Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant ...Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant and fibrinolytic activities,and relevant mechanisms have been confirmed through in vitro experiments.However,whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown.This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro.Methods:The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl3.Then,the protein bands of EPF3 incubated with simulated gastric fluid(SGF),simulated intestinal fluid(SIF),and homogenate of Caco-2 cells(HC2C)were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability.Finally,the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer.Results:EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid.It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl3.EPF3 was stable in SIF and HC2C and unstable in SGF.The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent.The efflux ratio was less than1.2 during transport,and the transport behavior was not affected by inhibitors.EPF3 could reversibly reduce the expression of tight junction-related proteins,including zonula occludens-1,occludin,and claudin-1 in Caco-2 cells.Conclusion:EPF3 could play a thrombolytic and antithrombotic role in zebrafish.It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction.This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation.Please cite this article as:Zhong WL,Yang JQ,Liu H,Wu YL,Shen HJ,Li PY,Du SY.Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong(Pheretima vulgaris Chen)and its transport mechanism in Caco-2 monolayer through cell bypass pathway.J Integr Med.2025;23(4):415–428.展开更多
To provide basic toxicity data for formulating risk characterization benchmarks, the effects of lead on survival, locomotion, and sperm morphology were investigated in the Asian earthworm Pheretima guillelmi. The LC50...To provide basic toxicity data for formulating risk characterization benchmarks, the effects of lead on survival, locomotion, and sperm morphology were investigated in the Asian earthworm Pheretima guillelmi. The LC50 of P. guiUelmi for 7 and 14 d were 4285＋339 mg/kg and 3207＋248 mg/kg, which shows P. guillelmi can tolerate a higher concentration of lead nitrate. The average weight of the surviving earthworms decreased at concentration of 2800 mg Pb/kg soil, and the locomotor ability of earthworms exposed to a range of soil Pb concentrations showed a general decrease with increasing Pb concentrations. We also presented data depicting the sperm morphology of earthworms, which shows potential as a sensitive biomarker for measuring the effects of heavy metal on reproduction.展开更多
Electroanalytical techniques could be a reliable and promising alternative to classical and sophisticated methods because of their simplicity（small and portable）,easy use,the ability to deliver fast response with hi...Electroanalytical techniques could be a reliable and promising alternative to classical and sophisticated methods because of their simplicity（small and portable）,easy use,the ability to deliver fast response with high sensitivity and selectivity.A square wave voltammetric method was developed for the assessment of organophosphorus（OPs） compound impact on acetylcholinesterase（AChE） of Pheretima with 2,6-dimethyl-p-benzoquinone（2,6DMBQ） as a redox indicator.The substrate of acetylthiocholine is hydrolyzed by AChE and the produced thiocholine reacts with 2,6-DMBQ to give an obvious shift of electrochemical signal.The reduction peak of 2,6-DMBQ is located at around 0.18 V which is far away from the oxidation potential of possible interference components often present in biosample.The decreased rate of reduction current was related with the activity of AChE.The inhibition of parathion-methyl on AChE was assessed.The inhibiton rate of OPs on AChE activity increased quickly during the first 10 min inhibition,and after that the value of inhibition rate approached to be constant.AChE lost almost 29.3% of activity after 10 min incubation with 1 μg/mL parathion-methyl and 67.5% of activity with 10 μg/mL parathion-methyl,while the activity that corresponds to 40 μg/mL parathion-methyl was nearly completely inhibited（94.9%）.Compared to cyclic voltammetry and amperometry,Square wave voltammetry（SWV） method is a high sensitive electroanalysis with fast scan-rate（only several seconds for one signal value） which is useful to prevent the electrodes from possible fouling or passivation.This method can be employed to assess the inhibition of organophosphate on AChE and investigate OPs impact on environmental animals.展开更多
Pheretima,also called“earthworms”,is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edition).However,its zool...Pheretima,also called“earthworms”,is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edition).However,its zoological origin is unclear,both in the herbal market and CPMs.In this study,a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms(restricted search,open search,and de novo)was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima,including Pheretima aspergillum(PA),Pheretima vulgaris(PV),and Metaphire magna(MM).We identified 10,477 natural peptides in the PA,7,451 in PV,and 5,896 in MM samples.Five specific signature peptides were screened and then validated using synthetic peptides;these demonstrated robust specificity for the authentication of PA,PV,and MM.Finally,all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills,revealing the inconsistent Pheretima species used in these CPMs.In conclusion,our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines,especially non-model species with poorly annotated protein databases.展开更多
目的探究威廉环毛蚓抗血栓蛋白DPf3经胃肠道消化后活性及成分变化,并结合同源建模预测其活性蛋白DPf3 ID NO.2的空间结构。方法体外模拟胃排空状态(pH 2)及半排空状态(pH 5)下胃蛋白酶(及胰蛋白酶)对DPf3的消化作用,昆明小鼠经灌胃给药D...目的探究威廉环毛蚓抗血栓蛋白DPf3经胃肠道消化后活性及成分变化,并结合同源建模预测其活性蛋白DPf3 ID NO.2的空间结构。方法体外模拟胃排空状态(pH 2)及半排空状态(pH 5)下胃蛋白酶(及胰蛋白酶)对DPf3的消化作用,昆明小鼠经灌胃给药DPf3考察体内酶解对其消化作用;纤维蛋白平板法联合十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析DPf3经体内外水解后活性及成分变化;I-TASSER联合SAVES v5.0同源建模并评价DPf3的活性蛋白成分DPf3 ID NO.2,并预测其二级和三级结构。结果DPf3经体外酶解后,在pH 2孵育条件下丧失纤溶活性,在pH 5条件下,DPf3经热灭活胃蛋白酶孵育后,纤溶活性较未经热灭活胃蛋白酶孵育物稍高;经体内酶解后,与对照组相比,仅胃部灌流液的纤溶活性具显著性差异;蛋白质谱图结果显示DPf3 ID NO.2的稳定存在或为DPf3具纤溶活性的重要原因;空间结构预测表明DPf3 ID NO.2属于典型的凝乳胰蛋白酶样丝氨酸蛋白酶,揭示其催化中心、酶原中心、重要的氨基酸残基及S1活性口袋。结论通过体内外水解为威廉环毛蚓抗血栓蛋白DPf3口服后蛋白表达谱及活性变化提供依据,同源建模可用于中药动物药大分子蛋白质中活性多肽的结构预测。展开更多
基金supported by grants from the Fundamental Research Funds for the Central Universities(No.2020-JYB-ZDGG-032)National Natural Science Foundation of China(No.82104352)。
文摘Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant and fibrinolytic activities,and relevant mechanisms have been confirmed through in vitro experiments.However,whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown.This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro.Methods:The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl3.Then,the protein bands of EPF3 incubated with simulated gastric fluid(SGF),simulated intestinal fluid(SIF),and homogenate of Caco-2 cells(HC2C)were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability.Finally,the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer.Results:EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid.It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl3.EPF3 was stable in SIF and HC2C and unstable in SGF.The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent.The efflux ratio was less than1.2 during transport,and the transport behavior was not affected by inhibitors.EPF3 could reversibly reduce the expression of tight junction-related proteins,including zonula occludens-1,occludin,and claudin-1 in Caco-2 cells.Conclusion:EPF3 could play a thrombolytic and antithrombotic role in zebrafish.It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction.This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation.Please cite this article as:Zhong WL,Yang JQ,Liu H,Wu YL,Shen HJ,Li PY,Du SY.Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong(Pheretima vulgaris Chen)and its transport mechanism in Caco-2 monolayer through cell bypass pathway.J Integr Med.2025;23(4):415–428.
基金supported by the Natural Science Fund of Zhejiang Province (No. Y506255)
文摘To provide basic toxicity data for formulating risk characterization benchmarks, the effects of lead on survival, locomotion, and sperm morphology were investigated in the Asian earthworm Pheretima guillelmi. The LC50 of P. guiUelmi for 7 and 14 d were 4285＋339 mg/kg and 3207＋248 mg/kg, which shows P. guillelmi can tolerate a higher concentration of lead nitrate. The average weight of the surviving earthworms decreased at concentration of 2800 mg Pb/kg soil, and the locomotor ability of earthworms exposed to a range of soil Pb concentrations showed a general decrease with increasing Pb concentrations. We also presented data depicting the sperm morphology of earthworms, which shows potential as a sensitive biomarker for measuring the effects of heavy metal on reproduction.
基金Supported by the Natural Science Foundation of Shanxi Province,China(No.20001057)
文摘Electroanalytical techniques could be a reliable and promising alternative to classical and sophisticated methods because of their simplicity（small and portable）,easy use,the ability to deliver fast response with high sensitivity and selectivity.A square wave voltammetric method was developed for the assessment of organophosphorus（OPs） compound impact on acetylcholinesterase（AChE） of Pheretima with 2,6-dimethyl-p-benzoquinone（2,6DMBQ） as a redox indicator.The substrate of acetylthiocholine is hydrolyzed by AChE and the produced thiocholine reacts with 2,6-DMBQ to give an obvious shift of electrochemical signal.The reduction peak of 2,6-DMBQ is located at around 0.18 V which is far away from the oxidation potential of possible interference components often present in biosample.The decreased rate of reduction current was related with the activity of AChE.The inhibition of parathion-methyl on AChE was assessed.The inhibiton rate of OPs on AChE activity increased quickly during the first 10 min inhibition,and after that the value of inhibition rate approached to be constant.AChE lost almost 29.3% of activity after 10 min incubation with 1 μg/mL parathion-methyl and 67.5% of activity with 10 μg/mL parathion-methyl,while the activity that corresponds to 40 μg/mL parathion-methyl was nearly completely inhibited（94.9%）.Compared to cyclic voltammetry and amperometry,Square wave voltammetry（SWV） method is a high sensitive electroanalysis with fast scan-rate（only several seconds for one signal value） which is useful to prevent the electrodes from possible fouling or passivation.This method can be employed to assess the inhibition of organophosphate on AChE and investigate OPs impact on environmental animals.
基金supported by the Key Program of the National Natural Science Foundation of China(Grant No.:82130111)the National Natural Science Foundation of China(Grant No.:81803716)+1 种基金the Qi-Huang Chief Scientist Project of the National Administration of Traditional Chinese Medicine,China(2020)the SIMM-SHUTCM Traditional Chinese Medicine Innovation Joint Research Program,China(Grant No.:E2G809H).
文摘Pheretima,also called“earthworms”,is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edition).However,its zoological origin is unclear,both in the herbal market and CPMs.In this study,a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms(restricted search,open search,and de novo)was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima,including Pheretima aspergillum(PA),Pheretima vulgaris(PV),and Metaphire magna(MM).We identified 10,477 natural peptides in the PA,7,451 in PV,and 5,896 in MM samples.Five specific signature peptides were screened and then validated using synthetic peptides;these demonstrated robust specificity for the authentication of PA,PV,and MM.Finally,all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills,revealing the inconsistent Pheretima species used in these CPMs.In conclusion,our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines,especially non-model species with poorly annotated protein databases.
文摘目的探究威廉环毛蚓抗血栓蛋白DPf3经胃肠道消化后活性及成分变化,并结合同源建模预测其活性蛋白DPf3 ID NO.2的空间结构。方法体外模拟胃排空状态(pH 2)及半排空状态(pH 5)下胃蛋白酶(及胰蛋白酶)对DPf3的消化作用,昆明小鼠经灌胃给药DPf3考察体内酶解对其消化作用;纤维蛋白平板法联合十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析DPf3经体内外水解后活性及成分变化;I-TASSER联合SAVES v5.0同源建模并评价DPf3的活性蛋白成分DPf3 ID NO.2,并预测其二级和三级结构。结果DPf3经体外酶解后,在pH 2孵育条件下丧失纤溶活性,在pH 5条件下,DPf3经热灭活胃蛋白酶孵育后,纤溶活性较未经热灭活胃蛋白酶孵育物稍高;经体内酶解后,与对照组相比,仅胃部灌流液的纤溶活性具显著性差异;蛋白质谱图结果显示DPf3 ID NO.2的稳定存在或为DPf3具纤溶活性的重要原因;空间结构预测表明DPf3 ID NO.2属于典型的凝乳胰蛋白酶样丝氨酸蛋白酶,揭示其催化中心、酶原中心、重要的氨基酸残基及S1活性口袋。结论通过体内外水解为威廉环毛蚓抗血栓蛋白DPf3口服后蛋白表达谱及活性变化提供依据,同源建模可用于中药动物药大分子蛋白质中活性多肽的结构预测。
