[Effect of heavy metal lead on anticoagulant activity of leech based on label-free quantitative proteomics].

Whitmania pigra is the most widely distributed species of leeches in the market. In this study, the effect of heavy metal lead pollution on the anticoagulant activity of Wh. pigra was studied and the potential mechanism was explored. Pb(NO_3)_2 was used to contaminate the breeding soil which was then used to rear Wh. pigra for 50 days(lead-contaminated group, LC group), and meanwhile the blank control group(CG group) was set. Proteins were extracted from the obtained leech samples, and the differentially expressed proteins between LC and CG groups were analyzed by label-free proteomics technology. In this study, a total of 152 differentially expressed proteins were screened out, of which 93 proteins were up-regulated and 59 proteins were down-regulated in LC group. Bioinformatics analysis showed that the biological processes enriched with the differentially expressed proteins were mainly vesicle-mediated transport and transport positive regulation; the enriched cell components were mainly endocytosis vesicles and apical plasma membrane; the enriched molecular functions mainly included carbohydrate binding. The differentially expressed proteins were enriched in 76 KEGG pathways, which mainly involved metabolic pathways, biosynthesis of secondary metabolites, and bacterial invasion of epithelial cells. In this study, two differentially expressed proteins with Antistasin domain were presumed, which provides reference for further exploring the regulatory mechanism and signal transduction underlying the effect of lead pollution on the anticoagulant activity of leech.

A study of the influence of lead pollution on the anticoagulant activity of Whitmania pigra based on pharmacodynamics and metabolomics research.

Whitmania pigra Whitman (leech, also called Shuizhi in China, abbreviated as SZ), which has been used as a traditional Chinese medicine in the treatment of blood stasis syndrome (BSS) for a long time, is vulnerable to lead pollution in aquaculture environments. SZ has good anticoagulant activity. However, there are few studies on the influence of lead pollution on it. Therefore, we carried out the following researches to explore the influence of lead pollution on the anticoagulant activity of SZ and its mechanism. Firstly, the acute blood stasis model of rats was established by subcutaneous injection of adrenaline hydrochloride and ice water bath. Then unpolluted SZ (UPS) and lead-polluted SZ (LPS) were extracted. Next, the blood stasis model rats were administrated by gavage and the rats in normal control (NC) group and blood stasis model (BM) group were given the same amount of normal saline. Finally, the blood of the rats was collected to detect the coagulation function and hemorheology indexes. The metabolomics of rat plasma was studied by ultra-high-performance liquid chromatography coupled with orbitrap mass spectrometry (UPLC-Orbitrap-MS) technology. Principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and Hierarchical clustering analysis (HCA) were used to perform metabolomics analysis. MetPA analysis was used to search for related metabolic pathways. The results of coagulation function and hemorheology showed that lead pollution could decrease the anticoagulant activity of SZ. The OPLS-DA score plots indicated that the plasma metabolites of rats in LPS group were close to BM group, while UPS group tended to be close to NC group both in the positive and negative ion mode. Hierarchical cluster analysis (HCA) suggested that UPS group and NC group were clustered into a branch, while LPS group and BM group were clustered into a branch. To sum up, lead pollution will reduce the anticoagulant activity of SZ. And lead pollution reduces the anticoagulant activity of SZ probably by influencing the metabolic pathways such as sphingolipid metabolism, amino acid metabolism and energy metabolism in rats.

Metabolomics-based study reveals the effect of lead (Pb) in the culture environment on Whitmania pigra.

Whitmania pigra, called Mahuang (MH) in Chinese, has been used as a traditional Chinese medicine for many years and is susceptible to Pb exposure in aquaculture environments. To understand the impact of Pb in the culture environment on MHs, we carried out a 50-day culture of MHs in environments with different levels of Pb pollution. Then, tissue samples of MHs reared in the different Pb-polluted environments were collected and analysed by UPLC-Q/TOF-MS. The results showed that the Pb residue in MHs increased with increasing Pb in the culture environment. There was no significant difference in MH Pb content (P < 0.05) between the low-Pb residue group (PbL) and the blank control group (BC), and those of the middle-Pb residue group (PbM) and the high-Pb residue group (PbH) were significantly different from that of the BC group. Metabolomics results showed significant changes in 24 metabolites in the PbL, PbM and PbH groups, some of which were dose-dependent. These metabolites were mainly lipids, nucleotides, and dipeptides, which are involved in metabolic pathways such as glycerophospholipid metabolism, sphingolipid metabolism, and nucleotide metabolism. Overall, the results proved that metabolomics can be an effective tool to understand the effects of Pb on the metabolic responses of MHs.

[Diversity of fungi on surface of Pheretima].

Traditional Chinese medicines( TCMs) are easily contaminated by fungi during planting,harvesting,processing,transportation and storage. The 2015 version of Chinese Pharmacopoeia stipulates the detection of aflatoxin in Dilong. After reviewing the literature,it has been found that there are no domestic and foreign scholars who have studied the surface fungi of Dilong. Pheretima,known as Dilong in China,is a commonly used TCMs in animal. In this experiment,8 batches of Dilong were collected from retail pharmacies in Beijing. The fungi on the surface of Dilong were cultured by traditional plate method and the single strain was obtained by the top purification method. The fungal colony morphology,microstructure characteristics and DNA barcode were used to isolate and identify the fungi. At the same time,based on Illumina Hi Seq 2500 high-throughput sequencing platform,the diversity of fungi on the surface of Dilong was analyzed. The results showed that 287 strains of 9 species of fungi were isolated and identified by plate method. Combined with 3 kinds of identification method,eight of nine fungi could be identified,respectively,Aspergillus niger,Penicillium,Alternaria nees,A. flavus,and Penicillium oxalicum,Humicola sp.,Talaromyces purpurogenus and A. insuetus,1 kind of fungi was not identified yet. Among them,Penicillium and Aspergillus were the dominant genus. The results of high-throughput sequencing belonged to 2 boundaries,6 gates,19 classes,44 orders,98 families,127 genus and 121 species in different classification levels. Wallemia,Aspergillus and Cordyceps were the dominant genus,and the relative abundances are 63. 33%,15. 28%,and 10. 28%,respectively. Through the diversity study on the surface fungi of Dilong in Beijing retail pharmacies,it can provide a reference for its safe storage and clinical use.

Analysis of Fungi on Coix (Coix lacryma-jobi) Seed and the Effect of Its Aqueous Extract on the Growth of Aspergillus flavus.

Coix (Coix lacryma-jobi) seeds are susceptible to fungal infections, making their surface fungi complex and diverse. Some fungi can produce mycotoxins under suitable conditions, and fungal growth is closely related to the production of mycotoxins. In this study, the surface fungi of coix seed were identified by Illumina HiSeq high-throughput sequencing. Simultaneously, the fungi cultured by the plate method were identified by microscopy and DNA barcoding; finally, the species of fungi were identified accurately and reliably by combining three methods. The aqueous extract of coix seed was cocultured with Aspergillus flavus spores, and the relationship between the aqueous extract and the growth of A. flavus was studied with the dry weight of mycelium as an indicator. The results showed that there were 89 genera and 96 species of fungi on coix seed, which were mainly distributed in Ascomycota (81.48%) and Basidiomycota (4.08%), and Xeromyces (8.50%), Gibberella (7.25%), and Aspergillus (4.74%) were the predominant genera. Four fungi were isolated from coix seed by plate culture and identified as Aspergillus fumigatus, A. flavus, Aspergillus oryzae, and Rhizopus oryzae by microscopy and DNA barcoding. The aqueous extract of coix seed at low concentrations has a promoting effect on the growth of A. flavus. When the concentration is 3.125%, the promotion effect is the most pronounced, and the promotion rate is 29.17%. These results reveal the diversity of fungi on the coix seed, which can provide a reference for the prevention and control of harmful fungi on coix seed.

[Effect of lead pollution on anticoagulant effect of Whitmania pigra based on in vitro anticoagulation experiment].

Leech has a good anticoagulant activity and is one of the raw materials for treatment of many cardiovascular and cerebrovascular diseases. This study was based on in vitro anticoagulant experiments( APTT and PT) to investigate the effects of lead contamination on the anticoagulant effect of leech. At present,the Hirudo circulating in the market are dominated by Whitmania pigra,therefore Wh. pigra were cultivated under a different lead pollution for 50 days. Then,the effects of Wh. pigra extract,extracting from different cultivating environment,on activated partial thrombin time( APTT) and prothrombin time( PT) were determined by automatic coagulation instrument. The results showed that the Wh. pigra extract significantly prolonged the APTT compared with the saline group.The APTT of the lead-high residual Wh. pigra was shorter than that of the blank Wh. pigra. The Wh. pigra extracts from different treatment groups had little effect on PT. The results showed that the lead residue in the Wh. pigra increased with the increase of lead in the cultured soil,the lead residual of the Pb-H group was( 10. 66±2. 79) mg·kg~(-1),which exceeded the lead limit specified in the 2015 edition of the Chinese Pharmacopoeia. The results indicated that growth environment pollution is one of the important factors causing excessive lead in Wh. pigra. Lead pollution will reduce the anticoagulant effect of Wh. pigra and affect its clinical efficacy.