[The medicinal serum of yi-shen ruan-jian san antagonized the effect of aristolochic acid on human proximal tubular epithelial cells in vitro].
OBJECTIVE
To evaluate whether the medicinal serum of Yi-shen Ruan-jian san can antagonize the fibrogenic effect of human proximal tubular epithelial cell line (HKC) activated by aristolochic acid (AA) in vitro.


METHODS
The HKC was incubated in the media containing 40 mg x L(-1) aristolochic acid sodium salt (AA-Na) with or without 10% concentration of Yi-shen Ruan-jian san medicinal serum. Then the cell proliferation and cytotoxicity of HKC were determined by MTF and lactate dehydrogenase (LDH) release assay respectively, the antigen expression of cytokeratin and alpha-smooth muscle actin on HKC was detected by immunocytochemistry, the mRNA expression of transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type I Collagen (Col I) of HKC was measured by RT-PCR, and their protein expression was measured by ELISA or Western blot.


RESULTS
No cytotoxic effect was found in HKC after stimulation of AA-Na with or without the medicinal serum of Yi-shen Ruan-jian san (P > 0.05). No epithelial-myofibroblast transdifferentiation was found in HKC after AA-Na stimulation. The mRNA and protein expression of TGF-beta1, CTGF, PAI-1 and TIMP-1 of HKC was significantly upregulated by AA-Na (P < 0.05). The above-mentioned enhanced mRNA and protein expression, except for PAI-1, was significantly downregulated by the medicinal serum of Yi-shen Ruan-jian san, compared with the control (normal rat serum in the same concentration) (P < 0.05).


CONCLUSIONS
The fibrogenic effects of HKC activated by AA are antagonized by Yi-shen Ruan-jian san, through downregulating the expression of promoting excellular matrix (ECM) synthesis factors (TGF-beta1, CTGF) and inhibiting ECM degradation factor (TIMP-1).
Fang
Jing
J
Center of Nephrology, China-Japan Friendship Hospital, Beijing 100029, China.
Chen
Yi-pu
YP
Yang
Yan-fang
YF
Zhang
Mei
M
D000818:Animals; D034341:Aristolochic Acids / Q000037:antagonists & inhibitors* / Q000633:toxicity; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D055513:Connective Tissue Growth Factor; D004338:Drug Combinations; D004365:Drugs, Chinese Herbal / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000633:toxicity; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics; D007687:Kidney Tubules, Proximal / Q000166:cytology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008419:Materia Medica / Q000494:pharmacology; D010946:Plants, Medicinal / Q000737:chemistry; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D044967:Serum; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics; D053773:Transforming Growth Factor beta1
D034341:Aristolochic Acids; C526317:CCN2 protein, human; C526316:CCN2 protein, rat; D004338:Drug Combinations; D004365:Drugs, Chinese Herbal; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008419:Materia Medica; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D007770:L-Lactate Dehydrogenase