Silibinin improves L-cell mass and function through an estrogen receptor-mediated antioxidative mechanism

Jinyu Wang; Luxin Zhang; Hao Cao; Xinyi Shi; Xiaorong Zhang; Zihao Gao; Katsumi Ikeda; Tingxu Yan; Ying Jia; Fanxing Xu

doi:10.1016/j.phymed.2022.154022

Silibinin improves L-cell mass and function through an estrogen receptor-mediated antioxidative mechanism

Phytomedicine. 2022 May:99:154022. doi: 10.1016/j.phymed.2022.154022. Epub 2022 Feb 27.

Authors

Jinyu Wang¹, Luxin Zhang², Hao Cao³, Xinyi Shi², Xiaorong Zhang², Zihao Gao², Katsumi Ikeda⁴, Tingxu Yan¹, Ying Jia⁵, Fanxing Xu⁶

Affiliations

¹ Faculty of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China.
² Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China.
³ School of Life Science and Bio-pharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China; School of Medicine, Tsinghua University, Beijing 100084, P.R. China.
⁴ School of Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya 663-8179, Japan.
⁵ Faculty of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China. Electronic address: jiayingsyphu@126.com.
⁶ Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China. Electronic address: fanxing0011@163.com.

PMID: 35255283
DOI: 10.1016/j.phymed.2022.154022

Abstract

Background: Silibinin, a major component of milk thistle extract silymarin, promotes hypoglycemia by activating estrogen receptor (ER) α and β-mediated pathways in pancreatic β-cells. Glucagon-like peptide-1 (GLP-1) is the enteroendocrine peptide produced in L-cells, and it controls glucose homeostasis through multiple pathways. The effect of silibinin on L-cell mass and function is still unknown.

Purpose: The protective effect of silibinin on palmitate (PA)-treated intestinal L-cell line GLUTag cells and the SHRSP•Z-Leprfa/Izm-Dmcr (SP•ZF) diabetic rat model was investigated in current study.

Methods: After pre-incubation with 50 μM silibinin for 4 h, GLUTag cells were treated with 0.125 mM PA. MTT, Annexin V/PI apoptosis, Hoechst 33342 staining, western blot, DCFH-DA, GLP-1 ELISA, qRT-PCR and immunofluorescence analyses were undertaken to determine ER-dependent protection of silibinin against PA-induced cellular damage. The differential protein expression of GLUTag cells under different treatments was examined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The SP•ZF diabetic rat model was chosen for in vivo study. After 4 weeks of gastric gavage with 100 or 300 mg kg^-1 of silibinin, the physiological indexes of the rats were measured. Cells expressing GLP-1, 8‑hydroxy-2'-deoxyguanosine (8-OHdG), ERα, and/or ERβ in duodenum tissues were detected by immunofluorescence.

Results: The current study showed that the GLUTag cells preincubated with silibinin activated the transcription factor nuclear erythroid-2 like factor-2 (Nrf2)-antioxidant pathway, reduced reactive oxygen species (ROS) generation, and improved cell survival and GLP-1 content, while the antioxidative effect of silibinin was blocked by the selective ERα antagonist MPP or ERβ antagonist PHTPP in GLUTag cells. Our proteomics data further revealed that ERα or β inactivation reduced glutathione peroxide and proteins associated with endocytosis and reproduction, thus at least partially reversing the protective effect of silibinin. SP•ZF rats received silibinin treatment showed increased serum GLP-1 content and improved glucose homeostasis. Furthermore, silibinin upregulated ERα and β levels and reduced the level of 8-OHdG in GLP-1-positive cells.

Conclusions: Our study showed that silibinin improved L-cell mass and function through an ER-mediated antioxidant pathway, and the proteomics analysis revealed for the first time the differential regulation of proteins by PA and silibinin in GLUTag cells.

Keywords: Estrogen receptors; GLP-1; L-cells; ROS.