广佛手多糖体外发酵降解物促NCI-H716细胞分泌GLP-1的作用及机制

钟诚; 吴宇箫; 方益虹; 周爱梅

doi:10.13386/j.issn1002-0306.2025050158

广佛手多糖体外发酵降解物促NCI-H716细胞分泌GLP-1的作用及机制

Effects and Mechanisms of Degradation Products of Citrus medica var. sarcodactylis Polysaccharides in Promoting GLP-1 Secretion from NCI-H716 Cells

摘要

摘要: 本研究旨在探讨未发酵的广佛手多糖FCP-2-1（Degradation products of FCP-2-1 from gut microbiota for 0 hours，DFPG-0）及其体外发酵8 h的降解物（Degradation products of FCP-2-1 from gut microbiota for 8 hours，DFPG-8）在NCI-H716细胞模型中促进胰高血糖素样肽-1（Glucagon-like peptide-1，GLP-1）分泌的作用及其机制。采用CCK-8试验确定DFPG-0和DFPG-8在50~200 μg/mL浓度范围内对细胞无毒性作用。随后，选择50、100和200 μg/mL浓度进行后续实验，评估DFPG-0和DFPG-8对GLP-1分泌、前胰高血糖素原（Preproglucagon，GCG）基因表达以及细胞内相关信号分子的影响。实验结果表明，DFPG-0和DFPG-8在50、100和200 μg/mL浓度下均能显著促进GLP-1分泌（P<0.05），且DFPG-8在相同浓度下的促进作用更为显著，分别提高了21.79%、10.94%和13.19%。在GCG基因表达方面，DFPG-0和DFPG-8均显著上调GCG基因的表达（P<0.05），且DFPG-8在100和200 μg/mL浓度下的效果分别比DFPG-0高出37.69%和80.24%。此外，两者还显著上调了环磷酸腺苷（Cyclic adenosine monophosphate，CAMP）、蛋白激酶A（Protein kinase A，PKA）、CAMP直接激活的交换蛋白（Exchange protein activated by CAMP，Epac）、环磷腺苷效应元件结合蛋白（CAMP-response element binding protein，CREB）、尾型同源框-2（Caudal type homeobox-2，Cdx-2）和GCG蛋白的表达，并且DFPG-8的效果均强于DFPG-0（P<0.05），分别提高了39.17%、39.41%、139.40%、45.16%、93.13%和45.34%。上述结果说明，DFPG-0和DFPG-8均具有促进细胞GLP-1分泌和提高GCG基因mRNA表达的作用，同时能显著上调CAMP、PKA、Epca、CREB、Cdx-2和GCG蛋白表达，说明广佛手多糖的体外降血糖机制可能是通过激活CAMP/PKA/GCG和CAMP/Epac/GCG通路，促进GLP-1的合成和分泌，从而发挥降血糖作用，并且体外发酵降解物的效果更为显著。

Abstract: The research was designed to examine how the unfermented FCP-2-1 (DFPG-0) and its degradation products after 8 h of in vitro fermentation (DFPG-8) stimulated GLP-1 secretion and to elucidate the underlying mechanisms using the NCI-H716 cell model. The CCK-8 test verified that DFPG-0 and DFPG-8 had been non-toxic to NCI-H716 cells within the 50~200 μg/mL concentration range. Thereafter, the study employed 50, 100, and 200 μg/mL doses in subsequent assays to assess how DFPG-0 and DFPG-8 affected GLP-1 release, GCG gene mRNA levels, and key intracellular signaling factors. Experimental data revealed that both DFPG-0 and DFPG-8 had markedly enhanced GLP-1 secretion at 50-200 μg/mL (P<0.05), with DFPG-8 demonstrating superior efficacy at the same doses, elevating GLP-1 levels by 21.79%, 10.94%, and 13.19%. In terms of GCG expression, both DFPG-0 and DFPG-8 had significantly raised mRNA levels (P<0.05), with DFPG-8 outperforming DFPG-0 at 100 and 200 μg/mL by 37.69% and 80.24%, respectively. Furthermore, DFPG-0 and DFPG-8 had both markedly enhanced the expression of cAMP, PKA, Epac, CREB, Cdx-2, and GCG proteins (P<0.05), and DFPG-8 had shown superior potency compared to DFPG-0, elevating each marker by 39.17%, 39.41%, 139.40%, 45.16%, 93.13%, and 45.34%. Collectively, the results had shown that DFPG-0 and DFPG-8 each facilitated GLP-1 secretion and elevated GCG gene expression, concurrently upregulating CAMP, PKA, Epac, CREB, Cdx-2, and GCG proteins, indicating that FCP-2-1’s in vitro glucose-lowering action was mediated via activation of the CAMP/PKA/GCG and CAMP/Epac/GCG cascades to drive GLP-1 production and secretion, with the fermented degradation products exerting a more pronounced effect.