Liu Na, Sun Yuzhuo, Liu Jieyun, Zhang Yangyang, Yi Xinyao, Yan Wenhui, Cui Xin, Guo Tingli, Zhao Wenzhuo, Han Shengli, Ma Weina, Cao Yapeng, Chen Lina
Department of Pharmacology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an, 710061, China.
School of Pharmacy, Xi'an Jiaotong University Health Science Center, Xi'an, 710061, China.
Apoptosis. 2025 Apr;30(3-4):842-860. doi: 10.1007/s10495-024-02067-9. Epub 2025 Jan 4.
Tangerine peel is a traditional Chinese herb and has been widely applied in foods and medicine for its multiple pharmacological effects. Erythropoietin receptor (EPOR), a member of the cytokine receptor family, is widely expressed in multiple tissues in especial kidney and plays protective effects in adverse physiological and pathological conditions. We hypothesized that it might be EPOR agonists existing in Tangerine peel bring such renal benefits. To test our hypothesis, an EPOR/cell membrane chromatography (CMC)-high performance liquid chromatography (HPLC)-mass spectrometry (MS) analytical system was developed to screen EPOR targeted compounds from tangerine peel extra out. A fraction was retained on the EPOR/CMC column, separated, and further identified as nobiletin. Frontal analysis, non-linear chromatography, and molecular docking assay were applied to determine the binding force and sites between nobiletin and EPOR. Intracellular Ca mobilization, cAMP accumulation, and phosphorylation of JAK2 and STAT5 were determined to confirm the EPOR activation effect of nobiletin. CoCl was applied to construct a renal hypoxic cell model, and cell viability and apoptosis of human glomerular mesangial cells (HMC) were carried out to assess the pharmacological effect of nobiletin. Apoptosis-related proteins including Bcl-2, Bcl-xL, Bax, Cleaved caspase 3, caspase 3, caspase 9, and Cytochrome C were determined. SiRNA and lentivirus were used to silence or overexpress EPOR. Our results indicated that nobiletin is a potential EPOR agonist, reflected on its explicit binding force and downstream signal activating effects. Furthermore, EPOR-overexpressing enhanced the hypoxia-tolerance of renal cells. Our mechanism research indicated that the protective effect of nobiletin against hypoxia was depended on its pro-proliferation and anti-apoptosis effects. In conclusion, nobiletin, a potential small molecular agonist of EPOR, protects HMC against hypoxia through positively activating EPOR.
陈皮是一种传统的中药材，因其多种药理作用而被广泛应用于食品和医药领域。促红细胞生成素受体（EPOR）是细胞因子受体家族的成员之一，在多个组织中广泛表达，尤其是在肾脏中，并在不良生理和病理条件下起保护作用。我们推测可能是陈皮中存在的EPOR激动剂带来了这种肾脏益处。为了验证我们的假设，开发了一种EPOR/细胞膜色谱（CMC）-高效液相色谱（HPLC）-质谱（MS）分析系统，以从陈皮提取物中筛选EPOR靶向化合物。一个馏分保留在EPOR/CMC柱上，进行分离，并进一步鉴定为川陈皮素。应用前沿分析、非线性色谱和分子对接试验来确定川陈皮素与EPOR之间的结合力和结合位点。测定细胞内钙动员、cAMP积累以及JAK2和STAT5的磷酸化，以确认川陈皮素的EPOR激活作用。应用氯化钴构建肾脏缺氧细胞模型，并检测人肾小球系膜细胞（HMC）的细胞活力和凋亡情况，以评估川陈皮素的药理作用。测定凋亡相关蛋白，包括Bcl-2、Bcl-xL、Bax、裂解的半胱天冬酶3、半胱天冬酶3、半胱天冬酶9和细胞色素C。使用小干扰RNA（SiRNA）和慢病毒来沉默或过表达EPOR。我们的结果表明，川陈皮素是一种潜在的EPOR激动剂，体现在其明确的结合力和下游信号激活作用上。此外，过表达EPOR增强了肾细胞的耐缺氧能力。我们的机制研究表明，川陈皮素对缺氧的保护作用取决于其促增殖和抗凋亡作用。总之，川陈皮素作为一种潜在的EPOR小分子激动剂，通过正向激活EPOR保护HMC免受缺氧影响。
