| Qingkailing Injection (QKLI) has several pharmacological effects, such as heat-clearing, detoxifying, resolving phlegm, dredging collaterals, tranquilizing and allaying excitement, restoring consciousness and inducing resuscitation. But as its clinical applications have been broadened, the incident frequency of QKLI’s adverse reaction (ADR) gradually increased. It was disclosed that that the ADRs of QKLI could occur within30min after drug treatment. The symptom can be drug fever, skin reactions, allergic shock, or ADRs associated with the blood, digestive and respiratory systems, which was recognized as acute hypersensitivity. Since the explosion of ADRs, the consequent concerns on safety caused a reduced usage of QKLI and increased questioning of its rationality, which ultimately leaded to the "crisis of confidence". Therefore, to find the most possible actors which cause QKLI’s ADR is the fundamental solution to ensure the security of QKLI.Currently, some studies have suggested that the extract liquids of honeysuckle might be the main allergen which induces pseudoallergy in QKLI. Meanwhile, the studies about IgE-mediated QKLI anaphylaxis are poor, which only established a BN rat model of acute hypersensitivity. Moreover, some studies reported that chlorogenic acid could be used as a hapten to induce immune response, while some other studies thought it could not.In the present work, we established Balb/c mice model of IgE-mediated Qingkailing injection anaphylaxis, which was the first step to find the sensibilisin which caused allergic shock. Meanwhile, we also demonstrated the allergenicity of chlorogenic acid by using the methods, providing the basis for follow-up study.Objection1. To establish Balb/c mice model of IgE-mediated Qingkailing injection anaphylaxis, providing the basis for follow-up study.2. To demonstrate the allergenicity of chlorogenic acid with the method which established the Balb/c mice model of IgE-mediated Qingkailing injection anaphylaxis, providing the basis for follow-up study.Methods1Establishment of Balb/c mice model of IgE-mediated Qingkailing injection anaphylaxisFor the first step, we used several strategies to prepare cationic bovine serum albumin (cBSA) and QKLI-cBSA conjugate. The preparation of QKLI-cBSA was based on the Mannich reaction. Briefly,4mg of QKLI and2mg of cBSA were dissolved in physiological saline and conjugation buffer (0.1M MES pH4.8), respectively. After dropwise addition of QKL solution into the protein solution, the mixture was stirred gently, then50μl of formaldehyde were added into the mixture and immediately stirred gently for24h at37℃. The native bovine serum albumin (nBSA), cBSA and QKLI-cBSA conjugate were separated and purified by cationic exchange method and also identified by ultraviolet-visible (UV-Vis) and Fourier transform infrared (FT-IR) spectrometry methods.6-week-old female18BALB/c mice were randomly divided into3groups:QKLI-cBSA with alum’s adjuvant group, Ovalbumin with alum’s adjuvant (OVA) group and physiological saline group,6mice in each group. QKLI-cBSA conjugate were dissolved in sterilized saline and emulsified with an equal volume of Alum’s adjuvant, The emulsion solutions were injected0.3mL of subcutaneously into Balb/c mice in QKLI-cBSA+Alum group; Balb/c mice of OVA group received subcutaneous injection of the same dosage respectively. Booster injections were carried out on the14th day after the primary doses. The the same quantity of immunogen emulsified with Alum’s adjuvant at14days intervals. Saline group were administered the same volume of physiological saline. Mice had their blood sampling from the retro-orbital plexus just at7,14,21days after booster injections For this, after collecting, serum was allowed to repose for30min at37℃and thus slightly centrifuged in a bench centrifuge during10min at4℃and the clean material stored at-20℃until use. Serum was analyzed for total IgE and histamine levels by enzyme-linked immunosorbent assays (ELISAs), respectively.the total IgE content in mice serum. The ELISA assay was performed according to the protocol provided by the supplier, respectively. Lung reactions were evaluated by analyzing lung pathologic changes. For assessment of pathologic alterations, dissected lung tissues were washed with normal saline and then placed in10%neutral-buffered formaldehyde for1week. After fixation, lung specimens were embedded in paraffin wax, and five-micrometer sections were cut and stained with hematoxylin and eosin dye for morphology. Images of selected sections were captured at X100magnification using a zoom digital camera.2Investigation of allergenicity of chlorogenic acidFor the first step, we used several strategies to prepare cBSA and CGA-cBSA conjugate. The preparation methods of cBSA and CGA-cBSA was used as previously described. The nBSA, cBSA and CGA-cBSA conjugate were separated and purified by cationic exchange method and also identified by UV-Vis, FT-IR and MALDI-TOF-MS methods.6Balb/c mice were randomly divided into2groups: CGA-cBSA conjugate1and CGA-cBSA conjugate2; and3mice were in each group. The conjugate was dissolved in sterilized saline and emulsified with an equal volume of Alum’s adjuvant, the emulsion solutions were injected0.3mL of subcutaneously into Balb/c mice in conjugate+Alum group, respectively. Booster injections were carried out at the14th day after the primary doses. The the same quantity of immunogen emulsified with Alum’s adjuvant at14days intervals. Mice were bled from the retro-orbital plexus just at28days after booster injections. For this, after collecting, serum was allowed to repose for30min at37℃and thus slightly centrifuged in a bench centrifuge during10min at4℃and the supernantant was stored at-20℃before testing. Serum was analyzed for the titers of antibodies. Results 1Establishment of Balb/c mice model of IgE-mediated Qingkailing injection anaphylaxisIn summary, the Balb/c mice model of IgE-mediated Qingkailing injection anaphylaxis was successfully established. Firstly, we separated, purificated and identified the nBSA, the cBSA and complete antigen (QKLI-cBSA). Remarkably, we separated and purified the cBSA and QKLI-cBSA conjugate by cationic ion exchange method, which was beneficial to eliminate the interference of impurities, while the previous method did not. Meanwhile, QKLI was successfully coupled with cBSA, which was identified by UV-Vis and FT-IR spectrometry methods. Secondly, Balb/c mice were then treated with subcutaneous injection of QKLI-cBSA plus alum. Thirdly, serum was analyzed for total IgE and histamine levels by ELISA, respectively. Lung reactions were evaluated by analyzing lung pathologic changes. QKLI-cBSA group mice were significantly and successfully sensitized. QKLI-cBSA conjugate significantly increased total IgE and histamine in Balb/c mice serum (P<0.05). Moreover, histological examination of QKLI-cBSA conjugate treated mice showed acute injury with pulmonary alveoli and rapture of trachea, as well as neutrophilic cell infiltration. In summary, we successfully established a murine model of IgE-mediated Qingkailing injection anaphylaxis.2Investigation of allergenicity of chlorogenic acidAllergenicity of chlorogenic acid (CGA) was further investigated. Firstly, we separated, purificated and identified the nBSA, the cBSA and complete antigen (CGA-cBSA). Remarkably, we separated and purified the cBSA and CGA-cBSA conjugate by cationic ion exchange method, which was beneficial to eliminate the interference of impurities, while the previous method did not. Meanwhile, CGA was successfully coupled with cBSA, which was identified by UV-Vis spectrometry, FT-IR spectrometry and MALDI-TOF-MS methods. Secondly, Balb/c mice were then treated with subcutaneous injection of CGA-cBSAs plus alum. After Balb/c mice were immunized with CGA-cBSAs, the similar sensitivity of antisera against CGA between the CGA-cBSA1group and CGA-BSA2group was compared. Both groups have high antibody titer, which were4000. Conclusion:The present study successfully cationized the nBSA and prepared QKLI-cBSA conjugate; and immunized the BALB/c mice with subcutaneous injection of QKLI-cBSA plus alum. After immunization, the model was verified by analyzing the total IgE and histamine levels in serum. In addition, I evaluated the lung reactions by analyzing lung pathologic changes. Finally, I successfully established a murine model of IgE-mediated Qingkailing injection anaphylaxis. The model might be enlighten allergen researchers on detecting allergens in QKLI and other Traditional Chinese Medical Injections (TCMIs). On one hand, the protocol is applicapable to validate allergenicity of CGA; on the other hand, the protocol can also be applied in establising anapylaxis animal models of other TCMIs. |
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