黄丸组cAMP、cAMP/cGMP、TNF-α、IL-6降低（P＜0.01），E2、E2/T下降（P＜0.05），VD3、EPO升高（P＜0.05，P＜0.01），胸腺指数升高（P＜0.01）；龙胆泻肝丸组cAMP/cGMP、E2下降（P＜0.05），TNF-α、IL-6下降（P＜0.01）。与六味地黄丸组比较，龙胆泻肝丸组胸腺指数下降（P＜0.05），EPO水平下降（P＜0.05）。结论 孕期腹腔注射VPA＋激素结合激怒法刺激干预可以构建ASD肝肾不足证病证结合动物模型。 |
| 关键词: 孤独症谱系障碍 六味地黄丸 病证结合 以方测证 肝肾不足 动物模型 |
| DOI：10.3969/j.issn.1674-070X.2025.07.004 |
| 投稿时间：2025-01-16 |
| 基金项目:湖南省自然科学基金项目（2024JJ8233）；湖南省中医药科研计划项目（A2023036）；湖南省卫生健康高层次人才项目（20230448）；湖南省教育厅科研项目（22A0263）；湖南省科技厅重点领域研发计划项目（2019SK2081）；湖南省卫生健康委员会科研课题（202206010043）；湖南省大学生创新创业训练计划（S202410541157）。 |
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| Construction and evaluation of a rat model combining autism spectrum disorder with liver-kidney deficiency pattern |
| WU Ji, PEI Fangyu, HUANG Pengjue, YIN Niya, OUYANG Lei, DAI Mingxuan, ZHU Qinquan, ZHANG Di |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Longgang District People's Hospital of Shenzhen of Guangdong Province, Shenzhen, Guangdong 518000, China;Yongzhou Vocational Technical College, Yongzhou, Hunan 425100, China;The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| Objective To construct a rat model combining autism spectrum disorder (ASD) with liver-kidney deficiency pattern and to evaluate the model. Methods Thirteen pregnant SD rats were randomly divided into the valproic acid (VPA) group (n=10) and the control group (n=3). On day 13 of pregnancy, the VPA group was intraperitoneally injected with VPA solution (600 mg·kg-1), while the control group was injected with an equal volume of normal saline. On postnatal days 7, 14, and 21, offspring body weight, body length, and tail length were measured to assess growth. On day 21, behavioral tests were conducted on male offspring from both groups to screen for 32 offspring exhibiting ASD-like behaviors. They were randomly divided into ASD group (equal volume of distilled water), ASD liver-kidney deficiency group (equal volume of distilled water), Liuwei Dihuang Pill (LWDHP) group (1.5 g·kg-1), and Longdan Xiegan Pill (LDXGP) group (2.33 g·kg-1). Additionally, normal male offspring were set as the blank group (equal volume of distilled water), with 8 rats in each group. Gavage administration was performed every morning, and in the afternoons, the ASD liver-kidney deficiency, LWDHP, and LDXGP groups received hormonal stimulation (levothyroxine sodium 150 μg·kg-1) combined with an irritation method for 14 days. After the intervention, behavioral tests were conducted. HE and Nissl stainings were used to observe pathological changes in the prefrontal lobe; HE staining was used to observe pathological changes in the femur; ELISA was used to measure the levels of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), estradiol (E2), testosterone (T), free triiodothyronine (FT3), free thyroxine (FT4), thyrotropin-releasing hormone (TRH), thyroid-stimulating hormone (TSH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), erythropoietin (EPO), and 1,25-dihydroxyvitamin D3 (VD3) in serum; the thymus index was measured by weighing. Results Compared with the blank group, the ASD group and the ASD liver-kidney deficiency group showed growth retardation, developmental malformations, abnormal behavioral manifestations to varying degrees, and pathological damage to the prefrontal lobe, along with a decreased thymus index (P<0.05, P<0.01). Pathological damage to the femur was also observed in the ASD liver-kidney deficiency group. Compared with the ASD group and the ASD liver-kidney deficiency group, the treatment groups showed varying degrees of improvement in behavioral abnormalities and damage to the prefrontal lobe and femur in the offspring rats, with the most significant improvement observed in the LWDHP group. ELISA results revealed that, compared with the blank group, the ASD group had decreased TSH levels (P<0.05), increased TNF-α and IL-6 levels (P<0.01), and decreased VD3 levels (P<0.05). The ASD liver-kidney deficiency group exhibited increased serum cAMP, cAMP/cGMP ratio, E2, and E2/T ratio (P<0.05, P<0.01), decreased cGMP, T, FT3, FT4, TRH, and TSH levels (P<0.05, P<0.01), increased TNF-α and IL-6 levels (P<0.01), and decreased VD3 and EPO levels (P<0.05, P<0.01). Compared with the ASD liver-kidney deficiency group, the LWDHP group had reduced cAMP, cAMP/cGMP ratio, TNF-α, and IL-6 levels (P<0.01), decreased E2 and E2/T ratio (P<0.05), increased VD3 and EPO levels (P<0.05, P<0.01), and an elevated thymus index (P<0.01). The LDXGP group showed decreased cAMP/cGMP ratio and E2 levels (P<0.05), along with reduced TNF-α and IL-6 levels (P<0.01). Compared with the LWDHP group, the LDXGP group had a decreased thymus index (P<0.05) and reduced EPO levels (P<0.05). Conclusion The animal model of ASD with liver-kidney deficiency pattern can be constructed by intraperitoneal injection of VPA during pregnancy combined with hormone treatment and irritation method. |
| Key words: autism spectrum disorder Liuwei Dihuang Pill disease-pattern combination formula-based pattern validation liver-kidney deficiency animal model |
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