Ameliorative Effects of Zingiber officinale Rosc on Antibiotic-Associated Diarrhea and Improvement in Intestinal Function.

The global increase in antibiotic consumption is related to increased adverse effects, such as antibiotic-associated diarrhea (AAD). This study investigated the chemical properties of Zingiber officinale Rosc (ZO) extract and its ameliorative effects using a lincomycin-induced AAD mouse model. Intestinal tissues were evaluated for the expression of lysozyme, claudin-1, and α-defensin-1, which are associated with intestinal homeostasis. The cecum was analyzed to assess the concentration of short-chain fatty acids (SCFAs). The chemical properties analysis of ZO extracts revealed the levels of total neutral sugars, acidic sugars, proteins, and polyphenols to be 86.4%, 8.8%, 4.0%, and 0.8%, respectively. Furthermore, the monosaccharide composition of ZO was determined to include glucose (97.3%) and galactose (2.7%). ZO extract administration ameliorated the impact of AAD and associated weight loss, and water intake also returned to normal. Moreover, treatment with ZO extract restored the expression levels of lysozyme, α-defensin-1, and claudin-1 to normal levels. The decreased SCFA levels due to induced AAD showed a return to normal levels. The results indicate that ZO extract improved AAD, strengthened the intestinal barrier, and normalized SCFA levels, showing that ZO extract possesses intestinal-function strengthening effects.

Cell Culture Systems for Studying Hepatitis B and Hepatitis D Virus Infections.

The hepatitis B virus (HBV) and hepatitis D virus (HDV) infections cause liver disease, including hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBV infection remains a major global health problem. In 2019, 296 million people were living with chronic hepatitis B and about 5% of them were co-infected with HDV. In vitro cell culture systems are instrumental in the development of therapeutic targets. Cell culture systems contribute to identifying molecular mechanisms for HBV and HDV propagation, finding drug targets for antiviral therapies, and testing antiviral agents. Current HBV therapeutics, such as nucleoside analogs, effectively suppress viral replication but are not curative. Additionally, no effective treatment for HDV infection is currently available. Therefore, there is an urgent need to develop therapies to treat both viral infections. A robust in vitro cell culture system supporting HBV and HDV infections (HBV/HDV) is a critical prerequisite to studying HBV/HDV pathogenesis, the complete life cycle of HBV/HDV infections, and consequently identifying new therapeutics. However, the lack of an efficient cell culture system hampers the development of novel antiviral strategies for HBV/HDV infections. In vitro cell culture models have evolved with significant improvements over several decades. Recently, the development of the HepG2-NTCP sec+ cell line, expressing the sodium taurocholate co-transporting polypeptide receptor (NTCP) and self-assembling co-cultured primary human hepatocytes (SACC-PHHs) has opened new perspectives for a better understanding of HBV and HDV lifecycles and the development of specific antiviral drug targets against HBV/HDV infections. We address various cell culture systems along with different cell lines and how these cell culture systems can be used to provide better tools for HBV and HDV studies.

Hepatitis B vaccine delivered by microneedle patch: Immunogenicity in mice and rhesus macaques.

Vaccination against hepatitis B using a dissolving microneedle patch (dMNP) could increase access to the birth dose by reducing expertise needed for vaccine administration, refrigerated storage, and safe disposal of biohazardous sharps waste. In this study, we developed a dMNP to administer hepatitis B surface antigen (HBsAg) adjuvant-free monovalent vaccine (AFV) at doses of 5 µg, 10 µg, and 20 µg, and compared its immunogenicity to vaccination with 10 µg of standard monovalent HBsAg delivered by intramuscular (IM) injection either in an AFV format or as aluminum-adjuvanted vaccine (AAV). Vaccination was performed on a three dose schedule of 0, 3, and 9 weeks in mice and 0, 4, and 24 weeks in rhesus macaques. Vaccination by dMNP induced protective levels of anti-HBs antibody responses (≥10 mIU/ml) in mice and rhesus macaques at all three HBsAg doses studied. HBsAg delivered by dMNP induced higher anti-HBsAg antibody (anti-HBs) responses than the 10 µg IM AFV, but lower responses than 10 µg IM AAV, in mice and rhesus macaques. HBsAg-specific CD4+ and CD8+ T cell responses were detected in all vaccine groups. Furthermore, we analyzed differential gene expression profiles related to each vaccine delivery group and found that tissue stress, T cell receptor signaling, and NFκB signaling pathways were activated in all groups. These results suggest that HBsAg delivered by dMNP, IM AFV, and IM AAV have similar signaling pathways to induce innate and adaptive immune responses. We further demonstrated that dMNP was stable at room temperature (20 °C-25 °C) for 6 months, maintaining 67 ± 6 % HBsAg potency. This study provides evidence that delivery of 10 µg (birth dose) AFV by dMNP induced protective levels of antibody responses in mice and rhesus macaques. The dMNPs developed in this study could be used to improve hepatitis B birth dose vaccination coverage levels in resource limited regions to achieve and maintain hepatitis B elimination.

Elucidation of protective effects of oxime derivatives against cisplatin-induced cytotoxicity in LLC-PK1 kidney cells.

This study aimed to explore the renoprotective effects of oxime derivatives against cisplatin-mediated cell death in LLC-PK1 porcine kidney epithelial cells. Treatment with compounds 161-A and 161-F improved cisplatin-mediated LLC-PK1 cell damage and increased cell viability by more than 80% of the control value when compared with that of cisplatin-treated cells. In addition, 161-A and 161-F reduced cisplatin-induced apoptosis. Analysis of the molecular mechanisms underlying the effects exerted by these compounds revealed that treatment with 161-A and 161-B inhibited the protein expression of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and cleaved caspase-3 in cisplatin-treated LLC-PK1 cells. Thus, these findings provide in vitro scientific evidence that oxime derivatives may be useful as pharmacological candidates for the prevention of cisplatin-mediated nephrotoxicity.

Beneficial Effect of Paeonol on Antibiotic-Associated Inflammatory Response in Mice with Diarrhea.

Diarrhea is a common adverse effect of antibiotics particularly that acts on anaerobes. Moutan Radicis Cortex (MRC) is an herbal medicine used for its anti-inflammatory and antibacterial actions. The purpose of this study was to analyze the active components of MRC to determine their effect on antibiotic-associated diarrhea (AAD) and anti-inflammatory effects. Of the various components of MRC, seven compounds (gallic acid, oxypaeoniflorin, paeoniflorin, ethyl gallate, benzoic acid, benzoylpaeoniflorin, paeonol) were identified and assessed for anti-inflammation effects. Paeonol was found to effectively reduce nitric oxide production and levels of IL-6 and TNF-α in a concentration-dependent manner. Paeonol also effectively reduced the mRNA expression level of IL-6, IL-1ß, and TNF-α. Western blotting analysis confirmed the reduction of COX-2 and NF-κB levels; p-p38 MAPK levels increased in the presence of a low concentration (25 µM) of paeonol but decreased in the presence of a high concentration (50 µM). In the mouse model of lincomycin-induced AAD, all experimental groups treated with paeonol (25, 50, and 100 mg/kg concentrations) showed diminished diarrhea status scores. Finally, the expression levels of TNF-α and IL-4 were reduced compared with those in the control group. Therefore, paeonol may have active compounds of MRC to alleviate the diarrhea symptoms of AAD and reduce inflammatory mediators. Other components of the MRC extract could contribute to its known anti-inflammatory and antibacterial activity and should be tested for their possible activity.

Natural antibody IgG levels are associated with HBeAg-positivity and seroconversion in chronic hepatitis B patients treated with entecavir.

B1 cell-derived natural antibodies are non-specific polyreactive antibodies and can activate the complement pathway leading to lysis of enveloped virus particles before activation of the adaptive immune response. We investigated the relationship between natural antibody levels and treatment outcomes of 126 treatment-naïve chronic hepatitis B (CHB) patients, who underwent entecavir (ETV) treatment. Serum IgG1-3 and complement C3 levels were significantly higher in HBeAg-positive patients. In pre-treatment, IgG1 (odd ratios [OR] 2.3, p < 0.0001), IgG2 (OR 9.8, p < 0.0001), IgG3 (OR 7.4, p < 0.0001), and C3 (OR 7.2, p < 0.0001) were associated with HBeAg-positive patients. At baseline, IgG2 (OR 10.2, p = 0.025), IgG4, (OR 3.4, p = 0.026), and complement C1q (OR 5.0, p = 0.0068) were associated with seroconverters. Post-treatment levels of IgG1-4 and C3/C1q were also associated with HBeAg-positive patients and seroconverters. High levels of IgG2-4 and C1q were observed in seroconverters but not in virological responders. Thus, high pretreatment and post-treatment levels of natural antibody IgG1-4, complement C3, and/or C1q were significantly associated with HBeAg-positivity and HBeAg seroconverters in CHB patients with ETV treatment. These results suggest that the presence of preexisting host immunity against chronic hepatitis B is closely related to outcome of ETV treatment.

Estrogenic activity of ethyl gallate and its potential use in hormone replacement therapy.

We aimed to compare the estrogenic activities of compounds isolated from Moutan Cortex Radicis (MRC, Paeonia suffruticosa Andrews) and identify their potential use in hormone replacement therapy. We quantified seven marker components (gallic acid, oxypaeoniflorin, paeoniflorin, ethyl gallate, benzoic acid, benzoylpaeoniflorin, and paeonol) in MRC using a high-performance liquid chromatography simultaneous analysis assay. To investigate the estrogenic activity of MRC and the seven marker components, an E-screen assay was conducted using the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line. Among them, ethyl gallate caused cell proliferation in a concentration-dependent manner at concentrations above 25 µM and was clearly suppressed by combination treatment with the ER antagonist ICI 182,780. Therefore, ethyl gallate may be a compound of MRC that can increase the estrogenic effect in ER-positive MCF-7 cells.

Discovery and optimization of novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides as bifunctional antidiabetic agents stimulating both insulin secretion and glucose uptake.

A novel series of 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides was designed, synthesized and evaluated for their biological activities on glucose-stimulated insulin secretion (GSIS). The cytotoxicity of all 41 novel compounds was screened to assess their pharmacological safety in pancreatic ß-cells. A two-step optimization process was carried out to establish the structure-activity relationship for this class and subsequently we identified the most active analogue 26. Further modification study of 26 evidenced the necessity of N-hydrogens in the core architecture. Protein expression analysis suggested that 26 increases insulin secretion via the activation of the upstream effector of pancreatic and duodenal homeobox 1 (PDX-1), which is an important factor promoting GSIS. Moreover, the administration of 26 effectively augmented glucose uptake in C2C12 myotube cells via the suppression of Mitsugumin 53 (MG53), an insulin receptor substrate 1 (IRS-1) ubiquitination E3 ligase.

Transcriptome analysis in rhesus macaques infected with hepatitis E virus genotype 1/3 infections and genotype 1 re-infection.

Hepatitis E virus (HEV) genotype 1 (gt1) and gt3 infections have distinct epidemiologic characteristics and genotype-specific molecular mechanisms of pathogenesis are not well characterized. Previously, we showed differences in immune response-related gene expression profiles of HEV gt1 and gt3 infections using qPCR. We hypothesize that HEV gt1 and gt3 infections induce transcriptome modifications contributing to disease pathogenesis. RNAseq analysis was performed using liver biopsy samples of naïve (baseline), HEV gt1, or gt3-infected rhesus macaques, and nine anti-HEV positive rhesus macaques re-inoculated with HEV gt1. All 10 primary HEV gt1/gt3 infected animals exhibited the typical course of acute viral hepatitis and cleared the infection between 27 to 67 days after inoculation. Viremic stages of HEV infection were defined as early, peak, and decline based on HEV RNA titers in daily stool specimens. During early, peak, and decline phases of infection, HEV gt1 induced 415, 417, and 1769 differentially expressed genes, respectively, and 310, 678, and 388 genes were differentially expressed by HEV gt3, respectively (fold change ≥ 2.0, p-value ≤ 0.05). In the HEV gt1 infection, genes related to metabolic pathways were differentially expressed during the three phases of infection. In contrast, oxidative reduction (early phase), immune responses (peak phase), and T cell cytokine production (decline phase) were found to be regulated during HEV gt3 infection. In addition, FoxO and MAPK signaling pathways were differentially regulated in re-infected and protected animals against HEV gt1 reinfection, respectively. Significant differences of hepatic gene regulation exist between HEV gt1 and gt3 infections. These findings reveal a new link between molecular pathogenesis and epidemiological characteristics seen in HEV gt1 and gt3 infections.

Feasibility of Hepatitis B Vaccination by Microneedle Patch: Cellular and Humoral Immunity Studies in Rhesus Macaques.

BACKGROUND: This study evaluated dissolvable microneedle patch (dMNP) delivery of hepatitis B vaccine in rhesus macaques and provides evidence that dMNP delivery elicits seroprotective anti-HBs levels comparable with human seroprotection, potentially useful for hepatitis B birth dose vaccination in resource-constrained regions. METHODS: Sixteen macaques were each vaccinated twice; they were treated in 4 groups, with dMNP delivery of AFV at 24 ± 8 µg (n = 4) or 48 ± 14 µg (n = 4), intramuscular injection of AFV (10 µg; n = 4), or intramuscular injection of AAV (10 µg; n = 4). Levels of antibody to hepatitis B surface antigen (HBsAg) (anti-HBs) and HBsAg-specific T-cell responses were analyzed. RESULTS: Six of 8 animals with dMNP delivery of AFV had anti-HBs levels ≥10 mIU/mL after the first vaccine dose. After dMNP delivery of AFV, interferon γ, interleukin 2, and interleukin 4 production by HBsAg-specific T cells was detected. A statistically significant positive correlation was detected between anti-HBs levels and cells producing HBsAg-specific interferon γ and interleukin 2 (T-helper 1-type cytokine) and interleukin 4 (T-helper 2-type cytokine) in all anti-HBs-positive animals. CONCLUSIONS: dMNP delivery of AFV can elicit seroprotective anti-HBs levels in rhesus macaques that are correlated with human seroprotection, and it could be particularly promising for birth dose delivery of hepatitis B vaccine in resource-constrained regions.

Analysis of IgG Anti-HEV Antibody Protective Levels During Hepatitis E Virus Reinfection in Experimentally Infected Rhesus Macaques.

BACKGROUND: Secondary spread of hepatitis E virus (HEV) infection occurs often in endemic settings in developing countries. The host immune signatures contributing to protection against subsequent HEV reinfection are unknown. METHODS: Twelve seroconverted rhesus macaques were reinoculated with homologous HEV genotype 1 (gt1, Sar-55) and followed for 115 days. HEV RNA, HEV-specific T-cell responses, IgG anti-HEV antibody, and the IgG anti-HEV avidity index were tested. RESULTS: Four animals with baseline IgG anti-HEV levels from 1.5 to 13.4 World Health Organization (WHO) U/mL evidenced reinfection as determined by HEV RNA in stool, and increase in IgG anti-HEV levels between 63- and 285-fold (P = .003). Eight animals with baseline IgG anti-HEV levels from 2.8 to 90.7 WHO U/mL did not develop infection or shed virus in feces, and IgG anti-HEV antibody levels were unchanged (P = .017). The 4 reinfected animals showed a lower HEV-IgG avidity index (average 35.5%) than the 8 protected animals (average 62.1%). HEV-specific interferon-gamma-producing T cells were 2-fold higher in reinfected animals (P = .018). CONCLUSIONS: Preexisting antibody and high IgG avidity index (>50%) are important factors for protection against HEV reinfection. HEV-specific T-cell responses were elevated in reinfected animals after subsequent exposure to HEV.

Hepatitis B vaccination using a dissolvable microneedle patch is immunogenic in mice and rhesus macaques.

Chronic Hepatitis B virus infection remains a major global public health problem, accounting for about 887,000 deaths in 2015. Perinatal and early childhood infections are strongly associated with developing chronic hepatitis B. Adding a birth dose of the hepatitis B vaccine (HepB BD) to routine childhood vaccination can prevent over 85% of these infections. However, HepB BD coverage remains low in many global regions, with shortages of birth attendants trained to vaccinate and limited HepB BD supply at birth. To address the challenges, we developed coated metal microneedle patches (cMNPs) and dissolvable microneedle patches (dMNPs) that deliver adjuvant-free hepatitis B vaccine to the skin in a simple-to-administer manner. The dMNP contains micron-scale, solid needles encapsulating vaccine antigen and dissolve in the skin, generating no sharps waste. We delivered HepB BD via cMNP to BALB/c mice and via dMNP to both mice and rhesus macaques. Both cMNP and dMNP were immunogenic, generating hepatitis B surface antibody levels similar to human seroprotection. Biomechanical analysis showed that at high forces the microneedles failed mechanically by yielding but microneedles partially blunted by axial compression were still able to penetrate skin. Overall, this study indicates that with further development, dMNPs could offer a method of vaccination to increase HepB BD access and reduce needle waste in developing countries.

The definition and diagnosis of cold hypersensitivity in the hands and feet: Finding from the experts survey.

BACKGROUND: Cold hypersensitivity in the hands and feet (CHHF) is a symptom patients usually feel cold in their hands and feet, but not dealt with a disease in western medicine. However, it is often appealed by patients at a clinic of Korean medicine (KM), considered to be a sort of key diagnostic indicator, and actively treated by physicians. Nevertheless, there is no standardized diagnostic definition for CHHF. Therefore, we surveyed KM experts' opinions to address the clinical definition, diagnostic criteria, and other relevant things on CHHF. METHODS: We developed a survey to assess the definition, diagnosis, causes, and accompanying symptoms on CHHF. 31 experts who work at specialized university hospitals affiliated with KM hospitals consented to participation. Experts responded to survey questions by selecting multiple-choice answers or stating their opinions. RESULTS: Vast majority of experts (83.8%) agreed with our definition on CHHF ("a feeling of cold as a symptom; that one's hands or feet become colder than those of average people in temperatures that are not normally perceived as cold"). 77.4% of experts considered subjective symptoms on CHHF were more important than medical instrument results. Constitution or genetic factors (87.1%) and stress (64.5%) were the most common causes reported for CHHF. CONCLUSIONS: This study offers an expert consensus regarding the themes, opinions, and experiences of practitioners with CHHF. Our results underscore the need for standardized definitions and diagnostic criteria for CHHF.

Banxia Xiexin tang for gastro-oesophageal reflux disease: A protocol for a systematic review of controlled trials.

BACKGROUND: Gastrooesophageal reflux disease (GORD) is one of the most common gastrointestinal diseases encountered in clinical practice. The aim of the present study is thus to systematically review the literature, including Asian studies, to assess the efficacy and safety of Banxia Xiexin tang (BXT) for the treatment of GORD. METHODS AND ANALYSIS: Eleven databases will be searched for studies conducted through March 2018. We will include randomized controlled trials (RCTs) of BXT as a treatment for GORD. All RCTs on BXT or related formulations will be included. The risk of bias will be assessed using the Cochrane Risk of Bias Assessment Tool, while confidence in the cumulative evidence will be evaluated using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) tool. ETHICS AND DISSEMINATION: This systematic review will be published in a peer-reviewed journal and will also be disseminated electronically and in print. The review will be updated to inform and guide healthcare practices. REGISTRATION NUMBER: CRD42018087056.

Protective effect of casuarinin against glutamate-induced apoptosis in HT22 cells through inhibition of oxidative stress-mediated MAPK phosphorylation.

Glutamate is the major excitatory neurotransmitter in the central nervous system and is involved in oxidative stress during neurodegeneration. In the present study, casuarinin prevented glutamate-induced HT22 murine hippocampal neuronal cell death by inhibiting intracellular reactive oxygen species (ROS) production. Moreover, casuarinin reduced chromatin condensation and annexin-V-positive cell production induced by glutamate. We also confirmed the underlying protective mechanism of casuarinin against glutamate-induced neurotoxicity. Glutamate markedly increased the phosphorylation of extracellular signal regulated kinase (ERK)-1/2 and p38, which are crucial in oxidative stress-mediated neuronal cell death. Conversely, treatment with casuarinin diminished the phosphorylation of ERK1/2 and P38. In conclusion, the results of this study suggest that casuarinin, obtained from natural products, acts as potent neuroprotective agent by suppressing glutamate-mediated apoptosis through the inhibition of ROS production and activation of the mitogen activated protein kinase (MAPK) pathway. Thus, casuarinin can be a potential therapeutic agent in the treatment of neurodegenerative diseases.

Interferon-alpha treatment rapidly clears Hepatitis E virus infection in humanized mice.

Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immunological determinants of viral persistence remain largely unexplored. We studied the antiviral potency of pegylated interferon-α (pegIFNα) against HEV infections in humanized mice and modelled intrahepatic interferon stimulated gene (ISG) responses. Human gene expression levels in humanized mouse livers were analyzed by qPCR and Nanostring. Human CXCL10 was measured in mouse serum. HEV genotype 3 (gt3) infections were cleared from liver and feces within 8 pegIFNα doses in all mice and relapsed after a single pegIFNα injection in only half of treated animals. Rapid viral clearance by pegIFNα was confirmed in HEV gt1, but not in Hepatitis B Virus infected animals. No ISG induction was observed in untreated HEV gt3 and gt1 infected humanized livers compared to control chimeric mice, irrespective of the human hepatocyte donor, viral isolate or HEV infection duration. Human specific ISG transcript levels in mouse liver increased significantly after pegIFNα treatment and induced high circulating human CXCL10 in mouse serum. In conclusion, HEV gt1 and gt3 infections do not elicit innate intrahepatic immune responses and remain highly sensitive to pegIFNα in immunocompromised humanized mice.

INHIBITORY EFFECT OF EMODIN ON RAW 264.7 ACTIVATED WITH DOUBLE STRANDED RNA ANALOGUE POLY I:C.

BACKGROUND: Emodin (3-methyl-1, 6, 8-trihydroxyanthraquinone) is a compound which can be found in Polygoni Multiflori Radix (PMR). PMR is the root of Polygonum multiflorum. PMR is used to treat dizziness, spermatorrhea, sores, and scrofula as well as chronic malaria traditionally in China and Korea. The anti-tumor property of emodin was already reported. However, anti-viral activity of emodin on macrophages are not fully reported. MATERIALS AND METHODS: Effects of emodin on RAW 264.7 mouse macrophages induced by polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA, were evaluated. RESULTS: Emodin restored the cell viability in poly I: C-induced RAW 264.7 at concentrations of up to 50 µM. Emodin significantly inhibited the production of nitric oxide, IL-1α, IL-Ιß, IL-6, GM-CSF, G-CSF, M-CSF, MCP-1, MIP-1a, MIP-Ιß, MIP-2, RANTES, and IP-10 as well as calcium release and mRNA expression of signal transducer and activated transcription 1 (STAT1) in poly I:C-induced RAW 264.7 (P < 0.05). CONCLUSION: This study shows the inhibitory effect of emodin on poly I: C-induced RAW 264.7 via calcium-STAT pathway.

Protective effect of lanostane triterpenoids from the sclerotia of Poria cocos Wolf against cisplatin-induced apoptosis in LLC-PK1 cells.

Cisplatin-induced nephrotoxicity is a serious adverse effect that limits the use of cisplatin in cancer patients. In the present study, we investigated the protective effect of lanostane triterpenoids (1-10) isolated from the ethanolic extract of Poria cocos Wolf against cisplatin-induced cell death in LLC-PK1 kidney tubular epithelial cells. Treatment of cisplatin induced significant cell death, which was suppressed by treatment with dehydroeburicoic acid monoacetate (1) and 3ß-acetoxylanosta-7,9(11),24-trien-21-oic acid (9). Compound 1 exhibited the highest efficacy among the tested compounds and was thus subjected to further mechanistic studies. The increase in the percentage of apoptotic cells induced by cisplatin reduced by 4.3% after co-treatment of cells with compound 1 (50 and 100µM). Furthermore, phosphorylation of the mitogen-activated protein kinases JNK, ERK, and p38, and caspase-3, which characterize oxidative stress-mediated apoptosis, increased significantly after treatment with cisplatin, and decreased after treatment with compound 1. These results indicate that the renoprotective effects of compound 1 may be mediated by its anti-apoptotic activity.

Inhibitory effect of Angelica gigas on cold­induced RhoA activation in vascular cells.

The herbal extract Angelica gigas (AG) has been applied as a vasodilating agent for patients suffering from vascular diseases for many years; however, the underlying mechanism has not been fully elucidated. The present study hypothesized that the anti­vasoconstrictive effect of AG may be effective in the treatment of abnormal cold­mediated vasospasms that occur in Raynaud's phenomenon (RP). The effect of AG on the activity of ras homolog gene family member A (RhoA) was investigated in cold­exposed vascular cells. Vascular cells were pretreated to AG, followed by a warm (37˚C) or cold (25˚C) incubation for 30 min and investigated with western blotting, ELISA and confocal microscopy. Cold treatment induced the activation of RhoA in pericytes and vascular endothelial cells, however this was reduced by treatment with AG. Furthermore, AG treatment reduced the endothelin­1 (ET­1)­mediated RhoA activation in pericytes; however, cold­induced ET­1 production by vascular endothelial cells was not affected by treatment with AG. In addition, AG treatment suppressed the formation of stress fibers and focal adhesion complexes, and the cold­induced phosphorylation of focal adhesion kinase, proto­oncogene tyrosine­protein kinase Src and extracellular signal­related kinase. Therefore, AG treatment demonstrated an ability to reduce cold­induced RhoA activation in pericytes and vascular endothelial cells, and attenuated ET­1­mediated RhoA activation in pericytes. In conclusion, the present study indicated that AG may be useful for the treatment of RP.