Combination of oxymatrine and diammonium glycyrrhizinate significantly mitigates mice allergic contact dermatitis induced by dinitrofluorobenzene

Chemicals and drugs

Oxymatrine (molecular formula, C₁₅H₂₄N₂O₂; molecular weight, 264.36; CAS, 16837–52-8; high performance liquid chromatography (HPLC) purity >98%) and diammonium glycyrrhizinate (molecular formula, C₄₂H₆₂O_16.2H₃N; molecular weight, 856.997; CAS, 79165–06-3; HPLC purity >98%) were purchased from Nanjing Zelang Biological Technology (Nanjing, China) and their structures are shown in Figure 1(a). DNFB (molecular formula, C₆H₃FN₂O₄; molecular weight, 186.10; CAS, 70–34-8; purity, 99%) was purchased from Shandong Xiya Chemical Industry (Jinan, China). Acetone (molecular formula, CH₃COCH₃; molecular weight, 58.08; CAS, 67–64-1; purity, 99%) and olive oil were purchased from Nanjing Zelang Biological Technology. Dexamethasone sodium phosphate injection was purchased from Xi’an Lijun Pharmaceutical (Xi’an, China). The IgE, IFN-γ, and IL-4 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shenzhen Xinbosheng Biological Technology (Shenzhen, China).

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Figure 1.

Combination of OMT and DG mitigates the DNFB-induced skin inflammation in mice. ICR mice were smeared with DNFB for five times and after first smearing, the mice were randomized and treated intraperitoneally with saline (model), or the indicated doses of OMT and/or DG daily for 14 days. Their body weights, skin inflammation and ear thicknesses were measured longitudinally. (a) The structures of OMT or DG. (b) The body weights of the different groups of mice. (c) Representative images of skin inflammation in different groups of mice (on day 14). (d) The inflammation scores of individual groups of mice. (e) The ear thicknesses. Data are representative images or expressed as the mean values of individual groups (n = 5 per group). The skin inflammatory scores and ear thicknesses of all ACD mice were significantly greater than the control while the skin inflammatory scores and ear thicknesses in all drug-treated groups were significantly less than that in the model group (statistical markers not shown in Figure 1(d) and (e)). Letters on the graphs indicate the following statistical significant group comparisons: ^aP< 0.05 vs. the control group; ^bP< 0.05 vs. the model group; ^cP< 0.05 vs. the DEX 2 mg·kg⁻¹ group; ^dP< 0.05 vs. the combination (OMT+DG, 45 + 45 mg·kg⁻¹). OMT: oxymatrine; DG: diammonium glycyrrhizinate; DEX: dexamethasone. (A color version of this figure is available in the online journal.)

Animals

Male ICR mice at six weeks of age and weighing 18–22 g were obtained from the Laboratory Animal Center of Ningxia Medical University (Yinchuan, China). The mice were housed in a specific pathogen-free facility at 23 ± 2°C, a relative humidity of 60% with a light–dark cycle of 12 h and free access to food and water. The mice were food-fasted overnight before experiment. The experimental protocol was approved by the Medical Ethics Committee of the Ningxia Medical University General Hospital (issue date: 6 April 2016; file number: 2016–177).

DNFB-induced ACD and treatment

Male mice (n = 55) were randomly divided into 11 groups (five mice each group). The mice were induced for ACD, as described previously.^33,34 Briefly, individual mice were topically applied with 25 µL of 0.15% DNFB in acetone/olive oil (3:1) onto the outer and inner surfaces of mouse right ear and 100 µL onto shaved back skin on days 1 and 4. On days 7, 10, and 13, the sensitized mice were challenged with 0.2% DNFB onto their skin surface. The mice were randomized one day after the first smearing and injected intraperitoneally with saline (Model), 45 or 90 mg·kg⁻¹ OMT and/or DG or 2 mg·kg⁻¹ dexamethasone (DEX, Positive control) daily for 14 consecutive days. The doses of OMT and DG were based on previous studies for their efficacy and toxicity.^32,35–38 A group of control healthy mice received vehicle smearing and injected with saline (n = 5). Their body weights were measured daily beginning one day before induction throughout the experimental period using an electronic balance (YP601N electronic balance, Shanghai Precision Science Instrument, Shanghai, China). The ear thicknesses of individual mice were measured ³⁹ using a gauge digital micrometer (Hengxing Measuring tools, Shanghai, China) on the day post each DNFB application. The severities of inflammation, such as erythema (hemorrhage), edema, excoriation (erosion), and dryness (scaling) in individual mice were scored as 0 (absent), 1 (mild), 2 (moderate), and 3 (severe) in a blinded manner.^33,40 A total score (minimum 0 and maximum 12) of each mouse was recorded daily. At the end of the experiment (on day 14), the mice were subjected to light anesthetization with diethylether, and their blood samples were withdrawn retro-orbitally for preparing plasma and serum samples, which were stored at −80°C until further processing. The levels of plasma sodium, potassium, creatinine, and urea were measured using a Siemens ADVIA 2400 Autoanalyzer (Siemens AG, Munich, Germany).

Enzyme-linked immunosorbent assay

The levels of serum IgE, IFN-γ, and IL-4 in individual mice were measured by ELISA using specific kits, according to the manufacturer’s instructions.

Histologic analysis

The ear and back skin samples were taken in the same manner at the end of the experiment, and fixed in 10% neutral buffered formalin, followed by paraffin-embedded. The tissue sections (3–4 μm) were stained with hematoxylin and eosin (H&E). The numbers of inflammatory infiltrates in individual samples were measured in a blinded manner. The number of inflammatory cells in five fields (magnification × 200) with the most infiltrates of each mouse was calculated in a blinded manner.

Statistical analysis

All data are expressed as the mean ± SD. The difference among groups was analyzed by repeated one-way ANOVA and least significant difference (LSD) test using the SPSS version 17.0 statistical software package (IBM, Armonk, NY). A P-value of <0.05 was considered statistically significant.

Combination of OMT and DG significantly mitigates the severity of inflammation in ACD mice

OMT and DG can benefit patients with dermatitis.^23,32 To investigate the impact of OMT and DG combination on the severity of inflammation, ICR mice were induced for ACD, randomized and treated with OMT and/or DG for 14 days. In comparison with the healthy control, treatment with vehicle in the model group did not significantly change the body weights (P > 0.05) while treatment with DG (90 mg·kg⁻¹) alone or DEX significantly increased the body weights beginning at five or seven days post induction, respectively (P < 0.05 for all, Figure 1(b)). In contrast, treatment with OMT (90 mg·kg⁻¹) alone or OMT (45 mg·kg⁻¹) significantly reduced the body weights in mice beginning at five or seven days post induction, respectively (P < 0.05 for all). However, treatment with both OMT and DG, regardless their doses, did not change the body weights in mice (P > 0.05), relative to that of either group of mice receiving monotherapy.

During the experimental period, we observed that some mice developed the skin inflammation, characterized by skin erythema, edema, dryness, and excoriation on day 8 post induction and some mice developed severe skin lesions at the end of the observation period (Figure 1(c)). Quantitative analysis indicated that all experimental groups of mice, but not the healthy control, developed varying degrees of skin inflammation beginning on day 8 post induction. The inflammation scores in the model group gradually increased throughout the observation period. In comparison with that in the model group, treatment with OMT or/and DG or DEX (2 mg·kg⁻¹) significantly decreased the skin inflammation scores beginning on day 8 post induction and later time points (P < 0.05 for all, Figure 1(d)). More interestingly, treatment with a high dose of OMT (90 mg·kg⁻¹) or any combination of both drugs significantly mitigated the skin inflammation scores, relative to the DEX group of mice beginning on day 11 and later time points (P < 0.05 for all). In addition, treatment with both OMT and DG significantly reduced the skin inflammation scores, compared with the mice receiving either OMT or DG monotherapy (P < 0.05 for all). A similar pattern of ear thicknesses was observed in the different groups of mice at varying time points (Figure 1(e)). These data clearly demonstrated that combination of OMT and DG additively enhanced their therapeutic effects on skin and ear inflammation in ACD mice.

Combination of OMT and DG decreases the DNFB-induced ear and skin inflammation in ACD mice

To understand the therapeutic action of OMT and DG, we measured inflammation in the skin and ear lesions of ACD mice by histology. As shown in Figure 2(a), the ear tissues in the control group displayed a clear structure of the epidermis without obvious inflammation in the superficial dermis. In contrast, the ear tissues in the model group exhibited hypertrophy, hyperkeratosis, partial crusts and ulceration, epidermal hyperplasia, obvious edema sponge, acanthosis, superficial dermal vessels with many inflammatory infiltrates, possibly by neutrophils and lymphocytes. Quantitative analysis indicated that the numbers of inflammatory infiltrates in the ear lesions of ACD mice were significantly greater than in the control mice (P < 0.05 for all) and the numbers of inflammatory infiltrates in the ear lesions of the model group of mice were also significantly higher than that of any of the drug-treated mice (P < 0.05 for all, Figure 2(b)). In comparison with that of the DEX group, treatment with a high dose of OMT or DG or with any of combination of OMT and DG, but not with a lower dose of OMT or DG, significantly reduced the numbers of inflammatory infiltrates in the ear lesions of mice (P < 0.05 for all). Interestingly, the inhibitory effect of treatment with both lower dose of OMT and DG on the numbers of inflammatory infiltrates was significantly stronger than that of treatment with a lower dose of OMT or DG as well as with a lower dose of OMT and higher dose of DG in mice (P < 0.05 for all). Similar patterns of histological changes and inflammatory cell infiltration were observed in the skin tissues of the different groups of mice, except that the anti-inflammatory effect of treatment with a high dose of DG was similar to that of DEX in mice (Figure 3). Collectively, such data demonstrated that combination of OMT and DG significantly mitigated the DNFB-induced ear and skin inflammation in ACD mice.

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Figure 2.

Histologic analysis of inflammatory infiltrates in the ear lesions of mice. At the end of the experiment (on day 14), the different groups of mice were sacrificed and their ear tissues were collected. The ear tissue sections were stained by H&E. The numbers of inflammatory infiltrates in individual sections were counted in a blinded manner. Data are representative images or expressed as the means ± SD of each group (n = 5 per group) from three separate experiments. (a) The representative images (magnification × 200). (b) Quantitative analysis of the inflammatory infiltrates. The numbers of inflammatory infiltrates in the ear tissues of all ACD mice were significantly greater than the control while the numbers of inflammatory infiltrates in all drug-treated groups were significantly less than that in the model group (statistical markers not shown). ^cP< 0.05 vs. the DEX 2 mg·kg⁻¹ group; ^dP< 0.05 vs. the combination (OMT+DG, 45 + 45 mg·kg⁻¹). (A color version of this figure is available in the online journal.)

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Figure 3.

Histologic analysis of inflammatory infiltrates in the skin lesions of mice. At the end of the experiment (on day 14), the different groups of mice were sacrificed and their skin tissues were collected. The skin tissue sections were stained by H&E. The numbers of inflammatory infiltrates in individual sections were counted in a blinded manner. Data are representative images or expressed as the means ± SD of each group (n = 5 per group) from three separate experiments. (a) The representative images (magnification × 200). (b) Quantitative analysis of the inflammatory infiltrates. The numbers of inflammatory infiltrates in the skin tissues of all ACD mice were significantly greater than the control while the numbers of inflammatory infiltrates in all drug-treated groups were significantly less than that in the model group (statistical markers not shown). ^cP< 0.05 vs. the DEX 2 mg·kg⁻¹ group; ^dP< 0.05 vs. the combination (OMT+DG, 45 + 45 mg·kg⁻¹). (A color version of this figure is available in the online journal.)

Combination of OMT and DG significantly decreases the levels of serum IgE, IFNγ, and IL-4 induced by DNFB in mice

High levels of IgE, Th1, and Th2 responses are associated with the development and progression of ACD.^2–4 To further understand the role of combined OMT and DG in regulating the DNFB-induced inflammation in mice, the levels of serum IgE, IFNγ, and IL-4 in individual mice were measured by ELISA. First, the levels of serum IgE in all ACD mice were significantly higher than that in the healthy control (P < 0.05 for all, Figure 4). Second, the levels of serum IgE in all drug-treated mice were significantly lower than that in the model group of mice (P < 0.05 for all), indicating that treatment mitigated the DNFB-induced IgE responses in mice. In comparison with the DEX-treated mice, treatment with a high dose of OMT or any combination of OMT and DG, but not other monotherapies significantly reduced the levels of serum IgE in ACD mice (P < 0.05 for all). More importantly, the inhibitory effect of combined lower dose of OMT and DG (45 mg·kg⁻¹ of each) was similar to that of combined OMT and DG (90 mg·kg⁻¹ of each, 90 mg·kg⁻¹ of OMT, and 45 mg·kg⁻¹ of DG), but significantly stronger than that of any of the monotherapies in ACD mice (P < 0.05 for all). Similar patterns of serum IFNγ and IL-4 were detected in the different groups of mice, except that treatment with a lower dose of OMT or each dose of DG had stronger inhibitory activity against the DNFB-induced IL-4 responses than DEX treatment (P < 0.05 for all). Thus, treatment with both OMT and DG significantly mitigated the DNFB-induced ACD-related IgE, IFNγ, and IL-4 responses in mice.

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Figure 4.

Measurement of serum IgE, IFN-γ, and IL-4 levels in individual mice. At the end of the experiment (on day 14), peripheral blood samples were collected from individual mice and the levels of serum IgE, IFN-γ, and IL-4 in individual mice were measured by ELISA using specific kits. Data are expressed as the means ± SD of each group (n=5 per group) from three separate experiments. The levels of serum IgE, IFNγ and IL-4 in all ACD mice were significantly higher than the control while the levels of serum IgE, IFNγ, and IL-4 in all drug-treated groups were significantly lower than that in the model group (statistical markers not shown). ^cP< 0.05 vs. the DEX 2 mg·kg⁻¹ group; ^dP< 0.05 vs. the combination (OMT+DG, 45 + 45 mg·kg⁻¹).

Effects of OMT, DG, and combination of OMT and DG on plasma levels of sodium, potassium, creatinine, and urea in mice

High doses of OMT or DG as well as DEX may change the balance of plasma electrolytes, such as sodium and potassium. Finally, we determined the levels of plasma sodium and potassium in individual mice. As shown in Figure 5, there was no significant difference in the levels of plasma sodium and potassium between the model and healthy control groups of mice. Treatment with a high dose of OMT significantly reduced the levels of plasma sodium while treatment with high doses of DG increased the levels of plasma sodium in mice, relative to that in the model or healthy control group (P < 0.05 for both). Furthermore, treatment with either DEX or a high dose of DG significantly decreased the levels of plasma potassium in mice, relative to that in the model or healthy control group (P < 0.05 for both). However, treatment with both OMT and DG did not change the levels of plasma sodium and potassium in mice (P > 0.05 for both). In addition, there was no significant difference in the levels of plasma creatinine and urea among the different groups of mice (P > 0.05 for all, Figure 6). Therefore, combination of OMT and DG mitigated the high dose of OMT- or DG-altered plasma sodium and reduced potassium levels in mice treated with high-dose DG.

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Figure 5.

Measurement of plasma sodium and potassium in individual mice. At the end of the experiment (on day 14), peripheral blood samples were collected from individual mice and the levels of plasma sodium and potassium in individual mice were measured using a Siemens ADVIA 2400 autoanalyzer. Data are expressed as the means ± SD of each group (n = 5 per group) from three separate experiments. There was no significant difference in the levels of plasma sodium and potassium between the model and control groups. ^aP< 0.05 vs. the control group; ^bP< 0.05 vs. the model group.

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Figure 6.

Measurement of serum creatinine and urea in individual mice. At the end of the experiment (on day 14), peripheral blood samples were collected from individual mice and the levels of serum creatinine and urea in individual mice were measured using a Siemens ADVIA 2400 autoanalyzer. Data are expressed as the means ± SD of each group (n = 5 per group) from three separate experiments. There was no significant difference among the different groups of mice.