Chinese medicine Di-long (Pheretima vulgaris) and its active fraction exhibit anti-rheumatoid arthritis effects by inhibiting CXCL10/CXCR3 chemotaxis in synovium.

ETHNOPHARMACOLOGICAL RELEVANCE: Di-Long (Pheretima vulgaris) is a classic animal sourced traditional Chinese medicine. It has been used for the treatment of joint inflammation and arthralgia for over two thousand years due to its effects of Tong-Luo-Zhi-Tong (dredging collaterals and alleviating pain). Our previous study showed that Chinese medicine Di-Long has significant anti-rheumatoid arthritis (RA) effects. AIM OF THE STUDY: Considering Di-Long as a potential source of active compounds with specific anti-RA therapeutic effects, this research was to obtain the anti-RA target-specific active fraction from Di-Long extracts (DL), and to further explore the chemical basis and verify the anti-RA mechanism of this active fraction. MATERIALS AND METHODS: Transcriptomic was applied to obtain the main anti-RA targets of DL on human RA fibroblast-like synoviocytes (FLS) and validated by qPCR. The target-corresponding active fraction was isolated from DL by ethanol precipitation and gel chromatography, and analyzed by nanoliter chromatography-mass spectrometry. Anti-RA effects of this active fraction was investigated by collagen-induced arthritis (CIA) in mice, and anti-RA mechanisms were verified in cocultured model of rat FLS and peripheral blood lymphocytes. RESULTS: We confirmed that CXCL10/CXCR3 was the main anti-RA target of DL. The active fraction - A (2182 - 890 Da) was isolated from DL based on its CXCL10 inhibiting effects in RA-FLS. Fraction A contains 195 peptides (192 were newly discovered), 26 of which might be bioactive and were considered to be the chemical basis of its anti-RA effects. Fraction A significantly ameliorated the joint destruction and overall inflammation in CIA mice, and downregulated CXCR3 expression in mice joint. Fraction A inhibited the chemotaxis of Th-cells in rat peripheral blood lymphocytes towards the TNF-α-induced rat FLS through CXCL10/CXCR3 pathway. CONCLUSIONS: Our work indicated that active fraction from DL containing small peptides exhibits promising therapeutic effects for RA through inhibiting CXCL10/CXCR3 chemotaxis.

Purification and identification of thrombolytic peptides from enzymatic hydrolysate of Pheretima vulgaris.

Pheretima vulgaris has been prescribed for the treatment of cardiovascular diseases in China for several hundred years in the form of dried powder in the clinic. However, the peptides with the potential antithrombotic activity of this source have never been reported. The total active proteins from Pheretima vulgaris were hydrolyzed by eight different commercial proteases and the alcalase hydrolysate showed the strongest thrombolytic activity. Four original thrombolytic peptides were isolated and characterized using bioactivity-directed fractionation of the active hydrolysate. The amino acid sequences were identified as HEPLPEP (m/z 818.40076), EYPLPEP (m/z 844.39648), LGEPSVP (m/z 698.39648), and LLAPP (m/z 510.28043) by nanoLC-ESI-Orbitrap mass spectrometry with PEAKS software. HEPLPEP and EYPLPEP, containing the common -PLPEP residue, showed superior thrombolytic activity in plasmin assay and fibrinogen-thrombin time assay. This research confirmed that Pheretima vulgaris was a potential source of active peptides with thrombolytic activities and provided novel candidates for the thrombolytic agents. PRACTICAL APPLICATIONS: Thrombosis has become the leading cause of mortality as it was the common underlying pathology of cardiovascular diseases, such as ischemic heart disease, and stroke. The demand for thrombolytics has increased gradually as the incidence trends of thrombosis-related diseases raise with the aging of the population. Four novel thrombolytic peptides were characterized from Pheretima vulgaris proteins hydrolysates, among which HEPLPEP and EYPLPEP could prevent the formation of thrombus and degrade existing thrombus in vitro. These peptides are promising to be meritorious templates for developing thrombolytic agents. The structure-function relationship of peptides resulting from the presence of specific residues in these sequences may contribute to extending the knowledge about their thrombolytic activity, which may be useful in designing novel thrombolytic agents. The present research based on a bioactivity-directed isolation strategy could also be applied to other animal-derived traditional Chinese medicines with proteins or peptides as their function basis.

Therapeutic effects of Chinese medicine Di-Long (Pheretima vulgaris) on rheumatoid arthritis through inhibiting NF-κB activation and regulating Th1/Th2 balance.

Chinese medicine Di-Long, the dried body of Pheretima vulgaris (Chen) has been used for the treatment of joint inflammation, arthralgia and numbness of limbs for many years. This study was to investigate the anti-rheumatoid arthritis (RA) effects of Di-Long and to explore its possible mechanisms. The identification and quantification of representative components in Di-Long extracts (DL) were carried out by HPLC analysis. The anti-RA effects and mechanisms of DL were studied in CIA mice, RAW 264.7 macrophages and spleen T lymphocytes. The Th1/Th2 cell ratio in CIA mice spleens were determined by Flow cytometry. The cytokine levels were determined by ELISA method. The expressions of p-NF-κB p65 in ankle joints of CIA mice were detected by Immunohistochemistry analysis. The phosphorylation of NF-κB signaling pathway in RAW 264.7 macrophages and expressions of T-bet and GATA-3 in CIA mice spleens were determined by Western blots. The treatment with DL significantly decreased the paw thickness, arthritis scores and inflammatory cells infiltration in CIA mice. The TNF-α, IL-6 concentrations in both mice serum and macrophages secretion were markedly reduced with the treatment of DL, as well as the phosphorylation of NF-κB pathway. DL inhibited the expressions of T-bet and GATA-3 and decreased Th1/Th2 cells ratio in CIA mice spleens. DL reduced IFN-γ, IL-2 levels in mice serum and spleen T lymphocytes, and increased IL-4 levels in CIA mice serum. Chinese medicine Di-Long have significant anti-RA effects. The mechanisms might be inhibiting the activation of NF-κB signaling pathway and regulating the balance of Th1/Th2 cells.

Bioevaluation of Pheretima vulgaris Antithrombotic Extract, PvQ, and Isolation, Identification of Six Novel PvQ-Derived Fibrinolytic Proteases.

Thrombosis is a disease that seriously endangers human health, with a high rate of mortality and disability. However, current treatments with thrombolytic drugs (such as recombinant tissue-plasminogen activator) and the oral anticoagulants (such as dabigatran and rivaroxaban) are reported to have a tendency of major or life-threatening bleeding, such as intracranial hemorrhage or massive gastrointestinal bleed with non-specific antidotes. In contrast, lumbrokinase is very specific to fibrin as a substrate and does not cause excessive bleeding. It can dissolve the fibrin by itself or convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots. Therefore, searching for potentially new therapeutic molecules from earthworms is significant. In this study, we first collected a strong fibrinolytic extract (PvQ) from the total protein of the Pheretima vulgaris with AKTA pure protein purification systems; its fibrinolytic bioactivity was verified by the fibrin plate assay and zebrafish thrombotic model of vascular damage. Furthermore, according to the cell culture model of human umbilical vein endothelial cells (HUVECs), the PvQ was proven to exhibit the ability to promote the secretion of tissue-type plasminogen activator (t-PA), which further illustrated that it has an indirect thrombolytic effect. Subsequently, extensive chromatographic techniques were applied to reveal the material basis of the extract. Fortunately, six novel earthworm fibrinolytic enzymes were obtained from the PvQ, and the primary sequences of those functional proteins were determined by LC-MS/MStranscriptome cross-identification and the Edman degradation assay. The secondary structures of these six fibrinolytic enzymes were determined by circular dichroism spectroscopy and the three-dimensional structures of these proteases were predicted by MODELLER 9.23 based on multi-template modelling. In addition, those six genes encoding blood clot-dissolving proteins were cloned from P. vulgaris by RT-PCR amplification, which further determined the accuracy of proteins primary sequences identifications and laid the foundation for subsequent heterologous expression.

A Novel Fibrinolytic Protein From Pheretima vulgaris: Purification, Identification, Antithrombotic Evaluation, and Mechanisms Investigation.

Thrombotic diseases have been considered major causes of death around the world. Treatments with thrombolytic drugs, such as recombinant tissue-plasminogen activator, urokinase, and streptokinase, are reported to have a life-threatening bleeding tendency. On the contrary, lumbrokinase, identified from Lumbricus rubellus, is specific to fibrin and does not cause excessive bleeding. It possesses fibrinolytic activity and activation of plasminogen to dissolve fibrin. Hence, the purification of fibrinolytic protein monomer from earthworm and antithrombotic evaluation and investigation of mechanisms are needed. In this study, a novel fibrinolytic protein EPF3, with strong fibrinolytic activity, was purified from Pheretima vulgaris by ion exchange and size exclusion chromatography. SDS PAGE, bottom-up proteomics analysis, de novo sequencing, and circular dichroism (CD) analysis were carried out for identification and characterization of it. EPF3, with a molecular weight of 25136.24 Da, consisted of 241 amino acids and contained various forms of secondary structures, including α-helix (3.9%), ß-sheet (42.8%), ß-turn (21.2%), and random coil (32.1%). It was a trypsin-like serine protease and stable at pH 7.0 to 11.0 and below 40°C. EPF3 was confirmed to possess an antithrombotic effect by ex vivo clot lysis test and fibrinogen-thrombin time (Fib-TT) assay. The three-dimensional structure of EPF3 was predicted by SWISS-MODEL. Molecular docking analysis predicted that EPF3 could directly interact with antithrombotic target proteins (fibrin, fibrinogen, and plasminogen), which was further confirmed by further studies. The antithrombotic mechanism of EPF3 was clarified to be outstanding direct fibrinolysis, fibrinogenolytic activity, and certain activation of plasminogen. EPF3 possesses the potential to be developed into a promising antithrombotic agent.

Rapid Enrichment and Activity Research of Antithrombotic Proteins from Pheretima vulgaris / 中国实验方剂学杂志

Objective:A strong antithrombotic protein component， named PvQ， was purified and enriched from total protein of <italic>Pheretima vulgaris</italic>，<italic> </italic>a<italic> </italic>traditional Chinese medicine. Moreover， we evaluated its fibrinolytic and anticoagulant activity， and expected to provide reference for the research on antithrombotic substances of Pheretima. Method:A rapid <italic>in</italic> <italic>vitro</italic> activity-oriented separation combined with the AKTA-Pure protein purification system conducted on <italic>P. vulgaris</italic>. Meanwhile， the fibrinolytic and anticoagulant activities of PvQ were measured by fibrin plate method and fibrinogen-thrombin time （Fibg-TT） method. And the <italic>in vitro</italic> thrombolysis assay was used for evaluating the lysis ability of PvQ to thrombus. Then the stability of PvQ was also analyzed for its anticoagulant activity at different pH and temperature. Result:The PvQ was successfully enriched and its activity was determined to have significant fibrinolytic and anticoagulant activities. And the result of <italic>in vitro</italic> thrombolysis assay revealed that PvQ could hydrolyze more than 80% of thrombus after 5 h of incubation at 37 ℃. In addition， the changes of temperature and pH had significant effects on antithrombotic activity， and this study showed that PvQ was rapidly inactivated at ≥60 ℃ or in acidic conditions （pH<7）. While， the activity of PvQ was unaffected or less affected at ≤50 ℃ and under alkaline conditions. Conclusion:A feasible preparation method of PvQ is established， and it can affect fibrin and fibrinogen at the same time， thus exerting a dual fibrinolytic effect and possessing significant fibrinolytic and anticoagulant activities. It provides a scientific interpretation for the treatment of thrombotic diseases by PvQ and a reference for the development of antithrombotic protein products of Pheretima.

Identification of Pheretima by Multiplex Allele-specific PCR / 中国实验方剂学杂志

Objective: In recent years,with the increase in the commodity price of medicinal pheretima,there have emerged increasing adulterates in the medicine market. Besides,the medicinal materials have mostly lost the main identification features, and are difficult to distinguish. Therefore,it is urgent to establish an accurate and stable method for the identification of pheretima. Method: According to the differences of COI gene DNA sequences among Pheretima aspergillum,Pheretima vulgaris,Pheretima guillelmi,Pheretima pectinifera and adulterants,the variation site was found,the specific primers were designed,the reaction conditions were optimized,and the polymerase Chain reaction(PCR) method for identification was explored and verified in terms of tolerance and feasibility in this study. The specific primers were combined to build multiple PCR systems. An effective,accurate,convenient,highly specific and repeatable Multiplex Allele-Specific PCR identification method was established for identifying medicinal pheretima and its common adulterants. Result: Through the established multiplex PCR reaction system, 366,487,487 and 475 bp of fragments were amplified from DNA templates of P. aspergillum,P. vulgaris,P. guillelmi and P.pectinifera respectively. All of the adulterants were negative by the multiplex PCR assay. The PCR amplification of specific alleles method established in this paper can accurately identify pheretima. Conclusion: The Multiplex Allele-Specific PCR identification method established in this paper can accurately identify medicinal pheretima and its adulterants.