Acceleration of Apoptosis by Extracellular Basic pH in a 3D Human Skin Equivalent System

Abstract

Previously, we have shown that extracellular basic pH plays a significant role in both the direct and indirect regulation of cellular processes in a wound; this in turn affects the wound-healing process. Several studies have demonstrated the importance of apoptosis modulation in the wound-healing process, especially in removing inflammatory cells and in inhibiting scar formation. However, the effects of extracellular basic pH on wound healing-related skin damage are yet to be examined. Therefore, we investigated the induction of accelerated apoptosis by extracellular basic pH in skin. Apoptosis-related protein levels were measured using an array kit, target protein expression levels were detected by immunostaining, lactate dehydrogenase was analyzed spectrophotometrically, and Annexin V levels were measured by fluorescence staining. Basic pH (8.40) strongly upregulated extrinsic apoptosis proteins (Fas, high temperature requirement A, and p21) and slightly upregulated intrinsic apoptosis proteins (cytochrome c, B-cell lymphoma 2 [Bcl-2], Bcl-2-associated death promoter, and Bcl-2-like protein 4) in a 3D human skin equivalent system. Moreover, basic pH (8.40) induced heat shock protein (HSP) 60 and 70. In addition, basic pH-exposed Fas- and HSP60-knockdown cells showed significantly decreased levels of apoptosis. Taken together, these results indicate that extracellular basic pH increases early-stage apoptosis through Fas/FasL via modulation of HSP60 and HSP70.

Enhanced Nrf2 up‐regulation by extracellular basic pH in a human skin equivalent system

“…Based on previous papers and the results of this paper, given that the antioxidant effect increases when the KGF inducer is processed, pH control and KGF inducer can play an important role in skin ageing or wound healing in the future (Figure 7). 6,7 Importantly, the inhibition of wound Future studies should perform metabolic profiling under different pH conditions. The analysis of NADPH activity is an important part of oxidative stress studies; we just briefly checked this in HaCaT cell line, and therefore, more detailed research is needed in the future (Figure S3).…”

“…Skin sections were prepared as previously described. 7 Briefly, 5μm-thick sections of 10% neutral formalin-fixed, paraffin-embedded tissues were cut on silane-coated glass slides, de-paraffinized three times with xylene and then dehydrated through a graded alcohol bath. Subsequently, the sections were washed with PBS before immunostaining and pretreated by blocking with 1% BSA for 30 minutes to prevent nonspecific binding of the antibodies.…”

“…In an in vitro study and a 3D human skin equivalent system (3HSE), basic pH conditions reportedly increased intracellular reactive oxygen species (ROS) generation and mitogen‐activated protein kinase signalling, whereas weakly acidic pH conditions slightly increased intracellular ROS generation and p38 kinase signalling, but not extracellular signal‐regulated kinases (ERK) and c‐Jun N‐terminal kinase signalling (JNK) 6 . Moreover, in an in vitro study and a 3HSE system, basic pH conditions increased early‐stage apoptosis through apoptosis antigen 1/apoptosis antigen 1 ligand via the modulation of heat‐shock protein (HSP)‐27, HSP‐60 and HSP‐70, which are an important effector of wound healing 7 . The inhibition of wound healing through pH imbalance and the effects of pH imbalance on the apoptotic processes involved in skin diseases such as atopic dermatitis suggest that the development of pH‐regulating or pH‐maintaining materials may provide effective therapeutic strategies for maintaining a healthy skin.…”

“…6 Moreover, in an in vitro study and a 3HSE system, basic pH conditions increased early-stage apoptosis through apoptosis antigen 1/apoptosis antigen 1 ligand via the modulation of heatshock protein (HSP)-27, HSP-60 and HSP-70, which are an important effector of wound healing. 7 The inhibition of wound healing through pH imbalance and the effects of pH imbalance on the apoptotic processes involved in skin diseases such as atopic dermatitis suggest that the development of pH-regulating or pH-maintaining materials may provide effective therapeutic strategies for maintaining a healthy skin.…”

Effects of the Higenamine, a Potent Compound from Aconitum, on UVB-Induced Photoaging in Hairless Mice

“…The cell culture system was established as per previous reports [ 14 , 15 ]. Human primary dermal fibroblast cells were purchased from TEGO Science (Seoul, Korea) and maintained in a fibroblast medium containing fibroblast growth supplement, 10% heat-inactivated FBS, and 1% penicillin-streptomycin.…”