Effects of 17ß-Estradiol on Colonic Permeability and Inflammation in an Azoxymethane/Dextran Sulfate Sodium-Induced Colitis Mouse Model.

Background/Aims: Intestinal barrier dysfunction is a hallmark of inflammatory bowel diseases (IBDs) such as ulcerative colitis. This dysfunction is caused by increased permeability and the loss of tight junctions in intestinal epithelial cells. The aim of this study was to investigate whether estradiol treatment reduces colonic permeability, tight junction disruption, and inflammation in an azoxymethane (AOM)/dextran sodium sulfate (DSS) colon cancer mouse model. Methods: The effects of 17ß-estradiol (E2) were evaluated in ICR male mice 4 weeks after AOM/DSS treatment. Histological damage was scored by hematoxylin and eosin staining and the levels of the colonic mucosal cytokine myeloperoxidase (MPO) were assessed by enzyme-linked immunosorbent assay (ELISA). To evaluate the effects of E2 on intestinal permeability, tight junctions, and inflammation, we performed quantitative real-time polymerase chain reaction and Western blot analysis. Furthermore, the expression levels of mucin 2 (MUC2) and mucin 4 (MUC4) were measured as target genes for intestinal permeability, whereas zonula occludens 1 (ZO-1), occludin (OCLN), and claudin 4 (CLDN4) served as target genes for the tight junctions. Results: The colitis-mediated induced damage score and MPO activity were reduced by E2 treatment (p＜0.05). In addition, the mRNA expression levels of intestinal barrier-related molecules (i.e., MUC2, ZO-1, OCLN, and CLDN4) were decreased by AOM/DSS-treatment; furthermore, this inhibition was rescued by E2 supplementation. The mRNA and protein expression of inflammation-related genes (i.e., KLF4, NF-κB, iNOS, and COX-2) was increased by AOM/DSS-treatment and ameliorated by E2. Conclusions: E2 acts through the estrogen receptor ß signaling pathway to elicit anti-inflammatory effects on intestinal barrier by inducing the expression of MUC2 and tight junction molecules and inhibiting pro-inflammatory cytokines.

Regulatory T Cells Contribute to the Inhibition of Radiation-Induced Acute Lung Inflammation via Bee Venom Phospholipase A2 in Mice.

Bee venom has long been used to treat various inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. Previously, we reported that bee venom phospholipase A2 (bvPLA2) has an anti-inflammatory effect through the induction of regulatory T cells. Radiotherapy is a common anti-cancer method, but often causes adverse effects, such as inflammation. This study was conducted to evaluate the protective effects of bvPLA2 in radiation-induced acute lung inflammation. Mice were focally irradiated with 75 Gy of X-rays in the lung and administered bvPLA2 six times after radiation. To evaluate the level of inflammation, the number of immune cells, mRNA level of inflammatory cytokine, and histological changes in the lung were measured. BvPLA2 treatment reduced the accumulation of immune cells, such as macrophages, neutrophils, lymphocytes, and eosinophils. In addition, bvPLA2 treatment decreased inflammasome-, chemokine-, cytokine- and fibrosis-related genes' mRNA expression. The histological results also demonstrated the attenuating effect of bvPLA2 on radiation-induced lung inflammation. Furthermore, regulatory T cell depletion abolished the therapeutic effects of bvPLA2 in radiation-induced pneumonitis, implicating the anti-inflammatory effects of bvPLA2 are dependent upon regulatory T cells. These results support the therapeutic potential of bvPLA2 in radiation pneumonitis and fibrosis treatments.

Apios americana Medik Extract Alleviates Lung Inflammation in Influenza Virus H1N1- and Endotoxin-Induced Acute Lung Injury.

Apios americana Medik (hereinafter Apios) has been reported to treat diseases, including cancer, hypertension, obesity, and diabetes. The therapeutic effect of Apios is likely to be associated with its anti-inflammatory activity. This study was conducted to evaluate the protective effects of Apios in animal models of acute lung injury induced by lipopolysaccharide (LPS) or pandemic H1N1 2009 influenza A virus (H1N1). Mice were exposed to LPS or H1N1 for 2-4 days to induce acute lung injury. The treatment groups were administered Apios extracts via oral injection for 8 weeks before LPS treatment or H1N1 infection. To investigate the effects of Apios, we assessed the mice for in vivo effects of Apios on immune cell infiltration and the level of pro-inflammatory cytokines in the bronchoalveolar lavage (BAL) fluid, and histopathological changes in the lung. After induction of acute lung injury, the numbers of neutrophils and total cells were lower in the Apios-treated groups than in the non-Apios-treated LPS and H1N1 groups. The Apios groups tended to have lower levels of tumor necrosis factor-a and interleukin-6 in BAL fluid. In addition, the histopathological changes in the lungs were markedly reduced in the Apios-treated groups. These data suggest that Apios treatment reduces LPS- and H1N1-induced lung inflammation. These protective effects of Apios suggest that it may have therapeutic potential in acute lung injury.

Expression levels of the hypothalamic AMPK gene determines the responsiveness of the rats to electroacupuncture-induced analgesia.

BACKGROUND: Although electroacupuncture (EA) relieves various types of pain, individual differences in the sensitivity to EA analgesia have been reported, causing experimental and clinical difficulties. Our functional genomic study using cDNA microarray identified that 5'-AMP-activated protein kinase (AMPK), a well-known factor in the regulation of energy homeostasis, is the most highly expressed gene in the hypothalamus of the rats that were sensitive to EA analgesia ("responder"), as compared to the rats that were insensitive to EA analgesia ("non-responder"). In this study, we investigated the causal relationship between the hypothalamic AMPK and the individual variation in EA analgesia. METHODS: Sprague-Dawley (SD) rats were divided into the responder and the non-responder groups, based on EA-induced analgesic effects in the tail flick latency (TFL) test, which measures the latency of the tail flick response elicited by radiant heat applied to the tail. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to quantify the expression levels of AMPK mRNA in the hypothalamus of the responder and non-responder rats. Further, we examined whether viral manipulation of the AMPK expression in the hypothalamus modulates EA analgesia in rats. RESULTS: The real-time RT-PCR analysis showed that mRNA expression levels of AMPK in the hypothalamus of the responder rats are significantly higher than those of the non-responder rats, validating the previous microarray results. Microinjection of dominant negative (DN) AMPK adenovirus, which inhibits AMPK activity, into the rat hypothalamus significantly attenuates EA analgesia (p < 0.05), whereas wild type (WT) AMPK virus did not affect EA analgesia (p > 0.05). CONCLUSIONS: The present results demonstrated that levels of AMPK gene expression in the rat hypothalamus determine the individual differences in the sensitivity to EA analgesia. Thus, our findings provide a clinically useful evidence for the application of acupuncture or EA for analgesia.

The effects of maekmoondong-tang on cockroach extract-induced allergic asthma.

Maekmoondong-tang (MMDT) has long been used in Asian countries to treat respiratory diseases. However, the precise mechanisms underlying its effects on asthma are unknown. This study was conducted to evaluate the protective effects of MMDT in a cockroach allergen (CKA-)induced animal model of allergic asthma. After being challenged with CKA, the number of macrophages, eosinophils, neutrophils, lymphocytes, and total cells in the bronchoalveolar lavage fluid (BALF) was evaluated. The Th2 specific cytokines IL-4, IL-5, and IL-13 were also analyzed in BALF along with IgE levels in serum. For histological analysis, hematoxylin and eosin (H&E) staining, periodic acid-Schiff (PAS) staining, and immunohistochemical staining were performed. In addition, airway hyperresponsiveness was assessed by noninvasive plethysmography. The cellular profiles and histopathologic analysis demonstrated that peribronchial and perivascular inflammatory cell infiltrates were significantly decreased in the MMDT-treated groups compared with the cockroach extract-injected (CKA) groups. In addition, the IgE, IL-4, IL-5, and IL-13 levels were significantly decreased in the MMDT group. MMDT treatment also significantly attenuated airway hyperresponsiveness. These results demonstrated that MMDT significantly reduced the hallmark signs of asthma: elevated serum IgE, airway eosinophilia, airway remodeling, mucus hypersecretion, and airway hyperresponsiveness. The remarkable antiasthmatic effects of MMDT suggest its therapeutic potential in allergic asthma treatment.

The effects of Gamijinhae-tang on elastase/lipopolysaccharide-induced lung inflammation in an animal model of acute lung injury.

BACKGROUND: Gamijinhae-tang (GJHT) has long been used in Korea to treat respiratory diseases. The therapeutic effect of GJHT is likely associated with its anti-inflammatory activity. However, the precise mechanisms underlying its effects are unknown. This study was conducted to evaluate the protective effects of GJHT in a porcine pancreatic elastase (PPE) and lipopolysaccharide(LPS) induced animal model of acute lung injury (ALI). METHODS: In this study, mice were intranasally exposed to PPE and LPS for 4 weeks to induce chronic obstructive pulmonary disease (COPD)-like lung inflammation. Two hours prior to PPE and LPS administration, the treatment group was administered GJHT extracts via an oral injection. The numbers of neutrophils, lymphocytes, macrophages and total cells in the bronchoalveolar lavage (BAL) fluid were counted, and pro-inflammatory cytokines were also measured. For histologic analysis, hematoxylin and eosin (H&E) stains and periodic acid-Schiff (PAS) stains were evaluated. RESULTS: After inducing ALI by treating mice with PPE and LPS for 4 weeks, the numbers of neutrophils, lymphocytes and total cells were significantly lower in the GJHT group than in the ALI group. In addition, the IL-1ß and IL-6 levels were significantly decreased in the GJHT group. The histological results also demonstrated the attenuation effect of GJHT on PPE- and LPS-induced lung inflammation. CONCLUSIONS: The results of this study indicate that GJHT has significantly reduces PPE- and LPS-induced lung inflammation. The remarkable protective effects of GJHT suggest its therapeutic potential in COPD treatment.

Microarray analysis of the gene expression profile of HMC-1 mast cells following Schizonepeta tenuifolia Briquet treatment.

It has long been believed that mast cells play a crucial role in the development of many physiological changes during immediate allergic responses. This study was conducted to evaluate the anti-inflammation mechanism of Schizonepeta tenuifolia (ST) extract and ST purified chemicals on the PMA plus A23187-induced stimulation of HMC-1 human mast cells. ST, rosmarinic acid, pulegone, and 2α,3α,24-thrihydrooxylen-12en-28oic acid treatment of HMC-1 cells led to significant suppression of pro-inflammatory cytokines (IL-6, IL-8, and TNF-α) in a dose dependent manner. In addition, the results of the microarray and real-time RT-PCR analyses revealed that ST regulates several pathways, including the cytokine-cytokine receptor interaction (CCRI), MAPK, and the Toll-like receptor (TLR) signaling pathways. ST may be useful for the treatment of inflammation disease via anti-inflammation activity that occurs through inhibition of the CCRI, MAPK, and TLR signaling pathways.

Electroacupuncture Attenuates Ovalbumin-Induced Allergic Asthma via Modulating CD4(+)CD25(+) Regulatory T Cells.

A mouse pulmonary hypersensitivity experimental model that mimics human asthma was developed, and electroacupuncture (EA) treatment was shown to reduce allergic inflammatory processes. In addition, we also assessed whether the beneficial effects of EA on allergic asthma could be correlated with CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg). Cellular profiles and histopathologic analysis demonstrated that peribronchial and perivascular inflammatory cell infiltrates were significantly decreased in the EA-treated groups when compared to the OVA and anti-CD25 Ab-injected (Treg depletion) groups. Furthermore, total BAL cells were reduced in the EA groups when compared to other groups. Interestingly, the population of CD4(+)CD25(+)Foxp3(+)Tregs in pneumonocytes increased in EA-treated group when compared to OVA and Treg depletion groups. These results imply that EA stimulation at ST 36 may affect CD4(+)CD25(+)Foxp3(+) Treg in an OVA-induced experimental model and may enhance Treg function by suppressing other T cells and limiting the immune response.

Safety and efficacy assessment of standardized herbal formula PM012.

BACKGROUND: This study was conducted to evaluate the efficacy of the herbal formula PM012 on an Alzheimer's disease model, human presenilin 2 mutant transgenic mice (hPS2m), and also to evaluate the toxicity of PM012 in Sprague-Dawely rats after 4 or 26 weeks treatment with repeated oral administration. METHODS: Spatial learning and memory capacities of hPS2m transgenic mice were evaluated using the Morris Water Maze. Simultaneously, PM012 was repeatedly administered orally to male and female SD rats (15/sex/group) at doses of 0 (vehicle control), 500, 1,000 and 2,000 mg/kg/day for 4 or 26 weeks. To evaluate the recovery potential, 5 animals of each sex were assigned to vehicle control and 2,000 mg/kg/day groups during the 4-week recovery period. RESULTS: The results showed that PM012-treated hPS2m transgenic mice showed significantly reduced escape latency when compared with the hPS2m transgenic mice. The repeated oral administration of PM012 over 26 weeks in male and female rats induced an increase and increasing trend in thymus weight in the female treatment groups (main and recovery groups), but the change was judged to be toxicologically insignificant. In addition, the oral administration of the herbal medicine PM012 did not cause adverse effects as assessed by clinical signs, mortality, body weight, food and water consumption, ophthalmology, urinalysis, hematology, serum biochemistry, blood clotting time, organ weights and histopathology. The No Observed Adverse Effects Levels of PM012 was determined to be 2,000 mg/kg/day for both sexes, and the target organ was not identified. CONCLUSION: These results suggest that PM012 has potential for use in the treatment of the Alzheimer's disease without serious adverse effects.

The genome-wide expression profile of Curcuma longa-treated cisplatin-stimulated HEK293 cells.

AIM: The rhizome of turmeric, Curcuma longa (CL), is a herbal medicine used in many traditional prescriptions. It has previously been shown that CL treatment showed greater than 47% recovery from cisplatin-induced cell damage in human kidney HEK 293 cells. This study was conducted to evaluate the recovery mechanisms of CL that occur during cisplatin induced nephrotoxicity by examining the genome wide mRNA expression profiles of HEK 293 -cells. METHOD: Recovery mechanisms of CL that occur during cisplatin-induced nephrotoxicity were determined by microarray, real-time PCR, immunofluorescent confocal microscopy and Western blot analysis. RESULTS: The results of microarray analysis and real-time PCR revealed that NFκB pathway-related genes and apoptosis-related genes were down-regulated in CL-treated HEK 293 cells. In addition, immunofluorescent confocal microscopy and Western blot analysis revealed that NFκB p65 nuclear translocation was inhibited in CL-treated HEK 293 cells. Therefore, the mechanism responsible for the effects of CL on HEK 293 cells is closely associated with regulation of the NFκB pathway. CONCLUSION: CL possesses novel therapeutic agents that can be used for the prevention or treatment of cisplatin-induced renal disorders.

The genome-wide expression profile of electroacupuncture in DNP-KLH immunized mice.

Previously, we demonstrated that electoracupuncture (EA) suppressed allergic reactions in DNP-KLH immunized mice. In this study, the mechanisms by which EA induces immunomodulation in the immunized mice were evaluated by genome-wide microarray analysis. The anti-allergic effects of EA in DNP-KLH immunized mice were confirmed by analyzing antigen specific IgE using ELISA. Microarray analysis, followed by real time RT-PCR validation, revealed that Th1 and Th17 cytokine-, opioid peptide-, and anti-apoptosis-related genes were up-regulated upon treatment with EA. In addition, significant decreases in Th2 cytokine-, MAPK signaling pathway-, and apoptosis-related genes were observed following EA treatment.

Inhibition effects of Vitex rotundifolia on inflammatory gene expression in A549 human epithelial cells.

BACKGROUND: Vitex rotundifolia has long been used in traditional medicine to treat asthma and other allergic diseases. OBJECTIVE: To evaluate the anti-inflammatory mechanisms of V rotundifolia in cultured A549 human alveolar epithelial cells. METHODS: In the present study, A549 cells were stimulated with tumor necrosis factor alpha, interleukin 4, and interleukin 1beta to induce expression of chemokines and adhesion molecules involved in eosinophil chemotaxis. The anti-inflammatory effects of V rotundifolia on stimulated A549 cells were then evaluated by analyzing eotaxin secretion and eosinophil migration. In addition, the effects of V rotundifolia on gene expression profiles in stimulated A549 cells were evaluated by oligonucleotide microarray and real-time reverse transcription-polymerase chain reaction (RTRP). RESULTS: The V rotundifolia-treated A549 cells had significantly suppressed eotaxin secretion and eosinophil migration in a dose-dependent manner. In addition, the results of the microarray analysis and RTRP revealed that inflammation-related genes and cell adhesion-related genes were down-regulated in V rotundifolia-treated A549 cells. Furthermore, several genes related to the mitogen-activated protein kinase pathway were down-regulated in V rotundifolia-treated A549 cells. CONCLUSIONS: The mechanism responsible for the effects of V rotundifolia on A549 cells is closely associated with regulation of the mitogen-activated protein kinase pathway. Thus, V rotundifolia may be useful in the treatment of asthma and other allergic diseases.

The genome-wide expression profile of Nelumbinis semen on lipopolysaccharide-stimulated BV-2 microglial cells.

This study was conducted to evaluate the protective mechanisms of Nelumbinis semen (NS) on lipopolysaccharide (LPS)-induced activation of BV-2 microglial cells. The anti-inflammatory effects of NS were determined by analyzing nitric oxide production and proinflammatory cytokines using enzyme-linked immunosorbent assay. The mechanism was evaluated in BV-2 cells with or without NS treated with LPS for various lengths of time using oligonucleotide microarray and real time reverse transcription-polymerase chain reaction. The oligonucleotide microarray analysis revealed that mitogen activated protein kinase (MAPK) signaling pathway-related genes such as Fgfr3, Fgf12, Rasal2, Nfkb2, Map2k5, Mapk1, Map3k7, and NFatc2 were down-regulated in LPS activated BV-2 cells by pretreatment with NS. In addition, significant decreases in Nos1ap gene expression were observed with NS pretreatment. Cluster linked pathway analysis using the Kyoto Encyclopedia of Genes and Genomes database revealed that the effects of NS were closely associated with the regulation of mitochondria functions. These results suggested that NS can affect the MAPK signaling pathway and mitochondrial functions in BV-2 cells activated with LPS.

Global gene analysis of Erigeron canadensis-treated TNF-alpha-, IL-4- and IL-1beta-stimulated A549 human epithelial cells.

BACKGROUND/AIMS: This study was conducted to evaluate the anti-inflammatory mechanisms of Erigeron canadensis (EC) on the tumor necrosis factor-alpha (TNF-alpha)-, interleukin (IL)-4- and IL-1beta-induced stimulation of A549 cells. METHODS: In the present study, the anti-inflammatory effects of EC on TNF-alpha-, IL-4- and IL-1beta-induced A549 cells were determined by analyzing eotaxin secretion using ELISA. In addition, the effects of ECon gene expression profiles in stimulated A549 cells were evaluated by microarray analysis. RESULTS: Oligonucleotide microarray analysis revealed that inflammatory-related genes such as NOS1, NOS2A, IL-1beta, IL-8 and CSF2 and cell adhesion-related genes such as SELE, MMP3, VCAM1, ICAM1, ITGA7 and ITGB2 were downregulated in EC-treated A549 cells that had been pretreated with TNF-alpha, IL-4 and IL-1beta. In addition, significant decreases in Eotaxin, ICAM, VCAM and IL-8 gene expression were observed in EC-treated A549 cells. CONCLUSIONS: EC has an anti-inflammatory effect in stimulated A549 cells. Microarray-based genomic survey is a high-throughput approach that is suitable for the evaluation of gene expression in cell lines that have been treated with EC.

The genome-wide expression profile of Scrophularia ningpoensis-treated thapsigargin-stimulated U-87MG cells.

The endoplasmic reticulum (ER) is a principal site for protein synthesis, protein folding, calcium storage, and calcium signaling. Thapsigargin (TG), an inducer of ER stress, inhibits ER-associated Ca(2+)-ATPase and disrupts Ca(2+) homeostasis. ER stress plays an important pathogenetic role in Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, and prion protein diseases. This study was conducted to evaluate the protective mechanisms of Scrophularia ningpoensis (SN) extracts and chemicals on TG-stimulated U-87MG cells. In this study, the recovery activities of E-harpagoside (EHA), harpagide (HA), 8-O-E-p-methoxycinnamoylharpagide (MH), aucubin (AB), cinnamic acid (CA), p-coumaric acid (pCA), p-methoxycinnamic acid methyl ester (MME), caffeic acid (CFA), ferulic acid (FA), and (E)-p-methoxycinnamic acid (MA) on TG-stimulated U-87MG cells were evaluated. The results revealed that SN, MME, CFA, and MH showed considerable recovery effects. Therefore, SN, MME, CFA, and MH were selected to evaluate the gene expression profile of U-87MG cells by using microarray analysis and real-time RT-PCR. The results of this analysis revealed that cell cycle, proliferation, protein folding, and anti-apoptosis-related genes were up-regulated in SN, MME, CFA, and MH-treated U-87MG cells. In addition, significant decreases in apoptosis, the MAPK signaling pathway, and mitochondria-related gene expressions were observed in SN-, MME-, CFA-, and MH-treated U-87MG cells. Thus, SN, MME, CFA, and MH might affect neurodegenerative diseases.

Screening of herbal medicines for recovery of acetaminophen-induced nephrotoxicity.

This study was conducted to quantitatively evaluate the recovery effects of herbal medicines on acetaminophen-induced nephrotoxicity. In the present study, the recovery effects of 251 herb medicines on HEK 293 cells that had been damaged by acetaminophen were evaluated using an MTS assay. HEK 293 cells were cultured in 96-well plates and then pretreated with or without 20µM acetaminophen (IC(50) value: 17.5±1.9) for 1h. Next, different herbal medicines were added to the wells, after which the cells were reincubated at 37°C for 24h. After the first round of screening, the candidate herbal medicines were selected based on a recovery rate of greater than 20% and their efficacy were then determined by dose response kinetic analysis. Among these extracts, 8 herbal medicines (Ledebouriella divaricata, Sparganium simplex, Panax ginseng, Aster tataricus, Citrus aurantium, Sanguisorba officianlis, Arisaema consanguineum, and Polygonum aviculare) had a strong recovery effect on acetaminophen-induced damage in HEK 293 cells. Dose response non-linear regression analysis demonstrated that P. aviculare showed the best recovery rate (98%), and that its EC(50) (0.1ng/mL) was the smallest among the screened candidate herbal medicines. Additional studies of these herbal medicines should be conducted to determine if they possess novel therapeutic agents for the prevention or treatment of renal disorders.

Screening of herbal medicines for the recovery of cisplatin-induced nephrotoxicity.

The goal of this study was to quantitatively determine the recovery effects of herbal medicines (HM) on the cisplatin-induced nephrotoxicity. In the present study, the recovery effects of 239 HM on HEK 293 cells that had been damaged by cisplatin were evaluated by a mitochondrial activity MTS assay. After the first round of screening, candidate HM were selected based on a recovery rate of greater than 20%. The efficacy of the selected herbs was then determined by dose response kinetic analysis. Of the extracts evaluated, 7 HM (Paeonia suffruticosa (PS), Curcuma longa (CL), Centipeda minima (CM), Loranthus parasiticus (LP), Pulsatilla dahurica (PD), Sinapis alba (SA), and Scutellaria barbata (SB)) had a strong recovery effect on cisplatin-induced damage in HEK 293 cells. An LDH assay showed that LP, CM, SB, CL, SA, and PS had the best recovery effect, whereas a comet assay indicated that PS, SB, SA, PD, and CL had the best recovery effect. Taken together, these results suggest that SB, CL, PS, and SA are the best candidate HM for the recovery of cisplatin-induced nephrotoxicity. Therefore, additional studies should be conducted to determine if these HM possess novel therapeutic agents that can be used for the prevention or treatment of renal disorders.