| This research is part of the two projects funded by the departments of GuangDong province as follows:Atopic Dermatitis(AD)-advantages of traditional Chinese medicine disease breakthrough projects (No. [2015] 19), and the clinical and basic research of Atopic Dermatitis(No. [2011]572). Our team developed Peituqingxin particulate granule (short for PTQX particulate) in the early stage of studies. We also established AD-like mice model, and preliminary analysis that the PTQX particulate could take the effect of reducing heart fire and repairing spleen deficiency, which have a good therapeutic effect in clinical.Based on the previous research, this research proposes establishing and assess the AD-like mice model, then investigate the role of PTQX particulate plays in the immune network system of AD-like mice. Research mainly divided into two parts. Part 1 Establishment and Assessment of Atopic Dermatitis-like mice model ObjectiveTo establish an AD model, consummate the systems of establishment and evaluation system after studying the literatures both domestic and foreign refers to the AD-like animal model. Methods1. Search the relevant database with the retrieving words such as "atopic dermatitis", "AD" or "animal model", then analyze these paper base on the data extraction and quality assessment. According to the AD animal model at home and abroad, we compare the animal species, sex selection and specific model construction methods, in order to screen suitable experimental animal species, animal sex and modeling methods.2. After established the AD-like model, we combine the evaluation indexes of both Delayed Type Hypersensitivity Chronic Eczema, then make an evaluation of lesion scores, behavioral observation score, the frequency of scratching, ear thickness and skin pathology. Results1. Combining with the above researches and laboratory existing conditions, female C57BL/6 mice were the better choice, while hapten stimulation could be the better method to establish AD-like mice model, so we use the 2,4-dinitro-f luorobenzene (DNFB) to induce the C57BL/6 mouse by multiple times to establish the AD-like model.2. Comparing with the normal control group, the lesion of back skin in the model group became thicken, the spinous layer cells increase, inflammatory cells scattered in the epidermis and dermis. And the IgE and IL-4 levels in the serum of AD-like mice model were significantly increased (P<0.01), while the level of IFN-y made no difference (P>0.05).3. The lesion scores, behavioral observation points, the scratching frequency, the thickness of the ear in the model group showed an upward trend. And they also make statistically significant when compared with the normal control group (P<0.05). Behavior score of the model group increased at first and then decreased, the frequency of scratching, skin lesions score, the ear thickness also increased significantly (P<0.01). Conclusions1. AD-like mice model could be established by using DNFB induced method, and this model showed typical lesions with chronic inflamminatory, depression and accentuation of immunity, and the serological performance tip skin inflammation is given priority to with type Th2 cells.2. The AD-like model showed obviously chronic inflammatory lesion, accompanied by itching, which are very similar to human AD. The skin lesion scores, ear thickness, skin tissue pathology and the serological changes and so could be used as an important criterion to estimate the success of the model. Part 2 Immune regulation of Peituqingxin particulate granule in Atopic Dermatitis-like miceObjectivesTo explore PTQX particles on immune regulation effects of AD-like mice, we observed the intervention of PTQX particles with AD-like mice, analyzed the serum levels of cytokines, checked the expression level of messenger RNA in the skin lesions and the changes of spleen Breg cells.Methods1. Female C57BL/6 mice were randomly divided into four groups, including blank control group, model group, high dose group of PTQX particles(the clinical double dose. PTQX particles 1.56g/kg·d), middle doses group of PTQX particles(the clinical equivalent dose. PTQX particles 0.78g/kg/kg·d). Every group was gave intergalactic administration for once per day and administrative 14 dags totally. The high and low of PTQX groups were administered PTQX solution respectively. The model group and the blank control group were treated with distilled water. Then we make an evaluation of the indexes such as skin lesions score, behavioral observation score, ear thickness, skin lesion scores in the experiment on dayl, day7 and day14, so as to analyze the characteristics of the model.2. DNFB repeated stimulation induced method was used to build AD-like model, and the animal skin should be propagated before experiment (size is about 2.5cm x 1.5cm). On the first day, the back of the model group and the PTQX groups were coated with 0.5% 25μ1 sensitization DNFB solution, and then on the following day5, day 8, day 11 and day14, both the back and the ear in mice were coated with 0.2% of 20μ1 DNFB solution, while the left ear was coated by the substrate solution. All the mice were sacrificed by eyeball removal method within 48 hours to the end of the experiment, and the skin lesions of back was taken to check the routine pathology and mast cell count test(toluidine blue stain).3. After administration of appropriate drug intervention, the expression of IgE in the serum of mice were detected by enzyme-linked immunosorbent assay (ELISA), and the expression of IFN-γ, IL-4, IL-10 in the serum of mice were tested by liquichip method.4. Extracting the total RNA in skin tissue of mice, and identify the concentration and purity of these RNA. Use the Quantitative real time polymerase chain reaction(Q-PCR) to identify the expression level of the mRNA of IFN-γ,IL-4 and IL-10.5. Picked off the spleens from all groups, then isolated and cultured the spleen lymphocytes. Flow cytometry were utilized to detect the number and percentage of regulatory B cells(Breg).Results1. After giving PTQX particulate, the frequency of scratching, skin lesions score and the ear thickness of the AD-like mice decreased significantly(P <0.01), the behavior score is usually normal (P> 0.05). The skin tissue pathology showed that the skin is generally normal and the inflammatory cell infiltration reduced, while the mast cell count was significantly lower than the model group(P<0.01).2. Comparing with the blank control group, the IgE, IL-4 in the serum of model group increased (P<0.01), while the level of IL-10 and IFN-γ made no differences (P>0.05). When giving PTQX particles, both high dose group of PTQX particles and low dose group of PTQX particles, the level of IgE in the serum decreased significantly(P<0.05), and the level of IL-4 in the serum also obviously decreased (P<0.05), while the high dose group only showed a downward trend. Compared with the model group, the cytokine IL-10 and IFN-γ in PTQX particles groups had no obvious change.3. The result of Q-PCR showed that the expression of ill0mRNA、ifnγ mRNA、 i14 mRNA were increased in the model group. After giving PTQX particles, comparing with the model group, the expression of ifnγ mRNA、i14mRNA in the PTQX particles groups decreased significantly(P<0.01). In addition, the expression of i110 mRNA in PTQX particles groups increased significantly, compare with the model group, their difference were significant (P<0.05).4. Compare with blank control group, the percentage of CD5+ B lymphocytes in AD-like mice spleen decreased significantly(P<0.01), when giving high and low dose of PTQX particles, the number of CD5+B lymphocytes increased significantly(P<0.05), pretend to be normal, but still lower than the normal group(P<0.01). At the same time, there were no difference in the percentages of CD4+ T lymphocytes, CD4+IL-4+T lymphocytes, CD4+IFN-γ+T lymphocytes, CD4+IL-17+T lymphocytes, CD5+CD19+ lymphocytes, CD5+CD1d+ B lymphocytes and Bregs between blank control group and the model group. When giving PTQX particles, the percentage of CD5+CDld+ B lymphocytes and Bregs in the high dose group were higher than the blank group and model group (P<0.05), and the low dose showed an increasing trend, but the trend was not clear(P> 0.05). Differences of the rest cells were not statistically significant{P> 0.05).Conclusions1. PTQX particles could treat AD by reducing the scores of skin lesions, relieving itching and declining the inflammatory cells in the skin.2. IgE、IL-4、IL-10 were the important targets for PTQX particles to treat AD, it may take effect through the following channels. PTQX particles could regulate the content of IgE、IL-4 in the serum, down-regulating the expression of ifn y mRNA、il4 mRNA, and up-regulatory the expression of i100 mRNA, in this way, it can restore and maintain immune homeostasis.3. PTQX particles may balance the immune system by stimulating lymphocyte proliferation, including CD5+ B lymphocytes, CD5+ CDld+ B lymphocytes and Breg cells, so that it could correct the immune cell disorder in the AD and achieve the purpose of treatment. |
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