Inhibition of Porphyromonas gingivalis-Induced Respiratory Inflammation by an Alkaline Extract of Sasa senanensis Leaves

TL;DR

A leaf extract from Sasa senanensis reduces inflammation caused by a periodontal bacterium in respiratory tissues, both in human cells and mice.

Contribution

The study demonstrates the anti-inflammatory effects of an alkaline leaf extract against P. gingivalis-induced respiratory inflammation.

Findings

SE reduced IL-6 and IL-8 mRNA and cytokine secretion in human bronchial epithelial cells.

SE inhibited NF-κB and MAPK pathways, including p38 and JNK, in a dose-dependent manner.

In mice, SE lowered lung cytokine levels and NF-κB activity after P. gingivalis exposure.

Abstract

Periodontal pathogens, including Porphyromonas gingivalis (P. gingivalis), are implicated in respiratory inflammatory conditions, and aspirated oral bacterial components may contribute to airway inflammation. This association has prompted the exploration of innovative therapeutic strategies in addition to conventional oral hygiene practices. We evaluated the anti-inflammatory efficacy of an alkaline extract of Sasa senanensis leaves (SE) against heat-inactivated P. gingivalis-induced inflammation in respiratory tissues. In human bronchial epithelial cells (BEAS-2B), SE reduced interleukin (IL)-6 and IL-8 mRNA expression and cytokine secretion in a dose-dependent manner. Moreover, SE attenuated nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs), including p38 and c-Jun N-terminal kinase (JNK), indicating broad anti-inflammatory actions. In mice, SE administration decreased early lung cytokine levels and reduced NF-κB activity following intratracheal challenge with heat-inactivated P. gingivalis. Together, these in vitro and in vivo findings indicate that SE suppresses proinflammatory signaling triggered by P. gingivalis components and may serve as a natural adjunct to mitigate bacteria-associated airway inflammatory responses.

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Full text

1. Introduction

Recently, the association between oral and systemic health has revitalized interest in the role of oral hygiene in overall health outcomes [1]. Among older adults, aspiration pneumonia remains an important clinical concern because it is associated with substantial morbidity and mortality [2]. Aspiration of oropharyngeal contents, including saliva and oral bacteria, can provoke inflammatory responses in the lower airway, characterized by increased recruitment of inflammatory cells and elevated levels of proinflammatory cytokines [3,4].

Periodontitis, a dental plaque bacteria-induced and host immune system-mediated breakdown of soft and hard tissues surrounding the teeth [5], is the sixth most prevalent chronic condition worldwide [6]. Porphyromonas gingivalis (P. gingivalis), a Gram-negative, black-pigmented anaerobe, is instrumental in disease development. This pathogen invades periodontal tissues, affecting epithelial, connective, and alveolar bone tissues [7]. Notably, P. gingivalis also colonizes respiratory airways, influences respiratory epithelial cells, increases inflammatory cytokines [8], and plays a role in aspiration pneumonia [9]. P. gingivalis has been isolated from the bronchoalveolar lavage fluid (BALF) or sputum of patients with pneumonia [10,11]. Management of periodontitis is crucial in maintaining oral health and protecting respiratory functions, particularly in individuals at high risk of aspiration pneumonia [8]. While conventional periodontal approaches, including brushing, flossing, and scaling with root planing, are vital, their effectiveness and applicability are limited in these patients, especially those with a physical handicap. Furthermore, the use of adjunctive antibiotics has led to the emergence of resistant bacterial strains, highlighting the need for alternative preventive strategies to combat the spread of periodontal pathogens linked to respiratory diseases.

The pursuit of safe and efficacious treatments for inflammatory disorders has intensified interest in natural plant and vegetable products [12]. Research has highlighted the anti-inflammatory properties of natural compounds, including curcumin, resveratrol, and cynaropicrin [13,14,15]. In particular, the leaves of Sasa senanensis, also known as Kumaizasa, an East Asian bamboo species native to Japan and China, have been reported to exhibit anti-HIV [16], anti-UV [17], and neuroprotective activities [18]. The alkaline extract from Sasa senanensis leaves (SE), an over-the-counter supplement, is available in drugstores in Japan. SE, with a dry weight concentration of 58.2 ± 0.96 mg/mL, comprises Fe (II)–chlorophyllin and lignin–carbohydrate complexes (LCCs), among other degradation products [19]. SE, known for its anti-inflammatory activities, is effective in suppressing nitric oxide and prostaglandin E_2_ production in mouse macrophages stimulated by lipopolysaccharide (LPS) [20]. However, whether SE can attenuate inflammatory responses triggered by periodontal pathogens (such as Porphyromonas gingivalis) in airway epithelial cells and whether it mitigates acute lung inflammatory responses induced by bacterial components in vivo remain unclear.

Based on these findings of the anti-inflammatory properties of SE, the present study aimed to evaluate the potential of SE in addressing P. gingivalis-induced inflammatory responses in bronchial epithelial tissue. We employed an in vitro assessment using human bronchial epithelial cells and in vivo experiments using mice intratracheally challenged with heat-inactivated P. gingivalis.

2. Materials and Methods

2.1. Cell and Bacterial Culture

Human bronchial epithelial cells (BEAS-2B) were purchased from ATCC (Manassas, VA, USA) and maintained at 37 °C in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Rockford, IL, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL). Following a 24 h incubation, which achieved 80–90% confluency, the cells were treated with different SE concentrations.

P. gingivalis ATCC 33277 was cultured in brain heart infusion broth (Becton, Dickinson and Company, Sparks, MD, USA) supplemented with 5 μg/mL hemin and 0.5 μg/mL menadione. The culture was incubated at 37 °C for 24–72 h and grown in an anaerobic chamber (TE-HER ANAEROBOX, Hirasawa, Tokyo, Japan) under anaerobic conditions of 10% H_2_, 10% CO_2_, and 80% N_2_. The bacterial cell density was adjusted to 1.0 × 10^10^ colony-forming units (CFU)/mL, and the bacterial suspension was heat-inactivated at 60 °C for 1 h and stored at −80 °C until use.

2.2. Reagents and Plasmid

The commercially available product of SE (SASA-Health^®^) was provided by Daiwa Biological Research Institute Co. Ltd., Kawasaki, Kanagawa, Japan. SE was prepared by iron ion substitution, alkaline extraction, and neutralization/desalting. Iron ion substitution was employed to obtain an iron-substituted chlorophyllin-related fraction with intended antioxidant and deodorizing properties. Alkaline extraction was used to efficiently obtain alkaline-soluble lignin-associated constituents (e.g., LCC-rich fractions) that are poorly extracted by hot water. Neutralization/desalting was performed to stop further alkaline hydrolysis during extraction and to obtain a near-neutral, low-salt preparation. Lyophilization and measurement of the dry weight of SE showed that it contained 58.2 ± 0.96 mg solid materials/mL [20,21,22]. All experiments were performed using the same lot of SE (Lot No. BCBC) to minimize lot-to-lot variability. The major components of SE have been characterized previously and include Fe (II)–chlorophyllin and LCCs, among other degradation products [19,22]. The SE components used in this study are summarized in Table 1.

p38- mitogen-activated protein kinase (MAPK) inhibitor (SB203580; 10 μM), c-Jun N-terminal kinase (JNK) inhibitor (SP600125; 10 μM), extracellular signal-regulated kinase (ERK) 1/2 inhibitor (U0126; 10 μM) (Merck, Darmstadt, Germany), and nuclear factor-κB (NF-ĸB) inhibitor (BAY11-7082; 10 μM) (Wako, Osaka, Japan) were added 60 min before the addition of P. gingivalis to allow sufficient time for cellular uptake and ensure effective pathway inhibition at the onset of bacterial stimulation. All inhibitor stocks were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. A reporter plasmid expressing firefly luciferase under the control of NF-κB (pGL3-5xκB-luc) was used as described previously [23]. Antibodies against p65, p-p65, IκBα, p-IκBα, p38, p-p38, JNK, p-JNK (Cell Signaling Technology, Danvers, MA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used. Horseradish peroxidase-conjugated secondary antibodies were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK).

2.3. Cell Viability Assay

Initially, the effects of SE on cell viability/metabolic activity were evaluated in BEAS-2B cells using a WST-1 assay (Roche Diagnostics, Basel, Switzerland). The medium was replaced with 1% FBS-containing medium supplemented with various concentrations of SE (1, 2, 4, 8, 16, 32, and 64%). After 15 h of incubation with SE, an WST-1 solubilization solution was added to each well. Four hours later, absorbance was subsequently quantified at 450 nm using a plate reader (SpectraMax ABS Plus; Molecular Devices, San Jose, CA, USA). Exposure for 15 h was used to capture potential delayed effects on cellular metabolic activity.

2.4. mRNA Expression of Inflammation-Related Genes

After a 24 h incubation reaching 80–90% confluency, BEAS-2B cells (4 × 10^5^ cells/mL) cultured in the medium containing 1% FBS were treated with various concentrations of SE (1, 2, and 4%). These concentrations were selected based on the WST-1 assay showing no overt reduction in cell viability/metabolic activity under these conditions and to allow for an evaluation of dose-dependent inhibitory effects. Following a 3 h incubation, P. gingivalis (1 × 10^8^ CFU/mL) was introduced. An additional incubation of 3 h was carried out before extracting total RNA using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). Total RNA was collected 3 h after P. gingivalis stimulation to capture early transcriptional responses. From 250 ng of DNase-treated total RNA, complementary DNA (cDNA) was synthesized using the PrimeScript RT Master Mix (Takara Bio, Shiga, Japan). Subsequently, this cDNA was analyzed via quantitative real-time polymerase chain reaction (qPCR) employing TB Green Premix Ex Taq II (Takara Bio). The 25 μL reaction mixture contained 12.5 μL of TB Green Premix Ex Taq™ II, 0.5 μL of each primer (10 μM), 9.5 μL of dH_2_O, and 2 μL of cDNA. The following primer sequences were used: IL-8 forward (5′-CTTGTCATTGCCAGCTGTGT-3′) and reverse (5′-TGA CTGTGGAGTTTTGGCTG-3′); IL-6 forward (5′-TTCGGTCCAGTTGCCTTCTC-3′) and reverse (5′-GAGGTGAGTGGCTGTCTGTG-3′); and GAPDH forward (5′-TGCACCACCAACTGCTTAGC-3′) and reverse (5′-GGCATGGACTGTGGTCATGAG-3′). PCR assays were conducted using a TP-800 Thermal Cycler Dice Real-Time System (Takara Bio) and the results were analyzed with software provided by the manufacturer. The thermal cycling conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. All real-time PCR experiments were performed in triplicate, with the product specificity confirmed through melting curve analysis. Gene expression levels were normalized against GAPDH mRNA.

2.5. Cytokine Measurements in BEAS-2B Cell Culture Supernatants

Cytokine concentrations were quantified in BEAS-2B cell culture supernatants following exposure to heat-inactivated P. gingivalis (1 × 10^8^ CFU/mL) using cytokine-specific enzyme-linked immunosorbent assay (ELISA) kits for IL-8 and IL-6 (Quantikine ELISA kits; R&D Systems, Minneapolis, MN, USA), adhering to the manufacturer’s protocols. Under our conditions (2 × 10^5^ cells per well in 0.5 mL), this dose corresponds to an approximate multiplicity of infection (MOI) of 250 (CFU equivalents per cell). ELISA analyses were performed to assess the effects of SE pretreatment (durations and concentrations) and pathway inhibitors (NF-κB, p38, JNK, and ERK), as detailed in the figure legends.

2.6. Transient Luciferase Assay

BEAS-2B (4 × 10^5^ cells/mL) were transfected with reporter plasmids using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. For each transfection, 200 ng of 5xκB-luc, a plasmid in which luciferase gene expression is under the control of NF-κB, and 10 ng of an internal control plasmid pRL-TK, which expresses Renilla reniformis luciferase under the control of TK promoter, were used. Twenty-four hours after transfection, the cells were treated with SE at two different concentrations (2 and 4%) for 3 h and then stimulated with P. gingivalis (1 × 10^8^ CFU/mL) for 12 h. Cells were harvested using a passive lysis buffer (Promega, Madison, WI, USA), and the extracts were assessed for luciferase activity using the dual-luciferase assay system (Promega) as described previously [23]. Luciferase activity was normalized to R. reniformis luciferase activity, which acted as an internal control for transfection efficiency.

2.7. Western Blotting

Western blotting (WB) was performed to analyze protein levels. Following a 24 h incubation, which achieved 80–90% confluency, BEAS-2B (4 × 10^5^ cells/mL) exposed to the medium with 1% FBS was supplemented with various concentrations of SE (1, 2, and 4%) for 3 h and subsequently stimulated with P. gingivalis (1 × 10^8^ CFU/mL) for 30 min. Experimental procedures for WB were performed as described previously [15]. Briefly, nuclear and cytoplasmic proteins were lysed using the nuclear/cytosolic fractionation assay kit from BioVision (Mountain View, CA, USA) following the manufacturer’s protocol. Equal amounts of protein (20 μg) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (EMD Millipore Corporation, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk and incubated with the following primary antibodies at 4 °C overnight: anti-p65 (1:500), anti-p-p65 (1:1000), anti-IκBα (1:1000), anti-p-IκBα (1:1000), anti-p38 (1:1000), anti-p-p38 (1:1000), anti-JNK (1:1000), anti-p-JNK (1:1000), and anti-GAPDH (1:1000) antibodies. Proteins on the membrane were visualized using a SuperSignal West Pico enhanced chemiluminescence kit (Thermo Fisher Scientific).

2.8. Animal Experiments Using Mice

The animal experiment was conducted in compliance with the ARRIVE guidelines and in accordance with the Regulations and Guidelines in Scientific and Ethical Care and Use of Laboratory Animals of the Science Council of Japan, enforced on 1 June 2006. The study protocol was approved by the Institutional Animal Care and Committee of Nihon University School of Dentistry. Specific pathogen-free male C57BL/6JJcl mice aged 7 weeks were obtained from CLEA Japan, Inc. (Tokyo, Japan). The mice were housed under standard conditions within the animal care facility at the Nihon University School of Dentistry, Tokyo, Japan. In the SE group mice, 40 mL of a 2% SE solution was added to 200 mL of drinking water (final concentration: 0.33% SE), which was then given to the mice as drinking water for 72 h. This concentration was selected based on the manufacturer’s recommended oral use for the commercially available preparation and to fall within a practically relevant dilution range. Based on the solid content of SE (58.2 mg/mL) and the final dilution in drinking water (0.33%, v/v), the SE-derived solid content in drinking water was approximately 0.192 mg/mL. Assuming a typical daily water intake of ~3–5 mL and a body weight of ~25 g, this corresponds to an estimated intake of ~23–38 mg/kg/day. Admittedly, considering water consumption was not measured in this study, the actual dose may vary. All mice were anesthetized with inhaled isoflurane and intratracheally instilled with heat-inactivated P. gingivalis (1 × 10^8^ CFU/mouse) as an acute sterile inflammatory challenge to bacterial components, as described previously [24]. Half of the mice in the SE group were euthanized 30 min later, and their lungs were extracted. The remaining half were euthanized 3 h later, and their lungs were also extracted. The extracted lungs were homogenized and centrifuged, and the supernatant was collected. WB and ELISA analyses were performed similarly to the ones described above. The lungs extracted after 30 min were used for WB and ELISA analyses (NF-ĸB), and those extracted after 3 h were used for ELISA analyses (keratinocyte-derived cytokine (KC) and IL-6).

2.9. Statistical Analysis

All statistical analyses were performed using KaleidaGraph (Synergy Software, Reading, PA, USA; version 5.0.3). One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis was used for comparisons of more than three groups. Significance level was defined by at least p < 0.05. All data are expressed as the mean ± standard deviation (SD).

3. Results

3.1. Effects of SE on IL-6 and IL-8 Expressions in P. gingivalis-Stimulated BEAS-2B

Initially, we evaluated the effects of SE on cell viability/metabolic activity in BEAS-2B cells using the WST-1 assay. The data indicated cell viability exceeding 80% at SE concentrations up to 4% (Figure 1A). Consequently, we investigated SE concentrations ranging from 0 to 4% in subsequent experiments. Further, we evaluated the influence of SE on IL-6 and IL-8 mRNA expression in BEAS-2B cells stimulated by P. gingivalis. Employing real-time PCR for mRNA analysis and ELISA for protein quantification, we determined the effect of SE on the expression of these inflammatory cytokines. P. gingivalis stimulation resulted in increased IL-8 and IL-6 expressions (Figure 1B). Pretreatment of cells with SE before P. gingivalis exposure significantly reduced IL-8 and IL-6 mRNA levels in a dose-dependent response (Figure 1B). This effect was also confirmed by measuring the secretion of both proteins through specific ELISA analysis (Figure 2A,B). SE markedly suppressed P. gingivalis-induced IL-8 production in BEAS-2B cells after 3 h of SE treatment and decreased IL-6 production in a time-dependent manner (Figure 2A). Even the small concentration of SE dramatically suppressed IL-8 production (Figure 2B). Regarding IL-6, suppression was observed with the high-concentration treatment, but the amount of suppression was about 1/10 of that of IL-8 (Figure 2B). Because SE was applied only during the pretreatment phase and the medium was replaced before P. gingivalis stimulation, SE carryover into supernatants collected for ELISA was expected to be minimal. Consistently, spike-and-recovery validation performed in the presence of SE showed acceptable recoveries (Table A1). In addition, to ensure that subsequent analyses performed under the same experimental schedule were not confounded by cytotoxicity, we confirmed that cell viability/metabolic activity remained above 80% of the control level across all treatment groups (Figure A1). These results confirm that SE could effectively suppress inflammatory cytokine levels induced by P. gingivalis without causing cytotoxic effects, affecting IL-8 production more significantly.

3.2. SE Inhibits P. gingivalis-Induced NF-κB Activity in BEAS-2B

Activation of MAPK and NF-κB pathways—common inflammatory signaling pathways—released proinflammatory cytokines including IL-8 and IL-6, leading to the inflammatory response. Under normal conditions, NF-κB is inactive, bound in the cytoplasm to the inhibitor of nuclear factor-κB (IκB) α. Exposure to periodontal pathogens, however, could result in the phosphorylation of IκBα, marking it for ubiquitination and subsequent degradation in periodontal tissues. This event liberates the NF-κB subunits p65/p50, allowing them to translocate into the nucleus and activate transcription. Moreover, phosphorylation of the NF-κB p65 subunit at Ser536 is crucial for augmenting its transcriptional efficacy. Given the established role of NF-κB in modulating IL-8 and IL-6 expression and the observed inhibition of these cytokines in human gingival cells treated with an NF-κB inhibitor, our study investigated whether SE similarly affects NF-κB activity in respiratory epithelial cells.

Initially, we assessed IL-8 production in BEAS-2B treated with inhibitors targeting NF-κB, p38, JNK, and ERK (Figure 3A). The ELISA results indicate a significant reduction in IL-8 production upon the addition of inhibitors against NF-κB, p38, and JNK, but not ERK. Among these inhibitors, NF-κB inhibition exerted the most pronounced suppressive effects. Consequently, these findings validate the pivotal roles of NF-κB, p38, and JNK pathways in mediating inflammatory cytokine secretion by P. gingivalis in BEAS-2B.

Further, we assessed the effect of P. gingivalis on NF-κB at the transcriptional level in BEAS-2B using a luciferase assay with an NF-κB-driven reporter plasmid. P. gingivalis exposure elevated NF-κB-mediated transcription approximately 1.8-fold (Figure 3B). However, pretreatment with SE diminished this enhancement in a dose-dependent manner (Figure 3B), suggesting that SE likely mitigates cytokine expression induced by P. gingivalis through NF-κB pathway inhibition. Because SE was applied only during the pretreatment phase and the medium was replaced before P. gingivalis stimulation, SE was not present during luminescence measurement and direct assay interference was unlikely. To exclude direct effects of SE on the luciferase readout, SE was spiked into reporter cell lysates immediately before measurement and did not measurably alter luminescence (Figure A2).

We also examined the NF-κB, p38, and JNK pathways by analyzing phosphorylation via WB. P. gingivalis stimulation led to the phosphorylation of NF-κB p65 subunit, IκBα, p38, and JNK in BEAS-2B (Figure 3C). SE treatment suppressed these phosphorylation events (Figure 3C), providing additional evidence that SE exerts its anti-inflammatory effects by interfering with the activation of NF-κB, p38, and JNK signaling pathways in P. gingivalis-stimulated BEAS-2B.

3.3. Determining the Inhibitory Effect of SE on P. gingivalis-Induced Inflammation In Vivo

To assess the inhibitory effect of SE on NF-κB activity induced by intratracheal challenge with heat-inactivated P. gingivalis, we further examined the production of KC, which is a mouse homolog of the neutrophil chemoattractant IL-8, and IL-6 in mice [24]. P. gingivalis stimulated the production of KC and IL-6 in the lungs of mice, and SE treatment significantly reduced the levels of both cytokines (Figure 4A). Subsequently, to confirm the pathways responsible for this cytokine secretion, we assessed the phosphorylation of NF-κB p65 subunit, IκBα, p38, and JNK in animal experiments through WB (Figure 4B). The results demonstrated that SE treatment inhibited the phosphorylation of NF-κB p65 subunit and IκBα in the mouse lung tissue following P. gingivalis challenge. However, phosphorylation bands for p38 and JNK were not detectable under these conditions. Furthermore, we validated the reduction in NF-κB activation following SE treatment using ELISA (Figure 4C).

4. Discussion

Pneumonia is a major lower respiratory tract infection and an increasing global health concern, with rising incidence and hospitalization rates worldwide. Periodontal pathogens play a crucial role in initiating pneumonia and other inflammatory disorders [8]. This study demonstrated that SE curtailed the production of inflammatory cytokines in bronchial cells triggered by P. gingivalis through the suppression of NF-κB p65 subunit, IκBα, p38, and JNK. Our mouse experiments validated that SE exhibited suppressive effects on P. gingivalis-induced inflammation. The NF-κB pathway appears to play an important role in mediating the anti-inflammatory effects listed above, although we were unable to visualize p38 and JNK phosphorylation in our WB analysis of mouse lung samples. The generally low expression levels of p38 and JNK in mouse lung tissue, combined with the tissue’s heterogeneous cellular composition, may have made the detection of these signaling proteins challenging by WB. The diverse cell populations within lung tissue may express variable amounts of these kinases, creating an overall dilution effect when analyzing whole-tissue homogenates. Additionally, the phosphorylation of these proteins occurs at different time points following stimulation, making simultaneous detection particularly difficult among in vivo models [25].

For older adults and individuals with medical conditions that impair voluntary movement, maintaining good oral hygiene itself can be challenging, and professional dental care is often inadequate during the perioperative period. The anti-inflammatory properties of SE observed in this study suggest that plant extracts may offer complementary approaches to prevention strategies for respiratory diseases, such as aspiration pneumonia.

SE is a commercial product derived from the leaves of the bamboo plant Sasa senanensis Rehder, which has traditionally been used as a herbal supplement in Japanese medicine due to its potential health benefits. When the LCC fractions of SE were purified via repeated cycles of acid precipitation and alkaline solubilization into four distinct fractions, anti-HIV activity was stepwise increased. We found that all LCCs and chlorophyllin showed higher anti-HIV and anti-inflammatory activity than the polysaccharide fraction [26]. Although this study did not determine which components exert an anti-inflammatory effect on bronchial epithelial cells, it may be reasonable to hypothesize that chlorophyllin and LCCs are responsible for the effect. Chlorophyllin can be classified as an “NF-κB DNA-binding inorganic complex” [27]. This categorization indicates that chlorophyllin disrupts NF-κB’s binding to DNA, inhibiting the transcription of inflammatory genes. Moreover, chlorophyllin also stabilizes IκBα, preventing NF-κB from entering the nucleus and suppressing proinflammatory gene expression. In hamsters, dietary chlorophyllin inhibited the NF-κB pathway and initiated apoptosis, reducing oral cancer onset by downregulating IKKβ, preventing IκB-α phosphorylation, and reducing nuclear NF-κB expression [28].

The LCC scavenges ROS [29], enhancing antioxidant capabilities through the Nrf2-ARE pathway and reducing vascular inflammation by inhibiting nicotinamide adenine dinucleotide phosphate oxidase. Alkaline extracts such as SE contain large amounts of LCCs and their decomposition products (phenylpropanoids such as p-coumaric acid) [20]. The former shows higher anti-HIV activity than the latter [30], while the latter shows stronger and more sustainable anti-UVC activity than vitamin C, correlating with hydroxyl radical scavenging activity [31]. The LCC strongly binds to influenza viruses and instantly inactivates them [32]. Exposure of HSV to SE (1 mg/mL) rapidly eliminated its infectivity within 3 min [21]. Decomposing the sugar portion of the LCC did not reduce the antiviral activity, but decomposing the lignin portion eliminated such activity, suggesting the importance of a highly polymerized lignified structure in the expression of antiviral activity [16]. This was confirmed by the finding that synthetic lignin (dehydrogenated polymer of phenylpropanoids) without a sugar moiety exhibits strong antiviral activity. Experiments using radiolabeled LCCs revealed that this fraction effectively binds to virions and cultures, leading to rapid inactivation of the virus [32]. On the other hand, hot-water extracts such as ten Kampo medicines (Byakkokaninjinto, Hangeshashinto, Hochuekkito, Juzentaihoto, Kikyoto, Ninjinyoeito, Rikkosan, Saireito, Shosaikoto, and Unseiin) showed no apparent anti-HIV activity [33]. Taken together, these data suggest that the LCC, which has both anti-inflammatory and antiviral effects, may be extremely effective against aspiration pneumonia and is expected to be used in clinical applications.

In the present study, we extended previous findings on the anti-inflammatory activity of SE by demonstrating its effects in an acute in vivo model. Proinflammatory cytokines play a central role in the lung inflammatory response. IL-6, a proinflammatory cytokine, is a key mediator of acute-phase responses and leukocyte recruitment in inflammatory conditions. In addition, IL-6 is involved in the stimulation of acute-phase protein synthesis, leukocyte recruitment, B-cell differentiation, and T-cell activation in many chronic inflammatory diseases [4,34]. Moreover, IL-6 levels are also increased in the plasma and BALF of patients with pneumonia [35]. Our results demonstrate a significant reduction in IL-6 levels in lung tissue following intratracheal challenge with heat-inactivated P. gingivalis. Furthermore, our findings indicate the suppression of KC production, a mouse homolog of the neutrophil chemoattractant that promotes neutrophil recruitment. IL-8 is also a potent neutrophil chemoattractant and activator in respiratory tissue, showing a significant correlation with bacterial load in patients with chronic bronchitis [36]. Our results align with a previous study on human gingival fibroblasts, which revealed a significant reduction in IL-8 production by SE, maintaining cell viability [37]. Natural substances that suppress respiratory inflammation have been rarely reported, but our results confirm a distinct effect of SE on bronchial cells inflamed by intratracheal challenge with heat-inactivated P. gingivalis. Importantly, while prior studies evaluated SE in non-airway cell types, our study demonstrates suppression of P. gingivalis-triggered inflammatory responses in bronchial epithelial cells and attenuation of early lung cytokine responses in vivo. We have previously reported that Japanese traditional Kampo medicines, such as Hangeshashinto, and their constituent plant extracts prepared by hot water extraction have low lignin content and therefore low antiviral effects [33]. Admittedly, considering that in the present study we did not directly compare SE with other herbal preparations, further comparative studies are warranted. Nevertheless, taken together, these findings suggest that SE may serve as a natural adjunct to attenuate bacteria-associated airway inflammatory responses.

It is worth mentioning that although SE pharmacokinetics after oral administration were not assessed in this study, the observed pulmonary effects may be mediated by systemic and/or immune mechanisms rather than direct delivery of intact components to the lung. Consistent with this concept, several researchers have documented the impact of oral administration of drugs or dietary supplements on respiratory tissues [38,39]. In particular, intestinal exposure to SE and LCC-rich fractions could modulate systemic inflammatory tone via the gut–lung axis, and absorbed low-molecular-weight constituents or intestinally processed fragments may also contribute to this outcome. In support of this plausibility, a previous biodistribution study using radiolabeled LCCs reported systemic handling after oral administration and distribution of radioactivity to multiple organs, including the lung, after intravenous administration [40]. In this regard, future studies measuring systemic exposure and tissue distribution are needed to identify which components and forms are responsible for the respiratory effects.

While this study demonstrates the anti-inflammatory effects of SE in respiratory models, several limitations should be noted. First, the in vivo experiment used heat-inactivated P. gingivalis and assessed early cytokine responses; thus, it does not recapitulate bacterial colonization and proliferation during infectious pneumonia and should be interpreted as demonstrating anti-inflammatory efficacy rather than protection against active infection. Second, SE is a complex extract and individual fractions/components were not isolated or tested in this model. Thus, the active constituents remain to be identified, including the possible contribution of LCCs.

Taken together, our observations indicate that SE represents a promising natural product-based adjunct for mitigating P. gingivalis-triggered airway inflammatory responses and could complement conventional oral hygiene-based approaches. Given that SE is already available as an orally administered preparation, future work should define clinically relevant exposure ranges and further standardize SE to ensure reproducible anti-inflammatory activity. Ultimately, these efforts should enable well-designed human studies to evaluate the feasibility, safety, and preliminary efficacy of SE in reducing bacteria-associated respiratory inflammatory biomarkers in at-risk populations.