Proteomics analysis of altered proteins in kidney of mice with aristolochic acid nephropathy using the fluorogenic derivatization-liquid chromatography-tandem mass spectrometry method.

Aristolochic acid (AA) causes interstitial renal fibrosis, called aristolochic acid nephropathy (AAN). There is no specific indicator for diagnosing AAN, so this study aimed to investigate the biomarkers for AAN using a proteomics method. The C3H/He female mice were given ad libitum AA-distilled water (0.5 mg/kg/day) and distilled water for 56 days in the AA and normal groups, respectively. The AA-induced proteins in the kidney were investigated using a proteomics study, including fluorogenic derivatization with 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide, followed by high-performance liquid chromatography analysis and liquid chromatography tandem mass spectrometry with a MASCOT database searching system. There were two altered proteins, thrombospondin type 1 (TSP1) and G protein-coupled receptor 87 (GPR87), in the kidney of AA-group mice on day 56. GPR87, a tumorigenesis-related protein, is reported for the first time in the current study. The renal interstitial fibrosis was certainly induced in the AA-group mice under histological examination. Based on the results of histological examination and the proteomics study, this model might be applied to AAN studies in the future. TSP1 might be a novel biomarker for AAN, and the further role of GPR87 leading to AA-induced tumorigenesis should be researched in future studies.

Anti-tumor enhancement of Fei-Liu-Ping ointment in combination with celecoxib via cyclooxygenase-2-mediated lung metastatic inflammatory microenvironment in Lewis lung carcinoma xenograft mouse model.

BACKGROUND: Fei-Liu-Ping (FLP) ointment is an oral prescription medication that has been widely applied to treat lung cancer patients in China. Regulation of the metastatic microenvironment is an important therapeutic approach for prevention and treatment of tumor recurrence and metastasis. The advantage of Traditional Chinese Medicine management of lung cancer lies in the prevention of recurrence and metastasis. Our previous study has demonstrated that FLP ointment could regulate lung inflammatory microenvironment in vitro. However, the effects of FLP on the tumor microenvironment in vivo are still poorly understood. The objective of this study is to investigate the effect of FLP alone or in combination with celecoxib in the prevention of lung cancer progression by Cyclooxygenase (Cox)-2 mediated tumor inflammatory microenvironment in vivo. METHODS: 120 Lewis lung carcinoma xenograft mice were divided equally into four groups: vehicle, FLP, celecoxib, and FLP plus celecoxib. The dynamic growth of the xenografted tumors was observed using an in vivo fluorescence imaging system. Mice were sacrificed on day 14, day 21, and day 28, and tumor specimens and lung tissues were harvested to detect the metastasis-associated protein expression. RESULTS: Tumor inhibition rate was 15.4, 44.2, 47.4 % at day 14, 37.3, 34.7, 61.5 % at day 21, and 15.5, 10.3, 32.5 % at day 28 after treatment of FLP, celecoxib, and FLP plus celecoxib, respectively. Upon treatment of FLP and celecoxib together, lung metastasis rate was 30 % (8 metastatic nodules) lower than other groups. FLP inhibited Cox-2 expression in a time-dependent manner. Moreover, FLP inhibited N-cadherin, matrix metalloproteinases (MMP)-9, and Vimentin expression. Treatment of FLP in combination with celecoxib was more effective than FLP or celecoxib alone in inhibiting vascular endothelial growth factor, platelet-derived growth factor receptors ß, microsomal Prostaglandin E synthase-1, MMP-2, MMP-9, N-cadherin, and Vimentin expression, but increased E-cadherin expression. CONCLUSIONS: FLP inhibited tumor growth and metastasis in a Lewis lung xenograft mice model through the Cox-2 pathway. FLP in combination with celecoxib enhanced the antitumor growth and anti-metastasis effects. Traditional Chinese herbs combined with anti-inflammatory drugs might offer a promising strategy to prevent tumor metastasis.

Extraction efficiency of shosaikoto (xiaochaihu tang) and investigation of the major constituents in the residual crude drugs.

Shosaikoto (Xiaochaihu Tang) is one of the frequently used traditional herbal medicines (Kampo medicines, Japan). To realize the effective use of precious crude drugs, we investigated the major constituents in the residual crude drugs after decoction and aimed for extraction efficiency of shosaikoto with regard to the extract, tannin content, and major constituents. We found that the residual crude drugs had large amounts of compounds, especially saikosaponin b(2), which had a 78.3% yield compared to that in the first decoction. The extraction efficiency increased when decoction time and volume of water increased. Both increases had an additive effect on the yield of the extract and saikosaponin b(2) in particular. We also found that the size of crude drug pieces that are available in Japanese markets is suitable for decoction because of quick permeation of water. From our study, the second decoction may be a valuable contribution to medical treatment and of effective use of crude drugs. Moreover, time and volume of water should be increased when patients have trouble in preparing a decoction. Our study revealing the factors that influence the extraction efficiency of shosaikoto will be the basis for empirical evidence about decocting Kampo medicine.

Cinobufagin inhibits proliferation of acute myeloid leukaemia cells by repressing c-Myc pathway-associated genes.

Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad, Bufo gargarizans. Bufanolides inhibit Na+/K+ ATPase and have similar effects as cardiac glycosides, such as digitoxin or ouabain derived from toxic herbs. Recently, the anti-cancer effects of bufanolides have gained attention, however the underlying molecular mechanisms remain unclear. Selecting cinobufagin as a candidate anti-leukaemia agent, we here conducted transcriptomic analyses on the effect of cinobufagin on human acute myeloid leukaemia (AML) cell lines, HL60 and Kasumi-1. Flow cytometry analysis showed that cinobufagin induced apoptosis in both cell lines. RNA-sequencing (RNA-seq) of the two cell lines treated with cinobufagin revealed commonly downregulated genes with enrichment in the term "Myc active pathway" according to Gene Ontology (GO) analysis. Gene Set Enrichment Analysis (GSEA) of genes downregulated by cinobufagin also showed "MYC_TARGETS_V2" with the highest normalised enrichment score (NES) in both cell lines. In contrast, hallmarks such as "TNFA_SIGNALING_VIA_NFKB", "APOPTOSIS", and "TGF_BETA_SIGNALING" were significantly enriched as upregulated gene sets. Epigenetic analysis using chromatin immunoprecipitation and sequencing (ChIP-seq) confirmed that genes encoding cell death-related signalling molecules were upregulated by gain of H3K27ac, whereas downregulation of c-Myc-related genes was not accompanied by H3K27ac alteration. Cinobufagin is an anti-proliferative natural compound with c-Myc-inhibiting and epigenetic-modulating activity in acute myeloid leukaemia.

Case Report: Acupuncture is an effective treatment for olfactory dysfunction in the post COVID-19 condition.

Olfactory dysfunction in the post COVID-19 condition reported worldwide are refractory for some patients. For this reason, appropriate treatment is desired. In this article, we describe two cases of olfactory dysfunction in the post COVID-19 condition that was improved by traditional acupuncture treatment. By using the Yingxiang point (LI20), which is said to improve the sense of smell since ancient times, acupuncture treatment was performed 1-2 times a week in two patients about 6 and 7 months after the diagnosis of COVID-19. Acupuncture needles with a body length of 30 mm and a body diameter of 0.16 mm were inserted about 10 mm deep into the skin. We stimulated LI20 of the right and left sides until the patients felt the de qi sensation (acupuncture resonance), and left needles in the points for about 15 min. Immediately after the acupuncture treatment, the symptoms of olfactory dysfunction were alleviated, and the improvement in olfactory dysfunction lasted for 2-4 days. As the number of acupuncture treatments increased, the time until the flareup of olfactory dysfunction was prolonged, and the symptoms tended to decrease. In our experience, the acupuncture treatment was effective in a short period for treating residual olfactory dysfunction of the post COVID-19 condition, suggesting that acupuncture may serve as an adjunct to modern medical treatment, and it may also be a new option for patients who are resistant to Western medical treatment or unable to continue treatment because of side effects. In conclusion, acupuncture may be a new option for patients who are resistant to modern medical treatment or who are unable to continue treatment because of side effects.

Repressor of GATA negatively regulates murine contact hypersensitivity through the inhibition of type-2 allergic responses.

Repressor of GATA (ROG) inhibits Th2 cell differentiation and allergic airway inflammation in the lung. To determine the role of ROG in the pathogenesis of contact hypersensitivity (CHS), a hapten-induced mouse model of CHS using ROG Tg and ROG-deficient (ROG(-/-)) was used. ROG Tg mice showed little ear swelling, while ROG(-/-) mice showed enhanced ear swelling in comparison to wild type mice. Interstitial edema and mast cell degranulation at the local inflammation sites were mild in ROG Tg mice and exacerbated in ROG(-/-) mice. In addition, the serum total IgE and hapten-specific IgG1 levels were increased in ROG(-/-) mice. Adoptive transfer of ROG(-/-) CD4(+) T cells exacerbated CHS in wild type mice, while transfer of ROG Tg CD4(+) T cells resulted in the attenuation of CHS. These results indicate ROG negatively regulates the induction of CHS by controlling the CD4(+) T cell-mediated allergic responses, including IgE generation and mast cell degranulation.

Combination Image Analysis of Tongue Color and Sublingual Vein Improves the Diagnostic Accuracy of Oketsu (Blood Stasis) in Kampo Medicine.

Aim: In tongue diagnosis, a dark purple tongue and enlarged sublingual vein are important findings of Oketsu (blood stasis). However, the association between the tongue color and the sublingual vein has not been reported. This study investigated the association between the tongue color values and the sublingual vein width using tongue image analyzing system (TIAS) for the objective assessment of blood stasis. Methods: A total of 38 patients (age 68.7 ± 11.3 years, 14 men and 24 women) who visited the Department of Kampo Medicine at Chiba University Hospital were included. Physical findings, blood test results, blood stasis score from medical records, and tongue images obtained with TIAS were analyzed. The patients were classified into two groups: patients with a sublingual vein width of ≤2.5 mm (20 patients) and those with a width of >2.5 mm (18 patients). The physical findings and the blood test results of the two groups were analyzed by Wilcoxon's rank-sum test or χ2-test, whereas logistic regression analysis was used to determine the association between the tongue color values and sublingual vein width. Receiver operating characteristic (ROC) analysis was used to differentiate blood stasis. Results: The color values significantly related to the sublingual vein width (mm) were the P1-L* and P4-L* (darkness of the tongue edge and tongue apex) and the P1-b* and P2-b* (blueness of the tongue edge and tongue posterior). The area under the curve was greater for the combination of the tongue color values and the sublingual vein width than that for either of them. Conclusion: This study demonstrated an objective evaluation of blood stasis in the tongue of patients with dark-blue discoloration and an enlarged sublingual vein. In addition, the combination of the tongue color and the sublingual vein is expected to facilitate a more reliable diagnosis of blood stasis.

Role of Blood Stasis Syndrome of Kampo Medicine in the Early Pathogenic Stage of Atherosclerosis: A Retrospective Cross-Sectional Study.

In Kampo medicine, blood stasis (BS) syndrome is strongly associated with microangiopathy and can lead to atherosclerosis. Vascular endothelial dysfunction (VED), evaluated through flow-mediated dilation (FMD), plays an important role in the early stages of atherosclerosis. However, the association of BS syndrome with VED, as determined using FMD, has not been reported. This study investigated the association between BS syndrome and VED using FMD. Forty-one patients with normal glucose tolerance or impaired glucose tolerance (IGT) and without macrovascular complications were evaluated using FMD from May 2017 to August 2017. Based on the BS score, the patients were divided into the non-BS (n = 19) and BS syndrome (n = 22) groups. Physical and background characteristics, physiological function test results, and laboratory data were compared. Univariate analysis revealed that FMD and a history of dyslipidemia/IGT were significantly different between the two groups (p < 0.05). Multiple logistic regression analysis showed that BS syndrome was significantly associated with FMD (odds ratio: 6.26; p=0.03) after adjusting for the history of dyslipidemia/IGT. The receiver operating characteristic curve showed that the area under the curve for BS syndrome (0.74; p < 0.001) and history of IGT (p < 0.007) provided good diagnostic accuracy for FMD. The area under the curve for "BS syndrome + IGT" showed very good accuracy (0.80; p < 0.0001) and was higher than that for BS syndrome or IGT alone. In conclusion, the results of this study suggest that the BS score in Kampo medicine could be a useful tool for detecting the early pathogenic stages of atherosclerosis.

Yokukan-san: a review of the evidence for use of this Kampo herbal formula in dementia and psychiatric conditions.

Japanese traditional herbal medicine (Kampo) has its origins in traditional Chinese medicine (TCM). It was introduced to Japan in the middle of the sixth century and has evolved over the past 1,400 years after combining with Japan's original folk remedies. While it retains some similarities to TCM, Kampo has evolved in Japan, resulting in a system of medicine that has many differences from TCM. Kampo medicine is considered to be very safe; in Japan, Kampo herbal formulas are manufactured by licensed pharmaceutical companies, prescribed by Western-trained medical doctors (usually as a freeze-dried extract), and have quality control standards similar to those of prescription drugs. The present study examined Yokukan-san (Yi-Gan San in TCM), a Kampo formula that has been used empirically in Japan for more than 400 years. Accumulating clinical trials have demonstrated Yokukan-san's efficacy in treating patients with behavioral and psychological symptoms of dementia, which has resulted in the Japanese Society of Neurology listing it in the Japanese Guidelines for the Management of Dementia 2010. Efficacy in other diseases and conditions, such as sleep disorders, tardive dyskinesia, aggression, and impulsivity has also been reported. This article reviews both clinical and basic studies of Yokukan-san, with the goal of clarifying its clinical indications.

Study of factors involved in tongue color diagnosis by kampo medical practitioners using the farnsworth-munsell 100 hue test and tongue color images.

In traditional Japanese medicine (Kampo medicine), tongue color is important in discerning a patient's constitution and medical conditions. However, tongue color diagnosis is susceptible to the subjective factors of the observer. To investigate factors involved in tongue color diagnosis, both color discrimination and tongue color diagnosis were researched in 68 Kampo medical practitioners. Color discrimination was studied by the Farnsworth-Munsell 100 Hue test, and tongue color diagnosis was studied by 84 tongue images. We found that overall color discrimination worsened with aging. However, the color discrimination related to tongue color regions was maintained in subjects with 10 or more years of Kampo experience. On the other hand, tongue color diagnosis significantly differed between subjects with <10 years of experience and ≥10 years of experience. Practitioners with ≥10 years of experience could maintain a consistent diagnosis of tongue color regardless of their age.

Orengedoku-to augmentation in cases showing partial response to yokukan-san treatment: a case report and literature review of the evidence for use of these Kampo herbal formulae.

BACKGROUND: Yokukan-san, a Japanese traditional herbal (Kampo) prescription, has recently gathered increasing attention due to accumulating reports showing its remarkable efficacy in treating a wide variety of diseases refractory to conventional medicine as well as the behavioral and psychological symptoms of dementia. As yokukan-san has become broadly integrated with conventional medicine, augmentation therapy with other Kampo prescriptions has become necessary when the yokukan-san has been only partially efficacious. In this paper, we report three cases in which the addition of orengedoku-to, another Kampo formula, to yokukan-san was remarkably effective. CASES: Case 1 was an 85-year-old man with Alzheimer-type dementia who had become aggressive during the past 2 years. Three milligrams of aripiprazole completely suppressed his problematic behaviors but had to be stopped because of extrapyramidal symptoms. In the second case, a 44-year-old man with methamphetamine-induced psychosis had suffered from serious tardive dystonia for 2 years. No conventional approach had improved his tardive dystonia. The third case was a 29-year-old engineer who often failed to resist aggressive impulses and was diagnosed with intermittent explosive disorder. He was prescribed 5 mg of olanzapine, which did not suppress his extraordinary anger and caused somnolence even though the dose was low. INTERVENTIONS AND OUTCOMES: Yokukan-san was complementarily added to the patients' regular medication and exerted a definitive but partial effect in all cases. The addition of orengedoku-to to yokukan-san exerted the same efficacy as aripiprazole in controlling aggressiveness in Case 1, improved the tardive dystonia by 80% in Case 2, and was completely effective in controlling the patient's aggressive impulses in Case 3. CONCLUSION: Together with empirical evidence demonstrating the effectiveness of both yokukansan and orengedoku-to in reducing irritability, impulsivity, and aggression, these three cases suggest that orengedoku-to augmentation can be an effective option in cases that are partially responsive to yokukan-san treatment.

A valid approach in refractory glossodynia: a single-institution 5-year experience treating with Japanese traditional herbal (kampo) medicine.

Glossodynia is often refractory to conventional medicine, and there is only limited evidence to guide clinicians in its management. Patients with refractory glossodynia are often introduced to Japanese traditional herbal (Kampo) medicine experts under such circumstances because Kampo medicine has become known in Japan to be effective in treating a wide variety of symptoms refractory to conventional medicine. Herein, we report our single-institution 5-year experience treating patients with Kampo medicine for primary glossodynia that was refractory to conventional medicine. We found that 69.2% of patients reported a beneficial effect of Kampo medicine on glossodynia, and the average onset of improvement was 8.0 ± 7.7 weeks after starting Kampo treatment. The top two frequently used Kampo medicines for glossodynia were seinetsuhokito and mibakuekkito among high responders who showed a decrease of severity by 50% or more. The top four most overlapped herbs among effective Kampo medicines for glossodynia were Glycyrrhiza Root, Ginseng Root, Hoelen, and Atractylodes (lancea) Rhizome, which compose an essential Kampo prescription called shikunshito. Although more research is required to further clarify the effectiveness of Kampo medicine, it has valid efficacy even in cases of glossodynia that remain incurable by conventional treatments.

Efficient preparation of Hangekobokuto (Banxia-Houpo-Tang) decoction by adding perilla herb before decoction is finished.

Hangekobokuto (banxia-houpo-tang) is a Kampo (traditional Japanese) medicine used mostly for anxiety disorder and consists of Pinellia Tuber, Poria Sclerotium, Magnolia Bark, Perilla Herb and Ginger. Perilla Herb contains an essential oil rich in perillaldehyde, which has a pleasant flavor, but this is easily lost due to heating. We therefore investigated how the major constituents of hangekobokuto, namely perillaldehyde, rosmarinic acid, magnolol and [6]-gingerol, varied with time during decoction and approached an optimal condition for decoction. Rosmarinic acid at 15 min after boiling, and magnolol and [6]-gingerol at 30 min were mostly extracted, while 0.09 mg of perillaldehyde remained at 15 min, but was not detected at 30 min. From these results, a decoction was prepared by adding Perilla Herb 1, 2, 5 and 10 min prior to the termination time of decoction at 30 min. When Perilla Herb was added 1 min beforehand, perillaldehyde was abundant (1.58 mg) and the amount of rosmarinic acid was already the same as that in the conventional decoction at 30 min, but the amount of total extract was inadequate. When Perilla Herb was added 5 min beforehand, perillaldehyde remained to some extent (0.61 mg) and rosmarinic acid was significantly increased compared with that in the conventional decoction at 30 min. From these results, we can conclude that the decoction should be prepared by boiling for not more than 30 min and, if possible, Perilla Herb should be added 5 min prior to the termination time. Considering the antidepressant-like activity of perillaldehyde and rosmarinic acid, the suggested method will not only achieve better treatment for anxiety, but also provide an effective use of crude drugs in the resource-limited environment.

A case of aromatase inhibitor (anastrozole)-induced side-effects successfully treated with Kampo medicines.

OBJECTIVES: Breast cancer is the fourth most frequent cause of death, and it is currently the most frequent cause of death among Japanese women. As to breast cancer therapy, lengthy hormonal therapy is very important for the treatment and prevention of recurrence. Aromatase inhibitors (AIs) are the initial drug of choice for postoperative adjuvant therapy of breast cancer in Japan. AIs require long-term use and occasionally cause serious side-effects. In this report, the effects of Kampo medicines (Japanese traditional medicines) on AIs-induced side-effects are described. SUBJECT: A 55-year-old woman visited the Kampo outpatient department of Chiba University Hospital for atypical genital bleeding and arthralgia. At the age of 54, she suffered from left breast cancer and underwent left total mastectomy followed by chemotherapy for 6 months. Afterwards, 1 mg/day of anastrozole, one of the AIs, was used for therapy. Three (3) months later, atypical genital bleeding from vaginal mucosa and joint pains of bilateral hands and knees occurred as side-effects of anastrozole. Her attending doctor could only prescribe nonsteroidal external medicine for the inflammation of vaginal mucosa and do close follow-up. However, her symptoms showed no improvement. INTERVENTIONS AND OUTCOME: Her deficiency of both ki (qi) and ketsu (Blood) was diagnosed based on Kampo diagnostics. Juzentaihoto was used for treatment. After taking juzentaihoto for 5 weeks, the atypical genital bleeding disappeared, and she no longer need topical medicine. Because her arthralgia showed no improvement, powdered processed aconitine root was added. After taking 3.0 g/day of this medication, her arthralgia almost completely disappeared. CONCLUSIONS: Controlling the side-effects is a clinical issue from the viewpoint of adherence to drug treatment. Kampo therapy should be considered one of the choices for side-effects in the process of cancer treatment.