Preliminary Study On Nature And Mechanism Of Qingkailing Injection's Adverse Reaction

Qingkailing Injection(QKLI) has effect of heat-clearing and detoxifying, resolving phlegm and dredging collaterals, tranquilizing and allaying excitement, restoring Consciousness and inducing Resuscitation, it's wildly used in clinical. But with the increase of clinical applications, the reports of QKLI's adverse reaction(ADR) had gradually increased. So, to find the most possible materials which cause QKLI's ADR is the fundamental solution to control the security of QKLI.Currently, some papers reported that the ADRs of QKLI could occur within 30min after drug treatment. These ADRs contained drug fever, skin reactions, allergic shock, or ADRs associated with the blood, digestive and respiratory systems, which appeared as acute hypersensitivity. However, there were many ADRs occurred in the first dosage without medication history. These reactions didn't have the incidence condition of TypeⅠallergic which has the sensitization process on the body. So many scholars classified such reactions as pseudoallergy. Pseudoallergy was caused by the non-IgE-mediated degraulations of mast cells(releasing a large number of biologically active substances), which is similar to allergy. There were two ways to cause pseudoallergy:1. Stimulating mast cells and basophils degranulation directly; 2. Increasing the number of anaphylatoxin C3a and C5a by the classical ways or the alternative pathways.Therefore, Identifying the nature and mechanism of QKLI's ADR was the first step to find the sensibilisin which caused ADRs. This paper started from toxic reaction of QKLI, and then studying on mast cells degranulation and tests of complement activation in vitro, providing the basis for follow-up study.Objection:1. To characterize the nature of QKLI's ADR in clinical and determine whether the ADRs is the toxic, allergic and pseudoallergic reactions or not.2. To determine the mechanism of QKLI's ADR in clinical and find out the relationship between the mechanism and the complement activation.Methods:1. Toxic reaction induced by single dose(1) Blood routine detection and histological examination:Kunming mice were randomly divided into 5 groups:4 groups of drugs which dose were 1,5,10,15 times of QKLI's clinical maximum dose and 1 group of saline,10 mice in each group. Every mouse was administrated through the tail vein. The mice were fasted but not deprived water for 12 h before administration, and were fed 14 d normally after administration. During the test, we observed the changes of animal weight, diet, appearance, behavior, secretions, excretions, death and toxic reactions.We dissected the dying and dead animals timely. We sampled blood of other animals for blood routine detection (counting red blood cells, white blood cells, platelets and hemoglobin, comparing the difference by Ons-Way ANOVA.) and also dissected them at the end of observation. We had histopathological examinations on the main organs which had any change of size, color, texture and so on. (2) Detection of serum electrolytes:Wistar rats were randomly divided into 2 groups:drug (10 times of QKLI's clinical maximum dose) group and saline group,8 rats in each group. Every rat was administrated through intraperitoneal. The mice were fasted but not deprived water for 12 h before administration. We anesthesiaed the rats by chloral hydrate at 5 min after administration,and then sampled blood by decollation to have tests for serum electrolytes (contents of K+n Na+,Ca2+), compared the difference by Independent-Samples T Test.2 Hemolysis test in vitro(1) Hemolysis in vitro:We prepared 2% red blood cell susupension with fresh rabbit blood according to Pharmacopoeia, detected the absorbance of hemoglobin by microplate reader at 570nm. We investigated the hemolysis of different lot numbers of QKLIs and different component extract liquids which were self-made or manufacturer's in vitro, every drug group had four concentrations. And we compared the difference by Univariate and LSD. (2)Hemolysis in vitro after multiple doses: Balb/C mice were randomly divided into experimental and blank group, administrated of QKLI (5 times of clinical maximum dose) and saline respectively every other day for 3 times totally,6 mice in each group.In the 15th day after administration, we prepared 2% red blood cell susupension with the blood sampled by decollation respectibely, and then investigated the hemolysis, compared the difference by Univariate.3 Mast cell degranulation test(1)We investigated the effect of PBS or QKLIs with differet concentration on the growth of RBL-2H3 cells by MTT after they were cultured for 24 h together. (2)We incubated the different liquids together with RBL-2H3 cells for 45 min, and then calculated the release of histamine orβ-heosaminidase in the supernatant, so as to investigate the effect on degranulation of Tyrode's liquid, QKLIs with different concentrations and lot numbers, also the different component extract liquids which were self-made or manufacture's. We compared the difference by One-Way AN OVA.4 Experiment of serum complement activation in vitroAfter QKLI or PBS together with human serum were incubated in vitro, we detected the content of SC5b-9 which was the final product of the complement activation, compared the difference by Paired-Samples T Tests,o as to study the effect of QKLI on the complement activation. Then we incestigated the effect of different concentrations and time for incubating on the content of SC5b-9, compared the difference by Univariate.Results:1 Toxic reaction induced by single doseWith dose increasing, mice quickly showed different responses such as hypodynamia,convulsion syncope and even death after administration. In 15 times of clinical maximum dose group, blood routine detection showed that mice had low counts of RBC and low contenst of hemoglobin(VS saline group, F=10.973, P= 0.008; F= 9.445, P=0.011); the pulmonary pathological sections of dead mice showed significant hyperemia. There was no significant difference in the contents of serum electrolytes (K+,Na+,Ca2+) between the experimental and blank group(t= 0.404, P=0.692;t=-0.352, P=0.730; t=-0.803, P=0.436).2 Hemolysis test in vitroIntegrating the observed and detected results, it showed that the extract liquids of honeysuckle, gardenia and the mixed liquid of buffalo horn & pearl shell could cause hemolysis, but the manufacture's baicalin liquid and both of the self-made and manufactur's honeysuckle, pearl shell, and radix liquid could cause red cells agglutinated in the high concentration(5 or more times of clinical maximum dose). In the different lot numbers of QKLI, excepting the drugs which lot number were 812903A and 811704A (both were got from the same pharmaceutical company), could cause hemolysis in the higher concentration(10 or more times of clinical maximum dose). The drugs which lot number were 811704A and 070702 caused red cells agglutinated mildly. There was no significant difference in the hemolysis test in vitro between the experimental and blank group of Balb/C mice after multiple dose(F =0.022, P=0.882).3 Mast cell degranulation testThere was significant difference in the effect of growth of RBL-2H3 cells between the groups of QKLIs and PBS(F=593.247, P=0.000). When the concentrations were 1/960～2/15(V/V), QKLI could promote the RBL-2H3 cells growth, while the concentration was greater than 2/15(V/V), QKLI could inhibit the RBL-2H3 cells growth. QKLIs of different concentration could increase the RBL-2H3 cells' release of histamine andβ-heosaminidase significantly (P<0.05). QKLIs of differentlot number could increase the release ofβ-heosaminidase significantly (P<0.05), and only 2/9 of them increased more than the positive group. The extract liquids which were self-made (concentration was 1/10(V/V)) could increase the RBL-2H3 cells' release ofβ-heosaminidase (especially the extract liquids of honeysuckle), excepting the pearl shell(F= 17024.728, P=0.000). All the extract liquids which were purchased from manufacturer (concentration was 1/10(V/V)) could increase the RBL-2H3 cells' release ofβ-heosaminidase, especially the honeysuckle and gardenia(F=2517.163, P=0.000).4 Experiment of serum complement activation in vitroCompare with the group of PBS, the QKLI group had significant difference in the content of SC5b-9 after incubating 30 min in vitro. The QKLI group was lower than the PBS group(t=2.897, P=0018). And we compared the groups of different incubated concentrations or time, F=3.436, P=0.066; F=1.97, P=0.182.Conclution:We should classify the ADRs of QKLI in clinical as not only TypeⅠallergy but also toxic and pseudoallergic reactions. Parts of symptoms of ADRs might be due to acute intravascular hemolysis which was caused by some honeysuckle and gardenia. The intravascular hemolysis phenomenon was independent of administration frequency. Parts of symptoms might be due to mast cells degranulation which was caused by the directly stimulating of honeysuckle, gardenia and radix, in other word, this phenomenon was pseudoallergy. QKLI could inhibit rather than activate the complement system in human serum, so we concluded that the mechanism of mast cell degranulation which was caused by QKLI had no relationship with complement activation.