EVALUATION OF WOUND HEALING AND ANTI-INFLAMMATORY PROPERTY OF 5, 3' DIHYDROXYFLAVONE AND GENE EXPRESSION OF JAK AND COX-2 IN LIPOPOLYSACCHARIDE INDUCED RAW264.7 CELL LINE
Keywords:
5,3'-dihydroxyflavone, Raw264.7 cell line, MTT, Scratch assay, Nitric oxide assay, Gene expression, Wound healingAbstract
ABSTRACT
Objective: The study aims to evaluate the wound healing potential and anti-inflammatory properties of 5,3'-dihydroxyflavone and investigate its regulatory effects on the expression of Janus kinase (JAK) and cyclooxygenase-2 (COX-2) genes in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cell line.
Methods: Cytotoxicity was evaluated using the MTT assay. RAW 264.7 cells were treated with 5,3'-dihydroxyflavone (1–100 µg/mL) for 24 hours. Absorbance was measured at 490 nm and 630 nm. Wound healing was assessed through a scratch assay. LPS-induced and DHF-treated groups (12.5, 25, 50 µg/mL) were analysed for scratch closure at 0 and 24 hours. Nitric oxide (NO) production was quantified using the Griess assay. Nitrite levels were measured at 520–550 nm. Gene expression of COX-2 and JAK1 was analysed using qPCR, with β-actin as a reference gene. Relative expression was determined by the 2^−ΔΔCt method.
Results: The MTT assay confirmed DHF's non-cytotoxicity, showing ≥90% viability at 1–100 µg/mL (p > 0.05). The scratch assay showed enhanced wound closure. At 50 µg/mL, DHF achieved ~75% closure, compared to 45% in the LPS group. NO estimation revealed a dose-dependent reduction. At 50 µg/mL, DHF significantly lowered NO levels (p < 0.01). qPCR analysis demonstrated downregulation of COX-2 (45%) and JAK1 (50%) at 50 µg/mL (p < 0.001). These findings confirm DHF’s anti-inflammatory and wound-healing potential.
Conclusion: This study demonstrates that 5,3'-dihydroxyflavone enhances wound healing in RAW 264.7 cells by downregulating JAK and COX-2 expression, highlighting its anti-inflammatory potential.
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References
1.Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res. 2010 Mar;89(3):219–29.
2.Li W, Kandhare AD, Mukherjee AA, Bodhankar SL. Hesperidin, a plant flavonoid accelerated the cutaneous wound healing in streptozotocin-induced diabetic rats: Role of TGF-ß/Smads and Ang-1/Tie-2 signaling pathways. EXCLI J. 2018 May 4;17:399–419.
3.H M. Chronic Wounds and Acute Wounds: Differences and Treatments [Internet]. Total Care Family Center. 2025 [cited 2025 July 23]. Available from: https://totalcarefc.com/blog/chronic-wounds-and-acute-wounds/
4.Chakraborty C, Gupta B, Ghosh SK. Mobile metadata assisted community database of chronic wound images. Wound Med. 2014 Sept 1;6:34–42.
5.Chronic Wound Care Market Size, Share | Global Report [2032] [Internet]. [cited 2025 July 14]. Available from: https://www.fortunebusinessinsights.com/industry-reports/chronic-wound-care-market-100222
6.India Wound Care Market Research Reports 2025: Industry Size, Share, Growth Trends, Forecast & Opportunities by 2033 - MARKET RESEARCH INDIA [Internet]. 2025 [cited 2025 July 23]. Available from: https://marketresearchindia.co.in/2025/07/16/india-wound-care-market-research-reports-2025-industry-size-share-growth-trends-forecast-opportunities-by-2033/
7. Che Zain MS, Lee SY, Sarian MN, Fakurazi S, Shaari K. In Vitro Wound Healing Potential of Flavonoid C-Glycosides from Oil Palm (Elaeis guineensis Jacq.) Leaves on 3T3 Fibroblast Cells. Antioxidants. 2020 Apr 17;9(4):326.
8.Zulkefli N, Che Zahari CNM, Sayuti NH, Kamarudin AA, Saad N, Hamezah HS, et al. Flavonoids as Potential Wound-Healing Molecules: Emphasis on Pathways Perspective. Int J Mol Sci. 2023 Feb 27;24(5):4607.
9.Chagas M do SS, Behrens MD, Moragas-Tellis CJ, Penedo GXM, Silva AR, Gonçalves-de-Albuquerque CF. Flavonols and Flavones as Potential anti-Inflammatory, Antioxidant, and Antibacterial Compounds. Oxid Med Cell Longev. 2022 Sept 6;2022:9966750.
10.Spinelli FR, Colbert RA, Gadina M. JAK1: Number one in the family; number one in inflammation? Rheumatol Oxf Engl. 2021 May 5;60(Suppl 2):ii3–10.
11.Umamaheswari S. Anti-Inflammatory Effect of Selected Dihydroxyflavones. J Clin Diagn Res [Internet]. 2015 [cited 2025 July 15]; Available from: http://jcdr.net/article_fulltext.asp?issn=0973-709x&year=2015&volume=9&issue=5&page=FF05&issn=0973-709x&id=5928
12.Park HY, Kim GY, Hyun JW, Hwang HJ, Kim ND, Kim BW, et al. 7,8-Dihydroxyflavone exhibits anti-inflammatory properties by downregulating the NF-κB and MAPK signaling pathways in lipopolysaccharide-treated RAW264.7 cells. Int J Mol Med. 2012 June 1;29(6):1146–52.
13.Banerjee S, Biehl A, Gadina M, Hasni S, Schwartz DM. JAK-STAT Signaling as a Target for Inflammatory and Autoimmune Diseases: Current and Future Prospects. Drugs. 2017 Apr;77(5):521–46.
14.View of Interference of Antioxidant Flavonoids with MTT Tetrazolium Assay in a Cell Free System [Internet]. [cited 2025 July 17]. Available from: https://journaljpri.com/index.php/JPRI/article/view/4142/8293
15.Abbas MM, Al-Rawi N, Abbas MA, Al-Khateeb I. Naringenin potentiated β-sitosterol healing effect on the scratch wound assay. Res Pharm Sci. 2019 Dec 11;14(6):566–73.
16.Gautam A, Kumar V, Azmi L, Rao CV, Khan MM, Mukhtar B, et al. Wound Healing Activity of the Flavonoid-Enriched Fraction of Selaginella bryopteris Linn. against Streptozocin-Induced Diabetes in Rats. Separations. 2023 Mar;10(3):166.
17.Bao Z, Huang Y, Chen J, Wang Z, Qian J, Xu J, et al. Validation of Reference Genes for Gene Expression Normalization in RAW264.7 Cells under Different Conditions. BioMed Res Int. 2019 May 16;2019:6131879.
18.Che Zain MS, Lee SY, Sarian MN, Fakurazi S, Shaari K. In Vitro Wound Healing Potential of Flavonoid C-Glycosides from Oil Palm (Elaeis guineensis Jacq.) Leaves on 3T3 Fibroblast Cells. Antioxidants. 2020 Apr 17;9(4):326.
19.KAPTANER İĞCİ B, AYTAÇ Z. An Investigation on the In Vitro Wound Healing Activity and Phytochemical Composition of Hypericum pseudolaeve N. Robson Growing in Turkey. Turk J Pharm Sci. 2020 Dec;17(6):610–9.
20.Park HY, Kim GY, Hyun JW, Hwang HJ, Kim ND, Kim BW, et al. 7,8-Dihydroxyflavone exhibits anti-inflammatory properties by downregulating the NF-κB and MAPK signaling pathways in lipopolysaccharide-treated RAW264.7 cells. Int J Mol Med. 2012 June;29(6):1146–52.
21.Kang YJ, Wingerd BA, Arakawa T, Smith WL. Cyclooxygenase-2 gene transcription in a macrophage model of inflammation. J Immunol Baltim Md 1950. 2006 Dec 1;177(11):8111–22.
22.Banerjee S, Biehl A, Gadina M, Hasni S, Schwartz DM. JAK–STAT Signaling as a Target for Inflammatory and Autoimmune Diseases: Current and Future Prospects. Drugs. 2017;77(5):521–46.
23.Carvalho MTB, Araújo-Filho HG, Barreto AS, Quintans-Júnior LJ, Quintans JSS, Barreto RSS. Wound healing properties of flavonoids: A systematic review highlighting the mechanisms of action. Phytomedicine. 2021 Sept 1;90:153636.
24.Vijayarajan K, Ramasamy K, Kadal KK. NEUROPROTECTIVE EFFECT OF 7,3´-DIHYDROXYFLAVONE AGAINST PACLITAXEL-INDUCED NEUROTOXICITY IN SH-SY5Y NEUROBLASTOMA CELL LINE: AN IN VITRO MODEL. Asian J Pharm Clin Res. 2025 Mar 7;130–5.
25.Nicholas C, Batra S, Vargo MA, Voss OH, Gavrilin MA, Wewers MD, et al. Apigenin blocks lipopolysaccharide-induced lethality in vivo and proinflammatory cytokines expression by inactivating NF-kappaB through the suppression of p65 phosphorylation. J Immunol Baltim Md 1950. 2007 Nov 15;179(10):7121–7.
26.Xagorari A, Papapetropoulos A, Mauromatis A, Economou M, Fotsis T, Roussos C. Luteolin inhibits an endotoxin-stimulated phosphorylation cascade and proinflammatory cytokine production in macrophages. J Pharmacol Exp Ther. 2001 Jan;296(1):181–7.
27.Vidyalakshmi K, Kamalakannan P, Viswanathan S, Ramaswamy S. Anti-inflammatory effect of certain dihydroxy flavones and the mechanisms involved. Anti-Inflamm Anti-Allergy Agents Med Chem. 2012;11(3):253–61.
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