目的 建立過敏和類過敏疊加的RBL-2H3細胞和ICR小鼠動物模型,對注射用血塞通(凍干)(XST)的過敏與類過敏反應(yīng)進行評價研究。方法 體外以RBL-2H3細胞的β-氨基己糖苷酶(β-Hex)和組胺釋放率為評價指標(biāo),確定抗二硝基苯單克隆抗體(DNP-IgE)、DNP-牛血清白蛋白(BSA)的劑量及與C48/80(30 μg·mL^-1)作用的最佳時間,篩選過敏和類過敏疊加模型的陽性條件,隨后考察XST (4、8、16 mg·mL^-1)對細胞活力及與DNP-IgE/BSA疊加后對細胞脫顆粒的影響。體內(nèi)以ICR小鼠為實驗對象,以(類)過敏反應(yīng)癥狀分值及血漿中免疫球蛋白E (IgE)、組胺、5-羥色胺、血管內(nèi)皮生長因子A (VEGF-A)、末端補體復(fù)合物(SC5b-9)含量為評價指標(biāo),篩選卵蛋白(OVA,2.5、5.0、10.0 mg·kg^-1)、C48/80(1、2、4 mg·kg^-1)過敏-類過敏模型陽性條件,最后對XST (60、120、240 mg·kg^-1)的致敏性及是否會產(chǎn)生過敏、類過敏反應(yīng)進行評價。結(jié)果 體外細胞實驗最終確定400 ng·mL^-1的DNP-IgE致敏后用50 ng·mL^-1的DNP-BSA激發(fā)的同時與C48/80共同作用30 min作為過敏與類過敏疊加陽性組;16 mg·mL^-1的XST與DNP-IgE/BSA聯(lián)合疊加時,與單給DNP-IgE/BSA或XST組比較均促進組胺和β-Hex的釋放(P<0.01)。體內(nèi)小鼠實驗中5、10 mg·kg^-1的OVA均會使小鼠體內(nèi)IgE顯著升高,依據(jù)過敏樣反應(yīng)分值和小鼠血漿內(nèi)組胺、VEGF-A和SC5b-9含量最終確定5 mg·kg^-1 OVA與1 mg·kg^-1 C48/80建立疊加模型,耳、肺及支氣管組織中可見明顯的水腫及炎性細胞浸潤。單純的XST不會對小鼠致敏,但是與OVA介導(dǎo)的過敏反應(yīng)疊加后,與單給DNP-IgE/BSA或XST組比較,會顯著提高血漿中內(nèi)組胺、VEGF-A和SC5b-9水平(P<0.05、0.01),與建立的過敏-類過敏疊加模型表現(xiàn)出較好地一致性。結(jié)論 體外體內(nèi)實驗均表明IgE介導(dǎo)的過敏反應(yīng)和C48/80引起的類過敏反應(yīng)會產(chǎn)生疊加作用,加劇過敏介質(zhì)的釋放和免疫反應(yīng)程度。同時此模型的建立也驗證了XST存在過敏與類過敏疊加現(xiàn)象,該模型可為中藥注射劑的臨床前安全性評價及合理用藥提供參考。

Objective To establish an allergic and anaphylactoid superimposed reaction RBL-2H3 cells and ICR mice model for the study of allergic and anaphylactoid reaction to Xuesaitong Injection (freeze-drying) (XST). Methods This study employed the β -hexosidase and histamine release rate of RBL-2H3 cells as evaluation indices in vitro to determine the optimal dose of DNPIgE, DNP-BSA, and the duration of interaction with C48/80. The study investigated the effects of various doses of XST on cell viability and degranulation after stacking. In vivo, ICR mice were used as experimental subjects, and the scores of (type) allergic reaction symptoms and the contents of immunoglobulin E (IgE), histamine, 5-hydroxytryptamine, vascular endothelial growth factor A (VEGF-A), and terminal complement complex (SC5b-9) in plasma were used as evaluation indicators to screen positive conditions for OVA (2.5, 5.0, 10.0 mg·kg^-1) and C48/80 (1, 2, 4 mg·kg^-1) allergy-like allergic models, and finally, the sensitization of XST (60, 120, 240 mg·kg^-1) and whether it would cause allergic or allergic-like reactions were evaluated. Results The positive group for anaphylactic-anaphylactoid superimposed reaction was determined to be 400 ng·mL^-1 DNP-IgE sensitised with 50 ng·mL^-1 DNPBSA and co-administered with C48/80 for 30 min. Additionally, the release of His and β-Hex was promoted by 4, 8, and 16 mg·mL^-1 of XST in combination with IgE. In the in vivo mouse experiment, 5 and 10 mg·kg^-1 OVA significantly increased IgE levels in mice. Based on the allergy-like reaction score and the contents of histamine, VEGF-A, and SC5b-9 in the plasma, the 5 mg·kg^-1 OVA and 1 mg·kg^-1 C48/80 were used to establish a stacked model of allergy-like reaction. There was obvious edema and inflammatory cell infiltration in the ear, lung, and bronchial tissue. XST alone did not sensitize mice, but when combined with OVA-induced allergic reaction, it significantly increased the levels of intra-plasma histamine, VEGF-A, and SC5b-9 compared to the single administration of DNP-IgE/BSA or XST groups (P < 0.05, 0.01), showing good consistency with the established allergy-like-allergy stacked model. Conclusion In vitro and in vivo experiments have shown that IgE-mediated allergic reactions and C48/80-induced anaphylactoid reactions produce a superimposed effect, exacerbating the release of allergic mediators and the degree of immune response. The establishment of this model also verified the existence of allergic and anaphylactoid superposition phenomenon of XST. This model can provide a reference for the preclinical safety evaluation and rational use of Chinese medicine injection.

R965.3

國家自然科學(xué)基金資助項目(82074006);江蘇省自然科學(xué)基金面上項目(BK20211303)