Abstract: Objective　To explore the protective mechanism of aqueous extract from Wenyanglishui Decoction（WD） on mouse podocyte injury caused by serum from idiopathic membranous nephropathy（IMN） patients with syndrome of Spleen and Kidney Yang deficiency（SSKYD）．Methods　The 58 participants were enrolled from Nephropathy Department，the First Affiliated Hospital of Guangzhou University of Chinese Medicine from February to March 2015，including 28 anti-PLA2R positive cases of IMN（diagnosed by initial renal biopsy） with SSKYD and 30 controls. Fasting venous blood samples were taken from both groups simultaneously early in the morning.The mouse podocyte injury models caused by serum from IMN patients with SSKYD were established in vitro. CCK-8 kit was applied to evaluate the influence of different concentrations（5%，7.5% and 10%） of serum from IMN patients with SSKYD on the proliferation ability of immortalized mouse podocytes cultured in vitro.Based on the testing results of CCK-8，5 groups including normal control，model，2 mg/ml，4 mg/ml，8 mg/ml aqueous extract from WD were divided，treated with 10% of serum from controls + RPMI-1640 culture medium，10% of serum from IMN patients with SSKYD + RPMI-1640 culture medium，10% of serum from IMN patients with SSKYD +RPMI-1640 culture medium + 2 mg/ml aqueous extract from WD，10% of serum from IMN patients with SSKYD +RPMI-1640 culture medium + 4 mg/ml aqueous extract from WD，10% of serum from IMN patients with SSKYD + RPMI-1640 culture medium + 8 mg/ml aqueous extract from WD，respectively.F-actin cytoskeleton of podocytes of the 5 groups was examined by immunofluorescence microscopy，and Caspase-3 mRNA was detected by qRT-PCR at 24 h and 48 h after incubation，respectively.Results　There were no significant differences between RPMI-1640 culture medium groups and RPMI-1640 culture medium + aqueous extract from WD groups in optical density（t=1.937，P>0.05）．The optical density in 0 mg/ml aqueous extract from WD group was higher than that of 20，40，60，80 and 100 mg/ml groups（P<0.05）．The optical density of 10 % serum of IMN patients group was lower than that of the control group，5% and 7.5% serum of IMN patients groups（P<0.05）．10 % serum of IMN patients destroyed the cytoskeleton of podocytes，down-regulated the expression of F-actin，and the filaments were redistributed inconsistently with the direction of the axis of the cells.Moreover，part of the filaments broken and cell bodies retracted.After 24 h and 48 h incubation，however，F-actin filaments showed up-regulated expression，and improved broken conditions and disordered arrangement in aqueous extract from WD groups when compared to model group.The relative expression level of Caspase-3 mRNA in model group was higher when compared to control group after 24 h incubation（P<0.05）．After 48 h incubation，the expression level of Caspase-3 mRNA in model group was higher than that of control group，2 mg/ml and 8 mg/ml aqueous extract from WD groups（P<0.05）．Among three decoction groups，the expression level of Caspase-3 mRNA was the lowest in 8 mg/ml group and showed a significant difference when compared with 2 mg/ml as well as 4 mg/ml groups（P<0.05）．Conclusion　The aqueous extract from WD can ameliorate the rearrangement of podocyte cytoskeleton F-actin injured by serum from IMN patients with SSKYD.This mechanism may be related to the inhibition and down-regulation of Caspase-3 mRNA expression，therefore the podocytes apoptosis is alleviated.

Key words: Glomerulonephritis, membranous；Podocytes；Spleen-Kidney Yang deficiency

摘要： 目的　探索温阳利水方水提物对脾肾阳虚特发性膜性肾病（IMN）患者血清致小鼠足细胞损伤的保护作用机制。方法　2015年2—3月，选取在广州中医药大学第一附属医院肾病科住院，首次肾穿刺活检确诊为IMN、血清抗M型磷脂酶A2受体（anti-PLA2R）阳性、脾肾阳虚的患者28例，采集清晨空腹静脉血。同时间段，采集健康志愿者30例清晨空腹静脉血。CCK-8试剂盒检测不同浓度（5%、7.5%、10%）脾肾阳虚IMN患者血清对体外培养小鼠永生化足细胞增殖能力的影响，构建脾肾阳虚IMN血清损伤足细胞体外模型。根据CCK-8检测结果，设置正常对照组、模型组、2 mg/ml温阳利水方水提物组、4 mg/ml温阳利水方水提物组和8 mg/ml温阳利水方水提物组共5组，依次予10%健康志愿者血清+RPMI-1640培养基、10%脾肾阳虚IMN患者血清+RPMI-1640培养基、10%脾肾阳虚IMN患者血清+RPMI-1640培养基+2 mg/ml温阳利水方水提物、10%脾肾阳虚IMN患者血清+RPMI-1640培养基+4 mg/ml温阳利水方水提物、10%脾肾阳虚IMN患者血清+RPMI-1640培养基+8 mg/ml温阳利水方水提物，分别孵育24 h和48 h。通过细胞免疫荧光观察足细胞骨架蛋白F-actin、qRT-PCR检测Caspase-3 mRNA。结果　RPMI-1640培养基组和RPMI-1640培养基加温阳利水方水提物组OD值比较，差异无统计学意义（t=1.937，P>0.05）。20、40、60、80、100 mg/ml温阳利水方水提物组OD值低于0 mg/ml温阳利水方水提物组（P<0.05）。10% IMN血清组OD值低于对照组、5% IMN血清组、7.5% IMN血清组（P<0.05）。10% IMN患者血清，可破坏足细胞骨架蛋白结构，F-actin表达减少，与细胞轴方向不一致，部分断裂，排列紊乱，细胞回缩。孵育24 h和48 h后，与模型组相比，温阳利水方水提物组F-actin表达增多，纤维断裂和排列紊乱情况均有所改善。孵育24 h，模型组Caspase-3 mRNA高于正常对照组（P<0.05）。孵育48 h，模型组Caspase-3 mRNA高于正常对照组、2 mg/ml温阳利水方水提物组、8 mg/ml温阳利水方水提物组（P<0.05）；孵育48 h，8 mg/ml温阳利水方水提物组Caspase-3 mRNA低于2、4 mg/ml温阳利水方水提物组（P<0.05）。结论　温阳利水方水提物可改善脾肾阳虚IMN患者血清致小鼠足细胞骨架蛋白F-actin重排，可能通过抑制Caspase-3 mRNA的表达，发挥抗凋亡效应。

关键词: 肾小球肾炎, 膜性, 足细胞, 脾肾阳虚