Mechansims of anti-asthmatic action of genistein in vivo and in vitro

• Main Authors: Ya-Hsin Lai, 賴雅欣
• Other Authors: Wun-Chang Ko, Ph. D.
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/13239116730725640898

碩士 === 臺北醫學大學 === 醫學研究所 === 91 === PART 1 In this present study, we tried to investigate that the structure-activity relationships between isoflavones and their inhibitory effects on PDE isozymes. Isolated guinea-pig lungs and hearts were separately homogenized and centrifuged. The supernatant was chromatographed over a column of Q-sepharose, and eluted with various concentrations of NaCl. In the following order, PDE subtype 1, 5, 2, 4 from lungs, and 3 from hearts were partially purified. Acccording to the method described by Thompson and Appleman in 1971, the activities of PDE isozymes were determined in the presence of cAMP and [3H]-cAMP or cGMP and [3H]-cGMP as substrate. The results revealed that in these isoflavones, genistein most potently inhibited the activity of PDE2, moderately inhibited the activities of PDE1, 3 and 4. However, genistein did not inhibit the activity of PDE5. Daidzein, an inactive analog of genistein, which has been reported to specifically inhibit protein tyrosine kinase, selectively inhibited the activity of PDE3. Biochanin A potently inhibited the activity of PDE4, moderately inhibited the activities of PDE1 and 2, but never inhibited the activities of PDE3 and 5. Prunetin almost did not inhibit these five PDE isozymes. Based on the similarity of structures of these isoflavones, therefore, we conclude that C-7 OH group is very important for PDE1~5, that C-4¢ OH group is very important for PDE3 and 5, subsequently for PDE1 and 2, and that C-5 OH group is very important for PDE1, 2, 4 and 5, subsequently for PDE3. PART 2 Genistein is a well known broad-spectrum protein tyrosine kinase (PTK) inhibitor. These isoflavones including genistein, are naturally occurring in the legume soybean. There have been numerous reports of isoflavones about their beneficial health effects, such as cardioprotection , antioxidant, anti-immune response, and anti-inflammation. Therefore we are interested in investigating its suppressive effects on ovalbumin (OVA)-induced airway hyperresponsiveness in vivo and in vitro. According to the method described by Kanehiro et al., in 2001, female BALB/c mice were actively sensitized by intraperitoneal injections of OVA and challenged by aerosolized OVA (1 %). After secondary challege, aerosolized methocholine (MCh, 6.25-50 mg/ml) induced increases of enhanced pause (Penh) values in conscious animals in a concentration-dependent manner. Genistein (10~100 mmol/kg, i.p.) markedly inhibited MCh (50 mg/ml)-induced increase of Penh value in the sensitized mice. In addition, genistein significantly reduced increases of total inflammatory cells, macrophages, neutrophils, and eosinophils, but not lymphocytes in bronchoalveolar lavage fluid, and markedly attenuated the release of cytokines, including IL-2, IL-4, IL-5, IFN-g and TNF-a with an exception that genistein at 30 mmol/kg did not significantly inhibit INF-g. Genistein (3~30 mM) significantly inhibited OVA-induced contrations in sensitized guinea pig isolated trachea. Genistein selectively and competitively inhibited PDE2 and 3, with a low Ki value of 4.32 and 11.47 mM, respectively. It subsequently inhibited PDE1 and 4, but not 5. The above results revealed that anti-asthmatic action of genistein is via inhibition of cAMP-PDE but not PTK activity, which has been reported to need higher concentration to inhibit.