有柄石韦中坝巴酸的通淋作用研究

Leaching effect of babaric acid in Pyrrosia petiolosa

目的研究黔产有柄石韦Pyrrosia petiolosa中坝巴酸的通淋作用。方法犬肾小管上皮细胞(MDCK)给予100μmol/L坝巴酸孵育24 h,MTT法检测坝巴酸对MDCK细胞活力以及脂多糖诱导的MDCK炎症模型细胞活力的影响。雄性昆明种小鼠随机分为正常组、模型组及坝巴酸低、高剂量(10、20 mg/kg)组和肾石通(4.5 g/kg)组,除正常组外,其余各组每日ig 1%乙二醇和1%NH_(4)Cl连续造模30 d,造模同时ig药物,末次给药后取小鼠尿液和肾脏,采用苏木素-伊红(HE)染色法检测肾组织病理变化;采用钙离子检测试剂盒检测尿钙、肾钙含量。利用Auto Dock软件对坝巴酸和过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorγ,PPARG)、WNK赖氨酸缺乏蛋白激酶1(WNK lysine deficient protein kinase 1,WNK1)、钙感应受体(calcium sensing receptor,CASR)、细胞色素P450家族24亚家族A成员1(cytochrome P450 family 24 subfamily A member 1,CYP24A1)蛋白进行分子对接并分析对接结果。结果体外实验结果显示,与正常组相比,100μmol/L坝巴酸对MDCK细胞活力无明显影响;在30 mg/mL脂多糖诱导的炎症细胞模型中,坝巴酸可以显著提高MDCK细胞活力(P<0.05)。体内实验结果显示,各给药组小鼠肾组织炎性细胞浸润得到改善,肾组织损伤评分显著降低(P<0.05);坝巴酸低剂量组和肾石通组小鼠肾钙含量较模型组显著降低(P<0.05),尿钙含量显著升高(P<0.05)。分子对接结果显示,PPARG、WNK1、CASR、CYP24A1蛋白与坝巴酸的分子对接能均小于-21 kJ/mol,对接效果均较好。结论黔产有柄石韦中坝巴酸具有较好的抗炎、抑制肾钙蓄积的作用,其通淋活性机制与PPARG、WNK1、CASR、CYP24A1蛋白的结合活性有关。

Objective To study the leaching effect of babaric acid in Pyrrosia petiolosa in Guizhou.Methods Canine renal tubular epithelial cells MDCK were incubated with 100μmol/L babaric acid for 24 h.MTT method was used to detect the effects of babaric acid on viability of MDCK cells and cell viability of MDCK inflammation model induced by lipopolysaccharide.Male Kunming mice were randomly divided into normal group,model group,low-,high-dose(10,20 mg/kg)babaric acid groups and Shenshitong(4.5 g/kg)group.Mice except normal group were daily ig 1%ethylene glycol and 1%NH_(4)Cl for 30 d to continue to model,at the same time mice were ig drugs,after the last administration,urine and kidney of mice were taken,hematoxylin-eosin(HE)staining method was used to detect the pathological changes of kidney tissue;Calcium ion detection kit was used to detect urinary calcium and renal calcium contents.Babaric acid and peroxisome proliferator activated receptorγ(PPARG),WNK lysine deficient protein kinase 1(WNK1),calcium sensing receptor(CASR)and cytochrome P450 family 24 subfamily A member 1(CYP24A1)protein were molecularly docked by Auto Dock software and docking results were analyzed.Results The results of in vitro experiments showed that compared with normal group,100μmol/L babaric acid had no significant effect on MDCK cell viability;In 30 mg/mL lipopolysaccharide-induced inflammatory cell model,babaric acid significantly increased MDCK cell viability(P<0.05).The results of in vivo experiments showed that inflammatory cell infiltration in kidney tissue of mice in each administration group was improved,and kidney tissue damage score was significantly reduced(P<0.05);Calcium content of kidneys in low-dose babatic acid group and Shenshitong group was significantly decreased than that of model group(P<0.05),and urinary calcium content was significantly increased(P<0.05).The results of molecular docking showed that molecular docking energy of PPARG,WNK1,CASR,CYP24A1 protein and babaric acid were all less than-21 kJ/mol,and docking effect was good.Conclusion Babaric acid in P.petiolosa in Guizhou has good anti-inflammatory and inhibiting effects on renal calcium accumulation.The mechanism of its leaching activity is related to the binding activity of PPARG,WNK1,CASR and CYP24A1 proteins.