In Vitro Immunotoxicity Evaluation Of 5-HMF And Its Dimer OMBF Using RBL-2H3 Cells And Immunogenicity Of Three Traditional Chinese Medicine Injections Using PLNA

5-Hydroxymethylfurfural(5-HMF)is the main product of Maillard or sugar caramelization.It is a low molecular weight compound(LMWC)with a molecular weight of 126.11 and widely found in sugar-containing drugs and foods.Previous researches had shown that 5-HMF has pharmacological effects such as antioxidant,anti-tumor and anti-ischemia activity.However,exposure to 5-HMF could lead to renal toxicity and potential genotoxic.The limit of 5-HMF in dextrose injection is 11.8μg·ml-1 stipulated in Pharmacopoeia of the People’s Republic of China.Our earlier study analyzed the content of 5-HMF in multiple batches of traditional Chinese medicine injections(TCMIs)sold on the market and the content of 5-HMF in some TCMIs was much higher than 11.8μg·ml-1.In addition,we also detected a noticeable compound in those injections containing much higher concentration of 5-HMF,and the compound was subsequently identified as the dimer of 5-HMF(i.e.5,5’-Oxydimethylenebis(2-furfural),OMBF).With the widespread use of TCMIs in clinical applications,reports of adverse reactions have also increased.The highest incidence was immune-mediated drug hypersensitivity(IDHR)including anaphylactoid and type I hypersensitivity reaction.5-HMF polymers are a common byproduct of sugar degradation and their potential toxicity has not attracted widespread attention.In the first part,we investigated the in vitro potential immunotoxicity of 5-HMF and its dimer OMBF,including anaphylactoid reaction and type I hypersensitivity,using Basophilic leukemia cell line 2H3(RBL-2H3)mast cells.Firstly,growth characteristics and degranulation excitation conditions of RBL-2H3 were explored,and in vitro mast cell activation and degranulation model was established.Under the optimal conditions,5-HMF and OMBF directly induced anaphylactoid reaction in RBL-2H3,and induced type I hypersensitivity reaction in sensitized RBL-2H3.The indicators for detection included RBL-2H3 cell morphology,release rate of β-Hexosaminidase(β-HEX)and histamine(His),Ca2+ levels,production of interleukin-6(IL-6)and Tumor necrosis factor(TNF-a).The main results are showed as follows:(1)The optimal density of plating was 1×105 cell/mL,and the whole experiment was completed within 72 hours.The IC50 of 5-HMF and OMBF were respectively 4.96 mM and 5.35×10-2 mM.The toxicity of OMBF was 90 times higher than that of 5-HMF.(2)In model group,0.5 μg/mL anti-DNP-IgE monoclonal antibody was incubated with RBL-2H3 cells overnight,100 ng/mL DNP-HSA induced cells to degranulate,and incubation time was selected for 2 h in follow-up experiments.(3)In vitro immunotoxicity evaluation of anaphylactoid reaction showed low dose of 5-HMF and low dose of OMBF significantly promoted the release of β-HEX and His and stimulated the secretion of IL-6.In high dose of OMBF group,the release of His,intracellular Ca2+ concentration and TNF-α,IL-6 secretion were significantly increased.(4)In vitro immunotoxicity evaluation of type I hypersensitivity reaction showed under high dose of 5-HMF stimulation,release rate of β-HEX and His,TNF-α and IL-6 secretion were significantly increased compared with control group.In high dose of OMBF group,the morphology changed,the release of β-HEX,His and secretion of TNF-α,and the intracellular Ca2+ concentration were significantly increased.We can conclude that low dose of 5-HMF,low dose and high dose of OMBF induced anaphylactic reaction,stimulated mast cell degranulation and released of bioactive media.BN rats were sensitized with 5-HMF and OMBF at a clinically equivalent 10-fold concentration.At the concentration of IC50,5-HMF and OMBF induced sensitized mast cells activation and cause type I hypersensitivity reaction.Our previous research found 5-HMF and its OMBF may play a role in causing IDHRs or autoimmune disorders by TCMIs containing saccharides.According the experimental results of first part,we chose three kinds of TCMIs that contain 5-HMF and OMBF:Mailuoning,Shengmai,Danxiangguanxin injection in the second part.We carried out direct popliteal lymph node assay(D-PLNA)and reporter antigen popliteal lymph node assay(RA-PLNA)to assess the immunogenicity of three test substances.We established D-PLNA and RA-PLNA mice models.The mice’s hind footpads were measured after they received a subcutaneous injection into hind footpad.The mice were sacrificed in D7 and popliteal lymph nodes were excised to prepare single cell suspensions.Cell number was counted and concentration of IgE and MCP-1 were measured after cell suspension was cultured with stimulant in plate.The main results are showed as follows(1)Immunogenicity of three kinds TCMIs using D-PLNA:Danxiangguanxin injection could increase hind footpads and PLN cellularity significantly;Mailuoning and Shengmai injection did not produce a significant positive reaction on D-PLNA.(2)Immunogenicity of three kinds TCMIs using RA-PLNA:each treatment group significantly increased PLN cellularity and the concentration of MCP-1 when co-injected with TNP-Ficoll;when co-injected with TNP-OVA,Mailuoning injection stimulated significant secretion of IgE while Danxiangguanxin increased the concentration of IgE and MCP-1.Mailuoning and Shengmai injection only caused positive reaction after RAs were added,this suggested they may contain low molecular weight compounds as hapten to induce type I hypersensitivity reaction.However,the composition of Danxiangguanxin injection is complex and may directly induce type I hypersensitivity reaction and may also act as a neo-antigen or neo-epitope to elicit immune response.In summary,we successfully established a RBL-2H3 cells degranulation model and systematically evaluated in vitro immunotoxicity of 5-HMF and OMBF.We found for the first time that 5-HMF and OMBF may simultaneously induce anaphylactoid reaction and type I hypersensitivity,and the formation of dimer OMBF increased the immunotoxicity of 5-HMF.The results of PLNA further proved that 5-HMF and OMBF in TCMIs may cause disorders of immune system as a hapten combining with in vivo protein.In vitro immunotoxicity evaluation of 5-HMF and OMBF and immunogenicity of three TCMIs provide a new basis for the prevention and treatment of clinical adverse reactions.In addition,it is necessary to carry out quality control and in-depth study of 5-HMF and its dimer OMBF in related TCMIs.