All six-week-old BALB/c mice were obtained from the Laboratory Animal Center of Southern Medical University, and were reared in the SPF facility at Southern Medical University. The animal experiments were carried out in the light of the approval and guidelines of Animal Care and Use Committee of Southern Medical University. All procedures in this study strictly complied with the Animal Welfare Act and principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996.

Cath-MH (APCKLGCKIKKVKQKIKQKLKAKVNAVKTVIGKISEHLG) and FITC-labeled Cath-MH were synthesized by GL Biochem Ltd. (Shanghai, China), and then were further purified and identified as described in our previous report (Chai et al., 2021).

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Cath-MH against P. acnes ATCC 6919 and ATCC 11827 were determined using two-fold broth dilution method as previously described by us (Ye et al., 2020). Specifically, two strains P. acnes ATCC 6919 and ATCC 11827 acquired from Guangdong Institute of Microbiology were grown in brain heart infusion (BHI) broth (HKM, China) under MGC Anaeropack systems (Mitsubishi, Gas Chemical, Japan) which provides anaerobic conditions. A two-fold serial dilution of peptide was added to 96-well plate (Costar, Corning, United States) at final concentrations of 0.78, 1.56, 3.13, 6.25, 12.5, 25, and 50 μM before an equal volume of bacteria in fresh BHI broth (the microbial loading was 10⁶ CFU/ml) was loaded. After 72 h incubation at 37°C, the absorbance measurement of P. acnes suspension solution was done at 600 nm with microplate reader (Infinite M1000 Pro, Tecan Company, Switzerland). As a positive control, clindamycin (Sigma, United States) was used. MIC was defined as the minimum concentration inhibiting visible growth. To determine the minimum concentration of Cath-MH causing bacterial death, MBC was then determined following the MIC assay. 10 μl of sample which exhibited no evident growth after 72 h incubation was inoculated onto BHI agar plates. These plates were placed at 37°C for another 72 h under an anaerobic atmosphere. The MBC was defined as the peptide concentration at which there was no colony growth (Andrä et al., 2005).

The bacterial killing kinetics of Cath-MH against P. acnes ATCC 6919 were carried out according to our previous method with minor modification (Ye et al., 2020). Briefly, Cath-MH at final concentration of 1× MIC was mixed with an equal volume of bacteria in fresh BHI broth under an anaerobic atmosphere. Duplicate samples were withdrawn at various timepoints (0, 15, 30, 60, 90, 120, 150, and 180 min) and spread on BHI agar plates. The 0 timepoint represents the sample withdrawn immediately after mixing. Viable colonies were counted after incubation of the plates for 72 h at 37°C under anaerobic conditions. Clindamycin at 1× MIC value and sterile saline were applied as the positive and negative control, respectively.

The bacterial agglutination assay was done according to the method previously reported by us (Wu et al., 2021). In brief, P. acnes ATCC 6919 at exponential phase were harvested, washed twice and diluted to 2.0 × 10⁸ CFU/ml of density with fresh BHI broth and incubated with BSA, Cath-MH (2× MICs), or Cath-MH (2× MICs) plus equal volume of 0.2 mg/ml LPS (L2880, Escherichia coli O55:B5, Sigma, United States) or 0.2 mg/ml LTA (L2512, Staphylococcus aureus, Sigma, United States) at 37°C for 30 min. The mixture was dropped on a glass slide and dyed with a Gram staining kit (Solarbio Technology, Beijing), and the results were observed under an oil microscope (Nikon Corporation, Japan).

The Circular Dichroism (CD) measurement was performed to study the interaction of Cath-MH with LTA or LPS. In brief, Cath-MH was prepared in H₂O or 30 mM SDS solution. Then, LTA or LPS (0.2 mg/ml) was loaded to the peptide solution (50 μM) for 1 h at room temperature, respectively. Binding of Cath-MH to LTA or LPS was studied by monitoring the change in its secondary structure. CD measurement was then carried out with Jasco-810 spectropolarimeter (Jasco, Japan). CD data were presented as the mean residue ellipticity (θ) of three consecutive scans per sample in deg·cm²·dmol⁻¹.

LTA binding of Cath-MH was further confirmed by measuring the inhibitory effects of LTA on the antimicrobial activity of Cath-MH against P. acnes ATCC 6919. In detail, LTA at concentrations (0, 0.0625, 0.125, 0.25, and 0.5 mg/ml) dissolved in sterile saline was mixed with 0.5, 1, and 2× MICs of Cath-MH for 30 min. Then, an equal volume of 10⁶ CFU/ml bacterial suspension in fresh BHI broth was added to the above mixture before coated on BHI agar plates. After anaerobic incubation for 72 h, the number of colonies were calculated. All experiments were repeated three times.

To ensure the underlying mechanism of action of Cath-MH against P. acnes ATCC 6919, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) experiments were performed for determination of the membrane permeability and morphological changes of P. acnes. Briefly, P. acnes ATCC 6919 at logarithmic growth phase were diluted to 10⁶ CFU/ml and incubated with Cath-MH (2× MICs) for 30 min at 37°C. SYTO9 and PI staining (LIVE/DEAD^® BacLight kit, Invitrogen, USA) was added to the bacterial suspensions, followed by incubation for 30 min at room temperature in the dark. CLSM (Leica TCS SP5, Leica Microsystems, Germany) was used to detect SYTO9 and PI with the excitation/emission spectrum of 480 and 635 nm, respectively. For SEM observation, bacterial suspensions at logarithmic growth phase were incubated with Cath-MH (2× MICs) for 30 min at 37°C. Then, P. acnes were harvested by centrifugation, sequentially fixed with 4 and 2.5% glutaraldehyde solution at room temperature for 4 h and 2.5% at 4°C overnight, respectively. After three times of wash with PBS, bacteria were dehydrated sequentially with 30, 50, 70, 85, 90, and 100% ethanol solution, followed by tert-Butyl alcohol, and dried in a freeze dryer (Quorum, UK). After gold coating, bacterial morphology was visualized by JSM-840 instrument (Hitachi, Japan) at the magnification of ×50,000.

The cytotoxicity of Cath-MH on RAW 264.7 cells was measured by the MTT method as reported previously by us (Zeng et al., 2020). In short, RAW 264.7 cells at a density of 5,000 cells per well were plated in 96-well plates and grown with medium DMEM in the presence or absence of continuous concentrations of Cath-MH (2.5, 5, 10, 20, and 40 μM) at 37°C for 24 h before MTT was added in the dark and the culture was continued for another 4 h. The supernatant was discarded and DMSO was loaded before the absorbance at 490 nm as measured. The experiment was repeated at least three times.

Membrane binding assays were undertaken with FITC-labeled Cath-MH. In short, RAW 264.7 murine macrophage cells in the logarithmic phase of growth were prepared in PBS at a density of 1 × 10⁵ cells/ml and incubation with FITC-labeled Cath-MH (0, 2, 4, 8, and 16 μM) at 37°C for 30 min. The unbound peptide was washed out with PBS containing 1% BSA in advance. Cell fluorescence intensity was detected with a FACscan flow cytometer (Becton Dickinson, United States), representing the ability to bind to cell membranes. Cells without peptide treatment were regarded as the negative control.

RAW 264.7 murine macrophage cells at the density of 1 × 10⁵ cells per well were added into 24-well plates and grown for 12 h for adherence. The cells were pretreated with Cath-MH (0, 1, 2, 4, and 8 μM) for 1 h and then co-incubated with LTA (10 μg/ml) or LPS (100 ng/ml) for 24 h. Then, the culture supernatants were used for analysis of NO production by Griess reagent (Beyotime Biotechnology, China) and IL-1β, IL-6, and TNF-α levels using enzyme linked immunosorbent assay (ELISA) (Thermo Fisher Scientific, United States) in light of the manufacturer’s manuals.

RAW 264.7 murine macrophage cells at the density of 1 × 10⁶ cells per well were added into 6-well plates and cultured for 12 h for adherence. The cells were pretreated with Cath-MH (0, 1, 2, 4, and 8 μM) for 1 h and then stimulated with LTA (10 μg/ml) or LPS (100 ng/ml) for 6 h at 37°C in 5% CO₂. Cells were subsequently harvested to measure the mRNA levels of iNOS, IL-1β, IL-6, TNF-α, TLR2, and TLR4 by qRT-PCR as reported previously by Zeng (Zeng et al., 2018). GAPDH gene was applied as a control to standardize the amount of the sample mRNA. Forty amplification cycles were required to complete exponential amplification.

RAW 264.7 murine macrophage cells were plated in 6-well plates at the density of 1 × 10⁶ cells/well and grown for 12 h for adherence. The cells were pretreated with Cath-MH (0, 1, 2, 4, and 8 μM) for 1 h and then stimulated with LTA (10 μg/ml) or LPS (100 ng/ml) at 37°C for 30 min in 5% CO₂. After that, the cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, China) and proteins were extracted using commercial kit (Cayman Chemical, United States) in light of the manufacturer’s recommendations. Primary antibodies of phospho-ERK/ERK, phospho-JNK/JNK, phosphor-p38/p38, NF-κB p65, Lamin A/C and GAPDH (1: 1,500, Cell Signaling Technology, United States) and horseradish peroxidase conjugated secondary antibodies (1: 2,000, Cell Signaling Technology, United States) were applied in western blot analysis. All experiments were repeated three times.

The in vivo anti-acne effect of Cath-MH was evaluated using the procedure described previously by us (Ye et al., 2020). Six-week-old BALB/c mice weighing about 22 g were randomly subdivided into four groups (n = 6). Approximately 25 μl of P. acnes (5 × 10⁸ CFU/ml) was intradermally administrated into the left ears of mice and the control mice received an equivalent volume of PBS. Clindamycin (10 μg) and Cath-MH (50 μg) were mixed in 50 mg of sterile vaseline and then were painted onto the ear surfaces, respectively. The ear thickness was measured at 24 h with a micro-caliper (Mitutoyo, Japan) after P. acnes injection. Afterwards, mice were sacrificed and ears were sampled for bacterial cell counts, histopathological assay, ELISA, qRT-PCR, and western blot detection.

qRT-PCR data were calculated with the 2^−ΔΔCT method. All data were expressed as mean ± SEM. Statistical analysis were carried out using one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant as compared to control.

The effect of Cath-MH on P. acnes was determined using MIC and MBC assays. As shown in Table 1, both MIC and MBC values of Cath-MH against P. acnes ATCC 6919 and ATCC 11827 were about 1.56 μM. However, the MICs of clindamycin against two stains were about 3.39 μM. Moreover, its MBCs against P. acnes ATCC 6919 and ATCC 11827 were approximately 6.77 and 13.54 μM, respectively. Therefore, Cath-MH has more potent anti-P. acnes activity than the positive control clindamycin.

To further explore the anti-P. acnes activity of Cath-MH, its bacterial killing kinetics against P. acnes ATCC 6919 was evaluated. As seen in Figure 1, Cath-MH (1× MIC) exhibited potent bactericidal activity within 120 min of incubation. However, under same circumstances, clindamycin (1× MIC) showed much slower killing kinetic, which could not completely kill bacteria in 180 min.

Cath-MH can agglutinate E. coli ATCC 25922 (Chai et al., 2021). Therefore, we measured its agglutination activity against P. acnes ATCC 6919. As shown in Figure 2A, Cath-MH displayed a high agglutinating activity after incubation with P. acnes for 30 min (panel b) when compared with the control treated with BSA (panel a). However, this agglutination was abolished by 0.2 mg/ml LPS (panel c) and 0.2 mg/ml LTA (panel d), respectively, indicating that Cath-MH could bind LPS and LTA.

The LTA/LPS-binding ability of Cath-MH was further supported by CD spectroscopy and antimicrobial assay. As presented in Figure 2B, in the presence of LTA or LPS, Cath-MH showed obviously different CD spectra which were similar to peptide dissolved in SDS solution. Furtherly, the suppression effects of increasing concentrations of LTA on antimicrobial activities of Cath-MH at 0.5, 1, and 2× MICs against P. acnes were examined. As expected, LTA alone did not change the proliferation of P. acnes. However, the anti-P. acnes activities of Cath-MH at 1× and 2× MICs were significantly decreased after incubation with LTA at concentrations ranging from 0.0625 mg/ml to 0.5 mg/ml for 30 min. Moreover, this inhibitory effect depended on the concentration of LTA (Figure 2C).

AMP generally starts their killing bacteria process promptly once their attraction and attachment to microbial surfaces (Vineeth Kumar and Sanil, 2017). CLSM was carried to ensure the permeabilization of bacteria caused by Cath-MH. The results showed the Cath-MH-treated group with intense red fluorescence, which confirmed the majority of P. acnes with damaged membranes, while the untreated group showed intense green fluorescence which indicated with intact cell membrane (Figure 2D). To better understand the mechanism of action of Cath-MH against P. acnes, SEM was used. As shown in Figure 2E, after Cath-MH treatment, P. acnes had changed in morphology including obviously wrinkling and cell contents releasing (areas indicated by arrow) compared to the control group. Altogether, these data demonstrated that the Cath-MH treatment could effectively destroy the bacterial cell membrane and then kill bacteria, like most AMPs.

Some AMPs can bind to their targets on the surface of macrophages, and then set off cellular signaling pathway and regulate the secretion of pro-inflammatory factors (Wang et al., 2011; Wei et al., 2015; Magrone et al., 2018; Zeng et al., 2018; Kasus-Jacobi et al., 2020; Chai et al., 2021). Therefore, the binding of Cath-MH to RAW 264.7 cells were evaluated with flow cytometry. As displayed in Figure 3A, Cath-MH could concentration-dependently bind to RAW 264.7 cells after co-incubation for 30 min. To define whether Cath-MH can affect the release of inflammatory factors in RAW 264.7 cells stimulated by LTA/LPS, we tested firstly its effect on the viability of RAW 264.7 cells. As shown in Figure 3B, Cath-MH at the concentrations of less than and equal to 10 μM had no cytotoxicity toward RAW 264.7 cells. Thus, we further investigated whether Cath-MH at the concentration of lower than 10 μM could inhibit the generation of inflammatory factors induced by LPS/LTA. As shown in Figures 3C–J, both 100 ng/ml LPS and 10 μg/ml LTA significantly increased the protein and mRNA contents of NO, IL-1β, IL-6, and TNF-α in the cell culture supernatants in comparison with the control without any treatment. However, the enhanced expressions induced by LPS/LTA were markedly reversed by Cath-MH in concentration-dependent manner. LTA/LPS as the TLR2/4 agonist can induce the TLR2/4-mediated inflammatory response and the expression elevation of TLR2/4 at mRNA and protein levels, respectively (Kwak et al., 2015). Therefore, we examined whether or not the suppressive effects of Cath-MH on LTA/LPS-induced cytokine production was correlated to TLR2/4 expression. The results showed that, with Cath-MH pre-treatment, TLR2/4 mRNA expression induced by LTA/LPS was downregulated (Figure 3K). However, without LTA/LPS stimulation, Cath-MH did not change TLR2/4 mRNA expression (Figure 3L), suggesting that the effect of Cath-MH on inflammatory factor expressions is associated with its suppression of TLR2/4 mRNA expression stimulated by LTA/LPS.

It has been well known that MAPK/NF-κB signaling pathways play a vital role in pro-inflammatory process by regulating the expression of inflammatory factors including IL-1β, IL-6, and TNF-α. Therefore, we evaluated the effects of Cath-MH on MAPK/NF-κB signaling pathways in LPS-stimulated RAW 264.7 cells using western blot. As presented in Figure 4, LPS (100 ng/ml) significantly increased the expression of phosphorylated ERK, JNK, p38, and the nuclear translocation of NF-κB p65 when compared to the control group. However, this upregulation induced by LPS was significantly repressed by Cath-MH in a dose-dependent manner.

The in vivo anti-acne activity of Cath-MH was explored using acne mouse model. As shown in Figure 5, following P. acnes injection, the ears of mice became red and swollen. However, like clindamycin, Cath-MH obviously relieved P. acnes-induced ear redness and swelling (Figures 5A,B). Moreover, as shown in Figure 5C, both Cath-MH and clindamycin substantially decreased the number of P. acnes colonized in the ear when compared with the model group only injected by P. acnes suspensions. To explore its effects on inflammation induced by P. acnes, histopathological analysis and cytokine expression measurement were also performed. As shown in Figure 5D , the infiltration of inflammatory cell and ear swelling were obviously increased in the ear injection of P. acnes when compared with the control sample. Nevertheless, it was significantly decreased after Cath-MH or clindamycin treatment. Consistently, following 24 h injection of P. acnes, both protein and mRNA expressions of TNF-α, IL-1β and IL-6 were all significantly upregulated. Yet, their upregulations were markedly reversed after treatment with Cath-MH or clindamycin (Figures 5E,F). To further clear the protective mechanism of Cath-MH in acne vulgaris mice, MAPK/NF-κB signaling pathways were investigated by western blotting (Figures 5G,H). Consistent with their effects on cytokine expression in vitro, injection of P. acnes significantly upregulated the expression of phosphorylated ERK, JNK, p38 and p65 translocated in nucleus of ear tissues when compared with vehicle. However, treatment with Cath-MH and clindamycin successfully suppressed these increases but had no influence on total ERK, JNK, and p38 expression. Together, these data demonstrated that the Cath-MH treatment can effectively improve acne vulgaris in mice.