Hiroaki Hikiami

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 Terasawas ‘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 Keishi-bukuryo-gan on vascular function and hemorheological factors in spontaneously diabetic (WBN/kob) rats

Keishi-bukuryo-gan (Gui-zhi-fu-ling-wan) is a formula used for the improvement of blood circulation. Recently it has often also been used for arteriosclerosis. One of the mechanisms involved is thought to be the improvement of endothelial dysfunction, but the details are still unclear. In this study, the effect of Keishi-bukuryo-gan on vascular function and hemorheological factors in spontaneously diabetic (WBN/kob) rats was studied. Rats were given Keishi-bukuryo-gan in chow for 30 weeks. Body weight, blood glucose, endothelium-dependent/-independent relaxation, vasocontraction by free radical-induced and contractive prostanoids, triglyceride, advanced glycation endproduct, lipid peroxides, serum NO2-/NO3- and blood viscosity were measured. The results indicated that Keishi-bukuryo-gan caused a decrease in endothelium-dependent relaxation by acetylcholine to become significantly increased, and vasocontraction induced by free radicals and contractive prostanoids was significantly decreased. Furthermore, serum NO2-/NO3- and blood viscosity were significantly decreased. From these results, it was supposed that Keishi-bukuryo-gan exerted a protective effect on the endothelium. The WBN/kob rat is a useful study model for the complications of human diabetes, and Keishi-bukuryo-gan showed a protective effect against vascular injury in the susceptible rat.

Keishibukuryogan (Gui-Zhi-Fu-Ling-Wan), a Kampo Formula, Decreases Disease Activity and Soluble Vascular Adhesion Molecule-1 in Patients with Rheumatoid Arthritis

An increasing death rate due to cardiovascular disease in patients with rheumatoid arthritis (RA) has been reported. Keishibukuryogan (KBG) is a traditional Chinese/Japanese (Kampo) formula that has been administered to patients with blood stagnation, e.g. thrombotic disease and atherosclerosis. The objective of this study was to evaluate the efficacy of KBG on disease activity and endothelial dysfunction in RA patients. Sixteen RA patients were enrolled and administered KBG (12 g per day) for 12 weeks in addition to continuing other drugs. The disease activity of RA was assessed by modified disease activity scores for 28 joints (DAS28). Plasma levels of adhesion molecules, soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were evaluated. C-reactive protein (CRP), inflammatory cytokines (IL-1β, IL-6 and TNF-α) and lipid peroxide (LPO) were also evaluated. Fourteen patients completed the study. The disease activity of RA, tender joint count, swollen joint count and DAS28 decreased significantly. Among adhesion molecules, only sVCAM-1 decreased significantly. LPO also decreased significantly, whereas CRP and inflammatory cytokines remained unchanged. These results suggest that KBG has insufficient anti-inflammatory or immunomodulating effect but does have a beneficial effect on articular symptoms and a protective effect against endothelial dysfunction in RA patients.

A Chinese Herbal Medicine, Tokishakuyakusan, Reduces the Worsening of Impairments and Independence after Stroke: A 1-Year Randomized, Controlled Trial

In post-stroke patients, the recurrence of stroke and progression of impairments lead to a bedridden state and dementia. As for their treatments, only anti-hypertension and anti-coagulation therapies to prevent the recurrence of stroke are available. In Asia, post-stroke patients with impairments are often treated with herbal medicine. The present study evaluated the effectiveness of tokishakuyakusan (TS) in improving the impairment and independence in post-stroke patients. Thirty-one post-stroke patients (mean age = 81.4 years) were recruited and enrolled. Participants were randomly assigned to the TS group (n = 16) or non-treatment (control) group (n = 15) and treated for 12 months. Impairments were assessed using the Stroke Impairment Assessment Set (SIAS). Independence was evaluated using the functional independence measure (FIM). For each outcome measure, mean change was calculated every 3 months. The results were that impairments according to SIAS did not significantly change in the TS group. In contrast, SIAS significantly worsened in the control group. There was a significant difference between the two groups. In each term of SIAS, affected lower extremity scores, abdominal muscle strength, function of visuospatial perception, and so forth. in the TS group were better than those in the control group. Independence according to FIM did not change significantly in the TS group. In contrast, FIM significantly worsened in the control group. There was also a significant difference between the two groups. In conclusion, TS was considered to suppress the impairments of lower limbs and to exert a favorable effect on cerebral function for post-stroke patients.

Clinical Evaluation of the Effect of Daio (Rhei Rhizoma) on the Progression of Diabetic Nephropathy with Overt Proteinuria

We studied the effect of traditional herbal medicines containing daio (Rhei Rhizoma) on the long-term progression of diabetic nephropathy with overt proteinuria in eight patients [mean age 60 (45-73) years; duration of diabetes 18 (7-36) years]. At the beginning of the study, mean HbA1c was 8.2% and mean serum creatinine was 1.0 +/- 0.3 mg/dl. Everypatient had diabetic neuropathy and retinopathy. Three of the patients had hypertension and four had ischemic heart disease. After 107 +/- 25 months, the mean serum creatinine level had significantly increased to 4.8 +/- 2.6 mg/dl. The mean serum creatinine levels of five patients not advancing to dialysis treatment increased from 1.2 +/- 0.3 to 3.2 +/- 1.0 mg/dl, and the three patients requiring dialysis increased from 0.8 +/- 0.1 to 7.5 +/- 2.1 mg/dl. In the control group, treated without traditional herbal medicines, the mean serum creatinine level had significantly increased from 1.0 +/- 0.3 to 9.5 +/- 1.9 mg/dl after 71 +/- 12 months. All of the control group required dialysis treatment. Diabetic nephropathy with overt proteinuria is reported to develop into renal failure after 6-7 years. In this retrospective study, traditional herbal medicines with Daio were considered to be effective in prolonging the pre-dialysis period of diabetic nephropathy.

Effect of Keishibukuryogan on Endothelial Function in Patients with at Least One Component of the Diagnostic Criteria for Metabolic Syndrome: A Controlled Clinical Trial with Crossover Design

We evaluated the effect of keishibukuryogan (KBG; Guizhi-Fuling-Wan), a traditional Japanese (Kampo) formula, on endothelial function assessed by reactive hyperemia peripheral arterial tonometry (Endo-PAT2000) in patients with metabolic syndrome-related factors by controlled clinical trial with crossover design. Ninety-two patients were assigned to group A (first KBG-treatment period, then control period; each lasting 4 weeks, with about one-year interval) or group B (first control, then KBG-treatment). In forty-nine (27, group A; 22, group B) patients completing all tests, the mean value of the natural logarithmic-scaled reactive hyperemia index (L_RHI) increased and those of serum nonesterified fatty acid (NEFA), malondialdehyde, and soluble vascular cell adhesion molecule 1 decreased significantly during the KBG-treatment period, but not during the control period, and 4-week changes of L_RHI, NEFA, and malondialdehyde between the 2 periods showed significance. These results suggest that KBG has beneficial effect on endothelial function in patients with metabolic syndrome-related factors.

Choto-san Prevents Occurrence of Stroke and Prolongs Life Span in Stroke-Prone Spontaneously Hypertensive Rats

The effects of long-term oral administration of choto-san (diao-teng-san in Chinese) extract on the occurrence of stroke and life span were investigated in stroke-prone spontaneously hypertensive rats (SHR-SPs). Twenty-four rats were ramdomized into three groups. From 8 weeks of age, 0.1% and 0.3% choto-san groups were given water containing 0.1% (150 mg/kg/day) and 0.3% (450 mg/kg/day) choto-san extract, respectively. A control group was given only water. The mean survival times of the control group, 0.1% and 0.3% choto-san groups were 122.1, 159.8 and 176.8 days, respectively. The percent survivals of both the 0.1% and 0.3% choto-san groups were significantly enhanced compared to the control (Kaplan-Meier analysis followed by log-rank test; 0.1% choto-san: p < 0.05; 0.3% choto-san: p < 0.05). Furthermore, the cumulative percent occurrence of neurological and behavioral signs accompying stroke in the 0.3% choto-san group was significantly inhibited compared to the control (p < 0.05). These results suggested that choto-san prevents the occurrence of stroke and prolongs the life span of SHR-SPs.

Traditional Japanese Formula Kigikenchuto Accelerates Healing of Pressure-Loading Skin Ulcer in Rats

We evaluated the effect of kigikenchuto (KKT), a traditional Japanese formula, in a modified rat pressure-loading skin ulcer model. Rats were divided into three groups, KKT extract orally administered (250 or 500 mg/kg/day for 35 days) and control. KKT shortened the duration until healing. Immunohistochemically, KKT increased CD-31-positive vessels in early phase and increased α-smooth muscle actin-(α-SMA-) positive fibroblastic cells in early phase and decreased them in late phase of wound healing. By Western blotting, KKT showed the potential to decrease inflammatory cytokines (MCP-1, IL-1β, and TNF-α) in early phase, decrease vascular endothelial growth factor in early phase and increase it in late phase, and modulate the expression of extracellular protein matrix (α-SMA, TGF-β1, bFGF, collagen III, and collagen I). These results suggested the possibility that KKT accelerates pressure ulcer healing through decreases of inflammatory cytokines, increase of angiogenesis, and induction of extracellular matrix remodeling.

The Long-Term Effects of a Kampo Medicine, Juzentaihoto, on Maintenance of Antibody Titer in Elderly People after Influenza Vaccination

We have performed a broad-ranging analysis of the adjuvant effect of a Kampo medicine, juzentaihoto (JTT), on influenza vaccination in a multicenter randomized controlled trial. In this study, the enhancing effect of JTT on antibody titer after influenza vaccination was studied for 28 weeks in elderly people who were in the high-risk group for influenza infection. In total, 91 subjects over 65 years old were recruited from four long-term-care facilities located in Chiba, Gunma, and Toyama prefectures in Japan. Participants were randomly assigned to the JTT and the control groups. Blood samples were taken at 4 weeks before vaccination, at the time of vaccination, and then at 4, 8, 12, and 24 weeks after vaccination. The hemagglutination inhibition (HI) titers against A/California/7/2009 (H1N1), A/Victoria/210/2009 (H3N2), and B/Brisbane/60/2008 were then manually measured. A significant increase in HI titer against H3N2 was observed at week 8 after vaccination in the JTT group compared with the control group (P = 0.0229), and the HI titer of the JTT group significantly increased from 4 to 24 weeks (P = 0.0468), compared with the control group. In conclusion, our results indicated that JTT increased and prolonged antibody production against A/Victoria/210/2009 (H3N2), in particular, after influenza vaccination.

Identification of candidate genes involved in endogenous protection mechanisms against acute pancreatitis in mice

We surveyed changes of the gene expression profile in caerulein-exposed pancreas using Affymetrix GeneChip system (39,000 genes). Up-regulation of genes coding for claudin 4, claudin 7, F11 receptor, cadherin 1, integrin beta 4, syndecan 1, heat shock proteins b1/90aa1, Serpinb6a, Serpinb6b, Serpinb9, Bax, Bak1, calpain 2, calpain 5, microtubule-associated protein 1 light chain 3 alpha, S100 calcium-binding proteins A4/A10 were found in mouse pancreas exposed to caerulein for 12h. In contrast, the anti-apoptotic gene Bcl2 was down-regulated. The functions of these genes concern tight junction formation, cell-cell/cell-matrix adhesions, stress response, protease inhibition, apoptosis, autophagy, and regulation of cytoskeletal dynamics. Caerulein-exposed pancreatic acinar cells were immunohistochemically stained for claudin 4, cadherin 1, integrin beta 4, heat shock protein b1, and Serpinb6a. In conclusion, we have newly identified a set of genes that are likely to be involved in endogenous self-protection mechanisms against acute pancreatitis.