Effects And Mechanisms Of Anthraxquinone Derivatives In Rhubarb On Aquaporins Expression In Rat Colon And In LoVo Cells

Rhubarb, famous for its outstanding cathartic effect, is prescribed widespread in clinic. The main active ingredients in rhubarb that bring about cathartic effect are anthraquinone derivatives in rhubarb and emodin, rhein belong to these active ingredients. Previous studies have confirmed that rhubarb could inhibit the water absorption from colonic enteric cavity and increase the water content in enteric cavity so as to bring about cathartic effect. Aquaporins (AQPs) discovered in the past few years belong to membrane protein families, which take responsibility of water transport. It was determined that aquaporin2 (AQP2) and aquaporin4 (AQP4) were expressed in colon and played important roles in water absorption. However it is not definite whether anthraquinone derivatives in rhubarb play cathartic effect through regulating AQP2 and AQP4 expression in colon epithelial cells and affecting water absorption from colonic enteric cavity.To begin with, the cathartic effect of total anthraquinones in rhubarb in SD rats and effects on AQP4 expression in rat colon were investigated. Moreover, LoVo cells, which origins from human colonic adenocarcinoma, were used as target cells in this study and the expression of AQP2 and AQP4 were detected with method of immuocytochemistry (ICC). To move forward, the regulative effects of emodin or rhein on AQP2, AQP4 expression in LoVo cells were explored with methods of western blot (WB) and RT-PCR. In the last of this study, the cell signaling passageway through which emodin down-regulated AQP4 expression was investigated by observing the change of PKA activity in LoVo cells cultured with emodin and/or excitomotor. Our main observations were reported as following.ExperimentⅠEffects of total anthraquinones in rhubarb on AQP4 expression in rat colonObjective: To investigate the cathartic effect of total anthraquinones in rhubarb in rats and the regulative effects on AQP4 expression in rat colon. Methods: 24 SD rats were randomly divided into Control group, Cathartic dose group and High dose group and were given intragastric administration in dose of 0 mg/(kg·d), 2500 mg/(kg·d), 4500 mg/(kg·d) respectively. After 5 d of treatment, stool water content in full length colon was detected and the expression of AQP4 in rat proximal colon was measured with WB and RT-PCR. Results: Stool water content in rats of Cathartic dose group and High dose group was higher than that of Control group, and the AQP4 expression decreased in Cathartic dose group(P<0.05) and in High dose group( P<0.01). ExperimentⅡEffects of emodin/rhein on AQP4 expression in LoVo cells in vitro Objective: To investigate the regulative effects of rhein/emodin on AQP4 in LoVo cells in vitro. Methods: LoVo cells were maintained with RPMI-1640 medium and treated with RPMI-1640 medium containing different concentration rhein/emodin for 24 h and were cultured with RPMI-1640 medium containing rhein/emodin (20 mg/L) for different time. The expressional location of AQP4 protein in LoVo cells was detected with method of ICC, and then WB and RT-PCR were adopted to detect AQP4 expression in LoVo cells. Results: AQP4 protein was mainly expressed in cytomembrane and partly expressed in cytoplasm of LoVo cells. The expression of AQP4 decreased with the increasing of rhein/emodin concentration and the AQP4 expression was attenuated gradually with the prolong of action time.ExperimentⅢEffects of emodin or rhein on AQP2 expression in LoVo cells in vitroObjective: To investigate the regulative effects of emodin or rhein on AQP2 expression in LoVo cells in vitro. Methods: LoVo cells were maintained and then were treated with RPMI-1640 culture medium containing different concentration emodin/rhein for 24 h or with RPMI-1640 culture medium containing emodin/rhein (20 mg/L) for different time. There were high dose group(emodin/rhein, 40 mg/L), midst dose group(emodin/rhein, 20 mg/L), low dose group(emodin/rhein, 10 mg/L) and control group in the first test while there were 0 h group, 3 h group, , 6 h group, 12 h group, 24 h group, 48 h group in the second test. The expressional location of AQP2 protein in LoVo cells was detected with method of ICC, and the expression of AQP2 protein and mRNA was detected with WB and RT-PCR respectively. Results: AQP2 protein was expressed in cytomembrane of LoVo cells. Emodin and rhein could inhibit the AQP2 expression in LoVo cells. In the aspect of dose-effect, the expression of AQP2 protein and mRNA continuously decreased and contrast with control group, AQP2 protein decreased in midst dose group and high dose group significantly(P<0.05; P<0.01); AQP2 mRNA decreased in high dose group and midst dose group significantly(midst dose group of rhein: P<0.05; the others: P<0.01). In the time-effect aspect, the AQP2 expression was so low that the values were not obtained. AQP2 protein increased in 3 h and 6 h groups insignificantly, however it decreased in the other groups(12 h, 24 h, 48 h groups of emodin: P<0.01; 24 h group of rhein: P<0.05). AQP2 mRNA decreased in all groups of emodin(P<0.01) while it decreased significantly in 12 h, 24 h groups of rhein(P<0.05; P<0.01).ExperimentⅣEmodin down-regulated the AQP4 expression in LoVo cells by inhibiting PKA activity in vitroObjective: To investigate the cell signaling passageway through which emodin down-regulated the AQP4 expression by observing the change of PKA activity in LoVo cells. Methods: LoVo cells were cultured with RPMI-1640 medium containing emodin (20 mg/L), 8-Bromo-cAMP (100μmol/L), both emodin (20 mg/L) and 8-Bromo-cAMP (100μmol/L) for 24 h. The activity of PKA in LoVo cells was detected with Non-Radioactive Protein Kinase Assays and the AQP4 expression was exammed with WB. Results: 8-bromo-cAMP, which can preferentially activate PKA, increased both AQP4 protein expression and the PKA activity significantly. The regulation of emodin on AQP4 and PKA activity in LoVo cells showed the opposite effects of 8-bromo-cAMP. When LoVo cells were affected together with emodin and 8-bromo-cAMP, 8-bromo-cAMP could extenuate the down-regulation of emodin on AQP4 and PKA activity.Conclusions: 1. Total anthraquinones in rhubarb could increase the water content in colon and inhibit AQP4 expression in rat colon significantly so as to bring about cathartic effect;2. AQP2 and AQP4 were expressed in cytomembrane of LoVo cells; LoVo cells could be used as target cells to investigate the effects of drugs on AQP2 and AQP4 expression; emodin, rhein could inhibit the genetic transcription and translation of AQP2 and AQP4 significantly; it is inferred that emodin, rhein can increase the water content in colon through inhibiting AQP2 and AQP4 expression in colonic endothelial cell so that cathartic effect takes place;3. PKA may participate these regulative effects of emodin on AQP4 in LoVo cells; which suggests that the inhibition of emodin on PKA activity may be one of action mechanisms through which emodin down-regulates AQP4 expression in LoVo cells;4. This study has illuminated the regulative effects of anthraquinone derivatives in rhubarb on AQP2 and AQP4 expression in rat CEC in vivo and in LoVo cells in vitro, and cell signaling molecule through which emodin down-regulates AQP4 in LoVo cells; this study provides a novel insight to explore the pharmacy-mechanisms of rhubarb playing cathartic effect by investigating AQPs expression.