Safflower yellow in Carthami Flos is responsible for Xuebijing Injection-induced immediate hypersensitivity reaction through activating complement C3.

Objective: Xuebijing Injection (XBJI) is mainly used for treating sepsis in China, and even COVID-19 recently. This study aimed to clarify the molecular mechanism(s) and identify the potential "common culprit(s)" for XBJI-caused immediate hypersensitivity reaction (IHR) which is the main type of its adverse reactions. Methods: Antiserum against XBJI was prepared by intraperitoneal immunization in combination with aluminum adjuvant for five weeks. Antagonistic experiments were performed by using several antagonists against different mediators in Evans Blue leakage model. Propranolol-pretreated mice were used to determine the capacity of XBJI to trigger systemic IHR. Serum total IgE (tIgE) and mouse mast cell protease 1 (MCPT-1) levels, complement activation, and the levels of supernatant inflammatory mediators were determined by ELISAs. Lipopolysaccharide (LPS)-activated RAW264.7 macrophages were used for evaluating the anti-inflammatory activity of XBJI, while human mast cells (LAD2) were used for assessing the effect of XBJI on mast cell degranulation. Results: Continuous treatment (i.p.) with XBJI along with aluminum adjuvant did not elevate the levels of serum tIgE and MCPT-1. In vitro, XBJI could not directly cause the degranulation of LAD2 cells. It induced a robust Evans Blue leakage after the first injection in mouse paw. Mechanism study demonstrated that antagonists for histamine H1/H2 receptors and complement C3a receptor counteracted XBJI-induced IHR. XBJI also directly activated complement C3 in human serum. Through screening five herbs of XBJI and the constituents, only safflower yellow (SY) in Carthami Flos was able to induce IHR. The discolored-XBJI not only did not induce IHR locally and systemically, but also could suppressing the production of proinflammatory mediators in LPS-activated RAW264.7 macrophages. Conclusion: XBJI failed to induce immune IHR, but potently triggered non-immune IHR through direct activating complement C3 to provoke histamine release. SY in Carthami Flos was the underlying "common culprit" responsible for XBJI-caused IHR. The anti-inflammatory action of XBJI can be retained after decolorization. Our study provides a scientific basis for not only preventing and treating XBJI-caused IHR clinically, but also improving its production process.