A Proteomic Approach for the Diagnosis of ‘Oketsu’ (blood stasis), a Pathophysiologic Concept of Japanese Traditional (Kampo) Medicine

‘Oketsu’ is a pathophysiologic concept in Japanese traditional (Kampo) medicine, primarily denoting blood stasis/stagnant syndrome. Here we have explored plasma protein biomarkers and/or diagnostic algorithms for ‘Oketsu’. Sixteen rheumatoid arthritis (RA) patients were treated with keishibukuryogan (KBG), a representative Kampo medicine for improving ‘Oketsu’. Plasma samples were diagnosed as either having an ‘Oketsu’ (n = 19) or ‘non-Oketsu’ (n = 29) state according to Terasawa's ‘Oketsu’ scoring system. Protein profiles were obtained by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and hierarchical clustering and decision tree analyses were performed. KBG treatment for 4 or 12 weeks decreased the ‘Oketsu’ scores significantly. SELDI protein profiles gave 266 protein peaks, whose expression was significantly different between the ‘Oketsu’ and ‘non-Oketsu’ states. Hierarchical clustering gave three major clusters (I, II, III). The majority (68.4%) of ‘Oketsu’ samples were clustered into one cluster as the principal component of cluster I. The remaining ‘Oketsu’ profiles constituted a minor component of cluster II and were all derived from patients cured of the ‘Oketsu’ state at 12 weeks. Construction of the decision tree addressed the possibility of developing a diagnostic algorithm for ‘Oketsu’. A reduction in measurement/pre-processing conditions (from 55 to 16) gave a similar outcome in the clustering and decision tree analyses. The present study suggests that the pathophysiologic concept of Kampo medicine ‘Oketsu’ has a physical basis in terms of the profile of blood proteins. It may be possible to establish a set of objective criteria for diagnosing ‘Oketsu’ using a combination of proteomic and bioinformatics-based classification methods

Effects of two formulations for overcoming oketsu on vascular function and expression patterns of plasma proteins in spontaneously diabetic rats

自然発症糖尿病モデルであるWBN/Kobラットに代表的な駆瘀血薬である桂枝茯苓丸と当帰芍薬散を長期間投与し, 血管機能とタンパク発現に及ぼす影響を検討した。方法は, WBN/Kobラット(雄, 24週令)を18週間飼育し糖尿病発症を確認した後, 対照群, 3%桂枝茯苓丸(KB)群, 3%当帰芍薬散(TS)群の3群に分け, さらに25週間飼育した。飼育後, 胸部大動脈を摘出しOrgan bath法を用いacetylcholine (Ach)による血管弛緩作用, xanthine/xanthine oxidase (X/XOD)投与による血管収縮作用等を検討した。同時に, 血液流動性, 血漿脂質, NO代謝物等の測定とSELDI-TOF-MSによる血漿プロテオーム解析を施行した。結果は, 対照群とKB, TS群の3群間において, 体重と血糖値に有意な差を認めなかった。Achによる内皮依存性血管弛緩率はKB群で対照群に対し有意に弛緩率の増加を認めた。X/XOD投与による血管収縮率はTS群で, PLA_2投与による血管収縮率はTS, KB群の両群で対照群に対し収縮率の減少を認めた。血液流動性はTS群で対照群に対し改善傾向を認め, NO代謝物はKB, TS群の両群で対照群に対し有意に減少した。血漿プロテオーム解析により, 対照群に比較しKB群では5個, TS群で8個のタンパク質の有意な変動を認めた。以上のことから, 2種類の代表的な駆瘀血薬は, 一部異なる作用機序で血流改善に影響を及ぼし, 発現するタンパク質にも差異が認められた。作用機序とタンパク質発現との関連は今後検討を要するが, これらの多成分系の方剤による生体の複雑な反応性の差異が「証」の成立に影響していると考えられた。We investigated the effects of keishibukuryogan and tokishakuyakusan, which are representative formulations for overcoming oketsu, on vascular function and expression patterns of plasma proteins in spontaneously diabetic rats. Twenty-one- to 24-week-old male WBN/Kob rats were maintained for 18 weeks on a diabetes-accelerated feed, and received standard (diabetes-accelerating) chow containing 3% (wt/wt) keishibukuryogan or tokishakuyakusan for 25 weeks. There was no significant change in body weight or blood glucose among the groups. Acetylcholine-induced endothelium-dependent relaxation of the keishibukuryogan group significantly increased compared to that of controls. Xanthine/xanthine oxidase-induced contraction of the tokishakuyakusan group and phospholipase A_2-induced contraction of the keishibukuryogan and tokishakuyakusan groups significantly decreased compared to the controls. Transit time of whole blood tended to decrease in the tokishakuyakusan group compared to controls. NO_2^-/NO_3^- in the keishibukuryogan and tokishakuyakusan groups significantly decreased compared to controls. A study using surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) demonstrated that five and eight peaks had significantly changed peak intensities in plasma of rats treated with keishibukuryogan and tokishakuyakusan, respectively, as compared to the controls. Thus, two representative formulations for overcoming oketsu with different mechanisms of action had favorable effects against vascular dysfunction. Altered plasma protein levels were commonly observed in the rats administered these two formulations. Our study using ProteinChip technology may be useful for the evaluation of the relationship between the efficacy and the profiling of plasma protein expression after administration Kampo medicines, thus leading to the understanding of "Sho" in Kampo medicine.The 21st Century COE Program, Toyama Medical and Pharmaceutical Universit