Division of Health and Applied Science (Biochemistry), Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla 90110, Thailand.
Center for Natural Rubber Latex Biotechnology Research and Innovation Development, Prince of Songkla University, Hat-Yai, Songkhla 90110, Thailand.
Molecules. 2024 May 2;29(9):2113. doi: 10.3390/molecules29092113.
Edible grey oyster mushroom, , β (1,3), (1,6) glucan possesses a wide range of biological activities, including anti-inflammation, anti-microorganism and antioxidant. However, its biological activity is limited by low water solubility resulting from its high molecular weight. Our previous study demonstrated that enzymatic hydrolysis of grey oyster mushroom β-glucan using β-1,3-glucanase isozymes obtains a lower molecular weight and higher water solubility, glucanoligosaccharide (Ps-GOS). Additionally, Ps-GOS potentially reduces osteoporosis by enhancing osteoblast-bone formation, whereas its effect on osteoclast-bone resorption remains unknown. Therefore, our study investigated the modulatory activities and underlying mechanism of Ps-GOS on Receptor activator of nuclear factor kappa-Β ligand (RANKL) -induced osteoclastogenesis in pre-osteoclastic RAW 264.7 cells. Cell cytotoxicity of Ps-GOS on RAW 264.7 cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and its effect on osteoclast differentiation was determined by tartrate-resistant acid phosphatase (TRAP) staining. Additionally, its effect on osteoclast bone-resorptive ability was detected by pit formation assay. The osteoclastogenic-related factors were assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), Western blot and immunofluorescence. The results revealed that Ps-GOS was non-toxic and significantly suppressed the formation of mature osteoclast multinucleated cells and their resorption activity by reducing the number of TRAP-positive cells and pit formation areas in a dose-dependent manner. Additionally, Ps-GOS attenuated the nuclear factor kappa light chain-enhancer of activated B cells' P65 (NFκB-P65) expression and their subsequent master osteoclast modulators, including nuclear factor of activated T cell c1 (NFATc1) and Fos proto-oncogene (cFOS) via the NF-κB pathway. Furthermore, Ps-GOS markedly inhibited RANK expression, which serves as an initial transmitter of many osteoclastogenesis-related cascades and inhibited proteolytic enzymes, including TRAP, matrix metallopeptidase 9 (MMP-9) and cathepsin K (CTK). These findings indicate that Ps-GOS could potentially be beneficial as an effective natural agent for bone metabolic disease.
食用灰树花，β（1,3），（1,6）葡聚糖具有广泛的生物活性，包括抗炎、抗微生物和抗氧化。然而，由于其分子量高，其生物活性受到低水溶性的限制。我们之前的研究表明，使用β-1,3-葡聚糖酶同工酶对灰树花β-葡聚糖进行酶解可获得低分子量和更高的水溶性，即葡寡糖（Ps-GOS）。此外，Ps-GOS 通过增强成骨细胞-骨形成有可能减少骨质疏松症，但其对破骨细胞-骨吸收的影响尚不清楚。因此，我们的研究调查了 Ps-GOS 对前破骨细胞 RAW 264.7 细胞中核因子 kappa-B 配体（RANKL）诱导的破骨细胞发生的调节活性及其潜在机制。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐（MTT）测定法测定 Ps-GOS 对 RAW 264.7 细胞的细胞毒性，并通过抗酒石酸酸性磷酸酶（TRAP）染色测定其对破骨细胞分化的影响。此外，通过陷窝形成测定法检测其对破骨细胞骨吸收能力的影响。通过定量逆转录聚合酶链反应（qRT-PCR）、Western blot 和免疫荧光测定法评估破骨细胞生成相关因子。结果表明，Ps-GOS 无毒，可显著抑制成熟破骨细胞多核细胞的形成及其在核因子 kappa-B（NF-κB）途径中通过减少 TRAP 阳性细胞数量和陷窝形成面积的方式抑制其吸收活性。此外，Ps-GOS 减弱了核因子 kappa-B 轻链增强子的 B 细胞 P65（NFκB-P65）表达及其随后的主要破骨细胞调节剂，包括激活 T 细胞核因子 c1（NFATc1）和 Fos 原癌基因（cFOS）。此外，Ps-GOS 显著抑制 RANK 表达，RANK 表达作为许多破骨细胞发生相关级联的初始递质，并抑制蛋白水解酶，包括 TRAP、基质金属蛋白酶 9（MMP-9）和组织蛋白酶 K（CTK）。这些发现表明，Ps-GOS 可能作为一种有效的天然骨代谢疾病治疗药物具有潜在益处。
