Abstract: BACKGROUND: Studies have shown that heme oxygenase-1 has anti-inflammatory and anti-apoptotic effects. However, its potential to exert anti-inflammatory protective effects in nucleus pulposus mesenchymal stem cells remains unclear.
OBJECTIVE: To explore the protective effects and mechanisms of heme oxygenase-1 on nucleus pulposus mesenchymal stem cells under an inflammatory microenvironment. 
METHODS: (1) Primary nucleus pulposus mesenchymal stem cells were isolated from the intervertebral disc of SD rat tails and identified by flow cytometry and trilineage differentiation. (2) Nucleus pulposus mesenchymal stem cells were infected with heme oxygenase-1 overexpression lentivirus, and green fluorescent protein expression was observed under a fluorescence microscope. Western blot assay was used to assess the heme oxygenase-1 protein expression levels and infection efficiency. (3) Nucleus pulposus mesenchymal stem cells were divided into four groups: the control group cells were cultured with DMEM/F-12 complete medium for 24 hours. The model group, empty vector group, and heme oxygenase 1 overexpression group were uninfected cells, LV-Ctrl cells infected, and LV-HO-1 cells infected, respectively, and cultured with DMEM/F-12 complete medium supplemented with 5 μg/mL lipopolysaccharide for 24 hours. Western blot assay and immunofluorescence were used to detect the expression of inflammation-related proteins. The expression levels of proteins related to the nuclear factor κB signaling pathway were assessed using western blot assay.  
RESULTS AND CONCLUSION: (1) Rat nucleus pulposus mesenchymal stem cells exhibited adherent growth, dense arrangement, vigorous proliferation, and spindle-shaped morphology. Flow cytometry results showed high purity of the cultured nucleus pulposus mesenchymal stem cells, and trilineage differentiation assay indicated the nucleus pulposus mesenchymal stem cells had good potential to differentiate into adipocytes, osteocytes, and chondrocytes. (2) The nucleus pulposus mesenchymal stem cells infected with heme oxygenase 1 overexpression lentivirus expressed green fluorescent protein, and heme oxygenase 1 was highly expressed. (3) Compared with the model group, the expression levels of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), matrix metalloproteinase 13, matrix metalloproteinase 3, and tumor necrosis factor alpha were significantly reduced in the heme oxygenase-1 overexpression group (P < 0.05). (4) Compared with the model group, the ratio of phosphorylated nuclear factor κB/nuclear factor κB was significantly decreased (P < 0.05), and phosphorylated nuclear factor κB inhibitor protein/nuclear factor κB inhibitor protein also decreased significantly (P < 0.05) in the heme oxygenase-1 overexpression group. The above results indicate that heme oxygenase 1 effectively reduces the lipopolysaccharide-induced inflammatory response of nucleus pulposus mesenchymal stem cells by inhibiting the activation of the nuclear factor κB signaling pathway. 

Key words: heme oxygenase-1,  , nucleus pulposus mesenchymal stem cell,  , lipopolysaccharide,  , inflammation,  , intervertebral disc degeneration,  , nuclear factor κB