Yu Ting, Wang Yun, Qian Dong, Sun Xiaomeng, Tang Yurong, Shen Xiaoxue, Lin Lin
Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Department of General Surgery, Affiliated Hospital of Nanjing University of TCM, Jiangsu Province Hospital of TCM, Nanjing, China.
Cell Physiol Biochem. 2017;43(4):1571-1587. doi: 10.1159/000482005. Epub 2017 Oct 16.
BACKGROUND/AIMS: Excessive production of advanced glycation end products (AGEs) has been implicated in diabetes-related complications. This study aimed to investigate the mechanism by which AGEs potentially contribute to diabetes-associated colonic dysmotility.
Control and streptozotocin (STZ)-induced diabetic groups were treated with aminoguanidine (AG). The colonic transit time and contractility of circular muscle strips was measured. ELISA, immunohistochemistry and western blotting were used to measure Nε-carboxymethyl-lysine (CML) levels. Primary cultured colonic smooth muscle cells (SMCs) were used in complementary in vitro studies.
Diabetic rats showed prolonged colonic transit time, weak contractility of colonic smooth muscle strips, and elevated levels of AGEs in the serum and colon tissues. cAMP levels, protein kinase-A (PKA) activities, and inositol 1,4,5-trisphosphate receptor type 3 (IP3R3) phosphorylation were increased in the colon muscle tissues of diabetic rats, whereas RhoA/Rho kinase activity and myosin phosphatase target subunit 1 (MYPT1) phosphorylation were reduced. The inhibition of the production of AGEs (AG treatment) reduced these effects. In cultured colonic SMCs, AGE-BSA treatment increased IP3R3 phosphorylation and reduced intracellular Ca2+ concentration, myosin light chain (MLC) phosphorylation, RhoA/Rho kinase activity, and MYPT1 phosphorylation. The PKA inhibitor H-89 and anti-RAGE antibody inhibited the AGE-BSA-induced impairment of Ca2+ signaling and cAMP/PKA activation.
AGEs/RAGE participate in diabetes-associated colonic dysmotility by interfering with Ca2+ signaling in colonic SMCs through targeting IP3R3-mediated Ca2+ mobilization and RhoA/Rho kinase-mediated Ca2+ sensitization via the cAMP/PKA pathway.
背景/目的：晚期糖基化终产物（AGEs）的过量产生与糖尿病相关并发症有关。本研究旨在探讨AGEs可能导致糖尿病相关性结肠运动障碍的机制。
将对照组和链脲佐菌素（STZ）诱导的糖尿病组用氨基胍（AG）治疗。测量结肠转运时间和环形肌条的收缩性。采用酶联免疫吸附测定（ELISA）、免疫组织化学和蛋白质印迹法检测Nε-羧甲基赖氨酸（CML）水平。原代培养的结肠平滑肌细胞（SMCs）用于补充体外研究。
糖尿病大鼠结肠转运时间延长，结肠平滑肌条收缩性减弱，血清和结肠组织中AGEs水平升高。糖尿病大鼠结肠肌肉组织中cAMP水平、蛋白激酶A（PKA）活性和1,4,5-三磷酸肌醇受体3型（IP3R3）磷酸化增加，而RhoA/Rho激酶活性和肌球蛋白磷酸酶靶向亚基1（MYPT1）磷酸化降低。抑制AGEs的产生（AG治疗）可减轻这些影响。在培养的结肠SMCs中，AGE-牛血清白蛋白（AGE-BSA）处理增加IP3R3磷酸化并降低细胞内Ca2+浓度、肌球蛋白轻链（MLC）磷酸化、RhoA/Rho激酶活性和MYPT1磷酸化。PKA抑制剂H-89和抗RAGE抗体抑制AGE-BSA诱导的Ca2+信号传导损伤和cAMP/PKA激活。
AGEs/RAGE通过cAMP/PKA途径靶向IP3R3介导的Ca2+动员和RhoA/Rho激酶介导的Ca2+致敏，干扰结肠SMCs中的Ca2+信号传导，从而参与糖尿病相关性结肠运动障碍。
