Transcriptomic and metabolomic insights into gabapentin’s therapeutic role in neurogenic inflammation of rosacea

Abstract

Rosacea is a prevalent skin disorder in which neurogenic inflammation plays a significant role in its pathogenesis. Gabapentin (GBP) has garnered attention as a therapeutic option; however, its precise mechanism of action in treating rosacea remains unclear. Through a comprehensive analysis of experimental and clinical data, the research team has elucidated the molecular mechanism by which GBP inhibits neurogenic inflammation, particularly through modulating the NF-κB signaling pathway to alleviate rosacea inflammation. Using a murine rosacea model induced by LL37, the efficacy of GBP was compared to Minocycline and Hydroxychloroquine combination therapy. Various techniques assessed marker expression, transcriptomic profiles, and in vitro cell experiments with BV2 cells. Clinical data from 60 rosacea patients were analyzed through a randomized trial, comparing GBP therapy to the combination treatment. Results showed GBP effectively reduced skin inflammation and facial redness in mice and patients. Metabolomic analysis indicated significant changes in metabolites post-GBP treatment, correlating with inflammatory factors. The study concludes that GBP mitigates rosacea progression by targeting neurogenic inflammation via NF-κB pathway regulation, shedding light on novel treatment mechanisms through transcriptomics and metabolomics for future clinical application in rosacea research.

Gabapentin alleviates rosacea by suppressing neurogenic inflammation via NF-κB pathway, improving erythema and inflammation in LL37-induced models and patients, revealed by integrated transcriptomic and metabolomic analyses.

Introduction

Rosacea is a common chronic skin condition characterized by chronic erythema, vascular dilation, and inflammatory reactions on the face^1–3. Patients often experience itching, burning sensations, and self-consciousness issues, causing disruptions in their daily lives and social interactions^4–6. Traditional treatments such as topical medications and phototherapy have limitations in fully addressing the concerns of rosacea patients, leading to potential side effects and discomfort with prolonged use^7,8. Currently, the focus of rosacea treatment mainly revolves around inflammation control and skin repair; however, a significant number of patients experience recurrent episodes or find it challenging to achieve effective relief, necessitating the exploration of novel therapeutic approaches^9,10.

The NF-κB pathway, as a crucial intracellular signaling pathway, plays a key role in inflammation regulation by controlling the expression of a range of inflammatory mediators^11,12. Neurogenic inflammation exerts a significant influence on the pathogenesis of rosacea, exacerbating symptoms such as skin inflammation and vascular dilation, with abnormal activation of the NF-κB pathway likely closely associated with this process^13,14. Therefore, investigating the role of the NF-κB pathway in rosacea inflammation is of paramount importance for a deeper understanding of the disease’s pathogenesis and progression.

Gabapentin (GBP), known as an antiepileptic drug, has been increasingly recognized in recent years for its anti-inflammatory properties in areas such as neuropathic pain and neuroinflammation^11,15,16. Previous studies have revealed that GBP modulates the NF-κB pathway, thereby inhibiting the occurrence of neurogenic inflammation^11,12. Thus, this study aims to explore the potential mechanism of action of GBP in treating rosacea, particularly its relationship with the NF-κB pathway, offering new insights and guidance for clinical treatment.

The methodology of this study includes establishing an LL37-induced rosacea-like mouse model and observing the therapeutic effects of either combined treatment with minocycline (MIN) and hydroxychloroquine (HCQ) or GBP treatment alone, with the combined MIN and HCQ treatment serving as the positive control. Histological observation, immunohistochemical staining, immunofluorescence staining, Western blotting, and RT-qPCR were used to measure relevant biomarkers to assess the therapeutic effects of GBP on rosacea^17,18. Additionally, transcriptomic high-throughput sequencing was utilized to acquire the post-treatment inflammatory transcriptomic profile and reveal key regulatory pathways and mechanisms of action. Furthermore, in the clinical study, data from 60 patients with rosacea were collected, and they were randomly assigned to either the combined treatment group of MIN and HCQ or the GBP treatment group. The expression levels of inflammatory factors in the serum were measured using ELISA techniques^19–21.

The aim of this study is to elucidate how GBP suppresses neurogenic inflammation by regulating the NF-κB pathway, thereby alleviating symptoms and inflammatory reactions of rosacea. This research outcome is expected to deepen the understanding of the pathological mechanism of the disease, provide scientific basis for the development of new treatment strategies, offer more effective treatment options for patients with rosacea, alleviate symptoms such as facial redness and swelling, and improve quality of life.

Result

Potential therapeutic effects of GBP in alleviating rosacea skin inflammation

Rosacea is a chronic skin condition characterized by skin inflammation and vascular dilation. Recent studies suggest that neuroinflammation may play a crucial role in the pathogenesis of rosacea, linking the disease mechanisms to neural components²². Traditional rosacea treatments often focus on anti-inflammatory medications, such as the combined use of MIN and HCQ, which have been proven effective for this condition²³.

GBP, a neuromodulator, has shown potential in inhibiting neuroinflammation²⁴. Given the emerging evidence linking neuroinflammatory processes with rosacea, exploring how GBP can alleviate these pathways provides a novel therapeutic approach. In this study, we aim to compare the efficacy of GBP with the combination treatment of MIN and HCQ, and to elucidate its molecular actions.

This study utilized an LL37-induced rosacea-like mouse model (Fig. 1A) to investigate the impact of GBP on skin inflammation. Experimental results demonstrated that compared to the control group, mice in the LL37 group exhibited pronounced rosacea-like skin inflammation, including vascular dilation and swelling (p < 0.0001). Treatment with either GBP or the combination of MIN and HCQ significantly improved these symptoms, reducing skin thickness, erythema index, and overall condition (p < 0.0001), with no significant differences between the GBP group and the MIN and HCQ combination treatment group (B: p > 0.9999; C: p = 0.7685; E: p > 0.8927)(Fig. 1B-E). Histopathological analysis revealed that both GBP and the MIN and HCQ combination treatment alleviated the LL37-induced pathological skin conditions, reduced inflammatory cell infiltration, and improved tissue structure disorganization (p < 0.0001) (Fig. 1F-G), with similar efficacy observed between the two treatment groups (p = 0.1654).

Fig. 1. GBP improves LL37-induced rosacea-like skin inflammation.

A Schematic representation of establishing a mouse model of rosacea-like skin inflammation using LL37 and treatment with GBP (Created by BioRender); B Administration of GBP to different groups of mice and observation of skin symptoms at the end of the final experiment; C Assessment of each group of mice’s skin thickness; D Evaluation of skin redness score in each group of mice; E Overall skin condition score for each group of mice; F Histopathological assessment using H&E staining to examine skin conditions of each group of mice, with a scale bar of 100 μm and a local magnification scale bar of 25 μm; G Analysis of inflammatory cell infiltration in different groups. All data are expressed as mean ± standard deviation (SD). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by post-hoc tests with Dunnett’s T3 and LSD-t methods. For animal experiments, n = 5. Statistical significance was defined as ^nsP > 0.05; ***P < 0.001.

Due to the upregulation of pro-inflammatory cytokines and chemokines in the skin lesions of rosacea patients, the severity of inflammation is closely related to symptoms²⁵. RT-qPCR was used to measure the mRNA expression levels of inflammatory factors in skin tissue samples. Results showed that compared to the control group, the expression of TNF-α, IL-6, and IL-1β mRNA was significantly increased in the LL37 group (p < 0.0001). In both the GBP group and the MIN and HCQ combination treatment group, the expression levels of these inflammatory factors were significantly reversed (p < 0.0001), confirming their potential therapeutic effect in alleviating LL37-induced skin inflammation (Supplementary fig. 1A-C).

In summary, GBP demonstrated equivalent effects to the combination treatment with MIN and HCQ in reducing inflammatory cell infiltration and the expression of inflammatory factors, significantly improving the symptoms of LL37-induced rosacea-like skin inflammation, including skin thickness, redness, and overall pathological state. This suggests that GBP has potential as a therapeutic drug for rosacea.

Modulation of neuroinflammation pathways and their impact on rosacea angiogenesis: the potential therapeutic role of GBP

Microvascular dilatation is considered the initial and fundamental sign of rosacea, which may progress to other clinical manifestations and symptoms¹. In recent years, mounting evidence has revealed its close association with neuroinflammation²⁶. The activation of neuroinflammation plays a crucial role in the development of rosacea, not only triggering skin erythema and paresthesia but also promoting vasodilation and increasing vascular permeability²⁷. Studies have suggested that modulating the neuroinflammation pathways can effectively alleviate microvascular dilatation, offering a potential therapeutic approach for rosacea²⁸. It is widely known that spicy foods are an significant trigger for microvascular dilatation.To further investigate the potential role of GBP in the treatment of rosacea, we topically applied capsaicin on mouse ear skin, followed by treatment with GBP (Fig. 2A).

Fig. 2. GBP inhibits angiogenesis in rosacea.

A Continuous treatment of mice with GBP for 4 days followed by application of capsaicin to the left and right ears 30 hours after the last dose to observe pathological changes (Created by BioRender); B Representative pathological images and redness scores of the left and right ears of mice after GBP treatment; C Immunohistochemical analysis of CD31 expression in the ear skin of mice in different groups, with a scale bar of 50 μm; D Western blot analysis of ICAM-1 and VCAM-1 protein expression levels in the ear tissues of different groups of mice. All data are expressed as mean ± standard deviation (SD). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by Dunnett’s T3 and LSD-t post hoc tests. For animal experiments, n = 5 per group. Statistical significance was indicated as ^ns På 0.05, *P <0.05, **P <0.01, ***P <0.001.

Our research results show that compared with the Ctrl group, the mice in the Capsaicin group exhibited significantly intensified erythema and vasodilation in the ear skin (p < 0.0001). Treatment with either GBP or the combination of MIN and HCQ significantly suppressed the signs of vasodilation and erythema in the ear skin after Capsaicin treatment (p = 0.0023) (Fig. 2B). CD31 is a marker of endothelial cells, and its upregulation is usually associated with increased angiogenesis. We examined the expression of CD31 in the ear tissues of mice in each group through immunohistochemical analysis, and the results (Fig. 2C) indicate that, compared with the Ctrl group, the expression level of CD31 in the dermal tissue of mice in the Capsaicin group was upregulated (p < 0.0001), while treatment with GBP and MIN + HCQ inhibited the expression of CD31 after Capsaicin treatment (p = 0.0002). Furthermore, we performed Western blot analysis to investigate the expression of ICAM-1 and VCAM-1, proteins related to angiogenesis, in the ear tissues of mice. The results revealed that, compared with the Ctrl group, the expression levels of ICAM-1 (p < 0.0001) and VCAM-1 (p < 0.0001) in the ear tissues of mice in the Capsaicin group significantly increased; after treatment with GBP and MIN + HCQ, the expression levels of these proteins were significantly downregulated compared to the Capsaicin group (p < 0.0001) (Fig. 2D). These results indicate that both GBP and the combination of MIN and HCQ can inhibit angiogenesis at the molecular level, which may be one of the mechanisms by which they alleviate rosacea symptoms.

In summary, similar to the combination treatment with MIN and HCQ, the application of GBP not only alleviated the capsaicin-induced vasodilation and erythema at the surface level but also significantly affected the expression of angiogenesis-related markers at the molecular level. These findings further confirm the potential role of GBP in treating rosacea and provide scientific rationale for its potential use as an anti-inflammatory and anti-angiogenic therapeutic agent.

GBP suppresses rosacea-like skin inflammation by modulating inflammation-related genes

In investigating the molecular mechanisms of GBP in treating rosacea, our team conducted a detailed analysis using high-throughput transcriptome sequencing technology on an LL37-induced rosacea-like skin inflammation mouse model (Fig. 3A). Previous studies have initially revealed the potential effects of GBP in alleviating LL37-induced rosacea-like skin inflammation. To further explore its mechanism of action, we compared the gene expression patterns between the LL37-treated group and the GBP-treated group. Using |Log2FC | >2 and P < 0.05 as significant selection criteria, we found that compared to the Ctrl group, the LL37 group had 4668 upregulated genes and 2158 downregulated genes (Fig. 3B). Taking the LL37 group as a reference, the GBP-treated group showed 2174 upregulated genes and 3767 downregulated genes (Fig. 3C). Principal component analysis (PCA) clearly separated the Ctrl, LL37, and GBP-treated samples into three distinct groups at the gene expression level, reflecting the significant differences in gene expression under different treatment conditions (Fig. 3D).

Fig. 3. Inhibition of rosacea angiogenesis by GBP.

A Collection of skin tissue from various groups of mice for transcriptome high-throughput sequencing (Created by BioRender); B Volcano plot analysis of DEGs between the Ctrl group and LL37 group from high-throughput transcriptome sequencing, where blue dots represent downregulated genes, red dots depict upregulated genes, and gray dots indicate insignificant genes, n = 3; C Differential gene analysis between LL37 and GBP groups by high-throughput transcriptome sequencing, with blue dots representing downregulated genes, red dots indicating upregulated genes, and gray dots showing insignificant genes, n = 3; D PCA analysis of differential genes between Ctrl, LL37, and GBP groups; E Venn diagram analysis of differential genes between Ctrl, LL37, and GBP groups, with blue circle representing genes differentially expressed in Ctrl and LL37 groups, and red circle indicating genes differentially expressed in LL37 and GBP groups; F Heat map showing the expression of selected inflammatory factors in the intersecting genes.

Furthermore, by intersecting the DEGs between the Ctrl and LL37 groups and the LL37 and GBP groups, a total of 4949 intersecting genes were identified (Fig. 3E). Heatmap analysis visualized the expression of some inflammatory factors among these intersecting genes. In the LL37-treated group, a series of inflammatory factors such as TNF, NF-κB, IL-18, STAT1, STAT4, MAPK1, and Wnk, were significantly upregulated, whereas compared to the LL37 group, the expression levels of these factors were reversed in the GBP group, indicating that GBP may exert its anti-inflammatory effects by influencing the expression of these inflammation-related genes (Fig. 3F).

In conclusion, the results suggest that GBP may suppress LL37-induced rosacea-like inflammatory response by modulating multiple inflammation-related signaling pathways and factors. These findings provide crucial molecular evidence for GBP as a potential drug for treating rosacea.

GBP alleviates rosacea neuroinflammation by modulating the NF-κB pathway

In the aforementioned study, it was observed that GBP significantly downregulates the expression of inflammatory factors in rosacea. Neuroinflammation is an inflammatory process involving the nervous system, typically encompassing the activation of astrocytes and inflammatory cells. Some studies suggest that rosacea patients may experience neurosystem-related abnormalities such as facial sensory alterations and pain. Concurrently, research indicates a potential association between neuroinflammation and the inflammatory papules and swelling in rosacea. In order to further investigate the mechanism of action of GBP in treating rosacea, the following experiments were conducted.

Analysis based on KEGG and GO results indicates a significant shift in the expression pattern of genes post-treatment with GBP compared to the LL37 group. Upregulated genes mainly involve processes such as the generation of precursor metabolites and energy, carbohydrate catabolic processes, carboxylic acid binding, and hydrolase activity acting on glycosyl bonds (Fig. 4A). Conversely, downregulated genes are predominantly related to pathways like positive regulation of response to external stimuli, response to viruses, regulation of T cell activation, NF-kappa B signaling pathway, and TNF signaling pathway (Fig. 4B). These findings suggest that GBP may exert its anti-inflammatory effects by influencing a wide range of gene expression and biological pathways.

Fig. 4. Regulation of the NF-κB pathway by GBP.

A KEGG and GO analyses reveal the upregulated DEGs controlling biological pathways in the LL37 and GBP groups; B KEGG and GO analyses show the downregulated DEGs controlling biological pathways in the LL37 and GBP groups; C, D GSEA analysis indicates the expression pattern of inflammatory factors regulated by Neuroinflammation and Glutamatergic Signaling; E, F GSEA analysis shows the expression pattern of inflammatory factors regulated by the NF-κB Signaling pathway.

Further insights from Gene Set Enrichment Analysis (GSEA) reveal the pivotal role of the NF-κB signaling pathway in the treatment of rosacea with GBP (Fig. 4C–E). The NF-κB pathway serves as a crucial factor in regulating immune and inflammatory responses, with its activation in various cell types closely linked to inflammatory states. Activation of NF-κB in neuroinflammation leads to the release of inflammatory mediators and neuronal damage. In the context of rosacea, this pathway likely plays a key role associated with pathological inflammatory responses. The heatmap of the expression of inflammatory factors from GSEA analysis (Fig. 4F) illustrates that post-LL37 treatment, numerous signal pathways linked to neuroinflammation and glial cell activation are activated, including IL-1β, TNF-α, Casp8, Stat1, NF-Κb, Myd88, among others. Notably, the treatment with GBP significantly reverses the activation status of these pathways, suggesting that GBP may exert its therapeutic effects by inhibiting the activation of these critical inflammatory and neuroinflammation pathways.

In conclusion, these analyses provide insights into the potential mechanisms of action of GBP in treating rosacea. GBP alleviates rosacea neuroinflammation by downregulating the inflammatory responses associated with the NF-κB pathway.

GBP suppresses skin neurogenic inflammation in rosacea mice by downregulating LL37-induced NF-κB pathway activation

Considering the crucial role of inflammation in the occurrence and progression of rosacea, we focused on neuroinflammation pathways and NF-κB signaling, combining the analysis results from KEGG and GSEA. In the realm of neuroinflammation, our attention was directed towards PGP9.5 (Protein Gene Product 9.5), also known as UCHL1, a protein widely expressed in the nervous system and extensively reported for its role in various neuro-related pathological conditions^29,30. Western blot results indicated a significant upregulation of PGP9.5, NF-κB p65, IKKα, and TNF-α expression in the dermal tissues of LL37-treated mice compared to the control group, along with a significant increase in nuclear NF-κB p65 and a notable decrease in IκB-α expression levels. Treatment with GBP following LL37 exposure led to a significant reversal in the expression levels of these proteins (all p < 0.0001) (Supplementary Fig. 2A), further supporting the hypothesis that GBP exerts its therapeutic effects by inhibiting the activation of the NF-κB signaling pathway. Moreover, correlation analysis revealed a positive correlation between nuclear NF-κB p65 and PGP9.5 protein expression (R² = 0.91, P < 0.05) (Supplementary fig. 2B), indicating a close relationship between neuroinflammation and the NF-κB pathway in the development of rosacea.

Subsequently, immunofluorescence results showed that the expression of NF-κB p65⁺PGP9.5⁺ cells in the dermal tissue of mice was significantly upregulated in the LL37 group compared to the Ctrl group, while GBP treatment significantly reduced the expression levels of NF-κB p65⁺PGP9.5⁺ cells compared to the LL37 group (all p < 0.0001) (Fig. 5A). Additionally, we revealed a correlation between the expression of NF-κB p65⁺PGP9.5⁺ and the Skin score index in mice. The correlation analysis showed a strong positive relationship between NF-κB p65⁺PGP9.5⁺ expression and Skin score with a coefficient of R² = 0.7310 and a significant level of P < 0.01 (Fig. 5B), highlighting the significant role of the NF-κB pathway in neuroinflammation in rosacea.

Fig. 5. Inhibition of the NF-κB pathway by GBP.

A Observation of NF-κB p65⁺ PGP9.5⁺ positive cells in the dermal tissues of mice from different groups using immunofluorescence staining, with a scale bar of 50 μm and a local magnification scale bar of 10 μm; yellow arrows represent NF-κB p65⁺ PGP9.5⁺ positive cells, while white arrows depict PGP9.5⁺ positive cells; B Correlation analysis between the expression of NF-κB p65⁺ PGP9.5⁺ positive cells in the epidermis of rosacea lesions in mice (immunofluorescence detection) and the total skin symptom score index (Skin score) of each group of mice (n = 10), using Spearman’s correlation coefficient for analysis; C Treatment of BV2 cells with different concentrations of GBP, followed by CCK-8 assay to evaluate cell viability after 48 hours; D Detection of NF-κB p65 translocation into the nucleus of BV2 cells in different groups using immunofluorescence staining, where white arrows indicate pathway activation, scale bar of 25 μm; E Western blot analysis of Nucleus p65, IKKα, and IKB-α expression in different cell groups. All data are expressed as mean ± standard deviation (SD). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by Dunnett’s T3 and LSD-t post hoc tests. Animal experiments with n = 5 and cell experiments were repeated three times. Statistical significance was indicated as ^ns På 0.05, *P <0.05, **P <0.01, ***P <0.001.

BV2 cells are a murine microglial cell line commonly utilized for modeling inflammatory responses in the central nervous system. In in vitro experiments, BV2 cells are stimulated with LPS to simulate neuroinflammation. Treatment with varying concentrations of GBP resulted in a dose-dependent increase in the inhibition of BV2 cell activity, as demonstrated by CCK-8 assay results (Fig. 5C). Immunofluorescence analysis revealed enhanced expression of NF-κB p65 in the nuclei of BV2 cells post-LPS stimulation compared to the Ctrl group (p = 0.0002), while the addition of GBP significantly suppressed NF-κB p65 expression in the nuclei of BV2 cells post-LPS stimulation (p = 0.0003) (Fig. 5D). Western blot analysis further confirmed these findings, showing increased expression of NF-κB p65 in the nuclei of BV2 cells post-LPS stimulation compared to the Ctrl group (p < 0.0001), which was inhibited by GBP treatment (p = 0.0003). Additionally, the experiment demonstrated enhanced expression of IKKα and downregulation of IKB-α post-LPS stimulation compared to the Ctrl group, and GBP treatment markedly reversed the expression levels of these proteins compared to the LPS group, further elucidating the inhibitory effect of GBP on the NF-κB pathway (all p < 0.0001) (Fig. 5E). ELISA results indicated increased expression of inflammatory factors IL-1β (p = 0.0002), TNF-α (p = 0.0004), IL-6 (p < 0.0001), IFN-γ (p < 0.0001), and IL-8 (p = 0.0002) in BV2 cells post-LPS stimulation compared to the Ctrl group, while the addition of GBP significantly downregulated the expression levels of these factors compared to the LPS group (all p < 0.0001) (Supplementary Fig. 2C-G).

In conclusion, it can be inferred that GBP may exert therapeutic effects on rosacea by suppressing neurogenic inflammation through the inhibition of the NF-κB axis.

GBP Alleviates Rosacea Inflammation by Inhibiting the NF-κB pathway

Based on the above observations, we noted that GBP, like the combination treatment with MIN and HCQ, exhibited significant improvement in reducing skin redness and inflammation in LL37-induced rosacea-like mice. GBP alleviated neurogenic inflammation in the skin by downregulating the NF-κB pathway induced by LL37. To further investigate whether GBP can also alleviate rosacea inflammation via the NF-κB pathway and thus effectively reduce facial symptoms in rosacea patients similar to the combination treatment with MIN and HCQ, we conducted a clinical study. We recruited 60 patients diagnosed with rosacea at our hospital and randomly assigned them to either the MIN and HCQ combination treatment group (MIN + HCQ group, n = 30) or the GBP group (GBP, n = 30). These patients received MIN and HCQ combination treatment or GBP treatment, twice daily for four weeks. After four weeks of treatment, compared to pre-treatment levels, patients in both the GBP group and the MIN + HCQ combination treatment group showed significant improvement in facial symptoms. The Investigator’s Global Assessment (IGA) and Clinical Erythema Assessment (CEA) scores were significantly reduced (all p < 0.0001) (Fig. 6), with no significant difference between the GBP group and the MIN + HCQ combination treatment group (p = 0.5368, p = 0.0553). This indicates that, like the combination treatment with MIN and HCQ, GBP can effectively reduce facial symptoms in rosacea patients.

Fig. 6. GBP alleviates facial symptoms in rosacea patients.

A Facial features of patients in each group before and after treatment; B IGA scores of patients in each group after 4 weeks of treatment; C CEA scores of patients in each group after 4 weeks of treatment. All data are expressed as mean ± standard deviation (SD). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by post hoc tests with Dunnett’s T3 and LSD-t methods. ***P <0.001, ^ns På 0.05.

Additionally, we used ELISA to measure the expression levels of inflammatory cytokines in the serum of patients from each group. The results showed that, compared to pre-treatment levels, the expression of vasoactive intestinal peptide (VIP) in the serum of rosacea patients in both the GBP group and the MIN and HCQ combination treatment group was upregulated. In contrast, the expression levels of NF-κB, NPY, substance P, IL-1β, IL-6, and IL-8 were significantly reduced (all p < 0.0001) (Supplementary Fig. 3A-H). This indicates that GBP significantly alleviates inflammation in rosacea patients by inhibiting the NF-κB pathway.

In summary, like the combination treatment with MIN and HCQ, GBP effectively alleviates facial symptoms in rosacea patients by modulating the NF-κB pathway. This study supports the potential of GBP as an alternative treatment for rosacea and underscores the importance of the NF-κB pathway in the pathogenesis of rosacea.

Metabolomics analysis and biomarker identification of GBP treatment for rosacea

To investigate the potential metabolites of GBP treatment for rosacea, serum samples from 30 rosacea patients were subjected to metabolomics analysis. A total of 306 metabolites were measured using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), with 183 metabolites detected, including important biomolecules such as fatty acids, amino acids, organic acids, and carbohydrates (Fig. 7A).

Fig. 7. Targeted metabolomics identification of serum metabolic profiles in rosacea patients.

A Statistical analysis of serum metabolites categories in rosacea patients before and after treatment with GBP; B Bar graph showing KEGG pathway enrichment analysis of serum metabolites in rosacea patients before and after treatment with GBP; C Correlation analysis of serum metabolites and inflammatory factors in rosacea patients before and after treatment with GBP. The color in the figure ranges from dark to light, indicating a correlation from 1 to -1. The larger the circle, the higher the correlation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to evaluate the metabolomics dataset aiming to discern chemical differences between the pre-treatment and post-treatment (0 week vs. 4 weeks) GBP groups of rosacea patients. The OPLS-DA analysis yielded model parameters of R²Y = 0.958 and Q² = 0.923, both exceeding 0.5, indicating good model fitting and predictive capability. The OPLS-DA score plot revealed distinct metabolomics features and successfully differentiated two main clusters (Supplementary Fig. 4A-C). Additionally, a volcano plot was used to visually demonstrate the overall distribution of metabolic differences in rosacea patients before and after treatment, showing significant changes in metabolites post-treatment (Supplementary Fig. 4D).

Based on variable importance in the projection (VIP-score) values generated after OPLS-DA analysis (VIP-score > 1) with a significance threshold set at a P-value of 0.05 for significantly different metabolites, 40 important biomarkers were identified for further study. Metabolic pathway analysis indicated that these 40 biomarkers mainly pertained to Glyoxylate and dicarboxylate metabolism, Glycine, serine, and threonine metabolism, Pyrimidine metabolism, Citrate cycle (TCA cycle), and other metabolic pathways (Fig. 7B).

Furthermore, correlations between IL-1β, IL-6, IL-8, TNF-α, IFN-γ, substance P, NPY, and serum metabolites were explored. The results revealed correlations of IL-1β with hydroxyurea, IL-6 with phenyl pyruvic acid, glutamyl valine, adenine, 2-methyl-5-(1-propenyl) pyrazine, TNF-α with dibutyl phthalate, substance P with uridine, 5-methylcytidine, 5-hydroxyindoleacetic acid, phenylalanyl-isoleucine, norophthalmic acid, malic acid, 9-decenoic acid, and dibutyl phthalate, and NPY with 5-methylcytidine and dibutyl phthalate (Fig. 7C).

In conclusion, these findings suggest that the therapeutic effects of GBP in treating rosacea may be attributed to its influence on specific metabolic pathways highlighted above, potentially ameliorating inflammatory conditions by modulating the levels of these metabolites.

Discussion

In this study, we employed a robust LL37-induced rosacea-like mouse model and conducted detailed histopathological observations to precisely demonstrate that the therapeutic effects of GBP are comparable to those of the combination treatment with MIN and HCQ, both of which significantly alleviate skin inflammation. Experimental results showed effective improvement in symptoms such as vasodilation and erythema, consistent with previous animal model experiments^31,32. Various techniques, including IHC staining, immunofluorescence, Western blot, and RT-qPCR, comprehensively evaluated the mechanism of action of GBP on inflammation regulation, providing a molecular-level explanation for treating rosacea^33,34.

Compared to previous studies, this research employed transcriptome high-throughput sequencing and delineated a comprehensive transcriptome profile of inflammation in the GBP-treated rosacea model, revealing potential key regulatory pathways and mechanisms^11,12. By leveraging this technology, the study presented the regulatory effect of GBP on the NF-κB pathway, offering a novel explanation for the inhibition of neurogenic inflammation and supplementing existing research uniquely³⁵.

In clinical studies, a significant reduction of facial erythema symptoms in patients further verified the efficacy observed in the laboratory. Measurement of serum inflammatory factors levels through ELISA technology confirmed the correlation between changes in metabolites identified in metabolomics analysis and inflammatory factors, providing stronger support for understanding the treatment mechanism³⁶. The consistency of these results further reinforces the efficacy and mechanistic understanding of GBP in treating rosacea.

The crucial role of the NF-κB pathway in inflammation regulation has been extensively studied, and our research further revealed that GBP positively regulates this pathway, achieving significant inhibition of neurogenic inflammation^37,38. This study delves deeper into the mechanism of the NF-κB pathway, offering a more detailed explanation for the treatment of neurogenic inflammation and holding some innovative value in the field.

In conclusion, this study utilized high-throughput transcriptome sequencing and metabolomics analysis to uncover the molecular mechanisms of GBP in treating rosacea, particularly its role in reducing neuropeptide release by regulating the NF-κB pathway, thus inhibiting neurogenic inflammation (Fig. 8). Specifically, GBP notably alleviated skin inflammation symptoms, such as vasodilation and erythema, in the rosacea-like mouse model and effectively reduced facial erythema in rosacea patients clinically. Transcriptome sequencing analysis revealed that GBP mainly influences the inflammatory pathways regulated by NF-κB, while metabolomics analysis showed significant differences in metabolites before and after GBP treatment, with these metabolites positively correlating with key inflammatory factors such as IL-1β, IL-6, IL-8, TNF-α.

Fig. 8. The mechanism of NF-κB regulating neurogenic peptide release to promote neurogenic inflammation aggravates the condition of rosacea (Created by BioRender).

The proposed working model of gabapentin (GBP) in the treatment of rosacea. In rosacea, activation of neurons promotes neurotransmitter release and triggers neurogenic inflammation through activation of the NF-κB signaling pathway, leading to vascular dilation, increased vascular permeability, and inflammatory responses in cutaneous blood vessels. GBP suppresses neuronal NF-κB activation, thereby reducing neuroinflammatory signaling, limiting neurotransmitter release, and subsequently alleviating vascular abnormalities and inflammation associated with rosacea.

This study holds scientific and clinical significance in elucidating the potential mechanisms of GBP in treating rosacea, offering new insights and pathways for disease management. Scientifically, experimental results demonstrate that GBP significantly alleviates skin inflammation in rosacea-like mice, and its efficacy in reducing facial redness and swelling in rosacea patients was confirmed in clinical studies, providing a novel mechanistic understanding for treating the disease. Furthermore, through transcriptomic and metabolomics analyses, its regulatory mechanisms on inflammatory factor expression were unveiled, laying a theoretical foundation for conducting more precise treatments and research in the future. Clinically, this research presents new avenues for the treatment strategy of rosacea, potentially improving patients’ clinical symptoms and quality of life.

This study has several limitations. First, interspecies differences between animal models and humans may limit the direct applicability of the findings. Second, the small sample size may undermine the reliability of clinical efficacy assessments, necessitating validation through larger-scale studies. Additionally, as gabapentin represents a novel application for rosacea, our pretreatment protocol (initiated before LL37-induced lesions), based on previous studies, assessed early therapeutic efficacy rather than direct reversing effect; future work will optimize dosing regimens to better model clinical treatment. While our data implicate the NF-κB pathway in gabapentin’s effect, pathway specificity requires further validation using inhibitors or gene editing. Furthermore, the neuroinflammation findings rely solely on BV2 cells; primary neuronal cultures are needed to confirm gabapentin’s role. Finally, the relative contributions of direct neuronal regulation versus indirect effects via immune cells (e.g., macrophages) to the observed anti-inflammatory effects remain undetermined.

Looking ahead, future exploration could delve into the therapeutic potential of GBP in other inflammatory skin conditions, deepen the understanding of its mechanisms of action, and expand its clinical applications. By integrating more omics technologies and clinical trial data, considering multiple factors comprehensively, and enhancing the level of individualized treatment, more effective treatment strategies can be provided to patients. In conclusion, this study offers new perspectives and pathways for a deeper understanding and treatment of rosacea, potentially paving the way for novel directions in the treatment and research of related inflammatory diseases.

Materials and methods

Animal experimentation

Female BALB/c mice (age 6-8 weeks, weight 23 ± 2 g) were purchased from Beijing VitalHD Animal Experimental Technology Co., Ltd. (strain code: 211). All mice were housed in an SPF-grade animal facility with a humidity of 60% ~ 65% and a temperature of 22 ~ 25 °C, with ad libitum access to food and water. The mice were acclimated for one week before the experiment, during which their health status was monitored. The experimental procedures and animal usage protocol were approved by the Institutional Animal Care and Use Committee.

To induce skin inflammation in a rosacea mouse model, the mice were depilated on their backs 24 hours prior to the experiment. Anesthetized with pentobarbital sodium (50 mg/kg), the mice were subcutaneously injected with 40 μL of 320 μM LL37 (HY-P1222, MCE) twice a day for two days, wild-type mice were used as controls. Skin inflammation in the rosacea mouse model was evaluated based on the severity of erythema and edema, as well as skin thickness measured using a vernier caliper^39–41.

Gabapentin was administered by oral gavage at a dose of 40 mg/kg once daily for 14 consecutive days. At the end of the treatment, skin tissues were collected from each group of mice and stored at -80 °C for subsequent experiments^42,43.

In the combination treatment group, minocycline (MIN) and hydroxychloroquine (HCQ) were administered by oral gavage at doses of 50 mg/kg and 40 mg/kg body weight, respectively. The dosing schedule was consistent with that of gabapentin: once daily for 14 consecutive days^44–46.

Regarding the treatment with capsaicin, on the 4th day following GBP treatment, capsaicin was applied to the surface of the left and right ears of the mice in each group. After 30 minutes, the pathological condition of the ears was observed, representative images were taken, and ear tissues were collected for subsequent experimental analysis.

Hematoxylin and eosin (H&E) staining

Skin or ear tissue samples were fixed in fresh 4% neutral buffered formalin for 24 hours, followed by gradient ethanol dehydration, clearing, and routine paraffin embedding. Tissue sections of 5 µm thickness were cut on a paraffin microtome, baked at 60 °C for 1 hour, and deparaffinized with xylene. After hydration, H&E staining (C0105S, Beyotime) was performed. The sections were initially stained in hematoxylin for 3 minutes, rinsed in distilled water for 10 seconds, and differentiated in 1% hydrochloric acid alcohol for 10 seconds. After a 1-minute distilled water rinse, the sections were stained with eosin for 1 minute, briefly rinsed in distilled water for 10 seconds, dehydrated in gradient alcohol, cleared in xylene, and mounted with neutral resin^47,48. The slides were then observed for pathological changes under an Olympus CK2 light microscope.

Immunohistochemistry

Ear tissues from each group were fixed and embedded in paraffin. Sections were deparaffinized and subjected to antigen retrieval by heating in citrate buffer (pH 6.0) (005000, Thermo Fisher) for 20 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (88597, Millipore) for 10 minutes at room temperature, followed by incubation with 5% BSA for 30 minutes. Sections were then incubated overnight at 4 °C with a specific CD31 antibody (ab182981, 1:2000, Abcam). Subsequently, sections were incubated with HRP-conjugated anti-rabbit secondary antibody (ab6721, 1:1000, Abcam) for 1 hour at room temperature. DAB solution (ab64238, Abcam, USA) was used for chromogenic detection, followed by mounting. For each group, 5 animals were used, and one section per animal was stained. Three random fields were selected for image acquisition. CD31 expression was quantified using ImageJ software²⁸.

Immunofluorescence staining

Mouse skin tissue sections were incubated in PBST (28352, ThermoFisher) at room temperature for 1 hour. Following PBS wash, samples were blocked with 5% goat serum (C0265, Beyotime) and then incubated overnight at 4 °C with primary antibodies against NF-κB p65 (ab32536, 1/100, Abcam) and PGP9.5 (ab108986, 1/500, Abcam). Subsequently, samples were washed 3 times with PBS, followed by incubation with goat anti-rabbit IgG (H + L) Alexa Fluor® 647 secondary antibody (ab190589, 1/100, Abcam) or goat anti-rabbit IgG (H + L) Alexa Fluor® 488 secondary antibody (ab281847, 1/100, Abcam) for 3 hours, and then washed 3 times with PBS again. Finally, the samples were mounted with an anti-fade mounting medium containing DAPI (4083, CST) or Hoechst (4082, CST), and images were acquired and observed under a laser scanning confocal microscope (LSM 980, ZEISS). Five animals were included in each group, with one slice stained per animal, and 3 fields of view were selected for image capture. After image acquisition, the number of positive cells was quantified using ImageJ software (National Institutes of Health)⁴⁹.

RT-qPCR

Total RNA was extracted from the skin tissues of each group using the Trizol reagent kit (T9424, Sigma-Aldrich). The quality and concentration of RNA were determined using a UV-visible spectrophotometer (ND-1000, Nanodrop, USA). For mRNA expression level detection, reverse transcription was performed using the PrimeScript™ RT kit (RR014B, TaKaRa, Japan). Real-time quantitative reverse transcription PCR (RT-qPCR) was performed with the TB Green Premix Ex Taq™ kit (RR420W, Takara, Japan) on an ABI 7500 PCR instrument (Applied Biosystems, USA). GAPDH was used as an internal control, and primer sequences are provided in Table 1. Relative quantification analysis was performed by comparing the Ct values using the 2^-ΔΔCt formula, where ΔΔCT = ΔCt experimental group - ΔCt control group, and ΔCt = target gene Ct - reference gene Ct⁵⁰.

Table 1.

RT-qPCR primer sequence

Gene Name	Primer Sequence
TNF-α(mouse)	Forward: 5’-GATCGGTCCCCAAAGGGATG-3’
	Reverse: 5’-CCACTTGGTGGTTTGTGAGTG-3’
IL-6(mouse)	Forward: 5’-TGGTCTTCTGGAGTACCATAGC-3’
	Reverse: 5’-TGTGACTCCAGCTTATCTCTTGG-3’
IL-1β(mouse)	Forward: 5’-TGCCACCTTTTGACAGTGATG-3’
	Reverse: 5’-TGATGTGCTGCTGCGAGATT-3’
GAPDH(mouse)	Forward: 5’-AAGAGGGATGCTGCCCTTAC-3’
	Reverse: 5’-GTTCACACCGACCTTCACCA-3’

Western blot experiment

Cellular or tissue total protein was extracted using the Protein Extraction Kit (BC3710, Solarbio), while the Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime) was used for isolating nuclear proteins. The protein concentration was determined using the BCA Protein Quantification Kit (P0010, Beyotime). Proteins were separated by 10% SDS-PAGE, transferred to a PVDF membrane (IPFL00010, Millipore), and blocked with 5% skim milk (P0216, Beyotime) at room temperature for 1 hour. The membranes were then incubated overnight at 4 °C with diluted primary antibodies: PGP9.5 (MA1-83428, 1/1000, Invitrogen), NF-κB p65 (ab32536, 1/1000, Abcam), ICAM-1 (#4915, 1/1000, CST), VCAM-1 (#32653, 1/2000, CST), IKB-α (ab32518, 1/1000, Abcam), IKKα (ab32041, 1/10000, Abcam), TNF-α (ab183218, 1/1000, Abcam), β-actin (ab8226, 1/1000, Abcam), Histone H1 (PA5-30055, 1/1000, Invitrogen). The membranes were then washed thrice with TBST (3×5 minutes) and incubated with Anti-Rabbit-IgG secondary antibody (7074, 1/1000, CST) or Anti-Mouse-IgG (ab205719, 1/2000, Abcam) at room temperature for 1 hour. After another three washes with TBST (3×5 minutes), excess TBST was removed. An appropriate amount of ECL working solution (WBULS0500, EMD Millipore, USA) was prepared, and the PVDF membrane was incubated in the ECL developing solution at room temperature for 1 minute. The excess ECL working solution on the PVDF membrane was removed, the membrane was sealed with plastic wrap, placed in a dark box, and exposed to X-ray film for 5-10 minutes, followed by visualization and fixing. The Western blot band intensities were quantified using Image J analysis software, with β-actin and Histone H1 used as internal controls⁵¹. Uncropped and unedited blot/gel images (Supplementary Fig. 5) are provided in the Supplementary Information.

ELISA detection

In this study, the expression levels of NF-κB (CSB-E08787h, CUSABIO), VIP (E-EL-H2155, Elabscience), NPY (CSB-E08168h, CUSABIO), Substance P (CSB-E08357h, CUSABIO), IL-1β (ab214025, Abcam), IL-6 (ab178013, Abcam), IL-8 (ab214030, Abcam), TNF-α (ab181421, Abcam), and IFN-γ (E-EL-M0048, Elabscience) in the serum of various patient groups were measured using ELISA assay kits. Antigens were first diluted to the appropriate concentration in coating diluent, and the diluted samples were added to the enzyme-linked immunosorbent assay plate wells. Subsequently, enzyme-labeled antibodies and substrate solution were added, followed by the addition of 50 μL stop solution to each well to terminate the reaction, with experimental results determined within 20 minutes. The plates were read at 450 nm using an ELISA reader (1681135, Bio-Rad, USA), standard curves were plotted, and data were analyzed⁵² according to the manufacturer’s instructions. Each sample was tested in triplicate to ensure result accuracy.

Transcriptome high-throughput sequencing and data analysis

Mouse skin tissue samples from each group were preserved in RNAlater storage solution (76104, Qiagen) to maintain RNA stability. Transcriptome high-throughput sequencing was performed using RNA sequencing. Specific steps included extracting total RNA from each sample using Trizol reagent (T9424, Sigma-Aldrich) following the manufacturer’s instructions. The quality and concentration of RNA were assessed using UV-visible spectrophotometry (840-343700, Thermo Scientific), with an A260/280 ratio between 1.8-2.0. The total RNA content of each sample was 3 μg, which served as the input material for RNA sample preparation. Following the manufacturer’s protocol, cDNA libraries were generated using NEBNext® UltraTM II RNA Library Prep Kit (E7770S, Gene Company, China) suitable for Illumina, and their quality was evaluated on an Agilent Bioanalyzer 2100 system. Subsequently, index-coded samples were clustered using TruSeq PE Cluster Kit v3 cBot HS (Illumina) on the cBot cluster generation system. Upon cluster generation, library preparation was sequenced on the Illumina HiSeq 550 platform, generating 125 bp/150 bp paired-end reads⁵³.

After obtaining the gene expression matrix, differential analysis was conducted using the R language limma package, with thresholds set at |log2FC | > 2 and P < 0.05 to identify differentially expressed genes (DEGs). The DEGs were visualized using the “pheatmap” package in the R software to create a volcano plot of DEG expression levels⁵⁴.

Venn diagrams and gene function enrichment analysis

The differential gene sets were subjected to Venn analysis using the Draw Venn Diagram tool to identify intersecting genes⁵⁵. Subsequently, the intersecting genes were analyzed for KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) enrichment pathways using the R software package clusterProfiler with a significance level set at P < 0.05. The results were visualized through bubble plots representing the biological processes (BP), cellular components (CC), and molecular functions (MF) within the GO categories⁵⁶.

Cellular experiments

BV2 cells (BFN608006363, BFB) were cultured in high-glucose DMEM (11965118, Gibco) supplemented with 10% FBS (10270106, Gibco) and 1% penicillin-streptomycin (15070063, Gibco). The cells were maintained in a humidified incubator at 37 °C with 5% CO₂. To induce inflammation, BV2 cells were stimulated with 1 μg/ml lipopolysaccharide (LPS) (HY-D1056, MCE) for 24 hours. Subsequently, cells were treated with 0, 5, 10, 15, and 20 μg/mL of GBP (XW601429632, China Pharmaceutical Group) for 48 hours before harvesting for further experiments.

CCK-8 assay

The CCK-8 assay (CK04, Dojindo Laboratories, Japan) was employed to determine cell viability. BV2 cells were seeded at a density of 5 × 10³ cells per well in a 96-well plate. Every 24 hours, CCK-8 solution (10 μL/well) was added, followed by the addition of 100 μL serum-free medium. After 2 hours of incubation at 37 °C, the optical density (OD) was measured at 450 nm. Cell viability was calculated as % cell survival = [(sample well - blank well) / (control well - blank well)] × 100%⁵⁷.

Clinical sample collection

We collected clinical samples from 60 newly diagnosed and untreated rosacea patients between January and December 2023. Participants were selected based on the 2017 diagnostic criteria set by the National Rosacea Society Expert Committee⁵⁸. Participants were randomly assigned into two groups: a control group receiving combined treatment with MIN and HCQ, and a treatment group receiving GBP. Each group consisted of 30 individuals, none of whom had any other metabolic comorbidities. Exclusion criteria for all participants included systemic diseases, other skin conditions, a history of systemic immunomodulators or antibiotics use, extreme diets in the past 12 weeks, pregnancy, and lactation. Clinical assessment utilized the Investigator’s Global Assessment (IGA) and Clinical Evaluator’s Assessment (CEA) scores, with detailed recording of demographic and baseline clinical characteristics (Table 2). The dosages for the medications were based on the literature⁵⁹. For the GBP treatment group, oral GBP (National Medicines Group XW601429632) was administered at a dose of 300 mg three times daily for a total of 4 weeks. For the combination treatment group, both MIN (National Medicine Approval Number H10950348) and HCQ (National Medicine Approval Number H19990263) were administered at a dose of 100 mg once a day and 200 mg twice daily, respectively, also for a duration of 4 weeks. Blood samples were collected at the initial diagnosis and follow-ups after fasting for 10 hours, followed by a 30-minute rest post-blood draw to obtain serum, which was immediately stored at -80 °C for subsequent analysis. All patients signed informed consent forms prior to enrollment.

Table 2.

Demographic and baseline clinical characteristics of healthy controls and patients with rosacea

Characteristics	Healthy Controls (n = 30)	Patients with Rosacea (n = 60)	P Value
Sex	100% female	100% female	1
Age, mean (SD)	31.41 (9.69)	32.67 (12.35)	0.317
BMI, mean (SD)	21.42 (3.88)	21.79 (3.15)	0.4721
Ethnicity	100% Chinese	100% Chinese	1
CEA,n (%)
Clear		1 (1.67)
Almost clear		13 (21.67)
Mild		23 (38.33)
Moderate		18 (30.00)
Severe		5 (8.33)
IGA,n (%)
Clear		2 (3.33)
Almost clear		11 (18.33)
Mild		28 (46.67)
Moderate		15 (25.00)
Severe		4 (6.67)

Metabolomics sequencing and data analysis

In this study, serum samples were collected from rosacea patients before (0 weeks) and after (4 weeks) receiving GBP treatment. Each sample had 25 μL of serum added to a 96-well plate and underwent sample processing with cold methanol (120 μL) containing internal standards. The internal standard components include L-phenylalanine (50 nM), L-tryptophan (50 nM), citric acid (10 μM), lactic acid (50 nM), uridine (50 nM), and tridecanoic acid (50 nM)⁶⁰. The samples were vigorously vortexed for 5 minutes and then centrifuged at 4000 g for 30 minutes. After transferring 30 μL of the supernatant to a new 96-well plate with 10 μL of internal standards, the samples were further diluted with 300 μL of a 50% ice-cold methanol solution and underwent repeat centrifugation. Finally, 135 μL of the supernatant was used for analysis, and the prepared samples were stored at -80 °C. Metabolomics analysis was carried out using a combination of an LC20A ultra-high-performance liquid chromatograph (Shimadzu) and a Triple TOF-6600 LC-MS/MS mass spectrometer (SCIEX). The chromatographic analysis was performed using a Waters ACQUITY UPLC HSS T3 C18 column (100 × 2.1 mm, 1.8 μm, Waters) at a column temperature of 40 °C and a flow rate of 0.4 mL/min. The mobile phase consisted of a solvent composition of 0.1% formic acid (A) and acetonitrile water (70:30, B). The gradient elution program of mobile phase B was as follows: 0-1 min (5% B), 1-11 min (5%-78% B), 11-13.5 min (78%-95% B), 13.5-14 min (95%-100% B), 14-16 min (100% B), 16-16.1 min (100%-5% B), 16.1-18 min (5% B); with a sample volume of 5.0 μL. Mass spectrometry conditions included an ionization voltage of 5500 V, capillary temperature of 550 °C, spray gas flow rate of 50 psi and auxiliary heating gas flow rate of 60 psi. Orthogonal partial least squares-discriminant analysis (OPLS-DA) and permutation tests (100 permutations) were applied to preprocess the data to prevent overfitting. Metabolites with VIP-score >1 and p-values < 0.05 in the OPLS-DA model were identified as differential metabolites (DMs). Additionally, combining univariate analysis, metabolites with fold changes ≥2 and ≤0.5, as well as a Student’s t-test p-value < 0.05, were selected as the final DMs. MetaboAnalyst (Version 5.0) was used to identify relevant metabolic pathways⁶¹.

Statistics and reproducibility

In our study, we utilized R version 4.2.1 and conducted data analysis in the RStudio integrated development environment (version 2022.12.0-353). Data were processed using GraphPad Prism 8.0, with quantitative data presented as mean ± standard deviation (Mean ± SD). Group comparisons were performed using unpaired t-tests for two-group analyses and one-way analysis of variance for multi-group comparisons. Levene’s test was employed to assess the homogeneity of variances. In the case of homogeneity, Dunnett’s T3 and LSD-t tests were carried out for pairwise comparisons, while Dunnett’s T3 test was used for heteroscedastic data⁶². Furthermore, χ2 tests and Spearman correlation analyses were conducted to examine the relationship between variables⁶³. A significance level of P < 0.05 was considered statistically significant for group-to-group comparisons. For clinical studies, a total of 60 newly diagnosed untreated rosacea patients were enrolled and randomized into two treatment arms (30 per group). Each participant represented one independent biological replicate for both clinical scoring (IGA, CEA) and serum metabolomic assays; no technical replicates were performed for the clinical scores. For animal experiments, each group contained 5 mice, and each mouse was treated as an independent biological replicate. For histology (H&E), immunohistochemistry, and immunofluorescence, one section per animal was prepared, and three randomly chosen fields of view per section were imaged for quantification.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Ethics approval and consent to participate

All animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University (No. IACUC-CQMU-2024-0832). This study was approved by the Clinical Ethics Committee of Chongqing Medical University (No. 2024-111-01) and adhered to the principles of the Helsinki Declaration, all patients signed informed consent forms. All ethical regulations relevant to human research participants were followed.

Author contributions

Z.Q.J., T.D., and Y.Z. contributed equally to study design, experimental work, and data analysis. M.L. and Y.T. assisted in conducting transcriptomic and metabolomic experiments and provided technical support. Y.X.Z. participated in clinical data collection and statistical analysis. B.W. conceived and supervised the study, provided critical revisions, and finalized the manuscript. All authors reviewed and approved the final version of the manuscript.

Peer review

Peer review information

Communications Biology thanks Marek Sanak and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Connie Wong and Joao Valente.

Data availability

The sequencing data has been deposited in the NCBI database with the following details: - Submission: SUB15316822 - BioProject: PRJNA1262076. The SRA IDs are listed below: Control group: - SRR33537231 - SRR33537230 - SRR33537229. LL37 group: - SRR33537228 - SRR33537227 - SRR33537226. GBP group: - SRR33537225 - SRR33537224 - SRR33537223. Uncropped and unedited blot/gel images (Supplementary fig. 5) are provided in the Supplementary Information. Numerical source data underlying the graphs and charts presented in the main figures can be found in Supplementary Data file.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-026-09662-3.