Abstract：
Tetramethylpyrazine (TMP) is one of the active ingredients of Chuan Xiong that has been reported to have effects on numerous diseases, including diabetic nephropathy (DN). Whereas, related molecular mechanisms are not fully elucidated. We aimed to explore circACTR2\'s role in TMP-mediated protective effects on DN. In vitro DN condition was established in human kidney cells (HK-2) by treating high glucose (HG). CCK-8 assay and flow cytometry assay were used to observe cell viability and survival. Oxidative stress was determined by the associated markers using kits. The release of inflammatory factors was detected using ELISA kits. Quantitative real-time PCR (qPCR) and western blot were utilized for expression analysis of cricACTR2, miR-140-5p, and GLI pathogenesis-related 2 (GLIPR2). The binding between miR-140-5p and circACTR2 or GLIPR2 was confirmed by dual-luciferase, RIP, and pull-down studies. HG largely induced HK-2 cell apoptosis, oxidative stress, and inflammation, which were alleviated by TMP. CircACTR2\'s expression was enhanced in HG-treated HK-2 cells but attenuated in HG + TMP-treated HK-2 cells. CircACTR2 overexpression attenuated the functional effects of TMP and thus restored HG-induced cell apoptosis, oxidative stress, and inflammation. CircACTR2 bound to miR-140-5p to enhance the expression of GLIPR2. MiR-140-5p restoration or GLIPR2 inhibition reversed the role of circACTR2 overexpression. CircACTR2 attenuated the protective effects of TMP on HG-induced HK-2 cell damages by regulating the miR-140-5p/GLIPR2 network, indicating that circACTR2 was involved in the functional network of TMP in DN.
摘要：
川雄的活性成分之一川芎嗪(TMP)已被报道对多种疾病有影响,包括糖尿病肾病(DN)。然而,相关的分子机制尚未完全阐明。我们旨在探讨circACTR2在TMP介导的DN保护作用中的作用。通过处理高葡萄糖（HG），在人肾细胞（HK-2）中建立了体外DN条件。CCK-8测定和流式细胞术测定用于观察细胞活力和存活。使用试剂盒通过相关标志物确定氧化应激。用ELISA试剂盒检测炎症因子的释放。定量实时PCR（qPCR）和蛋白质印迹用于cricACTR2，miR-140-5p的表达分析，和GLI发病机制相关2（GLIPR2）。miR-140-5p与cirACTR2或GLIPR2之间的结合通过双荧光素酶证实，RIP,和下拉研究。HG主要诱导HK-2细胞凋亡，氧化应激，和炎症，TMP缓解了这种情况。CircACTR2的表达在HG处理的HK-2细胞中增强,但在HG+TMP处理的HK-2细胞中减弱。CircACTR2过表达减弱了TMP的功能作用，从而恢复了HG诱导的细胞凋亡，氧化应激，和炎症。CircACTR2与miR-140-5p结合以增强GLIPR2的表达。MiR-140-5p恢复或GLIPR2抑制逆转了circACTR2过表达的作用。CircACTR2通过调节miR-140-5p/GLIPR2网络减弱TMP对HG诱导的HK-2细胞损伤的保护作用,表明circACTR2参与了DN中TMP的功能网络。
