健脾化湿法对脾虚湿困型溃疡性结肠炎大鼠水通道蛋白3、4表达的影响及机制研究
摘要
研究背景
中医治则治法对临床立法、处方、用药以及针灸治疗等都具有普遍的指导意义,开展对治则治法有效方剂的效用物质基础及作用机制研究,赋予治则治法新的内涵,有着重要的理论意义与科学价值。湿邪致病的广泛性、潜隐性、迁延性及兼挟性使湿病几乎存在于各系统的疾病中。溃疡性结肠炎(Ulcerative Colitis,UC)是一种病因不明的慢性非特异性炎性肠病,属中医的“肠澼”、“肠风下血”、“休息痢”等范畴,脾虚湿困是其主要病机。健脾化湿法治疗溃疡性结肠炎取得了很好的临床疗效,但是有关其作用机制的研究却鲜见报道。水通道蛋白在机体的各个组织广泛表达,提示其在维持体内水平衡过程中起重要作用。近年研究表明,水通道蛋白与水液代谢关系密切,与中医理论的肺、脾、肾等脏相关。有关水通道蛋白在脾胃湿热、脾虚湿困、湿阻中焦等方面的作用受到越来越多的重视,并有望成为相关疾病临床治疗的新靶点。深入研究水通道蛋白的作用,有望很好的解释水、湿、痰、饮等病理变化的机理。另外,在此基础上,还有可能阐明化湿药、利水渗湿药、燥湿药等的药理作用。因此,探讨湿病中水通道蛋白的变化和“湿”之间的内在联系,深入进行“湿”定性量化的研究,有着重要的理论与临床意义。诸多研究已证明水通道蛋白与水液代谢障碍性疾病有密切关系,但是健脾化湿法对脾虚湿困型UC的治疗作用是否通过水通道蛋白及相关信号转导通路来调节还有待于进一步研究。
研究目的
通过建立符合中医证候特点的脾虚湿困型UC大鼠动物模型,探讨健脾化湿法有效方剂参苓白术散对脾虚湿困型UC大鼠水通道蛋白3、4(AQP3、AQP4)的影响,并进一步探讨MAPK及NF-κB信号转导通路在健脾化湿法对水通道蛋白调节过程中所发挥的作用。旨在初步探讨健脾化湿法治疗脾虚湿困型UC的部分作用机制与途径,促进中医水液代谢障碍理论的深入研究,为进一步阐明健脾化湿法治疗脾虚湿困型UC的分子生物学机制奠定基础。
研究方法
(1)采用环境与饮食因素干预结合三硝基苯磺酸(TNBS)与乙醇复合物灌肠法建立脾虚湿困型UC大鼠模型,观察脾虚湿困型UC大鼠症状、体征以及结肠组织病理改变；并检测大鼠血清表皮生长因子(EGF)、超氧化物歧化酶(SOD)、丙二醛(MDA)及白细胞介素6(IL-6)、白细胞介素8(IL-8)、肿瘤坏死因子a(TNF-a)水平的变化及健脾化湿法的干预作用；
(2)采用免疫组织化学法检测脾虚湿困型UC大鼠结肠组织AQP3、AQP4的表达变化；应用免疫组织化学法、Western blot、荧光定量PCR法检测健脾化湿法对脾虚湿困型UC大鼠结肠组织AQP3、AQP4表达的影响；
(3)采用免疫组织化学法检测脾虚湿困型UC大鼠结肠组织细胞外信号调节激酶(ERK)、p38丝裂原激活蛋白激酶(p38MAPK)蛋白的表达；采用荧光定量PCR、Western blot去检测ERK阻断剂U0126和p38MAPK阻断剂SB203580对健脾化湿法改善脾虚湿困型UC大鼠AQP3、AQP4表达作用的影响；
(4)采用免疫组织化学法检测脾虚湿困型UC大鼠结肠组织NF-κB p56蛋白的表达情况；采用荧光定量PCR、Western blot法检测NF-κB阻断剂吡咯醛二硫氨基甲酸酯(PDTC)对健脾化湿法改善脾虚湿困型UC大鼠AQP3、AQP4表达作用的影响。
研究结果
(1)脾虚湿困型UC大鼠出现嗜睡懒动、大便变软、饮食减少、竖毛、易脱毛、腹泻、行走歪斜、肛周污秽、眯眼、神态极度萎靡等症状,经肛门灌入TNBS/乙醇出现黏液脓血便、饮食减少等。病理可见结肠黏膜充血、水肿,皱襞减少甚至消失,溃疡形成,黑黄色膜状物附着,或有息肉形成,肠管增粗,肠壁变薄,外壁与外周组织黏连,大鼠结肠组织损伤程度评分与正常对照组比较有显著性差异(P<0.01)。模型对照组大鼠EGF、SOD含量明显下降,MDA含量显著升高,与正常对照组比较差异有统计学意义(P<0.05或P<0.01)。健脾化湿组大鼠EGF含量与模型对照组比较,差异有统计学意义(P<0.05),SOD、MDA水平的差异有非常显著性(P<0.01)。柳氮磺吡啶组大鼠EGF、MDA与模型对照组的差异有统计学意义(P<0.05)。模型对照组大鼠血清IL-6、IL-8及TNF-α水平均显著升高,与正常对照组比较差异有统计学意义(P<0.05)。健脾化湿组大鼠IL-6、IL-8及TNF-a水平与模型对照组比较,差异有统计学意义(P<0.05)。柳氮磺吡啶组大鼠IL-6、IL-8及TNF-a与模型对照组的差异亦有统计学意义(P<0.05)
(2)免疫组织化学法检测发现,脾虚湿困型UC模型大鼠AQP3、AQP4阳性细胞表达较正常对照组少,着色浅,其阳性细胞数及平均光密度值显著下降,与正常对照组比较差异有统计学意义(P<0.05)。健脾化湿组和柳氮磺吡啶组大鼠结肠组织AQP3、AQP4表达量较模型对照组显著升高,其间差异有统计学意义(P<0.05),健脾化湿组大鼠结肠组织AQP4表达量与柳氮磺吡啶组比较差异亦有统计学意义(P<0.05)。Western blot检测结果显示模型对照组大鼠结肠组织中AQP3、AQP4表达较正常对照组明显降低,差异有统计学意义(P<0.01)。健脾化湿组及柳氮磺吡啶组较模型对照组均升高,差异有统计学意义(P<0.01)。FQ-PCR检测显示模型对照组大鼠结肠组织中AQP3 mRNA. AQP4 mRNA表达均明显降低,与正常对照组比较差异有统计学意义(P<0.05)。健脾化湿组大鼠结肠组织中AQP3 mRNA、AQP4 mRNA表达与模型对照组比较显著升高,其间差异有统计学意义(P<0.05)。
(3)免疫组织化学法检测发现,脾虚湿困型UC模型大鼠结肠组织ERK、p38 MAPK表达明显增多,与正常对照组比较差异有统计学意义(P<0.05)。同时健脾化湿组大鼠结肠组织ERK、p38 MAPK的表达与模型对照组比较具有显著性差异(P<0.05)。Western blot检测结果显示模型对照组大鼠结肠组织AQP3、AQP4蛋白表达显著降低(P<0.05),健脾化湿组大鼠结肠组织AQP3、AQP4蛋白表达较模型对照组明显升高(P<0.05),U0126+治疗组及SB203580+治疗组大鼠结肠组织AQP3、AQP4蛋白表达量与健脾化湿组及正常对照组比较差异有统计学意义(P<0.05)。FQ-PCR结果显示模型对照组大鼠结肠组织中AQP3 mRNA、AQP4 mRNA表达均明显降低,与正常对照组比较差异有统计学意义(P<0.05)。健脾化湿组大鼠结肠组织中AQP3 mRNA、AQP4 mRNA表达与模型对照组比较显著升高,其间差异有统计学意义(P<0.05)。U0126+治疗组与SB203580+治疗组大鼠结肠组织AQP3 mRNA、AQP4 mRNA表达与健脾化湿组及正常对照组比较差异均有统计学意义(P<0.05)(4)免疫组织化学法检测发现,脾虚湿困型UC模型大鼠结肠组织NF-κB p65表达的阳性细胞数、平均光密度均显著升高,与正常对照组比较有显著性差异(P<0.05)。而健脾化湿组大鼠结肠组织NF-κB p65表达的阳性细胞数、平均光密度明显低于模型对照组(P<0.05)。Western blot检测结果显示PDTC+治疗组大鼠结肠组织AQP3、AQP4蛋白表达与模型对照组比较无显著性差异(P>0.05),与正常对照组和健脾化湿组均有差异,且有统计学意义(P<0.05)。表明PDTC+台疗组大鼠经治疗后结肠组织AQP3、AQP4蛋白表达未能改善。FQ-PCR的检测结果亦显示PDTC可明显抑制健脾化湿法对UC大鼠结肠组织AQP3 mRNA、AQP4 mRNA表达的调节作用。
结论
(1)模拟中医传统病因,采用环境与饮食因素干预结合TNBS与乙醇复合物灌肠法建立脾虚湿困型UC动物模型,出现了与临床类似的症状、体征,符合人类自然发生的UC病变特点和中医证候特点,是较理想的脾虚湿困型UC大鼠模型。健脾化湿法能改善脾虚湿困型UC大鼠血清的EGF、SOD、MDA及IL-6、IL-8、TNF-α水平
(2)脾虚湿困型UC大鼠结肠组织AQP3、AQP4表达显著降低,健脾化湿治疗可以明显改善脾虚湿困型UC大鼠结肠组织中AQP3、AQP4蛋白及mRNA的表达,促进肠黏膜的修复。
(3)脾虚湿困型UC大鼠结肠组织ERK及p38 MAPK表达明显增强,健脾化湿治疗可显著降低脾虚湿困型UC大鼠结肠组织ERK及p38MAPK的表达水平。U0126及SB203580可以在一定程度上抑制健脾化湿法对脾虚湿困型UC大鼠结肠组织中AQP3、AQP4蛋白及mRNA的改善作用。ERK及p38 MAPK与健脾化湿法调控脾虚湿困型UC大鼠结肠组织水通道蛋白的作用相关。
(4)脾虚湿困型UC大鼠结肠组织中NF-κB p65的表达明显升高,健脾化湿法治疗可以显著改善脾虚湿困型UC大鼠结肠组织NF-κB p65的表达水平。NF-κB阻断剂PDTC能够部分阻断健脾化湿法对脾虚湿困型UC大鼠结肠组织AQP3、AQP4蛋白及mRNA表达的调控作用。NF-κB与健脾化湿法调控脾虚湿困型UC大鼠结肠组织水通道蛋白有一定相关性。
Background
Therapeutic principles and methods in Chinese medicine have universal guiding significance to prescription, medication and acupuncture.Studies on the material base and mechanism of representative prescription, and endow the methods with new connotation have theoretical significance and scientific value.Dampness disease exists in each system of human. Ulcerative colitis is an undetermined etiology of chronic nonspecific inflammatory bowel disease, and dampness stagnancy due to spleen deficiency is a common pathogenesis. Treatment of strengthening spleen and removing dampness has definite therapeutic effect on common disease in clinic.But the study on mechanism has not been reported before.In recent years,studies have found that aquaporin has close relation with dampness disease.It also has relation with lung, spleen, kidney of traditional Chinese medicine theory.The research on aquaporin has drawn more and more attentions, and is expected to become a new target for clinical treatment of related diseases. The mechanism of pathological changes of damp blockage of middle energizer, dampness-heat of spleen and stomach and syndrome of dampness stagnancy due to spleen deficiency may be explained by further study on aquaporin. In addition, on this basis, there is likely to clarify the pharmacological effects of drugs of dissipating dampness, inducing diuresis and excreting dampness.Therefore,the study on qualitative and quantitative of dampness has important theoretical and clinical significance. Many studies have demonstrated that the aquaporin is closely related to water metabolism disturbance,but the effects on aquaporin of UC with syndrome of dampness stagnancy due to spleen deficiency treated by Jianpihushi and the mechanism of related signal pathway also requires further study.
Objective
The ulcerative colitis rats with syndrome of dampness stagnancy due to spleen deficiency were established that according with the TCM syndrome characteristics.This research investigates the effects on AQP3 and AQP4 in UC rats with syndrome of dampness stagnancy due to spleen deficiency with Shenlinbaizhu powder that the effective prescription of Jianpihuashi,and further explore the role of MAPK and NF-κB signal pathway in the regulation on aquaporin with therapeutic method of Jianpihuashi.This research aims to clarify the part mechanism and pathway of treatment UC of syndrome of dampness stagnancy due to spleen deficiency with the therapeutic method of Jianpihuashi,to promote further study of the theory of water metabolism disturbance in TCM,and to lay a foundation for the studies of molecular biological mechanism on UC with syndrome of dampness stagnancy due to spleen deficiency by the therapeutic method of Jianpihuashi.
Methods
(1)Environmental and dietary factors intervention combined with TNBS and ethanol were used to establish syndrome of dampness stagnancy due to spleen deficiency of ulcerative colitis rats. The symptoms, signs and colon tissue pathological changes were observed in rats with syndrome of dampness stagnancy due to spleen deficiency of ulcerative colitis. And the change of serum EGF, the activities of SOD, MDA and IL-6, IL-8, TNF-a level were detected in model rats and the intervention effect with treatment of strengthening spleen and removing dampness.
(2) The expression changes of AQP3 and AQP4 in rats with syndrome of dampness stagnancy due to spleen deficiency of ulcerative colitis were detected by immunohistochemistry. The effects on expression changes of AQP3 and AQP4 in model rats with treatment of strengthening spleen and removing dampness were detected by immunohistochemistry, Western blot and fluorescence quantitative PCR.
(3) The expressions of phosphorylation ERK, p38MAPK in colon tissue of rats with syndrome of dampness stagnancy due to spleen deficiency of ulcerative colitis were detected by immunohistochemistry. The effects on expression changes of AQP3 and AQP4 in model rats which treatmented by strengthening spleen and removing dampness with U0126 and SB203580,were detected by fluorescence quantitative PCR and Western blot.
(4) The expressions of phosphorylation NF-κB in colon tissue of rats with syndrome of dampness stagnancy due to spleen deficiency of ulcerative colitis were detected by immunohistochemistry.The effects on expression changes of AQP3 and AQP4 in model rats which treatmented by strengthening spleen and removing dampness with PDTC,were detected by fluorescence quantitative PCR and Western blot.
Results
(1)The rats with syndrome of dampness stagnancy due to spleen deficiency of UC appeared symptoms such as lethargy,soft stool,decrease of diet, piloerection, unhairing, diarrhea, walk skew, perianal pollution, narrowing eyes, loss of energy et al.The bloody mucopurulent stool and emaciation appeared after TNBS and ethanol were injected through the anus.The pathological findings of colon shown that mucous hyperemia, edema, plica reduced or even disappeared, ulcer formation, black and yellow of membrane attachment, or the formation of polyps, bowel thickening, bowel wall thinning and tissue adhesion. The colonic tissue damage scores had significant difference compared with normal group (P<0.01).The EGF and SOD of model control group significantly decreased and the MDA content significantly increased, the difference was statistically significant compared with the normal control group (P<0.05 or P<0.01). The EGF level of Jianpihuashi group has significant difference compared with the model control group (P<0.05), the differences of SOD, MDA between two groups are very significant (P<0.01). The EGF, MDA of Sulfasalazine group has significant difference that compared with model control group (P<0.05). The level of serum IL-6, IL-8 and TNF-a in model control group significantly higher than normal control group (P<0.05).The level of serum IL-6, IL-8 and TNF-a in Jianpihuashi group and sulfasalazine group are significant than that in the model group (P<0.05).
(2) The results detected by immunohistochemistry shown that the expression of AQP3 and AQP4 positive cells in the rats with syndrome of dampness stagnancy due to spleen deficiency of UC was less than in normal group, lightly stained, the number of positive cells and the average optical density decreased significantly, which have very remarkable difference compared with the normal control group (P<0.05).The expression of AQP3 and AQP4 in Jianpihuashi group and sulfasalazine group significantly increased and showed significant difference compared with model group (P<0.05). There was significant difference between Jianpihuashi group and sulfasalazine group on the expression of AQP4 (P<0.05).Western blot results showed that the expression of AQP3 and AQP4 in model control group significantly decreased (P<0.05).The expression of AQP3 and AQP4 in Jianpihuashi group and sulfasalazine group significantly increased compared with the model group (P<0.05).FQ-PCR showed that the expression of AQP3 mRNA and AQP4 mRNA in model control group significantly decreased, compared with normal control group, difference was statistically significant (P<0.05).The expression of AQP3 mRNA and AQP4 mRNA in Jianpihuashi group increased significantly that compared with the model group, and there was a significant difference between two groups (P<0.05).
(3)The results detected by immunohistochemistry showed that the expression of ERK and p38 MAPK in the rats with syndrome of dampness stagnancy due to spleen deficiency of UC significantly increased,the difference compared with the normal control group was statistically significant (P<0.05).The expression of ERK and p38 MAPK in Jianpihuashi group have significant difference compared with model group (P<0.05).Western blot results showed that the expression of AQP3 and AQP4 in model control group significantly decreased (P<0.05).The expression of AQP3 and AQP4 in Jianpihuashi group significantly increased compared with the model group (P<0.05). The expression of AQP3 and AQP4 in U0126+treatment group and SB203580+ treatment group have significant difference compared with Jianpihuashi group and normal control group (P<0.05).FQ-PCR results showed that the expression of AQP3 mRNA and AQP4 mRNA in model control group significantly decreased compared with normal control group (P<0.05). The expression of AQP3 mRNA and AQP4 mRNA in Jianpihuashi group significantly increased compared with the model group (P<0.05). The expression of AQP3 mRNA and AQP4 mRNA in U0126+ treatment group and SB203580+treatment group have significant difference compared with Jianpihuashi group and normal control group (P<0.05).
(4) The results detected by immunohistochemistry indicated that the number of positive cells and the average optical density of NF-κB p65 in the rats with syndrome of dampness stagnancy due to spleen deficiency of UC significantly increased, the difference compared with the normal control group was statistically significant (P<0.05). The number of positive cells and the average optical density of NF-κB p65 in Jianpihuashi group were significantly lower than those of model control group (P<0.05). Western blot results showed that the expression of AQP3 and AQP4 in PDTC+treatment group have significant difference compared with Jianpihuashi group and normal control group (P<0.05), while no significant difference compared with the model group (P>0.05).FQ-PCR results also indicated that PDTC could inhibit the regulation effects on UC rats with syndrome of dampness stagnancy due to spleen deficiency with treatment of Jianpihuashi.
Conclusions
(1)Simulation of the etiology of traditional Chinese medicine, the UC rats with syndrome of dampness stagnancy due to spleen deficiency were built by the intervention of environmental and dietary factors combination with enema of TNBS and ethanol, the symptoms and signs that similar to the clinical were appeared, and according with the disease characteristics and TCM syndrome characteristics of UC,which is an ideal rat model of UC with syndrome of dampness stagnancy due to spleen deficiency.Therapeutic method of Jianpihuashi could improve the levels of EGF, SOD,MDA and IL-6,IL-8,TNF-a.
(2) The expressions of AQP3 and AQP4 in the UC rats with syndrome of dampness stagnancy due to spleen deficiency were significantly decreased.Therapeutic method of Jianpihuashi could significantly improve the expression of AQP3,AQP4 and AQP3 mRNA, AQP4 mRNA in the UC colon tissue, and promote the repair of the intestinal mucosa.
(3) The expressions of ERK and p38 MAPK in the UC rats with syndrome of dampness stagnancy due to spleen deficiency were significantly increased.Therapeutic method of Jianpihuashi could significantly decrease the expression of ERK and p38 MAPK in the UC colon tissue.And therapeutic method of Jianpihuashi could significantly improve the protein and mRNA expression of AQP3,AQP4,which could be inhibited by U0126 and SB203580 to a certain extent. The effects on AQP of UC rats with syndrome of dampness stagnancy due to spleen deficiency with therapeutic method of Jianpihuashi have a relationship with ERK and p38 MAPK.
(4) The expressions of NF-κB p65 in the UC rats with syndrome of dampness stagnancy due to spleen deficiency increased significantly. Therapeutic method of Jianpihuashi could significantly improve the expression of NF-κB p65 in the UC colon tissue. NF-κB blocker PDTC could partially block the effects on the protein and mRNA expression of AQP3,AQP4 of UC rats with syndrome of dampness stagnancy due to spleen deficiency with therapeutic method of Jianpihuashi. The effects on AQP of UC rats with syndrome of dampness stagnancy due to spleen deficiency with therapeutic method of Jianpihuashi have a relationship with NF-κB signal pathway.
中医治则治法对临床立法、处方、用药以及针灸治疗等都具有普遍的指导意义,开展对治则治法有效方剂的效用物质基础及作用机制研究,赋予治则治法新的内涵,有着重要的理论意义与科学价值。湿邪致病的广泛性、潜隐性、迁延性及兼挟性使湿病几乎存在于各系统的疾病中。溃疡性结肠炎(Ulcerative Colitis,UC)是一种病因不明的慢性非特异性炎性肠病,属中医的“肠澼”、“肠风下血”、“休息痢”等范畴,脾虚湿困是其主要病机。健脾化湿法治疗溃疡性结肠炎取得了很好的临床疗效,但是有关其作用机制的研究却鲜见报道。水通道蛋白在机体的各个组织广泛表达,提示其在维持体内水平衡过程中起重要作用。近年研究表明,水通道蛋白与水液代谢关系密切,与中医理论的肺、脾、肾等脏相关。有关水通道蛋白在脾胃湿热、脾虚湿困、湿阻中焦等方面的作用受到越来越多的重视,并有望成为相关疾病临床治疗的新靶点。深入研究水通道蛋白的作用,有望很好的解释水、湿、痰、饮等病理变化的机理。另外,在此基础上,还有可能阐明化湿药、利水渗湿药、燥湿药等的药理作用。因此,探讨湿病中水通道蛋白的变化和“湿”之间的内在联系,深入进行“湿”定性量化的研究,有着重要的理论与临床意义。诸多研究已证明水通道蛋白与水液代谢障碍性疾病有密切关系,但是健脾化湿法对脾虚湿困型UC的治疗作用是否通过水通道蛋白及相关信号转导通路来调节还有待于进一步研究。
研究目的
通过建立符合中医证候特点的脾虚湿困型UC大鼠动物模型,探讨健脾化湿法有效方剂参苓白术散对脾虚湿困型UC大鼠水通道蛋白3、4(AQP3、AQP4)的影响,并进一步探讨MAPK及NF-κB信号转导通路在健脾化湿法对水通道蛋白调节过程中所发挥的作用。旨在初步探讨健脾化湿法治疗脾虚湿困型UC的部分作用机制与途径,促进中医水液代谢障碍理论的深入研究,为进一步阐明健脾化湿法治疗脾虚湿困型UC的分子生物学机制奠定基础。
研究方法
(1)采用环境与饮食因素干预结合三硝基苯磺酸(TNBS)与乙醇复合物灌肠法建立脾虚湿困型UC大鼠模型,观察脾虚湿困型UC大鼠症状、体征以及结肠组织病理改变；并检测大鼠血清表皮生长因子(EGF)、超氧化物歧化酶(SOD)、丙二醛(MDA)及白细胞介素6(IL-6)、白细胞介素8(IL-8)、肿瘤坏死因子a(TNF-a)水平的变化及健脾化湿法的干预作用；
(2)采用免疫组织化学法检测脾虚湿困型UC大鼠结肠组织AQP3、AQP4的表达变化；应用免疫组织化学法、Western blot、荧光定量PCR法检测健脾化湿法对脾虚湿困型UC大鼠结肠组织AQP3、AQP4表达的影响；
(3)采用免疫组织化学法检测脾虚湿困型UC大鼠结肠组织细胞外信号调节激酶(ERK)、p38丝裂原激活蛋白激酶(p38MAPK)蛋白的表达；采用荧光定量PCR、Western blot去检测ERK阻断剂U0126和p38MAPK阻断剂SB203580对健脾化湿法改善脾虚湿困型UC大鼠AQP3、AQP4表达作用的影响；
(4)采用免疫组织化学法检测脾虚湿困型UC大鼠结肠组织NF-κB p56蛋白的表达情况；采用荧光定量PCR、Western blot法检测NF-κB阻断剂吡咯醛二硫氨基甲酸酯(PDTC)对健脾化湿法改善脾虚湿困型UC大鼠AQP3、AQP4表达作用的影响。
研究结果
(1)脾虚湿困型UC大鼠出现嗜睡懒动、大便变软、饮食减少、竖毛、易脱毛、腹泻、行走歪斜、肛周污秽、眯眼、神态极度萎靡等症状,经肛门灌入TNBS/乙醇出现黏液脓血便、饮食减少等。病理可见结肠黏膜充血、水肿,皱襞减少甚至消失,溃疡形成,黑黄色膜状物附着,或有息肉形成,肠管增粗,肠壁变薄,外壁与外周组织黏连,大鼠结肠组织损伤程度评分与正常对照组比较有显著性差异(P<0.01)。模型对照组大鼠EGF、SOD含量明显下降,MDA含量显著升高,与正常对照组比较差异有统计学意义(P<0.05或P<0.01)。健脾化湿组大鼠EGF含量与模型对照组比较,差异有统计学意义(P<0.05),SOD、MDA水平的差异有非常显著性(P<0.01)。柳氮磺吡啶组大鼠EGF、MDA与模型对照组的差异有统计学意义(P<0.05)。模型对照组大鼠血清IL-6、IL-8及TNF-α水平均显著升高,与正常对照组比较差异有统计学意义(P<0.05)。健脾化湿组大鼠IL-6、IL-8及TNF-a水平与模型对照组比较,差异有统计学意义(P<0.05)。柳氮磺吡啶组大鼠IL-6、IL-8及TNF-a与模型对照组的差异亦有统计学意义(P<0.05)
(2)免疫组织化学法检测发现,脾虚湿困型UC模型大鼠AQP3、AQP4阳性细胞表达较正常对照组少,着色浅,其阳性细胞数及平均光密度值显著下降,与正常对照组比较差异有统计学意义(P<0.05)。健脾化湿组和柳氮磺吡啶组大鼠结肠组织AQP3、AQP4表达量较模型对照组显著升高,其间差异有统计学意义(P<0.05),健脾化湿组大鼠结肠组织AQP4表达量与柳氮磺吡啶组比较差异亦有统计学意义(P<0.05)。Western blot检测结果显示模型对照组大鼠结肠组织中AQP3、AQP4表达较正常对照组明显降低,差异有统计学意义(P<0.01)。健脾化湿组及柳氮磺吡啶组较模型对照组均升高,差异有统计学意义(P<0.01)。FQ-PCR检测显示模型对照组大鼠结肠组织中AQP3 mRNA. AQP4 mRNA表达均明显降低,与正常对照组比较差异有统计学意义(P<0.05)。健脾化湿组大鼠结肠组织中AQP3 mRNA、AQP4 mRNA表达与模型对照组比较显著升高,其间差异有统计学意义(P<0.05)。
(3)免疫组织化学法检测发现,脾虚湿困型UC模型大鼠结肠组织ERK、p38 MAPK表达明显增多,与正常对照组比较差异有统计学意义(P<0.05)。同时健脾化湿组大鼠结肠组织ERK、p38 MAPK的表达与模型对照组比较具有显著性差异(P<0.05)。Western blot检测结果显示模型对照组大鼠结肠组织AQP3、AQP4蛋白表达显著降低(P<0.05),健脾化湿组大鼠结肠组织AQP3、AQP4蛋白表达较模型对照组明显升高(P<0.05),U0126+治疗组及SB203580+治疗组大鼠结肠组织AQP3、AQP4蛋白表达量与健脾化湿组及正常对照组比较差异有统计学意义(P<0.05)。FQ-PCR结果显示模型对照组大鼠结肠组织中AQP3 mRNA、AQP4 mRNA表达均明显降低,与正常对照组比较差异有统计学意义(P<0.05)。健脾化湿组大鼠结肠组织中AQP3 mRNA、AQP4 mRNA表达与模型对照组比较显著升高,其间差异有统计学意义(P<0.05)。U0126+治疗组与SB203580+治疗组大鼠结肠组织AQP3 mRNA、AQP4 mRNA表达与健脾化湿组及正常对照组比较差异均有统计学意义(P<0.05)(4)免疫组织化学法检测发现,脾虚湿困型UC模型大鼠结肠组织NF-κB p65表达的阳性细胞数、平均光密度均显著升高,与正常对照组比较有显著性差异(P<0.05)。而健脾化湿组大鼠结肠组织NF-κB p65表达的阳性细胞数、平均光密度明显低于模型对照组(P<0.05)。Western blot检测结果显示PDTC+治疗组大鼠结肠组织AQP3、AQP4蛋白表达与模型对照组比较无显著性差异(P>0.05),与正常对照组和健脾化湿组均有差异,且有统计学意义(P<0.05)。表明PDTC+台疗组大鼠经治疗后结肠组织AQP3、AQP4蛋白表达未能改善。FQ-PCR的检测结果亦显示PDTC可明显抑制健脾化湿法对UC大鼠结肠组织AQP3 mRNA、AQP4 mRNA表达的调节作用。
结论
(1)模拟中医传统病因,采用环境与饮食因素干预结合TNBS与乙醇复合物灌肠法建立脾虚湿困型UC动物模型,出现了与临床类似的症状、体征,符合人类自然发生的UC病变特点和中医证候特点,是较理想的脾虚湿困型UC大鼠模型。健脾化湿法能改善脾虚湿困型UC大鼠血清的EGF、SOD、MDA及IL-6、IL-8、TNF-α水平
(2)脾虚湿困型UC大鼠结肠组织AQP3、AQP4表达显著降低,健脾化湿治疗可以明显改善脾虚湿困型UC大鼠结肠组织中AQP3、AQP4蛋白及mRNA的表达,促进肠黏膜的修复。
(3)脾虚湿困型UC大鼠结肠组织ERK及p38 MAPK表达明显增强,健脾化湿治疗可显著降低脾虚湿困型UC大鼠结肠组织ERK及p38MAPK的表达水平。U0126及SB203580可以在一定程度上抑制健脾化湿法对脾虚湿困型UC大鼠结肠组织中AQP3、AQP4蛋白及mRNA的改善作用。ERK及p38 MAPK与健脾化湿法调控脾虚湿困型UC大鼠结肠组织水通道蛋白的作用相关。
(4)脾虚湿困型UC大鼠结肠组织中NF-κB p65的表达明显升高,健脾化湿法治疗可以显著改善脾虚湿困型UC大鼠结肠组织NF-κB p65的表达水平。NF-κB阻断剂PDTC能够部分阻断健脾化湿法对脾虚湿困型UC大鼠结肠组织AQP3、AQP4蛋白及mRNA表达的调控作用。NF-κB与健脾化湿法调控脾虚湿困型UC大鼠结肠组织水通道蛋白有一定相关性。
Background
Therapeutic principles and methods in Chinese medicine have universal guiding significance to prescription, medication and acupuncture.Studies on the material base and mechanism of representative prescription, and endow the methods with new connotation have theoretical significance and scientific value.Dampness disease exists in each system of human. Ulcerative colitis is an undetermined etiology of chronic nonspecific inflammatory bowel disease, and dampness stagnancy due to spleen deficiency is a common pathogenesis. Treatment of strengthening spleen and removing dampness has definite therapeutic effect on common disease in clinic.But the study on mechanism has not been reported before.In recent years,studies have found that aquaporin has close relation with dampness disease.It also has relation with lung, spleen, kidney of traditional Chinese medicine theory.The research on aquaporin has drawn more and more attentions, and is expected to become a new target for clinical treatment of related diseases. The mechanism of pathological changes of damp blockage of middle energizer, dampness-heat of spleen and stomach and syndrome of dampness stagnancy due to spleen deficiency may be explained by further study on aquaporin. In addition, on this basis, there is likely to clarify the pharmacological effects of drugs of dissipating dampness, inducing diuresis and excreting dampness.Therefore,the study on qualitative and quantitative of dampness has important theoretical and clinical significance. Many studies have demonstrated that the aquaporin is closely related to water metabolism disturbance,but the effects on aquaporin of UC with syndrome of dampness stagnancy due to spleen deficiency treated by Jianpihushi and the mechanism of related signal pathway also requires further study.
Objective
The ulcerative colitis rats with syndrome of dampness stagnancy due to spleen deficiency were established that according with the TCM syndrome characteristics.This research investigates the effects on AQP3 and AQP4 in UC rats with syndrome of dampness stagnancy due to spleen deficiency with Shenlinbaizhu powder that the effective prescription of Jianpihuashi,and further explore the role of MAPK and NF-κB signal pathway in the regulation on aquaporin with therapeutic method of Jianpihuashi.This research aims to clarify the part mechanism and pathway of treatment UC of syndrome of dampness stagnancy due to spleen deficiency with the therapeutic method of Jianpihuashi,to promote further study of the theory of water metabolism disturbance in TCM,and to lay a foundation for the studies of molecular biological mechanism on UC with syndrome of dampness stagnancy due to spleen deficiency by the therapeutic method of Jianpihuashi.
Methods
(1)Environmental and dietary factors intervention combined with TNBS and ethanol were used to establish syndrome of dampness stagnancy due to spleen deficiency of ulcerative colitis rats. The symptoms, signs and colon tissue pathological changes were observed in rats with syndrome of dampness stagnancy due to spleen deficiency of ulcerative colitis. And the change of serum EGF, the activities of SOD, MDA and IL-6, IL-8, TNF-a level were detected in model rats and the intervention effect with treatment of strengthening spleen and removing dampness.
(2) The expression changes of AQP3 and AQP4 in rats with syndrome of dampness stagnancy due to spleen deficiency of ulcerative colitis were detected by immunohistochemistry. The effects on expression changes of AQP3 and AQP4 in model rats with treatment of strengthening spleen and removing dampness were detected by immunohistochemistry, Western blot and fluorescence quantitative PCR.
(3) The expressions of phosphorylation ERK, p38MAPK in colon tissue of rats with syndrome of dampness stagnancy due to spleen deficiency of ulcerative colitis were detected by immunohistochemistry. The effects on expression changes of AQP3 and AQP4 in model rats which treatmented by strengthening spleen and removing dampness with U0126 and SB203580,were detected by fluorescence quantitative PCR and Western blot.
(4) The expressions of phosphorylation NF-κB in colon tissue of rats with syndrome of dampness stagnancy due to spleen deficiency of ulcerative colitis were detected by immunohistochemistry.The effects on expression changes of AQP3 and AQP4 in model rats which treatmented by strengthening spleen and removing dampness with PDTC,were detected by fluorescence quantitative PCR and Western blot.
Results
(1)The rats with syndrome of dampness stagnancy due to spleen deficiency of UC appeared symptoms such as lethargy,soft stool,decrease of diet, piloerection, unhairing, diarrhea, walk skew, perianal pollution, narrowing eyes, loss of energy et al.The bloody mucopurulent stool and emaciation appeared after TNBS and ethanol were injected through the anus.The pathological findings of colon shown that mucous hyperemia, edema, plica reduced or even disappeared, ulcer formation, black and yellow of membrane attachment, or the formation of polyps, bowel thickening, bowel wall thinning and tissue adhesion. The colonic tissue damage scores had significant difference compared with normal group (P<0.01).The EGF and SOD of model control group significantly decreased and the MDA content significantly increased, the difference was statistically significant compared with the normal control group (P<0.05 or P<0.01). The EGF level of Jianpihuashi group has significant difference compared with the model control group (P<0.05), the differences of SOD, MDA between two groups are very significant (P<0.01). The EGF, MDA of Sulfasalazine group has significant difference that compared with model control group (P<0.05). The level of serum IL-6, IL-8 and TNF-a in model control group significantly higher than normal control group (P<0.05).The level of serum IL-6, IL-8 and TNF-a in Jianpihuashi group and sulfasalazine group are significant than that in the model group (P<0.05).
(2) The results detected by immunohistochemistry shown that the expression of AQP3 and AQP4 positive cells in the rats with syndrome of dampness stagnancy due to spleen deficiency of UC was less than in normal group, lightly stained, the number of positive cells and the average optical density decreased significantly, which have very remarkable difference compared with the normal control group (P<0.05).The expression of AQP3 and AQP4 in Jianpihuashi group and sulfasalazine group significantly increased and showed significant difference compared with model group (P<0.05). There was significant difference between Jianpihuashi group and sulfasalazine group on the expression of AQP4 (P<0.05).Western blot results showed that the expression of AQP3 and AQP4 in model control group significantly decreased (P<0.05).The expression of AQP3 and AQP4 in Jianpihuashi group and sulfasalazine group significantly increased compared with the model group (P<0.05).FQ-PCR showed that the expression of AQP3 mRNA and AQP4 mRNA in model control group significantly decreased, compared with normal control group, difference was statistically significant (P<0.05).The expression of AQP3 mRNA and AQP4 mRNA in Jianpihuashi group increased significantly that compared with the model group, and there was a significant difference between two groups (P<0.05).
(3)The results detected by immunohistochemistry showed that the expression of ERK and p38 MAPK in the rats with syndrome of dampness stagnancy due to spleen deficiency of UC significantly increased,the difference compared with the normal control group was statistically significant (P<0.05).The expression of ERK and p38 MAPK in Jianpihuashi group have significant difference compared with model group (P<0.05).Western blot results showed that the expression of AQP3 and AQP4 in model control group significantly decreased (P<0.05).The expression of AQP3 and AQP4 in Jianpihuashi group significantly increased compared with the model group (P<0.05). The expression of AQP3 and AQP4 in U0126+treatment group and SB203580+ treatment group have significant difference compared with Jianpihuashi group and normal control group (P<0.05).FQ-PCR results showed that the expression of AQP3 mRNA and AQP4 mRNA in model control group significantly decreased compared with normal control group (P<0.05). The expression of AQP3 mRNA and AQP4 mRNA in Jianpihuashi group significantly increased compared with the model group (P<0.05). The expression of AQP3 mRNA and AQP4 mRNA in U0126+ treatment group and SB203580+treatment group have significant difference compared with Jianpihuashi group and normal control group (P<0.05).
(4) The results detected by immunohistochemistry indicated that the number of positive cells and the average optical density of NF-κB p65 in the rats with syndrome of dampness stagnancy due to spleen deficiency of UC significantly increased, the difference compared with the normal control group was statistically significant (P<0.05). The number of positive cells and the average optical density of NF-κB p65 in Jianpihuashi group were significantly lower than those of model control group (P<0.05). Western blot results showed that the expression of AQP3 and AQP4 in PDTC+treatment group have significant difference compared with Jianpihuashi group and normal control group (P<0.05), while no significant difference compared with the model group (P>0.05).FQ-PCR results also indicated that PDTC could inhibit the regulation effects on UC rats with syndrome of dampness stagnancy due to spleen deficiency with treatment of Jianpihuashi.
Conclusions
(1)Simulation of the etiology of traditional Chinese medicine, the UC rats with syndrome of dampness stagnancy due to spleen deficiency were built by the intervention of environmental and dietary factors combination with enema of TNBS and ethanol, the symptoms and signs that similar to the clinical were appeared, and according with the disease characteristics and TCM syndrome characteristics of UC,which is an ideal rat model of UC with syndrome of dampness stagnancy due to spleen deficiency.Therapeutic method of Jianpihuashi could improve the levels of EGF, SOD,MDA and IL-6,IL-8,TNF-a.
(2) The expressions of AQP3 and AQP4 in the UC rats with syndrome of dampness stagnancy due to spleen deficiency were significantly decreased.Therapeutic method of Jianpihuashi could significantly improve the expression of AQP3,AQP4 and AQP3 mRNA, AQP4 mRNA in the UC colon tissue, and promote the repair of the intestinal mucosa.
(3) The expressions of ERK and p38 MAPK in the UC rats with syndrome of dampness stagnancy due to spleen deficiency were significantly increased.Therapeutic method of Jianpihuashi could significantly decrease the expression of ERK and p38 MAPK in the UC colon tissue.And therapeutic method of Jianpihuashi could significantly improve the protein and mRNA expression of AQP3,AQP4,which could be inhibited by U0126 and SB203580 to a certain extent. The effects on AQP of UC rats with syndrome of dampness stagnancy due to spleen deficiency with therapeutic method of Jianpihuashi have a relationship with ERK and p38 MAPK.
(4) The expressions of NF-κB p65 in the UC rats with syndrome of dampness stagnancy due to spleen deficiency increased significantly. Therapeutic method of Jianpihuashi could significantly improve the expression of NF-κB p65 in the UC colon tissue. NF-κB blocker PDTC could partially block the effects on the protein and mRNA expression of AQP3,AQP4 of UC rats with syndrome of dampness stagnancy due to spleen deficiency with therapeutic method of Jianpihuashi. The effects on AQP of UC rats with syndrome of dampness stagnancy due to spleen deficiency with therapeutic method of Jianpihuashi have a relationship with NF-κB signal pathway.
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