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. 2017 Mar;118(3):629-639.
doi: 10.1002/jcb.25750. Epub 2016 Oct 12.

Sucrose, But Not Glucose, Blocks IL1-β-Induced Inflammatory Response in Human Chondrocytes by Inducing Autophagy via AKT/mTOR Pathway

Nazir M Khan  1 , Mohammad Y Ansari  1 , Tariq M Haqqi  1
Affiliations

Sucrose, But Not Glucose, Blocks IL1-β-Induced Inflammatory Response in Human Chondrocytes by Inducing Autophagy via AKT/mTOR Pathway

Nazir M Khan et al. J Cell Biochem. 2017 Mar.

Abstract

Pathogenesis of osteoarthritis (OA) is multifactorial but interleukin-1β (IL-1β) is known to be an important mediator of cartilage degradation. Autophagy is an essential cellular homeostasis mechanism and has been proposed to protect against cartilage degradation and chondrocyte death under pathological conditions. We investigated the role of autophagy activated by sucrose, a natural disaccharide, in suppressing inflammatory mediator's expression and cell death under pathological conditions in human chondrocytes. Autophagy activation was investigated by Western blotting for LC3 and Beclin-1, immunofluorescence staining for LC3 puncta, and measuring autophagic flux. Activation of mTOR, AKT, and P70S6K was evaluated by Western blotting. Chondrocyte apoptosis was evaluated by propidium iodide (PI) staining using flowcytometry, expression of Bax by Western blotting, gene expression by TaqMan assays and caspase 3/7 activity was measured using a luminescence-based assay. We found that sucrose-induced active autophagy in OA chondrocytes in vitro was dependent on the activation of AKT/mTOR/P70S6K signaling pathways but was independent of reactive oxygen species (ROS) production. Sucrose activated autophagy blocked IL-1β-induced apoptosis and mRNA expression of MMP-13, COX-2, and IL-6 in human OA chondrocytes. Glucose or fructose, the two metabolites of sucrose, failed to induce autophagy indicating that autophagy was specifically mediated by sucrose. In conclusion, sucrose attenuated IL-1β induced apoptosis and the expression of catabolic mediators by inducing autophagy, and the autophagy in part was mediated through the activation of AKT/mTOR/P70S6K signaling pathway in human OA chondrocytes. J. Cell. Biochem. 118: 629-639, 2017. © 2016 Wiley Periodicals, Inc.

Keywords: AUTOPHAGY; CHONDROCYTES; OSTEOARTHRITIS; SUCROSE.

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Figures

Fig. 1
Fig. 1. Sucrose is not toxic to human OA chondrocytes
(A) Human OA chondrocytes were treated with or without indicated concentration of sucrose for 24h and cells viability were analyzed using MTT assays with viability expressed relative to untreated control cells. Values are mean±SD (n=3). (B) Flow cytometric histograms of chondrocytes treated in presence or absence of sucrose and stained with PI. Cells were harvested at 24h of incubation and stained with PI. Twenty thousand cells in each group were acquired using a flowcytometer. The Sub-G1 region represents the percentage of cells undergoing apoptosis.
Fig. 2
Fig. 2. Sucrose induced autophagy in OA chondrocytes
(A) The expression of LC3-II and beclin-1 increased in a dosage dependent manner. Human OA chondrocytes were treated with 0, 10, 25, 50 and 100mM of sucrose for 24 h. LC3-II was measured by Western blotting. (B) LC3-II and beclin-1 increased in a time dependent manner. Chondrocytes were treated with 100mM sucrose for 0, 6, 12 and 24h. (C) LC3-II puncta increased after sucrose treatment. LC3 immunostaining in chondrocytes was performed at 24h after sucrose treatment (100mM). Significant increased green bright puncta showed the formation of autophagosome. (D) LC3 immunostaining in GFP-LC3 transfected chondrocytes treated with or without sucrose (100mM) for 24h and LC3-II puncta were visualized by confocal microscopy. (E) The mRNA expression of beclin-1 was up-regulated in sucrose treated chondrocytes (100mM, 24h). At the end of treatment chondrocytes were harvested, total RNA were isolated and mRNA expression were quantified using sybergreen qPCR assay. Expression of β-actin was used as endogenous expression control. *P≤ 0.05 compared control group, n=3
Fig. 3
Fig. 3. Sucrose induced autophagic flux in human OA chondrocytes
Human OA chondrocytes were left untreated or were treated with 100mM sucrose for 24h, with or without chloroquine or ammonium chloride. LC3-II was measured by Western blotting. Values are the mean± SD of 2 individual experiments.
Fig. 4
Fig. 4. Sucrose induced autophagy was independent of ROS
(A-B) DCFH2-DA (200µM, 0.5h) stained chondrocytes were treated with sucrose for 0.5 h at 37°C and twenty thousand cells in each group were acquired using a flowcytometer. Flow cytometric histograms (A) and corresponding bar graph showing DCF+ve cells were analyzed using FlowJo software. (C) DHR123 staining (5µM, 0.5h) staining was performed to estimate mitochondrial ROS levels induced by treatment with sucrose (100mM, 0.5h). Flow cytometric histograms showing DHR+ve cells were analyzed using FlowJo software.
Fig. 5
Fig. 5. Sucrose induced autophagy via activation of AKT/mTOR pathways
The phosphorylation of mTOR (Ser2448), AKT(Ser473) and P-70S6K was downregulated by sucrose treatment in a dosage dependent manner. Human OA chondrocytes were treated with 0, 25, 50 and 100mM of sucrose for 24 h and whole cell lysate were prepared and phosphorylation of mTOR, AKT and P-70S6K was measured by Western blotting.
Fig. 6
Fig. 6. Sucrose inhibit IL-1β induced apoptosis by in human OA chondrocytes
(A) Chondrocytes were treated with IL-1β (10ng/ml) in presence and absence of sucrose (100mM) for 24h. The expression of LC3-II slightly increased after IL-1β treatment, while it dramatically up-regulated in sucrose+IL-1β group. (B-C) Sucrose pretreatment protects chondrocytes apoptosis induced by IL-1β (10ng/ml, 48 h). Flow cytometric histograms and corresponding bar graph of sucrose treated cells stimulated with IL-1β. Cells were harvested at end of 48 h and stained with PI. Twenty thousand cells in each group were acquired using a flowcytometer. The Sub-G1 region represents the percentage of cells undergoing apoptosis. Each bar shows the mean±SD from two replicates, and two such independent experiments were carried out. *p≤0.05, compared to control, and #p≤0.05, compared to IL-1β treated group.(D)The findings of (B-C) were confirmed by the data of MTT assay. Viability was expressed relative to untreated control cells. *p≤0.05, compared to control, and #p≤0.05, compared to IL-1β treated group. Values are mean±SD (n=3).(E) Sucrose pretreatment inhibits caspases activation by IL-1β in chondrocytes. Activation of caspase-3/7 in response to IL-1β treatment was detected by luminescence measurement using caspase-glo 3/7 assay. *p≤0.05, compared to control, and #p≤0.05, compared to IL-1β treated group. Values are mean±SD (n=3). (F) Sucrose pretreatment suppresses the activation of pro-apoptotic protein in response to IL-1β in chondrocytes. Chondrocytes were treated with IL-1β (10ng/ml) in presence and absence of sucrose (100mM) for 24h and expression of Bax was measured by Western blotting.
Fig. 7
Fig. 7. Sucrose exert anti-apoptotic effects via induction of autophagy in human OA chondrocytes
(A) Bafilomycin (100nM) or 3-MA (5mM) was used to inhibit chondrocytes autophagy which was induced by sucrose (100mM). The expression of LC3-II drastically increased in sucrose+bafilomycin group, while it decreased in sucrose+3-MA group. (B) Inhibition of sucrose induced autophagy abolished its cytoprotective effects in preventing chondrocyte apoptosis. Chondrocytes viability decreased after the inhibition of sucrose induced autophagy by pretreatment with 3-MA. The cell viability was determined by MTT assay.*p≤0.05, compared to control, #p≤0.05, compared to IL-1β group and $p≤0.05, compared to sucrose+ IL-1β group. Values are represented as mean±SD (n=3).
Fig. 8
Fig. 8. Sucrose inhibited the expression of catabolic molecules in human OA chondrocytes
(A) Human OA chondrocytes were pretreated with or without sucrose (100mM) for 2h and then stimulated in the absence or presence of IL-1β (10ng/ml) for 24h. At the end of treatment chondrocytes were harvested, total RNA were isolated treated and mRNA expression were quantified using TaqMan assay. Expression of β-actin was used as endogenous expression control. (B). Sucrose inhibited the production of PGE2 in response to IL-1β (10ng/ml) as measured by ELISA in the culture supernatant of sucrose treated OA chondrocytes. (C) Inhibition of sucrose induced autophagy by pretreatment with 3-MA significantly abrogated sucrose mediated suppression of MMP-13, IL-6 and COX-2 in OA chondrocytes.*P≤ 0.05 compared to control group, #p≤0.05, compared to IL-1β group, $p≤0.05, compared to sucrose+ IL-1β group. n=3
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