原人参三醇 (PPT) 是由人参产生的一种重要的人参皂苷，人参是许多地区使用的滋补植物。 PPT 对许多疾病状态具有有益作用，包括炎症、糖尿病和癌症。然而，PPT 对皮肤完整性的保护作用却很少被研究。此前，我们报道过 PPT 可以通过激活核因子 kappa B (NF-κB) 和丝裂原激活蛋白激酶 (MAPK) 途径来维持皮肤水分。然而，通过 PPT 增强皮肤保湿效果的细胞靶点仍然未知。我们想要确定 PPT 上调保湿因子 (HAS-2) 表达的上游靶点。我们研究了 PPT 可以直接刺激哪些上游蛋白来调节 NF -κB、MAPK 和其他信号级联。然后，过表达目标蛋白以检查与 HAS-2 的关系。接下来，进行细胞热位移测定（CETSA）来检查目标蛋白和PPT之间的关系。利用人角质形成细胞HaCaT来测量保湿因子和PPT激活的信号蛋白的水平。使用表达表皮生长因子受体（EGFR）和人表皮生长因子受体2（HER2）的DNA构建体及其通过定点诱变制备的突变体建立转染条件。通过RT-PCR、荧光素酶报告基因检测、CETSA或Western blot等进一步研究分子机制。我们发现PPT可以激活EGFR和HER2的磷酸化。这些刺激引起 Src 磷酸化，从而激活磷酸肌醇 3 激酶 (PI3K)/丙酮酸脱氢酶激酶 1 (PDK1)/蛋白激酶 B (AKT)/NF-κB 和 MAPK 信号级联。此外，EGFR 和 HER2 激活导致信号转导子和转录激活子 3 (STAT3) 以及钙/钙调蛋白依赖性蛋白激酶 II (CaMKII) 磷酸化。这诱导了 AMP 激活的蛋白激酶 α (AMPKα) 信号通路。此外，PPT 还能阻断过氧化物酶体增殖物激活受体 γ (PPARγ)，这也有助于 Src 的磷酸化。总的来说，我们首先发现 PPT 对皮肤屏障和角质形成细胞中的氢供应具有出色的保护作用。此外，EGFR和HER2等生长因子受体被揭示是PPT直接靶向的中心酶。这些结果表明其作为化妆品成分具有潜在的价值。版权所有 © 2023 Elsevier GmbH。版权所有。

Protopanaxatriol (PPT) is an important ginsenoside produced by ginseng, a tonic plant used in many areas. PPT has beneficial effects against many disease states including inflammation, diabetes, and cancer. However, PPT's protective effects on skin integrity have been rarely studied. Previously, we reported that PPT can maintain skin moisture through activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) pathways. However, the cellular targets for enhancing skin moisturizing effects via PPT are still unknown.We wanted to identify the upstream targets of PPT on upregulating moisturizing factor (HAS-2) expression.We investigated which upstream proteins can be directly stimulated by PPT to modulate NF-κB, MAPKs and other signaling cascades. Then, the targeted proteins were overexpressed to check the relationship with HAS-2. Next, the cellular thermal shift assay (CETSA) was conducted to check the relationship between targeted proteins and PPT.A human keratinocyte HaCaT were employed to measure the levels of moisturizing factors and the signaling proteins activated by PPT. Transfection conditions were established with DNA constructs expressing epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) and their mutants prepared by site-directed mutagenesis. Further investigation on molecular mechanisms was conducted by RT-PCR, luciferase reporter gene assay, CETSA, or Western blot.We found that PPT can activate the phosphorylation of EGFR and HER2. These stimulations caused Src phosphorylation, which resulted in the activation of phosphoinositide 3-kinases (PI3K)/pyruvate dehydrogenase kinase 1 (PDK1)/protein kinase B (AKT)/NF-κB and MAPKs signaling cascades. Additionally, EGFR and HER2 activation resulted in phosphorylation of signal transducer and activator of transcription 3 (STAT3) and calcium/calmodulin-dependent protein kinase II (CaMKII). This induced the AMP-activated protein kinase alpha (AMPKα) signaling pathway. Additionally, PPT blocked peroxisome proliferator activated receptor gamma (PPARγ), which also contributed to the phosphorylation of Src.Overall, we first found that PPT offers excellent protection of the skin barrier and hydrogen supply in keratinocytes. Moreover, growth factor receptors such as EGFR and HER2 were revealed to be central enzymes to be directly targeted by PPT. These results suggest a potentially valuable role as a cosmetic ingredient.Copyright © 2023 Elsevier GmbH. All rights reserved.