关键词: 椎间盘退变, 西红花苷, 肿瘤坏死因子α, 炎症, NF-κB信号通路, 细胞凋亡

Abstract: Objective To investigate the therapeutic effects and possible mechanism of crocin on intervertebral disc degeneration(IVDD). Methods Human nucleus pulposus tissue Pfirrmann II samples were divided into two parts. One part was used to extract nucleus pulposus cells. According to the composition of the culture medium, the cells were divided into 4 groups, including blank control group, tumor necrosis factor α(TNF-α)stimulation group, low-concentration of crocin treatment group(10 μg/mL)and high-concentration of crocin treatment group(40 μg/mL). After 24 h of incubation, the mRNA expressions of inflammatory factors and metabolic markers in the 4 groups were measured with real-time quantitative PCR. After 0 min, 15 min, 30 min and 24 h of incubation, the protein expressions of inflammatory factors, metabolic markers, apoptotic markers and p-P65, a key protein of the nuclear factor-activated B-cell κ light chain enhancer(NF-κB)signaling pathway, were measured with Western blotting. The effects of crocin on the NF-κB signaling pathway was determined with dual luciferase reporter gene assay. Another part of the samples was cultured in vitro and divided into blank control group, TNF-α stimulation group and crocin treatment group(40 μg/mL). The protein expressions of inflammatory factors and cellular metabolic markers were detected with immunohistochemistry. Results The results of real-time quantitative PCR and Western blotting showed that the inflammatory response and extracellular matrix degradation in the TNF-α stimulation group intensified, and the gene transcription and protein expression levels of inducible nitric oxide synthase(iNOS), cyclo-oxygenase-2, a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTs-5), and matrix metalloproteinase 13(MMP-13)were elevated compared with those in the blank control group(P<0.05), while the gene transcription and protein expressions of aggrecan and collagen-2 were significantly decreased(P<0.05). Western blotting also showed that the protein expression of anti-apoptotic factor B lymphoblastoma-2 gene(Bcl-2)in the TNF-α stimulation group was significantly decreased(P<0.001)compared to the blank control group, while the protein expressions of Bcl-2-related X protein(P=0.004)and cleaved-caspase 3 were significantly higher(P=0.005). In contrast, the above changes induced by TNF-α were significantly suppressed in both the 10 μg/mL and 40 μg/mL crocin treatment groups(P<0.05). In addition, the protein expression of p-P65 was significantly increased in the TNF-α stimulation group compared with the blank control group(P<0.05), whereas it was significantly decreased in the 10 μg/mL and 40 μg/mL crocin treatment groups(P<0.05), and significantly suppressed in the 15 min and 30 min groups(P<0.05). The dual luciferase reporter gene assay showed that the fluorescence intensity increased in the TNF-α stimulation group compared with the blank group(P<0.001), and significantly decreased after crocin treatment(P=0.006). Conclusion Crocin can inhibit the inflammatory response, extracellular matrix degradation and apoptosis of nucleus pulposus cells, and the NF-κB signaling pathway plays an important role in the inhibition of IVDD by crocin.

Key words: Intervertebral disc degeneration, Crocin, Tumor necrosis factor α, Inflammation, NF-κB signaling pathway, Apoptosis