Homocysteine causes dysfunction of chondrocytes and oxidative stress through repression of SIRT1/AMPK pathway: A possible link between hyperhomocysteinemia and osteoarthritis - PubMed (original) (raw)

Ching-Hou Ma et al. Redox Biol. 2018 May.

Abstract

Emerging evidence has indicated that the perturbed expression of homocysteine (Hcy) may induce mitochondrial dysfunction and disturb bone metabolism. Sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK) are two critical sensors that regulate mitochondrial biogenesis and have been recognized as therapeutic targets in osteoarthritis (OA). This study was designed to test whether Hcy caused pro-osteoarthritic changes through modulation of SIRT1 and AMPK. Our results showed that administration of Hcy reduced the SIRT1/AMPK/PGC-1α signaling in chondrocytes, leading to mitochondrial dysfunction as a result of increased oxidative stress and apoptosis. Moreover, we demonstrated that the expression of NF-κB, COX-2, IL-8, and MMP-13 were elevated subsequent to inhibition of SIRT1/AMPK/PGC-1α/PPAR-γ pathway by homocysteine, thereby causing detrimental effects on chondrocytes. In the animal model of diet-induced hyperhomocysteinemia (HHcy), we observed the similar findings that SIRT1/PGC-1α/PPAR-γ cascades were downregulated with elevated MMP-13 and COX-2. Taken together, data from the current study revealed that the reduced SIRT1 by Hcy may contribute to degradative cartilage process, which provided insight into the etiology of OA.

Keywords: AMPK; Chondrocytes; Hyperhomocysteinemia; Osteoarthritis; SIRT1.

Copyright © 2018. Published by Elsevier B.V.

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Figures

Fig. 1

Fig. 1

Hcy mitigates SIRT1 expression in human chondrocytes. Human chondrocytes were treated with Hcy (100–25 μM) for 24 h (A) or treated with Hcy (100 μM) for 8, 12 or 24 h (B) followed by assessment of deacetylase activity using a commercial kit. The protein expression levels of SIRT1 (C) and quantification of the blot (D) in Hcy-stimulated chondrocytes were shown. (Data are presented as the mean ± SD of three different experiments. * p < 0.05 compared to control group).

Fig. 2

Fig. 2

Hcy represses AMPK and PGC-1α levels in human chondrocytes. Human chondrocytes were treated with Hcy (100–25 μM) for 24 h. The protein expression levels of AMPK and PGC-1α (A) and quantification of the blot (B,C) in Hcy-stimulated chondrocytes were shown. Human chondrocytes were treated with Hcy 100 μM for 24 h with SRT1720 or AICAR pretreatment. The protein expression levels of AMPK and PGC-1α (D) and quantification of the blot (E,F) in Hcy-stimulated chondrocytes were shown. (Data are presented as the mean ± SD of three different experiments. * p < 0.05 compared to control group; & p < 0.05 compared to Hcy-treated group).

Fig. 3

Fig. 3

Hcy causes mitochondrial dysfunction in human chondrocytes. Cells were treated with Hcy (100 μM) for 24 h. cDNAs, including SIRT1, AMPK and PGC-1 were transfected 48 h before Hcy stimulation. (A) ΔΨm was inspected with the signal from JC-1 fluorescence, as described previously. No treatment (right); Hct-treated cells (left). (B) Results were quantified by flow cytometry. (C) The mitochondrial copy number and (D) mitochondrial mass were tested to investigate the influence of Hcy on mitochondrial biogenesis. (Data are presented as the mean ± SD of three different experiments. *p < 0.05 compared with untreated control cells. & p < 0.05 compared to Hcy-treated group).

Fig. 4

Fig. 4

Hcy induces oxidative stress in human chondrocytes. Cells were treated with Hcy (100 μM) for 24 h. cDNAs of SIRT1, AMPK and PGC-1 were transfected for 48 h before Hcy stimulation. (B, C) (A) Fluorescent intensity of cells was measured using a fluorescence microplate reader to examine ROS concentrations. (B) Fluorescence distribution of MitoSOX is expressed as a percentage of increased intensity to quantify ROS levels in cells with Hcy. (C) SOD activity was tested using a SOD activity kit. (Data are presented as the mean ± SD of three different experiments. *p < 0.05 compared with untreated control cells. & p < 0.05 compared to Hcy-treated group).

Fig. 5

Fig. 5

Hcy increases apoptosis in human chondrocytes. Cells were treated with Hcy (100 μM) for 24 h. For overexpression, cDNAs of SIRT1, AMPK and PGC-1 were transfected for 48 h before Hcy stimulation. (A-C) Representative Western blots and quantification data showing that Hcy treatment upregulated pro-apoptotic protein (Bax) and downregulated anti-apoptotic (Bcl-2) protein as a result of inhibition of SIRT1/AMPK/PGC-1α. (D) In DPI intervention group, DPI was pretreated for 1 h before Hcy stimulation. A TUNEL assay was used for investigation of apoptosis. (Data are presented as the mean±SD of three different experiments. *p < 0.05 compared with untreated control cells. & p < 0.005 compared to Hcy-treated group).

Fig. 6

Fig. 6

Hcy induces inflammation in human chondrocytes. Cells were treated with Hcy (100 μM) for 24 h. (A-B) Representative Western blots and quantification data showing that Hcy treatment downregulated PPAR-γ expression. (C) NF-κB activity was tested using a NF-κB activity kit. (D-E) MMP-13 and IL-8 levels were tested by ELISA assay. (F, G) COX-2 levels were tested by Western blotting assay. (Data are presented as the mean ± SD of three different experiments. *p < 0.05 compared with untreated control cells. & p < 0.05 compared to Hcy-treated group).

Fig. 7

Fig. 7

Hcy causes inflammation in human chondrocytes by modulation of SIRT1/AMPK/PGC-1/PPAR-γ/NF-κB pathway Cells were treated with Hcy (100 μM) for 24 h. In PDTC intervention group, PDTC was pretreated for 1 h before Hcy stimulation. (A) NF-κB activity was tested using a NF-κB activity kit. (B, C) MMP-13 and IL-8 levels were tested by ELISA assay. (D, F) COX-2 levels were tested by Western blotting assay. (Data are presented as the mean± SD of three different experiments. *p 0 < 0.05 compared with untreated control cells. & p < 0.05 compared to Hcy-treated group).

Fig. 8

Fig. 8

Expression levels of SIRT1, PGC-1, PPAR-γ, MMP-13, COX-2 in HHcy animals. A total of 12 C57BL mice (6 in each group) were used in this study. The animals were fed with or without 1%

L

-methionine (w/w) in water for 4 months to induce HHcy. (A) The plasma Hcy levels in the control and HHCy animals. (B) Tissues from the control and the HHcy cartilage were collected for RNA expression test. SIRT1, PGC-1, PPAR-γ, MMP-13, COX-2 were tested by real-time PCR assay. (Data are presented as the mean ± SD of three different experiments. *p < 0.05 compared with untreated control.).

Fig. 9

Fig. 9

Schematic diagram of the major findings in this study.

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