Calvatia gigantea is a traditional Chinese medicinal fungus known for its diverse pharmacological effects, with polysaccharides being one of its main bioactive components. In this study, a novel homogeneous water-soluble polysaccharide (CGPA1) was successfully isolated and purified from the fruiting body of Calvatia gigantea. The molecular weight of CGPA1 was determined to be 11.69 kDa. Structural characterization was performed using a combination of high-performance gel permeation chromatography (HPGPC), Fourier-transform infrared spectroscopy (FTIR), methylation analysis, and nuclear magnetic resonance (NMR) spectroscopy. The analysis data revealed that CGPA1 is primarily composed of galactose, methyl-galactose, mannose, xylose, and glucose in a molar ratio of 60.01:27.16:10.34:1.83:0.67. The backbone structure predominantly consists of α-1,6-Gal-(1 → and α-1,6 (3-MeO)-Gal-(1→, with side chains of α-Gal and α-Man. Morphological analysis using SEM, XRD, and AFM, combined with Congo red experiments, suggesting that CGPA1 adopts an amorphous granular chain conformation. Both in vitro and in vivo tests confirmed that CGPA1 can promote the migration of MSF cells and accelerate the healing of acute injured wounds. It was found to facilitate skin re-epithelialization, and collagen deposition over the healing process.
Scaffolds mimicking the native annulus fibrosus (AF) extracellular matrix (ECM) structure are crucial to guide the seeding cells to regenerate aligned tissue, while fabricating such a scaffold by synthetic material is challengeable. Native acellular scaffolds derived from AF tissue certainly possess the advantages of natural structure and composition. Based on previous studies, we modified decellularization procedure and especially compared two drying methods, including gradient dehydration and freeze-drying. The decellularization process can effectively remove the host cells and antigens such as α-Gal, while maintaining the original ECM including GAG and collagen I. Compared with gradient dehydration, freeze-drying not only rendered the decellularized scaffold in dry state for storage but also gave the scaffold more aligned porous structure and hydrophilicity. And, the acellular porous scaffold manifested better capacity of supporting cell ingrowth when seeded human bone marrow mesenchymal stem cells (hBMSCs) or implanted in vivo. Furthermore, this optimized freeze-dried scaffold showed similar mechanical elastic modulus as native AF and demonstrated rare inflammatory granuloma and immune rejection as observed in HE staining and immunohistochemistry staining (IHC) of CD8 and MAC387 epitopes when implanted subcutaneously in vivo. To sum up, through our decellularization and freeze-drying procedure, an aligned porous three-dimensional scaffold derived from the natural AF ECM was successfully fabricated with good retention of ECM components and benign biocompatibility. It will be a promising scaffold for AF tissue engineering.
Rong T, Chunchun Z, Wei G, Yuchen G, Fei X, et al.
Biochimica et biophysica acta. Proteins and proteomics •
Protostane triterpenes in Alisma orientale (Sam.) Juz. have unique structural features with distinct pharmacological activities. Previously we have demonstrated that protostane triterpene biosynthesis could be regulated by methyl jasmonate (MeJA) induction in A. orientale. Here, proteomic investigation reveals the MeJA mediated regulation of protostane triterpene biosynthesis. In our study, 281 differentially abundant proteins were identified from MeJA-treated compared to control groups, while they were mainly associated with triterpene biosynthesis, α-linolenic acid metabolism, carbohydrate metabolism and response to stress/defense. Key enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), squalene epoxidase (SE), oxidosqualene cyclase (OSC) and cytochrome P450s which potentially involved in protostane triterpene biosynthesis were significantly enriched in MeJA-treated group. Basic Helix-loop-helix (bHLH), MYB, and GRAS transcription factors were enhanced after MeJA treatment, and they also improved the expressions of key enzymes in Mevalonate pathway and protostane triterpene. Then, MeJA also could increase the expression of α-galactosidase (α-GAL), thereby promoting carbohydrate decomposition, and providing energy and carbon skeletons for protostane triterpene precursor biosynthesis. As well, exogenous MeJA treatment upregulated 13-lipoxygenase (13-LOX), allene oxide synthase (AOS) and allene oxide cyclase (AOC) involved in α-linolenic acid metabolism, leading to the accumulation of endogenous MeJA and activation of the protostane triterpene biosynthesis transduction. Finally, MeJA upregulated stress/defence-related proteins, as to enhance the defence responses activity of plants. These results were further verified by quantitative real-time PCR analysis of 19 selected genes and content analysis of protostane triterpene. The results provide some new insights into the role of MeJA in protostane triterpene biosynthesis.