| 1 ObjectionLeech refer to Whitmania pigra Whitman,Hirudo nipponia Whitman and Whitmania acranulate Whitman.Modern pharmacological studies have shown that leech has the effects of anticoagulation,anti-atherosclerosis,anti-platelet aggregation and so on.Whitmania pigra Whitman,also known as mahuang(MH)in China.At present,the main source of medicinal leech on the market is cultured MH.MH are produced all over the country,but with the increase of its use and clinical dosage,coupled with serious environmental pollution and excessive capture,which has led to the reduction of its wild resources.Although the artificial culture of MH has been widely carried out,the relevant culture technology is not yet fully mature and will be polluted by heavy metals in the water during the growth process.In the previous study on heavy metal residues and anticoagulant activity of cultured MH,it was found that lead may have a greater impact on the anticoagulant activity of MH.Therefore,the accumulation of lead in the body of MH may reduce the efficacy and increase the threat of safe medication.Therefore,this experiment took MH as the research object,and used transcriptomics and proteomics to study the effect of lead on the anticoagulant activity of MH.The purpose of this study is to provide a theoretical basis for establishing the technical standard of leech culture environment and ensuring the quality of medicinal leech.2 Method2.1 Study on the relationship between the lead residues of MH and anticoagulant activitySimulating the pond culture environment of MH,by adding lead nitrate to the soil,causing soil lead pollution.The blank group and low,medium and high lead pollution group were obtained after 50 days.MHs were washed with clean water,some of them were dried at low temperature as experimental materials for the detection of lead residues and the evaluation of anticoagulant activity,and the rest of MHs were kept frozen.The lead residues of MH were detected by inductively coupled plasma mass spectrometry(ICP-MS).The anticoagulant activity of MH was evaluated by thrombin titration,thrombin time,activated partial thromboplastin time and prothrombin time.2.2 Transcriptomics study on the effect of lead on the anticoagulant activity of MHBased on the results of anticoagulant activity,the transcriptomics analysis methods of blank group and lead-contaminated group were established by Illumina sequencing platform.The total RNA quality was detected by Nanodrop and Agilent 2100.Computer sequencing was performed after the library was qualified,and the quality of sequencing data was evaluated.Key genes related to anticoagulant activity were mined through differential analysis.2.3 Proteomics study on the effect of lead on the anticoagulant activity of MHBased on the results of anticoagulant activity,the proteomics analysis methods of blank group and lead-contaminated group were established by Label Free technique.Through protein extraction,content determination,enzymolysis,Nano LC-MS/MS analysis and searching of protein database,the difference of protein expression between blank group and leadcontaminated group was obtained,and the target proteins related to anticoagulant activity were found.3 Results3.1 Study on the relationship between the lead residues of MH and anticoagulant activityThe lead residues of MH showed that there was no significant difference between the low lead pollution group,the medium lead pollution group and high lead pollution group had significant difference compared with the blank group,and the lead residues of the high lead pollution group exceeded the 10 mg·kg-1 stipulated in the 2015 pharmacopoeia.The results of thrombin titration showed that the antithrombin titers of blank,low,medium and high lead pollution group exceeded the 3 U,and the general trend was that the antithrombin titer decreased with the increase of lead residues.The results of TT showed that compared with the normal saline group,the TT values of blank,low,medium and high lead pollution group all had significant difference(P<0.01).Compared with the blank group,only the high lead pollution leech group had a significant difference(P<0.01).However,the TT values in the blank,low,medium and high lead pollution group showed a decreasing trend.3.2 Transcriptomics study on the effect of lead on the anticoagulant activity of MHBased on the results of anticoagulant activity,high-throughput sequencing technology was used to carry out transcriptomics on the blank group and the high lead pollution group.By evaluating the parameters such as the ratio of clean reads,the percentage of Q30 bases,content and mass distribution of bases,it shows that the quality of RNA sequencing was good and the data was reliable.Because it had no reference genome,denovo assembly was carried out.Functional annotation of the assembled Unigene:the results of GO annotation showed that Unigene was mainly related to cellular process,cell part and binding in biological process,cellular component and molecular function,respectively.The results of KOG annotation showed that among the 25 categories annotated to KOG database,the most genes were annotated to signal transduction mechanisms and the least to cell motility.A total of 1,110 differentially expressed genes were screened.Compared with the blank group,there were 694 up-regulated genes and 416 down-regulated genes in the high lead pollution group.In the GO enrichment analysis,intrinsic component of membrane and membrane part were significantly enriched in the cell component,oxidoreductase activity and cofactor binding were significantly enriched in the molecular function,and monocarboxylic acid metabolic process and carboxylic acid biosynthetic process were significantly enriched in the biological process.Six pathways were significantly enriched in KEGG analysis,involving arachidonic acid metabolism,lysosome and so on.A total of 6 differentially expressed genes related to anticoagulation were found.3.3 Proteomics study on the effect of lead on the anticoagulant activity of MHBased on the results of anticoagulant activity,Label Free quantitative technology was used to carry out proteomics on the blank group and the high lead pollution group.A total of 1,294 proteins were detected by LC-MS/MS.The molecular weight of the protein was mainly between 1 KDa and 80 KDa.The number of proteins with protein sequence coverage less than 20%was the highest,reaching 810.GO and KEGG functional annotation were performed on proteins:the results of GO annotation showed that biological process was mainly involved in cellular process and so on,cell component was mainly involved in cellular anatomical entity and so on,and molecular function was mainly involved in binding and so on.According to statistics,the KEGG pathway had the largest number of proteins involved in human diseases,which was 434.A total of 152 differentially expressed proteins were screened.Compared with the blank group,93 proteins were up-regulated and 59 proteins were down-regulated in the high lead pollution group.Bioinformatics analysis was performed on the differentially expressed proteins.The results of GO enrichment analysis showed that significant enrichment in biological process to vesicle-mediated transport and so on,significant enrichment in cell component to endocytic vesicle and so on,and significant enrichment in molecular function to carbohydrate binding and so on.Seventy-six pathways were identified by KEGG analysis,of which the number of proteins involved in metabolic pathway was the largest,but no significant enrichment pathway was found.In addition,two anticoagulant proteins with antistasin domain were presumed.4 Conclusion4.1 Based on the results of thrombin titration and TT assay,the TT values between the blank group and the high lead pollution group were significantly different,that is,there was a significant difference in anticoagulant activity.4.2 A total of six differentially expressed genes related to anticoagulation were found through transcriptomics and five anticoagulant proteins were presumed:Serpin,Bdellin,Granulin,Destabilase I and Eglin.4.3 It was presumed by proteomics that two anticoagulant proteins with antistasin domains,but further research is still needed.5 Innovation5.1 The effect of heavy metal lead on anticoagulant activity of MH was studied by transcriptomics for the first time.5.2 The effect of heavy metal lead on anticoagulant activity of MH was studied by proteomics for the first time. |
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