Hyperbaric oxygen protects mandibular condylar chondrocytes from interleukin-1β-induced apoptosis via the PI3K/AKT signaling pathway

Abstract

Objectives: Mandibular condylar chondrocyte apoptosis is mainly responsible for the development and progression of temporomandibular joint osteoarthritis (TMJ-OA). Interleukin-1β (IL-1β) generally serves an agent that induces chondrocyte apoptosis. Hyperbaric oxygen (HBO) treatment increases proteoglycan synthesis in vivo. We explore the protective effect of HBO on IL-1β-induced mandibular condylar chondrocyte apoptosis in rats and the potential molecular mechanisms. Methods: Chondrocytes were isolated from the TMJ of 3-4-week old Sprague-Dawley rats. The Cell Counting Kit-8 (CCK-8) assay was used to determine cell viability. The phosphorylated phosphoinositide-3 kinase (p-PI3K), phosphorylated AKT (p-Akt), type II collagen (COL2), and aggrecan (AGG) content was detected by immunofluorescence, immunocytochemistry and western blotting. The expression of Pi3k, Akt, Col2 and Agg mRNA was measured using real-time quantitative polymerase chain reaction (RT-qPCR). Results: HBO inhibited the cytotoxicity and apoptosis induced by IL-1β (10 ng/mL) in the mandibular condylar chondrocytes. HBO also decreased the IL-1β activity that decreased p-PI3K and p-AKT levels, and increased COL2 and AGG expression, with the net effect of suppressing extracellular matrix degradation. Conclusions: These data suggest that HBO may protect mandibular condylar chondrocytes against IL-1β-induced apoptosis via the PI3K/AKT signaling pathway, and that it may promote the expression of mandibular condylar chondrocyte extracellular matrix through the PI3K/AKT signaling pathway.

Keywords: Hyperbaric oxygen; IL-1β; PI3K/AKT signaling; extracellular matrix.

Figure 1

Identification of normal mandibular condylar chondrocytes. A: The morphology of the P2 chondrocytes was observed under a microscope. B: The immunohistochemical staining for type II collagen was positive in the chondrocytes. C: The immunofluorescence staining for type II collagen was positive in the chondrocytes.

Figure 2

Effects of HBO on IL-1β-induced chondrocyte proliferation. CCK-8 assay was used to examine the cell proliferation of each group, the absorbance was measured at 450 nm (n=5 per group). Bars represent the mean and SEM of each group. NC, normal control; IL-1β, cells with 10 ng/mL interleukin-1β; HBO, hyperbaric oxygen treated cells with 10 ng/mL interleukin-1β; H+I, hyperbaric oxygen treated cells with 10 ng/mL interleukin-1β and 25 μM LY294002 (the PI3K inhibitor). ** < 0.01, * < 0.05.

Figure 3

COL2, AGG, p-PI3K and p-AKT protein levels measured by immunohistochemistry and immunofluorescence. Comparison of the AGG, p-PI3K, p-AKT and COL2 protein levels in the different groups as determined by immunohistochemistry and immunofluorescence (n=6 per group).

Figure 4

Protein expression of COL2, AGG, PI3K, p-PI3K, AKT, and p-AKT. Western blot technique was used to examine the possible mechanism by which HBO protect chondrocytes. A: Comparison of the COL2, AGG, PI3K, p-PI3K, AKT and p-AKT protein levels in the different groups as determined by Western blot. B: Mean relative protein levels of COL2 and AGG in different groups (n=6 per group). C: p-PI3K levels were normalized to t-PI3K levels in different groups (n=6 per group). D: p-AKT levels were normalized to t-AKT levels in different groups (n=6 per group). Bars represent the mean and SEM of each group. ** < 0.01, * < 0.05.

Figure 5

The 2^-ΔΔCt method was adopted with GAPDH as the reference gene. Reverse transcription and real-time quantitative polymerase chain reaction technique was used to examine the mRNA expression COL2 and AGG in each group. A: The COL2 mRNA levels were normalized to GAPDH levels in different groups (n=6 per group). B: The AGG mRNA levels were normalized to GAPDH levels in different groups (n=6 per group). Bars represent the mean and SEM of each group. * < 0.05.