IL-1β induces apoptosis and autophagy via mitochondria pathway in human degenerative nucleus pulposus cells

Abstract

IL-1β has been reported highly expressed in degenerative intervertebral disc, and our previous study indicated IL-1β facilitates apoptosis of human degenerative nucleus pulposus (NP) cell. However, the underlying molecular mechanism remains unclear. We here demonstrate that IL-1β played a significantly pro-apoptotic effect under serum deprivation. IL-1β decreased Bcl-2/Bax ratio and enhanced cytochrome C released from mitochondria to cytosol, which proved mitochondria-meidated apoptosis was induced. Subsequently, mitochondria damage was detected under IL-1β stimualtion. In addition, IL-1β-mediated injuried mitochondria contributes to activate autophagy. However, pretreatment with the autophagy inhibitor 3-methyladenine showed the potential in further elevating the apoptosis rate induced by IL-1β in NP cells. Our results indicated that the mitochondrial pathway was involved in IL-1β-induced apoptosis of NP cells. Meanwhile, the damaged mitochondria-induced autophagy played a protective role against apoptosis, suggesting a postive feedback mechanism under inflammatory stress.

Figure 1. IL-1β expression is associated with cell apoptosis in NP tissues.

(A) Representative lumbar MRI of one patient with LVF (left) and the other with LDH (right) were classified according to Pfirrmann’s grading system. The red arrows represented the grade II disc in normal group and grade IV disc in degenerative group. (B) In situ apoptotic cells were determined by TUNEL staining in NP tissues. C and D, In situ IL-1β protein expressions from normal and degenerative NP tissues were determined by immunological histological chemistry and western blot. *P < 0.05 Vs. LVF group. Bars = 100 μm.

Figure 2. IL-1β induces cell apoptosis under serum deprivation.

(A) Morphologic changes in apoptotic NP cells. Left series is phase-contrast photomicrograph of NP cells and right series is apoptotic nuclei brightly stained by Hoechst 33258. Amplification: 200×. (B) Apoptotic cells were stained with Annexin V-PE and PI, and analyzed by flow cytometry. (C) Caspase-3 and -9 activity were determined by special Caspase Activity Assay Kits. Relative activity of caspase-3 and -9 was represented as fold changes to control (10%FBS + 0 ng/ml IL-1β group). *P < 0.05 and ^#P > 0.05 Vs. 10% FBS group, **P < 0.05 Vs. 0% FBS group.

Figure 3. Effect of IL-1β on apoptosis mediated through the mitochodrial pathway in NP cells.

(A) Western blot analysis for the protein expression of Bax, Bcl-2. The ratio of Bax/Bcl-2 was quantified. (B) Western blot analysis for the protein expression of cytochrome c from mitochondria and cytoplasm, respectively. The ratio of cytochrome c (mito)/cytochrome c (cyto) was quantified. (C) The intracellular ROS levels were measured by flow cytometry through DCFH-DA staining. The mean fluorescence intensity (MFI) of 10,000 cells was recorded. *P < 0.05 Vs. 0% FBS group.

Figure 4. IL-1β stress leads to mitochondrial dysfunction.

(A) The mitochondrial membrane potential was determined by staining with the mitochondrial dye JC-1. The quantitative ΔΨm was expressed as the ratio of red/green fluorescence intensity by flow cytometry. (B) Electron micrographs of mitochondria in NP cells treated with 10% FBS, 0% FBS and 0% FBS + 10 ng/ml IL-1β, respectively. Amplification: 2000×. (C) The intracellular ATP content was performed using an ATP Assay Kit by a microbeta counter. Results were normalized to cellular protein concentration for each sample. Three independent experiments were performed, and data of analysis represents the mean ± SD, *P < 0.05 and ^#P > 0.05 Vs. 0% FBS group.

Figure 5. IL-1β induced autophagy in NP cells.

(A) Western blot for the protein level of LC3 II/I and P62. (B) NP cells were transfected with adenovirus containing GFP-LC3. the formation and distribution of GFP-LC3 punctate were observed under confocal microscopy. Amplification: 800×. (C) Autophagy flux determination using Bafilomycin A1 pre-incubation with NP cells, western blot analyzed the exprssions of LC3 II/I and P62. (D) Morphological observation of autophagy under transmission electron microscope. Red arrows represent the characteristic double-membrane ultrastructural morphology of autophagic vacuoles. In the 0% FBS + 10 ng/ml IL-1β group, we observed double-membrane vesicles formed around pieces of mitochondrion (yellow arrow). *P < 0.05 Vs. 0% FBS group, ^#P < 0.05 Vs. IL-1β group.

Figure 6. The interactions between apoptosis and autophagy in NP cells under IL-1β stress.

NP cells were pre-treated 3MA for 2 h and then stimulated with IL-1β under serum deprivation. (A) Western blot analysis for LC II/I. (B) Apoptosis incidence quantified by flow cytometry by Annexin V and PI staining. (C) The mitochondria membrane potential was measured by flow cytometry through JC-1 staining. (D) Western blot analysis for the protein expression of Parkin in cytoplasm and mitochondria, respectively. The ratio of Parkin (mito)/Parkin (cyto) was quantified. *P < 0.05 Vs. IL-1β group, and ^#P > 0.05 Vs. 10% FBS group.