Berberine Acts As A Novel Autophagy Blocker To Protect Human müLler Cell From 4-hne Induced Cell Death

Abstract

Purpose: : 4-Hydroxynonenal (4-HNE, a component of oxidized lipoproteins) may mediate retinal injury and promote diabetic retinopathy (DR). Berberine (BBR) has favorable effects on glucose and lipid metabolism in animal and human clinical studies, but its effects on retinal cells exposed to modified lipoproteins (as occurs in DR) are unknown. Using 4-HNE to model effects of oxidized lipids and lipoproteins on retinal cells in DR, we investigated the potential protective effects of BBR.

Methods: : Confluent human retinal Müller cells were exposed to 4-HNE (5, 10, 20, or 40μM) for various periods (1h, 3h, 6h, 12h, or 24h) with/without BBR pretreatment (1, 5, 10, or 20μM for 1h). Cell viability wasdetected by CCK-8 assay. To investigate mechanisms of the potential protective effects of BBR, cells were pretreated with BBR (5μM, 1h) prior to 4-HNE treatment. Indices of autophagy and apoptosis were measured by western blot and immunocytochemistry (ICC).

Results: : 4-HNE induced dose- and time-dependent cell death (CCK-8 assay, n=3, p<0.001), which was partially attenuated by BBR pretreatment. Western blot and ICC demonstrated that 4-HNE induced markers of autophagy (LC3B) and apoptosis (PARP, BAX/BCL2 ratio, and thereafter, increased TUNEL positive cells). Pretreatment with BBR attenuated the over-expression of LC3B induced by 4-HNE. Such attenuation was also observed in cells pretreated with 3-Methyladenine (3-MA, an inhibitor of autophagy, as positive control) (5mM, 2hr). However, pretreatment with BBR did not inhibit apoptotic markers induced by 4-HNE.

Conclusions: : 4-HNE induced human retinal Müller cell death through both autophagy and apoptosis pathways. BBR attenuated autophagy, but had no effect on apoptosis. BBR may act as a novel inhibitor of autophagy, and has potential to prevent Müller cell death and inhibit DR.

Keywords: diabetic retinopathy