HBOA ameliorates CCl4-incuded liver fibrosis through inhibiting TGF-ß1/Smads, NF-κB and ERK signaling pathways.

An ingredient was isolated from Acanthus ilicifolius and identified as 4-hydroxy-2(3H)-benzoxazolone (HBOA). Its protective effects and underlying mechanism on liver fibrosis were investigated. Briefly, rats were intragastrically administrated with 50% CCl4 twice a week for 12 weeks to induce liver fibrosis. Meanwhile, the animals were treated with various medicines from weeks 8 to 12. Then the histological change, serum biochemical index, inflammatory factors and hepatocyte apoptosis were detected. Moreover, the TGF-ß1/Smads, NF-κB and ERK signaling pathways were also detected to illustrate the underlying mechanism. The results showed that HBOA significantly ameliorated CCl4-induced liver injury and collagen accumulation in rats, as evidenced by the histopathologic improvement. Moreover, HBOA markedly decreased hepatocyte apoptosis by regulating the expression levels of caspase-3, -9 and -12, as well as the Bcl-2 family. The mechanism study showed that HBOA significantly decreased the expressions of α-smooth muscle actin (α-SMA) and collagen and inhibited the generation of excessive extracellular matrix (ECM) components by restoring the balance between matrix metalloproteinases (MMPs) and its inhibitor (TIMPs). HBOA markedly alleviated oxidative stress and inflammatory cytokines through inhibiting the NF-κB pathway. In addition, HBOA significantly down-regulated the levels of TGF-ß1, Smad2/3, Smad4 and up-regulated the level of Smad7, inhibiting the TGF-ß1/Smads signaling pathway. Moreover, HBOA significantly blocked the ERK signaling pathway, leading to the inactivation of hepatic stellate cells. This study suggests that HBOA exerts a protective effect against liver fibrosis via modulating the TGF-ß1/Smads, NF-κB and ERK signaling pathways, which will be developed as a potential agent for the treatment of liver fibrosis.

Anti-Atopic Effect of Acorn Shell Extract on Atopic Dermatitis-Like Lesions in Mice and Its Active Phytochemicals.

To investigate the potential effects of acorn shells on atopic dermatitis (AD), we utilized oxazolone (OX)- or 2,4-dinitrochlorobenzene (DNCB)-induced AD-like lesion mouse models. Our research demonstrates that Acorn shell extract (ASE) improved the progression of AD-like lesions, including swelling, which were induced by oxazolone on Balb/c mouse ears. Additionally, ASE significantly decreased the ear thickness (OX: 0.42 ± 0.01 mm, OX-ASE: 0.32 ± 0.02 mm) and epidermal thickness (OX: 75.3 ± 32.6 µm, OX-ASE: 46.1 ± 13.4 µm). The continuous DNCB-induced AD mouse model in SKH-1 hairless mice demonstrated that ASE improved AD-like symptoms, including the recovery of skin barrier dysfunction, Immunoglobulin E hyperproduction (DNCB: 340.1 ± 66.8 ng/mL, DNCB-ASE: 234.8 ± 32.9 ng/mL) and an increase in epidermal thickness (DNCB: 96.4 ± 21.9 µm, DNCB-ASE: 52.4 ± 16.3 µm). In addition, we found that ASE suppressed the levels of AD-involved cytokines, such as Tumor Necrosis Factor α, IL-1ß, IL-25 and IL-33 in both animal models. Furthermore, gallic acid and ellagic acid isolated from ASE suppressed ß-hexosaminidase release and IL-4 expression in RBL-2H3 cells. The acorn shell and its active phytochemicals have potential as a new remedy for the improvement of atopic dermatitis and other inflammatory diseases.

Topical 'Sydney' propolis protects against UV-radiation-induced inflammation, lipid peroxidation and immune suppression in mouse skin.

BACKGROUND: Propolis is a honeybee product that has been used in traditional medicine for antioxidant, immune-stimulating, anti-inflammatory and anti-cancer effects. Here, the potential of the topical application of a crude ethanolic extract of Sydney propolis to protect against UV-radiation-induced impairments associated with an increased risk of photocarcinogenesis has been tested in the hairless mouse. METHODS: Solutions providing between 10 and 200 mg/kg propolis were applied to the skin following UV irradiation. The inflammation from exposure to UV (290-400 nm) was quantitated by measurement of increased skinfold thickness; lipid peroxidation was assayed by the induction of thiobarbituric acid reactive species in the skin; immune function was measured by the contact hypersensitivity (CHS) reaction and supported by the changes in epidermal cytokine expression. RESULTS: Propolis protected significantly and dose-dependently against both sunburn oedema and the suppression of CHS, and (at 100 mg/kg) against lipid peroxidation. The overexpression of IL-10 and the depletion of IL-12 characteristic of photoimmune suppression were markedly reduced by propolis. Further, the upregulation of IL-6 was decreased, and the associated induction of haem oxygenase was shown to play a role in propolis skin protection. CONCLUSIONS: Sydney propolis was able to effectively reduce cutaneous inflammation, immunosuppression and lipid peroxidation induced by UV exposure. It is concluded that Sydney propolis might have strong beneficial protective effects against photodamage and skin cancer development in humans.

The transcription factor NFATc2 controls IL-6-dependent T cell activation in experimental colitis.

The nuclear factor of activated T cells (NFAT) family of transcription factors controls calcium signaling in T lymphocytes. In this study, we have identified a crucial regulatory role of the transcription factor NFATc2 in T cell-dependent experimental colitis. Similar to ulcerative colitis in humans, the expression of NFATc2 was up-regulated in oxazolone-induced chronic intestinal inflammation. Furthermore, NFATc2 deficiency suppressed colitis induced by oxazolone administration. This finding was associated with enhanced T cell apoptosis in the lamina propria and strikingly reduced production of IL-6, -13, and -17 by mucosal T lymphocytes. Further studies using knockout mice showed that IL-6, rather than IL-23 and -17, are essential for oxazolone colitis induction. Administration of hyper-IL-6 blocked the protective effects of NFATc2 deficiency in experimental colitis, suggesting that IL-6 signal transduction plays a major pathogenic role in vivo. Finally, adoptive transfer of IL-6 and wild-type T cells demonstrated that oxazolone colitis is critically dependent on IL-6 production by T cells. Collectively, these results define a unique regulatory role for NFATc2 in colitis by controlling mucosal T cell activation in an IL-6-dependent manner. NFATc2 in T cells thus emerges as a potentially new therapeutic target for inflammatory bowel diseases.

Oxazolone and diclofenac-induced popliteal lymph node assay reactions are attenuated in mice orally pretreated with the respective compound: potential role for the induction of regulatory mechanisms following enteric administration.

The murine popliteal lymph node assay (PLNA) was examined as a preclinical assay with the potential to identify low-molecular-weight compounds (LMWCs) that are likely to be associated with immune-mediated drug hypersensitivity reactions (IDHRs) in humans. We hypothesized that the contact sensitizer oxazolone (OX) would cause a strong PLN reaction in naive mice and that the PLN reaction would be attenuated in mice orally pretreated with OX due to the induction of oral tolerance. In naive mice, OX induced a strong PLN reaction and caused dose-dependent increases in PLN size, weight, cellularity, percentage of CD4(+) PLN T cells, and percentage of PLN B cells, with a concomitant decrease in the percentage of CD8(+) PLN T cells. Next, the PLNA was conducted in mice gavaged three times with either OX or vehicle alone (olive oil). Mice pretreated with OX had suppressed PLN reactions following the footpad injection of OX (decrease in PLN size, weight, and cellularity), which was associated with an increase in the percentage of PLN CD8(+)T cells. In contrast, oral pretreatment with OX had no observable effect on the PLN reaction induced following footpad injection of the irrelevant hapten dinitrochlorobenzene (DNCB). Adoptive transfer studies were conducted to examine the mechanism of PLN hyporesponsiveness. It was found that either (1) unfractionated splenocytes or (2) purified CD8(+) splenocytes, but not (3) purified CD4(+) splenocytes isolated from mice gavaged with OX adoptively transferred PLN suppression to naive BALB/c mice. Because OX is not a pharmaceutical, we also examined the NSAID diclofenac (DF) (Voltaren). Like OX, DF caused dose-dependent increases in PLN size, weight, and cellularity in naive mice. Furthermore, like OX, the diclofenac-induced PLN reaction was attenuated in mice that had been orally pretreated three times with DF. However, splenocytes from mice orally treated with DF were not able to adoptively transfer PLN hyporesponsiveness. Collectively, these observations demonstrate that both OX and DF are potent immunostimulators in the PLNA. As importantly, these results demonstrate that the immunostimulating potential of OX and DF in the PLNA is significantly decreased in mice orally exposed to the respective drug, possibly due to the presence of a cellular mechanism of oral tolerance. For OX, the mechanism appears to involve, in part, CD8(+) T cells, whereas the mechanism(s) associated with PLN hyporesponsiveness using DF remain to be defined.

N,N'-diacetyl-L-cystine (DiNAC), the disulphide dimer of N-acetylcysteine, inhibits atherosclerosis in WHHL rabbits: evidence for immunomodulatory agents as a new approach to prevent atherosclerosis.

Oxidation of lipoprotein-derived lipids is generally accepted to be important in atherogenesis, and lipophilic antioxidants have been suggested as potential antiatherosclerotic agents. The antiatherogenic effects observed by certain antioxidants, especially probucol, in different animal models support this suggestion. There are however also cases where other lipophilic antioxidants have not been able to support this hypothesis. This has raised the question whether the effects of probucol and similar compounds are mainly due to some other property, unrelated to their antioxidant efficacy. For example, probucol is shown to possess immunomodulatory properties. Immune reactions are known to occur during atherogenesis. We therefore tested the dimer of N-acetylcysteine, DiNAC, which is a disulfide with immunomodulating properties and enhances oxazolone-induced contact sensitivity (CS) reactions in mice, for effects on atherosclerosis. When given to male heritable hyperlipidemic rabbit (WHHL) rabbits from 10 to 22 weeks of age, this compound reduced by 50% thoracic aorta atherosclerosis (p < 0.05), without affecting plasma lipid levels. Here we also show that probucol and a close chemical analog, both known to prevent atherosclerosis in WHHL rabbits, enhance the CS reaction in mice, while two other related antioxidants did not affect the CS reaction. At least one of these is also without effect on atherosclerosis in WHHL rabbits. The results show that DiNAC might represent a new treatment modality for atherosclerosis-related disease, and suggest that some antioxidants may have antiatherosclerotic properties more related to "immunomodulatory" properties than to antioxidant properties in general.

Chemical activation of innate and specific immunity in contact dermatitis.

Recent reports have suggested that chemical-induced allergic contact dermatitis may not be a traditional type IV hypersensitivity, in part due to the dual irritant and antigenic properties of sensitizing chemicals. In order to investigate the contribution of these properties to the molecular and cellular mechanism underlying allergic contact dermatitis, we evaluated oxazolone-induced changes in cell populations and cytokine production in the dermis of transgenic mice with impaired innate immunity (the FcgammaR subunit knockout mouse), and absent specific immunity (the athymic mouse), and the appropriate B6,129F2 and C57BL/6 control mice. Oxazolone and croton oil were applied in a single sensitizing dose, or in sensitizing and challenge doses, and the dermal response was evaluated by immunohistochemistry. In the wild type mice, with or without sensitization to oxazolone or croton oil, we observed mixed Th1/Th2 cytokine production and both CD4+ and CD8+ T lymphocytes; however, the neutrophil was the predominant cell in the dermis, even 72 h after final chemical application. Athymic mice displayed a similar neutrophil response with moderate Th1/Th2 cytokine production, and FcgammaR subunit knockout mice exhibited very mild dermatitis when treated with either oxazolone or croton oil. These results provide support for the hypothesis that allergic contact dermatitis is not a classic delayed type hypersensitivity, demonstrate the importance of the interaction between the irritant and antigenic properties of sensitizing chemicals in the development of allergic contact dermatitis, and suggest that the irritant effect of chemicals may be mediated through the cutaneous innate immune system.

Primary immune response in skin and skin-associated lymphoid tissue of interleukin-4 transgenic mice.

The interleukin-4 transgenic mice investigated here exhibit a ubiquitous expression of interleukin-4 in all organs, including the skin. In this study, the induction phase of oxazolone-induced local primary contact hypersensitivity and croton oil-induced irritant contact dermatitis in transgenic and wild-type mice was analysed. Compared to wild-type mice, the transgenic mice showed a decreased activation of the skin-draining lymph nodes but a strong hyperreactivity in the skin after topical sensitisation. In contrast to this, both the transgenic and the wild-type mice developed a strong and comparable inflammatory skin reaction after topical irritation. A striking increased expression level of tumour necrosis factor-alpha and macrophage inflammatory protein-2 genes were found in the skin of the transgenic mice during primary local contact hypersensitivity, while both the transgenic and the wild-type mice developed comparable expression levels of these cytokines during irritant contact dermatitis. Compared to wild-type mice, a strongly enhanced expression level of interleukin-6 transcripts derived from epidermal antigen presenting cells were detected in the skin of IL-4 transgenic mice, whereas in the skin-draining lymph nodes of transgenic mice significantly lower levels were detected. We conclude that the migration of epidermal antigen-presenting cells towards the skin-draining lymph nodes is reduced in transgenic mice, which could be due to the different cytokine balance in these mice strains. The atypical irritant-like reaction observed in transgenic mice after topical sensitisation is a phenomenon comparable to atopic diseases and therefore this transgenic strain might be a helpful model for investigating the immunopathophysiological features of these diseases.

The influence on the dermal cellular infiltrate of topical steroid applications and vehicles in guinea pig skin: normal skin, allergic and toxic reactions.

The mechanisms of the anti-inflammatory effects of corticosteroids are uncertain but could be explained by an influence on infiltrating leukocytes. Our method for the qualitative and quantitative investigation of the dermal cellular infiltrate makes it possible to study the effects of topically applied corticosteroid preparations and vehicles on the infiltrating leukocytes of normal skin, allergic and toxic reactions in guinea pig skin. Ointment and cream vehicles as well as corticosteroid cream and ointment preparations often caused erythema and increased mononuclear infiltrate after only short periods of application (24-72 h). The strongest steroid ointment gave the most marked macroscopic response and propylene glycol preparations the most marked cellular response. In both toxic and allergic reactions, application of steroid preparations after the provocation gave no beneficial result either macroscopically or microscopically. Macroscopic scores were worsened by cream and ointment preparations. Although steroid solutions had no beneficial effect, they caused no detrimental effect. The guinea pig seems to be extremely sensitive to irritants and has not proved to be a suitable model for this approach to the study of the efficacy of topically applied steroids.

Enhancement of contact sensitization in mice fed a diet enriched in vitamin A acetate.

Various methods have been used to enhance the ability of laboratory animals to develop contact sensitivity to so-called weak sensitizers. The present studies have examined whether diets supplemented with vitamin A acetate (VAA) could enhance induction and elicitation of contact sensitivity to oxazolone in the mouse. Application of 0.3% oxazolone to VAA-fed mice resulted in increased DNA synthesis in the draining lymph nodes and increased ear thickness after challenge, in comparison with mice on a stock (standard) diet. Moreover, VAA-fed (but not control) mice were sensitized to 0.03% oxazolone as shown by DNA synthesis, increased arrival of syngeneic 51Cr-labelled lymphocytes in the draining lymph nodes, and ear swelling after challenge. These findings demonstrate that vitamin A enhances delayed hypersensitivity responses, and point to a role for vitamin A in developing an animal model capable of detecting weak contact sensitizers after topical application.