【作者】 霍明东；

【导师】 丁义江；

【作者基本信息】 南京中医药大学 ， 中医外科学， 2013， 博士

【摘要】 研究目的：观察济川煎对“泻剂结肠”的治疗效果,探讨SCF/c-kit信号通路及AQP3、AQP4和AQP9在“泻剂结肠”发病机制中的作用。研究方法：1.临床研究：70例“泻剂结肠”患者进行临床研究。给予济川煎水煎液(肉苁蓉20克、当归30克、牛膝10克、泽泻10克、升麻10克、枳壳20克)治疗,每日一剂,分早、晚两次服用,十天为一疗程,共二个疗程。记录患者治疗前及治疗后一月便秘症状评分(包括排便频率评分、排便时间评分、粪便性状评分、腹胀情况评分、使用泻剂情况评分)及便秘患者生存质量自评量表(PAC-QOL)评分,观察治疗前、后的差异。2.实验研究：健康成年SPF级SD大鼠70只,随机分为对照组(对照组1：11只大鼠；对照组2：12只大鼠)和实验组(模型组1：11只大鼠；模型组2：12只大鼠；济川煎组：12只大鼠；聚乙二醇组：12只大鼠)。对照组大鼠生理盐水灌胃：对照组1与模型组1同时取材；对照组2与模型组2同时取材。实验组大鼠采用“大黄酸混悬液灌胃法”制作“泻剂结肠”动物模型。模型组1制模结束后后取材；模型组2：制模结束后,以生理盐水灌胃30天；济川煎组：制模结束后,以济川煎水煎液灌胃30天；聚乙二醇组：制模结束后,以聚乙二醇水溶液灌胃30天。检测首粒黑便排出时间及粪便干湿重比以反映肠道传输功能及粪便性质。应用逆转录聚合酶链反应(RT-PCR)检测结肠组织c-kit、SCF及AQP3、AQP4和AQP9、TFF3的mRNA水平表达,免疫组织化学方法进行c-kit、SCF的组织学定位,Western Blot法检测结肠组织中c-kit、SCF及AQP3、 AQP4和AQP9、TFF3的蛋白水平表达。研究结果：1.临床研究：“泻剂结肠”患者经济川煎治疗后便秘症状及生活质量均明显改善。排便频率提高,治疗前、后比较：2.37±1.18vs0.70±1.04,P<0.01；排便时间缩短,治疗前、后比较：1.66±0.56vs0.61±0.62,P<0.01；粪便性状变软,治疗前、后比较：2.22±0.84vs0.83±0.90,P<0.01；腹胀减轻,治疗前、后比较：1.23±0.49vs0.49±0.50,P<0.01；泻剂使用减少,治疗前、后比较：2.83±0.72vs0.83±0.61,P<0.01。PAC-QOL评分降低,治疗前、后比较：58.39±15.2vs32.10±13.97,P<0.01。2.实验研究：经过110天的大黄酸混悬液灌胃成功建立“泻剂结肠”大鼠动物模型。模型组1大鼠的首粒黑便排出时间显著延长,粪便干湿重比明显升高,与对照组1相比差别显著(P<0.01)。经过30天的治疗过程,济川煎组大鼠的首粒黑便排出时间明显缩短,粪便干湿重比明显降低,与模型组2相比均有显著差异(P<0.01),但与对照组2相比,首粒黑便排出时间仍延长,有统计学意义(P<0.05),而粪便干湿重比则没有明显差异(P>0.05)。而聚乙二醇组与模型组2相比,首粒黑便排出时间及粪便干湿重比均没有统计学差异(P>0.05)。免疫组化检测发现,c-kit分布于整个结肠肌层,以环肌层和肌间丛区域肌间神经丛最为明显,在ICC胞膜和突起上均有表达,模型组1中ICC呈淡棕黄色染色,呈微弱阳性,与对照组1相比,c-kit表达明显减弱。SCF表达于结肠壁全层,模型组1中SCF仅有轻度表达,与对照组1相比,表达明显降低。RT-PCR及Western blot检测发现,模型组1c-kit和SCF的mRNA和蛋白表达均较弱,与对照组1相比,差别显著(P<0.05)。在济川煎组,结肠组织中的c-kit和SCF的mRNA表达和蛋白表达均显著增高,与模型组2比较有统计学意义(P<0.05),与对照组2比较没有明显差异(P>0.05)。聚乙二醇组结肠组织中的c-kit和SCF的mRNA和蛋白表达与模型组2比较没有显著变化(P>0.05),与对照组2比较,差异显著(P<0.05)。与对照组2相比,模型组2结肠组织中AQP3的mRNA和蛋白表达均明显升高,AQP9和TFF3的mRNA和蛋白表达均明显降低,有统计学意义(P<0.05),而AQP4的mRNA和蛋白表达与对照组2相比则无明显差异(P>0.05)。济川煎组结肠组织中AQP3的mRNA和蛋白表达明显回调,AQP9和TFF3的mRNA和蛋白表达明显增加,与模型组2相比,有显著差异(P<0.05),而与对照组2相比,无明显差异(P>0.05)。聚乙二醇组AQP3, AQP9和TFF3mRNA和蛋白表达与模型组2相比均无统计学意义(P>0.05)。研究结论：1.济川煎治疗“泻剂结肠”可取得满意的疗效,患者便秘症状及生活质量均得到了明显改善。2.济川煎能够显著改善“泻剂结肠”大鼠的肠道传输功能及粪便干湿重比,从而起到治疗的作用。3.蒽醌类泻剂对大鼠肠道的长期刺激可引起结肠组织中c-kit, SCF及AQP3、AQP9、 TFF3的异常表达,这可能是“泻剂结肠”的发生机制之一。4.济川煎治疗“泻剂结肠”的机制可能与其修复SCF/c-kit信号通路及纠正结肠粘膜AQP3、AQP9、TFF3的异常表达有关。更多还原

【Abstract】 Objective:To observe the Jichuanjian therapeutic effect on cathartic colon, and discussing the function of the SCF/c-kit signal channel, AQP3,AQP4and AQP9on Cathartic Colon mechanism.Methods:1. Clinical studyThe70"cathartic colon" patients were given Jichuanjian Decoction treatment (cistanche20grams, angelica30grams, Radix10grams, alisma10grams, cohosh10grams,20grams of Fructus Aurantii). A agent daily, early and late two times daily, ten days as a course of treatment, a total of two courses. The patients’ constipation symptom score was recorded (including frequency of defecation score, defecation time score, characteristics of feces score, abdominal distension score and the use of laxatives score) in Prior treatment and Post treatment for one month and Patient Assessment of Constipation Quality of Life (PAC-QOL). What’s more, the differences in Prior treatment and Post treatment were observed.2. Experimental studyThe70healthy adult SD rats in SPF level were randomly divided into control group (Group1:11rats; group2:12rats) and experimental group (model group1:11rats; model group2:12rats; Jichuanjian group:12rats; polyethylene glycol group:12rats).The control group rats were perfused with physiological saline:the control group1and model group1, as well as control group2and model group2were drew materials at the same time. The "cathartic colon" animal model was made on experimental group rats by "Rhein suspension gavage method". For the model group1drawing materials after the end of the molding; for model group2perfused with physiological saline for30days after the end of the molding; for Jichuanjian group:after the end of the molding, perfused with Jichuanjian Decoction for30days; polyethylene glycol group:after the end of the molding, perfused with polyethylene glycol aqueous solutions for30days. Then I tested the first black stool time and fecal dry and wet weight ratio to reflect intestinal transmission function and the nature of stool. The level expression of mRNA of c-kit, SCF and AQP3and AQP4, AQP9, TFF3in colon tissue was detected by the application of reverse transcription polymerase chain reaction (RT-PCR). C-kit and SCF were positioned in histology by immunohistochemical method. The level expression of protein of c-kit, SCF and AQP3, AQP4and AQP9, TFF3in colon tissue was detected by Western Blot method. Results:1. Clinical researchThe symptoms of constipation and quality of life for the "Cathartic colon" patients were significantly improved after the treatment by Jichuanjian. Defecation frequency was increased in the comparison of prior treatment and post treatment:2.37±1.18vs0.70±1.04, P<0.01; defecation time was shorter in the comparison of prior treatment and post treatment:1.66±0.56vs0.61±0.62, P<0.01; the stool was softer in the comparison of prior treatment and post treatment:2.22±0.84vs0.83±0.90, P<0.01; abdominal distension was eased in the comparison of prior treatment and post treatment:1.23±0.49vs0.49±0.50, P<0.01; the use of laxative was reduced in the comparison of prior treatment and post treatment:2.83±0.72vs0.83±0.61, P<0.01. The PAC-QOL score was decreased in the comparison of prior treatment and post treatment:58.39±15.2vs32.10±13.97, P<0.01.2. Experimental studyAfter the rhein suspension gavage for110days, the first black stool time of rats in model group1was prolonged significantly, and the fecal dry and wet weight ratio was increased significantly, so the differences were significant comparing with the control group1(P<0.01). After the treatment process for30days, the first black stool time of rats in Jichuanjian group was shortened significantly, and the fecal dry and wet weight ratio was decreased significantly, so the differences were significant comparing with the model group2(P<0.01). But compared with the control group2, the first black stool time was still extended, so it has significance in statistic (P<0.05), however it has no obvious difference in the fecal dry and wet weight ratio (P>0.05). Compared polyethylene glycol group with model group2, it has no difference statistically in the first black stool time and fecal dry and wet weight ratio (P>0.05).Through the immunohistochemical detection, It was found that the c-kit was distributed in the whole colon muscle layer, in which the circular muscle layer and the myenteric nerve plexus in the myenteric plexus regional is the most obvious. It was expressed in ICC cell membrane and neuritis. In the model group1, ICC is light brown staining and weak positive, and in contrast to the control group1, the expression of c-kit was weakened significantly. The SCF was expressed in the whole colon wall, and it has only slight expression in model group l,so in contrast to the control group1, the expression was decreased obviously.Through the RT-PCR and Western blot detection, It was found that c-kit and mRNA of SCF and protein expression in model group1are weaker, and in contrast to the control groupl, there was significant difference (P<0.05). In Jichuanjian group, c-kit and mRNA of SCF and protein expression in the colonic tissues were increased significantly, which was significant statistically (P<0.05) compared with model group2, but it has no significant difference (P>0.05) compared with the control group2. In polyethylene glycol group, compared with model group2, c-kit and mRNA of SCF and protein expression in the colonic tissues have no significant changes (P>0.05) but it has significant difference (P<0.05) compared with the control group2.Compared with the control group2, the mRNA of AQP3and protein expression in colon tissue in model group2were significantly increased, yet the mRNA of AQP9and TFF3and protein expression were significantly lower, which has significance statistically (P<0.05). However compared with the control group2, the mRNA of AQP4and protein expressions have no significant difference (P>0.05). In Jichuanjian group, the mRNA of AQP3and protein expression in colon tissue was lower significantly, yet the mRNA of AQP9and TFF3and protein expression were significantly increased. Compared with the model group2, it has significant difference (P<0.05), but compared with the control group2, it has no significant difference (P>0.05). In polyethylene glycol group, compared with the model group2the mRNA of AQP3, AQP9, TFF3and protein expression have no significance statistically (P>0.05).Conclusion:1. The satisfactory curative effect can be obtained on the treatment of "cathartic colon" by JiChuanJian therapy, as well as the constipation symptoms and quality of life of the patients were improved significantly.2. The intestinal transmission function and fecal dry and wet weight ratio can be improved significantly for the "cathartic colon" rats by JiChuanJian therapy, so as to take the therapeutic effect.3. The abnormal expression of c-kit, SCF, AQP3, AQP9and TFF3in colon tissue can be caused by long-term stimulation on the rats’intestinal tract from anthraquinone laxative, which was perhaps the one of the "mechanism of cathartic colon".4. The mechanism of the treatment on "cathartic colon" by Jichuanjian therapy may be related to the repair of SCF/c-kit signal channel and the correct of abnormal expression of AQP3, AQP9, and TFF3in colonic mucosa.更多还原