Novel derivative of Danshensu acts as anti-thrombotic agent for modulation of protein disulfide isomerase family member ERp57

OBJECTIVE To identify the specific targets of a novel derivative of Danshensu ADTM as the protein disulfide isomerase(PDI)family proteins including ERp57.To further investigate the underlying mechanism of ADTM to modulate ERp57 to regulate platelet function with direct interaction withαⅡbβ3 integrin.METHODS To isolate the protein targets that bound to ADTM,a biotin-conjugated ADTM analogue(BAA)was designed and synthesized.BAA(300μmol·L-1)was incubated with rat blood platelet lysates and the BAA-protein complexes were pulled down with NeutrAvidin-agarose followed by protein profiling using LC-MS/MS.To determine platelet aggregation in vitro,rabbit platelets were incubated with the indicated concentrations of compounds and aggregation was induced by ADP(10μmol·L-1)or AA(200μmol·L-1)and measured using a platelet aggregometer.To determine platelet aggregation-induced by ADP in rat in vivo,ADTM(5-20mg·kg-1)in comparison with DSS(10mg·kg-1)and clopidogrel(18mg·kg-1)were administered daily by i.v.injection for 5d,respectively.To determine the action of ADTM on the ERp57/αⅡbβ3 interaction,it was examined by immunoprecipitation with anti-αⅡbβ3antibody,followed by detection of ERp57 immunoreactivity using immunoblotting.RESULTS BAA could bind to various proteins involved in platelet function.In particular,platelet aggregation-associated proteins were identified with>95% protein identification probability including ERp72,ERp57ERp5 and PDI,which are members of the protein disulfide isomerase(PDI)family related to platelet function and redox homeostasis.ADTM exhibited potent inhibition on the redox activity of ERp57 in a concentration-dependent manner(IC50=100 300μmol·L-1).In in vitro studies,ADTM exhibited concentration-dependent inhibition on ADP-induced and AA-induced platelet aggregation with comparable effects to aspirin and clopidogrel.In vivo study showed that ADP-induced platelet aggregation was significantly compromised(>40%reduction)in rats treated with ADTM(20mg·kg-1).Similarly,ADTM also exhibited significant anti-thrombotic effect in vivo as shown in the ferric chloride(FeCl3)-induced venous thrombosis.Immunoprecipitation with anti-αⅡbβ3antibody,followed by detection of ERp57 immunoreactivity using immunoblotting showed that ADTM disrupted the interaction of ERp57 with αⅡ bβ3.CONCLUSION These results demonstrated that ADTM exhibited broad-spectrum anti-platelet activities and ERp57 is a potential therapeutic target for anti-platelet therapy.

Transcriptome analysis of leaves,roots and flowers of Panax notoginseng on ginsenoside and alkaloid biosynthesis and amelioration of vascular insufficiency conditions by saponins from the flower buds

OBJECTIVE To investigate the transcriptomic details on the biosynthetic pathways in different parts of the Panax notoginseng and the pharmacological activity of the saponins extracted from the flower(FS)on vascular insufficiency conditions.METHODS RNA sequencing of three different Panax notoginseng tissues was performed using next generation DNA sequencing and differential gene expression was validated by real-time PCR.In order to determine pro-angiogenic and therapeutic effects of FS on myocardial infraction(MI),FS was examined on the endothelial cell migration assay,vascular insufficiency model in zebrafish and MI model in rats.RESULTS After assembling the high quality sequencing reads into 107 340 unigenes,biochemical pathways were predicted and 9 908 unigenes were assigned to 135 KEGG pathways.Among them,270 unigenes were identified to be involved in triterpene saponin biosynthesis as well as 350 and 342unigenes were predicted to encode cytochrome P450 sand glycosyltransferases,respectively.One unigene was annotated as CYP716A53v2,probably participates in the formation of protopanaxatriol from protopanaxadiol and the differential expression of this gene was confirmed by real-time PCR.In addition,the pharmacological evaluation demonstrate that FS significantly promoted vascular endothelial growth factor(VEGF)induced the migration of human umbilical vein endothelial cells(HUVECs)and partially restored defective intersegmental vessels in a chemically induced vascular insufficiency model of zebrafish larva.Moreover,the two week posttreatment of the rat MI model with FS(25-50mg·kg-1·d-1)induced approximately 3-fold upregulation of VEGF mRNA expression,with a concomitant increase in blood vessel density in the peri-infarct area of the heart by 50.7%,compared to 41.4%in the MI group.Furthermore,TUNEL analysis indicates a reduction in the mean apoptotic nuclei per field in peri-infarct myocardium upon FS treatment.CONCLUSION We have established a global transcriptome dataset for Panax notoginseng and provided additional genetic information for further genome-wide research and analyses.Candidate genes involved in ginsenoside biosynthesis,including putative cytochrome P450 sand glycosyltransferases were obtained.The transcriptomes in different plant tissues also provide invaluable resources for future study of the differences in physiological processes and secondary metabolites in different parts of P.notoginseng.And the significant pro-angiogenic effect of FS in multiple experimental models renders the purified saponin preparation as potential preventive and therapeutic agent for cardiovascular diseases yet to be developed.

In vivoand in vitrostructure and relationship analysis of andrographolide derivatives:Identifying AGL-2 as a novel anti-angiogenesis agent

OBJECTIVE To analyse structure and relationship of several andrographolide derivatives in multiple in vivo and in vitro angiogenesis assays,and to demonstrate a novel compound named AGL-2as a potential anti-angiogenesis agent.METHODS Human umbilical vein endothelial cells(HUVECs)in vitro and zebrafish(Danio rerio)in vivo models were used to screen and identify the anti-angiogenesis activities of six andrographolide derivatives;namely,AGL-1,AGL-2,AGS-72,AGS-72 a,AGS-79 and AGP-151.RESULTS AGL-2exhibited the strongest anti-angiogenic activity among all the derivatives in zebrafish model.Interestingly,another compound named AGS-72 showed stronger anti-angiogenic activity than AGL-2 in VEGF-induced HUVECs proliferation,migration,invasion and tube formation assays.In addition,AGL-2 was found to suppress the VEGF-induced VEGFR-2auto-phosphorylation and inhibit the activity of VEGFR-2 mediated signaling cascades in a dose-dependent manner.CONCLUSION AGL-2was demonstrated to be a promising anti-angiogenic agent among all the tested derivatives.The mechanism underlying the anti-angiogenic activity of AGL-2 probably involve VEGFR-2 signaling pathway.Even though,how some of chemical structure alterations result in discrepancy between in vivo and in vitro activities still remains to be resolved,this study shall provide new insight into how modification of the chemical structure of andrographolide affects this newly identified anti-angiogenesis activity.Meanwhile,AGL-2 can be exploited as a potential therapeutic agent for the treatment of angiogenesis-related diseases.

Cardioprotective and chemosensitizing effects of novel Danshensu derivatives

OBJECTIVE To investigate the cardioprotective effect of novel danshensu derivatives against doxorubicin(Dox)cardiotoxicity and their synergistic anti-tumor effect with Dox on breast cancer cells.METHODS Two new Danshensu derivatives were synthesized by conjugation with tetramethylpyrizine and/or4-(3-thioxo-3 H-1,2-dithiol-4-yl)-benzoic acid and tested for protective effects against Dox induced cardiotoxicity in cell and zebrafish.H9c2 cardiomyoblasts were co-treated with Dox and Danshensu derivatives for 24 h and then were measured for cell viability and cytotoxicity by MTT and LDH assays.The expression levels of mitochondrial biogenesis related proteins PGC-1α,NRF-1and Nrf2 were detected by Western blotting and qPCR.Moreover,in a Dox-induced cardiotoxicity model of zebrafish,zebrafish embryos were treated with Dox for 36 h,followed by measurement of numerous ventricular function parameters including heart rate,stroke volume,cardiac output and fractional shortening.In addition,the synergistic anti-tumor effects of the Danshensu derivatives and Dox had been studied in MCF-7 breast cancer cells.The effects of the Danshensu derivatives on the cell death and metabolism of MCF-7 cells were measured using apoptosis assay and Seahorse Metabolic Analyzers respectively.RESULTS Our results showed that the Danshensu derivatives were more potent than the parental compounds in ameliorating Dox-induced cytotoxicity in H9c2 cells and significantly preserving stroke volume of heart function in Dox-treated zebrafish.Further mechanistic studies identified that the danshensu derivatives increased mitochondrial copy numbers and protein expressions of PGC-1α,NRF-1 and Nrf2 in H9c2 cells.In addition,the Danshensu derivatives enhanced Dox-induced apoptosis,and decreased glycolysis and mitochondrial function in MCF-7 tumor cells.CONCLUSION Our results revealed that two new Danshensu derivatives displayed promising cardioprotective effects against Dox induced cardiotoxicity both in vivo and in vitro,at least partially through activating mitochondrial biogenesis.Also,the new Danshensu derivatives potentiated the anticancer effects of Dox in breast tumor cells involving induction of glycolytic inhibition and mitochondrial dysfunction.