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Acute lung injury (ALI) is often responsible for the high morbidity of critically ill patients. The present study aimed to investigate whether phillygenin (PHI) can inhibit inflammation and apoptosis of pulmonary epithelial cells by activating peroxisome proliferator-activated receptor γ (PPARγ) signaling. The
The lungs are one of the most vulnerable organs in the human body, and alveolar macrophages are the major inflammatory cells involved in the maintenance of the lung's defense against foreign pathogens (
Phillygenin (PHI; 4-[(3S,3aR,6R,6aR)-6(3,4-dimethoxyphenyl)-1,3,3a,4,6,6a-hexahydrofuro[3,4-c] furan-3yl]-2-methoxyphenol) (
A search of the SwissTargetPrediction website suggested that PHI can bind to and regulate the expression of MMP8. Previous studies have indicated that MMP8 and MMP9 expression levels are elevated in pediatric patients with ALI (
The clinical study protocol was approved by Gaolangang Hospital of Zhuhai People's Hospital (approval no. 2020-012; Zhuhai, China) between 2019/09 and 2020/02. Signed informed consent forms were obtained from the children's family for the collection and use of the specimens. Briefly, patients who were admitted to the pediatric intensive care unit of Gaolangang Hospital of Zhuhai People's Hospital were enrolled. Inclusion criteria were pediatric patients [age, 2–10 years; 28 (46.7%) females and 32 (53.3%) males] who were intubated and mechanically ventilated with a ratio of partial pressure of arterial oxygen (PaO2) to the fraction of inspired oxygen (FiO2) of ≤300 (adjusted to 253 in Salt Lake City due to altitude), bilateral pulmonary infiltrates and no clinical evidence of left atrial hypertension. Patients were excluded if they were <2 years of age; had respiratory failure from cardiac disease; had hypoxemia without bilateral infiltrates; had received a bone marrow or lung transplant; were supported on extracorporeal membrane oxygenation; had a non-pulmonary condition that could be exacerbated by the prone position; had participated in other clinical trials within the preceding 30 days; or if there was a decision to limit life support. Blood samples were collected within the first 24 h of diagnosis. The serum samples of pediatric patients with ALI (n=30) and normal subjects (n=30) were collected for further analysis following submission of the informed consent form.
The pulmonary epithelial cell line (BEAS-2B) was obtained from the American Type Culture Collection. The cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) containing 10% FBS (Thermo Fisher Scientific, Inc.) in an incubator (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO2. LPS was purchased from Sigma-Aldrich; Merck KGaA. For LPS induction, BEAS-2B cells were plated into 6-well plates and cultured for 48 h. LPS (100 ng/ml) or saline solution was added and the cells were incubated for 24 h to induce inflammatory response and apoptosis. PHI (purity 99%) was purchased from Chroma-Biotechnology Co., Ltd. (
The MMP8-overexpression plasmid vector (pcDNA3.1-MMP8) and empty vector (pcDNA3.1-NC) plasmid were purchased from Shanghai GenePharma Co., Ltd. In accordance with the manufacturer's instructions, following LPS treatment, 1 µg plasmids were transfected into BEAS-2B cells using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 6 h, the medium was replaced with RPMI-1640 medium with 10% FBS (Thermo Fisher Scientific, Inc). After 48 h of incubation, the cells were collected and the transfection efficiency was analyzed by reverse transcription-quantitative PCR (RT-qPCR).
Cells were seeded in 96-well plates at a density of 3,000/well and were subsequently treated by different concentrations of PHI for 24 h. A total of 10 µl MTT solution was added to the medium and the cells were cultured for an additional 4 h. Dimethyl sulfoxide was added into each well to dissolve the formazan particles. The absorbance was measured at 450 nm using a microplate reader (Molecular Devices, LLC).
BEAS-2B cells treated with PHI (12.5, 25 or 50 µg/ml; 24 h; 37°C) and LPS were fixed with 4% paraformaldehyde at 25°C for 15 min. Then, a TUNEL kit (Roche Applied Science) was used to detect apoptotic cells according to the manufacturer's protocol. Subsequently, cell nuclei were counterstained with 0.2 µg/ml DAPI at room temperature for 15 min and mounted with glycerol gelatin (Sigma-Aldrich; Merck KGaA). An Olympus fluorescence microscope (magnification, ×200) was used to acquire the images in ≥3 randomly selected fields of view.
BEAS-2B cells were plated in 96-well plates (8×104 cells/ml) and incubated with LPS in the presence or absence of PHI for 24 h. The concentration levels of IL-1β, IL-6 and TNF-α in the cell supernatant were measured by ELISA kits according to the manufacturer's recommendations. The following ELISA kits were purchased from Beyotime Institute of Biotechnology, IL-1β ELISA kit (cat. no. PI305), IL-6 ELISA kit (cat. no. PI330) and TNF-α ELISA kit (cat. no. PT518). The absorbance at 450 nm was detected by a microplate reader.
Total RNA in the cells and the serum of pediatric patients with ALI was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. Subsequently, the extracts were dissolved and the final RNA purity was measured by the Nucleic Acid/Protein Analyzer (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using 5X All-In-One Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocols. RT-qPCR reactions were performed using SYBR-Green Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.). The reaction conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 30 sec. The mRNA levels of the target genes were normalized to the levels of the GAPDH gene. The 2−ΔΔCq (
BEAS-2B cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) and the protein content was estimated using a BCA assay. The samples (30 µg) were subsequently loaded on SDS-PAGE gels (10, 12 or 15%), which were prepared previously. The gels were then transferred to nitrocellulose membranes. The membranes were first blocked using fat-free milk (5%) in TBS for 2 h at room temperature and then the membranes were incubated at 4°C for 24 h with primary antibodies against the following: Cox-2 (1:1,000; cat. no. sc-376861), Bcl-2 (1:1,000; cat. no. sc-7382), Bax (1:1,000; cat. no. sc-7480), caspase3 (1:1,000; cat. no. sc-7272), caspase9 (1:1,000; cat. no. sc-56076), MMP8 (1:1,000; cat. no. sc-137044), PPARγ (1:1,000; cat. no. sc-7273), GAPDH (1:2,000; cat. no. sc-47724; all purchased from Santa Cruz Biotechnology, Inc.), iNOS (1:1,000; cat. no. 13120), cleaved (c)-caspase3 (1:1,000; cat. no. 9661) and c-caspase9 (1:1,000; cat. no. 9509; all purchased from Cell Signaling Technology, Inc.). Following which, the membranes were incubated with HRP-conjugated secondary antibody (1:1,000; cat. nos. 7074 and 7076; Cell Signaling Technology, Inc.) for 50 min at 25°C.
TBS with Tween-20 (0.2%) was used to wash the membranes to remove non-specific binding of the antibodies and the ECL luminescence agent (Santa Cruz Biotechnology, Inc.) was added. The images were captured in a Bio-Rad chemiluminescence imager (Bio-Rad Laboratories, Inc.). The expression levels of the proteins were analyzed using ImageJ software version 1.4 (National Institutes of Health) and normalized to those of the control.
Data analysis was performed with SPSS version 23.0 (IBM Corp.) and GraphPad (version 5.0; GraphPad Software, Inc.). All experiments were repeated three times and the data are expressed as the mean ± standard deviation. Two group comparisons were performed using an unpaired Student's t-test was used for the comparison between two group of samples and statistical differences between groups were analyzed using one-way ANOVA followed by a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
To determine the effects of PHI on pulmonary epithelial cells, BEAS-2B cell viability was detected. Treatment of BEAS-2B cells with PHI (12.5, 25 and 50 µg/ml) indicated no apparent difference in cell viability, while significant differences were noted at 100 µg/ml PHI treatment (
Induction of cell apoptosis plays a critical role in the development of ALI (
Serum samples of normal subjects and pediatric patients with ALI were collected and assessed by RT-qPCR and western blot analyses. As demonstrated in
To directly assess the effects of MMP8 on LPS-induced BEAS-2B cells, which were treated with PHI, 50 µg/ml PHI was selected for subsequent experiments and MMP8-overexpression models were established. RT-qPCR and western blot analyses indicated significantly higher levels of MMP8 in the pcDNA3.1-MMP8 group compared with those of the control group (
The induction of apoptosis of LPS-treated BEAS-2B cells was detected by TUNEL staining. PHI decreased the number of apoptotic cells in LPS-induced BEAS-2B cells, while pcDNA3.1-MMP8 increased it, indicating that PHI treatment suppressed the induction of apoptosis in LPS-treated BEAS-2B cells (
Forsythiae Fructus is used as a single herb or added in compound prescriptions in Asia (
The generation of ROS and the reduction in MMP expression levels have been reported to be important events in triggering apoptosis. PHI was previously found to increase ROS and decrease MMP levels in promyelocytic leukemia HL-60 cells, indicating its effects on the suppression of cell apoptosis (
PPARs are ligand-activated transcription factors that exert anti-inflammatory effects on certain diseases, including brain injury (
In conclusion, the present study demonstrated that PHI inhibited inflammation and apoptosis of pulmonary epithelial cells by activating PPARγ signaling via downregulation of MMP8 expression, which may guide future studies on PHI and provide a theoretical basis for the therapeutic potential of PHI. However, there are numerous limitations of the present study. After validating the effect of PHI on LPS-induced inflammation and apoptosis in lung epithelial cells
Not applicable.
No funding was received.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
PY and YL collaborated on the manuscript, including designing the research, collecting and analyzing the experiments and data, and writing the manuscript. PY and YL confirmed the authenticity of all the raw data. Both authors read and approved the final manuscript.
The clinical study protocol was approved by Gaolangang Hospital of Zhuhai People's Hospital (Zhuhai, China; approval no. 2020-012). Signed informed consent forms were obtained from the children's guardians for the collection and use of the specimens.
Not applicable.
The authors declare that they have no competing interests.
PHI treatment suppresses the inflammatory response in LPS-induced BEAS-2B cells. (A) The chemical structure of PHI. (B) The effects of PHI on the viability of BEAS-2B cells were detected via an MTT assay. *P<0.05 vs. control. (C) The effects of PHI on the viability of BEAS-2B cells induced by LPS were detected via an MTT assay. ***P<0.001 vs. control; ##P<0.01 vs. LPS group. (D-F) The expression levels of inflammatory factors were measured by ELISA. ***P<0.001 vs. control; #P<0.05, ###P<0.001 vs. LPS group. (G) The levels of inflammation-associated proteins were detected via western blotting. ***P<0.001 vs. control; #P<0.05, ##P<0.01, ###P<0.001 vs. LPS group. PHI, phillygenin; LPS, lipopolysaccharide; cox-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase.
PHI treatment suppresses the induction of apoptosis in BEAS-2B cells. (A and B) Cell apoptosis of LPS-induced BEAS-2B cells was detected using a TUNEL assay (magnification, ×200; scale bar, 100 µm). ***P<0.001 vs. control; ###P<0.001 vs. LPS group. (C) Western blotting was used for the analysis of the expression levels of the apoptosis-associated proteins. ***P<0.001 vs. control; #P<0.05, ##P<0.01, ###P<0.001 vs. LPS group. PHI, phillygenin; LPS, lipopolysaccharide; c-, cleaved.
PHI treatment decreases the expression levels of MMP8 in LPS-induced BEAS-2B cells. (A) The mRNA and (B) protein levels of MMP8 were detected via reverse transcription-quantitative PCR and western blot analyses, respectively. ***P<0.001 vs. control. (C) The expression levels of MMP8 in LPS-induced BEAS-2B cells following PHI treatment were detected via western blotting. ***P<0.001 vs. control; ##P<0.01, ###P<0.001 vs. LPS group. PHI, phillygenin; LPS, lipopolysaccharide; ALI, acute lung injury.
PHI treatment suppresses inflammation of LPS-induced BEAS-2B cells. pcDNA3.1-MMP8 was constructed and the expression levels of MMP8 were measured via (A) reverse transcription-quantitative PCR and (B) western blot analyses. ***P<0.001 vs. control and pcDNA3.1-NC. (C-E) Following transfection of pcDNA3.1-MMP8 into LPS-induced BEAS-2B cells pretreated with PHI, the expression levels of the inflammatory factors were measured via ELISA. ***P<0.001 vs. control; ###P<0.001 vs. LPS group; ∆∆∆P<0.001 vs. LPS + PHI group. (F) Following transfection of pcDNA3.1-MMP8 into LPS-induced BEAS-2B cells pretreated with PHI, the expression levels of the inflammation-associated factors were measured via western blotting. ***P<0.001 vs. control; ###P<0.001 vs. LPS group; ∆P<0.05, ∆∆∆P<0.001 vs. LPS + PHI group. PHI, phillygenin; LPS, lipopolysaccharide; NC, negative control; cox-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase.
PHI treatment activates PPARγ signaling by downregulating MMP8 expression. (A and B) Following MMP8 overexpression, the induction of cell apoptosis of LPS-treated BEAS-2B cells, which were pretreated with PHI, was assessed using a TUNEL assay (magnification, ×200; scale bar, 100 µm). ***P<0.001 vs. control; ###P<0.001 vs. LPS group; ∆∆∆P<0.001 vs. LPS + PHI group. (C) Western blotting was used to determine the expression levels of the apoptosis-associated proteins in LPS-induced BEAS-2B cells treated with PHI and transfected with pcDNA3.1-MMP8. ***P<0.001 vs. control; ##P<0.01, ###P<0.001 vs. LPS group; ∆∆P<0.01 vs. LPS + PHI group. (D) Western blotting was used to determine the expression levels of PPARγ in LPS-induced BEAS-2B cells treated with PHI and transfected with pcDNA3.1-MMP8. ***P<0.001 vs. control; ###P<0.001 vs. LPS group. PHI, phillygenin; PPARγ, peroxisome proliferator-activated receptor γ; LPS, lipopolysaccharide; NC, negative control; c-, cleaved.