Effect Of Qiliqiangxin Capsule On AQP2 Expression In IMCD3 Cells And Its Mechanism

BackgroundChronic heart failure (CHF) is a serious threat to human health. Water retention is one of the most important pathological changes in chronic heart failure, and it is the direct cause of cardiac edema. Cardiac edema can further aggravate the degree of impairment of cardiac function.Aquaporin-2(AQP2) mainly exists in the renal collecting tube, which plays an important role in regulating water metabolism and electrolyte balance. The abnormal increase of AQP2 is an important reason for the poor prognosis of chronic heart failure caused by water fluid retention. Therefore, AQP2, as a new therapeutic target for the treatment of water imbalance, has attracted wide attention, and has become an important breakthrough point in the treatment of cardiac edema. Our previous study found that Qiliqiangxin capsule can inhibit the expression of AQP2, reduce the renal collecting duct reabsorption of water, so as to improve the water retention in chronic heart failure rats. This experiment further study of Qiliqiangxin capsule on expression of AQP2 induced by dDAVP in IMCD3 cells and its mechanism.ObjectiveTo investigate the effect of QLQX Capsule on the expression of AQP2 and expression mechanism and provide the experimental basis for the clinical treatment of chronic heart failure.Methods1.IMCD3 cell line was cultured in vitro,the cell proliferation activity was detected by CCK8 kit and the concentration of DDAVP were screened. The relative expression of AQP2 induced by dDAVP was detected by Western blot and the most appropriate concentration in subsequent experiments.2.Dissolve the QLQX capsule in the complete medium. Preparation of different concentration of QLQX solution,500ug/ml,750μg/ml, 1000μug/ml,1500ug/ml. The drug concentration, which had no significant effect on cell proliferation activity, was detected by CCK8 kit. To explore the effect of QLQX capsule on different concentrations of dDAVP induced expression of AQP2 protein in IMCD3 cells by Western blot detection.3.(1) Set the control group (blank), model group (dDAVP) and dosing groups (Low, medium and high concentration of QLQX capsules), After the cultivation of 24h, ELISA was used to detect the level of cAMP and Western blot was used to detect the expression of p-ERK1/2, Epacl. (2) Set the control group 1 (blank), model groupl (dDAVP), control group2 (8-CPT), model group2 (dDAVP+8-CPT), dosing groups (dDAVP+8-CPT+Low, medium and high concentration of QLQX capsules). After the cultivation of 24h, the Western blot experiment was conducted to test the expression of p-AQP2 and p-ERK1/2.Results1.The effect of different concentration gradient dDAVP on the proliferation activity of IMCD3 cells was not statistically significant (P> 0.05).2.Compared with the control group, the expression of AQP2 of dosing group is significantly higher when the dDAVP concentration in 10-7M,10-8M,10-9M,10010M (P< 0.05).What’s more, the expression of AQP2 was most significant in 10-9M, which could be used to build the model group.3.The effect of QLQX capsule at the concentration of 500ug/ml,750ug/ml, 1000μg/ml on cell proliferation activity was not statistically significant (P> 0.05). The drug concentration of 1500ug/ml has a significant inhibitory effect on cell proliferation, with statistical significance (P＜0.05).4.The data showed that the low, medium and high dose group of QLQX capsule all had inhibitory effect on the expression of AQP2 protein in IMCD3 cells induced by dDAVP (P<0.05). Inhibitory effect of QLQX capsule in the higher concentration on AQP2 expression is more obvious.5.Compared with the control group, dDAVP can obviously improve cAMP level in IMCD3 cells (P< 0.05)and QLQX solution can effectively reduce the level of cAMP (P< 0.05).6.dDAVP can obviously increase the expression of Epacl and p-ERK (p< 0.05). QLQX can reduce the expression of Epacl when the concentration in 750μg/ml, 1000μg/ml (P<0.05). Under the action of different concentrations of QLQX,the expression of p-ERK 1/2 were significantly decreased(P<0.05).7.After adding agonist 8-pCPT-2’-O-Me-cAMP, both the control group and the model group, the protein expression of p-ERK and p-AQP2 increased significantly, with statistical significance (p<0.05).And the protein expression of p-ERK and p-AQP2 was significantly down regulated by the QLQX capsule (P＜0.05)Conclusion1.DDAVP can induce the increase of AQP2 expression in IMCD3 cells.2.Qiliqiangxin capsule can effectively inhibit the expression of AQP2 induced by dDAVP.3.Qiliqiangxin capsule can be effectively regulated by cAMP/Epac/ERK signaling pathway on the expression of AQP2.