Lutein Attenuates Both Apoptosis and Autophagy upon Cobalt (II) Chloride-Induced Hypoxia in Rat Műller Cells

<div><p>Retinal ischemia/reperfusion injury is a common feature of various retinal diseases such as glaucoma and diabetic retinopathy. Lutein, a potent anti-oxidant, is used to improve visual function in patients with age-related macular degeneration (AMD). Lutein attenuates apoptosis, oxidative stress and inflammation in animal models of acute retinal ischemia/hypoxia. Here, we further show that lutein improved Műller cell viability and enhanced cell survival upon hypoxia-induced cell death through regulation of intrinsic apoptotic pathway. Moreover, autophagy was activated upon treatment of cobalt (II) chloride, indicating that hypoxic injury not only triggered apoptosis but also autophagy in our <i>in vitro</i> model. Most importantly, we report for the first time that lutein treatment suppressed autophagosome formation after hypoxic insult and lutein administration could inhibit autophagic event after activation of autophagy by a pharmacological approach (rapamycin). Taken together, lutein may have a beneficial role in enhancing glial cell survival after hypoxic injury through regulating both apoptosis and autophagy.</p></div

Number of apoptotic nuclei was attenuated in lutein-treated cells.

<p>Apoptotic nuclei were revealed by TUNEL assay after CoCl₂ treatment at different time points. <b>(A)</b> Representative images of TUNEL-positive nuclei (green) and DAPI-stained nuclei (blue) in rMC-1 cells. <b>(B)</b> Quantification of TUNEL-positive cells. n = 5 in each group. Lutein administration significantly reduced the number of TUNEL-stained nuclei. Scale bar, 100 μm. ^*<i>P</i><0.05, ^**<i>P</i>< 0.01, ^***<i>P</i><0.001 versus normal control group; ^#<i>P</i>< 0.05 versus vehicle-treated group.</p

Anti-apoptotic effects of lutein in rMC-1 cells upon CoCl₂-induced hypoxia.

<p>rMC-1 cells were exposed to CoCl₂ (300μM) with various concentration of lutein for 24 hours. <b>(A-D)</b> Protein levels of different apoptotic-related proteins including Bcl-2, Bcl-X_L, Bax, and cleaved caspase 3 were measured by Western blotting (normalized by β-actin) and quantified by densitometry. 20μM of Lutein was able to up-regulate protein expression of Bcl-2 and protein level of cleaved caspase 3 was inhibited by both 10μM and 20μM of lutein. <b>(E)</b> Densitometry analysis of ratio of Bax and Bcl-2 protein expression with different concentration of lutein. Lutein improved rMC-1 cell survival by decreasing Bax/Bcl-2 ratio. n = 5 in each group. ^*<i>P</i><0.05, ^***<i>P</i><0.001 versus normal control group; ^#<i>P</i>< 0.05, ^##<i>P</i><0.01 versus vehicle-treated group. N, Normal Control; Veh, Vehicle (0.01% DMSO).</p

The length of lutein treatment in rMC-1 cells upon CoCl₂-induced hypoxia.

<p>rMC-1 cells were exposed to CoCl₂ (300μM) with or without lutein (20μM) for different periods of time. <b>(A-D)</b> Protein levels of different apoptotic-related proteins including Bcl-2, Bcl-X_L, Bax, and cleaved caspase 3 were measured by Western blotting (normalized by β-actin) and quantified by densitometry. Lutein treatment was able to rescue cells upon CoCl₂-induced cell death by up-regulating protein expression of Bcl-2 and suppressing cleaved caspase 3 at 24 hours. <b>(E)</b> Densitometry analysis of ratio of Bax and Bcl-2 protein expression at different time points. Lutein improved rMC-1 cell survival by decreasing Bax/Bcl-2 ratio at 24 hours. n = 5 in each group. ^*<i>P</i><0.05, ^**<i>P</i>< 0.01, ^***<i>P</i><0.001 versus normal control group; ^#<i>P</i>< 0.05, ^###<i>P</i><0.001 versus vehicle-treated group.</p

Anti-autophagic property of lutein was involved in mTOR-mediated autophagy pathway.

<p><b>(A)</b> Western blotting and the densitometric analysis showed that the phosphorylated AMPK was up-regulated in CoCl₂-treated cells. <b>(B-D)</b> Protein levels of mTOR-associated proteins including P-mTOR, P-p70S6K and P-ULK1 (Ser757) were measured by Western blotting (normalized by β-actin) and quantified by densitometry. 20μM of lutein was able to restore the phosphorylation levels of P-mTOR and P-p70S6K upon CoCl₂-induced hypoxia. (E) Rapamycin was used to induce autophagy and chloroquine was also added to block the formation of autolysosome upon rapamycin-mediated autophagy. Densitometry analysis showed that LC3II protein expression was up-regulated in rapamycin-induced autophagy and accumulated in the presence of chloroquine (lane 6). Lutein treatment was able to decrease LC3II expression in rMC-1 cells upon rapamycin and chloroquine co-treatment (lane 7). n = 5 in each group. ^*<i>P</i>< 0.05, ^**<i>P<0</i>.<i>01</i>, ^***<i>P</i><0.001 versus normal control group; ^#<i>P</i>< 0.05, ^##<i>P</i><0.01 versus Lutein-treated group; ^#<i>P</i>< 0.05, rapamycin and chloroquine co-treatment group.</p

Lutein rescued rMC-1 cells from CoCl₂-induced hypoxic injury.

<p>rMC-1 cells were exposed to CoCl₂ (300μM) with or without lutein for various periods. Representative photographs of rMC-1 cells <b>(A-C)</b> normal control, <b>(D-F)</b> hypoxia with vehicle (0.01% DMSO), <b>(G-I)</b> hypoxia with lutein (20μM). <b>(J)</b> Percentage of cell viability. Treatment of lutein only without hypoxia did not affect the viability when compared with the normal control. Lutein-treated rMC-1 cells showed higher cell viability when compared with the vehicle-treated group at 24 hours. <b>(K)</b> Percentage of lactate dehydrogenase (LDH) release from damaged cells. Lutein attenuated LDH release after CoCl₂-induced injury when compared with that in vehicle-treated group at 24 hours. n = 5 in each group. ^**<i>P</i>< 0.01, ^***<i>P</i><0.001 versus normal control group; ^#<i>P</i>< 0.05, ^##<i>P</i><0.01 versus vehicle-treated group. Scale bar, 100 μm.</p

Sustained Delivery of Bioactive GDNF from Collagen and Alginate-Based Cell-Encapsulating Gel Promoted Photoreceptor Survival in an Inherited Retinal Degeneration Model - Fig 1

<p><b>Morphology of CAC ECT gel (A) without cell encapsulation and (B) with cell encapsulation (red dotted line) inside rat eyes under a surgical microscope.</b> The gel with no encapsulated cells was transparent and therefore could barely be seen. Photos were taken 1 week post-operation.</p

Concentration and bioactivity of GDNF delivered from the CAC ECT gel.

<p>(A) Box plot showing the accumulated concentration of GDNF secreted from the CAC ECT gel during the first and second week of culture. The ECT gel with cells showed a significantly higher GDNF release than the control group (p<0.045). (B) Photoreceptor survival after the implantation of ECT gel with 1x10⁴ cells (right) was compared to that of the non-treated control (left) at the central (top) and peripheral retinal regions (bottom). Better cell and spatial pattern retention in the ONL were detected after treatment with cell-containing ECT gel. Scale bar = 50μm.</p

Photoreceptor survival in dystrophic RCS/lav rats evaluated by H&E staining.

<p>(A, B) Six retinal areas studied were: peripheral, mid-central and central regions of the (a-d) superior and (e-h) inferior retina respectively. (A) H&E staining at p56 showing the retinal morphology of rats receiving: (a, e) no operation, (b, f) surgical sham operation, (c, g) ECT gel without cells, and (d, h) ECT gel with 2x10⁵ cells for 28 days. The ECT with cells group showed better-aligned and thicker ONL than the other control groups. Preservation of photoreceptors at the periphery was better than that of the central areas in all treatment groups. Straight red line denoted the inner boundary of the ONL, adjacent to the inner nuclear layer (INL). Dashed red line represented the outer boundary of the ONL, adjacent to the RPE. Transiting from the peripheral to central retinal regions, ONL cells became more dispersed and the outer ONL boundary became unclear, especially in the (b, f) sham and (g) ECT without cells groups. Scale bar = 25μm. (C) ONL count was significantly affected by both treatments received (p<0.0005) and retinal location (p<0.0005). Among the treatments received, ONL count in the ECT with cell group was significantly higher than the other treatment groups, namely ECT without cells (p = 0.006), sham (p<0.0005) and non-operated group (p<0.0005). As for the retinal location, ONL count in the peripheral retina was higher than the central retina.</p

Temporal changes in cell immobilization at different alginate concentrations evaluated by cell migration assay.

<p>1x10⁴ cell were encapsulated in a CAC matrix of (A-C) 0.7% or (D-E) 1% alginate and examined at day 0 (day of manufacturing), 7 and 28 (left to right). (A, D) The initial cell distribution was similar. (B) At day 7, cell colonies formation and mild migration of cells were observed in 0.7% alginate gels. (C) At day 28, colonies further expanded in size. Colonies located close to the outer regions of the gel or ones that had expanded to that region showed higher chances of cell migration from the gel to the surrounding (*, #). (D-F) Cell distribution in 1% alginate gel remained similar at day 0, 7 and 28. Scale Bar = 200μm.</p