齐墩果酸对脾虚水湿内停模型小鼠异常水液代谢的影响

Effect of Oleanolic Acid on Abnormal Water Metabolism of Mice with Water-dampness Retention Caused by Spleen Deficiency

目的:研究齐墩果酸(OA)对脾虚水湿内停模型小鼠的水液代谢功能的影响及其作用机制。方法:将60只SPF级昆明种小鼠随机分为空白组(10只)和造模组(50只),造模组采用“久居湿地+饮食失节”的方法建立脾虚水湿内停模型。模型复制成功后将造模组小鼠随机分为模型组、自然恢复组和OA低、中、高剂量组,共5组,每组10只。空白组、模型组、自然恢复组给予10 mL·kg^(-1)·d^(-1)生理盐水灌胃,OA低、中、高剂量组分别给予50、100、200 mg·kg^(-1)·d^(-1)OA灌胃,持续给药7 d。造模和给药前后观察小鼠的一般体征,测定小鼠体质量,使用烘干法测定小鼠粪便含水率和组织含水量,称量法检测水负荷指数和脏器系数,酶联免疫吸附测定法(ELISA)检测尿D-木糖排泄量、血清胃泌素(GAS)、总蛋白(TP)、白蛋白(ALB)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、白细胞介素-6(IL-6)、抗利尿激素(AVP)、肾髓水通道蛋白1(AQP1)及肝脏Na^(+)-K^(+)-ATP酶等指标,并将OA与检测指标中的ALB、IL-6、AQP1和Na^(+)-K^(+)-ATP酶进行分子对接。结果:与空白组比较,模型组小鼠毛发疏松、消瘦、倦怠懒惰、动作迟缓,体质量增长缓慢、自主活动次数减少、粪便含水率和组织含水量增加、水负荷小鼠体质量下降值减少、各脏器系数明显升高(P<0.05,P<0.01);尿D-木糖排泄量明显减少,血清中GAS、TP、ALB和HDL-C的水平均明显降低,TC、LDL-C、AVP、IL-6的水平均明显升高,肝脏Na^(+)-K^(+)-ATP酶表达明显降低,肾髓AQP1表达明显升高(P<0.05,P<0.01)。与模型组比较,OA各剂量组的一般体征明显好转,体质量增长加快,自主活动次数增加,粪便含水率和组织含水量降低,水负荷小鼠体质量下降值增加、各脏器系数明显降低(P<0.05,P<0.01);尿D-木糖排泄量增加,血清中GAS、TP、ALB和HDL-C的水平升高,TC、LDL-C、AVP、IL-6的水平降低,肝脏Na^(+)-K^(+)-ATP酶表达明显升高,肾髓AQP1表达明显降低(P<0.05,P<0.01)。OA各剂量组的各指标恢复效果均强于自然恢复组。分子对接结果也表明OA与ALB、IL-6、AQP1和Na^(+)-K^(+)-ATP酶具有较强的结合亲和力。结论:OA能改善脾虚水湿内停小鼠的水液代谢异常,为其潜在临床新用途提供科学依据。

Objective:To explore the effect of oleanolic acid(OA)on water metabolism in mice with water-dampness retention caused by spleen deficiency and the mechanism.Method:The 60 SPF Kunming(KM)mice were randomized into blank group(n=10)and modeling group(n=50).Through long-term living in damp place and irregular diet,water-dampness retention caused by spleen deficiency was induced in modeling mice.Then the model mice were randomly classified into model group,natural recovery group,and low-dose,medium-dose,and high-dose OA groups.The mice in the blank group,model group,and natural recovery group were given(ig)10 mL·kg^(-1)·d^(-1)normal saline,and mice in the low-dose,medium-dose,and high-dose OA groups received 50,100,200 mg·kg^(-1)·d^(-1)OA,respectively.The intervention lasted 7 days.Before and after modeling and administration,the general conditions of the mice were observed and body weight of mice was measured.The water content in feces and tissues was detected with the oven-drying method,and water load index and organ coefficient were measured with the weighing method.Enzyme-linked immunosorbent assay(ELISA)was employed to detect the urinary D-xylose excretion,serum gastrin(GAS),total protein(TP),albumin(ALB),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),interleukin-6(IL-6),antidiuretic hormone(AVP),aquaporin 1(AQP1)in renal medulla,and liver Na^(+)-K^(+)-ATPase.At the same time,OA was docked with ALB,IL-6,AQP1,and Na^(+)-K^(+)-ATPase.Result:Compared with the blank group,the model group showed withered hair,emaciation,laziness,bradykinesia,slow weight growth,infrequent spontaneous activities,high water content in feces and tissues,low weight loss after water loading,high coefficient of each organ(P<0.05,P<0.01).Moreover,the model group had less urinary D-xylose excretion,lower serum levels of GAS,TP,ALB,and HDL-C,higher levels of TC,LDL-C,AVP,and IL-6,lower expression of Na^(+)-K^(+)-ATPase in the liver,and higher expression of AQP1 in renal medulla than the blank group(P<0.05,P<0.01).The three OA groups demonstrated better general conditions,faster weight gain,more frequent spontaneous activities,lower water content in feces and tissues,larger weight loss after water loading,and lower coefficient of each organ than the model group(P<0.05,P<0.01).Moreover,compared with the model group,the three OA groups had high D-xylose excretion,high serum levels of GAS,TP,ALB,and HDL-C,low serum levels of TC,LDL-C,AVP,and IL-6,high expression of Na^(+)-K^(+)-ATPase in liver,and low expression of AQP1 in renal medulla(P<0.05,P<0.01).The recovery in each OA group was better than that in natural recovery group.Molecular docking results also confirmed that OA had high binding affinity with ALB,IL-6,AQP1,and Na^(+)-K^(+)-ATPase.Conclusion:OA can alleviate the abnormal water metabolism in mice with water-dampness retention caused by spleen deficiency,which lays a basis for its potential clinical application.