Study On Immunosensitizing Potential Of Shuanghuanglian Injection Using Popliteal Lymph Node Assay

Traditional Chinese medicine injection (TCMI), as a unique Chinese Medical formulation invented by our country and a developmental direction of the modernization of traditional Chinese medicine, has broken down the limitations of Traditional Chinese Medicine regiments. With features like high bioavailability and fast-acting, TCMI has already overcome the weakness of traditional Chinese medicine (cannot be used to cure the severe diseases) and played an important or even irreplaceable role in the treatment of conditions ranging from cardiovascular and cerebrovascular diseases, respiratory diseases and cancer. However, with the wide application of TCMI, the prevalence of adverse reactions related to TCMI appears to have increased significantly in China over the past ten years. Immediate hypersensitivity reactions (IHR), generally being considered as the most frequent and severe adverse reactions induced by TCMI, may cause anaphylactic shock or even be life-threatening. Those safety concerns caused a reduced use of TCMI and increasingly questioned its rationality, and thus ultimately lead to the "crisis of confidence" to the whole traditional Chinese medicine industry. In order to solve this crisis, the adverse reactions and risks induced by TCMI must be comprehensively, objectively and scientifically evaluated and the safety of clinical use should be ensured. Among those, the key point which needs to be further elucidated is to understand the cause and mechanisms of adverse reactions induced by TCMI. TCMI usually contains 3 to 7, sometimes even up to 12, kinds of raw material medicines. Thus TCMI does contain complex mixtures with a number of unknown constituent. Moreover the constituent that induced the adverse reactions and what mechanisms to be involved are poorly understood yet. Notably, all TCMIs approved for sale in the market and used in clinical are products which have been performed pre-clinical safety evaluations well. The results from safety evaluations showed that TCMIs did not induce any adverse reactions in animals. For this reason TCMIs were regarded as safe drugs in human, In fact, since it remains difficult for regulatory safety studies to predict the immunosensitizing potential of TCMI, there is poorly correlation between animal experiments results and clinical adverse reactions. Traditionally the animal model used to predict hypersensitivity reaction are the active cutaneous anaphylaxis assay (ACA), the passive cutaneous anaphylaxis assay (PCA) and the active systemic anaphylaxis assay (ASA), which are mainly used to detect the sensitization of macromolecular protein antigen. As far as low molecular weight compound(LMWC) is concerned, ASA, ACA, and PCA's positive results suggests that LMWC has immunosensitizing potential, but it is not always true for the negative results because of the possibility of false negative results in the test. In brief, the lack of suitable experimental models is the bottleneck that restricts the study on mechanism. Therefore, there is a great need for new animal models to study adverse reactions and mechanisms caused by TCMI prior to crisis of TCMI to be resolved.Popliteal lymph node assay (PLNA) is regarded as a unique reliable and promising screening methods to indicate immunosensitization by LMWC. It is simple, time-saving, sensitive, high specific and with less uses of laboratory animals and samples. Five different versions of the PLNA have been proposed according to the different local treatments factors:the direct, secondary, adoptive, reporter antigen (RA) and Adjuvant PLNA. The direct PLNA (d-PLNA) and RA-PLNA are the most commonly used in drug immunosensitizing studies. Up to now, more than 130 LMWCs have been detected by PLNA in different laboratories and the findings showed good correlation, consistency and repeatability. However, existing research results are all about single-component LMWCs. it has not been reported that whether PLNA could be used to study the immunosensitizing potential of complex TCMI components. In addition, the standard system of PLNA has not been established at present, and there is no research report on the immunosesentizhing potential of chemicals or Chinese medicine allergens detected by PLNA in China. Moreover, animal species used in experiment reported in the foreign literatures, such as CBA mice, C57BL mice, F344 rats, and BN rats, were so expensive in China and not easily obtained. What's more, both the experience of animal's husbandry and the information of experimental background are scarce. For these reasons and combining the actual situation in our country, the aims of this study were to establish d-PLNA and RA-PLNA experimental model firstly; choose and define sensitive and appropriate animals from widely used laboratory animal species in China to define; secondly, establish a standardized, simple and feasible PLNA Experimental Operation procedures; thirdly, use d-PLNA and RA-PLNA methods that have been established to evaluate the immunosensitizing potential of TCMI and its single-components. We are trying to establish a special and sensitive method to evaluate the immunosensitizing potential of TCMI, and to clarify its mechanism and determine the allergen, which will provide new ideas and experimental evidences for TCMI safety evaluation, promote our immunosensitizing studies on drugs with international standards and have great economic and practical values on new drugs' R&D.Shuanghuanglian injection (SHLI) is a representative of heat-clearing and detoxifying TCMI with a wide range of applications and good efficacy in clinical use. SHLI (lyophilized) was included in The People's Republic of China Pharmacopoeia Supplement (2005) in July,2009. It is the only TCMI that was put into the Pharmacopoeia and the first one to use fingerprint atlas to control its quality. However, just like other types of TCMIs, the reports of clinical adverse reactions related to SHLI are increasing. Analysis of data from the existing literatures report confirmed that IHR induced by SHLI or Houttuynia injection accounted for the most adverse reactions of all TCMI and came in the forefront. The adverse reaction induced by SHLI is allergy-based and the common symptoms are mild skin etthema and rash, etc.; serious adverse reactions include systemic damage, respiratory damage as the main ones. The main causes to death are anaphylactic shock and anaphylactoid reactions. National Adverse Drug Reaction Monitoring Center reported the hypersensitivity reactions related to SHLI twice in 2001 and 2009. This means that SHLI is also facing the danger of being eliminated from the market. SHLI was the first successful development of TCMI and was made up of three kinds of Chinese herbal medicines, including skullcap, honeysuckle and forsythia. Chlorogenic acid (CGA), which was highly suspected as a hapten, was rich in SHLI. In addition, the injection does not contain any excipient and the main component is LMWC. Therefore, allergens can be determined by studying the mechanisms of SHLI-induced allergic reactions through d-PLNA and RA-PLNA. This has great significance for SHLI as well as all TCMI in clinical drug safety.1 Construction of animal models for popliteal lymph node assay Specific pathogen-free female adult KM mice, BABL/c mice, C57BL/6J mice, Wistar rats and BN rats were randomly divided into Group Veh, negetive control group and model group. Animals were injected subcutaneously on the right hind footpad with Mercury chloride (HgCl2), Diclofenac sodium salt (DF), D-Penicillamine (D-pen), Diphenylhydantoin (DP) and Streptozotocin (STZ) in saline or 20%DMSO/saline. A volume of 25μl or 50μl was used for mice while 50μl or 100μl for rats. Phenobarbital (PB) was used for negative control. Normal animal were only injected on the right hind footpad with saline. After being injected for seven days, mice were sacrificed by inhalating CO2. Popliteal lymph node (PLN) was extracted and placed on ice in PBS-BSA (1%). Weighted PLN after the rid of adherent adipose tissues and being dried. Took preparation of single-cell suspension and got the weight index (WI= weight PLN treated side/weight PLN untreated side) and cellullarity index (CI). WI>2 or CI>5 is considered as a positive response. During the study, do the hypodermic on the toes of right hind leg with test material and TNP-OVA or TNP-Ficoll together for 7 days. After the administration, executed the animals and extracted the PLN for single cell susponsion. Use the Elispot method for testing the number of AFCs. The increase of AFCs is considered as a positive reaction. The study results showed that the C57BL/6J mice, BALB/c mice as well as BN rats could structure the d-PLNA models successfully. We found that mice only have one PLN on each side. PLN of mouse is easy to be located and removed than rat. So BABL/c mice and C57BL/6J are the preferred animals for PLNA. Based on those, we chose BABL/c mice to structure the RA-PLNA models and used on the following two SHLI studies.2 Assessment of Immunosensitizing Potential of Shuanghuanglian Injection Using Direct Popliteal Lymph Node Assay in C57BL/6J MiceSixty C57BL/6J mice were randomly divided into 6 groups:vehicle (Veh), PB (1 mg/mouse), HgCl2 (50μg/mouse), D-pen(2 mg/mouse), SHLI low dose (1 mg/mouse) and high dose(5 mg/mouse),10 mice/group. Articles were injected subcutaneously into one hind foot pad of SPF female mice. On day 8 after injection, animals were sacrificed, the left (untreated side) and right (treated side) PLN were isolated, weighed respectively, and then PLN mass index (MI) was calculated. PLNs of 4 mice in each group were fixed in 4%paraformaldehyde solution, routinely stained with HE for histopathologic examination. The others (n=6) were prepared into single-cell suspensions, and then PLN cells index (CI) was calculated. PLN changes after administration of SHLI were observed and compared. We founded that MI and CI of SHLI treated group increased with the average MI≥2 and the average CI≥5,and the difference was significant when compared with group Veh (P<0.05) in C57BL/6J mice. Pathological examination showed that the volume of the right PLNs treated with SHLI became larger than those in left. Germinal center of PLN lymph was evident and vice cortex HEV cross-section increased. The results suggested that SHLI could induce significant PLN response that suggests it has the immuosensitizing potential.3 Assessment of Immunosensitizing Potential of Shuanghuanglian Injection and its Refined products Prepared according to Chinese Pharmacopoeia Using Direct Popliteal Lymph Node AssayTest articles, including 10 different pharmaceutical samples, were obtained from SHLI and its extracts after being refined by macroporous resin (D101).Randomly grouped the BALB/c mice and sterilized the right hind toes by alcohol. Use the insulin syringe or 1 ml syringe for injecting the sensitizer (D-pen), test material and dissolvent as the negative control. The treated volume of mice was 50μl. We used d-PLNA method to study on the reactions of the dose of 5 mg/mouse at first. At this moment, cut down the dose to 2.5mg/mouse when the sample had the PLNA positive reaction. When the negative reaction occurred, raise the dose to 10 mg/mouse. Dose-responses relationship could be studied through these methods. We found that SHLI prepared according to Pharmacopoeia procedure obtained positive results at high-dose, the rest did not respond; the refined products'PLNA reactions reduced. This suggested that the Pharmacopoeia procedure of SHLI was badly needed an improvement. What's more, the refined products were good in water-solubility and even the increase of dose wouldn't lead to positive reactions. This suggested that the adverse reactions may be induced by poor water-soluble components, In this sense, in order to study on the sensitized components of SHLI, new production procedure should be used for the separation of three medicinal compositions.4 Assessment of Immunosensitizing Potential of Shuanghuanglian Injection Prepared by new Production Procedure Using Direct Popliteal Lymph Node Assay d-PLNA was used for the assessment of immunosensitizing potential of 16 samples separated by new production procedure. The BALB/c mice were randomly grouped and the right hind toes were sterilized by 70%alcohol. The insulin syringe was used for injecting. The treated volume of mice was 50μl. The dose was 10 mg/mouse. For those constituents that were insoluble in 0.9%saline, were dissolved by pure DMSO of 10μl per mouse, and then diluted to the corresponding concentration with 0.9%saline for administration. For those compounds with PLNA positive results, kept on further reduction from 5 mg/mouse,2.5 mg/mouse or 0.25 mg/mouse to 0 mg/mouse for administration to get the dose-effect relationship. We found that 7/16 samples induced PLNA positive reactions at high dose (10 mg/animal); those suggested that those samples maybe contain harmful components. PLNA positive reactions caused by those components were found in the extracts of three herbs, including 2 extracts from honeysuckle,3 extract from Forsythia, and 2 extracts from skullcap, indicating that there may exist harmful components in all the 3 medicinal plants. Low dose (2.5 mg/animal) failed to induce the positive PLN reactions. Non-specific inflammatory stimulate may lead to d-PLNA false positive results, so further study on mechanism should be done for defining the sensitization of these compositions.5 Evaluation of the Immunosensitizing Potential of Shuanghuanglian injection Using a Reporter Antigen Popliteal Lymph Node Assay in miceThe immunosensitizing mechanisms of SHLI were investigated using RA-PLNA in mice. The results showed that high-dose of SHLI combined with TNP-OVA (T cell dependent antigen) or TNP-Ficoll (T cell independent antigen) could induce PLN positive response (WI>2 or CI>5), but the intense of positive reaction were similar to those induced by high-dose of SHLI alone. Mixed with TNP-OVA, high-dose of SHLI could significantly increase the number of the TNP-specific antibody-forming cells (AFCs), but with TNP-Ficoll and while Low-dose of SHLI with 2 RA failed to do so. Those results suggested that high-dose-SHLI provided costimulatory signals which could induce inflammatory reaction and this effect would disappear at low dose. Moreover, it has been demonstrated via flow cytometry that high-dose-SHLI co-injected with TNP-OVA could activate macrophages in vivo. Activated macrophages could release cytokines which played an important role in induction of inflammation. However, the results did not support that SHLI activated neoantigen-specific T cells in BALB/c mice, which indicated that SHLI by itself do not have immunosesensitizing potential. Thus SHLI did not induced immune response resulting from specific antigen. As a result we postulated that adverse reactions of SHLI may be due to anaphylactoid reaction rather than IgE-mediated hypersensitivity reactions, which is congruent with conclusions obtained by other researchers in our laboratory.6 Evaluation of the immunosensitizing potential of Monomer constituents in Shanghuanglian Injection6.1 Evaluation of the immunosensitizing potential of Chlorogenic acid, Baicalin and Phillyrin Using Direct Popliteal Lymph Node AssayThe great value of d-PLNA has been proved by several labs in predicting the immunosensitizing potential of pure LMWC. However, the value of d-PLNA has not been adequately validated in predicting TCMI which contain complex mixtures with a number of unknown constituents. Therefore, it is more reliable to evaluate the monomer constituents contained in SHLI by using d-PLNA. CGA, Baicalin and phillyrin are main constituents of honeysuckle, skullcap and forsythia respectively, which are composed crude drug of SHLI. Most researchers thought that those monomer constituents are the basis of pharmacodynamics of SHLI. The others thought that they are related to ADR induced by SHLI. To determine whether the CGA, Baicalin and phillyrin are associated with ADR, we detected their immunosensitizing potential using d-PLNA firstly. The results found that a dose of 1 mg CGA, baicalin and phillyrin per animal failed to induce BALB/c mice d-PLNA positive reaction, suggesting that they do not have immunosensitizing potential.6.2 Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c miceIt has yet to be established whether CGA, a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs).Previous results from the d-PLNA demonstrated that CGA failed to induce positive response. To further determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both PLNA combined with flow cytomtry and RA-PLNA method. CGA was selected since many investigators postulated that it is a hapten. Another aim of present study is to validate the accuracy and the reliability of results using d-PLNA to predict the immunosensitizing potential of CGA, baicalin and phillyrin.Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP-OVA or TNP-Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. The results form RA-PLNA confirmed those from d-PLNA. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.In conclusion, we successfully established models of d-PLNA and RA-PLNA, and confirmed BALB/c mice and C57 BL/6J mice as the preferred sensitive animals of both methods in our laboratory; d-PLNA and RA-PLNA are suitable methods to determine the immunosensitizing potential of SHLI and its constituents, which could also reveal the primary mechanism involved in adverse reaction related to SHLI. Our results showed that high-dose-SHLI could provide costimulatory signals to induce inflammatory reactions, but could not activate antigen-specific T-lymphocytes. Those results indicated that SHLI or its constituents do not have the immunosensitizing potential. Form the results, we concluded that SHLI by itself can not directly stimulate BALB/c mice to produce IgE-mediated hypersensitivity reactions. As a result we propose that ADR related to SHLI may be due to anaphylactoid reaction rather than IgE-mediated hypersensitivity reactions, which is congruent with conclusions obtained by other researchers in our laboratory. The release of other active mediators from basophils or mast cells directly stimulated by SHLI may also be the primary mechanism of ADR in human.