By modulating pAMPK/iNOS2 and MMP signalling, Immunogrit drives cellular resilience against D-galactose-induced senescence-associated stress markers in human keratinocytes

Abstract

Cellular senescence is a multifactorial, progressive phenomenon that disrupts biological homeostasis by increasing stress markers, causing morphological changes, and inducing inflammation, ultimately leading to irreversible cell cycle arrest. Natural plant-based bioactive products are frequently preferred over synthetic chemicals for their safety and efficacy. This study aims to assess the therapeutic effectiveness of a botanical formulation, Immunogrit, in mitigating stress-associated senescence markers in the skin. d-Galactose (D-Gal) has been used to induce senescence-associated stress markers in HaCaT human keratinocytes. Additionally, a thorough chemical characterization employing ultra-performance liquid chromatography-mass spectrometry coupled with quadrupole time-of-flight (UPLC/MS-QToF) and high-performance thin layer chromatography (HPTLC) was performed to generate a comprehensive phytochemical profile of Immunogrit. In HaCaT cells, d-Gal-induction increased oxidative and nitrosative stress with a perturbed expression of cell cycle biomarkers. Enhanced SA-β-galactosidase activity and subsequent downregulation of nuclear protein, LMNB1, further established the suitability of d-Gal-induced HaCaT cells as the relevant skin model for investigating senescence-associated interventions at the molecular levels. Treatment of Immunogrit with d-Gal induction, prevented the upregulation of p16, p21, and p53 levels; and maintained the basal SA-β-galactosidase activity. This study also identified pAMPK and iNOS2 as the targets of d-Gal orchestrating oxidative and nitrosative stress. Immunogrit mitigated the d-Gal-modulated levels of pAMPK and iNOS2. Moreover, collagen degradation and upregulation of metalloprotease (MMP1 and MMP9) in d-Gal-induced HaCaT cells were ameliorated by Immunogrit treatment. Our findings highlight Immunogrit as a promising botanical therapy for alleviating the key cellular markers of senescence, in human keratinocytes.

Keywords: Aging; D-Galactose; Immunogrit; Keratinocytes; MMP1/9; Senescence; iNOS2; pAMPK.