| Background:In recent years,reports of drug-induced liver injury after taking Polygonum multiflorum/polygonum multiflorum preparations have increased,which has also attracted the attention of some experts in the field of pharmacy.According to literature reports,animal experiments on the toxicity of Polygonum multiflorum have found that there is no toxic reaction.Therefore,liver disease experts concerned consider taking Polygonum multiflorum preparations to cause drug-induced liver injury,its underlying causes.Because of individual differences.Because of its low incidence,difficult to detect,and no obvious relationship with the dosage of drugs,the type of drug-induced liver injury caused by Polygonum multiflorum/polygonum multiflorum preparations belongs to the special heterogenous liver injury type.Two kinds of hypotheses were put forward,one is the hypothesis of immunological mechanism,including the hypothesis of Immunopharmacology(p-i),gene polymorphism and hapten,the other is the hypothesis of "three-cause poisoning" caused by immune stress of traditional Chinese medicine.Immunological mechanisms lead to liver injury in two ways:one is to stimulate T cells directly by binding with small molecules in vivo after entering the body with drugs,or to change the antigenicity of molecule surface to be recognized by T cells,and then lead to liver injury.The other is that the immune state of the body itself is active,and then after drug stimulation,the immune homeostasis is unbalanced,which results in liver injury.At present,the study of this immunological mechanism is mainly reflected in the liver injury caused by chemical drugs.The hypothesis of"three-cause toxicity" of immune stress in traditional Chinese medicine lays emphasis on the theory of "non-toxicity" of traditional Chinese medicine.Some studies have established a low-dose LPS model to prove the attributes of heterogenous liver injury in Polygonum multiflorum Thunb.and found that the heterogenous liver injury induced by Polygonum multiflorum Thunb.is abnormal immune function,immunoactive ingredient(cis-stilbene glycoside,trans-SG)and potential susceptible ingredient(trans-SG)of liver injury The synergistic effect of stilbene glycoside and cis-SG is the result of which the abnormal immune function is the basis of liver injury.Therefore,this study aims to obtain the immune characteristics of the body.Research purposes:By analyzing the characteristics of TCR immunohistochemcal bank in peripheral blood of patients with or without liver injury after taking Polygonum multiflorum preparations,it was found that patients with liver injury were prone to take Polygonum multiflorum preparations,which could provide a reference for screening high-sensitive people with liver injury caused by Polygonum multiflorum preparations and reducing the occurrence of adverse reactions after taking Polygonum multiflorum preparations.Materials and Methods:1.This study is based on the data collection form and blood samples of patients with drug-induced liver injury after taking Polygonum multiflorum and its preparations collected by the participating units of the National Science and Technology Major Project of "Major New Drug Creation".Nested case-control method was used to match patients with liver injury who took the preparation containing Polygonum multiflorum with age(+5 years old),sex and basic diseases similar.Patients who took the preparation containing Polygonum multiflorum multiflorum orally and did not show liver injury were matched with a ratio of 1:1.A total of 92 patients were enrolled.Nested case-control design is mainly used in data collection.Nested case-control design combines the advantages of cohort study and control design.It is often used in rare disease research in clinical trials.Drug-induced liver injury is a small probability event,so this method can be used in this experiment.Immunohistology library is the sum of T or B cells in human immune system in a specific time.To study the characteristics of immunohistology library,we need to determine the TCR sequence and gene information in immune system.This method obtains complementary determinant regions of T cell diversity by PCR amplification technology,and then comprehensively evaluates the immune system through the second generation high-throughput sequencing technology.Diversity,in-depth exploration of the relationship between immunohistochemical library and the occurrence of specific heterogenous liver injury.The detection of samples was carried out by the Chinese big gene company,including lymphocyte extraction,library construction,quality detection and sequencing of the library Sample detection mainly detected the concentration and integrity of samples.The fluorescence quantification mainly quantified the sample concentration.Agarose gel electrophoresis used 1%agarose gel to electrophoretic 40min at 150V voltage.Integrity QIAGEN kit was used to construct the library.Agilent 2100 Bioanalyzer was used to detect the fragment range of the library.ABI Step One Plus Real-Time PCR System(TaqMan Probe)was used to quantify the concentration of the library.Qualified libraries were detected,and NaOH was added to the single chain.According to the expected confidentiality,the denatured and diluted libraries were added to FlowCell,and contacted with the connectors on FlowCell.Then the bridge-type PCR expansion was completed through cluster generation platform cBot,and sequencing was completed on Illumina HiSeq2000 platformVDJtools software was used for analysis after comparison,basic statistical analysis, clonal diversity assessment,clonal overlap between samples,clonal population similarity between samples and other analysis modulesResult:1.General data results:A total of 92 patients were included,the ratio of male to female was 1:1.All patients were between 38 and 85 years old,the average age was 64.45 years old,and the median age was 63 years old.According to the frequency of occurrence,the top five diseases in the observation group were hypertension,coronary heart disease,hyperlipidemia,diabetes mellitus and lacunar cerebral infarction;the top five diseases in the control group were hypertension,coronary heart disease,arrhythmia,type 2 diabetes mellitus and lacunar cerebral infarction;the diseases included in the treatment group were treated with Polygonum multiflorum preparation.Statistical analysis showed that coronary heart disease,hypertension and hyperlipidemia were the top three diseases treated with Polygonum multiflorum preparations in the observation group,while coronary heart disease,hypertension and arrhythmia were the top three diseases treated with Polygonum multiflorum preparations in the control group.All of them were Chinese patent medicines,and 6 cases of unknown type of Radix Polygoni Multiflori were Chinese herbal decoction.2.Immunohistological library results(1)The number and types of CDR3 clones in the observation group were significantly higher than those in the control group,and there were statistical differences.The depth of sequencing is not different.(2)The length of amino acid sequence in CDR3 region was analyzed.Statistical differences were found at 17,20,46,62 and 63 sites.(3)By analyzing the abundance of V gene,we found that there were significant differences in the abundance of TRBV13,TRBV29,TRBV5-5 and TRBV6-8 between the two groups.(4)Analysis of J gene abundances showed that there were significant differences in TRBJ2-4 and TRBJ2-5 abundances between the two groups.(5)The same pair of V-J genome combinations between the two groups were analyzed. There were differences in the recombination of TRBV13,TRBV29,TRBV5-5,TRBV6-8, TRBJ2-4 and TRBJ2-5 genes between the two groups.(6)Seven indicators,clone number,resampled diversity,extrapolate_diversity,chaoE, chaol,Shannon Wiener Index,inverse Simpson Index,reflect the diversity of samples.It can be concluded that,except Shannon Wiener Index and inverse Simpson Index,the diversity of the two groups is higher than that of the control group,and there are statistical differences. AO1 index showed significant statistical difference.Conclusion:(1)The diversity of CDR3 sequence in the observation group was higher than that in the control group,and there were statistical differences.It is suggested that the occurrence and deterioration of extra-heterogenous liver damage are closely related to the diversity of the immunohistochemical library.(2)By comparison,it was found that there were many differences in recombination of V-J gene in TCR CDR3 immunohistochemical Library of patients with liver injury,and the differences were mainly reflected in TRBJ2-4 and TRBJ2-5 genes,suggesting that abnormal recombination of TRBJ2-4 and TRBJ2-5 affects antigen receptors. |
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