Exploration of hapten-induced atopic dermatitis murine models for non-clinical pharmacodynamics study of drugs / 药学学报

Atopic dermatitis (AD) is a chronic, relapsing, inflammatory dermatosis with a variety of clinical manifestations and difficult to cure. Currently, many AD drug candidates have entered the research and development pipeline. In order to provide technical specifications for the clinical development of AD drugs, the Center for Drug Evaluation of National Medical Products Administration released the "Technical Guidelines for Clinical Trials of Drugs for AD Treatment" (Draft for Comments) in November 2022. Non-clinical pharmacodynamics evaluation is an important research before the drug enters clinical trials. Oxazolone (OXA)- and 2,4-dinitro-fluorobenzene (DNFB)-induced models are the most popular classical hapten-induced AD murine models, but variations of modeling are existing in the methods from different studies, including sensitization sites, haptens' dosages, the period of challenges, and the skin lesions severity evaluation as well. In this study, the investigation of OXA- and DNFB-induced AD murine models with various conditions of modeling was performed to compare the characteristics of hapten-induced AD murine models in the pathological process and severity according to the appearance of AD patients, and the guidance of pharmacodynamics evaluation of AD-therapeutic drugs in clinical trials as well, which may provide a proposal for AD treatment drug candidates in the non-clinical pharmacodynamics evaluation. All animal experiments were approved by the Animal Care & Welfare Committee of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (approval No.: 00007782 and 00007784).

Periplaneta americana extract promotes intestinal mucosa repair of ulcerative colitis in rat

Abstract Purpose: To investigate the mechanism of Periplaneta americana extract promoting intestinal mucosal repair of OXZ-induced colitis in rat. Methods: All experiments used an equal number of male and female SD rats (n=48). We injected OXZ into the colon to induce UC rat model. To determine the optimal concentration of P. Americana's extract (PA-40), it was classified into low (L), medium (M), and high (H) doses. After OXZ treatment, each drug was administered by enema for 7 consecutive days. Rats were divided into the following 6 groups: (1) Saline treatment group (NC), (2) OXZ treatment UC model group (MC), (3) OXZ + budesonide group (BUN), (4) OXZ + PA-40 L group, (5) OXZ + PA-40 M group, (6) OXZ + PA-40 H group. Disease activity index (DAI) scores, colon length, histopathological score, serum cytokine level (IL-4, IL-10, iNOS, tNOS), and amount of MPO, EGF, IL-13 in colonic mucosa were measured. Results: PA treatment had a significant healing effect on the OXZ-colitis model and significantly reduced the lesioned area, especially in the PA-40H groups. PA treatment did not alter the expression of IL-10 and MPO level, but increased EGF (epidermal growth factor) and decrease IL-13 in the colonic tissue. PA inhibited the rise of NOSs (nitric oxide synthase) and decreased the serum IL-4 level. Conclusions: The data suggest that Periplaneta americana extract may be a potential compound for the treatment of colonic lesions. The mechanism may be related to inhibiting the secretion of IL-13 and promoting the formation of EGF.

Dioscorea japonica Thunb. Ethanolic Extract Attenuated Oxazolone-Induced Atopic Dermatitis-like Skin Lesions in BALB/c Mice

The rhizomes of Dioscorea japonica Thunb. are widely consumed as food and also used to treat diabetes and polyuria in Korea. This study was undertaken to study the anti-atopic dermatitis effects of a 95% ethanolic extract (DJE) of D. japonica in an oxazolone-stimulated murine model of atopic dermatitis (AD). The therapeutic effects of DJE on AD-like skin lesions were assessed on both ears. DJE (1%) or dexamethasone (0.5%; the positive control) were applied to skin lesions for three weeks. Serum levels of IgE and IL-4 were assessed by ELISA (enzyme-linked immunosorbent assay). Histopathological examinations were performed by hematoxylin and eosin (H&E) and toluidine blue staining and revealed DJE significantly reduced dermal thickness and inflammatory cell infiltration when applied to oxazolone-treated ear skin. DJE-treated AD mice also showed lower serum levels of IgE and IL-4 than oxazolone-stimulated controls. Our findings demonstrate DJE might be a useful safe, topical agent for the treatment of atopic diseases.

Levels of Th1/Th2 in spleen lymphocyte of colitic mice without or with Trichinella spiralis infection / 中国免疫学杂志

Objective:To study the levels of Th1 and Th2 in spleen lymphocyte of TNBS and OXZ colitis without or with Trichinella spiralis(T.spiralis)infection.Methods:Female BALB/c mice were randomly divided into 3 groups:50％ Ethanol,TNBS or OXZ,T.spiralis+TNBS or OXZ(at least 6 in each group when mice were killed).The ratio of Th1/Th2 lymphocyte in spleen was detected by FCM with three color intracellular cytokine staining.Results:In spleen lymphocyte of TNBS colitic mice with prior nematode infection,the ratio of Th1/Th2 were not significantly different compared with that seen in colitic mice without prior nematode infection on day 3 post-induction of colitis(P＞0.05),the ratio of Th1/Th2 was significantly lower than which on day 7 post-induction of colitis (P＜0.05).In spleen lymphocyte of OXZ colitic mice with prior nematode infection on days 3 and 7 post-induction of colitis,the ratio of Th1/Th2 was significantly higher than that seen in colitic mice without prior nematode infection(P＜0.05).Conclusion:Infection of mice with T.spiralis distracts the mucosal immune system response from a Th1 response toward a protective Th2 response and up-regulate Tr1-cytokines with an attendant reduction in the severity of colonic damage and balance of Th1/Th2.Prior T.spiralis infection doesn't reduce the severity of OXZ-induced colitis,but without aggravating colitis.

Changes of Expressions of Claudin-1,-2,-4 in Experimental Colitis Rats / 胃肠病学

Background:Ulcerative colitis (UC)is a chronic inflammatory disorder. Studies have shown that intestinal injury of UC is related to changes of tight junction proteins. Aims:To investigate the expressions and localizations of tight junction protein claudin-1,-2,-4. Methods:Forty female Wistar rats were randomly divided into normal control group and model group. Rats in model group received 7. 5 mg/ mL oxazolone enema to induce experimental colitis. Rats received 0. 9%NaCl solution enema were served as normal controls. Macroscopic score and histological score were assessed. ELISA was used to determine serum and colon tissue inflammatory factor TNF-α,IL-4,IL-5,IL-10 levels. Protein expressions of tight junction protein claudin-1,-2,-4 were measured by immunohistochemistry and Western blotting. mRNA expressions of claudin-1,-2,-4 were determined by real-time PCR. Results:Compared with normal control group,macroscopic score and histological score were significantly increased (P < 0. 05),serum and colon tissue IL-4 and IL-5 levels were significantly increased (P < 0. 05)in model group,however,no significant differences in TNF-α and IL-10 levels were found between the two groups (P > 0. 05). mRNA and protein expressions of claudin-1,-4 were significantly decreased in model group than in normal control group (P < 0. 05),while mRNA and protein expressions of claudin-2 were significantly increased (P < 0. 05). Conclusions:Changes of distributions and expressions of tight junction protein claudin-1,-2,-4 are found in experimental colitis rats,which may lead to impaired epithelial barrier,and might be served as potential target for treatment of UC.

Changes of Expressions of Claudin-1,-2,-4 in Experimental Colitis Rats / 胃肠病学

Background:Ulcerative colitis (UC)is a chronic inflammatory disorder. Studies have shown that intestinal injury of UC is related to changes of tight junction proteins. Aims:To investigate the expressions and localizations of tight junction protein claudin-1,-2,-4. Methods:Forty female Wistar rats were randomly divided into normal control group and model group. Rats in model group received 7. 5 mg/ mL oxazolone enema to induce experimental colitis. Rats received 0. 9%NaCl solution enema were served as normal controls. Macroscopic score and histological score were assessed. ELISA was used to determine serum and colon tissue inflammatory factor TNF-α,IL-4,IL-5,IL-10 levels. Protein expressions of tight junction protein claudin-1,-2,-4 were measured by immunohistochemistry and Western blotting. mRNA expressions of claudin-1,-2,-4 were determined by real-time PCR. Results:Compared with normal control group,macroscopic score and histological score were significantly increased (P < 0. 05),serum and colon tissue IL-4 and IL-5 levels were significantly increased (P < 0. 05)in model group,however,no significant differences in TNF-α and IL-10 levels were found between the two groups (P > 0. 05). mRNA and protein expressions of claudin-1,-4 were significantly decreased in model group than in normal control group (P < 0. 05),while mRNA and protein expressions of claudin-2 were significantly increased (P < 0. 05). Conclusions:Changes of distributions and expressions of tight junction protein claudin-1,-2,-4 are found in experimental colitis rats,which may lead to impaired epithelial barrier,and might be served as potential target for treatment of UC.

Ethanol Extract of Peanut Sprout Exhibits a Potent Anti-Inflammatory Activity in Both an Oxazolone-Induced Contact Dermatitis Mouse Model and Compound 48/80-Treated HaCaT Cells

BACKGROUND: We developed an ethanol extract of peanut sprouts (EPS), a peanut sprout-derived natural product, which contains a high level of trans-resveratrol (176.75 microg/ml) and was shown to have potent antioxidant activity. OBJECTIVE: We evaluated the potential anti-inflammatory activity of EPS by measuring its antioxidant potential in skin. METHODS: The anti-inflammatory activity of EPS was tested using two models of skin inflammation: oxazolone (OX)-induced contact dermatitis in mice and compound 48/80-treated HaCaT cells. As biomarkers of skin inflammation, cyclooxygenase-2 (COX-2) and nerve growth factor (NGF) levels were measured. RESULTS: OX-induced contact dermatitis was suppressed markedly in mice that were treated with an ointment containing 5% EPS as evidenced by a decrease in the extent of scaling and thickening (p<0.05) and supported by a histological study. COX-2 (messenger RNA [mRNA] and protein) and NGF (mRNA) levels, which were upregulated in the skin of OX-treated mice, were suppressed markedly in the skin of OX+EPS-treated mice. Consistent with this, compound 48/80-induced expression of COX-2 (mRNA and protein) and NGF (mRNA) in HaCaT cells were suppressed by EPS treatment in a dose-dependent manner. As an inhibitor of NF-kappaB, IkappaB protein levels were dose-dependently upregulated by EPS. Fluorescence-activated cell sorting (FACS) analysis revealed that EPS scavenged compound 48/80-induced reactive oxygen species (ROS) in HaCaT cells. CONCLUSION: EPS exerts a potent anti-inflammatory activity via its anti-oxidant activity in both mouse skin and compound 48/80-treated HaCaT cells in vitro. Compound 48/80-treated HaCaT cells are a useful new in vitro model of skin inflammation.

A Review on Chemical-Induced Inflammatory Bowel Disease Models in Rodents

Ulcerative colitis and Crohn's disease are a set of chronic, idiopathic, immunological and relapsing inflammatory disorders of the gastrointestinal tract referred to as inflammatory bowel disorder (IBD). Although the etiological factors involved in the perpetuation of IBD remain uncertain, development of various animal models provides new insights to unveil the onset and the progression of IBD. Various chemical-induced colitis models are widely used on laboratory scale. Furthermore, these models closely mimic morphological, histopathological and symptomatical features of human IBD. Among the chemical-induced colitis models, trinitrobenzene sulfonic acid (TNBS)-induced colitis, oxazolone induced-colitis and dextran sulphate sodium (DSS)-induced colitis models are most widely used. TNBS elicits Th-1 driven immune response, whereas oxazolone predominantly exhibits immune response of Th-2 phenotype. DSS-induced colitis model also induces changes in Th-1/Th-2 cytokine profile. The present review discusses the methodology and rationale of using various chemical-induced colitis models for evaluating the pathogenesis of IBD.

The Effect of Adipose-Derived Stem Cell-Cultured Media on Oxazolone Treated Atopic Dermatitis-Like Murine Model

BACKGROUND: A stem cell is an undifferentiated cell that has the potential for self-renewal and differentiation. Adipose-derived stem cells (ADSCs) have advantages in accessibility and abundance compared to other kinds of stem cells and produce many growth factors and hormones. OBJECTIVE: We investigated whether ADSC cultured media could be used as a therapy for atopic dermatitis. METHODS: ADSC cultured media was topically applied twice daily for 5 days to oxazolone-treated atopic dermatitis-like hairless mice. RESULTS: Topical application of ADSC cultured media improved the epidermal permeability barrier and keratinocyte differentiation, and restored the predominant Th2 phenotype when compared to vehicle. ADSC cultured media-treated epidermis also showed an increase in the expression of antimicrobial peptides cathelin-related antimicrobial peptide, mouse beta-defensein 3. CONCLUSION: Topical ADSC cultured media could be useful in the treatment of atopic dermatitis.

The effect of laser irradiation on oxazolone-induced ulcerative colitis / 中华物理医学与康复杂志

Objective To observe any therapeutic effect of laser irradiation on pathological inflammatory reactions in ulcerative colitis (UC) induced by oxazolone,and to investigate possible mechanisms.Methods Six rats were selected as a normal control group.Another 24 rats with UC induced by oxazolone were randomly assigned to a UC model group (n =8),a 400 mW laser group (161.3 mW/cm2,n =8),and a 200 mW laser group (80.6 mW/cm2,n =8).All the rats were fixed in custom-built devices.Those in the therapy groups were treated daily,10 min per time,for 10 days.After the end of the last irradiation session,disease activity indexes (DAIs)were observed.Rats from every group were sacrificed 24 h after the last irradiation in order to observe any pathological changes in colon tissue,the weight of fresh ulcerated tissues,and gross changes in morphology.Colon segments were stained with hematoxylin-eosin and histological lesion scoring was performed under a light microscope.Any changes in inflammatory edema in the colonic mucous membrane were observed before and after laser irradiation.Results The UC model was successfully established.Average body weight in the 400 mW laser group increased significantly more than in the UC model group,approaching that of the normal control group.DAI decreased significantly.The thickness of the epithelial mucous membrane,lamina propria and submucosa was basically restored.Histological lesion scores also improved significantly,and the weight of fresh ulcerative tissue was significantly lower.Mucous membrane ulcers,submucosa edema and inflammatory cell infiltration all were alleviated significantly,merely presenting a few inflamed cells and small amounts of periphlebitis.In the 200 mW laser group all these outcome measures improved significantly compared with the UC model group,but were not as good as in the 400 mW laser group,and the rats needed longer to recover.Conclusion Laser irradiation at 400 mW has advantages over 200 mW,and could significantly relieve the pathological inflammatory response in colonic tissue,decrease submucosa edema and ameliorate other symptoms of ulcerative colitis,at least in rats.

Paeoniflorin increases ?-defensin expression and attenuates lesion in the colonic mucosa from mice with oxazolone-induced colitis / 药学学报

Previous studies have demonstrated that the Chinese medicine paeoniflorin, derived from the Ranunculaceae plant peony, peony, purple peony root, was able to have anti-inflammatory, anti-ulcer, anti- hypersusceptibility and anti-oxidation activity. In order to elucidate the pesticide effect and the mechanisms by which paeoniflorin exerts its effect of anti-inflammation and immunoregulation on oxazolone-induced colitic mice, disease activity index (DAI) and histological grading of colitis (HGC) were evaluated in animal model. Moreover, the expressions of HBD-2, IL-6 and IL-10 of mice with experimental colitis were observed with immunohistochemistry and RT-PCR in this study. Results showed that DAI and HGC of oxazolone control group was significantly higher than that of normal control group, and that paeoniflorin groups and 5-ASA group, compared with oxazolone control group, could alleviate the symptoms and histological damages of colitic mice (P

The expressions of nuclear factor-κB and activator protein-1 in oxazolone induced colitis in mice / 中华消化杂志

Objective To investigate the expression changes of nuclear factor(NF)-κB and activator protein (AP)-1 in oxazolone induced colitis in mice and their mechanisms. Methods Twenty-four mice were randomly divided into normal group and model group with 12 each. Experimental colitis was induced with skin sensitization of 3% oxazolone for 5 days, then rectal administration of 0.15 ml of 0. 5% oxazolone solution in mice. All mice were sacrificed on day 3. Peripheral blood mononuclear cells (PBMC), spleen mononuclear cells (SMC) and lamina propria mononuclear cells (LPMC) were isolated from the colon tissues. Expression of NF-κB and AP-1 in SMC, LPMC and PBMC were determined by fluorescence quantitative polymerase chain reaction (PCR). The colitis was evaluated histologically. Results The expressions of NF-κB and AP-1 in SMC, LPMC,PBMC of model groupwere significantly higher than those in normal group(NF-κB : 5.62±0.78 vs. 3.16±0.59,5.46±0.38 vs. 3.18±0.58, 5.65±0.56 vs. 3.36±0.59, P＜0.01; AP-1; 5.61±0.54 vs. 3.22±0.50, 5.50±0.69 vs. 3.19± 0.40,5.67±0.44 vs. 3. 27±0.41, P＜0.01). Conclusion The activation of NF-κB and AP-1 are involved in the mechanisms of ulcerative colitis.

Effect of pioglitazone on oxazolone-induced colitis in mice / 中华消化杂志

Objective To investigate the effect of pioglitazone on oxazolone-induced colitis and to clarify the mechanism of apoptosis by Fas/Fas ligand(FasL). Methods Eighteen BALb/c mice of ulcerative colitis induced by oxazolone enema were equally allocated into 3 groups. The mice in group 1 were sacrificed 3 days after enema,lamina propria mononuclear cells(LPMC) were isolated from freshly obtained colonic specimens and treated in vitro with pioglitazone (40 ?mol/L),the annexin-V-FITC was used for detection of apoptosis,and Fas/FasL expression was assayed by flow cytometry. Meanwhile,untreated mice were served as normal controls. Mice in group 2 (control group) and 3 (experiment group) were treated with methylcellulose or pioglitazone(20 mg?kg -1 ?d -1 ) 3 days after enema and were administrated for consecutive 7 days. The colonic inflammation including disease activity index (DAI),macroscopic and histological changes,myeloperoxidase(MPO) activity and levels of interleukin (IL)-4,IL-5 were evaluated. Results Apoptosis of LPMC,expression of Fas and FasL in normal and colitis mucosa were 12.89?1.23,70.63?6.24,8.59?5.47 and 4.25?0.84,62.60?5.85,23.75?10.23,respectively. After treated with pioglitazone,both apoptosis of LPMC and expression of Fas increased,while FasL expression decreased (40.58?10.32,83.98?11.38 and 10.04?5.21 respectively). In group 2 and group 3,the macroscopic and microscopic score,MPO activity,IL-4 and IL-5 level were 2.50?0.55,8.83?0.75, 3.81?0.17,216.46?34.32,102.28?25.74 and 0.33 ?0.52,4.00?0.63,1.25?0.16,179.36?18.15,61.65?17.45,respectively. Conclusion The results indicate that pioglitazone treatment can significantly attenuate colonic inflammation,and it might be related to the apoptosis of LPMC through Fas and FasL.

Effect of long-term nicotine on oxazolone induced murine colitis model / 中华消化杂志

Objective To investigate the long term effect of nicotine on oxazolone (OXZ) induced murine colitis models pathologically and analyse the cytokine profile produced by colonic mucosa and splenocytes. Methods BALB/c mice was used, colitis model was induced by intrarectal injection of oxazolone; nicotine (0.5 mg?kg -1 ?d -1 ) was injected subcutaneously for three weeks. After being sacrificed, the colon and spleen were removed. Colonic changes were examined pathologically. The IFN ? and IL4 produced by colonic mucosa and splenocytes was analyzed with ELISA. The intracellular IFN ? and IL 4 produced by splenocytes were tested by FACScan. Results In nicotine group, the histological score was significantly lower than that of OXZ group (19.8 vs 23.7, P

Significance of transforming growth factor receptors expressing in oxazolone-induced colitis model in mice / 中华消化杂志

Objective To observe the expression of TGF?RⅠ,TGF?RⅡ and Smad7 in oxazolone-induced colitis of mice and to investigate the role of the TGF? signal transduction on pathogenesis of colitis. Methods Balb/c mice were pre-sensitized by skin painting with 0.2 ml 3% oxazolone on day 0 and 1 followed by intrarectal administration of 0.15 ml 1% oxazolone on day 7. The mice were sacrificed after 3 days. Colitis was evaluated by macroscopic and microscopic examination. The expressions of TGF?RⅠ, TGF?RⅡ and Smad 7 were examined by immunohistochemical study and Western blot respectively. All the results were compared with the controls. Results Twenty-four hours after intrarectal administration of oxazolone, the mice presented anorexia, less moving, loose stool, hematochezia or occult blood(+) and weight loss. The macroscopic and microscopic scores in two groups were 0.17?0.41, 2.67?1.03 and 2.33?0.52, 8.17?0.75, respectively. In the normal intestine, TGF?RⅠ, TGF?Ⅱ and Smad7 were mainly co-localized on the upper part of the villus. However, their expression was not only throughout the villus including fundus of crypts, but also in the mononuclear cells of the lamina propria and submucosa in the experimental intestine. The amounts of TGF?RⅠ, TGF?Ⅱ, Smad7 and the ratio of TGF?RⅠ/Ⅱ in control and colitis groups were 3.40?1.25, 21.71?6.97, 8.95?2.12, 0.16?0.01 and 6.49?3.18, 4.40?3.34, 17.92?6.80, 2.14?1.61, respectively. Conclusions Decreased TGF?RⅡ and increased Smad7 expressions indicate the abnormality of TGF? signal transduction in oxazolone-induced colitis. These pathologic and immunologic characteristics may resemble human ulcerative colitis.

Multi-glycosidorum triptery suppresses production of interleukin-4 by splenocytes in oxazolone-induced murine colitis / 中华消化杂志

Objective To investigate the in vitro effect of multi glycosidorum triptery (MGT) on cytokine production by splenocytes of oxazolone (OXZ) induced colitis in murine model. Methods Six mg of OXZ (in 50% of ethanol) was administered in male SJL/J mice intrarectally to induce colitis and mice were sacrificed 3 days later. Isolated splenocytes were cultured for 24 hours in the presence of PMA and ionomycin, MGT of 0.1 mg/ml or 0.01 mg/ml was added to the culture medium of splenocytes. Production of IFN ? and interleukin 4 (IL 4) in the supernatant was measured by ELISA. Results The production of IFN ? was suppressed by both 0.01 mg/ml and 0.1 mg/ml of MGT [normal control ( 1.24 ? 0.13 ) pg/ml→(0.97?0.26) pg/ml→(0.87?0.18) pg/ml, P