Mechanism of emodin modulating pain behavior in mouse model of osteoarthritis

doi:10.11958/20240056

Abstract

Abstract:

Objective To explore the regulatory mechanism of emodin on pain behavior in a mouse model of osteoarthritis based on mitochondrial key genes. Methods Thirty C57BL/6J mice were randomly divided into the control group, the osteoarthritis (OA) model group and the emodin-treated (OA+emodin) group, 10 mice per each group. The mice in the OA group and the OA+emodin group were intra-articular injection of complete Freund’s adjuvant (20 μL) in knee to establish the OA model, and mice in the OA+emodin group were treated by intraperitoneal emodin (10 mg/kg) injection. After behavioral testing, knee tissue of mice was collected for hematoxylin-eosin staining. Western blot analysis was used to detect expression levels of proinflammatory factors interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and mitochondria-related proteins NADH dehydrogenase (ubiquinone) flavoprotein 1 (NDUFV1), cytochrome C oxidase subunit 5B (COX5B), cytochrome C oxidase assembly protein COX15 homolog (COX15), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 10 (NDUFA10) in knee tissue. Results Compared with the control group, mice in the OA group showed decreased mechanical nociceptive threshold (PWT), reduced latency and distance in rotarod test (P<0.05). Compared with the OA group, mice in the OA+emodin group showed increased PWT, latency, and distance (P<0.05). In the control group, the structures of cartilage and subchondral bone were intact, while in the OA group, the cartilage was thinner and the subchondral trabeculae was deteriorated. The treatment with emodin alleviated cartilage degeneration. The expression levels of IL-1β, TNF-α, COX15 and NDUFA10 were increased while expression levels of NDUFV1 and COX5B were decreased in the OA group compared with the control group. The emodin treatment restored the above-mentioned protein expression levels (P<0.05). Conclusion Emodin can alleviate pain behavior in OA mice by regulating the expressions of inflammatory factors and mitochondrial related proteins.

Key words: osteoarthritis, emodin, mitochondria, computational biology, inflammatory pain

CLC Number:

R965.1，R684.3

Figures/Tables 8

Fig.1 Schematic diagram of experimental procedures of emodin regulating pain behavior in a mouse model of osteoarthritis

Tab.1 Comparison of behavioral tests between different groups of mice

组别	机械痛阈值/g	转棒停留时间/s	运动距离/m
对照组	1.48±0.25	543.92±46.50	23.89±2.17
OA组	0.21±0.13^a	215.18±50.45^a	8.42±2.16^a
OA+大黄素组	0.78±0.20^b	353.49±23.94^b	14.98±1.14^b
F	100.531^＊＊	154.783^＊＊	169.678^＊＊

Fig.2 HE staining of mouse knee cartilage tissue in each group

Fig.3 Correlation analysis of OA differentially expressed genes and mitochondria

Fig.4 Protein expression of mitochondria-associated proteins in each group detected by Western blot assay

Tab.2 Comparison of mitochondria-associated proteins NDUFV1, COX5B, COX15 and NDUFA10 expression between different groups of mice

组别	NDUFV1	COX5B	COX15	NDUFA10
对照组	1.00±0.04	1.00±0.04	1.00±0.09	1.00±0.03
OA组	0.53±0.03^a	0.55±0.05^a	1.77±0.08^a	2.40±0.09^a
OA+大黄素组	0.67±0.03^b	1.12±0.03^b	1.31±0.05^b	0.55±0.03^b
F	148.151^＊＊	133.223^＊＊	82.022^＊＊	767.383^＊＊

Fig.5 Protein expressions of IL-1β and TNF-α in knee joint tissue of each group detected by Western blot assay

Tab.3 Comparison of IL-1β and TNF-α protein expression between different groups of mice

组别	n	IL-1β	TNF-α
对照组	3	1.00±0.17	1.00±0.04
OA组	3	2.19±0.16^a	1.52±0.04^a
OA+大黄素组	3	1.14±0.16^b	1.15±0.05^b
F		47.405^＊＊	104.231^＊＊

References 30

[1]	ABRAMOFF B, CALDERA F E. Osteoarthritis:pathology,diagnosis,and treatment options[J]. Med Clin North Am, 2020, 104(2):293-311. doi:10.1016/j.mcna.2019.10.007.
[2]	LONG H, LIU Q, YIN H, et al. Prevalence trends of site-specific osteoarthritis from 1990 to 2019:findings from the global burden of disease study 2019[J]. Arthritis Rheumatol, 2022, 74(7):1172-1183. doi:10.1002/art.42089.
[3]	罗锟, 王智, 王柯. 山姜素调节VEGF/SphK1/S1P信号通路对膝骨关节炎大鼠血管生成的影响[J]. 天津医药, 2023, 51(1):1-6.
	LUO K, WANG Z, WANG K. Impacts of alpinetin on angiogenesis in knee osteoarthritis rats by regulating the VEGF/SphK1/S1P signaling pathway[J]. Tianjin Med J, 2023, 51(1):1-6. doi:10.11958/20230892.
[4]	CHEN Y, WU Y Y, SI H B, et al. Mechanistic insights into AMPKSIRT3 positive feedback loop-mediated chondrocyte mitochondrial quality control in osteoarthritis pathogenesis[J]. Pharmacol Res, 2021, 166:105497. doi:10.1016/j.phrs.2021.105497.
[5]	LI M, LUO X, LONG X, et al. Potential role of mitochondria in synoviocytes[J]. Clin Rheumatol, 2021, 40(2):447-457. doi:10.1007/s10067-020-05263-5.
[6]	MAO X, FU P, WANG L, et al. Mitochondria:potential targets for osteoarthritis[J]. Front Med (Lausanne), 2020, 7:581402. doi:10.3389/fmed.2020.581402.
[7]	LIU Q, WANG J, SUN Y, et al. Chondroitin sulfate from sturgeon bone protects chondrocytes via inhibiting apoptosis in osteoarthritis[J]. Int J Biol Macromol, 2019, 134:1113-1119. doi:10.1016/j.ijbiomac.2019.05.110.
[8]	LIU B, CHENG Y, WU Y, et al. Emodin improves alveolar hypercoagulation and inhibits pulmonary inflammation in LPS-provoked ARDS in mice via NF-κB inactivation[J]. Int Immunopharmacol, 2020, 88:107020. doi:10.1016/j.intimp.2020.107020.
[9]	GAO Y, LIU H, DENG L, et al. Effect of emodin on neuropathic pain transmission mediated by P2X2/3 receptor of primary sensory neurons[J]. Brain Res Bull, 2011, 84(6):406-413. doi:10.1016/j.brainresbull.2011.01.017.
[10]	SHELTON L R. A closer look at osteoarthritis[J]. Nurse Pract, 2013, 38(7):30-36; quiz 36-37. doi:10.1097/01.NPR.0000431178.
[11]	GLYN-JONES S, PALMER A J, AGRICOLA R, et al. Osteoarthritis[J]. Lancet, 2015, 386(9991):376-387. doi:10.1016/S0140-6736(14)60802-3.
[12]	RICHARD M J, DRIBAN J B, MCALINDON T E. Pharmaceutical treatment ofosteoarthritis[J]. Osteoarthritis Cartilage, 2023, 31(4):458-466. doi:10.1016/j.joca.2022.11.005.
[13]	MARTEL-PELLETIER J, BARR A J, CICUTTINI F M, et al. Osteoarthritis[J]. Nat Rev Dis Primers, 2016, 2:16072. doi:10.1038/nrdp.2016.72.
[14]	王柯, 叶寒露. 大黄素对膝骨关节炎大鼠软骨细胞铁死亡的影响及机制研究[J]. 天津医药, 2023, 51(10):1090-1097.
	WANG K, YE H L. Effect and its mechanism of emodin on the ferroptosis of chondrocytes in rats with knee osteoarthritis[J]. Tianjin Med J, 2023, 51(10):1090-1097. doi:10.11958/20221976.
[15]	BAKER-LEPAIN J C, LANE N E. Role of bone architecture and anatomy in osteoarthritis[J]. Bone, 2012, 51(2):197-203. doi:10.1016/j.bone.2012.01.008.
[16]	ZANETTE V, VALLE D D, TELLES B A, et al. NDUFV1 mutations in complex Ideficiency:case reports and review of symptoms[J]. Genet Mol Biol, 2021, 44(4):e20210149. doi:10.1590/1678-4685-GMB-2021-0149.
[17]	FINSTERER J, ZARROUK-MAHJOUB S. Phenotype of NDUFV1-related disease[J]. J Pediatr Neurosci, 2019, 14(3):175-176. doi:10.4103/jpn.JPN_124_18.
[18]	ALFADHEL M, LILLQUIST Y P, WATERS P J, et al. Infantile cardioencephalopathy due to a COX15 gene defect:report and review[J]. Am J Med Genet A, 2011, 155A(4):840-844. doi:10.1002/ajmg.a.33881.
[19]	GALATI D, SRINIVASAN S, RAZA H, et al. Role of nuclear-encoded subunit Vb in the assembly and stability of cytochrome c oxidase complex:implications in mitochondrial dysfunction and ROS production[J]. Biochem J, 2009, 420(3):439-449. doi:10.1042/BJ20090214.
[20]	ANTONICKA H, MATTMAN A, CARLSON C G, et al. Mutations in COX15 produce a defect in the mitochondrial heme biosynthetic pathway,causing early-onset fatal hypertrophic cardiomyopathy[J]. Am J Hum Genet, 2003, 72(1):101-114. doi:10.1086/345489.
[21]	张琳, 周雯静, 黄建廷, 等. 缺血后处理心肌细胞线粒体比较蛋白质组学的研究[J]. 遵义医学院学报, 2017, 40(4):369-373.
	ZHANG L, ZHOU W J, HUANG J T, et al. Comparative proteomics study on myocardial mitochondrial proteins in rats following ischemic postconditioning[J]. J Zunyi Med Univ, 2017, 40(4):369-373. doi:10.14169/j.cnki.zunyixuebao.2017.0082.
[22]	YANG X Y, HE K, PAN C S, et al. 3, 4-dihydroxyl-phenyl lactic acid restores NADH dehydrogenase 1 α subunit 10 to ameliorate cardiac reperfusion injury[J]. Sci Rep, 2015, 5:10739. doi:10.1038/srep10739.
[23]	MOLINA-GRANADA D, GONZÁLEZ-VIOQUE E, DIBLEY M G, et al. Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit[J]. Commun Biol, 2022, 5(1):620. doi:10.1038/s42003-022-03568-6.
[24]	WANG Y, LIU Q, CAI J, et al. Emodin prevents renal ischemia-reperfusion injury via suppression of CAMKII/DRP1-mediated mitochondrial fission[J]. Eur J Pharmacol, 2022, 916:174603. doi:10.1016/j.ejphar.2021.174603.
[25]	LIU D, CAI Z J, YANG Y T, et al. Mitochondrial quality control in cartilage damage and osteoarthritis:new insights and potential therapeutic targets[J]. Osteoarthritis Cartilage, 2022, 30(3):395-405. doi:10.1016/j.joca.2021.10.009.
[26]	BLANCO F J, REGO I, RUIZ-ROMERO C. The role of mitochondria in osteoarthritis[J]. Nat Rev Rheumatol, 2011, 7(3):161-169. doi:10.1038/nrrheum.2010.213.
[27]	ZHONG G, MADRY H, CUCCHIARINI M. Mitochondrial genome editing to treat human osteoarthritis-a narrative review[J]. Int J Mol Sci, 2022, 23(3):1467. doi:10.3390/ijms23031467.
[28]	MARCHI S, GUILBAUD E, TAIT S, et al. Mitochondrial control of inflammation[J]. Nat Rev Immunol, 2023, 23(3):159-173. doi:10.1038/s41577-022-00760-x.
[29]	HA M K, SONG Y H, JEONG S J, et al. Emodin inhibits proinflammatory responses and inactivates histone deacetylase 1 in hypoxic rheumatoid synoviocytes[J]. Biol Pharm Bull, 2011, 34(9):1432-1437. doi:10.1248/bpb.34.1432.
[30]	马园强. 大黄素对大鼠软骨细胞氧化应激损伤保护作用的研究[D]. 哈尔滨: 东北农业大学, 2020.
	MA Y Q. Stress damage in rat chondrocytstudy on the protective effect of emodin on oxidative[D]. Harbin: Northeast Agricultural University, 2020.