Nobiletin: a potential erythropoietin receptor activator protects renal cells against hypoxia

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 Ca²⁺ mobilization, cAMP accumulation, and phosphorylation of JAK2 and STAT5 were determined to confirm the EPOR activation effect of nobiletin. CoCl₂ 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.