Genipin suppression of fibrogenic behaviors of the alpha-TN4 lens epithelial cell line.
OBJECTIVE
To determine in a lens epithelial cell line, alpha-TN4, whether genipin, an intestinal metabolite component of the herbal medicine inchin-ko-to, suppresses profibrogenic myofibroblast generation and upregulation of fibrogenic cytokines and to evaluate the potential benefit of the medicine in preventing posterior capsule opacification (PCO).


METHODS
Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan.


METHODS
In this study, alpha-TN4 cell proliferation, migration, and expression of alpha-smooth muscle actin (alpha-SMA), the hallmark of myofibroblast generation, were assayed with a colorimetric assay, scratch wound assay, immunohistochemistry, and Western blot analysis. Gene expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) was characterized with real-time reverse transcription-polymerase chain reaction. In addition, p38 mitogen-activated protein kinase (p 38 MAPK), extracellular signal-regulated kinase (ERK) limb, and Smad signalings were evaluated by Western blotting and immunohistochemistry. Cytotoxicity of genipin was evaluated using a commercial colorimetric assay kit for nuclear matrix protein 41/7 (NMP41/7) in culture medium.


RESULTS
Genipin suppressed cell proliferation and migration in association with inhibition of Smad and p38 MAPK phosphorylation, although ERK signaling was enhanced. Genipin suppressed mRNA expression of TGF-beta1 and CTGF. Cytoplasmic fiber formation declined based on less intense alpha-SMA immunocytochemical staining. However, alpha-SMA protein expression was actually not altered. This negative result suggests that genipin attenuated formation of alpha-SMA-containing cytoskeleton. Treatment of the cells with genipin for 48 hours did not increase the release of NMP41/7 to the medium, indicating this compound is not cytotoxic.


CONCLUSIONS
Because genipin suppressed alpha-TN4 lens cell fibrogenic behaviors, it may be of therapeutic value in preventing PCO.
Kitano
Ai
A
Department of Ophthalmology, Wakayama Medical University, Kimiidera, Wakayama, Japan. aikitano@wakayama-med.ac.jp
Saika
Shizuya
S
Yamanaka
Osamu
O
Reinach
Peter S
PS
Ikeda
Kazuo
K
Okada
Yuka
Y
Shirai
Kumi
K
Ohnishi
Yoshitaka
Y
D000199:Actins / Q000378:metabolism; D034961:Antigens, Nuclear / Q000378:metabolism; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D055513:Connective Tissue Growth Factor; D004365:Drugs, Chinese Herbal / Q000494:pharmacology* / Q000633:toxicity; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D057889:Iridoid Glycosides; D039823:Iridoids; D007908:Lens, Crystalline / Q000166:cytology*; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D011714:Pyrans / Q000494:pharmacology* / Q000633:toxicity; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics; D053773:Transforming Growth Factor beta1; D015854:Up-Regulation; D014945:Wound Healing / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000199:Actins; D034961:Antigens, Nuclear; C526317:CCN2 protein, human; D018797:Cell Cycle Proteins; D004365:Drugs, Chinese Herbal; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D057889:Iridoid Glycosides; D039823:Iridoids; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D011714:Pyrans; D012333:RNA, Messenger; D051785:Smad Proteins; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; C007834:genipin; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases