Pheretima extract ameliorates nucleus pulposus cell degeneration in the intervertebral disc by inhibiting nuclear factor-kappa B pathway

doi:10.3969/j.issn.2095-4344.2983

Abstract

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.

Key words: bone, intervertebral disc, degeneration, nucleus pulposus cells, Pheretima, extract, nuclear factor-κB, factor

CLC Number:

Fu Yuanfei, He Shenghua, Lai Juyi, Sun Zhitao, Feng Hualong, Lan Zhiming . Pheretima extract ameliorates nucleus pulposus cell degeneration in the intervertebral disc by inhibiting nuclear factor-kappa B pathway [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(2): 264-268.

Figures/Tables 5

References

[1] GBD 2016 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet. 2017;390(10100):1211-1259.
[2] BRINJIKJI W, DIEHN FE, JARVIK JG, et al. MRI findings of disc degeneration are more prevalent in adults with low back pain than in asymptomatic controls: a systematic review and meta-analysis. AJNR Am J Neuroradiol. 2015;36(12):2394-2399.
[3] VERGROESEN PP, KINGMA I, EMANUEL KS, et al. Mechanics and biology in intervertebral disc degeneration: a vicious circle. Osteoarthritis Cartilage. 2015;23(7):1057-1070.
[4] YAMAMOTO J, MAENO K, TAKADA T, et al. Fas ligand plays an important role for the production of pro-inflammatory cytokines in intervertebral disc nucleus pulposus cells. J Orthop Res. 2013;31(4):608-615.
[5] LE MAITRE CL, HOYLAND JA, FREEMONT AJ. Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1beta and TNFalpha expression profile. Arthritis Res Ther.2007;9(4):R77.
[6] SEGUIN CA, PILLIAR RM, ROUGHLEY PJ, et al. Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue. Spine (Phila Pa 1976). 2005;30(17):1940-1948.
[7] RISBUD MV, SHAPIRO IM. Role of cytokines in intervertebral disc degeneration: pain and disc content. Nat Rev Rheumatol. 2014;10(1): 44-56.
[8] SEGUIN CA, PILLIAR RM, ROUGHLEY PJ, et al. Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue. Spine (Phila Pa 1976). 2005;30(17):1940-1948.
[9] 石召华,陈立军,黄文芳,等.地龙活性组分提取物干燥方式的筛选[J]. 中药材, 2015,38(7):1363-1365.
[10] 万明, 杨新, 张涛, 等. 沪地龙抗凝血活性部位分离研究[J]. 时珍国医国药, 2018,29(1):49-53.
[11] ADAMS MA, ROUGHLEY PJ. What is intervertebral disc degeneration, and what causes it? Spine. 2006;31(18):2151-2161.
[12] WANG SZ, RUI YF, LU J, et al. Cell and molecular biology of intervertebral disc degeneration: current understanding and implications for potential therapeutic strategies. Cell Prolif. 2014;47(5):381-390.
[13] FRIEDMANN A, GOEHRE F, LUDTKA C, et al. Microstructure analysis method for evaluating degenerated intervertebral disc tissue. Micron. 2017;92:51-62.
[14] WANG Y, ZUO R, WANG Z, et al. Kinsenoside ameliorates intervertebral disc degeneration through the activation of AKT-ERK1/2-Nrf2 signaling pathway. Aging (Albany NY). 2019;11(18):7961-7977.
[15] XU X, WANG D, ZHENG C, et al. Progerin accumulation in nucleus pulposus cells impairs mitochondrial function and induces intervertebral disc degeneration and therapeutic effects of sulforaphane. Theranostics. 2019;9(8):2252-2267.
[16] ZHU J, XIA K, YU W, et al. Sustained release of GDF5 from a designed coacervate attenuates disc degeneration in a rat model. Acta Biomater. 2019;86:300-311.
[17] ZHOU X, WANG J, HUANG X, et al. Injectable decellularized nucleus pulposus-based cell delivery system for differentiation of adipose-derived stem cells and nucleus pulposus regeneration. Acta Biomater. 2018;81:115-128.
[18] LI Z, ZHANG K, LI X, et al. Wnt5a suppresses inflammation-driven intervertebral disc degeneration via a TNF-alpha/NF-kappaB-Wnt5a negative-feedback loop. Osteoarthritis Cartilage. 2018;26(7):966-977.
[19] 王东, 杨欢, 王瑞辉, 等. 多层纳米地龙蛋白复合物的制备及在小鼠创伤中的应用[J]. 中华中医药学刊,2019,38(5): 1-7.
[20] 何红,车庆明,孙启时. 地龙提取物的抗凝血作用[J]. 中草药,2007, 38(5):733-735.
[21] 何升华,谭伟伟,孙志涛,等. 腰突颗粒对人髓核细胞核因子κB信号通路的影响[J]. 中国组织工程研究, 2016,20(33):4933-4939.
[22] HAYDEN MS, GHOSH S. Shared principles in NF-kappaB signaling. Cell. 2008;132(3):344-362.
[23] BALDWIN AJ. Series introduction: the transcription factor NF-kappaB and human disease. J Clin Invest. 2001;107(1):3-6.
[24] LIU Y, QU Y, LIU L, et al. PPAR-γ agonist pioglitazone protects against IL-17 induced intervertebral disc inflammation and degeneration via suppression of NF-κB signaling pathway. Int Immunopharmacol. 2019;72:138-147.
[25] WU X, LIU Y, GUO X, et al. Prolactin inhibits the progression of intervertebral disc degeneration through inactivation of the NF-κB pathway in rats. Cell Death Dis. 2018;9(2):98.
[26] TISHERMAN R, COELHO P, PHILLIBERT D, et al. NF-κB Signaling Pathway in Controlling Intervertebral Disk Cell Response to Inflammatory and Mechanical Stressors. Phys Ther. 2016;96(5):704-711.