Study On Changes Of TLR2 And TLR4 Expression In Tissues Of Guinea Pigs After Anaphylactic Shock And The Effect Of Different Environmental Temperature On Its Detection

Objective : Anaphylactic shock is a serious and life-threatening systemic multiple-system hypersensitivity reaction.In recent years,the incidence of serious allergic reactions induced by allergens such as drugs and food is increasing all over the world.In particular,medical malpractice caused by drug-induced death in medical treatment often leads to medical disputes.These cases by autopsy and histopathological examination are difficult to find specific pathological changes.Therefore,the identification of death cases caused by anaphylactic shock is a major challenge in forensic identification work.In the practice of forensic science,we must exclude the causes of death such as lethal diseases,poisoning and other mechanical injuries,combined with the contact history of the antigen of the deceased,history of allergies,clinical symptoms and signs,autopsy and histopathological examination diagnosis of death from anaphylactic shock will be made.Therefore,to find reliable diagnostic indicator of anaphylactic shock and accurate diagnostic methods,has become an important study of forensic pathology.Toll-like receptors(TLRs),belonging to the family of pattern recognition receptors(PRRs),recognize the highly conserved microbial component pathogen-associated molecular patterns(PAMPS).The specific immune response against pathogens is a bridge linking innate and adaptive immune responses.Toll-like receptor 2(TLR2)is widely distributed in cardiovascular system,respiratory system,reproductive system,digestive system and nervous system.It is mainly expressing in monocytes,macrophages,neutrophils and mast cells,especially in the spleen and peripheral blood leukocyte is highly expressed.Toll-like receptor 4(TLR4)tissue distribution more widely found in vascular endothelial cells,monocytes,neutrophils,eosinophils,mast cells,dendritic cells,intestinal epithelial cells,human gingiva fibroblasts,respiratory epithelial cells.It has been reported that TLR2 and TLR4 play relevant role in some autoimmune diseases.TLR2 plays different roles in the activation of human mast cells,and TLR4 plays an important role in promoting specific cellular immune responses,activating mast cells,promoting neutrophil migration and leukotriene synthesis in monocytes.TLR2,TLR4 and myeloid differentiation factor 88(MyD88)play different roles in the accumulation of eosinophils in BALF in the ovalbumin-mediated allergic airway disease model.TLR2,TLR4 and MyD88 are also key molecules that cause airways hyperresponsiveness(AHR).This article will study the changes of TLR2 and TLR4 expression in tissues of guinea pigs killed by anaphylactic shock under different storage conditions,which will be helpful for the diagnosis of anaphylactic shock.Methods:60 healthy guinea pigs,weighing 250g~300g,and 56 guinea pigs remained a week after adaptive feeding and were divided into control group and experimental group.The experimental group was divided into immediate death group of anaphylactic shock,cold storage 48 h group(4 ?),freezing 7d group(-20 ?),room temperature(20 ?-23 ?)12h group,24 h group at room temperature,48 h group at room temperature,72 h group at room temperature,7 subgroups,7 guinea pigs in each group.Adaptable feeding one week,each experimental group was subcutaneously injected with 0.1 mL of diluted human serum(human mixed serum: saline = 1: 10)subcutaneously into the posterior fossa of guinea pigs,and the control group was replaced with an equal volume of saline.Two weeks after feeding,the experimental group in the intracardiac injection of 1m L diluted mixed serum,induced allergic reactions.Control group with saline instead.Extract larynx,trachea,lung,stomach and intestinal tissue.Part of the organization fixed,making paraffin sections for histological examination.The expression of TLR2 and TLR4 was observed by microscopic image analysis.Lung tissues were harvested and ground with high-throughput tissue grinder.Western blot and ELISA were performed to detect the expression of TLR2 and TLR4.qRT-PCR was used to detect TLR2 mRNA and TLR4 mRNA.Results:Though HE staining,we found that the larynx,trachea,lung,stomach and intestinal tissue of the experimental group were congested and edematous,and a large number of eosinophils infiltrated.There are no significant changes in the control group.Immunohistochemical staining show that the TLR2 and TLR4 positive cells were mainly expressing in the larynx,the trachea submucosa,the lung interstitium and the mucosal and submucosal layers of the stomach wall and the intestinal wall.The number of TLR2 positive cells in guinea pigs tissue immediately after death was significantly increased,(p<0.05).The numbers of TLR4 positive cells in guinea pigs in each experimental group were significantly increased compared with the control group(p<0.05),and there was no statistical difference between the experimental groups.Immunofluorescence double staining of TLR2 and TLR4 expression cells were located and found that mast cells express both two receptors.Western blot analysis showed that the expression of TLR2 protein in guinea pigs immediately after death increased significantly(p<0.05);the expression of TLR4 protein in lung tissue of each experimental group was significantly higher than that of control group(p<0.05),no significant difference between the experimental groups.The results of ELISA showing that the expressions of TLR2 and TLR4 in the immediately death group were significantly higher than that in control group(p<0.05).The results of qRT-PCR showed that the expression of TLR2 mRNA in the lungs of the immediately death group,the cold preservation group and the frozen group was significantly higher than that of control group(p<0.05).The TLR4 mRNA expression in the lung tissue of each experimental group was higher than that of control group,with statistical significance(p<0.05).There are no differences of TLR2 mRNA and TLR4 mRNA expression between the experimental groups.Conclusion: 1.The expressions of TLR2 and TLR4 protein in the larynx,trachea,lung,stomach and intestine of guinea pigs with anaphylactic shock increased significantly,which may be used as an auxiliary diagnosis index of anaphylactic shock;2.The influence of ambient temperature on the detection of TLR4 protein is small.Tip TLR4 is more stable,with greater forensic diagnostic value for anaphylactic shock than TLR2.