Polyphyllin I Inhibits Propionibacterium acnes-Induced IL-8 Secretion in HaCaT Cells by Downregulating the CD36/NOX1/ROS/NLRP3/IL-1β Pathway

Acne vulgaris (AV) is a chronic skin disease involving inflammation of the pilosebaceous units. Propionibacterium acnes (P. acnes) hypercolonization is one pathogenic factor for AV. P. acnes that triggers interleukin-1β (IL-1β) by activating the pyrin domain-containing 3 protein (NLRP3) inflammasome of the NOD-like receptor family in human monocytes. Reactive oxygen species (ROS) acts as a trigger for the production of IL-8 and activates theNLRP3 inflammasome. IL-8 promotes the metastasis and multiplication of different cancerous cells, whereas keratinocyte proliferation and migration contribute to the progression of AV. A steroidal saponin called polyphyllin I (PPI) that is extracted from Paris polyphylla's rhizomes has anti-inflammatory properties. This study investigates the regulatory role of P. acnes in the secretion of IL-8 mediated by the CD36/NADPH oxidase 1 (NOX1)/ROS/NLRP3/IL-1β pathway and the effects of PPI on the CD36/NOX1/ROS/NLRP3/IL-1β/IL-8 pathway and human keratinocyte proliferation and migration. HaCaT cells were cultured and stimulated with 108 CFU/ml of P. acnes for 0, 6, 12, 18, 24, 30, and 36 hours. P. acnes induced IL-8 secretion from HaCaT cells via the CD36/NOX1/ROS/NLRP3/IL-1β pathway. PPI inhibited the CD36/NLRP3/NOX1/ROS/IL-8/IL-1β pathway and HaCaT cell proliferation and migration. PPI alleviates P. acnes-induced inflammatory responses and human keratinocyte proliferation and migration, implying a novel potential therapy for AV.


Introduction
e skin is the primary stress perceiving organ; hence, it is prone to inflammatory dermatoses [1,2]. Acne vulgaris (AV) is a chronic skin disease that involves the inflammation of the pilosebaceous unit that affects adults as well as teens, but it is not fatal. However, affected individuals may be socially maladjusted due to severe acne and ongoing psychological effects [3]. Conventional treatments include hormonal therapy, oral antibiotics, and isotretinoin, but these strategies have different side effects including chapped lips and teratogenicity that limit isotretinoin medication [4]. In addition, isotretinoin is also known for causing inflammatory bowels in adolescents and children [5], and it is also reported to cause depression [6]. Drug resistance is another major concern due to the long-term and extensive use or overuse of antibacterial drugs [7]. erefore, a more effective and safer AV therapy targeted to all age groups is in demand.
is study is aimed at exploring the underlying molecular and cellular mechanisms regarding the pathogenesis of AV. Previous studies have revealed that the hypercolonization of Propionibacterium acnes (P. acnes) is one mechanism of AV pathogenesis [8,9]. P. acnes is a rodshaped, anaerobic, Gram-positive pathogen mostly inhabitates the deep microaerophilic parts of healthy follicles, where it comes into contact with follicular keratinocytes and cells in the proximal part of the sebaceous duct. Clinical isolates of P. acnes (889) are capable of inducing primary human epidermal keratinocyte growth in vitro [10]. In addition, P. acnes mediates keratinocyte proliferation and elevates inflammation in the region where it resides, making it a key factor in causing inflammatory acne lesions and the development of microcomedos in the initial acne stages [11,12]. e NOD-like receptor family, pyrin domain-containing 3 protein (NLRP3) inflammasome contains a variety of sensor molecules including NLRP3, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and procaspase-1. NLRP3 has a pyrin domain (PYD) and an ASC that harbors both PYD and CARD domains. After its activation, NLRP3 interaction with ASC occurs through PYD and CARD domains of procaspase-1 to form the NLRP3-ASC-procaspase-1 complex, which is also referred to as the NLRP3 inflammasome [13]. By promoting caspase-1 activation, the NLRP3 inflammasome enhances the proteolytic maturation of the proinflammatory cytokine interleukin-1β (IL-1β) to facilitate its attachment to the IL-1 receptor [14]. IL-1β further induces the expression of the canonical IL-1 target gene interleukin-8 (IL-8) in human macrophages [15] and human endometrial stromal cells [16]. P. acnes activates IL-8 secretion by interacting with toll-like receptor 2 and toll-like receptor 4 (TLR-2 and TLR-4) present on the surface of human epidermal keratinocytes, contributing to the development of inflammatory lesions [10,17]. erefore, the assembly of NLRP3 inflammasome promotes caspase-1-mediated IL-1β secretion [18]. Previous studies show that P. acnes activates NLRP3 inflammasome and caspase-1 which promotes IL-1β secretion in human monocytes and sebocytes [19,20]. is indicates the role of P. acnes in activating the NLRP3 inflammasome-mediated IL-8 secretion in human keratinocytes.
P. acnes activates NADPH oxidase 1 (NOX1) that triggers the release of reactive oxygen species (ROS) and secretion of IL-8 which is an essential proinflammatory cytokine in the pathogenesis of AV, by recognizing the thrombospondin receptor (CD36) on the surface of human keratinocytes (HaCaT cells) [21]. CD36 and other scavenger receptors bind to a range of microbial and endogenous cargoes and mediate their internalization [22,23]. Several of these cargoes activate NLRP3 inflammasome. Based on this fundamental data, CD36 is likely to activate NLRP3 inflammasome through NOX1 following P. acnes stimulation in human keratinocytes.
ROS serves as a trigger that activates the NLRP3 inflammasome, facilitating the pathological processes. External stimuli such as a bacterial infection may trigger the activation of NLRP3 inflammasome [24,25], causing the release of cytokines and O − 2 effectors, which leads to chronic sterile inflammation and tissue injury [26,27]. If the ROS is more than their scavengers during the local inflammatory response, it results in the increasing intracellular and extracellular oxidative stress and IL-8 secretion [28,29], bringing about the progression of AV [30,31]. Additionally, IL-1β directly acts on keratinocytes to suppress the expression of functional protein and nitric oxide availability to further contribute to local hyperkeratosis [32,33].
Paris polyphylla is a traditional medicinal herb widely known in the Shennongjia National Nature Reserve of China, which has a wide range of beneficial components that act against immune disorders, infectious diseases, cardiovascular diseases, and cancer [34][35][36][37][38]. One of the medicinal elements of P. polyphylla is polyphyllin I (PPI), which is a steroidal saponin derived from the rhizomes of P. polyphylla, and it is known for its anti-inflammatory properties. For example, PPI reduces the severity of collagen-induced arthritis by suppression of the nuclear factor kappa B (NF-κB) pathway in macrophages that causes the suppression of the intracellular inflammatory response [39]. Our previous study also shows that PPI reduces the secretion of inflammatory cytokines, including tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), and IL-8, and suppresses the expression of NF-κB, TLR-2, and the mitogen-activated protein kinase (MAPK) signal transduction pathways in HaCaT cells infected by P. acnes [40]. Furthermore, PPI has been found to possess preclinical anticancer efficacy by inhibiting proliferation and invasion of various cancer types, including gastric cancer [41], prostate cancer [42], and hepatocellular carcinoma [43]. However, the effect of PPI on IL-8 secretion through modulating the NLRP3 inflammasome is unknown.
We, therefore, aimed to elucidate the ability of P. acnes to regulate IL-8 secretion via the NLRP3/CD36/NOX1/ROS/ IL-1β pathway and the effects of PPI on the NLRP3/CD36/ NOX1/ROS/IL-8/IL-1β pathway and human keratinocyte proliferation and migration. Our results will further explain the role of P. acnes in the pathogenesis of AV and supply basic in vitro experimental data for the application of PPI in AV therapy.

Western
Blot. Lysis of HaCaT cells was carried out using radioimmunoprecipitation assay (RIPA) and lysis buffer supplemented with phenylmethanesulfonyl fluoride (PMSF) 2 Evidence-Based Complementary and Alternative Medicine after their isolation.

Enzyme-Linked Immunosorbent Assay (ELISA).
A 96well plate was used to seed 5 × 10 3 HaCaT cells which were then cultured at 37°C (5% CO 2 ) according to the set protocols. e supernatant from cultures cells was collected, and the concentration of IL-8 was calculated by the human IL-8 ELISA kit ( # KHC0081; ermo Fisher) following the manufacturer's protocols. e absorbance reading was done at a wavelength of 450 nm using a microplate photometer supplied by ermo Scientific ( # VLBL00D1).

siRNA Transfection and Reagent
Usage. Human CD36 siRNA, NOX1 siRNA, and NC siRNA were synthesized by GenePharma (China) ( Table 1). HaCaT cells were plated in a dish of 6 cm in size (5 × 10 5 cells) a day before transfection with the Lipofectamine 2000 transfection reagent ( # 12566014; ermo Fisher). Separate dilution of fifteen microliters (10 nM) of siRNA and 20 μl Lipofectamine 2000 in 500 μl of DMEM were mixed after 5 minutes followed by an incubation period of 20 minutes at 25°C. en, the mixture was poured into the cell culture dishes with 5 ml of DMEM. After 6 h, the medium was substituted by fresh medium, and cells were harvested after24 hours of transfection.

Detection of ROS Levels.
Flow cytometry detected the intracellular ROS accumulation using the cell-permeable fluorogenic probe 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA; # S0033; Beyotime, China). e supernatant was removed from cultured cells that were treated with different reagents, following incubation in the dark in a fresh medium and 10 μM DCFH-DA at 37°C for 1 h. Afterwards, the media from the culture was removed, and cold PBS was used to wash the cells. Next, after harvesting the cells, the pallets were suspended in 500 μl of PBS. Sample analysis was performed at 480 nm excitation wavelength and an emission wavelength of 525 nm by a flow cytometer supplied by FACScan.

EdU Proliferation Assay.
To determine whether PPI affects HaCaT cell proliferation, an EdU incorporation assay was carried out. 48 hours after cell seeding, 10 μM EdU was poured into the culture following incubation for some time followed by fixation using 4% paraformaldehyde in PBS for 15 minutes. EdU labeling with an azide derivative of Apollo 643 was performed using a Cell-Light ™ EdU Apollo 643 In Vitro Imaging Kit ( # C10310-2, RiboBio Co., Ltd., China). A 652 nm laser was used for the excitation of Apollo 643. Microscopic images were obtained with a FluoView FV1000 confocal laser scanning microscope (Olympus, Japan). ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to get composite images.

Transwell Migration Assay.
For the migration assay, HaCaT cells were exposed to P. acnes and PPI (0.9 μg/ml) or the same volume of 0.1% DMSO, and then, 5 × 10 4 cells were transferred into each transwell insert pore of about 8 μm ( # 3422; Corning, USA). e cells were then incubated for 24 hours followed by the removal of cells adhered to the upper surface of the insert, while the ones that adhered to the lower surface were stained with crystal violet (0.2%). Afterwards, isopropanol was used to dissolve the migratory cells, and the wavelength at 595 nm was considered to detect their optical density.
Evidence-Based Complementary and Alternative Medicine 2.8. Statistical Analysis. Data interpretation was performed by one-way analysis of variance, followed by the Bonferroni post hoc test for comparing the mean values of multiple groups using GraphPad Prism 6 (GraphPad Software, USA), and p < 0.05 was considered a statistically significant difference. e results were presented as the mean ± SEM.
e results indicate that P. acnes induces the activation of NLRP3 inflammasome in HaCaT cells.

PPI Inhibits HaCaT Cell Proliferation and Migration.
Keratinocyte proliferation and migration contribute to the progression of AV [44,45]. Moreover, PPI exhibits inhibitory effects on human ovarian cancer HO-8910PM cell growth, promotes apoptosis, and inhibits cell migration [46]. erefore, the PPI effect of HaCaT cell proliferation and migration was evaluated. Consistent with its previously reported roles, PPI inhibited HaCaT cell proliferation (Figures 4(a) and 4(c)) and migration (Figures 4(b) and 4(d)) compared to those of the P. acne's group. e data suggested that PPI might alleviate AV by downregulating keratinocyte proliferation and migration.

Discussion
Human keratinocytes are cultured frequently for in vitro studies of and understanding of their inflammatory responses and immunological role. However, due to variable results from different donors, short culture time and passage variations may cause difficulty in interpreting the collected data. HaCaT cells are a sustainable cell line of spontaneously immortalized human keratinocytes. e previous study highlights the influence of cell density, different concentrations of Ca 2+ ions in the medium, and the presence of serum at different levels on the secretion of proinflammatory mediators by these cells. Moreover, HaCaT cells survived in the low Ca 2+ ionic medium and showed 80% resemblance to normal keratinocytes in their cytokine secretion suggesting that HaCaT cells are useful anti-inflammatory therapeutic agents for the investigation of dermatological ailments [47]. Additionally, HaCaT has been widely used for the research of acne vulgaris, such as the molecular mechanism study [48,49], and potential drug screen [50,51]. erefore, HaCaT cells were used for this research.
Herein, we found that P. acnes activated the NLRP3 inflammasome in human keratinocytes. e NLRP3 inflammasome is a molecular platform that assembles in response to various stimuli, including excessive ROS levels, and has been extensively studied [52,53]. P. acnes can produce ROS during the infection process [54,55]. erefore, whether P. acnes activates the NLRP3 inflammasome in human keratinocytes by upregulating intracellular ROS needs further investigation. e combined effect of tumor necrosis factor-α and interleukin-17 enhances ROS release and improves NOX1 activity which in turn triggers the expression of lipocalin-2 (LCN-2) by controlling the expression of a major LCN-2 inducer called IkappaBzeta (IκBζ). In addition, mice models that were deficient in NOX1 had lowered levels of LCN-2 production and colon damage during TNBS-induced colitis [3]. Furthermore, UVA activates NOX1-based NADPH oxidase to release ROS that further stimulates the synthesis of prostaglandin E2. Hence, NOX1 presents as an appropriate target for the components that are designed to protect from UVA-induced skin injury [56]. Moreover, the NOX1 gene is knocked down using RNA interference for the          ROS triggers not only the NLRP3 inflammasome but also the downstream effector molecule of this inflammasome. It is clearly understood that the ROS initiated oxidative stress and is directly or indirectly responsible for causing AV [31,57]. us, antioxidant therapies have been applied for AV and have shown the expected efficacy [58,59]. For example, topical or oral zinc, the vitamin C precursor sodium ascorbyl phosphate, and nicotinamide were reported to be effective against acne in multiple studies [60,61]. Whether PPI plays an antioxidant role in human keratinocytes must be elucidated in future research.
In the previous study, researchers have identified that caspase-1, ASC, and NLRP3 knockdown or knockout inhibits P. acnes-induced IL-1β production in acne [33]. IL-1β drives inflammatory responses in Propionibacterium acnes both in vitro and in vivo. P. acnes activates the NLRP3 inflammasome of monocyte-macrophages and promotes the processing and secretion of IL-1β. Additionally, ultraviolet irradiation stimulates the NLRP3 inflammasome activation in keratinocytes [62]. erefore, we did not perform similar experiments to confirm that P. acnes induces the NLRP3/ ASC/caspase-1/IL-1β pathway in keratinocytes. Upon the stimulation of pattern recognition receptors, keratinocytes secrete IL-1β that causes rapid initiation of the immune response, leading to the expression of other cytokines, including IL-8 [63]. In the present study, we also found that IL-1β promoted IL-8 secretion by HaCaT cells following P. acnes treatment.
In summary, P. acnes induced the production of ROS, activation of the NLRP3 inflammasome, and IL-8 release in HaCaT keratinocytes, while PPI inhibited ROS  Figure 4: PPI inhibiting proliferation and migration of HaCaT cells. (a) e proliferation of HaCaT cells following P. acnes treatment with PPI (0.9 μg/ml) or 01% DMSO exposure detected by the EdU incorporation assay. e red color represents EdU positive, and the blue color represents DAPI (nucleus). e average ratio of EdU positive HaCaTcells is calculated by ImageJ. (b) e migration of HaCaTcells following P. acnes treatment with PPI (0.9 μg/ml) or 0.1% DMSO exposure detected by the transwell assay. (d) e average number of migrated HaCaT cells calculated by ImageJ. n � 5/each group. * * P < 0.01 vs. control group. ## P < 0.01 vs. P. acnes treatment group. production, NLRP3 activation, IL-8 secretion, and HaCaT keratinocyte proliferation and migration, therefore suggesting a potential treatment strategy for AV. Certainly, there are some limitations of our study that were worthy of being addressed in future studies; for example, the detailed mechanisms of NLRP3 inflammasome activation and human AV lesion observation should be studied in the future.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
is research was authorized by the Ethics Committee of the People's Hospital of Baoshan.

Consent
Not applicable. Evidence-Based Complementary and Alternative Medicine 9