Allergic Reactions of Guinea Pigs Induced by Several Chinese Herbal Injections

Abstract

In order to explore the feasibility to predict allergic reactions induced by several Chinese herbal injections (CHIs) earlier, guinea pigs were sensitized by injecting different CHIs for three times, serum IL-4 and IgE were detected by method of enzyme linked immunosorbent assay (ELISA) before challenged once by injecting the same drug intravenously. The results were compared with that of routine method of intraperitoneal sensitization. We found the contents of serum IL-4 and IgE increased significantly before guinea pigs were challenged, either in eighth day (d8) after intravenous sensitization or in d14 and d21 after intraperitoneal sensitization by Qingkailing injection or Xiangdan injection. Allergic reactions of guinea pigs occurred after challenged by the same drug, while not seen in Chuankezhi injection. It suggests that CHIs-induced allergic reactions can be predicted earlier by detecting serum IL-4 and IgE in d8 after sensitization, the examination period is reduced by 1-2 weeks compared with the routine method. It has a good application value in drug emergency test.

Keywords: IL-4; IgE; Chinese herbal injections; Allergic reaction; Guinea pigs

Introduction

Type I allergy induced by several Chinese herbal injections (CHIs), showed an increasing trend recently [7,2,4]. This dangerous type of allergic reactions was commonly thought to be mediated by IgE antibodies [1,9,3,5]. Some studies showed that IL-4 and IL-13 were responsible for resulting in production of allergen-specific IgE antibodies [11,13,10]. Thus, anti-IgE and anti-IL-4 therapy had been used as a new strategy in allergy [6,16,15].

The criterion of test for allergen had not been employed in current Pharmacopeia of United States, European Pharmacopeia, Japanese Pharmacopeia and British Pharmacopeia. Although the current method of test for allergen with guinea pigs was employed in Chinese Pharmacopeia (Ch.P.), the main limitation lied with its long examination period (2-3 weeks) and subjective indexes of assessment (including hair-pricking, shivering, retching and so on), which limited its application in drug emergency test for allergen. It was necessary to find new methods for early prediction of allergic reactions induced by CHIs. In this study, we try to propose the biomarkers of serum IL-4 and IgE to predict allergic reactions induced by CHIs.

Materials and Methods

Drugs and reagents

Qingkailing injection (lot No.090118-6, specification: 10 ml/ bottle; Jilin, China). Xiangdan injection (lot No.080524, specification: 10 ml/bottle; Zhejiang, China). Chuankezhi injection (lot No. 20071102, specification: 2.0 ml/bottle; Guangdong, China). All these CHIs above mentioned were produced according to the requirements of GMP and Ch.P.. Horse serum (lot No.090318, specification: 100 ml/bottle) was provided by Guangzhou Rueite Biological Technology Co., Ltd of China. Normal Saline (N.S, lot No.09121103, specification: 100 ml/bottle) was provided by Kunming Yusi Pharma. Co., Ltd of China, The kit for determination of serum IL-4 and total IgE were purchased from Shenzhen Jingmei Biological Engineering Co., Ltd of China. Elx-808 microplate reader was purchased from Bio-tek Company of USA.

Experimental animals

Hartley guinea pigs (qualified number is 2008A0026, half male and half female) were purchased from medical experimental animal center of Guangdong province, with an average weight of 280- 320 g and similar age. The guinea pigs were bred and housed in environmentally controlled rooms with 12 h light/dark cycle and allowed free access to food and water. Experiments followed the criteria of the National Research Council for the care and use of laboratory animals in research and guidelines by the Animal Ethics Committee of Guangdong Institute for Food and Drug Control, respectively.

Determination of serum IL-4 and total IgE by ELISA method

Venous blood were centrifuged at 1200 g for 15 min, serum was isolated and transferred into another tube and stored under -30°C condition, contents of serum IL-4 and total IgE were detected in a month with ELISA method followed by the instructions manual.

Detection of allergic reactions provoked by CHIs (intravenous sensitization)

Forty guinea pigs were randomly divided into five groups (N.S, 10% horse serum, Qingkailing injection, Xiangdan injection, Chuankezhi injection), half male and half female. Horse serum was dissolved in N.S with final concentration of 10% (v/v), other CHIs were applied respectively without dilution. Eight guinea pigs in one group were sensitized successively by injecting corresponding drugs (0.5 ml each) intravenously once a day for three times (d0, d1, d2), venous blood were collected respectively at the same time on d8 before eight guinea pigs in same group were challenged. After that, each guinea pig was challenged once by injecting the same drug (1.0 ml each) intravenously; allergic reactions were checked and recorded. Serum IL-4 and total IgE were detected.

Detection of allergic reactions induced by CHIs (intraperitoneal sensitization)

Ninety guinea pigs were randomly divided into five groups (N.S, 10% horse serum, Qingkailing injection, Xiangdan injection, Chuankezhi injection), half male and half female. Horse serum was dissolved in N.S with final concentration of 10% (v/v), other CHIs were applied respectively without dilution. Eighteen guinea pigs of the same big group were sensitized by injecting corresponding drugs intraperitoneally (0.5 ml each) every other day for three times (d0, d2, d4), venous blood were collected respectively at the same time on d8, d14 and d21 before six guinea pigs in each small group were challenged in that day. After that, six guinea pigs in each small group were challenged once by injecting the same drug (1.0 ml each) intravenously; allergic reactions were checked and recorded. Serum IL-4 and total IgE were detected.

Data processing and analyzing

Data of ELISA detection were presented as mean ± S.D. All data in this paper were analyzed by SPSS 18.0. T-test was used for comparison with two sets of data, and single factor ANOVA was used for comparison with three or more sets of data.

Results

Results of CHIs induced allergic reactions (intravenous sensitization)

After sensitized successively by injecting different drugs intravenously for three times (d0, d1, d2), no guinea pigs showed allergic reactions in groups of N.S and Chuankezhi injection after challenged in d8, and all guinea pigs showed allergic reactions in groups of 10% horse serum, Qingkailing injection, Xiangdan injection in d8 (Table 1), the onset time of allergic reactions was at about 5 min after challenged.