Effects of Heibu Ointment on the growth and mitochondrial apoptosis of keloid fibroblasts under hypoxia

doi:10.19803/j.1672-8629.20250922

Abstract

Abstract: Objective To investigate the effects of Heibu ointment on the proliferation, migration, apoptosis and expressions of hypoxia-inducible factor-1α (HIF-1α), and key proteins of the mitochondrial apoptotic pathway in keloid fibroblasts (KFs) under hypoxic conditions. Methods A human KFs cell line was cultured and divided into the following groups: normoxia, hypoxia, and hypoxia treated with Heibu ointment at concentrations of 500, 250, 125, 62.5, and 31.25 μg·mL^-1. After 24 hours of culture, the MTT assay was performed to evaluate the proliferation of KFs and to select the optimal concentration for subsequent experiments. The KFs were then regrouped into normoxia, normoxia + 125 μg·mL^-1 extract, hypoxia, and hypoxia + 125 μg·mL^-1 extract. MTT assay, flow cytometry, scratch wound assay, immunofluorescence, and Western blot were used to evaluate the effects of the extract on the proliferation, apoptosis, migration and HIF-1α expression of KFs, and on the protein levels of key mitochondrial apoptosis factors (Bax, Bcl-2, Caspase-3). Results Compared with normoxic conditions, hypoxia significantly enhanced KFs’ proliferation (P<0.01), suppressed apoptosis (P<0.01), and promoted cell migration (P<0.01) while upregulating both the fluorescence intensity and protein expression of HIF-1α (P<0.01). Within the mitochondrial apoptotic pathway, hypoxia downregulated the expression levels of Bax and Caspase-3 (P<0.01) while upregulating Bcl-2 expressions (P<0.01). Treatment with Heibu ointment inhibited KFs’ proliferation (P<0.01), increased the apoptosis rate (P<0.01), and reduced migratory capacity P<0.01) compared to the hypoxia alone group. Concurrently, this ointment decreased both HIF-1α fluorescence intensity and protein expressions (P<0.01), elevated the expression levels of Bax and Caspase-3, but lowered Bcl-2 expression (P<0.01). Conclusion Our findings indicate that Heibu ointment can exert anti-scarring effects by inhibiting proliferation/migration, inducing apoptosis, downregulating HIF-1α, and activating the mitochondrial apoptotic pathway in hypoxic KFs.

Key words: Heibu Ointment, Keloid, Human Keloid Fibroblasts (KFs) Line, Hypoxia, Fibroblasts, Mitochondrial Apoptosis

CLC Number:

Ma Hui, Li Linchang, Zhang Tianlei, Chen Weiwen, Zheng Haiyun. Effects of Heibu Ointment on the growth and mitochondrial apoptosis of keloid fibroblasts under hypoxia[J]. Chinese Journal of Pharmacovigilance, 2026, 23(7): 775-782.

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URL: https://zgywjj.magtechjournal.com/EN/10.19803/j.1672-8629.20250922

https://zgywjj.magtechjournal.com/EN/Y2026/V23/I7/775

References

[1] Tan A, Glass Da 2nd. Patient-reported outcomes for keloids: a systematic review[J]. G Ital Dermatol Venereol, 2019, 154(2): 148-165.
[2] Oswaldo Jie, Tripodi D, Al-Shaqsi Y, et al.Effects of mechanical forces on the formation of cutaneous wounds during skin expansion and emerging therapies for wound healing and scar prevention[J]. Saudi Med J, 2023, 44(1): 106-109.
[3] Li Q, Wu P, Xia Y, et al.Dihydroartemisinin-induced mitochondrial mRNA degradation and apoptosis in keloid fibroblasts[J]. Chinese Medical Journal, 2022, 135(13): 1613-1615.
[4] Tan S, Khumalo N, Bayat A.Understanding Keloid pathobiology from a quasi-neoplastic perspective: less of a scar and more of a chronic inflammatory disease with cancer-like tendencies[J]. Front Immunol, 2019, 10: 1810.
[5] Haj M, Levon A, Frey Y, et al.Accelerated replicative senescence of ataxia-telangiectasia skin fibroblasts is retained at physiologic oxygen levels, with unique and common transcriptional patterns[J]. Aging Cell, 2023, 22(8): e13869.
[6] Yang Z, Huang Y, Zhu L, et al.SIRT6 promotes angiogenesis and hemorrhage of carotid plaque via regulating HIF-1alpha and reactive oxygen species[J]. Cell Death Dis, 2021, 12(1): 77.
[7] Lei R, Li J, Liu F, et al.HIF-1alpha promotes the keloid development through the activation of TGF-beta/Smad and TLR4/MyD88/NF-kappaB pathways[J]. Cell Cycle, 2019, 18(23): 3239-3250.
[8] Cha S, Kim H G, Jang H, et al.Steppogenin suppresses tumor growth and sprouting angiogenesis through inhibition of HIF-1alpha in tumors and DLL4 activity in the endothelium[J]. Phytomedicine, 2023, 108: 154513.
[9] Lee S Y, Kim E K, Seo H B, et al.IL-17 Induced stromal cell-derived factor-1 and profibrotic factor in keloid-derived skin fibroblasts via the stat3 pathway[J]. Inflammation, 2020, 43(2): 664-672.
[10] Lei R, Shen J, Zhang S, et al.Inactivating the ubiquitin ligase Parkin suppresses cell proliferation and induces apoptosis in human keloids[J]. J Cell Physiol, 2019, 234(9): 16601-16608.
[11] Dirand Z, Tissot M, Chatelain B, et al.Is spheroid a relevant model to address fibrogenesis in keloid research?[J]. Biomedicines, 2023, 11(9): 2350.
[12] Dai S, Xu M, Pang Q, et al. Hypoxia macrophage-derived exosomal miR-26b-5p targeting PTEN promotes the development of keloids[J]. Burns Trauma, 2024, 12: tkad036.
[13] Kong Y X, Li Z S, Liu Y B, et al.FOXO4-DRI induces keloid senescent fibroblast apoptosis by promoting nuclear exclusion of upregulated p53-serine 15 phosphorylation[J]. Commun Biol, 2025, 8(1): 299.
[14] Li Y M, Rasheed M, Liu J K, et al.Deciphering the molecular nexus: an indepth review of mitochondrial pathways and their role in cell death crosstalk[J]. Cells, 2024, 13(10): 863.
[15] Zhu X D, Li S L.Ferroptosis, necroptosis, and pyroptosis in gastroin-testinal cancers: the chief culprits of tumor progression and drug resistance[J]. Adv Sci (Weinh), 2023, 10(26): e2300824.
[16] Yfantis A, Mylonis I, Simos G.Direct Interaction between mortalin and HIF-1alpha at the mitochondria inhibits apoptosis by blocking recruitment of bax[J]. FEBS J, 2023, 290(15): 3764-3780.
[17] Si L, Zhang M, Guan E, et al.Resveratrol inhibits proliferation and promotes apoptosis of keloid fibroblasts by targeting HIF-1α[J]. Journal of Plastic Surgery and Hand Surgery, 2020, 54(5): 290-296.
[18] Wang Q, Yang X, Ma J, et al.PI3K/AKT pathway promotes keloid fibroblasts proliferation by enhancing glycolysis under hypoxia[J]. Wound Repair Regen, 2023, 31(2): 139-155.
[19] Lindenmann J, Kamolz L, Graier W, et al.Hyperbaric oxygen therapy and tissue regeneration: a literature survey[J]. Biomedicines, 2022, 10(12): 3145.
[20] Fu J D, Yao J J, Wang H, et al.Effects of EGCG on proliferation and apoptosis of gastric cancer SGC7901 cells via down-regulation of HIF-1alpha and VEGF under a hypoxic state[J]. European Review for Medical and Pharmacological Sciences, 2019, 23(1): 155-161.
[21] Lin S, Qin H Z, Li Z Y, et al.Gallic acid suppresses the progression of triple-negative breast cancer HCC1806 cells via modulating PI3K/AKT/EGFR and MAPK signaling pathways[J]. Front Pharmacol, 2022, 13: 1049117.
[22] Arsianti A, Nur Azizah N, Erlina L.Molecular docking, ADMET profiling of gallic acid and its derivatives (N-alkyl gallamide) as apoptosis agent of breast cancer MCF-7 cells[J]. F1000Res, 2024, 11: 1453.
[23] Wang X, Liu K, Ruan M, et al.Gallic acid inhibits fibroblast growth and migration in keloids through the AKT/ERK signaling pathway[J]. Acta Biochimica et Biophysica Sinica, 2018, 50(11): 1114-1120.