Effect of Tongxie Yaofang combined with acupoint catgut embedding on 5-HT and SP in ulcerative colitis rats with liver stagnation and spleen deficiency
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摘要: 目的 观察痛泻要方联合穴位埋线对肝郁脾虚型溃疡性结肠炎大鼠结肠、海马组织中5-羟色胺(5-hydroxytryptamine，5-HT)、神经递质P物质(neurotransmitter substance P，SP)的影响，并探讨其作用机制。方法 将48只SPF级SD雄性大鼠随机分为空白组(12只)、造模组(36只)。采用2，4，6-三硝基苯磺酸/乙醇混合剂灌肠+束缚+饮食失节+夹尾刺激制备肝郁脾虚型溃疡性结肠炎模型。将成模大鼠随机分为模型组、痛泻要方组、痛泻要方联合穴位埋线组；痛泻要方联合埋线组予以痛泻要方汤剂灌胃，连续14 d，同时于足三里、天枢、肝俞、期门、脾俞、大肠俞、膈俞各穴位处埋线，每7 d进行1次，共2次；痛泻要方组予痛泻要方汤剂灌胃；空白组和模型组予等体积纯净水进行灌胃，1次/d，干预14 d。采用苏木精-伊红染色观察结肠组织病理变化，免疫组织化学法检测大鼠结肠、海马组织中5-HT、SP的表达；ELISA法检测大鼠血清、海马组织中5-HT、SP的表达; RT-qPCR法检测结肠组织中肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)、IL-6、IL-1β、IL-23含量的变化。结果 与空白组相比，余3组血清、海马及结肠组织中5-HT、SP的表达量明显增加(P < 0.05)，结肠组织中TNF-α、IL-6、IL-1β、IL-23的含量显著升高(P < 0.05)；与模型组比较，痛泻要方组、痛泻要方联合穴位埋线组血清、海马及结肠组织中5-HT、SP的表达量明显减少(P < 0.05)，结肠组织中TNF-α、IL-6、IL-1β、IL-23的含量显著降低(P < 0.05)。结论 痛泻要方联合穴位埋线可以显著下调大鼠血清、海马及结肠组织中5-HT、SP的表达水平，并抑制炎性因子的表达，达到对肝郁脾虚型溃疡性结肠炎大鼠的治疗作用。Abstract: Objective To observe the effects of Tongxie Yaofang combined with acupoint catgut embedding on 5-hydroxytryptamine(5-HT) and neurotransmitter substance P(SP) in colonic and hippocampal tissues of rats with ulcerative colitis of liver depression and spleen deficiency, and to explore its mechanism.Methods Totally 48 SPF SD male rats were randomly divided into blank group(12 rats) and modeling group(36 rats). The ulcerative colitis models of liver depression and spleen deficiency pattern is established by 2, 4, 6-tritrobenzene sulfonic acid/ ethanol mixture enema combined with physical restraint, disorder diet and tail-clipping. The successfully established rat models were randomly divided into model group, Tongxie Yaofang group and Tongxie Yaofang combined with embedding group. The Tongxie Yaofang group and the combination group were treated with Tongxie Yaofang decoction by gavage for 14 days, and the combination group was also treated with acupoint embedding at"Zusanli", "Tianshu", "Ganshu", "Qimen", "Pishu", "Dachangshu"and "Geshu", seven days/time, a total of two times; and the blank group and model group were given same amount of purified water by gavage for 14 days. HE staining was used to observe the pathological changes of colon tissue and the expression of 5-HT and SP in colonic and hippocampal tissues of rats was detected by immunohistochemistry. 5-HT and SP in serum and hippocampal tissue were detected by ELISA, and tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), IL-1β and IL-23 in colon tissue were detected by Real-time quantitative Polymerase Chain Reaction(RT-qPCR).Results Compared with blank group, the expression levels of 5-HT and SP in serum, hippocampus and colon tissues were significantly increased(P < 0.05), and the contents of TNF-α, IL-6, IL-1β and IL-23 in colon tissues were significantly increased(P < 0.05). Compared with model group, the expressions of 5-HT and SP in serum, hippocampus and colon tissue of Tongxie Yaofang group and Tongxie Yaofang combined with acupoint catgut group were significantly decreased(P < 0.05), and the contents of TNF-α, IL-6, IL-1β and IL-23 in colon tissue were significantly decreased(P < 0.05).Conclusion Tongxie Yaofang combined with acupoint catgut can significantly down-regulate the expression levels of 5-HT and SP in serum, hippocampus and colon tissue, and reduce the expression of inflammatory factors, so as to treat ulcerative colitis of liver depression and spleen deficiency.
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表 1 临床四诊信息与大鼠宏观表征转化
临床四诊信息 大鼠宏观表征 主症 主要依据 胃脘或胁肋胀痛，腹胀 喜蜷卧，行动迟缓 食少纳呆 体重增长缓慢，进食量少 便溏不爽 大便稀溏 次症 主要依据 情绪抑郁或急躁易怒，善太息 抓取反抗微弱或激烈 肠鸣矢气，腹痛即泻、泻后痛减 拉尾排便次数增加 舌苔白或腻 舌体多津 脉弦或细 
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表 2 各基因PCR引物序列
基因名称 引物序列(5′-3′) 产物长度
/ bpGAPDH F：ACAGCAACAGGGTGGTGGAC 252 R：TTTGAGGGTGCAGCGAACTT TNF-α F：CCCCTCTATTTATAATTGCACCT 167 R：CTGGTAGTTTAGCTCCGTTT IL-6 F：TCACTATGAGGTCTACTCGG 141 R：CATATTGCCAGTTCTTCGTA IL-1β F：CAGCAGCATCTCGACAAGAG 123 R：AAAGAAGGTGCTTGGGTCCT IL-23 F：CCTGCTGGACTCGGACAT 85 R：GCCCAGTAGGGAGGTATGAA
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表 3 各组大鼠DAI、CMDI及TDI评分的比较
分，X±S 组别 例数 DAI CMDI TDI 空白组 9 0.53±0.17 0 0 模型组 11 2.57±0.161) 4.10±0.701) 4.16±0.381) 痛泻要方组 11 1.91±0.231)2) 2.92±0.351)2) 2.83±0.321)2) 痛泻要方联合穴位埋线组 11 1.09±0.251)2)3) 1.82±0.441)2)3) 1.77±0.341)2)3) 与空白组相比，1)P < 0.05；与模型组相比，2)P < 0.05；与痛泻要方组相比，3)P < 0.05。
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表 4 各组大鼠海马组织及血清中5-HT、SP含量的比较
pg/mL，X±S 组别 例数 海马组织 血清 5-HT SP 5-HT SP 空白组 3 1.27±0.13 55.88±1.46 5.34±0.80 14.59±2.80 模型组 3 2.09±0.171) 89.11±2.901) 20.26±2.061) 32.93±1.011) 痛泻要方组 3 1.76±0.051)2) 75.33±0.901)2) 9.99±0.481)2) 26.02±0.711)2) 痛泻要方联合穴位埋线组 3 1.50±0.051)2)3) 63.96±3.451)2)3) 7.53±0.421)2)3) 21.03±1.391)2)3) 与空白组相比，1)P < 0.05；与模型组相比，2)P < 0.05；与痛泻要方组相比，3)P < 0.05。
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表 5 各组大鼠海马、结肠组织中5-HT、SP表达的比较
X±S 组别 例数 海马组织 结肠组织 5-HT SP 5-HT SP 空白组 3 0.09±0.01 0.08±0.03 0.13±0.03 0.09±0.01 模型组 3 0.30±0.031) 0.30±0.021) 0.65±0.111) 0.37±0.031) 痛泻要方组 3 0.22±0.041)2) 0.23±0.011)2) 0.40±0.051)2) 0.26±0.001)2) 痛泻要方联合穴位埋线组 3 0.15±0.021)2)3) 0.13±0.011)2)3) 0.25±0.041)2)3) 0.14±0.011)2)3) 与空白组相比，1)P < 0.05；与模型组相比，2)P < 0.05；与痛泻要方组相比，3)P < 0.05。
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表 6 各组大鼠结肠组织中TNF-α、IL-6、IL-1β、IL-23含量的比较
pg/mL，X±S 组别 例数 TNF-α IL-6 IL-1β IL-23 空白组 3 1.30±0.38 1.07±0.37 1.22±0.22 1.15±0.24 模型组 3 7.04±0.741) 9.73±1.751) 7.90±1.361) 9.59±1.121) 痛泻要方组 3 4.25±0.351)2) 6.50±0.671)2) 5.62±1.141)2) 7.03±0.341)2) 痛泻要方联合穴位埋线组 3 3.26±0.351)2)3) 3.74±1.721)2)3) 3.77±0.491)2)3) 4.44±0.911)2)3) 与空白组相比，1)P < 0.05；与模型组相比，2)P < 0.05；与痛泻要方组相比，3)P < 0.05。
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