基于NaCl重吸收抑制作用的石韦利尿活性成分筛选

Screening of active components from Pyrrosia petiolosa based on inhibition of NaCl reabsorption

目的建立基于抑制NaCl重吸收作用的利尿药物体外筛选模型,研究21个从石韦中分离的化合物的利尿活性,并通过体内实验进行验证。方法采用犬肾小管上皮细胞MDCK对石韦中21个化合物进行体外利尿活性筛选,确定NaCl体外转运实验的最佳NaCl剂量;将MDCK细胞接种于Transwell小室内,设置对照组、石韦化合物(100μmol/L)组、氢氯噻嗪(100μmol/L)组,给药孵育24 h后,加入NaCl溶液(15 mg/mL),于30 min、2 h、4 h分别取Transwell下室培养液,检测Cl-和Na+吸光度。昆明小鼠随机分为对照组、绿原酸甲酯(10 mg/kg)组、山柰酚(10 mg/kg)组、氢氯噻嗪(10 mg/kg)组,给予药物干预5 d后,检测各组小鼠5 h内的尿量。结果与对照组比较,加入NaCl 30 min,氢氯噻嗪组MDCK细胞Cl-和Na+水平显著降低(P<0.05),绿原酸甲酯组MDCK细胞Na+水平显著降低(P<0.05);加入NaCl 2h,绿原酸甲酯组MDCK细胞Cl-水平显著降低(P<0.05)。与对照组比较,绿原酸甲酯组和氢氯噻嗪组小鼠给药后5 h内的总尿量显著增加(P<0.05);氢氯噻嗪组和绿原酸甲酯组小鼠尿量峰值分别为给药后1.5、2.5h,随后尿量均呈下降趋势。结论绿原酸甲酯可抑制肾小管氯化钠转运,对小鼠具有利尿作用,是石韦中的利尿活性成分。

Objective To establish an in vitro screening model of diuretic drugs based on the inhibition of NaCl reabsorption, study and verify the diuretic activity of 21 compounds isolated from Pyrrosia petiolosa. Methods Canine renal tubular epithelial MDCK cell was used to screen for diuretic activity of 21 compounds isolated from P. petiolosa in vitro to determine the optimal NaCl dose for the transport experiment of NaCl;MDCK cells were inoculated into Transwell chamber, and control group;Compound from P. petiolosa(100 μmol/L) group and hydrochlorothiazide(100 μmol/L) group were set up, after 24 h of incubation, NaCl solution(15 mg/mL) was added, the Transwell lower chamber culture medium was respectively taken at 30 min, 2 h, and 4 h, and the Cl- and Na+ absorbance were measured. KM mice were randomly divided into control group, methyl chlorogenate(10 mg/kg) group, kaempferol(10 mg/kg) group, and hydrochlorothiazide(10 mg/kg) group. After drug intervention for 5 d, urine volume of mice in each group was tested within 5 h. Results Compared with control group, after adding NaCl for 30 min, the levels of Cl- and Na+ in MDCK cells of hydrochlorothiazide group were significantly reduced(P < 0.05), and the levels of Na+ in MDCK cells in methyl chlorogenate group were significantly reduced(P < 0.05);After adding NaCl for 2 h, the level of Cl- in MDCK cells in methyl chlorogenate group was significantly reduced(P < 0.05). Compared with control group, the total urine output of mice in methyl chlorogenate group and hydrochlorothiazide group were significantly increased within 5 h after administration(P < 0.05);The peak urine output of the mice in hydrochlorothiazide group and methyl chlorogenate group were respectively 1.5 h and 2.5 h after administration, the urine output showed a downward trend. Conclusion Methyl chlorogenate, the diuretic active ingredient in P. petiolosa, could inhibit the transport of sodium chloride in the renal tubules and has a diuretic effect on mice.