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Sci Rep
2016 Dec 15;6:38693. doi: 10.1038/srep38693.
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Tissue Extract Fractions from Starfish Undergoing Regeneration Promote Wound Healing and Lower Jaw Blastema Regeneration of Zebrafish.
Dai Y
,
Prithiviraj N
,
Gan J
,
Zhang XA
,
Yan J
.
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Natural bioactive materials provide an excellent pool of molecules for regenerative therapy. In the present study, we amputate portions of the arms of Archaster typicus starfish, extract and separate the active biomaterials, and compare the effects of each fraction on in vitro wound healing and in vivo lower jaw regeneration of zebrafish. Compared with crude extract, normal hexane fractions (NHFs) have a remarkable effect on cellular proliferation and collective migration, and exhibit fibroblast-like morphology, while methanol-water fractions (MWFs) increase cell size, cell-cell adhesion, and cell death. Relative to moderate mitochondrialand lysosomal aggregation in NHFs-cultured cells, MWFs-cultured cells contain more and bigger lysosomal accumulations and clump detachment. The in vivo zebrafish lower jaw regeneration model reveals that NHFs enhance blastema formation and vasculogenesis, while MWFs inhibit fibrogenesis and induce cellular transformation. Gene expression analyses indicate that NHFs and MWFs separately activate blastema-characteristic genes as well as those genes-related to autophagy, proteasome, and apoptosis either during cell scratch healing or ganciclovir-induced apoptosis. Our results suggest that bioactive compounds from NHFs and MWFs could induce blastema formation and remodeling, respectively, and prevent tissue overgrowth.
Figure 1. Anatomical features and histological processes in A. typicus arm regeneration.(a) Vertical sections of intact arm; (b) wound healing; (c) formation of blastema-like structure. a2–c2 are magnifications of a1–c1, respectively. Cc, coelomic canal; CE, coelomic epithelium; Cg, coelomic granule elements; ctf, constricted tube foot or coelomic canal; DCT, dense connective tissue; Ep, epidermis; LCT, loose connective tissue; Os, ossicle; rEP, regenerating epidermis; rtf, regenerating tube foot; Sp, spine epidermis; tf, tube foot; WE, wound epidermis. Scale bars: 250 μm (a1–c1) and 50 μm (a2–c2).
Figure 3. Fluorescence images of mitochondrion and lysosome staining.PAC2 cellswere exposed to the extracts (100 μg/ml) or the same volume of DMSO. (a) DMSO control, (b) NHFs treatment, (c) MWFs treatment. Mitochondria and lysosomes are stained green and red, respectively. Insets show magnification of individual cells. Scalebar: 50 μm.
Figure 4. Analyses of gene expression in response to wound healing.(a–d) Show PAC2 cell wound healing, as determined by in vitro scratch assay; (e) shows lower jaw wound healing at 2 dpa. The autophagy-proteasome pathway genes were largely activated (a), and all tested apoptosis genes were down regulated (b). Further, the genes associated with the mTOR and TGF-beta pathways were selectively activated (c,d). (e) Effects of MWFs and NHFs on gene expression during zebrafish wound healing at 2 days after amputation of the lower jaw. Same statistical analyses as in Fig. 2. Significance between NHFs vs. MWFs is also labeled.
Figure 5. Starfish biomaterials affect wound epidermis reconstitution and blastema formation of the zebrafish lower jaw at 5 dpa.The upper row shows HE staining, while the bottom row shows the Flk-GFP signal in flk1 promoter-driven GFP transgenic zebrafish. The dotted red line demarcates the blastema region. The blue circle indicates cell clusters or presumed chondrogenic centers. The dotted blue circle shows chondrocytes. WE, wound epidermis; Md, mandible; Mc, Meckel’s cartilage. Scale bars: 100 μm.
Figure 6. A hypothesized model of NHFs and MWFs acting in blastema regeneration.Wounding stimulates autolysis and tissue repair via lysosome and mitochondrion axis mechanisms, followed by blastema formation and remodeling. During the regenerative processes, TGF-β signaling systems modulate the autophagy, proteasome, and apoptosis pathways to control cell phenotypes, including cell proliferation, migration, and transformation. With the involvement of tissue-specific factors, wound repairs go through blastema formation and tissue remodeling. NHFs and MWFs, as a cocktail of multiple bioactive materials, act at the multiple regenerative steps in different ways. Generally, NHFs promote proteasome-autophagy toward blastema formation, while MWFs inducein complete apoptosis to orchestrate blastema remodeling.
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