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.

Proteomic identification of haptoglobin as a stroke plasma biomarker in spontaneously hypertensive stroke-prone rats.

AIMS: We investigated changes in the expression of plasma proteins in spontaneously hypertensive stroke-prone rats (SHRSP) to identify stroke biomarkers. MAIN METHODS AND KEY FINDINGS: The present analysis using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) demonstrated that three peaks at mass/charge ratios (m/z) of 9330, 9480 and 9700 decreased in intensity during the development and progression of hypertensive stroke in SHRSPs, but not in age-matched control SHR and Wistar rats. Administration of verapamil, an L-type calcium channel blocker which was effective for hypertension in SHRSP rats, prevented the decrease in plasma protein expression. A candidate biomarker protein (m/z 9330) was identified using LC-MS/MS as haptoglobin (Hp). Immunoblotting with anti-Hp antibody demonstrated the decreased expression of both Hpalpha and Hpbeta chains in SHRSP. In contrast, haptoglobin mRNA expression in the liver of SHRSPs slightly increased as compared with control rats. SIGNIFICANCE: These findings suggest that Hp is a biomarker candidate for discriminating pathogenic alterations of stroke.

Identification of a predictive biomarker for the beneficial effect of a Kampo (Japanese traditional) medicine keishibukuryogan in rheumatoid arthritis patients.

OBJECTIVES: Kampo (Japanese traditional herbal) medicines are now ethically used in Japan as pharmaceutical grade prescription drugs. However, there are distinct groups of responders and non-responders to Kampo medicines. We searched for biomarker candidates to discriminate responders from non-responders to keishibukuryogan (KBG); one of the most frequently used Kampo medicines. DESIGN AND METHODS: A combination of SELDI technology and a decision tree analysis with proprietary developed bioinformatics tools was applied to 41 (32 for tree construction and 9 for validation test) plasma samples obtained from rheumatoid arthritis (RA) patients. A candidate biomarker protein was identified using LC-MS/MS. RESULTS: The constructed tree with measurable reliability contained only a single peak which was identified as haptoglobin alpha 1 chain (Hpalpha1). CONCLUSION: Hpalpha1 is a biomarker candidate for discriminating responders from non-responders to KBG treatment for RA. The present results may open the way to the establishment of "evidence-based" complementary and alternative medicine.

A newly devised formulation for self-medication enhances interferon-gamma production and proliferation of splenic lymphocytes.

A newly devised formulation for self-medication in Toyama, PanaWang, is a new herbal medicine (so called Toyama original brand formulation) developed based on traditional philosophy and scientific evidence. We here tried to examine the effect of oral administration of PanaWang on the balance of type I helper T cells (Th1) and Th2 cells. Splenic lymphocytes from normal mice were stimulated with Concanavalin A (Con A) in vitro and the secretion of Th1- and Th2-type cytokines, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) respectively, was investigated. Con A-induced production of IFN-gamma from spleen cells, but not IL-4, was enhanced by the administration of PanaWang. Increased production of IFN-gamma was also detected in splenic lymphocytes from Th2-predominant BALB/c mice after DNP-immunization, without a change in antigen-specific IgE levels in vivo. Antigen-specific proliferative responses were also increased in lymphocytes from PanaWang-treated mice. These findings raise the possibility that PanaWang has Th1-stimulating activity and induces Th1-predominant immunity.

Expression patterns of plasma proteins in spontaneously diabetic rats after oral administration of a Kampo medicine, Hachimi-jio-gan, using SELDI ProteinChip platform.

We investigated the changes (increase or decrease in peak intensity) in the expression of plasma proteins in spontaneously diabetic WBN/Kob rats that were with complicated diabetic nephropathy, to determine multiple biomarkers in the plasma of diabetic rats. The present study using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) demonstrated that six peaks at mass/charge ratios (m/z) of 4678, 4732, 4808, 9058, 9323, and 9465, among approximately 80 peaks per spectrum in the 2000-10000 Da mass range, had increased peak intensities with the development or progression of diabetic nephropathy in plasma of spontaneously diabetic WBN/Kob rats as compared with those of normal Wistar rats. Administration of the Kampo medicine Hachimi-jio-gan was effective at reducing the expression of diabetic nephropathy but not at reducing blood glucose levels. It also improved the increased levels of these plasma proteins. Other biomarker peaks at m/z 5067, 5279, 7598, and 7917 were not affected by Hachimi-jio-gan administration. Further study will be needed to identify these positive biomarkers and to evaluate the relationship between the efficacy and expression patterns of the plasma proteins in greater detail. The expression patterns of proteins and molecular-related ions revealed that several proteins in plasma may be involved in the development and/or progression of diabetic nephropathy in WBN/Kob rats and the efficacy of Hachimi-jio-gan. This study using ProteinChip technology may provide a useful basis in the search for multiple biomarkers in plasma for the diagnosis of disease and therapeutic evaluation of Kampo medicines.