|摘要: || 糖尿病患者因體內葡萄糖代謝異常，導致細胞膜上之高度醣化終產物受器 (receptor for advanced glycation endproduct, RAGE) 活化，增加糖尿病併發症之機率。近期研究指出，sRAGE (soluble RAGE) 透過抑制 RAGE 下游訊息活化而減緩糖尿病症狀，被認為是具改善糖尿病之潛力蛋白。sRAGE 分為 esRAGE (endogenous secretory RAGE) 與 cRAGE (cleaved RAGE)，其生成作用皆受鈣離子調控。而鈣離子通道 TRPC (canonical transient receptor potential channels) 被發現與胞內氧化壓力有密切關係，亦與糖尿病併發症有重要關連性，但其於糖尿病中所扮演之角色仍未十分明瞭。研究指出甘草其代謝後之主要成分為甘草次酸 (18β-glycyrrhetinic acid, 18βGA)，具有良好之抗潰瘍、抗發炎與改善糖尿病等生理活性。因此，本研究以高糖 (25 mM) 培養人類單核球 THP-1 細胞，探討高糖下鈣離子對 esRAGE 與 cRAGE 釋放作用之調節機制，及介入 18βGA 分析其於此機制中之影響。|
結果發現，高糖誘發 TRPC3 和 TRPC6 基因與蛋白質表現升高，並藉由提升胞內鈣離子濃度，促進酵素 Nox2 (NADPH oxidase 2) 與 iNOS (inducible nitric oxide synthase) 蛋白表現，並抑制粒線體抗氧化防禦蛋白 UCP2 (uncoupling protein 2) 之表現，造成胞內 ROS (reactive oxygen species) 超載，因而降低 esRAGE 釋放量。而高糖引起之胞內高鈣離子濃度，促進培養初期主導 cRAGE 剪切釋放之 ADAM10 (a disintegrin and metalloprotease 10) 蛋白表現 (≦ 6 h)；但持續維持胞內高鈣離子濃度，卻使得後期 ADAM10 蛋白表現趨於下降 (≧ 12 h)，以致測得較低之 sRAGE 與 cRAGE 釋放量。透過抑制 TRPC3 或 TRPC6 可減緩部分胞內鈣離子濃度，有效提升高糖下 sRAGE 分泌量降低之現象，顯示 TRPC 參與 sRAGE 生成作用。18βGA 透過抑制高糖下 TRPC3 與 TRPC6 通道開啟，降低胞內鈣離子濃度，進而減緩 Nox2 與 iNOS 酵素活化，並提升粒線體內 UCP2 蛋白表現，因而減少胞內 ROS 生成量與恢復 esRAGE 之釋放量；此外，18βGA 亦能降低發炎現象而增加 cRAGE 分泌量。綜合上述，18βGA 藉由減緩 TRPC 通道活化，而提升 sRAGE 總分泌量，顯示甘草具有改善糖尿病及其併發症之潛力。
Impairment of blood glucose is a major cause of receptor for advanced glycation endproducts (RAGE) activation, increasing the probability of diabetic complications. Recent studies have suggested that soluble RAGE (sRAGE) improved diabetes mellitus by down-regulation of RAGE signaling cascades. For this reason, sRAGE is considered as a potential protein for diabetes therapy. sRAGE is an isoform of RAGE, which is classified into esRAGE (endogenous secretory RAGE) and cRAGE (cleaved RAGE). The mechanisms of sRAGE secretions are regulated by calcium. Calcium channels, canonical transient receptor potential channels (TRPC), were associated with oxidative stress-activated diabetic complications. Nevertheless, the role of TRPC channels is unkown in diabetes mellitus. 18β-glycyrrhetinic acid (18βGA), which is poduced form glycyrrhizin in intestines, has been shown to possess several beneficial pharmacological activities, such as anti-ulcerative, anti-inflammatory and anti-diabetic properties. Thus, the aim of this study was to investigate the regulatory effect of 18βGA on esRAGE and cRAGE secretions via TRPC channels in high glucose (HG)-induced THP-1 cells.
The results showed that high glucose enhanced the mRNA and protein expressions of TRPC3 and TRPC6, resulting in elevation of the intracellular [Ca2+]. Moreover, high glucose increased the protein expressions of NADPH oxidase 2 (Nox2) and inducible nitric synthase (iNOS), while decreased the protein expression of uncoupling protein 2 (UCP2). The excessive intracellular reactive oxygen species (ROS) led to attenuation of esRAGE secretion in HG-induced THP-1 cells. On the other hand, elevating intracellular [Ca2+] induced the innitial protein expression of ADAM10 (≦ 6 h), while decreased the final protein expression of ADAM10 (≧ 12 h). Meanwhile, the productions of sRAGE and cRAGE were diminished. In addition, the inhibititions of TRPC3 and TRPC6 retarded the intracellular [Ca2+] to raise the levels of sRAGE. The results implied that TRPC played an important role in the production of sRAGE. 18βGA suppressed the protein expressions of TRPC3 and TRPC6 to retarded the intracellular [Ca2+]. Lower intracellular [Ca2+] decreased the protein expressions of Nox2 and iNOS, but increased the protein expression of UCP2. The intracellular ROS was also decreased, resulting in esRAGE enhancement. Moreover, 18βGA increased the level of cRAGE through inhibition of HG-induced inflammation. Taken together, our research demonstrates that 18βGA enhances the secretion of sRAGE via suppressing TRPC channels in HG-induced THP-1 cells. Therefore, 18βGA provides a potential natural complement to diabetes mellitus and its complications.
Keywords: sRAGE, TRPC, UCP2, ADAM10, 18β-glycyrrhetinic acid