Screening Of Natural Small-molecule Compounds Capable Of MaintainingStem Cell Pluripotency And Attenuating Endoplasmic Reticulum Stress

Pluripotent stem cells are capable of unlimited self-renewal and potential differentiation into cells of all three germ layers.For these reasons,pluripotent stem cells play an important role in fundamental research,drug discovery and regenerative medicine.However,the trend toward differentiation of stem cells in vitro culture creates problems for their large scale production as well as for their clinical applications.The maintenance of pluripotency and self-renewal in pluripotent cells relies on a complex network of transcription factors,with Oct4 playing a key role.Natural products have been recognized as rich resources for drug discovery.In this study,we constructed a firefly luciferase reporter driven by the Oct4 gene promoter to screen natural small-molecule compounds capable of maintaining the self-renewal and pluripotency of pluripotent stem cells.In primary screening assay,we found that 6(SA5,SA28,SA79,SA95,SA129 and SA138)out of 209 tested compounds could effectively enhance the promoter activity of Oct4 gene.The results of secondary screening assay showed that only SA79(EPMC)could effectively enhance Oct4 promoter activity.Further results showed that EPMC could promote the expression of Oct4 in both mRNA and protein levels.Next we investigated the effects of EPMC on stem cell self-renewal and pluripotency.The results showed that EPMC could promote the colony formation of P19 cells and UC-MSCs(umbilical cord mesenchymal stem cells),moreover,EPMC had little effect on cell proliferation,indicating that EPMC could enhance the self-renewal of P19 cells and UC-MSCs.Further results showed that the pluripotency markers Oct4,Sox2 and Nanog were expressed at higher levels in EPMC-induced colonies,suggesting that EPMC might have the ability to enhance pluripotency.So we investigated the effect of EPMC on the pluripotency of P19 cells.The results showed that the markers of entoderm(AFP),mesoderm(GATA4 and cTnT)and ectoderm(Tuj1)were expressed at higher levels in the teratomas generated by EPMC-treated P19 cells,suggesting that EPMC could enhance the pluripotency of P19 cells.Mechanistic studies showed that EPMC could significantly activate NF-?B(nuclear factor kappa B)signaling pathway.EPMC-induced up-regulation of Oct4 was reversed after blocking NF-?B signaling pathway with PDTC or p65 shRNA.These results suggested that NF-?B signaling pathway was essential to EPMC-induced Oct4 expression,and EPMC promoted Oct4 expression at least partially through NF-?B signaling pathway.TRAF6(TNF receptor associated factor 6)is a key activator of NF-?B.Co-immunoprecipitation results showed that EPMC could activate TRAF6.Previous results revealed that TRAF6 couldintegrate upstream signals from the members of the TNFR(TNF receptor)family and MyD88(myeloid differentiation factor 88)-dependent TLR(toll-like receptor)/IL-1R(interleukin-1receptor)family resulting in NF-?B activation.Our results showed that knockdown of MyD88 by shRNA effectively suppressed EPMC-induced NF-?B activation and Oct4 expression,indicating that EPMC activated NF-?B signaling via MyD88-dependent pathway.Overall,our results showed that EPMC activated NF-?B signaling pathway via MyD88-dependent signaling,then the activated NF-?B promoted Oct4 expression,and finally the up-regulation of Oct4 enhanced self-renewal and pluripotency.This study provided a natural small molecule capable of enhancing self-renewal and pluripotency,and provided a material basis for the research and application of stem cells.Prolonged and severe ER stress leads to tissue injury and serious diseases,such as neurodegenerative disease,cancer and diabetes.Thus,it is important to identify drugs that can attenuate ER stress for disease treatment.Natural products continue to provide lead compounds for drug discovery,because they have good bioactivity and biocompatibility.Previous studies suggest that selenoprotein S(Sel S)is a sensitive and ideal maker of ER stress.In this study,we used a firefly luciferase reporter driven by the Sel S gene promoter(p Sel S-luc)to screen natural compounds capable of attenuating ER stress.In primary screening assay,we transfected the luciferase reporter constructs(pSel S-luc)into HEK293 T cells,and screened 361 purified natural compounds.The results showed that54 tested compounds could effectively inhibit Sel S promoter activity(fold induction < 1.00,P< 0.05).The results of secondary screening assay showed that paclitaxel and 25-OCH3-PPD with fold induction of 0.368 and 0.370(P < 0.05)exhibited the most obviously inhibitory effect on Sel S promoter activity.Further results revealed that paclitaxel and 25-OCH3-PPD also significantly inhibited tunicamycin-induced up-regulation of Sel S in Hep G2 and HEK293 T cells.Moreover,paclitaxel and 25-OCH3-PPD could efficiently inhibit tunicamycin and dithiothreitol induced high expression of GRP78,suggesting that paclitaxel and 25-OCH3-PPD could reverse ER stress.This study provides two natural small molecules for attenuating ER stress,and reveals new insight into the therapeutic application of paclitaxel.