Notes
Development of an Assay Method to Search for Compounds Inhibiting Stress-Enhanced Allergy
2016 Volume 39 Issue 5 Pages 874-878
Details
2016 Volume 39 Issue 5 Pages 874-878
Stress exacerbates allergic disorders such as atopic dermatitis and asthma. It is also an important factor affecting blood flow (BF). Allergic reactions also affect blood flow. For example, we observed that mice sensitized with hen egg-white lysozyme (HEL) have decreased BF during the allergy induction phase. Based on this finding, we established a model for evaluating chronic restraint stress-enhanced allergies. Mice were sensitized with 12.5 µg/head of HEL on day 0, then restrained for 90 min daily on days 1–3, 5, and 6 in a modified 50 mL polystyrene conical centrifuge tube with multiple air holes for ventilation. We used the decrease in BF during that time as a guide for developing an in vivo assay for substances that can inhibit stress-enhanced allergies. Finally, we demonstrated the utility of the new method by testing crude drugs that are used solely or in combination with other crude drugs to treat stress-related illness and neuropsychiatric symptoms. Our model should be useful for identifying potential anti-stress-enhanced allergy drugs.
Allergic diseases, such as atopic dermatitis and asthma, become severe or relapse due to stress. However, there have been no systems that can evaluate allergy enforced by stress.
As stress is an important factor affecting blood flow (BF), we have developed a new assay system based upon our findings1–3) on BF decrease in the induction phase of allergy in the tail vein of mice sensitized by use of hen egg-white lysozyme (HEL). We previously developed an in vivo assay to search natural resources for allergy-preventive substances. We used it to assess BF during the allergy induction phase in the tail veins of mice that have been sensitized to HEL.1–3) Blood flow in the model appears to be regulated by various factors such as nitric oxide, thromboxane A2 (TXA2), prostacyclin (PGI2), and endothelin-1 (ET-1) together with cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and constitutive nitric oxide synthase (cNOS).1) A reduction of BF occurs via two pathways. One is an iNOS-independent response involving TXA2 and ET-1; and the other is an iNOS-dependent response involving cyclooxygenase COX-1, COX-2, and PGI2.2) Its clinical condition is also similar to the “oketsu” (the state of blood stagnation) of the Kampo medicine (a traditional medicine practiced in Japan, based on ancient Chinese medicine).3) In our continuing search for allergy-preventive substances from natural sources using this assay method.4–7) On the other hand, allergic disorders become severe or relapse due to stress.8,9) It also affects the production of substances such as interleukin and tumor necrosis factor, which cause allergies and inflammation.10) Additionally, stress is an important factor affecting BF.11–13) However, assay for compounds that inhibit stress-enhanced allergy have been unavailable. Therefore, we developed a novel assay system based upon our finding on BF decrease. In addition, we applied this assay method to crude drugs that used solely or in combination with other crude drugs for stress to examine usefulness of the method. We believe this method can be used to analyze the mechanism of stress-enhanced allergies and will contribute to the search for new treatments.
Complete Freund’s adjuvant (CFA), HEL, disodium cromoglycate (DSCG), and crude drugs were purchased from DIFCO (MI, U.S.A.), Sigma Co., Ltd. (St. Louis, MO, U.S.A.), Funakoshi Co., Ltd. (Tokyo, Japan), and Tochimoto Tenkaido Co., Ltd. (Osaka, Japan), respectively.
AnimalsMale ddY mice (SPF grade), 5 weeks old, were obtained from Japan SLC, Inc. (Shizuoka, Japan) and housed at 26±2°C. Food and water were available ad libitum. All animal experiments were performed in accordance with the Guidelines for Animal Experiments of Mukogawa Women’s University.
HEL SensitizationSensitization with HEL was performed as previously described1) with slight modifications. On day 0, male ddY mice were injected intraperitoneally with 12.5, 25, and 50 µg of HEL in 50% CFA.
BF MeasurementSubcutaneous BF in mice tail veins was monitored using a laser Doppler blood flow meter (FLO-C1; Neuroscience, Tokyo, Japan) as previously reported.2) Each mouse was warmed for 15 min at 36°C prior to the experiment and then placed on a holder (Muromachi Kikai Co., Ltd., Tokyo, Japan) in a measuring chamber kept at 36°C throughout the measurement. The systolic microcirculatory BF in the tail veins of unanesthetized mice was measured (n=5). Normal blood flow was measured for 10 min 1 d before studies began. The BF of the sensitized mice was measured for 10 min every other day up to 7–10 d after sensitization. The results were expressed as a relative percent of normal BF for each mouse.
Protocol and Schedules of Chronic Restraint StressThe stress load was applied using a modification of the method described by Castilla-Ortega et al.14) Mice in the stressed groups were restrained in a modified 50 mL clear polystyrene conical centrifuge with multiple air holes for ventilation. Three schedules of restraint were used. Schedule I: Mice were restrained 90 min daily for 10 consecutive days. Schedule II: Mice were restrained 90 min daily on days 1–3 and 5–8. Schedule III: Mice were restrained 60 min daily on days 1–3 and 5–9. Unstressed mice remained undisturbed in their home cages.
Measurement of Serum Corticosterone LevelsAfter the final day of stress, blood from the eyeground venous plexus of each mouse was collected and centrifuged (3000 rpm, 15 min, 20°C). The corticosterone level (pg/mL) in the supernatant serum was measured using a Cortisol Express EIA Kit (Cayman Chemical Co., MI, U.S.A.).
Crude DrugsCrude drugs (20 g each), Poria sclerotium (sclerotium of Poria cocos; PS, lot No. 009507001), cinnamon bark (bark of Cinnamomum cassia; CB, lot No. 002814004), bupleurum root (root of Bupleurum falcatum; BR, lot No. 004210011), magnolia bark (bark of Magnolia obovate; MB, lot No. 003608005), Japanese valerian (root and rhizome of Valeriana fauriei; JV, lot No. 001807001), and Japanese peppermint (herb of Mentha arvensis var. piperascens; JP, lot No. 00130504) were extracted overnight with 30% EtOH (60 mL) at room temperature, then filtered. The filtrate was evaporated in vacuo to yield crude drug extracts (0.41, 0.66, 0.73, 3.39, 6.33, and 6.41 g, respectively). These extracts were suspended in distilled water and used for bioassays.
Effects of Crude Drugs and an Anti-allergic AgentAll of the crude drugs were administered orally (200 mg/kg) at 0 (1 h before sensitization), 3, and 6 d as previously reported.3) An anti-allergic agent (DSCG, 10 mg/kg) was injected on days 0, 3, and 6 as previously reported.1) Water was administered to the control group on the same schedule.
Statistical AnalysisStatistical analysis was performed using Dunnett’s multiple range test coupled with Bonferroni inequality for significant differences between each test group and the control group. For the Bonferroni test, 5 points were used 4 d after the HEL sensitization, because a significance difference was observed between the BF of untreated and sensitized mice after 4 d.
Mice sensitized with HEL (50 µg/head) and restrained according to schedule I showed a decrease in BF, but the effect was not significantly different from BF decrease in unrestrained sensitized mice (Fig. 1). The BF decrease in the unrestrained sensitized group was too drastic to evaluate the effect of stress on allergy. Then, we experimented to find an amount of dose of antigen for the induction of a milder decrease of BF to evaluate effects of the stress in the BF.
Each point with bars represents the mean±S.E. of BF of five mice. ◇, only HEL-sensitized mice (sens.); ■, chronic restraint stress enhanced HEL-sensitized mice (sens.+stress).
The BF decrease for mice sensitized with HEL 12.5, 25, and 50 µg/head is shown in Fig. 2. While BF decrease was small but partial in the untreated group, it increased as the dose of HEL was raised, demonstrating that BF was dependent on the dose of HEL. We concluded that stress-enhanced allergy could be evaluated by reducing the dose of HEL to 12.5 µg/head.
Each point with bars represents the mean±S.E. of BF of five mice. HEL concentration is 0 (○, normal), 12.5 (●), 25 (△), and 50 (▲) µg/head. * p<0.05 compared with normal (Dunnett’s test with Bonferroni).
As shown in Fig. 3, the BF of mice that were sensitized with HEL (12.5 µg/head) and restrained (schedule I) gradually decreased. By day 10, it was significantly decreased compared to the BF in the untreated group. The BF in mice that were only sensitized or only stressed (schedule I) was the same as that of the untreated group. These results suggest that we can evaluate stress-enhanced allergy using the decrease in BF as a guide.
Each point with bars represents the mean±S.E. of BF of five mice. ▲, untreated mice (normal); ○, only stress enhanced mice; △, only HEL-sensitized mice; ●, stress enhanced HEL-sensitized mice. * p<0.05 compared with the normal (Dunnett’s test with Bonferroni).
We studied the stress schedule, looking for ways to provide relief for the mice and expedite the method. Schedule II reduced the number of days of stress, and schedule III reduced the number of days of stress and shortened the daily duration of the tests. For both schedules II and III, mice did not receive restraint stress on day 4. If restraint stress was canceled on days 2 or 3, we were concerned about the possibility of BF recovering from chronic stress as enough decrease of BF was not yet established. We decided to cancel days 4 and 5 (the early stage in the second half) when enough decrease of BF was established. For both schedules, the BF of mice in the stressed groups was significantly decreased compared with the untreated group (Fig. 4). Mice subjected to schedule II experienced a significant decrease in BF on day 5. For mice on schedule III, a significant decrease in BF was observed on day 10. The concentration of corticosterone (a stress hormone) was measured at the end of the experiment. Only mice restrained according to schedule II had a significant increase in serum corticosterone level (Fig. 5). Furthermore, the corticosterone level significantly differed between schedules II and III. In addition, the weight of stress-enhanced mice was impartial until the day 6, and a significant difference was seen on day 7 (Fig. 6). These results suggest that schedule II can shorten the duration of the experiment. Thus, mice may be immobilized in the tube for 90 min/d for five days and the BF tested on day 6.
Each point with bars represents the mean±S.E. of BF of five mice. △, untreated mice (normal); ○, schedule II stress enhanced HEL-sensitized mice; ●, schedule III stress enhanced HEL-sensitized mice. * p<0.05 compared with the normal (Dunnett’s test with Bonferroni).
Data represent the mean±S.E. of five mice. Untreated (normal), schedule II stress enhanced HEL-sensitized (II), and schedule III stress enhanced HEL-sensitized mice (III) were serum measurement corticosterone levels at day 10. * p<0.05 (Dunnett’s test).
Data represent the mean±S.E. of five mice. Body weight of untreated (normal), schedule II stress enhanced HEL-sensitized (II), and schedule III stress enhanced HEL-sensitized mice (III) on days 5 and 7. * p<0.05 compared with the normal (Dunnett’s test).
We restrained HEL-sensitized mice (n=5 mice per group) for 90 min daily on days 1–3, 5, and 6. Five groups were tested. Significant BF was found on days 5 or 7 for all groups, which confirmed reproducibility (Fig. 7).
Data represent the mean±S.E. of five mice. The reproducibility could be confirmed, since significant BF decrease was found on day 5 (a) or day 7 (b) for all experiments. a, b: p<0.05 compared with the normal BF of each mouse (Dunnett’s test with Bonferroni).
We sensitized mice with HEL (12.5 µg/head) on day 0. They were restrained for 90 min daily on days 1–3, 5, and 6 d in a modified 50 mL polystyrene conical centrifuge tube with multiple air holes for ventilation. The microcirculatory BF in the tail veins of the unanesthetized mice was monitored every other day up to 7 d after sensitization. Using BF decrease during that time as a guide, we developed an in vivo assay method to search for substances that can inhibit a chronic stress.
Effects of Crude Drugs on Stress-Enhanced AllergySeveral crude drugs have been used solely or in combination with other crude drugs to treat stress-related illnesses and neuropsychiatric symptoms. PS,15,16) CB,17,18) BR,19,20) MB,21,22) and JV23) are reported scientifically proven to have an effect on those clinical condition. Here, these drugs significantly improved BF in stress-enhanced mice (Figs. 8A, B). JP15) of the negative control, did not improve BF. Other assay methods based only on sensitization did not detect an appreciable effect on BF by PS, CB, and BR,3) because those crude drugs have different effects on BF depending on the presence or absence of stress. In a previous report, the anti-allergic agent DSCG significantly inhibited HEL-sensitized BF reduction in the absence of stress.3) However in our study, it did not suppress stress-enhanced BF reduction (Fig. 8C). This novel assay is appropriate for evaluating the complex interaction of allergy, stress, and inflammation.
Each point with bars represents the mean±S.E. of BF of five mice. Two hundred milligrams per kilogram of poria sclerotium (PS), cinnamon bark (CB), bupleurum root (BR), magnolia bark (MB), Japanese valerian (JV), and Japanese peppermint (JP) were orally administrated on days 0, 3, and 6. Ten milligrams per kilogram of DSCG and water (control) were administered on the same schedule. * p<0.05, ** p<0.01 compared with the control (Dunnett’s test with Bonferroni).
We could develop a new in vivo assay system for evaluation of the effect of the stress on allergy. Crude drugs that used solely or in combination with other crude drugs as remedy for the disease caused by stress did not show an appreciable effect on the BF decrease by the assay method based only on the sensitization established previously, but the present assay method showed a significant improvement of the BF decrease. Therefore, it indicates that the present system is applicable to evaluate the complex form of allergy, stress, and inflammation.
Thus, this system would contribute to the analysis of the mechanism of allergy enhanced by stress and the search for new substances effective to stress-enhanced allergy. Furthermore, it would be applicable to the analysis of the mechanisms of the enhancement of allergy by stress.
The work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
