Activation of JNK/p38 MAPK Signaling Pathway by lncRNA DGCR5 Promotes Nucleus Pulposus Cell Degeneration and Pyroptosis in Intervertebral Disk Degeneration.

Abstract

Currently, the role of long noncoding RNA (lncRNA) DiGeorge syndrome critical region gene 5 ( DGCR5 ) in intervertebral disk degeneration (IDD) remains unclear. This study explored the molecular mechanisms of how lncRNA DGCR5 promoted human nucleus pulposus cells (NPCs) degeneration and pyroptosis during IDD. NPCs were identified by microscopic observation, flow cytometry, and immunofluorescence. An in vitro NPC degeneration model was induced using lipopolysaccharides treatment. SP600125 and SB203580 were used to inhibit the c-Jun N-terminal kinase (JNK) and p38 MAPK signaling, respectively. Quantitative real-time polymerase chain reaction or Western blot was performed to detect lncRNA DGCR5 , JNK, phospho-JNK, p38 MAPK, and phospho-p38 MAPK expressions in nucleus pulposus tissues and NPCs. Cell Counting Kit-8 assay was conducted to detect NPC activity. Western blot was performed to detect the expression levels of extracellular matrix-associated proteins (including collagen II, aggrecan, and matrix metalloproteinase 3 [MMP3]) and pyroptosis-associated proteins (including nucleotide oligomerization domain-like receptors family pyrin domain containing 3), cleaved caspase-1, lactate dehydrogenase, and N-terminal fragment of Gasdermin D (GSDMD) in NPCs. Enzyme-linked immunosorbent assay was conducted to detect the expressions of interleukin (IL)-1beta (IL-1β) and IL-18 in NPC supernatants. DGCR5 was upregulated, and the JNK/p38 MAPK signaling was activated both in nucleus pulposus tissues of IDD patients and lipopolysaccharides-induced NPCs. Inhibition of the JNK/p38 MAPK signaling enhanced NPC proliferation, promoted collagen II and aggrecan expression, while inhibited MMP 3 expression. Silencing of DGCR5 suppressed JNK/p38 MAPK signaling activity, inhibited NPC proliferation, and reduced collagen II and aggrecan expression but promoted MMP 3 expression. Similar findings were also observed for the expressions of nucleotide oligomerization domain-like receptors family pyrin domain containing 3, cleaved caspase-1, N-terminal fragment of GSDMD, IL-1β, and IL-18 as well as the released lactate dehydrogenase level. However, overexpression of DGCR5 yielded opposite results. These data suggest that lncRNA DGCR5 regulates NPC degeneration and pyroptosis to promote IDD through the JNK/p38 MAPK signaling pathway.