| Locoweed is a kind of poisonous plants including many Astragalus and Oxytropisspecies which caused locoism. These plants possess a wide range of geographical distributionand seriously threat prairie animal husbandry all over the world. Previous studies haveconformed that swainsonine (1,2,8-trihyroxyindolizidine, SW) is the main toxic ingredient inlocoweeds. In vivo investigations conducted on pregnant goats have demonstrated thatingestion of SW-containing plants can induce lesion of placental trophoblast cells, retardedplacental development, placentae congestion and ultimately lead to abortion. As we know,placental trophoblast cells play vital roles in the processes of embryonic implantation andplacentation. Previous studies have also demonstrated the apoptotic effects of some pathogensand toxicological compounds on trophoblast cells are responsible for trophoblasts lesion andabortion occurrence. Therefore, in this study, we used goat trophoblast cells (GTCs) to detectethe effects of SW on cell cycle and cell invasiveness, and investigate the possible mechanismsinvolved in these processes in vitro. Finally, the signal transduction pathways of SW-inducedapoptosis were demonstrated rigorously. The results are as follows:1. Purified GTCs were obtained from pregnant Saanen dairy goat uteri (45to60days ofpregnancy) under aseptic conditions. Purified GTCs grow as epithelial-like monolayers withtypical cobblestone morphology and exhibited contact inhibition. In addition, most of thepurified cells were uninucleate trophoblast cells, with minor binucleate cells. The purifiedprimary GTCs were transfected at passage3by electroporation with pCI-neo-hTERT plasmid.After G418selection, the positive cells were gradually propagated and cultured in completeDMEM/F12medium. The morphology of transfected cells (designated hTERT-GTCs) atpassage50was similar to primary GTCs and the results of immumofluorescence assayshowed that hTERT-GTCs and primary GTCs are positive for cytokeratin7(CK-7). Resultsfrom RT-PCR, Western blot and TRAP-ELISA assay indicated that hTERT-GTCscontinuously expressed hTERT and maintained a relative higher telomerase activity. Thepopulation doubling time (PDT) of hTERT-GTCs was about1h less than that of primaryGTCs. Taken together, our results demonstrated that exogenous hTERT gene was successfully integrated into primary GTCs genome, and recovered the activity of telomerase, leading to theimmortalization of primary GTCs.2. The epithelial-origin characteristic of hTERT-GTCs and primary GTCs was confirmedfor the expression of E-cadherin by RT-PCR. Results from immumofluorescence and RT-PCRshowed that hTERT-GTCs and primary GTCs expressed vimentin and non-classical MHCclass I antigen, which were related to invasiveness. The invasive property of hTERT-GTCsand primary GTCs was further confirmed by Transwell assay. Further more,radioimmunoassay (RIA) showed that hTERT-GTCs and primary GTCs secreted CG-β andPL, and there were no significant differences in secretory levels between hTERT-GTCs andprimary GTCs. In addition, soft agar assay and the tumorigenicity assay in nude miceconfirmed that malignant transformation was not taken place in hTERT-GTCs. These resultsdemonstrated that hTERT-GTCs retained some key biological properties of normal primaryGTCs, and without any features of neoplastic transformation. The immortalized goattrophoblast cells could serve as a valuable model for study of trophoblast biological functions,and interactions with pathogens and toxicant.3. MTT assay and trypan blue exclusion assay showed that GTCs cell viability was notsignificantly varied after treatment with low concentrations of SW (0、0.4、0.8or1.2μg/mL)for24h, while GTCs viability was significantly reduced after treatment with1.6μg/mL ofSW for24h. SW induced cell cycle arrest was detected by flow cytometry, and the resultsshowed that SW significantly suppressed GTCs cell cycle in G0/G1phase after treatment with0.8,1.2and1.6μg/mL of SW for24h, and the expression of cyclin E, cyclin D1, CDK2wasdecreased. Cell adhesion assay showed that the adhesive ability of GTCs was significantlysuppressed after SW treatment, and the expression of cellular adhesion molecule, integrin αvβ3,was reduced in a concentration-dependent manner. Results from Transwell assay showed thatthe ability of migration and invasiveness of GTCs was significantly suppressed after SWtreatment. In addition, Western blot showed that the expression of MMP-2secreted by GTCsand the ratio of MT1-MMP/TIMP-2were decreased with the increase of SW.4. MTT assay showed that GTCs viability was significantly reduced after treatment with1.6μg/mL of SW for24h or2.4μg/mL of SW for12h. Results from AO/EB double stainingshowed that GTCs appeared typical apoptosis features, such as obvious chromatincondensation and slight nuclear fragmentation, at24h after1.6μg/mL of SW treatment or12h after2.4μg/mL of SW treatment. Besides the morphological changes of apoptosis inSW-treated GTCs, DNA fragmentation assay showed characteristic ladder patterns appearedin GTCs treated with2.4μg/mL of SW for24h, and were more evident with the increasing ofSW treatment times and concentrations. Finally, SW-induced apoptosis was further quantified by flow cytometry, and the results showed that the average proportion of Annexin V-stainingpositive cells increased with the SW concentration and SW-treated time. Taken together, theseresults suggested that SW suppressed viability of GTCs by inducing GTCs apoptosis.5. Results from caspase activity measurement and Western blot showed that SWtreatment resulted in a significant activation of caspases-9and-3, but not caspase-8during thetreated period. The substrates of caspase-3, PARP, was also cleaved in response to SWtreatment. Moreover, caspase-9inhibitor (LEHD) and caspase-3inhibitor (DEVD) showedsignificant inhibitory effects on SW-induced apoptosis to a certain extent, suggestingSW-induced apoptosis was dependent on activation of caspase-9and-3. However, SWtreatment did not affect the expression of Fas and FasL. Western blot assay showed that SWtreatment resulted in an increased level of Bax expression in a time-dependent manner,whereas the level of Bcl-2decreased gradually with time. Thus, SW treatment increased theratio of Bax/Bcl-2. Next, we observed the translocation of Bax from cytosol to mitochondria,which in turn caused the release of cytochrome c. In addition, results from TUNEL assayindicated that SW selectively promoted GTCs apoptosis but not CK-7negative cells in goatplacenta cotyledons.These results suggested that SW-induced apoptosis in GTCs viamitochondrial pathway.Taken together, we established an immortalized goat placental trophoblast cell line,which retained some key biological properties of normal primary goat trophoblast cells.Furthermore, this study also illustrated that low concentrations of SW could induce cell cyclearrest and suppress adhesive, migrative and invasive ability of GTCs. However, highconcentrations of SW triggered caspase-dependent apoptosis in GTCs via mitochondrialpathway. These findings revealed the mechanisms of SW-caused goats abortion, and prividednew insights into understanding the mechanisms of reproductive disorder caused bySW-containing plants... |
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