Results

The high-salt microenvironment promotes AD-like dermatitis development in murine models

To investigate the potential impact of a high-salt microenvironment on the development of AD, we conducted a study using AD mouse models. To establish these models, we subjected BALB/c mice to continuous treatment with DNFB and OVA for four consecutive weeks according to previously reported methods.17 Simultaneously, a smear of PBS or NaCl solution was applied to the backs of the mice representing the control and treatment groups, respectively (Fig. 1A). After the four-week treatment period, we photographed the mice and then sacrificed them to assess the effects of the NaCl solution on the development of AD. As shown in Figure 1B and C, NaCl-treated mice exhibited more pronounced and histologically evident skin lesions compared with PBS-treated mice. Furthermore, the dermatitis scores and scratching scores were significantly higher in NaCl-treated mice compared with their control counterparts (Fig. 1D and E). In addition, we observed a significant up-regulation of AD-related inflammatory cytokines (IL-4, IL-25, IL-33, IgE, and TSLP) at both the mRNA and protein levels in the NaCl-treated mice, as shown in Figure 1E and F. Taken together, these findings provide strong evidence that a high-salt microenvironment can indeed promote the development of AD in vivo.

Figure 1: The high-salt microenvironment promotes disease development in mice with DNCB/OVA-induced atopic dermatitis (A) Schematic of the animal experiment procedure (B) Representative photos of dorsal skin in DNCB/OVA-induced atopic dermatitis in PBS- or NaCl-treated mice (C) Representative mouse model skin tissues are shown by hematoxylin–eosin staining (D) Time course of total skin lesion scores of PBS- or NaCl-treated mice (n = 5) (E) Scratching behavior examination (F) Quantitative reverse transcription PCR assay of the related target mRNA levels in the dorsal skin lesion. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (G) Related target molecules secretion (ELISA) of the mouse model blood (n = 5). Statistical analysis was performed using Student's t-test. Data are expressed as mean ± standard deviation. ∗∗p < 0.01 and ∗∗∗p < 0.001.

NaCl disrupts epidermal barrier function in AD and suppresses keratinocyte differentiation

Skin barrier dysfunction is increasingly recognized as a pivotal factor in the pathogenesis of AD, with keratinocytes playing a critical role in maintaining skin barrier homeostasis.8,13 To further understand the mechanisms behind the progression of AD induced by a high-salt microenvironment, we conducted experiments to evaluate the skin barrier function. The results showed an increase in TEWL levels in the NaCl-treated mice, indicating a disruption of skin barrier function (Fig. 2A). Furthermore, Western blotting analysis revealed a significant decrease in the expression of the epidermal barrier proteins FLG and LOR in the NaCl-treated AD mice (Fig. 2B). To investigate the effects of high-salt microenvironment on keratinocytes in vitro, we treated the human keratinocyte cell line HaCaT with different concentrations of NaCl. The results obtained by quantitative PCR and Western blotting assays showed that both mRNA and protein levels of FLG and LOR decreased in a dose- and time-dependent manner (Fig. 2C–F). Furthermore, the inhibitory effects of NaCl on FLG and LOR expression were confirmed by immunofluorescence assays in vitro (Fig. 2G) and immunohistochemistry assay results in vivo (Fig. 2H). Taken together, these results indicate that NaCl suppresses keratinocyte differentiation and disrupts skin barrier function in AD.

Figure 2: NaCl disrupts skin barrier function in atopic dermatitis and suppresses keratinocyte differentiation (A) Trans-epidermal water loss (TEWL) values of mice dorsal skin (n = 5) (B) Immunoblotting analysis of filaggrin (FLG) and loricrin (LOR) expression levels in skin lesions (C) Western blotting analysis of FLG and LOR expression levels at the indicated concentrations of NaCl in keratinocytes for 24 h (D) Western blotting analysis of FLG and LOR expression levels at different time points in keratinocytes treated with 20 mM NaCl (E) Quantitative reverse transcription PCR assay analysis of FLG and LOR mRNA expression levels in keratinocytes treated with different concentrations of NaCl (0 mM, 10 mM, 20 mM, 40 mM) (F) Quantitative reverse transcription PCR assay analysis of FLG and LOR mRNA expression levels at different time points in keratinocytes treated with 20 mM NaCl (G) Representative images of immuno-fluorescent staining of lysosome (stained by Lysotracker, red) and LOR or FLG (green) in keratinocytes treated with PBS and 20 mM NaCl. Scale bar: 10 μm (H) Representative immunohistochemistry images of skin lesions for LOR and FLG in PBS- or NaCl-treated mice. Scale bar: 50 μm. Data are shown as mean ± standard deviation from at least three independent experiments. Statistical analysis was performed using Student's t-test. Data are expressed as mean ± standard deviation. ∗∗p < 0.01 and ∗∗∗p < 0.001.

Cell viability and apoptosis in keratinocytes treated with NaCl

HaCaT cells were exposed to solutions containing or devoid of mannitol, NaCl, Na-gluconate, and choline chloride for 24 h, after which cell viability was evaluated. The findings indicated that, in comparison to the control group, treatment with the four hyperosmotic solutions at 380 mOsm/kg H2O did not significantly impact cell viability (Fig. S1A). Furthermore, apoptosis levels were analyzed by flow cytometry, and the high percentage of viable cells across all groups suggested that the cells maintained normal viability (Fig. S1B). These findings indicate that 380 mOsm/kg H2O and the associated ionic conditions are biocompatible with HaCaT cells. Additionally, we examined the effects of different isotonic solutions on keratinocyte barrier proteins (Fig. S2). The results revealed that, under 380 mOsm/kg H2O conditions, the levels of osmotic pressure-related proteins, including NFAT5 and phosphorylated p38, were significantly elevated. In contrast, the expression of barrier proteins was reduced in the NaCl group, and similar effects were observed in the sodium and chloride ion exchange groups. These findings suggest that the impact of NaCl on skin barrier function is not due to osmotic effects but rather to the specific microenvironmental changes induced by NaCl.

NaCl activates the SGK-1–STAT3 pathway in keratinocytes

In the present study, we aimed to investigate the activation of STAT3 in keratinocytes under high-salt conditions, as it is known to play a critical role in regulating keratinocyte differentiation in AD.18 We first performed Western blotting assays on HaCaT cells to analyze the levels of phosphorylated STAT3 when exposed to different concentrations of NaCl. The results showed a dose-dependent increase in phosphorylated STAT3, indicating that high-salt milieu activated STAT3 in keratinocytes (Fig. 3A). Next, we investigated whether there was a potential synergy between IL-4/IL-13, two canonical Th2 cytokines and established inducers of STAT3 phosphorylation in AD, and NaCl in activating STAT3. We treated HaCaT cells with IL-4/IL-13, NaCl, or a combination of both and analyzed the levels of phosphorylated STAT3. Interestingly, both IL-4/IL-13 and NaCl treatment individually up-regulated phosphorylated STAT3, and their combination had a synergistic effect on activating STAT3 (Fig. 3B). This finding suggests that NaCl induces STAT3 phosphorylation through a different mechanism than IL-4/IL-13. Further investigation into the mechanism of IL-4/IL-13-induced STAT3 activation in AD led us to the JAK–STAT3 pathway. Previous studies have reported that IL-4/IL-13 activates STAT3 through this pathway in AD.19 To confirm our hypothesis, we investigated the effects of delgocitinib, a pan-JAK inhibitor approved for AD treatment in Japan,20 on NaCl-induced STAT3 phosphorylation and blockade of keratinocyte differentiation. Western blotting analysis showed that delgocitinib partially alleviated NaCl-induced STAT3 phosphorylation and decreased the level of marker protein LOR and FLG (Fig. 3C). In addition, we performed further experiments using IL-4/IL-13 and NaCl in combination with delgocitinib. Interestingly, delgocitinib almost completely rescued STAT3 phosphorylation and keratinocyte differentiation when HaCaT cells were exposed to IL-4/IL-13 alone. However, when NaCl was added to the medium, the effects of delgocitinib were significantly reduced (Fig. 3D). This suggests that NaCl activates STAT3 in a manner different from the canonical IL-4/IL-13–JAK–STAT3 pathway.

Figure 3: NaCl activates the SGK-1–STAT3 pathway in keratinocytes (A) Western blotting analysis of STAT3 and phosphorylation of STAT3 protein expression levels in keratinocytes were treated with different concentrations of NaCl (0 mM, 10 mM, 20 mM, 40 mM) (B) The effect of NaCl and IL-4/13 on Stat 3 and Stat3 phosphorylation was determined by Western blotting (C) The protein levels of STAT3, phosphorylated STAT3, FLG, and LOR were detected in HaCaT cells incubated with 40 mM NaCl and/or JAK2 inhibitor delgocitinib for 24 h (D) Representative immunoblotting performed to assess protein expression of LOR and FLG in keratinocytes treated with JAK2 inhibitor, NaCl, and/or IL4/13 recombinant protein (E) Western blotting analysis of SGK-1 and phosphorylated SGK-1 in HaCaT cells treated with the indicated concentrations of NaCl (F) Western blotting analysis of SGK-1 and phosphorylated SGK-1 in HaCaT cells treated with 40 mM NaCl and/or IL4/13 recombinant protein (G) The protein levels of SGK-1, STAT3, phosphorylated STAT3, FLG, and LOR were detected in control and SGK-1 KD HaCaT cells treated with or without 40 mM NaCl (H) Western blotting analysis of SGK-1, STAT3, phosphorylated STAT3, FLG, and LOR in control and SGK-1 KD HaCaT cells treated with or without 40 mM NaCl and/or IL4/13. Data are shown as mean ± standard deviation from at least three independent experiments. Statistical analysis was performed using Student's t-test. Data are expressed as mean ± standard deviation. ∗∗p < 0.01 and ∗∗∗p < 0.001.

SGK-1 is a well-characterized kinase known to be sensitive to salt in mammalian cells.21 To gain a deeper understanding of how NaCl inhibits keratinocyte differentiation, we conducted a study to determine whether this specific kinase mediated the effects of NaCl on HaCaT cells. Our results from Western blotting assays showed that both the expression and phosphorylation of SGK-1 increased in a dose-dependent manner with NaCl treatment (Fig. 3E). This suggests that the presence of a high-NaCl microenvironment up-regulates and activates SGK-1. Interestingly, our experiments also showed that exposure to IL-4/IL-13 did not affect the expression and activation of SGK-1 (Fig. 3F), suggesting that SGK-1 functioned as a specific target for NaCl in keratinocytes in AD. Furthermore, when we knocked down SGK-1 in HaCaT cells, it substantially reversed the NaCl-induced phosphorylation of STAT3 as well as the decrease in LOR and FLG (Fig. 3G and H). Taken together, these results demonstrate that NaCl activates the SGK-1–STAT3 signaling pathway to suppress keratinocyte differentiation in the AD microenvironment.

SGK-1 promotes STAT3 activation via mTOR

To gain a better understanding of how SGK-1 modulates STAT3 activation, we performed a co-immunoprecipitation assay to investigate the direct interaction between SGK-1 and STAT3 (Fig. S3A). Surprisingly, our results demonstrated that SGK-1 did not directly bind to STAT3 in HaCaT cells, consistent with previous observations in melanoma cells.22 This intriguing discovery suggests the presence of another protein targeted by SGK-1 that might influence STAT3 phosphorylation. Moreover, our investigation unveiled that SGK-1 was capable of activating mTOR through the phosphorylation of TSC complex subunit 2 (TSC-2), which plays a crucial role in regulating the mTOR pathway.23 SGK-1 inhibits the activation of TSC2, thereby activating mTORC1 and promoting the phosphorylation of STAT3 (Fig. 4A and B). We also found that treatment with the mTOR inhibitor rapamycin successfully rescued the NaCl-induced activation of STAT3 and alleviated the blockade of keratinocyte differentiation (Fig. 4C). Furthermore, the results of our SGK-1 knockdown experiment further confirmed that the activation of STAT3 induced by NaCl occurs via the SGK-1–mTOR–STAT3 pathway (Fig. 4D).

Figure 4: SGK-1 promotes STAT3 activation via mTOR (A) Immunoprecipitation analysis of TSC-2 and SGK-1 in HaCaT cells. Precipitation samples and lysates were analyzed by immunoblotting. Precipitation samples and lysates were analyzed by immunoblotting (B) Western blotting analysis of P70 and phosphorylated P70 protein expression levels in HaCaT cells treated with different concentrations of NaCl (0 mM, 10 mM, 20 mM, 40 mM) (C) The protein levels of phosphorylated STAT3, STAT3, phosphorylated STAT3, FLG, and LOR were detected in control and SGK-1 KD HaCaT cells treated with or without 40 mM NaCl and/or rapamycin (D) Western blotting analysis of SGK-1 and phosphorylated SGK-1 protein expression levels in control and SGK-1 KD HaCaT cells treated with or without indicated concentrations of NaCl (–0 mM, +20 mM, ++40 mM). Data are shown as mean ± standard deviation from at least three independent experiments. Statistical analysis was performed using Student's t-test. Data are expressed as mean ± standard deviation. ∗∗p < 0.01 and ∗∗∗p < 0.001.

NaCl promotes the production of inflammatory cytokines in keratinocytes through the SGK-1–mTOR–NFκB pathway

Recently, keratinocytes have been found to play a critical role in the production of inflammatory cytokines that contribute to the progression of AD.24 Following this finding, we decided to investigate whether the high concentration of NaCl in the AD microenvironment could also affect the production of these inflammatory cytokines in keratinocytes. Our results, as shown in Figure 5A and B, demonstrated that NaCl treatment resulted in a concentration- and time-dependent increase in the mRNA and protein levels of IL-25, IL-33, and TSLP. Moreover, when SGK-1 was knocked down in HaCaT cells, the effects of NaCl on the production of these inflammatory cytokines were almost completely abolished (Fig. 5C). Furthermore, administration of rapamycin, a specific inhibitor of the mTOR pathway, was able to rescue NaCl-induced cytokine production (Fig. 5D). This suggests that the NaCl–SGK-1–mTOR pathway plays an important role in the secretion of inflammatory cytokines by keratinocytes. Nuclear factor-kappa B (NFκB) is widely recognized as a downstream target of the mTOR pathway.25 Therefore, we sought to determine whether NFκB was involved in this process. Our results from Western blotting assays showed that NFκB was indeed activated upon exposure to NaCl (Fig. 5E), and this activation was reduced when SGK-1 was knocked down (Fig. 5F). These results provide further evidence that NaCl promotes the secretion of inflammatory cytokines by keratinocytes through the SGK-1–mTOR–NFκB pathway.

Figure 5: NaCl promotes the production of inflammatory cytokines in keratinocytes through the SGK-1–mTOR–NFκB pathway (A) Representative data of quantitative PCR analysis of IL-25, IL-33, and TSLP mRNA normalized to GAPDH in HaCaT cells treated with indicated concentrations of NaCl (0, 10, 20, 40 mM) for 24 h and 20 mM NaCl at indicated time points (0, 12 h, 24 h, 48 h). Each bar represents the mean ± standard error (n = 3 in each group) (B) HaCaT cells were treated with indicated concentrations of NaCl (0, 10, 20, 40 mM) for 24 h and 20 mM NaCl at indicated time points (0, 12 h, 24 h, 48 h), and IL-25, IL-33, and TSLP levels in the culture supernatants were measured using ELISA. Each bar represents the mean ± standard deviation of three independent experiments (C) Representative data of quantitative PCR analysis and ELISA analysis of IL-25, IL-33, and TSLP in control and SGK-1 KD HaCaT cells treated with 40 mM NaCl for 24 h (D) HaCaT cells were treated with NaCl or NaCl combined with rapamycin (E) HaCaT cells were treated for 24 h with or without 40 mM NaCl before Western blotting analysis of NFκB signaling molecules. Bar graphs depict the quantification of p50 and p65 in the cytosolic or nuclear fraction relative to the total amount of actin and H2B, respectively, by Western blotting densitometry (F) Control and SGK-1 KD HaCaT cells were treated for 24 h with or without 40 mM NaCl before Western blotting analysis of NFκB signaling molecules. Bar graphs depict the quantification of p50 and p65 in the cytosolic or nuclear fraction relative to the total amount of actin and H2B, respectively, by Western blotting densitometry. Data are shown as the mean ± standard deviation from at least three independent experiments. Statistical analysis was performed using Student's t-test. Data are expressed as mean ± standard deviation. ∗∗p < 0.01 and ∗∗∗p < 0.001.

In conclusion, our study has shown that the presence of a high concentration of NaCl in the AD microenvironment can stimulate keratinocytes to produce inflammatory cytokines. This effect is mediated by activation of the SGK-1–mTOR–NFκB pathway. These findings shed light on the pathogenesis of AD and may provide potential targets for the development of therapeutic interventions.

Inhibiting SGK-1 alleviates NaCl-induced differentiation blockade and inflammatory cytokine production in keratinocytes

Our research has shown that a high level of NaCl in the AD microenvironment can activate the SGK-1–mTOR pathway. This activation inhibits keratinocyte differentiation and leads to the production of inflammatory cytokines. In light of these findings, we decided to investigate whether a small molecule SGK-1 inhibitor called GSK 650394 could be repurposed as a viable treatment option for AD. To begin the study, we conducted experiments to assess the effect of GSK 650394 on rescuing NaCl-induced blockade of keratinocyte differentiation. Results from Western blotting and quantitative PCR assays indicated that GSK 650394, the SGK-1 inhibitor, was able to increase the expression of LOR and FLG at both the mRNA and protein levels in the high-salt environment (Fig. 6A and B). This suggests that the inhibitor has the potential to restore normal differentiation in keratinocytes affected by high NaCl levels. Subsequent Western blotting assays showed that the SGK-1 inhibitor was also effective in suppressing the activation of mTOR–STAT3 induced by NaCl (Fig. 6C). This further supports the potential therapeutic use of the inhibitor in the treatment of AD. Furthermore, we observed that the SGK-1 inhibitor could reduce the production of inflammatory cytokines at the mRNA and protein levels in keratinocytes exposed to NaCl (Fig. 6E and F). In addition, the inhibitor was found to inhibit the activation of NFκB, another key player in the inflammatory process (Fig. 6G). These findings suggest that the SGK-1 inhibitor can alleviate the inflammatory symptoms associated with AD.

Figure 6: NaCl promotes the production of inflammatory cytokines in keratinocytes through the SGK-1–mTOR–NFκB pathway (A) Western blotting analysis of FLG and LOR in HaCaT cells treated with or without 40 mM NaCl and/or SGK-1 inhibitor GSK 650394 at indicated concentrations (–0 μM, +5 μM, ++10 μM, +++20 μM) (B) Quantitative PCR analysis of FLG and LOR mRNA normalized to GAPDH in HaCaT cells treated with 40 mM NaCl and/or SGK-1 inhibitor GSK 650394 at indicated concentrations (–0 μM, +5 μM, ++10 μM, +++20 μM) for 24 h (C) The protein levels of P70, phosphorylated P70, STAT3, and phosphorylated STAT3 in HaCaT cells treated with 40 mM NaCl and/or SGK-1 inhibitor GSK 650394 at indicated concentrations (–0 μM, +5 μM, ++10 μM, +++20 μM) for 24 h (D) Quantitative PCR analysis of IL-25, IL-33, and TSLP mRNA normalized to GAPDH in HaCaT cells treated with 40 mM NaCl and/or SGK-1 inhibitor GSK 650394 at indicated concentrations (0 μM, 5 μM, 10 μM, 20 μM) for 24 h (E) HaCaT cells were treated with 40 mM NaCl and/or SGK-1 inhibitor GSK 650394 at indicated concentrations (0 μM, 5 μM, 10 μM, 20 μM) for 24 h, and IL-25, IL-33, and TSLP levels in the culture supernatants were measured using ELISA. Each bar represents the mean ± standard deviation of three independent experiments (F) HaCaT cells were treated for 24 h with or without 40 mM NaCl and/or 10 μM GSK 650394 for 24 h before Western blotting analysis of NFκB signaling molecules. Bar graphs depict the quantification of p50 and p65 in the cytosolic or nuclear fraction relative to the total amount of actin and H2B, respectively, by Western blot densitometry. Data are shown as the mean ± standard deviation from at least three independent experiments. Statistical analysis was performed using Student's t-test. Data are expressed as mean ± standard deviation. ∗∗p < 0.01 and ∗∗∗p < 0.001.

Taken together, these results demonstrate that the SGK-1 inhibitor GSK 650394 is a promising potential drug for the treatment of AD. By effectively restoring differentiation blockade, suppressing mTOR–STAT3 activation, and reducing inflammatory cytokine production, this small molecule inhibitor may provide a rational therapeutic approach for patients suffering from AD.

Inhibiting SGK-1 relieves AD-like symptoms in vivo and exerts synergized effects with delgocitinib

To further investigate the effects of the SGK-1 inhibitor in vivo, we conducted experiments using mouse models of AD. The mice were divided into four groups: one group received PBS (control), another group received the SGK-1 inhibitor, a third group received the pan-JAK inhibitor delgocitinib, and the fourth group received a combination of the SGK-1 inhibitor and delgocitinib. After four weeks of treatment, we evaluated the effects of the different treatments on AD by photographing the mice and then sacrificing them for further evaluation. Upon visual inspection (Fig. 7A and B), it was evident that the skin lesions in the control group treated with PBS were more severe compared with the groups treated with delgocitinib and SGK-1 inhibitor alone. Notably, the combination therapy of SGK-1 inhibitor and delgocitinib showed the most significant improvement in AD-like symptoms. This observation was consistent with the results obtained using a scoring system called dermatitis scores and TEWL (Fig. 7C and D). In addition, we analyzed the protein expression levels by Western blotting. The results showed that the combination therapy of delgocitinib and SGK-1 inhibitor led to a significant up-regulation of FLG and LOR proteins (Fig. 7E). These proteins are known to be involved in skin barrier function, and their up-regulation suggests an improvement in skin integrity and barrier function. In addition, we examined the levels of AD-related inflammatory cytokines at both mRNA and protein levels. Our data showed that the combination therapy of SGK-1 inhibitor and delgocitinib significantly decreased the levels of IL-4, IL-25, IL-33, IgE, and TSLP (Fig. 7F and G). These cytokines play a critical role in the pathogenesis of AD, and the reduction in their levels suggests that the combination therapy has an anti-inflammatory effect.

Figure 7: SGK-1 inhibitor relieves atopic dermatitis symptoms in vivo and exerts synergistic effects with JAK inhibitor (A) Effect of delgocitinib and/or GSK 650394 on DNCB/OVA-induced atopic dermatitis in dorsal skin (B) Hematoxylin-eosin staining of dorsal skin tissue sections (C) Effects of delgocitinib and/or GSK 650394 on dermatitis scores in DNCB-induced atopic dermatitis (D) Effects of delgocitinib and/or GSK 650394 on trans-epidermal water loss (TEWL) in DNCB/OVA-induced atopic dermatitis (E) Representative immunohistochemistry images of skin lesions for LOR and FLG in delgocitinib and/or GSK 650394-treated mice. Scale bar: 50 μm (F) Western blotting analysis of FLG and LOR in dorsal skin tissue of DNCB/OVA-induced atopic dermatitis mice treated with delgocitinib and/or GSK 650394 (G) Quantitative reverse transcription PCR assay of the related target mRNA levels in skin lesions from mice receiving different treatments. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (H) Related target molecules secretion (ELISA) of mouse model blood from different groups (n = 5) (I) Proposed working model of how NaCl modulates keratinocytes' function in atopic dermatitis. Data are shown as the mean ± standard deviation from at least three independent experiments. Statistical analysis was performed using Student's t-test. Data are expressed as mean ± standard deviation. ∗∗p < 0.01 and ∗∗∗p < 0.001.

Taken together, these results provide strong evidence that the SGK-1 inhibitor effectively suppresses the development of AD in mice and shows a synergistic effect when combined with the JAK inhibitor delgocitinib. This suggests that dual inhibition of the SGK-1 and JAK pathways may be a promising therapeutic strategy for the future treatment of AD in humans.