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Study of jianpihuashi on splenasthenic Hygrosis type ulcerative colitis rat aquaporin 3, 4 expression and its mechanism

Author: LiZiHui
Tutor: WangJian
School: Beijing University of Traditional Chinese Medicine
Course: Basic Theory of Traditional Chinese Medicine
Keywords: AQP3 AQP4 MAPK NF-κB Therapeutic method of Jianpihuashi Syndrome of dampness stagnancy due to spleen deficiency Ulcerative colitis
CLC: R259
Type: PhD thesis
Year: 2012
Downloads: 394
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Abstract

BackgroundTherapeutic 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.ObjectiveThe 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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