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. 2025 May 16;20(5):e0323598. doi: 10.1371/journal.pone.0323598

Minocycline inhibits rosacea-like inflammation through the TLR4-mediated NF-κB signaling pathway in vivo and in vitro

Piyan Hua 1,#, Ying Tu 1,#, Zhenghui Yang 1, Yunting He 1, Li He 1, Qiuyan Yao 1, Hua Gu 1,*
Editor: Divakar Sharma2
PMCID: PMC12083832  PMID: 40378103

Abstract

Background

Rosacea is a chronic inflammatory skin disease characterized by multiple intricate pathogenic factors. Previous studies have substantiated the anti-inflammatory properties of minocycline and its potential therapeutic efficacy in treating rosacea. However, further elucidation of the underlying mechanism is warranted.

Methods

HaCaT cells and BALB/c mice were treated with LL37. Moreover, the effect of minocycline on rosacea was explored through the addition of an NF-κB inhibitor (PDTC) or overexpression of Toll-like receptor 4 (TLR4). The expression of related markers was detected by western blotting, immunofluorescence, ELISA, flow cytometry, etc.

Results

Minocycline suppressed dermal infiltration of inflammatory cells in rosacea-like mice and reduced the expression of inflammatory cytokines in rosacea-like mice and cells. Moreover, minocycline downregulated the expression of TLR4 and p-NF-κB thereby inhibiting ROS production. However, overexpression of TLR4 or the addition of PDTC counteracted the effects of minocycline by promoting cellular inflammation and ROS production. Mechanistically, minocycline hinders TLR4/TNF-α activation induced by LL37 in skin and cells to suppress the expression of inflammatory cytokines.

Conclusion

Minocycline alleviates inflammation progression in rosacea by downregulating TLR4 and inhibiting the activation of the NF-κB pathway, providing a scientific basis for subsequent clinical treatment.

1 Introduction

Rosacea is a recrudescent chronic inflammatory skin condition that is characterized by recurrent episodes of erythema, telangiectasia, and papulopustular lesions involving predominantly on the convexities of the central face [1]. However, the etiology and pathogenesis of rosacea have not been fully elucidated. Currently, it is believed that genetics, immune and neurovascular dysregulation, microbial infection, skin barrier dysfunction, and other factors play important roles in the occurrence and exacerbation of rosacea [2]. Due to its tendency to recur easily along with poor compliance and limited treatment efficacy, the quality of life for patients with rosacea is often significantly impacted [3]. Though there is no cure for rosacea, its manifestations may be reduced or controlled with a series of topical, and oral therapies, light devices, appropriate skincare, and lifestyle management [4].

Minocycline, a second-generation derivative of the tetracycline family, possesses antibacterial, anti-inflammatory, immunoregulatory, neuroprotective, and anti-angiogenic properties. It is commonly utilized for the treatment of respiratory infections, genital tract infections, and skin disorders such as acne and rosacea [5–10]. Eady EA et al. [11] have confirmed that minocycline effectively improves acne through its regulation of inflammatory cytokines. β-Hydroxybutyrate and minocycline demonstrate synergistic efficacy in attenuating heat stress-induced neuroinflammation through targeted modulation of the Toll-like receptor 4 (TLR4)/p38 mitogen-activated protein kinase (MAPK) and Nuclear Factor-κ B (NF-κB) signaling cascades [12]. A meta-analysis has shown that 100mg minocycline in the treatment of rosacea was more effective than 40mg doxycycline, 40mg minocycline, topical ivermectin, and 0.75% metronidazole [13]. However, the specific mechanism of minocycline treatment of rosacea has not been clarified.

Toll-like receptor 4 (TLR4) plays a pivotal role in the signaling pathways associated with chronic inflammation [14]. TLR4, as an intact transmembrane protein, initiates downstream signaling cascades via kinases to activate transcription factors like Activator Protein-1 (AP-1) and NF-κB, thereby exerting its specific function in inflammation through the NF-κB signaling pathway [15]. The NF-κB signaling pathway has two distinct activation pathways, with the canonical pathway inducing the expression of genes that regulate immune and inflammatory responses [16]. In epithelial cells, NF-κB plays a significant role in maintaining skin immune homeostasis. A study by Pasparakis M et al. [17] confirmed that IkappaBkinase (IKK)/NF-κB signaling in epidermal keratinocytes is crucial for regulating skin immune homeostasis. Multiple studies have indicated a close association between the TLR4/NF-κB signaling pathway and inflammatory response [18,19]. However, the precise molecular mechanism underlying how the TLR4/NF-κB signaling pathway mediates skin inflammation in rosacea remains incompletely understood. Here, the potential therapeutic role and the possible mechanism of minocycline on rosacea in vivo and in vitro have been investigated in this study.

2 Methods

2.1 Cell culture

HaCaT cells (immortalized human keratinocyte cell line) were purchased from the American Type Culture Collection (ATCC) and incubated in DMEM (Gibco, USA) supplemented with 10% fetal calf serum at 37 °C and 5% CO2 and 95% humidity in the incubator.

2.2 Cell transfection

TLR4 overexpression plasmid and its negative control were purchased from GeneChem (Shanghai, China). Lipofectamine 2000 (Invitrogen) was used to transfect cells. The overexpression efficiency of the constructs was confirmed by western blotting 48 h after transfection, after which the oe-TLR4 cells were used in subsequent experiments.

2.3 Cell grouping

HaCaT cells in the Negative Control (NC) group were cultured normally without any intervention. One group of HaCaT cells was stimulated with Leucine-Leucine-37 (LL37) at 8 μM for 1 h. The cells in the LL37 + Minocin group were treated with 10 μM minocycline for 2 h and then stimulated with 8 μM LL37 for another 1 h. The oe-TLR4 cells in the LL37 + Minocin+oe-TLR4 group were treated the same as the LL37 + Minocin group. For the LL37 + Minocin+oe-TLR4 + PDTC group, NF-κB inhibitor (PDTC, 60 μmol/L) was added in the oe-TLR4 cells and then treated as the LL37 + Minocin group.

2.4 Animal

Seven-week-old BALB/c mice were purchased from Shanghai SLAC Experimental Animal Co. Ltd. (Shanghai). All animal experiments were carried out under specific pathogen-free conditions. In the process of feeding mice, keep the feeding environment clean, and quiet, temperature (22 ± 2) °C, humidity (50 ± 5) % constant, and provide adequate food and clean drinking water, to reduce the stress and pain caused by external environmental factors on mice. All studies and experimental procedures were approved by the Animal Ethics Committee of The First Affiliated Hospital of Kunming Medical University(kmmu20231144). The mice were divided into five groups (the NC group, the LL37 group, the LL37 + Minocin group, the LL37 + Minocin+oe-TLR4 group, and the LL37 + Minocin+oe-TLR4 + PDTC group) and treated differently. The mice were shaved 24 h before treatment and then injected subcutaneously with 40 μL of LL37 peptide twice a day for 3 days. The mice in the LL37 + Minocin group were administered an intraperitoneal injection of minocycline at a dose of 50 mg/kg/day for 3 days. For the LL37 + Minocin+oe-TLR4 group, Minocin-treated mice were injected with 100 μg of oe-TLR4 plasmid via the tail vein. For the LL37 + Minocin+oe-TLR4 + PDTC (5108–96–3, AbMole) group, the mice were intraperitoneally injected with PDTC at a dose of 50 mg/kg. The experiment was performed 72 h after the initial injection of LL37. In order to alleviate the pain of mice, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40mg/kg) before the experimental operation. After the experiment, the mice were euthanized by cervical dislocation. Skin inflammation was evaluated by the severity of erythema and edema. Skin tissues were collected for protein extraction, hematoxylin-eosin staining, and immunofluorescence analysis.

2.5 Western blot

Cells and skin tissues were collected, total proteins were extracted using RIPA buffer containing 1% protease inhibitors, and the total protein concentration was determined by the BCA method. The proteins were heated in a boiling water bath for 5 min to fully denature the proteins, after which the target bands were separated via 10% SDS‒polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to PVDF membranes using a Bio-Rad standard wet transfer apparatus. After the membrane was completely transferred, the membranes were blocked with 5% skim milk for 1 h at room temperature by shaking slowly on a shaker. After blocking, the primary antibodies anti-TLR4 (1:1000, ab217274, Abcam), anti-NF-κB (1:1000, ab207297, Abcam), anti-p-NF-κB (1:1000, ab76302, Abcam) and anti-β-actin (1:1000, ab8226, Abcam) were diluted at 4 °C in a shaker with gentle shaking overnight. The next day, the HRP-labeled secondary antibody (1:5000, ab205718, Abcam) was added at an appropriate concentration, and the samples were incubated at room temperature with gentle shaking for 1 hour. The protein bands were visualized by enhanced chemiluminescence (ECL) solution and analyzed by ImageJ. The experiment was repeated three times.

2.6 Immunofluorescence staining

The expression of tumor necrosis factor-α (TNF-α) in cells and skin tissues was detected via immunofluorescence staining. Cells and frozen skin tissue sections were fixed with 4% paraformaldehyde (Thermo Fisher Scientific, Shanghai, China) for 20 min, blocked with PBS containing 5% bovine serum albumin for 1 h, and incubated with primary antibodies anti-TNF-α (1:250, ab96879, Abcam) overnight at 4 ° C, and then incubated with FITC-labeled secondary antibodies at room temperature for 1 h. The nucleus was stained with DAPI for 30 min, and the results were imaged by a fluorescence microscope.

2.7 Reactive oxygen species (ROS) detection

The expression of ROS in cells was detected by fluorescence staining according to the instructions of the ROS detection kit (Thermo Fisher Scientific, Shanghai, China). The level of tissue ROS was detected by flow cytometry and analyzed by FlowJo software.

2.8 Enzyme-linked immunosorbent assay (ELISA)

The levels of TNF-α, Interleukin-6(IL-6), Interleukin-1α(IL-1α), and Interleukin-1β(IL-1β) in cells were quantified using an ELISA kit (Thermo Fisher Scientific, Shanghai, China) following the manufacturer’s instructions. The absorbance of both standards and samples was measured using a microplate reader, and the expression levels were calculated according to the manufacturer’s instructions.

2. 9 HE staining

The mouse skin tissues were fixed in 4% paraformaldehyde, then embedded in paraffin, dehydrated, and sectioned into slices. The tissue sections were stained with hematoxylin or Eosin solution for observation and analysis.

2.10 Statistical analysis

All the experimental data in this paper are expressed as the mean ± standard deviation (mean ± SD). GraphPad Prism 7 was utilized for data analysis and visualization. A t-test was employed for comparisons between two groups, while one-way ANOVA was used for comparisons among multiple groups. Pairwise comparisons between groups were conducted using two-way ANOVA. Statistical significance was defined as P < 0.05.

3 Results

3.1 Minocycline suppressed the expression of NF-κB signaling pathway-associated proteins and downstream inflammatory signaling factors

To further investigate the role of minocycline on rosacea, Western blot analysis was employed to assess the expression levels of TLR4 and p-NF-κB/ NF-κB. Compared to the NC group, upregulation of TLR4 and p-NF-κB/ NF-κB expression was observed in both LL37-treated HaCaT cells and skin tissue from rosacea-like mice, which was partially attenuated following minocycline treatment (Figs 1A and 2B). The levels of inflammatory factors such as TNF-α, IL-6, IL-1α, and IL-1β were up-regulated after LL37 intervention, which inversely reduced upon minocycline treatment (Figs 1B, 1C 2C, and 2F). ROS production was increased in LL37-treated cells and mice, while decreased after minocycline treatment (Figs 1D and 2D). Moreover, following subcutaneous injection of LL37, marked inflammatory lesions were observed in the dorsal skin of BALB/c mice. Notably, minocycline treatment significantly attenuated these inflammatory responses compared to the LL37-only group (Fig 2A). Similarly, the infiltration of inflammatory cells in BALB/c mice induced by LL37 was increased, which decreased following minocycline intervention (Fig 2E).

Fig 1. Minocycline suppressed the expression of NF-.

Fig 1

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κB signaling pathway-associated proteins and downstream inflammatory signaling factors in vitro. A: The expression of TLR4 and p-NF-κB/NF-κB was detected by western blot; B: The expression of TNF-α was detected by immunofluorescence staining (Scale bar:100μm); C: The levels of the inflammatory cytokines TNF-α, IL-6, IL-1α, and IL-1β were detected by ELISA; D: ROS expression was detected by immunofluorescence (Scale bar:50μm); * P < 0.05, ** P < 0.01, and *** P < 0.001.

Fig 2. Minocycline suppressed the expression of NF-.

Fig 2

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κB signaling pathway-associated proteins and downstream inflammatory signaling factors in vivo. A: The appearance of dorsal skin in three groups of mice; B: The expression of TLR4 and p-NF-κB/NF-κB was detected by western blot; C: The levels of inflammatory factors (TNF-α, IL-6, IL-1α, and IL-1β) were detected by ELISA; D: The level of ROS was detected by flow cytometry; E: Histopathological changes observed with HE staining; F: TNF-α expression was detected by immunofluorescence staining (Scale bar:50μm); * P < 0.05, ** P < 0.01, and *** P < 0.001.

3.2 Minocycline inhibited rosacea-like inflammation response by regulating TLR4

TLR4 was overexpressed in vivo and in vitro to further explore whether minocycline exerts its anti-inflammatory effect through TLR4. Interestingly, when TLR4 was overexpressed, the efficacy of minocycline was attenuated and the expression of TLR4 was increased. This was accompanied by an increased p-NF-κB/ NF-κB, though the elevation observed in vitro did not reach statistical significance. (Figs 3A and 4B). TNF-α, IL-6, IL-1α, and IL-1β levels were down-regulated following both minocycline and LL37 intervention compared to the LL37 group. However, overexpression of TLR4 weakened the effect of minocycline observing an increase in inflammatory factors including TNF-α, IL-6, IL-1α, and IL -1β (Figs 3B, 3C, 4C, and 4F). Similarly, a significant increase in ROS production was observed while TLR4 was overexpressed (Figs 3D and 4D). When TLR4 was overexpressed, an aggravation of the inflammation manifestation and an increase in inflammatory infiltrating cells were observed in rosacea-like mice treated with minocycline (Figs 4A, and 4E).

Fig 3. Minocycline inhibited rosacea-like inflammation response by regulating TLR4 in Hacat cells.

Fig 3

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A: The expression of TLR4 and p-NF-κB/NF-κB were detected by western blot; B: The expression of TNF-α was detected by immunofluorescence staining (Scale bar: 100μm); C: Inflammatory cytokines (TNF-α, IL-6, IL-1α, and IL-1β) were detected by ELISA; D: ROS expression was detected by immunofluorescence (Scale bar: 50μm); * P < 0.05, *** P < 0.001.

Fig 4. Minocycline inhibited rosacea-like inflammation response by regulating TLR4 in BALB/c mice.

Fig 4

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A: The appearance of dorsal skin in four groups of mice; B: The expression of TLR4 and p-NF-κB/NF-κB was detected by western blot; C: The levels of inflammatory factors (TNF-α, IL-6, IL-1α, and IL-1β) were detected by ELISA; D: The level of ROS was detected by flow cytometry; E: HE-stained skin tissue; F: TNF-α expression was detected by immunofluorescence staining (Scale bar: 100μm); * P < 0.05, ** P < 0.01, and *** P < 0.001.

3.3 Minocycline affects LL37-induced HaCaT cells and rosacea-like mice through the TLR4-mediated NF-κB signaling pathway

Following combined treatment with TLR4 overexpression and the NF-κB signaling pathway inhibitor PDTC, TLR4 expression remained unaltered in rosacea-like cells, while the p-NF-κB/NF-κB was significantly reduced (Fig 5A). In addition, both TLR4 expression and the p-NF-κB/NF-κB showed no marked changes in rosacea-like animal models (Fig 6B). However, the results showed that minocycline treatment decreased levels of inflammatory cytokines like TNF-α, IL-6, IL-1α, and IL-1β. Conversely, TLR4 overexpression increased their levels. Notably, further addition of PDTC attenuated TLR4-overexpression’s impact on minocycline and reduced inflammatory cytokine levels (Figs 5B, 5C, 6C, and 6F). Similarly, with the addition of the NF-κB signaling pathway inhibitor PDTC, the impact of TLR4 overexpression on minocycline was attenuated leading to decreased ROS production (Figs 5D, and 6D). Furthermore, the significant alleviation of skin inflammation and the reduced infiltration of inflammatory cells were observed in minocycline-treated rosacea-like mice subjected to TLR4 overexpression and PDTC administration (Figs 6A and 6E).

Fig 5. Minocycline affects LL37-induced HaCaT cells through the TLR4-mediated NF-.

Fig 5

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κB signaling pathway. A: The expression of TLR4 and NF-κB was detected by western blot; B: The expression of TNF-α was detected by immunofluorescence staining (Scale bar: 100μm); C: The expression of TNF-α, IL-6, IL-1α, and IL-1β was detected by immunofluorescence staining; D: ROS expression was detected by immunofluorescence (Scale bar: 50μm); * P < 0.05, ** P < 0.01, and *** P < 0.001.

Fig 6. Minocycline affects rosacea-like mice through the TLR4-mediated NF-.

Fig 6

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κB signaling pathway. A: The appearance of dorsal skin in four groups of mice; B: The expression of TLR4 and NF-κB was detected by western blot; C: The levels of inflammatory factors (TNF-α, IL-6, IL-1α, and IL-1β) were detected by ELISA; D: ROS were detected by flow cytometry; E: HE staining of skin tissues; F: TNF-α expression was detected by immunofluorescence staining (Scale bar: 50μm); * P < 0.05, ** P < 0.01, and *** P < 0.001.

4 Discussion

Rosacea is a chronic inflammatory skin disease primarily affecting the midface, exerting a significant impact on patients’ psychological well-being and overall quality of life. The pathogenesis of rosacea is complex and varied, in which inflammation plays a pivotal role [4]. Therefore, the administration of anti-inflammatory medications assumes critical importance in its treatment.

Minocycline has been extensively utilized in the treatment of rosacea [19]. However, limited studies exist regarding the specific mechanism of action in this context. The human antibacterial peptide LL37 plays a crucial role in the pathogenesis of rosacea, often used to induce rosacea-like models in vivo and in vitro [20,21].In this study, we explored the possible mechanisms of minocycline treatment of rosacea. The results revealed that minocycline effectively suppressed the expression levels of inflammatory cytokines as well as TLR4 and p-NF-κB in LL-37-treated cells and rosacea-like mice. Notably, overexpression of TLR4 attenuated the inhibitory effect exerted by minocycline. Nevertheless, the addition of the NF-κB signaling pathway inhibitor PDTC based on overexpression of TLR4 significantly restored the efficacy of minocycline.

Toll-like receptors (TLRs) play a crucial role in skin inflammation [22]. Both TLR4 and TLR2 are highly expressed Toll-like receptors in sebaceous glands, regulating genes involved in inflammation, wound healing, and chemotaxis. They play pivotal roles in acne development [23]. Similarly, Yamasaki K’s study demonstrated an increased expression of TLR2 in rosacea patients, along with elevated levels of tumor necrosis factor-α (TNF-α), the main cytokine induced by TLR signaling [24]. Wladis EJ et al. reported increased NF-kB protein levels in eyelid specimens and inflamed skin tissues from patients with rosacea [25]. Reactive oxygen species (ROS), as signaling molecules, participate in diverse biological processes and contribute to cellular homeostasis maintenance [26]. Excessive ROS levels have been reported to mediate inflammatory signaling pathways leading to pathophysiological conditions such as rosacea [27]. TNF-α, IL-6, IL-1α, and IL-1β are well-characterized inflammatory mediators in rosacea pathogenesis, driving key pathological processes including inflammatory cell infiltration, vascular hyperreactivity, and neuroimmune disorder [28,29]. The dysregulation of these cytokines has been consistently associated with disease severity, with elevated expression observed in both lesional skin and serum samples from rosacea patients, making them critical biomarkers for assessing inflammatory progression [29–32]. Consistent with these findings, we demonstrated that TLR4 and p-NF-κB expression, along with inflammatory factor levels, were significantly upregulated, while ROS levels were markedly increased in both rosacea-like cells and mice. Furthermore, Minocycline effectively inhibited the expression of inflammatory cytokines, TLR4, and p-NF-κB in LL-37 treated cells. Minocycline has a significant therapeutic effect on rosacea-like mice, and the inflammatory reaction of skin lesions and inflammatory cell infiltration of mice are significantly improved after intraperitoneal injection of minocycline. The expressions of TLR4 and p-NF-κB in skin tissue were down-regulated, and the levels of TNF-α, IL-6, IL-1α, IL-1β, and ROS were decreased.

Previous research has shown that NF-κB signaling is associated with various inflammatory skin diseases and observed significant activation during dermatitis exacerbation [33]. TLR4 can regulate infection induction or the inflammatory response through endogenous molecules and apoptotic processes [34,35]. The TLR4-NF-κB pathway is the main regulatory pathway involved in cellular inflammation [36]. Additionally, a study revealed that Dendrobium polysaccharides could inhibit the activation of the TLR4/NF-κB pathway in mouse skin, thereby reducing inflammation and exerting a protective effect against rosacea in mice [37]. In our study, Minocycline significantly downregulated the expression of TLR4 and inflammatory cytokines while inhibiting ROS production. However, when TLR4 was overexpressed, minocycline’s effect was weakened. The efficacy of minocycline was substantially restored upon the addition of the NF-κB signaling pathway inhibitor PDTC. Therefore, we propose that minocycline suppresses NF-κB pathway activation by downregulating TLR4, thereby reducing the expression of inflammatory cytokines and inhibiting ROS production.

Our findings indicate that minocycline modulates rosacea development via the TLR4-mediated NF-κB signaling pathway. Overexpression of TLR4 stimulates cellular inflammation, thwarting the therapeutic effect of minocycline. The TLR4 receptor is capable of regulating downstream NF-κB signaling activity. NF-κB phosphorylation regulates the transcriptional activity of NF-κB in the nucleus, triggering the production of inflammatory cytokines [38]. In this study, HaCaT cells were treated with the NF-κB signaling pathway inhibitor PDTC and found that inhibition of NF-κB activity attenuated the impact of TLR4 overexpression while suppressing inflammation and ROS levels. These results suggest that minocycline suppresses inflammation, reduces ROS production, and alleviates inflammatory progression in rosacea by downregulating TLR4 expression and inhibiting the NF-κB signaling pathway.

However, since we did not directly validate the relationship between TLR4 and NF-κB in HaCaT cells, our study provides evidence for an association rather than causation. Future investigations should be conducted to elucidate specific mechanisms underlying minocycline-induced downregulation of TLR4 expression in rosacea.

Furthermore, It is critical to emphasize that minocycline’s therapeutic effects on rosacea likely extend beyond TLR4/NF-κB modulation. Preclinical evidence for other diseases indicates its capacity to inhibit matrix metalloproteinases (MMPs) [39–41], regulate macrophage polarization [42], and exert antioxidant/neuroprotective effects [43–45]. While these pleiotropic mechanisms could synergistically contribute to the observed anti-inflammatory outcomes, the precise mechanistic contributions require further investigation.

5 Conclusion

Minocycline alleviates inflammation progression in rosacea by downregulating TLR4 and inhibiting the activation of the NF-κB pathway, providing a scientific basis for subsequent clinical treatment.

Data Availability

All data files are available from the Figshare repository (DOI：https://doi.org/10.6084/m9.figshare.28464782).

Funding Statement

This work was supported by the Yunnan Revitalization Talent Support Program (Recipient: Hua Gu).

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Decision Letter 0

Helen Howard

16 Jan 2025

PONE-D-24-43618Minocycline inhibits rosacea-like inflammation through the TLR4-mediated NF-κB signaling pathway in vivo and vitroPLOS ONE

Dear Dr. Gu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript has been evaluated by three reviewers, and their comments are available below.

The reviewers have raised a number of concerns that need attention. In particular, they request additional information on methodological aspects of the study, the inclusion of images to show the changes in the macroscopic appearance of skin lesions, and further discussion.

Could you please revise the manuscript to carefully address the concerns raised?

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

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Reviewer #1: The authors conducted in-vitro and in-vivo experiments to demonstrate the effect of minocycline on the TLR4/NF-kB pathway in LL37-induced models of rosacea. The results showed that minocycline downregulated TLR4 and inhibited NF-kB pathway activity, proposing a molecular basis for the efficacy of minocycline in treating rosacea. This study adds to our understanding of the mechanism of action of minocycline in rosacea. Thank you for including me in the peer review of this manuscript, please see my comments below:

1. Introduction: “However, the 34 etiology and pathogenesis of rosacea remain have not been fully elucidated”. Remove “remain”.

2. Introduction: “immunoregulatory, neuroprotective and angiogenic properties”. Angiogenic or anti-angiogenic? Given the clinical features of rosacea, it would seem that anti-angiogenic properties would be beneficial.

3. Introduction: “TLR4/p38 MAPK and NF κB pathways”. Abbreviations not defined. Several other abbreviations also not defined at first appearance, e.g. IKK, NC, IL, TNF, etc.

4. Discussion: “Toll-like receptors (TLRs) playing a crucial role in skin inflammation”. Change “playing” to “play”.

5. Discussion: “YAMASAKI K’ study”. This does not need to be in all capitals.

6. Discussion: “Future investigations should be conducted to elucidate specific mechanisms 310 underlying minocycline-induced upregulation of TLR4 expression in rosacea.” Upregulation or downregulation?

7. Why were TNF-α, IL-6, IL-1α, and IL-1β chosen to be measured? If these cytokines have been shown to be particularly important in rosacea pathogenesis, please introduce and cite references in the introduction to explain the rationale.

8. The term “minocin” is used instead of “minocycline” throughout the results and other places in the manuscript. Minocin is a brand name.

9. While the results demonstrate the effects of minocycline on the TLR4/NK-kB pathway, minocycline is not a specific inhibitor of this pathway. Another limitation could be potential confounding from inhibition of other pro-inflammatory factors by minocycline. Please briefly discuss other potential targets of minocycline.

Reviewer #2: This is a good and orginal study calling attention to the pathogenesis and treatment mechanisms of acne rosacea. The manuscript is written well and on a scientific base. However, there are some grammatical mistakes in the main text that need to be corrected. Thanks to the authors for their work.

Reviewer #3: 1. "Minocycline-only group" was not included in the control settings of all experiments.

2. In the in vivo experimental results, no images were provided to show the changes in the macroscopic appearance of skin lesions. Based on the current results, it is difficult to confirm whether the model was successfully established and whether the minocycline treatment was effective.

**********

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Reviewer #2: No

Reviewer #3: No

**********

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PLoS One. 2025 May 16;20(5):e0323598. doi: 10.1371/journal.pone.0323598.r003

Author response to Decision Letter 1

25 Mar 2025

Responses to Journal Requirements

Comment 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response: Thank you for highlighting this important requirement. We have carefully reviewed and adjusted the manuscript to comply with PLOS ONE's style guidelines.

Comment 2: To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

Response: In order to comply with the requirements submitted by PLOS ONE, the completed modeling methods are provided in the Methods section, supplemented with additional information about animal experiments, including sacrificial methods, anesthesia and/or analgesia methods, and pain relief efforts. The supplementary content has been marked in red font in the article.

‘Seven-week-old BALB/c mice were purchased from Shanghai SLAC Experimental Animal Co. Ltd. (Shanghai). All animal experiments were carried out under specific pathogen-free conditions. In the process of feeding mice, keep the feeding environment clean, quiet, temperature (22 ± 2) ℃, humidity (50 ± 5) % constant, provide adequate food and clean drinking water, to reduce the stress and pain caused by external environmental factors on mice. All studies and experimental procedures were approved by the Animal Ethics Committee of The First Affiliated Hospital of Kunming Medical University. The mice were divided into five groups (the NC group, the LL37 group, the LL37+Minocin group, the LL37+Minocin+oe-TLR4 group, and the LL37+Minocin+oe-TLR4+PDTC group) and treated differently. The mice were shaved 24 hours before treatment and then injected subcutaneously with 40 μL of LL37 peptide twice a day for 3 days. The mice in the LL37+Minocin group were administered an intraperitoneal injection of minocycline at a dose of 50 mg/kg/day for 3 days. For the LL37+Minocin+oe-TLR4 group, Minocin-treated mice were injected with 100 μg of oe-TLR4 plasmid via the tail vein. For the LL37+Minocin+oe-TLR4+PDTC (5108–96-3, AbMole) group, the mice were intraperitoneally injected with PDTC at a dose of 50 mg/kg. The experiment was performed 72 hours after the initial injection of LL37. In order to alleviate the pain of mice, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40mg/kg) before the experimental operation. After the experiment, the mice were euthanized by cervical dislocation. Skin inflammation was evaluated by the severity of erythema and edema. Skin tissues were collected for protein extraction, hematoxylin-eosin staining, and immunofluorescence analysis.’

Comment 3: Thank you for stating the following financial disclosure: This work was supported by the Yunnan Revitalization Talent Support Program. Hua Gu is the Fund recipient.

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Response: Thank you for raising this important point. We have revised the manuscript to explicitly clarify the funders’ role in the Funding section: The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

Comment 4: In the online submission form, you indicated that the data that support the findings of this study are available from the corresponding author upon reasonable request. All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either 1. In a public repository, 2. Within the manuscript itself, or 3. Uploaded as supplementary information. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval.

Response: Thank you for your comments. We have complied with the journal’s requirements by uploading all relevant data associated with this study to the Figshare public repository and have explicitly stated the data access methods in the revised manuscript. The specific updates are detailed below:

Data Storage and Access

All raw data have been uploaded to Figshare (DOI: 10.6084/m9.figshare.28464782). The dataset has been set to public access, ensuring that readers can freely download and use the materials.

Manuscript Updates

In the "Data Availability Statement" section of the manuscript, we have added the following declaration:

“All relevant data from this study has been uploaded to Figshare and can be accessed at https://figshare.com/s/659c253d0b649844765f. This dataset is licensed under CC BY 4.0, allowing free reuse with attribution. (DOI: 10.6084/m9.figshare.28464782)”

Comment 5: Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

Response: Thank you for your valuable comments. We have carefully revised the manuscript according to your suggestions. The ethics statement has been moved exclusively to the Methods section (specifically in the Animal). All duplicate ethics statements previously appearing in DECLARATION have been completely removed.

Response to reviewer1

Comment 1-6: 1. Introduction: “However, the 34 etiology and pathogenesis of rosacea remain have not been fully elucidated”. Remove “remain”. 2. Introduction: “immunoregulatory, neuroprotective and angiogenic properties”. Angiogenic or anti-angiogenic? Given the clinical features of rosacea, it would seem that anti-angiogenic properties would be beneficial. 3. Introduction: “TLR4/p38 MAPK and NF κB pathways”. Abbreviations not defined. Several other abbreviations also not defined at first appearance, e.g. IKK, NC, IL, TNF, etc. 4. Discussion: “Toll-like receptors (TLRs) playing a crucial role in skin inflammation”. Change “playing” to “play”. 5. Discussion: “YAMASAKI K’ study”. This does not need to be in all capitals. 6. Discussion: “Future investigations should be conducted to elucidate specific mechanisms 310 underlying minocycline-induced upregulation of TLR4 expression in rosacea.” Upregulation or downregulation?

Response: We would like to express our sincere gratitude for your positive acknowledgment of our work and constructive comments. Regarding the grammatical and typographical issues identified in the Introduction and Discussion sections, we fully concur with your insightful recommendations and have accordingly implemented the suggested amendments throughout the manuscript.

Comment 7: Why were TNF-α, IL-6, IL-1α, and IL-1β chosen to be measured? If these cytokines have been shown to be particularly important in rosacea pathogenesis, please introduce and cite references in the introduction to explain the rationale.

Response: We sincerely appreciate the reviewer’s insightful comment. The selection of TNF-α, IL-6, IL-1α, and IL-1β as key biomarkers in this study was based on their well-established roles in rosacea pathogenesis, particularly in driving inflammation, vascular dysfunction, and immune dysregulation. We have added the corresponding introduction and citations in the Discussion sections, see the red font section of the manuscript.

TNF-α, IL-6, IL-1α, and IL-1β are well-characterized inflammatory mediators in rosacea pathogenesis, driving key pathological processes including inflammatory cell infiltration, vascular hyperreactivity, and neuroimmune disorder [28, 29]. The dysregulation of these cytokines has been consistently associated with disease severity, with elevated expression observed in both lesional skin and serum samples from rosacea patients, making them critical biomarkers for assessing inflammatory progression [29-32].

Comment 8: The term “minocin” is used instead of “minocycline” throughout the results and other places in the manuscript. Minocin is a brand name.

Response: We sincerely appreciate the reviewer's meticulous attention to terminology consistency. In accordance with scientific writing standards requiring the use of generic drug names, we have systematically replaced "Minocin" with "minocycline" throughout the Results, Discussion, and other sections of the manuscript. The initial use of "Minocin" in the Methods section was intentionally retained to specify the exact commercial formulation used in our experiments, as drug bioavailability and excipient composition may vary between brands. All instances of "Minocin" outside the Methods section have now been revised to "minocycline" to maintain scientific objectivity. We have additionally performed a full-text verification to ensure no unintended usage remains.

Comment 9: While the results demonstrate the effects of minocycline on the TLR4/NK-kB pathway, minocycline is not a specific inhibitor of this pathway. Another limitation could be potential confounding from inhibition of other pro-inflammatory factors by minocycline. Please briefly discuss other potential targets of minocycline.

Response: We sincerely thank the reviewer for raising this critical point regarding the multifunctional nature of minocycline. As rightly noted, while our study focused on its effects on the TLR4/NF-κB pathway, we fully acknowledge that minocycline is not a pathway-specific inhibitor and may exert broader anti-inflammatory actions. In the revised Discussion section, we have added the following content to address this limitation:

" Furthermore, It is critical to emphasize that minocycline's therapeutic effects on rosacea likely extend beyond TLR4/NF-κB modulation. Preclinical evidence for other diseases indicates its capacity to inhibit matrix metalloproteinases (MMPs)[39-41], regulate macrophage polarization[42], and exert antioxidant/neuroprotective effects[43-45]. While these pleiotropic mechanisms could synergistically contribute to the observed anti-inflammatory outcomes, the precise mechanistic contributions require further investigation."

Response to Reviewer 2

Comment: This is a good and original study calling attention to the pathogenesis and treatment mechanisms of acne rosacea. The manuscript is written well and on a scientific base. However, there are some grammatical mistakes in the main text that need to be corrected. Thanks to the authors for their work.

Response: We sincerely appreciate the reviewer's encouraging evaluation of our work and their keen attention to manuscript quality. We have conducted a full-scale grammatical revision of the manuscript with Grammarly Premium.

Response to Reviewer 3

Comment 1: "Minocycline-only group" was not included in the control settings of all experiments.

Response: We sincerely appreciate your meticulous review of our manuscript and the valuable comments you've provided. Regarding your concern about the absence of a "Minocycline-only group" in the control settings of all experiments, we would like to present the following considerations.

Firstly, the core objective of this study is to explore the effect of Minocycline or its combination with other intervention factors (e.g.oe-TLR4, PDTC) on disease progression in the LL37-induced rosacea-like cell and animal model, and to clarify the mechanism of action of TLR4 and NF-κB signaling pathways. However, the minocycline-alone group was not included in the experimental design because it was not associated with the core purpose of the study.

Secondly, a number of targeted control groups have been set up in the current experiment, such as the NC group as the normal physiological state control, the LL37 group as the disease model control, and the LL37 + Minocycline group to evaluate the role of Minocycline in the disease model. These control groups can fully meet the logical needs of experimental design and provide a reliable reference for the research results.

Finally, considering the resource and time constraints in actual scientific research, the additional Minocycline single-use group will significantly increase the experimental cost and time investment. On the basis of ensuring that the research objectives can be achieved, the experimental design has been optimized to achieve the efficient use of resources. The existing control group was set up to meet the needs of the study, so the Minocycline-only group was not included.

Comment 2: In the in vivo experimental results, no images were provided to show the changes in the macroscopic appearance of skin lesions. Based on the current results, it is difficult to confirm whether the model was successfully established and whether the minocycline treatment was effective.

Response: We sincerely appreciate your insightful review and constructive feedback on our manuscript. We fully understand your concerns regarding the lack of images depicting the macroscopic appearance changes of skin lesions in the in-vivo experimental results. To address this issue, we have now added a set of high-quality images in the revised manuscript. These images clearly show the skin lesions in different groups. From these images, it is evident that in the LL37 group, there are obvious signs of erythema, papule, and other typical manifestations of the established model, which strongly indicates the successful establishment of the rosacea-like model. In the LL37 + Minocycin group, a significant improvement in the macroscopic appearance of the skin lesions can be observed compared to the LL37 group, demonstrating the effectiveness of Minocycline treatment. Please see Figures 2A, 4A, 6A. We believe that these added images will greatly enhance the comprehensibility and persuasiveness of our study. Once again, we are grateful for your valuable suggestions, which have helped us to improve the quality of our manuscript.

Attachment

Submitted filename: Response to reviewers.docx

pone.0323598.s002.docx (26.2KB, docx)

Decision Letter 1

Divakar Sharma

11 Apr 2025

Minocycline inhibits rosacea-like inflammation through the TLR4-mediated NF-κB signaling pathway in vivo and in vitro

PONE-D-24-43618R1

Dear Dr. Gu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Divakar Sharma

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Accept

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

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Reviewer #1: I would like to thank the authors for their efforts in further elucidating the pathogenesis mechanisms of rosacea. My comments have been addressed satisfactorily.

Reviewer #4: This is a revised version of manuscript reviewed by other experts, I have went through the comments and the reponses and I beleive authors have made satisfactory revison. Now this manuscript can be accepted in PLOS One.

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Reviewer #1: No

Reviewer #4: No

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Acceptance letter

Divakar Sharma

PONE-D-24-43618R1

PLOS ONE

Dear Dr. Gu,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

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Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Divakar Sharma

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Attachment

    Submitted filename: Response to reviewers.docx

    pone.0323598.s002.docx (26.2KB, docx)

    Data Availability Statement

    All data files are available from the Figshare repository (DOI：https://doi.org/10.6084/m9.figshare.28464782).

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