Melatonin Prevents Chondrocyte Matrix Degradation in Rats with Experimentally Induced Osteoarthritis by Inhibiting Nuclear Factor-κB via SIRT1

Abstract

Osteoarthritis (OA) is a common degenerative joint disease characterized by an imbalance of cartilage extracellular matrix (ECM) breakdown and anabolism. Melatonin (MT) is one of the hormones secreted by the pineal gland of the brain and has anti-inflammatory, antioxidant, and anti-aging functions. To explore the role of MT in rats, we established an OA model in rats by anterior cruciate ligament transection (ACLT). Safranin O-fast green staining showed that intraperitoneal injection of MT (30 mg/kg) could alleviate the degeneration of articular cartilage in ACLT rats. Immunohistochemical (IHC) analysis found that MT could up-regulate the expression levels of collagen type II and Aggrecan and inhibit the expression levels of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), and ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS-4) in ACLT rats. To elucidate the mechanism of MT in protecting the ECM in inflammatory factor-induced rat chondrocytes, we conducted in vitro experiments by co-culturing MT with a culture medium. Western blot (WB) showed that MT could promote the expression levels of transforming growth factor-beta 1 (TGF-β1)/SMAD family member 2 (Smad2) and sirtuin 2-related enzyme 1 (SIRT1) and inhibit the expression of levels of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibi-tor (p-p65) and phosphorylated IκB kinase-α (p-IκBα). In addition, WB and real-time PCR (qRT-PCR) results showed that MT could inhibit the expression levels of MMP-3, MMP-13, ADAMTS-4, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in chondrocytes induced by interleukin-1β (IL-1β), and up-regulate the expression of chondroprotective protein type II collagen. We found that in vivo, MT treatment protected articular cartilage in the rat ACLT model. In IL-1β-induced rat chondrocytes, MT could reduce chondrocyte matrix degradation by up-regulating nuclear factor-kB (NF-κB) signaling pathway-dependent expression of SIRT1 and protecting chondrocyte by activating the TGF-β1/Smad2 pathway.

Keywords: NF-κB pathway; SIRT1; TGF-β1/Smad2 pathway; chondrocytes; melatonin.

Figure 1

The circulating levels of melatonin (MT) and inflammatory factors in the serum of rats in each group. (A–E) Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the circulating levels of MT, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and tumor necrosis factor-alpha (TNF-α) in the serum of each group. All results represent mean ± standard deviation (SD) (n = 3), * p < 0.05, ** p < 0.01 (compared to the model group). MT group: 30 mg/kg/2d MT.

Figure 2

Melatonin (MT) inhibited chondrocyte matrix degradation in the rat anterior cruciate ligament transection (ACLT) model. (A) The surface of cartilage in each group was observed by Safranin O staining 12 weeks after the operation and scored by Mankin score. (B) Immunohistochemistry (IHC) staining and quantification of IHC staining for matrix metalloproteinase-3 (MMP-3), Aggrecan, matrix metalloproteinase-13 (MMP-13), ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS-4) and collagen type II. * p < 0.05, ** p < 0.01 (compared to the model group).

Figure 3

Melatonin (MT) increased chondrocyte viability and up-regulated sirtuin 2-related enzyme 1 (SIRT1) expression in interleukin 1-beta (IL-1β)-treated chondrocytes. (A) The effect of MT on the activity of rat chondrocytes for 24 and 48 h. (B) The transcription level of SIRT1 messenger RNA (mRNA) was detected by real-time PCR (qRT-PCR). (C,D) Western blot (WB) to detect the expression level of SIRT1 protein. Gray values were analyzed with ImageJ. All values represent mean ± standard deviation (SD) (n = 3), * p < 0.05, ** p < 0.01 (compared with the model group).

Figure 4

Melatonin (MT) inhibited the nuclear factor κB (NF-κB) signaling and activated the transforming growth factor-beta 1 (TGF-β1)/SMAD family member 2 (Smad2) pathways in interleukin 1-beta (IL-1β)-treated chondrocytes. (A,B) Western blot (WB) was used to detect protein expression levels of p65, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (p-p65), alpha (IκBα), p-IκBα, TGFβ1, and Smad2. * p < 0.05, ** p < 0.01 (compared to the model group).

Figure 5

Melatonin (MT) inhibited the degradation of the interleukin 1-beta (IL-1β)-induced chondrocyte extracellular matrix. (A,C) Western blot (WB) detected the expression levels of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS-4), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and collagen II. (B,D) Real-time PCR (qRT-PCR) detected the transcript levels of MMP-3, MMP-13, ADAMTS-4, COX-2, and collagen II. * p < 0.05, ** p < 0.01 (compared to the model group).

Figure 6

EX527 abrogated melatonin (MT)-induced sirtuin 2-related enzyme 1 (SIRT1) activation in interleukin 1-beta (IL-1β)-treat chondrocytes. (A) Western blot (WB) was used to detect the expression level of SIRT1 protein. (B) Transcription levels of SIRT1 messenger RNA (mRNA) were detected by real-time PCR (qRT-PCR). (C) Immunofluorescence (IF) staining was used to detect the number of SIRT1-positive chondrocytes in each group. ** p < 0.01 (compared to the model group). EX527: Classic SIRT1 inhibitor; DAPI: 4’,6-diamino-2-phenylindole.

Figure 7

Effects of EX527 on the nuclear factor kappa B (NF-κB) and transforming growth factor beta (TGF-β)/SMAD family member 2 (Smad2) pathways in melatonin (MT)-treated interleukin 1-beta (IL-1β)-treated chondrocytes. (A,B) Western blot (WB) was used to detect the protein expression levels of p65, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor(p-p65), alpha (IκBα), p-IκBα, transforming growth factor beta (TGF-β1), and Smad family member 2 (Smad2). (C,D) Immunofluorescence (IF) staining was used to check the nuclear entry of p65 and Smad2 in each group. * p < 0.05, ** p < 0.01 (compared to the model group).

Figure 8

EX527 abrogated the inhibitory influence of MT on IL-1β-induced chondrocyte matrix degradation. (A,C) Western blot (WB) was used to detect the levels of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS-4), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and type II collagen. (B,D) Real-time PCR (qRT-PCR) was used to detect the levels of MMP-3, MMP-13, ADAMTS-4, COX-2, and type II collagen. (E) The intensity of collagen type II alpha 1 chain (COL2A1) in each group was detected by IF staining. ** p < 0.01 (compared to the model group).

Figure 9

Interaction between melatonin (MT)/sirtuin 2-related enzyme 1 (SIRT1)/nuclear factor kappa B (NF-κB)/transforming growth factor-beta 1 (TGF-β1)/SMAD family member 2 (Smad2).