Materials
Zinc Oxide nanoparticles (ZnO NPs) and Oleic Acid (OA) were provided by Sigma (St Louis, MO). Orlistat, Roswell Park Memorial Institute (RPMI) medium 1640, phosphate buffered saline (PBS), Cell Counting Kit-8 (CCK8), Fluo-3 AM, Calcein AM/PI Double Staining Kit, Lipase (LPS) Activity Assay Kit, Amplex Red Free Fatty Acid(FFA) Assay Kit, Reactive Oxygen Species (ROS) Assay Kit, GSH Assay Kit and Hydrogen Peroxide (H2O2) Assay Kit were provided by Beijing Solarbio Science & Technology Co., Ltd. Mito Tracker Red CMXRos, Hoechst 33,342, Trypan Blue Staining Cell Viability Assay Kit and BCECF AM were provided by Beyotime, China. Cell Malondialdehyde (MDA) assay kit and Lactate dehydrogenase (LDH) assay kit were provided by Nanjing Jiancheng Biotechnology Research Institute Co., Ltd. Anti-MMP-9, anti-TGF-β1, anti-SMAD-4 and goat anti-rabbit secondary antibody were provided by Wuhan Service-bio Technology Co., Ltd. MMP-9-IN-1 and β-Neo-Endorphin were provided by Shanghai Aladdin Biochemical Technology Co., Ltd.
Microbial/cell culture and animals
Cutibacterium acnes (C. acnes) was provided by Shanghai Microbiological Culture Collection Co., Ltd. Activated C. acnes were inoculated into the Brucella blood AGAR culture plate. The Bradner blood AGAR culture plates inoculated with C. acnes were immediately placed into anaerobic gas-producing bags, which were incubated in a constant temperature incubator (37℃, 5% CO2).
Preparation of the bacterial suspension: Cultured C. acnes were diluted in gradient (105 CFU/mL, 106 CFU/mL, 107 CFU/mL, 108 CFU/mL, and 109 CFU/mL) with FT. The bacterial suspension obtained was used as the bacterial solution for cell experiments. In addition, the bacterial solution was diluted to 109 CFU/mL with PBS buffer for animal experiments.
NCTC clone 929 cells (L929) were provided by BeyoClickTM, China. At 37℃ and 5% CO2, L929 cells were cultured using RPMI 1640 medium, which contained 1% double antibodies (100 µL·mL-1 penicillin and 100 mg·mL-1 streptomycin) and 10% fetal bovine serum.
Sprague-Dawley rats (3 w, 160–180 g) were provided by Qingdao Darenfucheng Animal Technology Co., Ltd. Each rat was randomly placed in a cage with free access to food and water. After one week of adaptive feeding, rats were used for the preparation of an acne animal model and other treatments at later stages.
Characterization
A transmission electron microscope (TEM, HT7700, Hitachi, Japan) was used to observe the microstructure and morphology of the nanoparticles at 80 kV. Scanning electron microscopy (SEM, JSM-7500 F, JEOL, Japan) was used to analyze the surface morphology of the nanoparticles at 2 kV. A Zeta Simeter (ZEN3700, Malvern, Germany) was used to measure the Zeta potential and dynamic light scattering (DLS) of the nanoparticles. X-ray powder diffractometer (XRPD; Crystal phase analysis was performed using a Bruker D8 Advance X-ray diffractometer. A TCS SP5 laser scanning microscope (CLSM, LEICA, Germany) was used to acquire fluorescence images of cells or tissues. The CCK-8 method was used to detect cell viability after treatment of nanoparticles at 450 nm on Microplate Reader (Thermo Scientific Multiskan™ SkyHigh). Fluorescence microscopy (Olympus, Japan) was used to obtain immunofluorescence images of the cells.
Determination of pH and lipase content of bacterial supernatants
Activated C. acnes were seeded on FT at 4℃ at concentrations of 105 CFU/mL, 106 CFU/mL, 107 CFU/mL, 108 CFU/mL and 109 CFU/mL respectively. C. acnes supernatant was obtained by centrifugation after being cultured for 6, 12, and 24 h in a cell incubator at 37℃ and 5% CO2. The pH of the supernatant at different time periods was measured by a pH meter (PB-10). In addition, FT inoculated with C. acnes (109 CFU/mL) was simultaneously supplemented with 10 µM Orlistat, which was labeled as C.+ Orlistat group. And C.+ Orlistat group was cultured in a cell incubator at 37℃ for 6, 12, and 24 h, respectively. The supernatant was obtained by centrifugation, and the lipase content secreted by C. acnes at each time point was detected by lipase test kit.
Construction of an in vitro model of acne-like L929 cells
2 mL of L929 cell suspension (1 × 105 cells/well) was seeded into 12-well plates for culture. At 70–80% confluence of the cells, the old medium was replaced with 2 mL of a new medium pH 6.4, which was labeled as the pH 6.4 group. Then, 1 mL C. acnes suspension (1 × 109 CFU/mL) was inoculated in transwell chambers, which were immersed in 12-well plates of L929 cells cultured with normal medium for 6 h. therefore, an in vitro model of acne-like L929 cells was established. which was labeled as C. acnes (C.) group. At the same time as C. acnes suspension was inoculated in a transwell chamber, 10 µL of 1 mM Orlistat was added and then immersed in 12-well plates of L929 cells cultured with normal medium for 6 h and labeled as C.+ Orlistat group.
Analysis of the effect of C. acnes treatment on free fatty acid content
L929 cells were treated and grouped according to the above method: control group, pH 6.4 group, C. Group and C. + Orlistat group. L929 cells in all groups were treated for 6 h and then were treated with amplex red free fatty acid assay kit respectively.
Analysis of the effect of C. acnes and ZnO NPs treatment on intracellular ROS production
L929 cells were given different treatments: control group, pH 6.4 group, C. group, C.+ Orlistat group, control + ZnO NPs (50 µg/mL) group and C. + ZnO NPs (50 µg/mL) group. L929 cells in the first four groups were treated for 6 h. L929 cells in the last two groups were treated for 0, 3, 6, 9, 12 h and then were treated with Reactive Oxygen Species Assay Kit respectively.
Analysis of the influence of C. acnes and ZnO NPs treatment on intracellular MDA and GSH
L929 cells were given different treatments: control group, pH 6.4 group, C. group, C. + Orlistat group, control + ZnO NPs (50 µg/mL) group and C. + ZnO NPs (50 µg/mL) group. L929 cells in the first four groups were treated for 6 h. L929 cells in the last two groups were treated for 0, 3, 6, 9, 12 h and then were treated with malondialdehyde kit and glutathione kit respectively.
Analysis of the influence of C. acnes treatment on LDH content in cell supernatant
L929 cells were given different treatments: control group, pH 6.4 group, C. Group with different concentrations (105 CFU/mL, 106 CFU/mL, 107 CFU/mL, 108 CFU/mL, 109 CFU/mL) and C. + Orlistat group (109 CFU/mL C. acnes + 10 µL/1 mM Orlistat). L929 cells in each group were treated for 6, 12, 24 h and then were treated with Lactate dehydrogenase assay kit respectively.
Analysis of the influence of C. acnes treatment on membrane permeability
L929 cells were given different treatments: control group, pH 6.4 group, C. Group with different concentrations (105 CFU/mL, 106 CFU/mL, 107 CFU/mL, 108 CFU/mL, 109 CFU/mL) and C. + Orlistat group (109 CFU/mL C. acnes + 10 µL/1 mM Orlistat). L929 cells in each group were treated for 6, 12, 24 h and then were treated with Trypan Blue Staining Cell Viability Assay Kit respectively.
Analysis of the influence of C. acnes treatment on intracellular pH
L929 cells were given different treatments: control group, pH 6.4 group, C. Group and C. + Orlistat group. L929 cells in all groups were treated for 6 h and then were treated with BCECF AM respectively.
BioTEM for ZnO NPs endocytosis
L929 cells were given different treatments: control group, pH 6.4 group, ZnO NPs Group and ZnO NPs + Orlistat group (ZnO NPs: 50 µg/mL). L929 cells in all groups were treated for 6 h. The cells were washed by PBS and collected in a centrifuge tube, and then were fixed with 2.5% (v/v) glutaraldehyde. Finally cells in each group were subjected to bioTEM and ZnO NPs endocytosis was observed.
Cytotoxicity evaluation
200 µL L929 cells (1 × 104 cells/well) were cultured in 96-well plates for 24 h. The cells are divided into the following five groups: Control group, C. group, Orlistat group, ZnO NPs group, C. + ZnO NPs group. For the control group, L929 cells were cultured under standard conditions. For C. group, 200 µL of C. acnes suspensions with different concentrations (105CFU/mL, 106 CFU/mL, 107 CFU/mL, 108 CFU/mL, 109 CFU/mL) were inoculated into 96-well plates which had been cultured with L929 cells, and the C. acnes/cells mixed solutions were incubated for 3, 6, 9, 12 h, 24 h. For the Orlistat group, 100 µL C. acnes suspension (2 × 109 CFU/mL) and 100 µL of Orlistat (20 µM) solution were added to 96-well plates containing L929 cells, and all the above components were incubated for 6, 12, and 24 h. for ZnO NPs group, 12.5 µg·mL-1, 25 µg·mL-1 and 50 µg·mL-1 ZnO NPs were added to L929 cell culture medium and incubated for 3, 6, 9 and 12 h, respectively. For the C. +ZnO NPs group, 200 µL C. acnes suspension (1 × 109 CFU/mL) was inoculated on a 96-well plate in which L929 cells had been cultured, and the mixed solutions were incubated for 6 h. After that, the old mediums were removed and all the cells were cleaned twice with PBS. L929 cells were then treated with 12.5 µg·mL-1, 25 µg·mL-1 and 50 µg·mL-1 ZnO NPs for 3, 6, 9 and 12 h, respectively. All groups were treated with Cell Counting Kit-8 respectively.
Inductively coupled plasma (ICP) for Zn2+ content analysis
L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 µg·mL-1) group, C. +ZnO NPs (50 µg·mL-1) group. L929 cells in all groups were treated for 0, 3, 6, 12 h. The cells were broken under an ice water bath with an ultrasonic cell breaker. The intracellular Zn2+ content was detected by inductively coupled plasma emission spectrometer. There were three parallels in each group.
Analysis of intracellular Zn2+ content
L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 µg·mL-1) group, C. +ZnO NPs (50 µg·mL-1) group. L929 cells in all groups were treated for 0, 3, 6, 12 h and then were treated with Zinquin ethyl ester respectively.
Evaluation of mitochondrial membrane potential and apoptosis of L929 cells
L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 µg·mL-1) group, C. + ZnO NPs (50 µg·mL-1) group. L929 cells in all groups were treated for 0, 3, 6, 9, 12 h and then were treated with Mito Tracker Red CMXRos and Calcein AM/PI Double Staining Kit respectively.
Detection of type I collagen and type III collagen secretion
L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 µg·mL-1) group, C. +ZnO NPs (50 µg·mL-1) group. L929 cells in all groups were treated for 6 h. All cells were then incubated overnight with COL-I and COL-III antibodies at 4℃, then were incubated with Cyanine 3-labeled goat anti-rabbit immunoglobulin G (IgG) at room temperature away from light. The cells were then added to 4’, 6-diaminidine 2-phenylindole dye and incubated at room temperature away from light. The cells were encapsulated and photographed by fluorescence microscopy.
Western-blotting analysis of MMP9, TGF-β1和SDAD4
L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 µg·mL-1) group, C. + ZnO NPs (50 µg·mL-1) group. L929 cells in all groups were treated for 6 h. Nucleoprotein and cytoplasmic protein extraction kits containing 1 mM phosphatase inhibitors were used to treat cells after PBS washing. The cell lysate was heated in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer. The proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane, where the separation process was then blocked by skim milk. The polyvinylidene fluoride film was incubated with primary antibodies including anti-MMP9, anti-TGF-β1 and anti-SDAD4 at 4℃ overnight. After washing with TBS containing Tween-20 (TBST) buffer, polyvinylidene fluoride film coupled with horseradish peroxidase (HRP) was incubated at room temperature. Enhanced chemiluminescent reagent (ECL) kits and automated chemiluminescence image analysis systems were used to detect the expression of MMP9, TGF-β1 and SDAD4 in the cells of above groups.
Transcriptome analysis
L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 µg·mL-1) group, C. +ZnO NPs (50 µg·mL-1) group. L929 cells in all groups were treated for 6 h. The cells in each group were added with total RNA extraction reagent (TRIzol) reagent. The subsequent RNA sequencing was performed by eukaryotic mRNA sequencing based on Illumina sequencing platform by Shanghai NOhe Zhiyuan Bioinformation Technology Co., LTD. DESeq2 software was used for ZnO NPs group VS control group and C. +ZnO NPs group VS C. group transcriptional behavior differential analysis (DEGs) and further functional analysis of DEGs.
After the differential genes were obtained through gene expression analysis, the functions of differential genes were enriched and analyzed to find the signaling pathways and molecular regulatory mechanisms that play a key role in the differential biological behavior. ClusterProfiler software was used to complete GO functional enrichment analysis and KEGG path enrichment analysis of DEGs sets. Among them, GO analysis, which is based on the GO database (Gene Ontology), was responsible for the classification of genes according to Biological process (BP), Cellular component (Cellular component), biological process (BP). CC) and Molecular function (MF). KEGG pathway enrichment, which is based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database, was responsible for statistical analysis of the differential signal pathways.
After the target genes were identified, Pearson or Spearman algorithm was used to obtain the correlation coefficient between genes, which provided support for the drawing of the visual network map. In this work, STRING database was used for interaction network analysis of target proteins. In addition, Cytoscape software was adopted for Protein-protein interaction (PPI) analysis.
Preparation of ZnO NPs supported gels
0.1 g of carbomer powder was added to 10 mL of PBS buffer. The solution was stirred evenly, and 1% (m/v) carbomer gel was prepared. In addition, 50 µg/mL PBS solution of ZnO NPs was prepared and ultrasounded for 30 min. After 10 mL of the above ZnO NPs suspension was added with 0.1 g carbomer powder and the solution was stirred evenly, ZnO NPs (50 µg/mL) carbomer gel was prepared.
Evaluation of in vivo effects of ZnO NPs on acne skin tissue
In this study, all animal procedures were conformed to the Guide for the Care and Use of Laboratory Animals and performed following the guidelines and the protocol approved by the application of the experimental animal ethical project of Qingdao Agricultural University (Approval No.20220072). After the rats were fed adaptively for one week and the rats’ back hair was shaved, 0.5 mL oleic acid was applied once a day to the back skin for 7 days. From day 8, 0.2 mL C. acnes (1 × 109 CFU/mL) was injected into the dermis for 14 days, and acne-like inflammatory lesion model was established. The rats were randomly divided into six groups: Control group (PBS, n = 5), Carbomer + ZnO NPs group (CZ, n = 5), C. acnes group (C., n = 5), C. acnes + Carbomer + ZnO NPs group (C. CZ, n = 5), C. acnes + Carbomer + ZnO NPs + MMP-9-IN-1 group (CCZ + M, n = 5), C. acnes + Carbomer + ZnO NPs + β-Neo-Endorphin group (C. CZ + N, n = 5). From the 15th day to the 21st day of modeling, rats in different groups were treated as follows: for control group and C. acnes group, 1 mL PBS was applied on the back of rats every day; for the CZ group and the C. Z group, the back of rats was coated with 1 mL ZnO NPs carbomer gel every day. for C. CZM group, the back of rats was coated with 1 mL ZnO NPs and MMP-9-IN-1 carbomer gel every day. for the C. CZβ group, rats were coated with 1 mL ZnO NPs and β-Neo-Endorphin carbomer gel daily on their backs. The acne wounds were photographed on day 15 and day 21 respectively, and the area of acne wounds was recorded. On day 22, all anesthetized rats were killed by neck amputation and then microsurgical scissors were used to separate the acne wounds on the back of the rats. Skin pH value, Bacterial colony count, Zn level, HE staining, Masson staining, Sirius red staining, and immunohistochemical analysis were respectively performed on the back acne wounds.
Specifically, the wound tissues of the Control group, CZ group, C. group and C. CS group on the last day of treatment were placed in 4% paraformaldehyde, dehydrated by alcohol gradients of different concentrations, embedded in paraffin wax and were prepared into tissue sections with thickness of 5 mm. When measuring the skin’s pH, the test strip was first moistened in distilled water, then applied to both normal skin and acne areas. It was removed after five seconds, and the color was compared with the reference chart provided on the test strip container. When the number of bacteria in the wound tissue was measured, the wound tissue of the C. and C.C groups was placed in sterile PBS on the last day. After homogenizing and continuously diluting with sterile PBS, the diluent was inoculated on Columbia blood AGAR plate and incubated at 37℃ for 36 h. Finally, C. acnes was selectively isolated and the total number of colonies was counted.
In order to determine the pathological condition of the acne tissue, the wound tissue was analyzed by HE staining. After dewaxing and H&E staining, the tissue sections were viewed microscopically and photographed in full scan.
In order to evaluate the effects of ZnO NPs on collagen fibers and muscle fibers in acne tissues, acne tissues were subjected to Masson’s trichrome staining analysis. After undergoing dewaxing and Masson staining, sections of acne tissue were observed and photographed.
To evaluate the effects of ZnO NPs on type I collagen and type III collagen in acne tissues, the acne tissues were stained with Sirius red. After dewaxing, Sirius red staining, the tissue sections were placed under polariscope for observation and photography of collagen fibers.
In addition, the distribution of type I collagen, type III collagen and smooth actin in acne tissues was also confirmed by immunohistochemical analysis. The acne tissue sections of each group were labeled with Collagen I, Collagen III and α-SMA monoclonal antibodies, respectively, and then incubated with second anti-antibody. Finally, the section samples were viewed under a fluorescence microscope and photographed.
To determine Zn levels in acne tissues, acne tissues of each group were placed on sterile PBS on the last day of treatment to remove residual ZnO NPs. After that, inductively coupled plasma analyzer was used to determine the content of Zn in the tissues.