RESUMO
BACKGROUND Shen Qi Wan (SQW) as a well-known formula for the amelioration of kidney yang deficiency syndrome (KYDS), and it has been widely employed in traditional Chinese medicine (TCM). This study aimed to investigate the effect and underlying mechanism of SQW medicated serum on proliferation and migration in NRK-52E cells. MATERIAL AND METHODS We employed the real-time cell analysis (RTCA) system to investigate the effect of SQW medicated serum on proliferation and migration in NRK-52E cells. In addition, the migration was further investigated by using a wound-healing assay. The mRNA and protein expression level of aquaporin 1 (AQP1) of NRK-52E cells with SQW medicated serum-treated were quantified by real-time quantitative polymerase chain reaction (q-PCR) and western blot assay, respectively. Furthermore, NRK-52E cells were transfected with lentivirus AQP1-RNAi to assess migratory cell abilities in vitro. RESULTS The migratory abilities of NRK-52E cells were significantly increased after SQW medicated serum treatment (P<0.05), and no significant difference in cell proliferation. In addition, SQW medicated serum was significantly upregulated the mRNA and protein expression level of AQP1 in NRK-52E cells (P<0.05). Additionally, the in vitro metastasis test proved that knockdown of AQP1 suppressed migratory abilities according to RTCA and wound healing test while was reversed by SQW medicated serum (P<0.05). CONCLUSIONS Our study demonstrates that SQW medicated serum effectively promotes the migration of NRK-52E cells by increasing AQP1 expression, and AQP1 may be as a therapeutic target of SQW for renal injury treatment under KYDS.
Assuntos
Aquaporina 1/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Nefropatias/tratamento farmacológico , Deficiência da Energia Yang/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Aquaporina 1/biossíntese , Aquaporina 1/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Terapia de Alvo Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Deficiência da Energia Yang/genética , Deficiência da Energia Yang/metabolismo , Deficiência da Energia Yang/patologiaRESUMO
This study was aimed to investigate the effect and mechanism of Zhenwu Tang on AVP-V2R-AQP2 pathway in NRK-52E cells in vitro. Forty eight male SD rats were randomly divided into eight groups with 6 animals in each group. Distilled water or 22.68 g·kg⻹·d⻹ Zhenwu Tang(calculated by raw drug dosage meter) was given by gavage. Blood samples were collected by cardiac punctureï¼ and the medicated serum was centrifuged from the blood by 3 000 r·min⻹. NRK-52E cells were treated with different medicated serum or dDAVP. The condition of cell proliferation was detected by RTCA. The distribution of V2R and AQP2 in cells were detected by immunofluorescence. The expression of V2Rï¼ PKA and AQP2 were detected by Western blot and AQP2 mRNA level was detected by real-time PCR. Results showed that the level of AQP2 mRNA(P<0.01) and protein expression of V2Rï¼ PKA and AQP2(P<0.05ï¼ P<0.01ï¼ P<0.05) of Z7d group which was treated with Zhenwu Tang medicated serum for 24 h were significantly higher than that of normal rat serum group. And the expression level of V2Rï¼ p-AQP2 and AQP2(P<0.01ï¼ P<0.05ï¼ P<0.01) of Z7d+dDAVP group were significantly increased comparing to normal rat serum group. The results indicate that the applying of Zhenwu Tang medicated serum could increase the expression level of V2Rï¼ PKA and AQP2 which exist in AVP-V2R-AQP2 pathway in NRK-52Eï¼ and there is synergistic effect between Zhenwu Tang medicated serum and dDAVP. So the pathway of AVP-V2R-AQP2 may be one of the mechanism for which Zhenwu Tang regulate balance of water transportation.