The use of medicinal leech(Hirudo medicinalis) has been increased during the past years and is a very common procedure for treatment of venous congestion in flaps and replanted part in plastic surgery. One of the main complication in using leeches is infection. We used the medicinal leeches in venous congestion after total ear reconstruction(8 cases) and replanted finger(1 case). A bacteriologic study of the leeches is undertaken before applying it to the patient and an antibiotic susceptibility test was also performed. In 6 patients. 6 pathogenic organisms were identified, and in 2 cases 2 different organisms were identified simultaneously.
Ear ; Humans ; Hyperemia ; Leeches ; Surgery, Plastic

OBJECTIVETo study the anticoagulant constituents in dried leech (Whitmania pigra Whitman).

METHODThe plasma recalcification time (PRT) as index, the constituents with anticoagulant activity were isolated and purified by anion-exchange chromatography on Sephadex DEAE A-50, gel permeation chromatography on Sephadex G-25 and Sephadex LH-20 columns, and then reversed phase high-performance liquid chromatography successively.

RESULTThree anticoagulant polypeptides were isolated and purified. Compounds 1 and 2 can be translated each other in natural conditions, and their molecular weights are 7100 and 5531, respectively. Compound 3 was identified as a pure polypeptide by HPLC and SDS-PAGE, and its molecular weight was determined as 8 608 by MALDI-TOF-MS. The amino acid composition of compound 3 was also determined.

CONCLUSIONCompound 3 was inferred to be a novel anticoagulant, and named whitmanin.

Animals ; Anticoagulants ; analysis ; chemistry ; isolation & purification ; Leeches ; chemistry ; Molecular Weight

Animals ; Bronchi ; Child ; Child, Preschool ; Foreign Bodies ; Humans ; Leeches ; Male

Protein kinase C(PKC) is a kind of kinase which is widely involved in cell proliferation and development. PKC(Wp-PKC) in Whitmania pigra body belongs to classic PKC. In order to investigate the effect of Wp-PKC on the development of Wh. pigra germ cells, 17β-estradiol(17β-E2)(100 ng·mL~(-1)) and methyltestosterone(MT)(150 μg·L~(-1)), 150 μg·L~(-1)(MT)+0.5 mg·L~(-1) PKC, 0.5 mg·L~(-1) PKC inhibitor were added to Wh. pigra culture water, and no addition group(control group) was added, and the effects on the development of Wh. pigra germ cells and the expression of Wp-PKC were observed. The results showed that: Wp-PKC in male gonads was always higher than that in female gonads; MT promoted the development of male gonads in Wh. pigra, while the expression of Wp-PKC was significantly higher than that in the control; 17β-E2 promoted the development of female gonads in Wh. pigra and Wp-PKC expression significantly lower than that of the control; while the development of the female and male gonads in the PKC inhibitor group was inhibited, the expression of Wp-PKC was significantly lower than that of the control. In summary, Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads.
Animals ; Estradiol ; Female ; Gonads ; Leeches ; Male ; Methyltestosterone ; Ovary

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.
Animals ; Anticoagulants/pharmacology* ; Environmental Pollution ; Leeches ; Metals, Heavy ; Proteomics

The objectives of study were to explore the effects of exogenous methyltestosterone( MT) on the growth and gonadal development of overwintering Whitmania pigra. Before overwintering,0. 1,1. 0,10. 0,100. 0,150. 0 μg·L^-1 of MT were added to the aquaculture water for 6 weeks. The changes of growth performance,gonad index,endogenous steroid hormones level and internal quality were measured after hibernate for 60 days. Then the tissue slice technique was used to observe the spermary( ovary) of Wh. pigra.The results showed that the body weight,survival rate and gonadal index increased first and then decreased with the increase of exogenous MT concentration; the male gland index was found the highest at the concentration of MT 10. 0 μg·L^-1 and the female gland index was the highest at the concentration of MT 1. 0 μg·L^-1. The survival rate of Wh. pigra peaked at the concentration of MT 10. 0 μg·L^-1.The weight reaches a peak at a concentration of MT 100. 0 μg·L^-1( P<0. 05). The number of primary spermatocytes in the testis was negatively correlated with the concentration of exogenous MT. The number of secondary spermatocytes and sperm cells increased first and then decreased. The concentration of secondary spermatocytes was the highest when the concentration of MT was 100. 0 μg·L^-1.The number and volume of oocytes in the ovary and the yolk granules increased first and then decreased with the increase of exogenous MT concentration,and the highest was observed at the MT concentration of 100. 0 μg·L^-1. The endogenous steroid hormone of Wh.pigra increased first and then decreased with the increase of exogenous MT concentration. The concentration of androgen and progesterone was the highest in MT 100. 0 μg·L^-1 treatment( P<0. 05),and the concentration of estrogen was found the highest in MT 10. 0 μg·L^-1 treatment( P<0. 05). After adding exogenous MT,Wh. pigra moisture content,acid-insoluble ash content,p H and anti-thrombin activity met the quality criterion of medicinal Wh. pigra in Chinese Pharmacopoeia( 2015 edition). In conclusion,the short-term addition of 1. 0-100. 0 μg·L^-1 exogenous MT before hibernation can promote the growth,the development of sperm cells and the antithrombin activity of Wh. pigra.
Animals ; Estrogens ; Female ; Gonads ; Leeches/physiology* ; Male ; Methyltestosterone ; Ovary ; Progesterone

HSP90 is a widely distributed molecular chaperone that participates in a variety of cellular processes and plays an important role in the meiosis of germ cells. However, its role in the gonadal development of hermaphroditic Whitmania pigra is not yet clear. To explore the effect of HSP90 on the germ cell development of Wh. Pigra, this study cloned the wpHSP90 gene, performed bioinformatics analysis, and measured its expression levels. The results showed that the cloned wpHSP90 was 2 592 bp in length, with an open reading frame(ORF) of 2 373 bp, encoding 790 amino acids. Prediction analysis revealed 85 phosphorylation modification sites on serine, threonine, and tyrosine residues of the wpHSP90 protein. Structural domain prediction and multiple sequence alignment results showed that wpHSP90 contained two conserved domains of HSP90 and exhibited the highest homology with Helobdella robusta, with a sequence similarity of 80.72%. RT-qPCR results showed that the relative expression level of wpHSP90 in the gonads of 5-month-old Wh. pigra was positively correlated with temperature within the range of 12 ℃ to 28 ℃. The expression level in the female gonads was significantly higher than in the male gonads and correlated with the trend of germ cell development in the ovaries and testes. In conclusion, wpHSP90 may be involved in regulating the development of germ cells, particularly oocytes, in Wh. pigra. This study provides a reference for further research on the gonadal development mechanism in Wh. pigra.
Animals ; Female ; Male ; Temperature ; Ovary ; Gonads ; Testis ; Leeches ; Cloning, Molecular

Animal medicines mainly contain protein which was organic molecule with quaternary structure and had the property of thermal denaturation. When suffering from heat for a consistent time, the native conformation of protein would be destroyed. After denaturation the biological activity of protein will lose and some physicochemical and biochemical properties will be changed. Leech was a classical animal medicine in the views of traditional Chinese medicine (TCM) which had the functions of breaking stagnant and eliminating blood stasis. In the usage history, it was processed for a long time. No matter stir-frying leech with talc powder embodied in Chinese Pharmacopoeia or stir-baking with wine as a distinctive method in Beijing district, the process procedure was basically performed under high temperature. The purposes and intentions of process are mostly limited to technology conditions at specific historical period. In this article, based on existing processing procedure and its character of Leech, the changes of active components and pharmacological activities before and after processing under high temperature were summarized. The results demonstrate that the protein of leech would be denaturated; some active peptide such as hirudin were partly or totally destroyed; some toxic mineral elements, such as Pb, Hg, Cd, were decreased; at the same time, heating can promote some chemical components transforming into hypoxanthine which had the function of antihypertensive, antiasthmatic and antalgic. Consequently, after processed under high temperature, the purpose of decreasing toxicity and alleviating the strong property was achieved. Pharmacological changes of leech processed under high temperature were mainly manifested in the anticoagulant and antithrombotic activity, etc. Based on current processing research status about animal medicine leech, future research methods and directions on scientific connotation of leech processed under high temperature were put forward in this article.
Animals ; Biological Factors ; chemistry ; isolation & purification ; pharmacology ; Chemistry, Pharmaceutical ; methods ; Hot Temperature ; Humans ; Leeches ; chemistry

OBJECTIVEUsing HPLC To determine hypoxanthine in co-hirudo injection for establishing its HPLC fingerprint, and evaluating its internal quality.

METHODThe chromatographic separation was performed on a Kromasil C18 column (4.6 mm x 250 mm,5 microm). A linear gradient elution with A (0.01 mol x L(-1) x KH2PO4) and B (50% methanol) was used, the flow rate was 0.8 mL x min(-1), the detection wavelength was set at 254 nm, and the column temperature was at normal.

RESULTHypoxanthine was used as the reference substance in the fingerprint of co-hirudo injection, it showed 15 common peaks and theirs similarity threshod was 0.97.

CONCLUSIONThis method was accurate, repeatable and useful for the quality control of co-hirudo injection.

Animals ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Hypoxanthine ; chemistry ; Leeches ; chemistry ; Reproducibility of Results

OBJECTIVETo determine the best spawning temperature and weight of Whitmania pigra and the hatching temperature of its eggs.

METHODObserve the state of W. pigra spawning and hatching were observed under different temperatures and weights conditions.

RESULT AND CONCLUSION25 degrees C is the most appropriate temperature for W. pigra spawning and incubation, the best spawning weight of W. pigra was around 20 g. The temperature influenced the lost weight after spawning significantly and was proportion to the lost weight. The body weight showed no substantial influence to the lost weight after spawning. The egg weight of W. pigra which body weight was below 2.0 g was proportion to the hatching rate. The same relationship was not observed in W. pigra over 2.0 g.

Animals ; Body Weight ; Breeding ; methods ; Female ; Leeches ; growth & development ; physiology ; Male ; Ovum ; growth & development ; Temperature