Nuezhenide Exerts Anti-Inflammatory Activity through the NF-κB Pathway

Author(s): Qin-Qin Wang, Shan Han, Xin-Xing Li, Renyikun Yuan, Youqiong Zhuo, Xinxin Chen, Chenwei Zhang, Yangling Chen, Hongwei Gao*, Li-Chun Zhao*, Shilin Yang

Journal Name: Current Molecular Pharmacology

Volume 14 , Issue 1 , 2021

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Graphical Abstract:


Background: Nuezhenide (NZD), an iridoid glycoside isolated from Ilex pubescens Hook. & Arn. var. kwangsiensis Hand.-Mazz., used as a traditional Chinese medicine for clearing away heat and toxic materials, displays a variety of biological activities such as anti-tumor, antioxidant, and other life-protecting activities. However, a few studies involving anti-inflammatory activity and the mechanism of NZD have also been reported. In the present study, the anti-inflammatory and antioxidative effects of NZD are illustrated.

Objective: This study aims to test the hypothesis that NZD suppresses LPS-induced inflammation by targeting the NF-κB pathway in RAW264.7 cells.

Methods: LPS-stimulated RAW264.7 cells were employed to detect the effect of NZD on the release of cytokines by ELISA. Protein expression levels of related molecular markers were quantitated by western blot analysis. The levels of ROS, NO, and Ca2+ were detected by flow cytometry. The changes in mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were observed and verified by fluorescence microscopy. Using immunofluorescence assay, the translocation of NF-κB/p65 from the cytoplasm into the nucleus was determined by confocal microscopy.

Results: NZD exhibited anti-inflammatory activity and reduced the release of inflammatory cytokines such as nitrite, TNF-α, and IL-6. NZD suppressed the expression of the phosphorylated proteins like IKKα/β, IκBα, and p65. Besides, the flow cytometry results indicated that NZD inhibited the levels of ROS, NO, and Ca2+ in LPS-stimulated RAW264.7 cells. JC-1 assay data showed that NZD reversed LPS-induced MMP loss. Furthermore, NZD suppressed LPS-induced NF-B/p65 translocation from the cytoplasm into the nucleus.

Conclusion: NZD exhibits anti-inflammatory effects through the NF-κB pathway on RAW264.7 cells.

Keywords: Nuezhenide, RAW264.7 cells, LPS, anti-inflammation, NF-κB pathway, cytotoxicity.

Jiang, W-Y. Therapeutic wisdom in traditional chinese medicine: A perspective from modern science. Trends Pharmacol. Sci., 2005, 26(11), 558-563.
[] [PMID: 16185775]
Mohan, H. Textbook of pathology. Jaypee Brothers; Medical Publishers Pvt. Limited, 2018.
Gieseck, R.L., III; Wilson, M.S.; Wynn, T.A. Type 2 immunity in tissue repair and fibrosis. Nat. Rev. Immunol., 2018, 8(1), 62-76.
Valesini, G.; Gerardi, M.C.; Iannuccelli, C.; Pacucci, V.A.; Pendolino, M.; Shoenfeld, Y. Citrullination and autoimmunity.Mosaic of autoimmunity; Elsevier, 2019, pp. 117-126.
May, M.J.; Ghosh, S. Signal transduction through nf-κb. Immunol. Today, 1998, 19(2), 80-88.
[] [PMID: 9509763]
Haque, M.A.; Jantan, I.; Harikrishnan, H.; Ghazalee, S. Standardized extract of Zingiber zerumbet suppresses lps-induced pro-inflammatory responses through nf-κb, mapk and pi3k-akt signaling pathways in u937 macrophages. Phytomedicine, 2019, 54, 195-205.
[] [PMID: 30668369]
Pearson, J.A.; Wong, F.S.; Li, W. The importance of the non obese diabetic (NOD) mouse model in autoimmune diabetes. J. Autoimmun., 2016, 66, 76-88.
[] [PMID: 26403950]
Dwivedi, M.; Kumar, P.; Laddha, N.C.; Kemp, E.H. Induction of regulatory t cells: A role for probiotics and prebiotics to suppress autoimmunity. Autoimmun. Rev., 2016, 15(4), 379-392.
[] [PMID: 26774011]
Chen, L.; Deng, H.; Cui, H.; Fang, J.; Zuo, Z.; Deng, J.; Li, Y.; Wang, X.; Zhao, L. Inflammatory responses and inflammation-associated diseases in organs. Oncotarget, 2018, 9(6), 7204-7218.
Zhang, H.; Tsao, R. Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects. Current opinion in food science; Marangoni, A.G.; Sant'Ana, Anderson, Eds.; Elsevier Science B. V: Amsterdam, 2016, 8, pp. 33-42.
Wu, X.; Gao, H.; Hou, Y.; Yu, J.; Sun, W.; Wang, Y.; Chen, X.; Feng, Y.; Xu, Q-M.; Chen, X. Dihydronortanshinone, a natural product, alleviates lps-induced inflammatory response through nf-κb, mitochondrial ros, and mapk pathways. Toxicol. Appl. Pharmacol., 2018, 355, 1-8.
Herrington, F.D.; Carmody, R.J.; Goodyear, C.S. Modulation of nf-κb signaling as a therapeutic target in autoimmunity. J. Biomol. Screen., 2016, 21(3), 223-242.
[] [PMID: 26597958]
Ning, S.; Friedrich, T.; Guo, L. Nfκb and kidney injury. Front. Immunol., 2019, 10, 815.
[] [PMID: 31040851]
Sparg, S.; Light, M.; Staden, J.V. Biological activities and distribution of plant saponins. J. Ethnopharmacol., 2004, 94(2-3), 219-243.
[] [PMID: 15325725]
Thring, T.S.; Hili, P.; Naughton, D.P. Anti-collagenase, anti-elastase and anti-oxidant activities of extracts from 21 plants. BMC Complement. Altern. Med., 2009, 9(1), 27.
[] [PMID: 19653897]
Bai, J.; Kwok, W.C.; Thiery, J-P. Traditional Chinese medicine and regulatory roles on epithelial–mesenchymal transitions. Chin. Med., 2019, 14(1), 1-13.
[] [PMID: 31558913]
Zhao, Y-C.; Xue, C-H.; Zhang, T-T.; Wang, Y-M. Saponins from sea cucumber and their biological activities. J. Agric. Food Chem., 2018, 66(28), 7222-7237.
[] [PMID: 29932674]
Minghetti, L. Cyclooxygenase-2 (cox-2) in inflammatory and degenerative brain diseases. J. Neuropathol. Exp. Neurol., 2004, 63(9), 901-910.
[] [PMID: 15453089]
Dhir, A. An update of cyclooxygenase (cox)-inhibitors in epilepsy disorders. Expert Opin. Investig. Drugs, 2019, 28(2), 191-205.
[] [PMID: 30521407]
Zhong, Z.; Zhang, Q.; Tao, H.; Sang, W.; Cui, L.; Qiang, W.; San Cheang, W.; Hu, Y.; Yu, H.; Wang, Y. Anti-inflammatory activities of sigesbeckia glabrescens makino: Combined in vitro and in silico investigations. Chin. Med., 2019, 14(1), 35.
Shin, W-B.; Dong, X.; Kim, Y-S.; Park, J-S.; Kim, S-J.; Go, E-A.; Kim, E-K.; Park, P-J. Anti-inflammatory effects of batillaria multiformis water extracts via nf-кb and mapk signaling pathways in lps-induced raw 264.7 cells Taurine 11. 2019, , 1001-1014.
Wu, C-T.; Deng, J-S.; Huang, W-C.; Shieh, P-C.; Chung, M-I.; Huang, G-J.; Longevity, C. Salvianolic acid c against acetaminophen-induced acute liver injury by attenuating inflammation, oxidative stress, and apoptosis through inhibition of the keap1/nrf2/ho-1 signaling. Oxid. Med. Cell. Longev., 2019, 2019, 1-13.
Sugimoto, M.A.; Vago, J.P.; Perretti, M.; Teixeira, M. Mediators of the resolution of the inflammatory response. Trends Immunol., 2019, 40(3), 212-227.
Mantovani, A.; Dinarello, C.A.; Molgora, M.; Garlanda, C. Interleukin-1 and related cytokines in the regulation of inflammation and immunity. Immunity, 2019, 50(4), 778-795.
Ahn, C-B.; Cho, Y-S.; Je, J-Y. Purification and anti-inflammatory action of tripeptide from salmon pectoral fin byproduct protein hydrolysate. Food Chem., 2015, 168, 151-156.
Alam, Q.; Zubair Alam, M.; Mushtaq, G.; Haque, A. Inflammatory process in alzheimer’s and parkinson’s diseases: Central role of cytokines. Curr. Pharm. Des., 2016, 22(5), 541-548.
Del Giudice, M.; Gangestad, S. Behavior, immunity: Rethinking il-6 and crp: Why they are more than inflammatory biomarkers, and why it matters. Brain Behav. Immun., 2018, 70, 61-75.
Du, H.; Kuang, T-T.; Qiu, S.; Xu, T.; Huan, C-L.G.; Fan, G.; Zhang, Y. Fecal medicines used in traditional medical system of china: A systematic review of their names, original species, traditional uses, and modern investigations. Chin. Med., 2019, 14(1), 31.
Tafani, M.; Sansone, L.; Limana, F.; Arcangeli, T.; De Santis, E.; Polese, M.; Fini, M.; Russo, M.A. The interplay of reactive oxygen species, hypoxia, inflammation, and sirtuins in cancer initiation and progression. Oxid. Med. Cell. Longev., 2016, 2016, 3907147.
Zhang, Y.; Dai, M.; Yuan, Z. Methods for the detection of reactive oxygen species. Anal. Methods, 2018, 10(38), 4625-4638.
Garcia, A.J., III; Viemari, J. C. Khuu M.A. Neurobiology: Respiratory rhythm generation, hypoxia, and oxidative stress-implications for development. Respir. Physiol. Neurobiol., 2019, 103259.
Dominguez-Andres, J.; Netea, M. Long-term reprogramming of the innate immune system. J. Leukoc. Biol., 2019, 105(2), 329-338.
Bols, N.C.; Brubacher, J.L.; Ganassin, R.C.; Lee, L. Ecotoxicology and innate immunity in fish. Dev. Comp. Immunol., 2001, 25(8-9), 853-873.
Yuan, R.; Huang, L.; Du, L-J.; Feng, J-F.; Li, J.; Luo, Y-Y.; Xu, Q-M.; Yang, S-L.; Gao, H.; Feng, Y-L. Dihydrotanshinone exhibits an anti-inflammatory effect in vitro and in-vivo through blocking tlr4 dimerization. Pharmacol. Res., 2019, 142, 102-114.
Perry, S.W.; Norman, J.P.; Barbieri, J.; Brown, E.B.; Gelbard, H.A. Mitochondrial membrane potential probes and the proton gradient: A practical usage guide. Biotechniques, 2011, 50(2), 98-115.
Sena, L.A.; Chandel, N. Physiological roles of mitochondrial reactive oxygen species. Mol. Cell, 2012, 48(2), 158-167.
Poli, G.; Schaur, R.J.; Leonarduzzi, GJMrr. 4-hydroxynonenal: A membrane lipid oxidation product of medicinal interest. Med. Res. Rev., 2008, 28(4), 569-631.
Lecaille, F.; Brömme, D.; Lalmanach, G. Biochemical properties and regulation of cathepsin k activity. Biochimie, 2008, 90(2), 208-226.
Jault, C.; Pichon, L.; Chluba, J. Toll-like receptor gene family and tir-domain adapters in danio rerio. Mol. Immunol., 2004, 40(11), 759-771.
He, J.; Han, S.; Li, X-X.; Wang, Q-Q.; Cui, Y.; Chen, Y.; Gao, H.; Huang, L.; Yang, S. Diethyl blechnic exhibits anti-inflammatory and antioxidative activity via the tlr4/myd88 signaling pathway in lps-stimulated raw264.7 cells. Molecules, 2019, 24(24), 4502.
Medzhitov, R. Origin and physiological roles of inflammation. Nature, 2008, 454(7203), 428.
Bäck, M.; Yurdagul, A.; Tabas, I.; Öörni, K.; Kovanen, P.T. Inflammation and its resolution in atherosclerosis: Mediators and therapeutic opportunities. Nat. Rev. Cardiol., 2019, 16(7), 389-406.
Perkins, D.J.; Were, T.; Davenport, G.C.; Kempaiah, P.; Hittner, J.B.; Ong'echa, J.M. Severe malarial anemia: Innate immunity and pathogenesis. Int. J. Biol. Sci., 2011, 7(9), 1427.
Shapouri-Moghaddam, A.; Mohammadian, S.; Vazini, H.; Taghadosi, M.; Esmaeili, S.A.; Mardani, F.; Seifi, B.; Mohammadi, A.; Afshari, J.T.; Sahebkar, A. Macrophage plasticity, polarization, and function in health and disease. J. Cell. Physiol., 2018, 233(9), 6425-6440.
Munford, R.S.; Pugin, J. Normal responses to injury prevent systemic inflammation and can be immunosuppressive. Am. J. Respir. Crit. Care Med., 2001, 163(2), 316-321.
Lenz, A.; Franklin, G.A.; Cheadle, W.G. Systemic inflammation after trauma. Injury, 2007, 38(12), 1336-1345.
Mauriz, J.L.; Collado, P.S.; Veneroso, C.; Reiter, R.J. A review of the molecular aspects of melatonin’s anti-inflammatory actions: Recent insights and new perspectives. J. Pineal Res., 2013, 54(1), 1-14.
Tian, R.; Hou, G.; Li, D.; Yuan, T-F. A possible change process of inflammatory cytokines in the prolonged chronic stress and its ultimate implications for health. Scientific World Journal, 2014, 2014, 780616.
Kunz, M.; Ceresér, K.M.; Goi, P.D.; Fries, G.R.; Teixeira, A.L.; Fernandes, B.S.; Belmonte-de-Abreu, P.S. Serum levels of il-6, il-10 and tnf-Α in patients with bipolar disorder and schizophrenia: Differences in pro-and anti-inflammatory balance. Br. J. Psychiatry, 2011, 33(3), 268-274.
Steensberg, A.; Fischer, C.P.; Keller, C.; Møller, K. Metabolism: Il-6 enhances plasma il-1ra, il-10, and cortisol in humans. 2003, 285(2), E433-E437.
Ferrucci, L.; Fabbri, E. Inflammageing: Chronic inflammation in ageing, cardiovascular disease, and frailty. Nat. Rev. Cardiol., 2018, 15(9), 505-522.
Hu, X.; Liu, S.; Zhu, J.; Ni, H. Dachengqi decoction alleviates acute lung injury and inhibits inflammatory cytokines production through tlr4/nf-κb signaling pathway in vivo and in vitro. J. Cell. Biochem., 2019, 120(6), 8956-8964.
Alcorn, J.L. Innate immunity and pulmonary inflammation: A balance between protection and disease. Translational inflammation; Elsevier, 2019, pp. 153-175.
Bannenberg, G.; Serhan, C.N. Specialized pro-resolving lipid mediators in the inflammatory response.An update. Biochim. Biophys. Acta, 2010, 1801(12), 1260-1273.
[PMID: 20708099]
Salvemini, D.; Cuzzocrea, S.J.Ccm. Therapeutic potential of superoxide dismutase mimetics as therapeutic agents in critical care medicine. 2003, 31(1), S29-S38.
Epidemiology Working Group for NCIP Epidemic Response, Chinese Center for Disease Control and Preventio. The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in China. Zhonghua Liu Xing Bing Xue Za Zhi, 2020, 41(2), 145-151.
Supuran, C.T.; Casini, A.; Mastrolorenzo, A.; Scozzafava, A. Cox-2 selective inhibitors, carbonic anhydrase inhibition and anticancer properties of sulfonamides belonging to this class of pharmacological agents. Mini Rev. Med. Chem., 2004, 4(6), 625-632.
Kawanishi, S.; Ohnishi, S.; Ma, N.; Hiraku, Y.; Murata, M. Crosstalk between DNA damage and inflammation in the multiple steps of carcinogenesis. Int. J. Mol. Sci., 2017, 18(8), 1808.
Jia, Z.; He, J. Paeoniflorin ameliorates rheumatoid arthritis in rat models through oxidative stress, inflammation and cyclooxygenase 2. Exp. Ther. Med., 2016, 11(2), 655-659.
Lee, S-B.; Lee, W.S.; Shin, J-S.; Jang, D.S.; Lee, K.T. Xanthotoxin suppresses lps-induced expression of inos, cox-2, tnf-α, and il-6 via ap-1, nf-κb, and jak-stat inactivation in raw 264.7 macrophages. Int. Immunopharmacol., 2017, 49, 21-29.
Gilroy, D.W.; Colville-Nash, P.; Willis, D.; Chivers, J.; Paul-Clark, M.; Willoughby, D. Inducible cyclooxygenase may have anti-inflammatory properties. Nat. Med., 1999, 5(6), 698.
Chen, J-Y.; Ye, Z-X.; Wang, X-F.; Chang, J.; Yang, M-W.; Zhong, H-H.; Hong, F-F.; Yang, S-L. Nitric oxide bioavailability dysfunction involves in atherosclerosis. Biomed. Pharmacother., 2018, 97, 423-428.
Yu, X.; Ge, L.; Niu, L. longevity c: The dual role of inducible nitric oxide synthase in myocardial ischemia/reperfusion injury: Friend or foe? Oxid. Med. Cell. Longev., 2018, 7
Kapoor, Y.; Kumar, K. Medicinal and chemical perspectives of nitric oxide: An overview. SF J. Pharm. Anal. Chem., 2019, 2(1), 1015.
Sasaki, M.; Kodama, Y.; Shimoyama, Y.; Ishikawa, T.; Kimura, S. Aciduricity and acid tolerance mechanisms of streptococcus anginosus. J. Gen. Appl. Microbiol., 2018, 64(4), 174-179.
Nafees, M.; Fahad, S.; Shah, A.N.; Bukhari, M.A.; Ahmed, I.; Ahmad, S.; Hussain, S. Reactive oxygen species signaling in plants. Plant abiotic stress tolerance; Springer, 2019, pp. 259-272.
Ozcan, A.; Ogun, M. Biochemistry of reactive oxygen and nitrogen species. Basic principles and clinical significance of oxidative stress; , 2015, 3, pp. 37-58.
Forrester, S.J.; Kikuchi, D.S.; Hernandes, M.S.; Xu, Q. Reactive oxygen species in metabolic and inflammatory signaling. Circ. Res., 2018, 122(6), 877-902.
Grimm, A.; Friedland, K.; Eckert, A.J. Mitochondrial dysfunction: The missing link between aging and sporadic alzheimer’s disease. Biogerontology, 2016, 17(2), 281-296.
Han, S.; Gao, H.; Chen, S.; Wang, Q.; Li, X.; Du, L-J.; Li, J.; Luo, Y-Y.; Li, J-X.; Zhao, L.C.; Feng, J.; Yang, S. Procyanidin a1 alleviates inflammatory response induced by lps through nf-κb, mapk, and nrf2/ho-1 pathways in raw264.7 cells. Sci. Rep., 2019, 9(1), 1-13.
Afonina, I.S.; Zhong, Z.; Karin, M.; Beyaert, R. Limiting inflammation-the negative regulation of nf-κb and the nlrp3 inflammasome. Nat. Immunol., 2017, 18(8), 861-869.
Cantrell, D. Signaling in lymphocyte activation. Cold Spring Harb. Perspect. Biol., 2015, 7(6), a018788.
Yang, Y.; Wang, J.K. The functional analysis of micrornas involved in nf-κb signaling. Eur. Rev. Med. Pharmacol. Sci., 2016, 20(9), 1764-1774.
Shi, J.; Hu, H.; Harnett, J.; Zheng, X.; Liang, Z.; Wang, Y-T.; Ung, C.O.L. An evaluation of randomized controlled trials on nutraceuticals containing traditional Chinese medicines for diabetes management: a systematic review. Chin. Med., 2019, 14(1), 54.
[] [PMID: 31798675]
Dedic, N.; Chen, A.; Deussing, J.M. The crf family of neuropeptides and their receptors - mediators of the central stress response. Curr. Mol. Pharmacol., 2018, 11(1), 4-31.
[PMID: 28260504]
Wu, B.; Wang, R.; Li, S.; Wang, Y.; Song, F.; Gu, Y.; Yuan, Y.J.P.R. Antifibrotic effects of fraxetin on carbon tetrachloride-induced liver fibrosis by targeting nf-κb/iκbα, mapks and bcl-2/bax pathways. Pharmacol. Rep., 2019, 71(3), 409-416.
Chandra, S.; Stanford, D.; Fletcher, E.; Walker, L.A. Raw materials production and manufacturing process control strategies. The science and regulations of naturally derived complex drugs; Springer, 2019, pp. 175-190.
Chen, Z.; Zhang, C.; Gao, F.; Fu, Q.; Fu, C.; He, Y.; Zhang, J.J.F. A systematic review on the rhizome of ligusticum chuanxiong hort.(chuanxiong). Food Chem. Toxicol., 2018, 119, 309-325.
Ye, L.; Cao, Z.; Wang, W.; Zhou, N. New insights in cannabinoid receptor structure and signaling. Curr. Mol. Pharmacol., 2019, 12(3), 239-248.
[] [PMID: 30767756]
Mothes, J.; Busse, D.; Kofahl, B.; Wolf, J.J.B. Sources of dynamic variability in nf-κb signal transduction: A mechanistic model. BioEssays, 2015, 37(4), 452-462.

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Year: 2021
Published on: 11 June, 2020
Page: [101 - 111]
Pages: 11
DOI: 10.2174/1874467213666200611141337

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