Reactive Oxygen Species Regulate Endoplasmic Reticulum Stress and ER-Mitochondrial Ca²⁺ Crosstalk to Promote Programmed Necrosis of Rat Nucleus Pulposus Cells under Compression

Figure 5

Increased ROS levels relate to compression-induced ERS, [Ca²⁺]_m overload, and programmed NP cell death. (a) Representative flow cytometry images of PI uptake analysis for NP cells that were treated with 5 mM NAC for 1 h and then exposed to compression for 36 h. (b) Quantitative analysis of flow cytometry for cellular PI uptake. NP cells were treated as described in (a). The values are expressed as the from three biological replicates ( vs. control, ^### vs. compression alone, ANOVA/LSD). (c) Representative western blot graphs of the levels of HMGB1 content in the media. NP cells were treated as shown in (a). (d) Histogram for statistical analysis of the LDH leakage in compression-treated NP cells. NP cells were treated as in (a). The values are expressed as the from three biological replicates ( vs. control, ^### vs. compression alone, ANOVA/LSD). (e) Representative western blot graph of the levels of p-PERK, GRP78, and CHOP protein expression in NP cells exposed to compression. NP cells were treated as in (a). (f) The mRNA levels of PERK, GRP78, and CHOP detected by RT-qPCR in NP cells under compression conditions. NP cells were treated as in (a). The values are expressed as the from three biological replicates (^### vs. compression alone, Student’s -test). (g, h) Representative histograms and statistical analysis of [Ca²⁺]_m in compression-treated NP cells detected by flow cytometry with Rhod-2 AM. NP cells were treated as in (a). The values are expressed as the from three biological replicates ( vs. control, ^## vs. compression alone, ANOVA/LSD). (i) Typical fluorescence photomicrograph of in situ [Ca²⁺]_m staining with Rhod-2 AM under a fluorescence microscope. NP cells were treated as in (a).

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)