PMC

Schematic illustration showing that ROS-mediated LMP is involved in bupivacaine-induced death of IVD cells.

Observations of the morphological ultrastructural appearance by TEM. The IVD cells exposed to saline displayed a nearly normal cell morphology, including plasma membrane integrity, abundant surrounding microvilli, and slight swelling of organelles. The IVD cells exposed to 0.5% bupivacaine showed typical necrotic morphological changes, including severe swelling of organelles, severe disruption of the plasma membrane, and cellular lysis. AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine.

ROS level increases in IVD cells after 60 min of treatment with bupivacaine. (A) Representative graphs obtained from flow cytometry analysis after fluorescent labeling with DCFH-DA. FL1-H: green. (B) Histogram analysis showing the intracellular ROS levels. Data are presented as the means ± SD of three independent experiments (^*P < 0.05 vs. saline control). AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine.

Effect of bupivacaine on lysosomal compartments. After exposure to the indicated concentrations of bupivacaine, the lysosomal compartments in IVD cells was analyzed by LTR staining. (A) Representative graphs obtained from flow cytometry analysis. (B) Histogram analysis showing the MFI of LTR staining. Data are presented as the means ± SD of three independent experiments (^*P < 0.05 vs. saline control). (C) Typical confocal images of in situ LTR staining (Magnification ×400, scale bars = 50 µm). AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine.

ROS is an important mediator of bupivacaine-induced LMP and cell death. Prior to bupivacaine treatment, IVD cells were preincubated with or without 5 mM NAC for 60 min. (A, B) Effect of NAC on ROS production. (C, D) Effect of NAC on lysosomal compartments by LTR staining. (E, F) Effect of NAC on LMP by AO uptake experiments. (G, H) Effect of NAC on bupivacaine-induced cell death by Annexin V/PI staining. Data are presented as the means ± SD of three independent experiments (^*P < 0.05, NAC-treated vs corresponding untreated cells). AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine.

Cytotoxic effects of bupivacaine on rabbit IVD cells. Cell viability was determined by CCK-8 assays. (A) CCK-8 assay dose-course. To determine the dose-dependent effects of bupivacaine, cells were treated with various concentrations of bupivacaine for 60 min or 0.9% saline as a control. (B) CCK-8 assay time-course. To illuminate the time-dependent effects of bupivacaine, cells were treated with 0.375% bupivacaine for 0, 30, 60, 90, and 120 min, and 0.9% saline was used as a control. The results are expressed as the means ± SD of three independent experiments (^*P < 0.05 vs saline control). AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine.

Bupivacaine induces necrotic cell death in IVD cells. (A) Representative graphs obtained from flow cytometry analysis. After 60 min of exposure to bupivacaine, Annexin V/PI staining was performed to determine the type of cell death. Annexin-/PI- represents live cells, Annexin-/PI+ and Annexin + /PI+ represents necrotic cells, and Annexin + /PI- represents apoptotic cells. (B) Histogram analysis shows the percentage of PI-positive IVD cells. Data are presented as the means ± SD of three independent experiments (^*P < 0.05 vs. saline control). (C) Typical fluorescence photomicrograph of live/dead cell staining (Scale bars: 100 µm). Green fluorescence represents live cells, whereas red fluorescence represents necrotic cells. Photographs of green and red fluorescence were obtained under the same field and then merged. AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine.

Implication of lysosomal cathepsins in bupivacaine-induced cell death. (A) Typical confocal images of cathepsin D immunofluorescence staining in IVD cells after exposure to saline or bupivacaine (Magnification ×800, scale bars = 20 µm). (B) Representative graphs obtained from flow cytometry analysis after Annexin V/PI staining. Before exposure to bupivacaine, the IVD cells were pretreated with or without the cathepsin B inhibitor CA074-Me (20 μM) or the cathepsin D inhibitor pepstatin A (20 μM) for 60 min. After bupivacaine treatment, cell death was assessed by flow cytometry. (C) Histogram analysis showing the percentage of PI-positive cells. Data are presented as the means ± SD of three independent experiments (^*P < 0.05, CA-treated or PstA-treated vs. corresponding untreated cells). AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine; CA, CA074-Me; PstA, pepstatin A.

Cytotoxicity of bupivacaine involves the necroptosis pathway. (A) Representative Western blots of the expression of RIPK1, RIPK3, and MLKL.
After exposure to bupivacaine for 60 min, the protein expression of RIPK1, RIPK3, and MLKL in IVD cells was determined by Western blot. (B) Histogram analysis showing the relative protein levels of RIPK1, RIPK3, and MLKL. Data are presented as the means ± SD of three independent experiments (^*P < 0.05 vs. saline control). (C) Protection of necroptosis inhibitors against bupivacaine-induced cytotoxicity. Before exposure to bupivacaine, IVD cells were preincubated with or without the RIPK1 inhibitor Nec-1 (20 μM), the RIPK3 inhibitor GSK872 (4 μM), or the MLKL inhibitor NSA (4 μM) for 60 min. After bupivacaine treatment, we assessed cell viability by CCK-8 assays. Data are presented as the means ± SD of three independent experiments (^*P < 0.05, Nec-1-treated, GSK872-treated, or NSA-treated vs. corresponding untreated cells). AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine.

Bupivacaine induces permeabilization of the lysosomal membrane. (A) Histogram representing the statistical analysis shows the MFI of green fluorescence in the AO relocation experiment. IVD cells were first preincubated with 5 μg/ml AO solution for 15 min with complete medium and then exposed to bupivacaine for 60 min. Green fluorescence was determined by flow cytometry. Data are presented as the means ± SD of three independent experiments (^*P < 0.05 vs. saline control). (B) Representative graphs of the AO uptake experiment obtained by flow cytometry analysis. IVD cells were first treated with bupivacaine for 60 min and then stained with 5 μg/ml AO solution in complete medium. Cells deficient in intact lysosomes (“pale cells”) were determined by flow cytometry. (C) Histogram analysis showing the percentage of “pale cells”. Data are presented as the means ± SD of three independent experiments (^*P < 0.05 vs. saline control). (D) Typical confocal images of in situ AO staining (Magnification ×400, scale bars = 50 µm). AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine.