Effects of Xinfeng capsule on pulmonary function based on treg-mediated notch pathway in a rat model of adjuvant arthritis

Objective

To observe the impact of xinfeng xapsule (XFC) on pulmonary function in a rat model of adjuvant arthritis (AA) and to investigate the mechanism of action.

Methods

Forty rats were randomly divided into four groups of ten: normal control (NC); model control (MC); tripterygium glycosides tablet (TPT); and xinfeng capsule (XFC). Except for the NC group, AA was induced in all rats by intracutaneous injection of 0.1 mL Freund's complete adjuvant in the right paw on the 19th day. NC and MC groups were given (0.9%) physiological saline. The TPT and XFC groups were given TPT (10 mg/kg) and XFC (1.2 g/kg), respectively. Thirty days after administration, changes in paw edema (E), the arthritis index (AI), pulmonary function, levels of regulatory T-cells (Treg), ultrastructure of lung tissue, and expression of Notch receptors and ligands in lung tissue were observed.

Results

In the MC group, E and the AI were increased and pulmonary function significantly decreased; the structure of alveolar type-II cells was damaged; ratios of Treg in peripheral blood were reduced; and expression of Notch receptors such as Notch3 and Notch4 and ligands such as Delta1 in lung tissue were significantly increased whereas expression of Notch1, Jagged1 and Jagged2 were significantly decreased. After intervention with XFC, E and the AI were decreased; pulmonary function was enhanced; the structure of alveolar type-II cells was improved; and expression of Treg, Notch1, Jagged1, Jagged2 was elevated, whereas that of Notch3, Notch4 and Delta1 was reduced.

Conclusion

XFC can not only inhibit E and the AI and improve joint symptoms, it can also improve pulmonary function and reduce inflammation in lung tissue. These actions could be carried out through increases in the expression of Treg, Notch receptors (Notch1) and ligands (Jagged1, Jagged2), and reductions in the expression of Notch3, Notch4 and Delta1. These phenomena would reduce the deposition of immune complexes and the inflammatory response in lung tissue, thereby improving joint symptoms and pulmonary function.     

Triptolide promotes generation of FoxP3+ T regulatory cells in rats

Ethnopharmacological relevance

Triptolide (TPT), a component of the Chinese herb Triptergium wilfordii, has potent immunosuppressive and anti-inflammatory activity and is used clinically in recipients of kidney transplantation.

Aim of the study

This work aimed to investigate the effect of TPT on the differentiation of regulatory T lymphocytes (Tregs) from CD4+ cells in rats.

Materials and methods

MACS-purified rat CD4+ cells were costimulated with anti-CD3 and anti-CD28 in the presence of TGF-β to induce the expression of FoxP3, which was detected by flow cytometry. TPT and cyclosporine A (CsA) were separately added into the cultures to observe the effect on the expression of FoxP3. Kidney transplantation was performed in rats that either received no treatment or were treated with TPT after transplantation.

Results

TPT treatment enhanced the expression of FoxP3 in CD4+ cells, whereas CsA inhibited the FoxP3 expression. In the rat kidney transplantation model, the recipient rats treated with TPT survived longer than the control rats (18–19.83 vs 6.83 days, P < 0.05). Meanwhile, the FoxP3+ T cells in the spleens of treated rats were higher than those from the untreated rats (12.4% vs 4.7%, P < 0.05).

Conclusions

These data suggest that TPT may promote the differentiation of CD4+ cells to FoxP3+ Tregs. This would be at least one of the pathways responsible for the immunosuppressive activity of TPT.     

Immunosuppressive effects of an ethyl acetate extract from Urtica dentata Hand on skin allograft rejection

Aim of the study

To investigate the immunosuppressive effects of HPLC qualitied ethyl acetate extract (EAE) from Urtica dentate Hand on skin allograft rejection in a murine model.

Materials and methods

Allo-skin transplantation model was established by placing skin allograft of C57BL/6 mice in the wound bed which was on the back of Balb/c mice. We used FACS to study the effects of EAE on dendritic cells (DCs) maturation and CD4⁺CD25⁺T regulatory cells (Tregs) differentiation. We also studied spleen lymphocyte proliferation and T-bet gene expression in DCs. Concentration of Th1/Th2 cytokines was monitored as markers of Th1/Th2 responses by ELISA.

Results

A significant prolongation of skin allografts survival was observed as a dose-dependent manner in the animals treated with EAE. By FACS, we found that treatment with EAE (200 mg kg⁻¹) resulted in an immature statement of DCs and stimulated the differentiation of CD4⁺CD25⁺Tregs. Additionally, the expression of T-bet gene and the proliferation of spleen lymphocytes were efficiently abated in EAE treated mice. Comparing to the model control, EAE-treated recipients showed a significant down-regulation (P < 0.01) of Th1 cytokines (IL-2, IFN-γ) and an obviously increase (P < 0.01) of Th2 cytokine (IL-10) in the serum, which presented in a dose-related way.

Conclusions

The anti-allograft rejection effect of EAE by enhancing CD4⁺CD25⁺Tregs differentiation and sustaining DCs immaturation makes EAE to be a possible choice for treating autoimmune diseases in a way of inducing a stable immunological tolerance state.     

The effect of the extract of Crocus sativus on tracheal responsiveness and plasma levels of IL-4, IFN-γ, total NO and nitrite in ovalbumin sensitized Guinea-pigs

Ethnomedical relevance

Anti-inflammatory, anti oxidant and effect of Crocus sativus (C. sativus) on Th₁/Th₂ balance were described previously.

Aim of the study

The preventive effects of the extract of Crocus sativus on tracheal responsiveness and plasma levels of IL-4, IFN-γ, total NO and nitrite were examined on sensitized guinea pigs.

Materials and methods

Five groups of sensitized guinea pigs to ovalbumin (OVA), were given drinking water containing three concentrations of the extract of Crocus sativus, dexamethasone (S+D) or alone (group S). Tracheal responses (TR) of control animals (group C) and sensitized guinea pigs (n=6, for each group) to methacholine, OVA and the levels of IL-4, IFN-γ, total NO and nitrite in serum were examined.

Results

The TR to both methacholine and OVA, the levels of serum IL-4, total NO and nitrite in S guinea pigs were significantly increased but that of IFN-γ and IFN-γ/IL-4 ratio (Th₁/Th₂ balance) were decreased compared to the controls (p<0.05 to p<0.001). In the treated animals with dexamethasone and all concentrations of the extract, TR to both methacholine and OVA, IL-4, total NO and nitrite were significantly decreased but IFN-γ and IFN-γ/IL-4 ratio increased compared to S group (p<0.05 to p<0.001). The effects of the highest concentration of the extract was greater than those of other concentrations and the effect of dexamethasone (p<0.05 to p<0.01).

Conclusions

These results not only showed a preventive effect of C. sativus extract on tracheal responses and serum levels of inflammatory mediators in sensitized guinea pigs but also showed increased Th₁/Th₂ balance.     

Th1/Th2 cytokines’ expression and production by propolis-treated mice

Aim of the study

Propolis is a natural product extensively used in food and beverages to improve health and to prevent diseases, showing immunomodulatory properties. The goal of this work was to evaluate the effect of propolis administration over a short-term to mice on Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-10) cytokines’ expression and production.

Materials and methods

Propolis was administered for 3 days to mice by gavage, spleens were removed and RNA was extracted to assess cytokines’ expression by real-time PCR. Supernatants of spleen cell cultures were used for cytokines determination by ELISA.

Results

Propolis administration to mice did not affect IL-2, IL-4 and IL-10 expression and production, while IFN-γ production was inhibited in the splenocyte cultures stimulated or not by Con A.

Conclusions

Since IFN-γ is a pro-inflammatory cytokine, our data suggest that propolis administration over a short-term to mice may be associated with anti-inflammatory effects in vivo, and further assays could check propolis efficiency in inflammatory diseases.     

Total coumarins from Urtica dentata Hand prevent murine autoimmune diabetes via suppression of the TLR4-signaling pathways

Ethnopharmacological relevance

Urtica dentata Hand (UDH), the root of Laportea bulbifera (Sieb. et. Zucc.) Wedd, has been traditionally used in traditional Chinese medicine as an anti-inflammatory and immuno-regulatory agent for rheumatoid arthritis and some other autoimmune diseases treatment. And the coumarins are the major components of UDH.

Aim of the study

To investigate the effect of total coumarins (TC) isolated from UDH on the development of autoimmune diabetes.

Materials and methods

Eight-week-old non-obese diabetic (NOD) mice were randomly divided into four groups: control group, low-dose (37.5 mg/kg), middle-dose (75 mg/kg), and high-dose (150 mg/kg) TC-treatment groups. NOD mice were then given with a suspension of TC or saline by intragastric (i.g.) administration every other day. After 4 weeks of treatment, 8 mice at 12-weeks of age per group were randomly selected to be sacrificed to perform intraperitoneal glucose tolerance test, examine histopathological insulitis, spleen T lymphocyte proliferation, the percentage of CD4+CD25+Foxp3+ T regulatory cell (Treg), dendritic cell (DC) surface molecules, toll-like receptor (TLR)4 expression and signal pathways involved. The remaining 10 mice per group were kept until 26 weeks of age to assess the incidence of diabetes. We also studied the direct effect of TC on DC and CD4+CD25+ Tregs in vitro.

Results

Treatment with TC for 4 weeks significantly inhibited insulitis, increased pancreatic islet number, delayed the onset and decreased the development of diabetes by 26 weeks of age in NOD mice, compared with the untreated control mice. TC suppressed spleen T lymphocyte proliferation, induced Th2-biased cytokine response, the generation of CD4+CD25+Foxp3+ Tregs and Foxp3 mRNA expression. And TC-treated DCs were characterized as low expression of MHC class II and CD86 molecules. TLR4 gene and protein expressions in the spleen, thymus and pancreas were down-regulated in TC-treated groups. The key molecules in the downstream signaling cascades of TLR4, including myeloid differentiation factor (MyD)88, nuclear factor (NF)-κB, IL-1β, Toll–IL-1 receptor domain-containing adaptor inducing interferon-β(TRIF), TRIF-related adaptor molecule (TRAM), interferon regulatory factor (IRF)-3 and IFN-β, all decreased significantly in TC groups, suggesting that TC inhibits both MyD88-dependent and -independent pathways of TLR4. At the cellular level, however, TLR4 protein expression in DCs, but not in Tregs, was downregulated by TC. And TC strengthened the role of DC, not Treg, in negative immune regulation in vitro. In contrast, anti-TLR4 antibody could block the effect of TC on DCs immune function.

Conclusion

These results suggest that TC extracted from UDH prevent the development of autoimmune diabetes in NOD mice via suppression of the TLR4-signaling pathways. TC maintain the DCs in an immature tolerogenic state, at least in part, mediated by down-regulating TLR4-signaling pathways in DCs, then enhance Treg differentiation, shift toward Th2 and suppress T lymphocyte proliferation.     

Regulatory effects of stage-treatment with established Chinese herbal formulas on inflammatory mediators in pediatric asthma

Objective

To explore the regulatory effects of established Chinese herbal formulas on inflammatory mediators released during asthma attacks, and to elucidate the molecular mechanism of Traditional Chinese Medicine in the treatment of asthma.

Methods

Seventy-five asthmatic children were randomly divided into a Chinese medication group (45 cases) and a Western medication control group (30 cases). Patients in the Chinese medication group were treated with a series of established Chinese herbal formulas, whereas the Western medication control group received a leukotriene receptor antagonist and a bronchial relaxant. Real-time PCR was used to determine the mRNA expression levels of interleukin (IL)-4, cysteinyl leukotriene receptor 1 (CysLTR1), and interferon (IFN)-γ in peripheral blood mononuclear cells before and after treatment. Enzyme-linked immunosorbent assay was used to measure the peripheral blood levels of IL-4, leukotriene (LTE)-4, and INF-γ before and after treatment.

Results

After treatment, the mRNA expression levels of IL-4 and CysLTR1 were down-regulated (P< 0.01) and the mRNA expression levels of IFN-γ were up-regulated (P<0.05) in the Chinese medication and Western medication groups; no significant difference was found between the two groups. In the Chinese medication group, IL-4 blood level was decreased and it was significantly different from that in the Western medication group (P<0.05); there was also a significant increase in IFN-γ blood levels after treatment with Chinese medication (P<0.05). There were no significant differences in LTE-4 blood levels between the two groups before and after treatment (P>0.05).

Conclusion

Chinese medication has a regulatory effect on leukotriene receptor gene expression and the imbalance of Th1/Th2 immune cells during asthma attacks in pediatric patients.     

Anti-inflammatory and antinociceptive activities of the ethanolic extract of Bougainvillea xbuttiana

Ethnopharmacological relevance

Bougainvillea xbuttiana is widely distributed in Mexico and it is used as an analgesic in folk medicine.

Aim of the study

In the present study the in vivo antinociceptive and anti-inflammatory effects of the Bougainvillea xbuttiana ethanolic extract have been studied in mice.

Materials and methods

The phytochemical analysis was performed. Antinociceptive activity was evaluated through writhing and formalin test in mice. The anti-inflammatory activity was determined with the carrageenan-induced mice paw oedema model. IL-6, IL-10 and IFN-γ levels were determined by enzyme-like immunosorbent assay, whereas TNF and nitrite levels were detected by standard assay with L929 cells and colorimetric Griess reactive, respectively.

Results

The results showed that the ethanolic extract of the Bougainvillea xbuttiana has significant anti-inflammatory and antinociceptive activities, by inhibition of nociception induced by acetic acid and paw oedema. This extract also induced a decrease in TNF levels and an increase of IL-6, IFN-γ and NO levels that we observed up to 2 h. The highest levels of IL-10 were observed up to 4 h. The ratios of pro-/anti-inflammatory cytokines in sera from mice injected with the ethanolic extract, may be manifesting an anti-inflammatory status.

Conclusions

The present study provides convincing evidences that Bougainvillea xbuttiana extract possesses significant anti-nociceptive and anti-inflammatory effects.     

Topical application of an ethanol extract prepared from Illicium verum suppresses atopic dermatitis in NC/Nga mice

Ethnopharmacological relevance

Illicium verum is a traditional herbal medicine with anti-inflammatory properties used in Asia. However, its usefulness in the treatment of allergic diseases remains unclear. This study evaluated the anti-inflammatory and antiallergic effects of I. verum extract (IVE) in a mouse model of atopic dermatitis.

Materials and methods

We investigated the effects of IVE on compound 48/80-induced histamine release, and phorbol 12-myristate13-acetate and calcium ionophore A23187-stimulated cytokines secretion in MC/9 mast cells. Atopic dermatitis was induced in NC/Nga mice by exposure to extract of house dust mite (Dermatophagoides farinae). After a topical application of IVE on ear and skin lesions, we evaluated the severity of skin symptoms, ear thickness, inflammatory cell infiltration, and serum levels of immunoglobulin E (IgE), histamine, interleukin (IL)-6, and intercellular adhesion molecule (ICAM)-1. In addition, we determined the expression of IL-4, IL-6, tumor necrosis factor (TNF)-α, interferon (IFN)-γ thymus- and activation-regulated chemokine (TARC), regulated on activation, normal T cell expressed and secreted (RANTES), ICAM-1, and vascular cell adhesion molecule (VCAM)-1 in ear tissues.

Results

IVE inhibited secretion of histamine, IL-4, IL-6, and TNF-α from mast cells in a dose-dependent manner. Topical application of IVE significantly reduced dermatitis scores, ear thickness, and serum levels of IgE, histamine, IL-6, and ICAM-1. Histopathological analysis demonstrated decreased epidermal thickening and dermal infiltration by inflammatory cells. In the ear lesions, IVE treatment reduced expression of IL-4, IL-6, TNF-α, TARC, RANTES, ICAM-1, and VCAM-1, but not IFN-γ.

Conclusions

These results indicate that IVE inhibits atopic dermatitis-like skin lesions by suppressing the expression of cytokines, chemokines, and adhesion molecules. These results suggest that IVE may be a potential therapeutic candidate for atopic dermatitis.     

Oral administration of Uncariae rhynchophylla inhibits the development of DNFB-induced atopic dermatitis-like skin lesions via IFN-γ down-regulation in NC/Nga mice

Ethnopharmacological relevance

Uncariae rhynchophylla (UR) is an herb which has blood pressure lowering and anti-inflammatory effects and has been prescribed traditionally to treat stroke and vascular dementia.

Aim of study

In the present study, we examined whether UR suppress Atopic dermatitis (AD)-like skin lesions in NC/Nga mice treated with 2, 4-dinitrofluorobenzene (DNFB) under SPF conditions.

Material and methods

The effect of UR in DNFB- treated NC/Nga mice was determined by measuring the skin symptom severity, levels of serum IgE, and of the amounts of IL-4 and IFN-γ secreted by activated T cells in draining lymph nodes.

Results

Oral administration of UR to DNFB-treated NC/Nga mice was found to inhibit ear thickness increases and the skin lesions induced by DNFB. IFN-γ production by CD4+ T cells from the lymph nodes of DNFB-treated NC/Nga mice was significantly inhibited by UR treatment, although levels of IL-4 and total IgE in serum were not.

Conclusion

UR may suppress the development of AD-like dermatitis in DNFB-treated NC/Nga mice by reducing IFN-γ production.     

Attenuation of airway hyperreactivity and T helper cell type 2 responses by coumarins from Peucedanum praeruptorum Dunn in a murine model of allergic airway inflammation

Ethnopharmacological relevance

The root of Peucedanum praeruptorum Dunn (PPD) is a commonly used traditional Chinese medicine for the treatment of asthma. Its major constituents, coumarins, were presumed to be responsible for its efficacy.

Aim of the study

The potential of coumarins from PPD (CPPD) as anti-asthma agent was investigated.

Materials and methods

Female BALB/c mice were sensitized and challenged with ovalbumin (OVA) to induce allergic airway inflammation. CPPD was administered intragastrically before every OVA challenge. Airway reactivity to the intravenous administration of acetylcholine chloride was measured 48 h after final OVA inhalation. Airway inflammation was evaluated by leukocyte counts of bronchoalveolar lavage fluid (BALF) and histopathological analysis of lung lesions. Levels of interleukin (IL)-4, IL-5, IL-10, IL-13 and IFN-γ in BALF and OVA-specific immunoglobulin (Ig) E in serum, and activity of eosinophil peroxidase (EPO) in lung was measured. The percentage of CD4⁺CD25⁺Foxp3⁺ regulatory T cells among CD4⁺ T cells in spleen was analyzed by flow cytometry.

Results

Compared with model group, CPPD significantly reduced airway hyperreactivity and airway eosinophilic inflammation, improved pathologic lesion of the lungs, reduced levels of IL-4, IL-5, IL-13 in BALF and OVA-specific IgE in serum, inhibited the activities of EPO in lung, and up-regulated levels of IL-10 and IFN-γ in BALF as well as the percentage of CD4⁺CD25⁺Foxp3⁺ regulatory T cells in spleen.

Conclusion

CPPD can significantly suppress OVA-induced airway inflammation, airway hyperreactivity and Th2 predominant response in mice, showing great therapeutic potential for the treatment of allergic asthma.     

Effects of Catalpa ovata stem bark on atopic dermatitis-like skin lesions in NC/Nga mice

Ethnopharmacological relevance

The stem bark of Catalpa ovata has been used as a traditional herbal medicine for the treatment of various inflammatory diseases such as itching and scabies.

Aim of the study

In the present study, we investigated the anti-AD effects of Catalpa ovata stem bark on Dermatophagoides farinae-induced AD in a NC/Nga mouse AD model. We determined dermatitis score, histology, IgE, cytokines, and chemokines related to hypersensitive immune responses in AD. The mechanism of action was also investigated using HaCaT cells.

Materials and methods

We investigated the topical effects of Catalpa ovata stem bark on AD-like skin lesions in NC/Nga mice. Five category-experiments were performed, including assessment of dermatitis score; histological analysis of dorsal skin lesions; quantitative measurement of serum total IgE; quantitative measurement of cytokines (IL-1β, IL-4, IL-5, IL-6, IL-13, TNF-α) from dorsal tissue; and RT-PCR analysis of for TSLP and TARC mRNA expression in HaCaT cells.

Results

The clinical dermatitis score was significantly lower in Catalpa ovata extract (COE) groups than in the control group. Histological analysis showed that COE inhibited hypertrophy and hyperkeratosis of the epidermis, intracellular edema, and reduced the infiltration of inflammatory cells. COE significantly inhibited serum total IgE; Th2 cytokines IL-4, IL-5 and IL-13; pro-inflammatory cytokines IL-1β, IL6 and TNF-α; the Th2 chemokine TARC and the pro-Th2 cytokine TSLP.

Conclusion

These results demonstrate that Catalpa ovata stem bark may be a useful external medicine for treatment of AD. Further investigation is necessary to determine appropriate COE dosage and to evaluate the safety of this medicinal herb.     

Effects of Korean red ginseng extract on the prevention of atopic dermatitis and its mechanism on early lesions in a murine model

Ethnopharmacological relevance

Korean red ginseng (KRG) has been shown to possess various biological activities including anti-inflammatory properties.

Aim of the study

We aimed to investigate the effects and mechanism of KRG on the prevention of atopic dermatitis (AD) using a mouse model.

Materials and methods

The effect of KRG in trinitrochlorobenzene (TNCB)-treated NC/Nga mice was assessed by measuring ear thickness, transepidermal water loss (TEWL), total serum IgE, histologic changes of lesional skin, mRNA and protein expression of thymic stromal lymphopoietin (TSLP) and tumor necrosis factor (TNF)-α, immunohistochemistry for tissue interleukin (IL)-4, IL-17, and interferon (IFN)-γ.

Results

KRG significantly reduced ear thickness. Oral administration of KRG significantly prevented the increase in TEWL induced by TNCB. The serum IgE level was significantly lower in the KRG group. Histologically, lymphocyte infiltration was markedly decreased by KRG. CD1a positive (CD1a+) cells were diminished by KRG. Immunohistochemically, KRG significantly suppressed the protein expression of TSLP and TNF-α. The mRNA expression of TSLP in the lesions was significantly reduced by KRG. These results demonstrate that oral administration of KRG may inhibit the development of AD-like skin lesions in NC/Nga mice by modifying TSLP, DCs, and at least in part, the Th2 response.

Conclusion

KRG may be a potential therapeutic modality for the prevention of AD.     

The impact of Zataria multiflora Boiss extract on in vitro and in vivo Th1/Th2 cytokine (IFN-γ/IL4) balance

Ethnopharmacological relevance

Anti-inflammatory, anti-oxidant and effect of Zataria multiflora on Th₁/Th₂ balance were previously described. Different therapeutic effects of this plant have been described in Iranian traditional medicine. To evaluate the immune modulatory effects of Zataria multiflora on Th1/Th2 balance, which may be implicated in inflammatory disorders, in vitro and in vivo studies were carried out.

Materials and methods

The effects of three concentrations of the extract, dexamethasone, and saline on interleukin 4 (IL-4) and interferon γ (IFN-γ) gene expression were evaluated in phytohemagglutinin (PHA) stimulated and non-stimulated human peripheral blood mononuclear cells (hPBMCs). RNA was extracted from the hPBMCs to make cDNA for real time PCR relative quantification. Furthermore, the effect of the extract on serum level of IL-4 and IFN-γ was assessed in ovalbumin (OA) sensitized guinea pigs (n=6 for each group).

Results

Dexamethasone showed significant inhibitory effect on both IFN-γ and IL-4 gene expression and serum level of the cytokines and significantly enhanced IFN-γ/IL-4 ratio (p<0.05–p<0.001). The extract inhibited IL-4 and enhance IFN-γ gene expression and IFN-γ/IL-4 ratio too (p<0.05–p<0.001). In sensitized animals also serum level of IL-4 were significantly decreased after treatment with both dexamethasone and extract, but serum level of IFN-γ and IFN-γ/IL-4 ratio were significantly increased due to extract treatment (p<0.01 for medium and p<0.001 for high concentration).

Conclusions

These results indicated consistent in vitro and in vivo data for selective immune modulatory effect of the extract of Zataria multiflora which increased IFN-γ, decreased IL-4, and enhanced the ratio of IFN-γ to IL-4 (Th₁/Th₂ balance). Therefore, the extract of Zataria multiflora may have therapeutic value in inflammatory responses such as allergy, autoimmunity and infectious diseases associated with Th₁/Th₂ imbalance.