关键词: 肾小球肾炎, 膜性, 足细胞, 脾肾阳虚

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