Humic Acid Impairs Key Cellular Functions in Fibroblasts: Implications for Supplement Safety

Year 2025, Volume: 14 Issue: 3, 574 - 580, 31.10.2025

Başak Işıldar

Abstract

Objective: Humic acid, the primary bioactive constituent of the traditional compound Shilajit, has been widely promoted for its therapeutic benefits. However, despite extensive interest in its anticancer effects, there remains a substantial lack of evidence regarding its cellular impact on non-cancerous cells. This study addresses this gap by evaluating the cytotoxic, apoptotic, migratory, and autophagic responses of 3T3 fibroblasts following humic acid exposure. Materials and Methods: 3T3 fibroblast cells were cultured under standard conditions and treated with various concentrations of humic acid for 24 hours to assess cytotoxicity using the MTT assay. The effects of humic acid on cell migration were evaluated using an in vitro scratch assay and apoptosis and autophagy were assessed by immunocytochemical detection of cleaved caspase-3 and Beclin-1, respectively. Data were analyzed statistically using appropriate tests. Results: Humic acid significantly reduced the viability of 3T3 fibroblast cells in a dose-dependent manner, with the lowest tested concentration (25 µg/mL) selected for further experiments. At this concentration, cell migration was markedly inhibited at 24 hours, although no significant difference was observed at 6 hours. Immunocytochemical analysis showed a significant increase in apoptosis, while autophagic activity tended to decrease, although the change was not statistically significant. Conclusion: These findings suggest that humic acid may have detrimental cellular effects and underline the importance of careful evaluation before its use in dietary supplements.

Keywords

Humic acid , 3T3 fibroblast cells , Cytotoxicity , Cell migration , Apoptosis , Autophagy

Ethical Statement

The authors declare that they have no competing interests.

Supporting Institution

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Thanks

The author gratefully acknowledges the contributions of those involved in the establishment of the Meryem Şeremet Cell Culture Laboratory, Faculty of Medicine, Balıkesir University, where this study was conducted.

Humik Asit, Fibroblastlarda Anahtar Hücresel Fonksiyonları Bozar: Diyet Takviyelerinin Güvenliği İçin Çıkarımlar

Abstract

Amaç: Geleneksel bir bileşik olan Shilajit’in başlıca biyolojik etkili bileşeni humik asit, terapötik yararları nedeniyle yaygın şekilde kullanılmaktadır. Ancak antikanser etkilerine yönelik ilgi artmış olsa da, bağ dokusu gibi kanser dışı hücreler üzerindeki etkilerine dair literatürde ciddi bir bilgi eksikliği bulunmaktadır. Bu çalışma, humik asite maruz bırakılan 3T3 fibroblastlarında sitotoksik, apoptotik, migrasyon ve otofaji yanıtlarını değerlendirerek bu boşluğu doldurmayı amaçlamaktadır. Gereç ve Yöntem: 3T3 fibroblast hücreleri standart koşullarda kültüre edilerek 24 saat boyunca farklı konsantrasyonlardaki humik asit ile muamele edilmiştir. Sitotoksisite MTT testi ile, hücre göçü in vitro yara iyileşme testi ile; apoptoz ve otofaji ise sırasıyla kesilmiş kaspaz-3 ve Beclin-1'in immünositokimyasal tespiti ile değerlendirilmiştir. Veriler uygun istatistiksel testlerle analiz edilmiştir. Bulgular: Humik asit, 3T3 fibroblast hücrelerinin canlılığını doza bağlı olarak anlamlı şekilde azaltmıştır ve ileri deneylerde en düşük test edilen doz olan 25 µg/mL kullanılmıştır. Bu konsantrasyonda, 24. saatte hücre göçü belirgin şekilde baskılanmış, ancak 6. saatte anlamlı bir fark gözlenmemiştir. İmmünositokimyasal analizlerde apoptozda anlamlı bir artış saptanırken, otofajide gözlenen azalma istatistiksel olarak anlamlı bulunmamıştır. Sonuç: Elde edilen bulgular, humik asidin hücresel düzeyde zararlı etkiler oluşturabileceğini göstermekte ve bu maddenin diyet takviyesi olarak kullanılmadan önce dikkatli bir şekilde değerlendirilmesi gerektiğini ortaya koymaktadır.

Keywords

Humik asit , 3T3 fibroblast hücreleri , Sitotoksisite , Hücre göçü , Apoptoz , Otofaji

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