Regulating Effects And Mechanisms Of AQP2, AQP4 Expression In LoVo And NRK Cells By Total Anthraquinone In Rheum And Its Derivatives

It is recorded by Chinese Medical Literature, besides its decanta effect, Rhubarb also has strong diuresis effect. Aquaporines (AQPs) are one group of conveying membrane proteins associated with water permeability which affects the transmembrane aquatic transport. Among all the Aquaporines, AQP2 and AQP4 are confirmed to be expressed in colon and kidney, and the decanta and diuresis effects of Rhuarb were found to be associated with its regulation of AQP2, AQP4 expression in colon and kidney by tentative confirmation.Our research focused on the LoVo cells oringinated from human colon cancer and NRK cells oringinated from normal rat kidney cells as target cells. We observed the regulation effect of Total Anthraquinone in Rheum and its derivatives on the expression of AQP2 and AQP4 in LoVo and NRK cells in vitro. First we treated SD rats with different dosages of Total Anthraquinone in Rheum by intragastric administration, and the serum containing Total Anthraquinone in Rheum was prepared for culturing LoVo and NRK cells. The expression of AQP2 and AQP4 were tested by immunofluorescence, western blot and RT-PCR to confirm the regulaion effect. Then we tested the regulation of chrysophanol on the expression of AQP2, 4 in LoVo cells to consummate the previous research. Finally, we investigated the regulation effect of emodin,rhein,chrysophanol on the expression of AQP2, 4 in NRK cells.ExperimentⅠRegulating effects and mechanisms of AQP2, 4 expression in LoVo cells by Total Anthraquinone in RheumObjective: To investigate the regulating effects and mechanisms of serum containing on the expression of AQP2, 4 in cultured LoVo cells in vitro. Methods: 16 SD rats were randomly divided into 4 groups respectively treated with 0, 1400, 2500 and 4500㎎·㎏^-1·d^-1 of Total Anthraquinone in Rheum by intragastric administration. After 7 days of treatment, the rats were anesthetized and celiotomized to prepare the serum containing Total Anthraquinone in Rheum. LoVo cells cultured in vitro were treated with different concentrations of Total Anthraquinone in Rheum for 24 hrs.The localization of AQP2, 4 were tested by immunofluorescence techniques. The expression levels of protein and mRNA of AQP2, 4 were decided by Western Blot and semiquantive RT-PCR. Test the activity of PKA after 24 hrs of pharmaceutically treated LoVo cells with non-radioactive detection method. Results:AQP2, 4 is located at the cell membrane of LoVo cells. Western Blot and semiquantive RT-PCR showed that serum containing rhubarb prepared from rats treated with 4500㎎·㎏^-1·d^-1 of Total Anthraquinone in Rheum could inhibit the expression of mRNA and protein levels of AQP2, 4(P<0.01). The expression level of PKA significantly decreased in LoVo cells of 4500㎎·㎏^-1·d^-1 of Total Anthraquinone in Rheum group(P<0.05).ExperimentⅡRegulating effects and mechanisms of AQP2, 4 expression in NRK cells by Total Anthraquinone in RheumObjective: To investigate the regulating effects and mechanisms of serum containing total anthraquinone in rheum on the expression of AQP2, 4 in cultured NRK cells in vitro. Methods: 16 SD rats were randomly divided into 4 groups respectively treated with 0, 1400, 2500 and 4500㎎·㎏^-1·d^-1 of total anthraquinone in rheum by intragastric administration. After 7 days of treatment, the rats were anesthetized and celiotomized to prepare the serum containing total anthraquinone in rheum. NRK cells cultured in vitro were treated with serum containing total anthraquinone in rheum for 24 hrs. The localization of AQP2, 4 were tested by immunofluorescence techniques. The expression levels of protein and mRNA of AQP2,4 were decided by Western Blot and semiquantive RT-PCR. Test the activity of PKA after 24 hrs of pharmaceutically treated NRK cells with non-radioactive detection method. Results:AQP2, 4 is located at the cell membrane of NRK cells. Administration of 2500,4500㎎·㎏^-1·d^-1 of culture medium with serum containing total anthraquinone in rheum can inhibit the protein and mRNA levels of AQP2,4 in NRK cells, which were significantly different from control group (P<0.05). The expression level of PKA significantly decreased in NRK cells of 4500㎎·㎏^-1·d^-1 of Total Anthraquinone in Rheum group(P<0.05).ExperimentⅢRegulating effects and mechanisms of AQP2, 4 expression in LoVo cells by chrysophanolObjective: To investigate the regulating effects and mechanisms of chrysophanol on the expression of AQP2, 4 in cultured LoVo cells in vitro.Methods: LoVo cells cultured with RPMI-1640 medium in vitro were divided into two steps. The first step was randomly divided into 4 groups: control group, 10, 20, 40 mg/L chrysophanol groups. The expression levels of protein and mRNA of AQP2, 4 after 24hrs of treatment were decided by Western Blot and semiquantive RT-PCR; The second step was randomly divided into control group, 10 mg/L 8-Bromo-cAMP group, 40 mg/L chrysophanol group and 40 mg/L chrysophanol together with 10 mg/L 8-Bromo-cAMP group. Test the activity of PKA after 24 hrs of pharmaceutically treated LoVo cells with non-radioactive detection method. Results:Administration of 20, 40 mg/L chrysophanol groups can inhibit the protein and mRNA levels of AQP2,4 in LoVo cells, which were significantly different from control group (P<0.01). The expression level of PKA significantly decreased in LoVo cells of 40 mg/L chrysophanol group (P<0.05).ExperimentⅣRegulating effects and mechanisms of AQP2, 4 expression in NRK cells by emodin or rhein or chrysophanolObjective: To investigate the regulating effects and mechanisms of emodin or rhein or chrysophanol on the expression of AQP2, 4 in NRK cells in vitro. Methods: NRK cells cultured withα-DMEM medium in vitro were divided into two steps. The first step was randomly divided into 4 groups: control group, 5, 10, 20 mg/L emodin or rhein or chrysophanol groups. The expression levels of protein and mRNA of AQP2, 4 after 24hrs of treatment were decided by Western Blot and semiquantive RT-PCR; The second step was randomly divided into control group, 10 mg/L 8-Bromo-cAMP group, 20 mg/L emodin or rhein or chrysophanol group and 20 mg/L emodin or rhein or chrysophanol together with 10 mg/L 8-Bromo-cAMP group. Test the activity of PKA after 24 hrs of pharmaceutically treated NRK cells with non-radioactive detection method. Results:With the results of Western Blot and semiquantitive RT-PCR, we found AQP2, 4 protein and mRNA expression were significantly decreased in NRK cells of 10, 20 mg/L emodin or rhein or chrysophanol treated groups (P<0.05).There aren't significantly different among emodin, rhein and chrysophanol (P>0.05). The expression level of PKA significantly decreased in NRK cells of 20 mg/L emodin or rhein or chrysophanol group (P<0.05).Conclusions:1. Serum containing total anthraquinone in rheum and chrysophanol can inhibit the genetic transcription and the translation of AQP2, 4 genes in LoVo cells;It is inferred that Serum containing total anthraquinone in rheum and chrysophanol can increase the water content in colon through inhibiting AQP2, 4 expression in colonic endothelial cell, which demonstrates that the regulation of AQP expression may be corrected with the cathartic effect of rhubarb.2. Serum containing total anthraquinone in rheum, emodin, rhein and chrysophanol can inhibit the genetic transcription and the translation of AQP2, 4 genes in NRK cells;It is inferred that Serum containing total anthraquinone in rheum, emodin, rhein and chrysophanol can inhibit the water reabsorb in kidney through inhibiting AQP2, 4 expression in renal endothelial cell, which demonstrates that the regulation of AQP expression may be corrected with the diuresis effect of rhubarb.3. It is likely that the expression of AQP2 is regulated by rhubarb through PKA signal pathway. 4. The regulation of AQP2 and AQP4 expression in colon and kidney by Total Anthraquinone in Rheum and its derivatives indicate that Rhubarb can regulate the water metabolism of both colon and kidney, which might contribute to the mechanism of Rhubarb's multiply effects.