Pheretima extract ameliorates nucleus pulposus cell degeneration in the intervertebral disc by inhibiting nuclear factor-kappa B pathway / 中国组织工程研究

ABSTRACT

BACKGROUND: Inflammatory microenvironment of nucleus pulposus cells is an important factor to promote nucleus pulposus degeneration. Inflammatory factors amplify the cascade of inflammation by activating the nuclear factor-κB signaling pathway to form a vicious cycle and accelerate the process of nucleus pulposus degeneration. It could delay the progression of intervertebral disc degeneration by inhibiting the activity of nuclear factor-κB signaling pathway. OBJECTIVE: To investigate the mechanism of Pheretima extract on nucleus pulposus cell degeneration. METHODS: Pheretima active ingredients were extracted by cold dipping. The cell counting kit-8 method was used to detect the effects of 0, 25, 50, 100, 200, 400 mg/L Pheretima extract on rat nucleus pulposus cell activity, and then suitable drug concentrations were chosen for following research. Tumor necrosis factor-α (TNF-α) was used to stimulate nucleus pulposus cells to induce the nucleus pulposus cell degeneration model. Pheretima extracts (50, 100, 200 mg/L) were used to treat the cell model. Western blot was used to detect the expression of Aggrecan, Collagen II, TNF-α and interleukin-6. RT-qPCR was used to detect mRNA expression of TNF-α and interleukin-6. Immunofluorescence was used to detect the nuclear expression of P65 in the nucleus pulposus cells. RESULTS AND CONCLUSION: Compared with the blank group (0 mg/L), 400 mg/L Pheretima extract inhibited the activity of nucleus pulposus cells, and 50, 100, 200 mg/L extracts of Pheretima were used for subsequent experimental research. Compared with the normal group, the expression of Aggrecan and Collagen II in the model group was reduced, the protein and mRNA expression levels of TNF-α and interleukin-6 were up-regulated, and P65 expression increased in the nucleus. Compared with the model group, Pheretima extract could increase the expression of Aggrecan and Collagen II, down-regulate the protein and mRNA levels of TNF-α and interleukin-6, and reduce P65 expression in the nucleus. To conclude, Pheretima extract can reduce the expression of inflammatory factors, increase the synthesis of extracellular matrix and inhibit nuclear factor-κB pathway activity. Pheretima extract may ameliorate nucleus pulposus cell degeneration in the intervertebral disc via the nuclear factor-κB signaling pathway.