Protective effect of metformin against lipopolysaccharide-induced chondrocyte injury by activating nuclear factor E2 and its relevant signaling pathway

doi:10.12307/2023.441

Abstract

Abstract: BACKGROUND: Previous studies have shown that the nuclear factor E2-related factor 2/heme oxygenase 1 signaling pathway is crucial for the development of osteoarthritis, and metformin has a certain protective effect on chondrocytes.
OBJECTIVE: To investigate the protective effect and mechanism of metformin on chondrocyte injury induced by lipopolysaccharide.
METHODS: Sprague-Dawley rat chondrocytes were isolated, cultured, and identified in vitro. Passage 3 chondrocytes were selected and treated with different concentrations of lipopolysaccharide (0, 10, 50, 100, 500, 1 000, and 5 000 μg/L) and metformin (0, 50, 100, 500, 1 000, 5 000, and 10 000 μmol/L) for 24 hours. Cell viability was then detected by cell counting kit-8 method, and the optimal mass concentrations of lipopolysaccharide and metformin were screened. According to different treatment factors, the chondrocytes were divided into blank group, lipopolysaccharide (5 000 μg/L) induced group, and 5 000 μg/L lipopolysaccharide +1 000 μmol/L group. Cell apoptosis was detected by flow cytometry. Oxidative stress was detected by reactive oxygen species, malondialdehyde, and superoxide dismutase kits. The mRNA and protein expressions of interleukin-1β, cyclooxygenase 2, type II collagen, nuclear factor E2-related factor 2, and heme oxygenase were detected by RT-PCR and western blot, respectively.
RESULTS AND CONCLUSION: Lipopolysaccharide reduced the activity of chondrocytes and induced apoptosis of chondrocytes, while metformin increased the activity of chondrocytes and decreased the apoptosis rate. The levels of reactive oxygen species and malondialdehyde were increased and the activity of superoxide dismutase was decreased in lipopolysaccharide-induced chondrocytes, while treatment with metformin decreased the levels of reactive oxygen species and malondialdehyde and increased the activity of superoxide dismutase in lipopolysaccharide-induced chondrocytes. Lipopolysaccharide increased the expressions of interleukin-1β and cyclooxygenase-2 in chondrocytes, and decreased the expressions of type II collagen, nuclear factor E2-related factor 2, and heme oxygenase 1 in chondrocytes. After metformin intervention, the expressions of interleukin-1β and cyclooxygenase-2 were decreased, and the expression of type II collagen, nuclear factor E2-related factor 2, and heme oxygenase 1 were increased. To conclude, metformin can inhibit lipopolysaccharide-induced chondrocyte injury, possibly through the activation of nuclear factor E2-related factor 2/heme oxygenase 1 signaling pathway

Key words: metformin, osteoarthritis, oxidative damage, Nrf2/HO-1 pathway

CLC Number:

Wang Xudong, Han Junzhu, Wang Wenrui, Xia Qixin, Guo Cheng. Protective effect of metformin against lipopolysaccharide-induced chondrocyte injury by activating nuclear factor E2 and its relevant signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(26): 4147-4153.

Figures/Tables 7

References

[1] LI J, ZHANG B, LIU WX, et al. Metformin limits osteoarthritis development and progression through activation of AMPK signalling. Ann Rheum Dis. 2020;79(5):635-645.
[2] ANSARI MY, AHMAD N, HAQQI TM. Oxidative stress and inflammation in osteoarthritis pathogenesis: Role of polyphenols. Biomed Pharmacother. 2020;129:110452.
[3] WANG T, HE C. Pro-inflammatory cytokines: The link between obesity and osteoarthritis. Cytokine Growth Factor Rev. 2018;44:38-50.
[4] WANG Y, ZHANG JJ, YANG WR, et al. Lipopolysaccharide-induced expression of FAS ligand in cultured immature boar sertoli cells through the regulation of pro-inflammatory cytokines and miR-187. Mol Reprod Dev. 2015;82(11):880-891.
[5] HENNIG P, GARSTKIEWICZ M, GROSSI S, et al. The Crosstalk between Nrf2 and Inflammasomes. Int J Mol Sci. 2018;19(2):562.
[6] LOBODA A, DAMULEWICZ M, PYZA E, et al. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cell Mol Life Sci. 2016;73(17):3221-3247.
[7] CHEN Z, ZHONG H, WEI J, et al. Inhibition of Nrf2/HO-1 signaling leads to increased activation of the NLRP3 inflammasome in osteoarthritis.Arthritis Res Ther. 2019;21(1):300.
[8] PAN X, CHEN T, ZHANG Z, et al. Activation of Nrf2/HO-1 signal with Myricetin for attenuating ECM degradation in human chondrocytes and ameliorating the murine osteoarthritis. Int Immunopharmacol. 2019;75:105742.
[9] JOO CHOI R, CHENG MS, SHIK KIM Y. Desoxyrhapontigenin up-regulates Nrf2-mediated heme oxygenase-1 expression in macrophages and inflammatory lung injury. Redox Biol. 2014;2:504-512.
[10] CAI D, FENG W, LIU J, et al. 7,8-Dihydroxyflavone activates Nrf2/HO-1 signaling pathways and protects against osteoarthritis. Exp Ther Med. 2019;18(3):1677-1684.
[11] LEE TS, CHAU LY. Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat Med. 2002;8(3):240-246.
[12] PANENI F, LüSCHER TF. Cardiovascular Protection in the Treatment of Type 2 Diabetes: A Review of Clinical Trial Results Across Drug Classes.Am J Cardiol. 2017;120(1S):S17-S27.
[13] YEREVANIAN A, SOUKAS AA. Metformin: Mechanisms in Human Obesity and Weight Loss. Curr Obes Rep. 2019;8(2):156-164.
[14] LUO F, DAS A, CHEN J, et al. Metformin in patients with and without diabetes: a paradigm shift in cardiovascular disease management. Cardiovasc Diabetol. 2019;18(1):54.
[15] YANG BY, GULINAZI Y, DU Y, et al. Metformin plus megestrol acetate compared with megestrol acetate alone as fertility-sparing treatment in patients with atypical endometrial hyperplasia and well-differentiated endometrial cancer: a randomised controlled trial. BJOG. 2020;127(7):848-857.
[16] FAN KJ, WU J, WANG QS, et al. Metformin inhibits inflammation and bone destruction in collagen-induced arthritis in rats. Ann Transl Med. 2020;8(23):1565.
[17] KATILA N, BHURTEL S, PARK PH, et al. Metformin attenuates rotenone-induced oxidative stress and mitochondrial damage via the AKT/Nrf2 pathway. Neurochem Int. 2021;148:105120.
[18] WEN Y, VECHETTI IJ JR, VALENTINO TR, et al. High-yield skeletal muscle protein recovery from TRIzol after RNA and DNA extraction. Biotechniques. 2020;69(4):264-269.
[19] SRINIVAS US, TAN B, VELLAYAPPAN BA, et al. ROS and the DNA damage response in cancer. Redox Biol. 2019;25:101084.
[20] CLAVIJO-CORNEJO D, MARTíNEZ-FLORES K, SILVA-LUNA K, et al. The Overexpression of NALP3 Inflammasome in Knee Osteoarthritis Is Associated with Synovial Membrane Prolidase and NADPH Oxidase 2. Oxid Med Cell Longev. 2016;2016:1472567.
[21] ZHAO M, WANG Y, LI L, et al. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance. Theranostics. 2021; 11(4):1845-1863.
[22] AHMAD N, ANSARI MY, HAQQI TM. Role of iNOS in osteoarthritis: Pathological and therapeutic aspects. J Cell Physiol. 2020;235(10): 6366-6376.
[23] KATZ JN, ARANT KR, LOESER RF. Diagnosis and Treatment of Hip and Knee Osteoarthritis: A Review. JAMA. 2021;325(6):568-578.
[24] WANG C, YAO Z, ZHANG Y, et al. Metformin Mitigates Cartilage Degradation by Activating AMPK/SIRT1-Mediated Autophagy in a Mouse Osteoarthritis Model. Front Pharmacol. 2020;11:1114.
[25] YAN Y, JUN C, LU Y, et al. Combination of metformin and luteolin synergistically protects carbon tetrachloride-induced hepatotoxicity: Mechanism involves antioxidant, anti-inflammatory, antiapoptotic, and Nrf2/HO-1 signaling pathway. Biofactors. 2019;45(4):598-606.
[26] WANG G, WANG Y, YANG Q, et al. Metformin prevents methylglyoxal-induced apoptosis by suppressing oxidative stress in vitro and in vivo. Cell Death Dis. 2022;13(1):29.
[27] SUZUKI T, MOTOHASHI H, YAMAMOTO M. Toward clinical application of the Keap1-Nrf2 pathway. Trends Pharmacol Sci. 2013;34(6):340-346.
[28] HOETZENECKER W, ECHTENACHER B, GUENOVA E, et al. ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression. Nat Med. 2011;18(1):128-134.
[29] REDDY NM, KLEEBERGER SR, KENSLER TW, et al. Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice.J Immunol. 2009;182(11):7264-7271.
[30] LI D, SHI G, WANG J, et al. Baicalein ameliorates pristane-induced lupus nephritis via activating Nrf2/HO-1 in myeloid-derived suppressor cells. Arthritis Res Ther. 2019;21(1):105.
[31] LI J, LU K, SUN F, et al. Panaxydol attenuates ferroptosis against LPS-induced acute lung injury in mice by Keap1-Nrf2/HO-1 pathway. J Transl Med. 2021;19(1):96.
[32] LUO JF, SHEN XY, LIO CK, et al. Activation of Nrf2/HO-1 Pathway by Nardochinoid C Inhibits Inflammation and Oxidative Stress in Lipopolysaccharide-Stimulated Macrophages. Front Pharmacol. 2018; 9:911.