Trigonelline, a naturally occurring alkaloidal agent protects ultraviolet-B (UV-B) irradiation induced apoptotic cell death in human skin fibroblasts via attenuation of oxidative stress, restoration of cellular calcium homeostasis and prevention of endoplasmic reticulum (ER) stress
Introduction
Skin photodamage is growing rapidly with a high rate of prevalence and has become an impregnable problem world over [1].Ultraviolet (UV) radiation, especially UV-B radiations from sun is the most hazardous environmental factor causing 90% of skin related pathologies from photo-damage to photo carcinogenesis [2].On the basis of the wavelength and energy, UV radiations are categorized into three regions: UV-A region of wavelength 320–400 nm, UV-B region of wave length 280–320 nm, and UV-C region of wavelength 100–280 nm. Ultraviolet C region has shorter wavelength, thus doesn't reach earth surface and is mostly absorbed by the ozone layer in the stratosphere [3]. It has been reported that 90–95% of UV-A and 5–10% of UV-B radiation reaches the earth's surface [3]. Overexposure of ultraviolet radiation particularly UV-B radiations due to high energy and shorter wavelength causes most of the pathological states of skin [4]. UV-B exposure to skin leads to generation of reactive oxygen species (ROS) as byproducts of photochemical reactions in various cytosolic organelles like mitochondria and endoplasmic reticulum (ER) [5,6]. We have previously reported that UV-B irradiation impairs cellular homeostasis and promotes apoptosis in human dermal immortalized keratinocytes (HaCaT) and human dermal immortalized fibroblasts (HS68) cells via induction of ROS-mediated ER stress [7].
ER is critical membrane bound cytosolic organelle, plays a central role in maintaining the cellular homeostasis, protein synthesis and folding [8]. A number of insults including physical, chemicals and physiological, lead to accumulation of misfolded proteins in the ER, which results in unfolded protein response (UPR) via the modulation of signal transduction pathways [9]. Misfolded proteins in the ER lumen cause ER-stress that result in release of ER calcium to cytosol by Ca2+ATPases. The release of ER Ca2+ has been reported as apoptoginic [10]. The lumen of the ER maintains the higher concentration of Ca2+ to maintain an oxidative environment, essential for disulfide bond formation and proper protein folding [11]. The protein folding in the lumen is ensured by molecular chaperones like glucose regulated protein (Grp78/Bip). However, disturbances in cellular redox regulation cause agglomeration of nonfunctional proteins in the lumen of ER leading to the development of ER stress that subsequently triggers unfolded protein response (UPR) to restore cellular homeostasis [12]. UPR is regulated by ER transmembrane sensor proteins that are inositol-requiring enzyme 1 alpha (IRE1a), double-stranded RNA-dependent protein kinase-like ER kinase (PERK), and activating transcription factor (ATF)6 and their up-regulation is prevented by GRP78/Bip during normal conditions. Unresolved and continued ER stress activates PERK signaling pathway that shifts the adoptive response towards cell death via up-regulation of CHOP that on its own part increases oxidative stress, energy depletion, and finally apoptosis [13]. Recently, efforts have been made to identify and develop plant-based products against photodamage. Trigonelline (TG) is a heterocyclic alkaloidal phytochemical majorly found in the seeds of fenugreek, coffee and possess broad spectrum of health benefits [14]. In this study we explored the photo-protective activities of TG on UV-B irradiated human dermal fibroblasts. We found that TG treatment significantly attenuated the UV-B induced oxidative stress mediated ER- stress, restored Ca2+ homeostasis and prevented from cell death in UV-B exposed Hs68 cells. We have also found that topical application of TG to UV-B exposed areas of mice skin (Balb/C) prevents photodamage. Over all, this report supports the therapeutic role of trigonelline for the attenuation of UV-B mediated photo-damage.
Section snippets
Chemicals and Reagents
Trigonelline, DMEM media were purchased from Sigma Aldrich, Fetal Bovine Serum were purchased from GIBCO USA. Anti-phospho-elf2α was purchased from Cell Signaling Technology (Beverly, MA, USA), ER-tracker and Fura-4 AM from thermofisher and β-Actin from sigma Aldrich. All other phospho-antibodies and their non-phosphorylated control antibodies in this study were obtained from scanta Cruz (USA).
Cell Culture and UV-B Treatment
We used human foreskin fibroblasts Hs68 that were purchased from American Type Culture Collection
TG Treatment Attenuates ultraviolet-B Irradiation Mediated Photo Toxicity in Hs68 Cells
Trigonelline didn't alter the cellular viability of Hs68 cells and HaCaT up to the concentrations of 100 μM at 24 h. (Fig. 1A). UV-B -irradiation induced cell death in Hs68 cells dose dependently (Fig. 1B). 10 mJ/cm2 has been considered as environmental exposure level of UV-B radiation [18], and this radiation level has been used to study the photo protective effect of TG in Hs68 cells. We observed TG has protective effect against the UV-B radiation induced photo-toxicity in a dose dependent
Discussion
Skin is the outer most covering of human body that serves as barrier from extrinsic etiological challenges including physical, chemical and biological insults [25]. Ultraviolet (UV)-B (280–320 nm) is the lead environmental agent responsible for causing skin pathologies with highest incidence rate [26].The signs and symptoms of photo-damaged skin after the exposure to ultraviolet radiation, includes ROS generation, inflammation, ER stress, apoptosis, degradation of extracellular matrix
Conclusion
From our results we can conclude that Trigonelline might be a potential therapeutic/cosmaceutic agent against UV-B induced photo damages.
Declaration of Competing Interest
The authors have no conflict of interest.
Acknowledgments
This work was supported by counsel of scientific and industrial research (CSIR), New Delhi, India vide project No GAP 2166. Senior Research Fellowship (SRF) to authors LAN, and MAT under NET-JRF scheme by University Grant commission (UGC), New Delhi, India. Junior Research Fellowhip to author NHS by CSIR under CSIR-JRF scheme, and Junior Research Fellowship to author RRS by Department of science and technology (DST), New Delhi, India under DST-INSPIRE scheme.
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