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. 2022 Jan 31;9:803132. doi: 10.3389/fcell.2021.803132

FIGURE 5.

FIGURE 5

ER stress plays a critical role in cholesterol-induced pyroptosis and matrix degradation in NP cells. (A) Representative images of GRP78 and CHOP staining in mild and severe degenerative NP tissues, as determined by IHC (bar = 20 μm). (B) Quantification of positive cells showing increased expressions of GRP78 and CHOP in severely degenerative NP tissues (n = 8–12). (C) Immunofluorescence images of NP cells stimulated with cholesterol and stained with an antibody against GRP78 (bar = 100 μm). (D, E) Protein expression levels of GRP78 and CHOP were upregulated, as revealed by immunoblotting and subsequent quantification (n = 3). (F) TEM images of NP cells. Control cells showed slender tube-like rough ERs. Cholesterol-stimulated cells featured swelling and enlarged rough ERs (upper panel, bar = 500 nm; lower panel, bar = 5 μm). (G) 4μ8C (20 μM) suppressed cholesterol (10 μg/ml) induced LDH increase in the culture supernatants of NP cells (n = 3). (H) Western blot and quantitative analysis showing that the increased pyroptosis biomarkers, including NLRP3, p20 and GSDMD-NT, induced by cholesterol were downregulated by 4μ8C (20 μM) in NP cells. (I) Western blot analysis revealed that 4μ8C (20 μM) increased the expression of COL2A and aggrecan in NP cells administrated with 10 μg/ml cholesterol. The data are presented as the mean ± SD. *p < 0.05 compared with control group; # p < 0.05 compared with cholesterol group.