The energy metabolism of the freshwater leech Whitmania pigra in response to fasting.

Non-blood-feeding leeches, Whitmania pigra, have evolved unique digestive structures and physiological mechanisms to cope with fasting. However, the metabolic changes and molecular mechanisms induced by fasting remain unclear. Therefore, this study recorded the weights of leeches during the fasting process. The weight changes were divided into two stages: a rapid decline period (1-9 weeks) and a fluctuating decline period (9-24 weeks). Leeches fasted for 4 (H4), 11 (H11), and 24 (H24) weeks were selected for transcriptome sequencing. Compared to the control group (H0), 436, 1157, and 337 differentially expressed genes (DEGs) were identified, which were mainly related to glycolysis/gluconeogenesis, amino acid metabolism, and the lipid metabolism pathway. The 6-phosphofructokinase (Pfk), pyruvate kinase (PK), and phosphoenolpyruvate carboxykinase (Pck) transcription levels revealed glycolysis/gluconeogenesis activation during the early stage of fasting and peaked at 11 weeks. Decreased expression of the rate-limiting enzyme acetyl-CoA carboxylase (ACC) in fatty acid synthesis during fasting may impede fatty acid synthesis. These results indicated that the nutrient storage and energy-supplying pathways in W. pigra were modified to improve fasting resistance. The findings of this study provided guidance for exploring the mechanism underlying fasting metabolism and laid a foundation for artificial breeding to improve the resistance of leeches.

Exploring a Hirudin variant from nonhematophagous leeches: Unraveling full-length sequence, alternative splicing, function, and potential as a novel anticoagulant polypeptide.

ETHNOPHARMACOLOGICAL RELEVANCE: Leeches exhibit robust anticoagulant activity, making them useful for treating cardiovascular diseases in traditional Chinese medicine. Whitmania pigra, the primary source species of leech-derived medicinal compounds in China, has been demonstrated to possess formidable anticoagulant properties. Hirudin-like peptides, recognized as potent thrombin inhibitors, are prevalent in hematophagous leeches. Considering that W. pigra is a nonhematophagic leech, the following question arises: does a hirudin variant exist in this species? AIM OF THE STUDY: In this study we identified the hirudin-encoding gene (WP_HV1) in the W. pigra genome. The goal of this study was to assess its anticoagulant activity and analyze the related mechanisms. MATERIALS AND METHODS: In this study, a hirudin-encoding gene, WP_HV1, was identified from the W. pigra genome, and its accurate coding sequence (CDS) was validated through cloning from cDNA extracted from fresh W. pigra specimens. The structure of WP_HV1 and the amino acids associated with its anticoagulant activity were determined by sequence and structural analysis and prediction of its binding energy to thrombin. E. coli was used for the expression of WP_HV1 and recombinant proteins with various structures and mutants. The anticoagulant activity of the synthesized recombinant proteins was then confirmed using thrombin time (TT). RESULTS: Validation of the WP_HV1 gene was accomplished, and three alternative splices were discovered. The TT of the blank sample exceeded that of the recombinant WP_HV1 sample by 1.74 times (0.05 mg/ml), indicating positive anticoagulant activity. The anticoagulant activity of WP_HV1 was found to be associated with its C-terminal tyrosine, along with the presence of 9 acidic amino acids on both the left and right sides. A significant reduction in the corresponding TT was observed for the mutated amino acids compared to those of the wild type, with decreases of 4.8, 6.6, and 3.9 s, respectively. In addition, the anticoagulant activity of WP_HV1 was enhanced and prolonged for 2.7 s when the lysine-67 residue was mutated to tryptophan. CONCLUSION: Only one hirudin-encoding variant was identified in W. pigra. The active amino acids associated with anticoagulation in WP_HV1 were resolved and validated, revealing a novel source for screening and developing new anticoagulant drugs.

Antithrombotic pharmacodynamics and metabolomics study in raw and processed products of Whitmania pigra Whitman.

Objective: As a traditional Chinese medicine, leech has obvious pharmacological activities in anticoagulantion and antithrombosis. Whitmania pigra Whitman (WP) is the most commonly used leech in the Chinese market. It is often used in clinical applications after high-temperature processing by talcum powder to remove the fishy taste and facilitate crushing. The anticoagulant and thrombolytic active ingredients are protein and polypeptide, which may denaturate and lose activity after high-temperature processing. The rationality of its processing has been questioned in recent years. This study aims to investigate the effect of talcum powder scalding on the antithrombotic activity of WP in vivo and to discuss its pharmacodynamic mechanism in vivo. Methods: Raw and talcum-powdered processed WP were administered intragastrically for 14 days, and carrageenan was injected intraperitoneally to prepare a mouse model of tail vein thrombosis. The incidence rate of tail vein thrombosis and the thrombus area under pathological tissue sections were calculated to evaluate the antithrombotic effect between raw and processed WP. Non-targeted metabolomics was conducted using UPLC-Q-TOF/MS technology to analyze the changes of small molecule metabolites in the body after administration of WP. Results: After intragastric administration, both the raw product and the processed product of WP could inhibit the thrombosis induced by carrageenan, and the processed product had a more apparent antithrombotic effect than the raw product. The administration of WP could regulate the changes of some small molecular metabolites, such as amino acids, lipids, and steroids, in Sphingolipid metabolism and Glycerophospholipid metabolism. Conclusions: Based on the results of pharmacodynamics and metabolomics, processed WP will not reduce the antithrombotic activity of WP. This study provided a scientific basis for the rational use of leeches.

[Cloning and expression analysis of wpHSP90 gene from Whitmania pigra at different temperatures].

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.

Division of developmental phases of freshwater leech Whitmania pigra and key genes related to neurogenesis revealed by whole genome and transcriptome analysis.

The freshwater leech Whitmania pigra (W. pigra) Whitman (Annelida phylum) is a model organism for neurodevelopmental studies. However, molecular biology research on its embryonic development is still scarce. Here, we described a series of developmental stages of the W. pigra embryos and defined five broad stages of embryogenesis: cleavage stages, blastocyst stage, gastrula stage, organogenesis and refinement, juvenile. We obtained a total of 239.64 Gb transcriptome data of eight representative developmental phases of embryos (from blastocyst stage to maturity), which was then assembled into 21,482 unigenes according to our reference genome sequenced by single-molecule real-time (SMRT) long-read sequencing. We found 3114 genes differentially expressed during the eight phases with phase-specific expression pattern. Using a comprehensive transcriptome dataset, we demonstrated that 57, 49 and 77 DEGs were respectively related to morphogenesis, signal pathways and neurogenesis. 49 DEGs related to signal pathways included 30 wnt genes, 14 notch genes, and 5 hedgehog genes. In particular, we found a cluster consisting of 7 genes related to signal pathways as well as synapses, which were essential for regulating embryonic development. Eight genes cooperatively participated in regulating neurogenesis. Our results reveal the whole picture of W. pigra development mechanism from the perspective of transcriptome and provide new clues for organogenesis and neurodevelopmental studies of Annelida species.

Cloning and expression analysis of wpHSP90 gene from Whitmania pigra at different temperatures / 中国中药杂志

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.

The genome of medicinal leech (Whitmania pigra) and comparative genomic study for exploration of bioactive ingredients.

BACKGROUND: Leeches are classic annelids that have a huge diversity and are closely related to people, especially medicinal leeches. Medicinal leeches have been widely utilized in medicine based on the pharmacological activities of their bioactive ingredients. Comparative genomic study of these leeches enables us to understand the difference among medicinal leeches and other leeches and facilitates the discovery of bioactive ingredients. RESULTS: In this study, we reported the genome of Whitmania pigra and compared it with Hirudo medicinalis and Helobdella robusta. The assembled genome size of W. pigra is 177 Mbp, close to the estimated genome size. Approximately about 23% of the genome was repetitive. A total of 26,743 protein-coding genes were subsequently predicted. W. pigra have 12346 (46%) and 10295 (38%) orthologous genes with H. medicinalis and H. robusta, respectively. About 20 and 24% genes in W. pigra showed syntenic arrangement with H. medicinalis and H. robusta, respectively, revealed by gene synteny analysis. Furthermore, W. pigra, H. medicinalis and H. robusta expanded different gene families enriched in different biological processes. By inspecting genome distribution and gene structure of hirudin, we identified a new hirudin gene g17108 (hirudin_2) with different cysteine patterns. Finally, we systematically explored and compared the active substances in the genomes of three leech species. The results showed that W. pigra and H. medicinalis exceed H. robusta in both kinds and gene number of active molecules. CONCLUSIONS: This study reported the genome of W. pigra and compared it with other two leeches, which provides an important genome resource and new insight into the exploration and development of bioactive molecules of medicinal leeches.

Purification and characterization of a novel thermostable anticoagulant protein from medicinal leech Whitmania pigra Whitman.

ETHNOPHARMACOLOGICAL RELEVANCE: The prevalence of cardiovascular disease (CVD) is increasing worldwide. Despite significant improvements in novel targeted treatment agents, natural products purified from medicinal animals with minimal side effects have attracted much attention. Several native proteins explored from suck-blood leeches, such as non-thermostable hirudin and its variants, revealed potent anticoagulant activity. Traditional Chinese medicine clinics have proved that non-suck-blood leech Whitmania pigra Whitman (W. pigra) also played notable roles in CVD treatments even after decoction. However, only a few natural proteins and peptides have been identified from the fresh material of this medicinal species. AIM OF THE STUDY: We aimed to purify and characterize thermostable anticoagulant proteins from W. pigra for further development of a therapeutic agent for thrombosis. MATERIALS AND METHODS: W. pigra crude extract was prepared by decoction in water. Anticoagulant proteins were purified by DEAE cellulose DE-52, Sephadex G-75, and reversed-phase liquid chromatography sequentially and analyzed by SDS-PAGE and LC-MS/MS for structural information. In addition, we conducted in vitro anticoagulant experiments, including plasma recalcification time (PRT) assay, fibrinolytic assay, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fib) assay, and cell viability assays. Furthermore, a carrageenan-induced chronic thromboembolism model was employed in ICR mice, and four coagulation factors (APTT, PT, TT, and Fib) activities were determined after intragastric administration. RESULTS: The anticoagulant protein WP-77 has a relative molecular weight of ca. 20.8 kDa. It was effective over a broad temperature range from 20 °C to 100 °C and a pH 2-8 condition. The anticoagulant activity of WP-77 was retained after incubation with pepsin but was greatly inhibited by trypsin (P < 0.01). It significantly prolonged APTT and TT (P < 0.05) but had little effect on PT and Fib in vitro. Furthermore, WP-77 of a low concentration resulted in the recovery of injured EA.hy926 by thrombin. The protein also significantly prolonged APTT and TT (P < 0.01) and inhibited thrombus formation in carrageenan-induced thrombosis mice, demonstrating its antithrombotic effect in vivo. CONCLUSION: Our results suggest that WP-77 from W. pigra plays a distinct role in treating thrombotic diseases, and it is an essential substance of anticoagulant activity of non-suck-blood medicinal leeches. This thermostable anticoagulant protein could be a promising candidate for the development of clinical antithrombosis medicines.

WPK5, a Novel Kunitz-Type Peptide from the Leech Whitmania pigra Inhibiting Factor XIa, and Its Loop-Replaced Mutant to Improve Potency.

Kunitz-type proteins or peptides have been found in many blood-sucking animals, but the identity of them in leeches remained elusive. In the present study, five Kunitz-type peptides named WPK1-WPK5 were identified from the leech Whitmania pigra. Recombinant WPK1-WPK5 were expressed in Pichia pastoris GS115, and their inhibitory activity against Factor XIa (FXIa) was tested. WPK5 showed inhibitory activity against FXIa with an IC50 value of 978.20 nM. To improve its potency, the loop replacement strategy was used. The loop 1 (TGPCRSNLER) and loop 2 (QYGGC) in WPK5 were replaced by loop 1 (TGPCRAMISR) and loop 2 (FYGGC) in PN2KPI, respectively, and the resulting peptide named WPK5-Mut showed an IC50 value of 8.34 nM to FXIa, which is about 100-fold the potency of FXIa compared to that of WPK5. WPK5-Mut was further evaluated for its extensive bioactivity in vitro and in vivo. It dose-dependently prolonged APTT on both murine plasma and human plasma, and potently inhibited FeCl3-induced carotid artery thrombosis in mice at a dose of 1.5 mg/kg. Additionally, WPK5-Mut did not show significant bleeding risk at a dose of 6 mg/kg. Together, these results showed that WPK5-Mut is a promising candidate for the development of an antithrombotic drug.

[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.

[Effects of Wp-PKC on gonadal development of Whitmania pigra].

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, 17ß-estradiol(17ß-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; 17ß-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.

[Cloning and expression analysis of protein kinase C gene from Whitmania pigra].

Protein kinase C(PKC) is a type of protein kinase widely involved in cell proliferation and development, but the developmental mechanism in the gonads of androgynous animals is still unclear. In order to explore the role of protein kinase C in the development of Whitmania pigra germ cells, the Wh. pigra PKC(Wp-PKC) gene was cloned, bioinformatics analysis was conducted, and fluorescent quantitative PCR was used to analyze the expression of female and male gonads. The results showed that:(1)The cloned Wp-PKC had a full length of 2 580 bp, a relative molecular weight of 76 555.19, and contains an open reading frame encoding 670 amino acids, Wp-PKC was closely related to Danio rerio PKC-α and rat PKC-γ. The similarity of amino acid sequence was 55% and 58%.(2)The protein encoded by Wp-PKC had no signal peptide and was a hydrophilic protein. The secondary structure is mainly composed of random coils, α-helices, extended chains, folds and folds, with the largest proportion of random coils and α-helices. Wp-PKC protein does not contain a transmembrane domain. Multiple sequence alignment and domain prediction analysis show that Wp-PKC contains 4 conserved domains of classical protein kinase C.(3)Fluorescence quantitative results showed that the expression of Wp-PKC in Wh. pigra gonads was positively correlated with the development of germ cells, and the expression in male gonads was significantly higher than that in female gonads. In summary, Wp-PKC is a classic PKC, and Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads, and provide references for further research on the developmental mechanisms of Wh. pigra.

Aqueous extract of Whitmania pigra Whitman ameliorates ferric chloride-induced venous thrombosis in rats via antioxidation.

Whitmania pigra Whitman (W. pigra) has been widely employed in decoction for the treatment of blood stasis syndrome for many years in China. The aim of the present study was to explore the anti-venous thrombosis (VT) mechanism of the aqueous extract of W. pigra (AEW) in rats. Rats were orally administered with AEW. A inferior vena cava (IVC) thrombosis model was established. Thrombosed IVC was weighed and histopathological analyses were performed. Blood coagulation, blood fibrinolysis, blood cell count, blood viscosity and platelet activity were evaluated. Reactive oxygen species (ROS) accumulation was analyzed. Malondialdehyde (MDA) content in thrombosed IVC and antioxidants in serum were detected. Protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in thrombosed IVC was determined. AEW significantly reduced thrombus weight. It did not affect blood coagulation, blood fibrinolysis, blood cell count, platelet activity, or whole blood viscosity. However, AEW remarkably alleviated vascular injury, reduced ROS accumulation and MDA content, enhanced the total antioxidant capacity and the activities of superoxide dismutase, glutathione peroxidase and glutathione reductase. It increased the glutathione/oxidized glutathione ratio and the protein expression levels of Nrf2 and HO-1. In summary, W. pigra may prevent VT via Nrf2-mediated antioxidation.

Effects of Wp-PKC on gonadal development of Whitmania pigra / 中国中药杂志

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.

Cloning and expression analysis of protein kinase C gene from Whitmania pigra / 中国中药杂志

Protein kinase C(PKC) is a type of protein kinase widely involved in cell proliferation and development, but the developmental mechanism in the gonads of androgynous animals is still unclear. In order to explore the role of protein kinase C in the development of Whitmania pigra germ cells, the Wh. pigra PKC(Wp-PKC) gene was cloned, bioinformatics analysis was conducted, and fluorescent quantitative PCR was used to analyze the expression of female and male gonads. The results showed that:(1)The cloned Wp-PKC had a full length of 2 580 bp, a relative molecular weight of 76 555.19, and contains an open reading frame encoding 670 amino acids, Wp-PKC was closely related to Danio rerio PKC-α and rat PKC-γ. The similarity of amino acid sequence was 55% and 58%.(2)The protein encoded by Wp-PKC had no signal peptide and was a hydrophilic protein. The secondary structure is mainly composed of random coils, α-helices, extended chains, folds and folds, with the largest proportion of random coils and α-helices. Wp-PKC protein does not contain a transmembrane domain. Multiple sequence alignment and domain prediction analysis show that Wp-PKC contains 4 conserved domains of classical protein kinase C.(3)Fluorescence quantitative results showed that the expression of Wp-PKC in Wh. pigra gonads was positively correlated with the development of germ cells, and the expression in male gonads was significantly higher than that in female gonads. In summary, Wp-PKC is a classic PKC, and Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads, and provide references for further research on the developmental mechanisms of Wh. pigra.

Effect of heavy metal lead on anticoagulant activity of leech based on label-free quantitative proteomics / 中国中药杂志

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.

Proteome and phosphoproteome profiling reveals the regulation mechanism of hibernation in a freshwater leech (Whitmania pigra).

Hibernation is an energy-saving and adaptive strategy adopted by leech, an important medicinal resource in Asia, to survive low temperature. Reversible protein phosphorylation (RPP) plays a key role in the regulation of mammalian hibernation processes but has never been documented in freshwater invertebrate such as leech. In this study, we detected the effects of hibernation on the proteome and phosphoproteome of the leech Whitmania pigra. A total of 2184 proteins and 2598 sites were quantified. Deep-hibernation resulted in 85 up-regulated and 107 down-regulated proteins and 318 up-regulated and 204 down-regulated phosphosites using a 1.5-fold threshold (P<0.05). Proteins involved in protein digestion and absorption, amino acid metabolism and N-glycan biosynthesis were significantly down-regulated during deep-hibernation. However, proteins involved in maintaining cell structure stability in hibernating animals were up-regulated. Differentially phosphorylated proteins provided the first global picture of a shift in energy metabolism, protein synthesis, cytoprotection and signaling during deep hibernation. Furthermore, AMP-activated protein kinase and protein kinase C play major roles in the regulation of these functional processes. These data significantly improve our understanding of the regulatory mechanisms of leech hibernation processes and provides substantial candidate phosphorylated proteins that could be important for functionally adapt in freshwater animals. SIGNIFICANCE: The leech Whitmania pigra as an important medicinal resource in Asia is an excellent model freshwater invertebrate for studies of environmentally-induced hibernation. The present study provides the first quantitative proteomics and phosphoproteomic analysis of leech hibernation using isobaric tag based TMT labeling and high-resolution mass spectrometry. These data significantly improve our understanding of the regulatory mechanisms when ectotherm animals face environmental stress and provides substantial candidate phosphorylated proteins that could be important for functionally adapt in freshwater animals.

[Effects of constant temperature on growth and gonad development of overwintering Whitmania pigra].

The present study was conducted to explore the effect of constant temperature overwintering on the growth, gonadal development and internal quality of the Wh. pigra which were in overwintering period. Wh. pigra which in overwintering period were placed in light incubator at 21 ℃, and the Wh. pigra that overwintered under natural conditions were used as control. That the changes of growth performance, gonad index, internal quality of two groups of Wh. pigra were measured at the end of the overwintering. Simultaneously the tissue slice technique was used to observe the morphological structure of the spermary and ovary of the two groups. The results showed that the body weight of constant temperature overwintering Wh. pigra which were placed in light incubator at 21 ℃ was 2.35 times that of natural overwintering Wh. pigra which overwintered under natural conditions, and the weight of female gonads of the Wh. pigra which were placed in light incubator at 21 ℃ was 11.54% higher than that of Wh. pigra which overwintered under natural conditions, and the weight of male gonads of the Wh. pigra which were placed in light incubator at 21 ℃ was 48.33% higher than that of the Wh. pigra which overwintered under natural conditions. At the same time, that vitellogenesis cells and film forming cells which in ovarian vesicles were significantly higher than those of the Wh. pigra which overwintered under natural conditions, and that spermatogonia and primary spermatocytes which in seminal vesicle were significantly higher than those of Wh. pigra which overwintered under natural conditions. Most important of all, the anti-thrombin activity of Wh. pigra which were placed in light incubator at 21 ℃ increased by 27.85% compared with the Wh. pigra that overwintered under natural conditions. In conclusion, that constant temperature can promote the growth, the development of sperm and egg cells, and the increase of anti-thrombin activity of Wh. pigra which were in overwintering period.

Purification and characterization of a novel fibrinolytic enzyme from Whitmania pigra Whitman.

Developing an effective fibrinolytic drug for treating thrombolysis with minimal undesirable side effects is of great importance. In the current study, an optimum solvent was selected for the extraction of fibrinolytic active components. Furthermore, a strong fibrinolytic enzyme named WPI01 was purified from Whitmania pigra Whitman through various chromatographic steps. WPI01 has a molecular mass of 27044.297 Da, and the N-terminal 8 amino acid sequence was determined as VVGGVEAR. WPI01 was stable within the pH range of 6.0-10.0 and with maximum fibrinolytic activity at 40 °C and a pH of 8.0. At 500 U/mL, WPI01 induced 50.59% blood clot reduction in vitro within 6 h, which was higher than that induced by urokinase at 1000 U/mL. In an analysis of the plasminogen activator activity, WPI01 produced obvious halos on heated and unheated fibrin plates, suggesting that WPI01 may not only act as a plasminogen activator but also degrade fibrin clots directly, and more study is needed to support this. In conclusion, WPI01 is obviously different from known fibrinolytic enzymes in terms of substrate specificity and fibrinolytic mode of action, suggesting that it is a novel fibrinolytic enzyme with potential applications in the treatment and prevention of thrombosis.