Summary
Acute lung injury (ALI) caused by lipopolysaccharide (LPS) is a common, severe clinical syndrome. Injury caused by inflammation and oxidative stress in vascular endothelial and alveolar epithelial cells is a vital process in the pathogenesis of ALI. Toll-like receptor 9 (TLR9) is highly expressed in LPS-induced ALI rats. In this study, Beas-2B human pulmonary epithelial cells and A549 alveolar epithelial cells were stimulated by LPS, resulting in the upregulation of TLR9 in a concentration-dependent manner. Furthermore, TLR9 overexpression and interference vectors were transfected before LPS administration to explore the role of TLR9 in LPS-induced ALI in vitro. The findings revealed that inhibition of TLR9 reduced inflammation and oxidative stress while suppressing apoptosis of LPS-induced Beas-2B and A549 cells, whereas TLR9 overexpression aggravated these conditions. Moreover, TLR9 inhibition resulted in downregulated protein expression of myeloid differentiation protein 88 (MyD88) and activator activator protein 1 (AP-1), as well as phosphorylation of nuclear factor-κB (NF-κB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). The phosphorylation of extracellular-regulated protein kinases 1/2 was upregulated compared to that of cells subjected to only LPS administration, and this was reversed by TLR9 overexpression. These results indicate that inhibition of TLR9 plays a protective role against LPS-induced inflammation and oxidative stress in Beas-2B and A549 cells, possibly via the MyD88/NF-κB and MyD88/MAPKs/AP-1 pathways.
Keywords: Acute lung injury, Toll-like receptor 9, Inflammation, Oxidative stress, MyD88
Introduction
Acute lung injury (ALI) is a complex clinical syndrome induced by many factors, such as inhalation of toxic gases, burns, severe sepsis, severe pneumonia, and severe trauma, resulting in substantial morbidity and mortality [1,2]. Injury caused by inflammation and oxidative stress in vascular endothelial and alveolar epithelial cells is a vital process in the pathogenesis of ALI [2,3]. Although the pathophysiological changes in ALI have been thoroughly studied and treatment methods, such as intensive care and mechanical ventilation, have made great progress, the mortality rate of ALI remains as high as 40 %, exerting a significant impact on public health [2,4]. Lipopolysaccharide (LPS) is an important component of the cell wall of Gram-negative bacteria and can cause ALI by targeting vascular endothelial cells, alveolar epithelial cells, neutrophils, and alveolar macrophages [5]. For this reason, LPS is commonly used to induce and construct ALI models for basic research [6–9].
Toll-like receptors (TLRs) are an important component of the innate immune system and widely distributed in epithelial, endothelial, dendritic, and immune cells [10]. TLRs are involved in downstream signaling via myeloid differentiation protein 88 (MyD88)-dependent and toll-interleukin 1 receptor domain-containing adapter inducing interferon-β-dependent pathways; these induce the production of interferon (IFN)-stimulated genes and pro-inflammatory cytokines [11]. As one of the subfamilies of TLRs, TLR9 regulates the downstream signaling molecules nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs), mainly through the MyD88 signaling pathways, ultimately affecting cellular physiopathological processes [12]. Accumulating evidence has demonstrated that several TLRs, including TLR9, TLR4, and TLR2, are involved in the regulation of ALI, and the effects of TLR4 and TLR2 and their potential mechanisms have been well studied [13–15]. Previous studies have also revealed that TLR9 is highly expressed in rat models of LPS-induced ALI, and inhibition of its expression attenuated the degree of ALI [16]. However, the protective effect of high or low expression of TLR9 on LPS-induced ALI has seldom been reported. In this study, human pulmonary epithelial cells Beas-2B and A549 alveolar epithelial cells were stimulated with LPS to construct a lung injury model to investigate the role of TLR9 in vitro through the MyD88 pathway.
Methods
Cell culture and treatment
The Human Pulmonary Epithelial cell line Beas-2B and Elveolar Epithelial cell line A549 were provided by the Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China). After recovery, the cells were grown in RPMI 1640 (Hyclone, Logan, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco, New York, USA), 100 U/ml penicillin (Solarbio, Beijing, China), and 100 μg/ml streptomycin (Solarbio) at 37 °C in a humidified atmosphere containing 5 % CO2. When the cells reached the logarithmic growth phase, the culture medium was discarded. The cells were washed with phosphate-buffered solution (PBS, Bioswamp, Wuhan, China) and digested with 0.25 % trypsin (Bioswamp, Myhalic Biotechnology Co., Ltd., Wuhan, China) for passaging. The medium was changed every three days.
Cell counting kit-8 (CCK-8) assay
Beas-2B and A549 cells were seeded into 96-well plates at a density of 1 × 103 cells/well. After pretreatment with LPS (Sigma, St. Louis, MO, USA) at 0, 10, 100, 200, 800, or 1000 ng/ml for 24, 48, or 72 h, 10 μl of CCK-8 solution (Bioswamp) was added to each well for 4 h to measure cell viability. A Spectra Max 190 microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA) was used to measure the absorbance at 450 nm. All experiments were performed in triplicates.
LPS preparation method: LPS (1 mg) was resuspended in 1 ml of sterile balanced salt solution or cell culture medium and gently vortexed and shaken until the powder was completely dissolved to obtain 1 mg/ml of storage solution.
Plasmid and transfection
The TLR9 overexpression vector pcDNA3.1 HISA (TLR9 sequence: F: 5′-CGGAATTCCGATGGGTTTCTGCCGCAGC-3′, R: 5′-CCCTCGAGGGCTATT-CGGCCGTGGGTCCC-3′) and interference vector pSIREN (shRNA1 sequence: 5′-CCTACAACCGCATCGTCAAAC-3′, shRNA2 sequence: 5′-GCTGCCCAAATCCCTCATATC-3′, shRNA3 sequence: 5′-GCTAGACCTGTCCCACAATAA-3′) were purchased from Addgene (Cambridge, MA, USA). Cells (4 × 105 cells/well) were seeded in 24-well plates and cultured until they reached 70–80 % confluence, then transfected with pcDNA3.1-TLR9 (TLR9 overex-pression, OV-TLR9), empty pcDNA3.1 HISA vectors (empty TLR9 overexpression vector, OV-TLR9-EV), pSIREN-shTLR9 (TLR9 interference, sh-TLR9), or empty pSIREN vectors (empty TLR9 interference vector, sh-TLR9-EV) using Lipofect-amine2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. Non-transfected Beas-2B and A549 cells were used as controls (CON). After transfection for 24 h, the transfection rate was detected by western blot and the cells were treated with LPS for 24 h. Experiments using each cell type were divided into four groups: LPS (only LPS treatment), LPS+OV-TLR9 (TLR9 overexpression with LPS treatment), LPS+sh-TLR9 (TLR9 interference with LPS treatment), and LPS+EV (TLR9 overexpression empty vector with LPS treatment).
Enzyme-linked immunosorbent assay
After transfection for 24 h, Beas-2B and A549 cells were treated with 100 ng/ml LPS for 24 h, and the expression levels of inflammation-related factors were measured. The levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, monocyte chemotactic protein 1 (MCP-1), and monocyte chemotactic protein 5 (MCP-5) in the culture supernatant were detected using ELISA kits (IL-6, HM10205; TNF-α, HM1001; IL-1β; HM10206; MCP-1, HM10894; MCP-5, HM10983; Bioswamp) according to the manufacturer’s protocols. The absorbance was measured at 450 nm using a Labsystems Multiskan MS-352 microplate reader (Vantaa, Finland). The levels of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and nitric oxide (NO) were measured according to the instructions of the respective kits designed by Nanjing Jiancheng Bioengineering Institute (iNOS, A014-1; eNOS, H195; NO, A012-1, Nanjing, China). All experiments were performed in triplicates.
Flow cytometry
After transfection for 24 h, Beas-2B and A549 cells were treated with 100 ng/ml LPS for 24 h. Cell apoptosis and reactive oxygen species (ROS) levels were evaluated using flow cytometry (Beckman Coulter, Brea, CA, USA). Cell apoptosis was evaluated using an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (Bioswamp) according to the manufacturer’s protocol. Cells were suspended at a concentration of 1 × 105 cells in 200 μl of binding buffer. Annexin V-FITC and PI (10 μl of each) were added and the cells were incubated for 30 min in the dark, after which the cells were analyzed by flow cytometry. For the ROS level assay, the collected cells (1 × 107/ml) were resuspended in diluted dihydroethidium for 20 min at 37 °C and subjected to flow cytometry.
Western blot assay
Total proteins were extracted from Beas-2B and A549 cells using radioimmunoprecipitation assay lysis buffer (Bioswamp) containing a cocktail of protease and phosphatase inhibitors. Protein concentration was quantified using a bicinchoninic acid kit (Bioswamp). Protein samples (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Boston, Massachusetts, USA). The membranes were blocked with 5 % skim milk for 2 h at room temperature then incubated overnight at 4 °C with primary antibodies against TLR9 (Ab37154, 1:1000, Abcam, Cambridge, UK), MyD88 (Ab2064, 1:1000, Abcam), nuclear factor (NF)-κB (Ab16502, 1:2000, Abcam), p-NF-κB (Ab86299, 1:5000, Abcam), activator protein 1 (AP-1, Ab31419, 1:1000, Abcam), extracellular-regulated protein kinases (ERK)1/2 (Ab17942, 1:1000, Abcam), p-ERK1/2 (Ab50011, 1:10000, Abcam), c-Jun N-terminal kinase (JNK, Ab179461, 1:1000, Abcam), p-JNK (Ab124956, 1:5000, Abcam), p38 mitogen-activated protein kinase (MAPK, Ab31828, 1:1000, Abcam), p-p38MAPK (Ab47363, 1:1000, Abcam), caspase 8 (Ab25901, 1:1000, Abcam), Bcl-2 (Ab196495, 1:1000, Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 2118, 1:1000; Cell Signaling Technology, Danvers, MA, USA). After washing with PBS/Tween 20, the membranes were incubated with goat anti-rabbit IgG secondary antibody (PAB160011, 1:10000, Bioswamp) for TLR9 and goat anti-mouse IgG (PAB150009, 1:10000, Bioswamp) for 1 h at room temperature. Immunoreactivity was visualized by a colorimetric reaction using an enhanced chemiluminescence substrate buffer (Millipore, Massachusetts, USA). The membranes were then detected using an automatic chemiluminescence analyzer (Tanon-5200, Shanghai, China), and the relevant band gray values were read using the Tanon Gel Imaging Software. All experiments were performed in triplicate.
Statistical analysis
Data are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA) to compare differences between groups, and the least significant difference (LSD) method was used to detect differences between two pairs. Statistical significance was set at P<0.05.
Results
Effect of LPS on Beas-2B and A549 cell survival and TLR9 expression
As shown in Fig. 1A and 1B, LPS exerted similar cytotoxic effects on Beas-2B and A549 cells by reducing cell viability in a concentration-dependent manner. In addition, cell viability decreased with the same concentration of LPS for up to 72 h.
Fig. 1.
Effect of LPS on cytotoxicity and TLR9 expression in A549 and Beas-2B cells. The viability of A549 (A) and Beas-2B (B) cells in response to treatment with different concentrations of LPS at 24, 48, and 72 h. TLR9 protein expression in A549 (C) and Beas-2B (D) cells in response to treatment with different concentrations of LPS at 24, 48, and 72 h. Data represent mean ± SD (n = 3). ***P<0.001 compared to 0 ng/ml.
Western blot (Fig. 1C and 1D) analysis demonstrated that LPS increased the protein expression of TLR9 in a concentration- and time-dependent manner in both A549 and Beas-2B cells. These results indicated that LPS induced cell damage and increased the expression of TLR9 in both cell types. Based on these results, the selected concentration of LPS was 100 ng/ml and the selected treatment time was 24 h for subsequent experiments. Under these conditions, the viability of both Beas-2B and A549 cells was significantly lower than that of the control (0 ng/ml LPS) (P<0.001), and the expression of TLR9 was relatively high.
Detection of TRL9 protein expression after transfection
Western blot analysis was used to evaluate the transfection efficiency (Fig. 2). Both A549 and Beas-2B cells transfected with OV-TLR9 exhibited higher TLR9 expression than those in the CON and OV-TLR9-EV (P<0.001). Transfection with sh-TLR9 resulted in a remarkably lower expression of TLR9 than that of CON and sh-TLR9-EV. The cells transfected with OV-TLR9-EV exhibited no significant difference in the expression of TLR9 compared to cells transfected with sh-TLR9-EV. Hence, OV-TLR9-EV cells were chosen as the comparison group in subsequent experiments (termed LPS+EV).
Fig. 2.
Detection of TLR9 protein expression after transfection. TLR9 protein expression in A549 (A) and Beas-2B (B) cells. Data represent mean ± SD (n = 3). ***P < 0.001 compared to CON, ###P < 0.001 compared to OV-TLR9-EV, &&&P < 0.001 compared to sh-TLR9-EV.
Effect of TLR9 on the expression of inflammation-associated cytokines in LPS-stimulated A549 and Beas-2B cells
Furthermore, the expression levels of inflammation-associated factors IL-1β, IL-6, TNF-α, MCP-1, and MCP-5 in the culture were measured. As shown in Fig. 3A, 3B, 3C, and 3D, both A549 and Beas-2B cells transfected with OV-TLR9 displayed higher expression of IL-1β (P<0.001), IL-6 (P<0.01), TNF-α (P<0.001), MCP-1 (P<0.001), and MCP-5 (P<0.001) compared to those in the LPS and LPS + EV groups, and transfection with sh-TLR9 displayed remarkably lower expression of these proteins. These results indicated that inhibiting the expression of TLR9 attenuated LPS-induced inflammation, whereas TLR9 overexpression aggravated it.
Fig. 3.
Effect of TLR9 on the expression of cytokines in supernatants of LPS-challenged A549 and Beas-2B cells. The levels of IL-1β, IL-6, and TNF-α in A549 (A) and Beas-2B (B) cells. The levels of IFN-γ, IFN-α, and IFN-β in A549 (C) and Beas-2B (D) cells. The levels of MCP-1 and MCP-5 in A549 (E) and Beas-2B (F). Data represent mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001
Effect of TLR9 on oxidative stress in LPS-stimulated A549 and Beas-2B cells
As NO is a vital indicator of oxidative stress, the production of NO and the expression of the related enzymes iNOS and eNOS were detected (Fig. 4). Both Beas-2B and A549 cells transfected with OV-TLR9 exhibited significantly higher expression of iNOS (P<0.01) and lower expression of eNOS (P<0.001) compared to those of the LPS and LPS + EV groups. Inhibition of TLR9 expression in LPS-stimulated A549 and Beas-2B cells attenuated the levels of iNOS and increased those of eNOS compared to those in the LPS group (Fig. 4A–4D). NO and ROS generation was dramatically increased in the LPS + OV-TLR9 group compared with the LPS group, while transfection with sh-TLR9 markedly reduced NO and ROS production in the LPS group (Fig. 4E, 4F, 4G, and 4H). These results indicated that TLR9 inhibition attenuated LPS-induced oxidative stress, whereas TLR9 overexpression aggravated it.
Fig. 4.
Effect of TLR9 on iNOS, eNOS, NO, and ROS production in supernatants of LPS-stimulated A549 and Beas-2B cells. iNOS level in A549 (A) and Beas-2B (B) cells. eNOS level in A549 (C) and Beas-2B (D) cells. NO generation in A549 (E) and Beas-2B (F) cells. The ROS level in A549 (G) and Beas-2B (H) cells. Data represent mean ± SD (n = 3). **P < 0.01, ***P < 0.001.
Effect of TLR9 on LPS-induced apoptosis of A549 and Beas-2B cells
Next, the influence of TLR9 on LPS-induced apoptosis of A549 and Beas-2B cells was assessed. Flow cytometry showed that, compared to the LPS and LPS + EV groups, the percentage of apoptotic cells in the LPS + OV-TLR9 group was higher, whereas that in the LPS + sh-TLR9 group was lower (Fig. 5A and 5B). The expression of the apoptosis-related proteins caspase 8 and Bcl-2 was measured by western blotting (Fig. 5C and 5D) and showed the same tendency in A549 and Beas-2B cells. Compared to the LPS and LPS + EV groups, the expression of the pro-apoptotic protein caspase 8 in the LPS + OV-TLR9 group was upregulated (P<0.001) and that in the LPS + sh-TLR9 group was downregulated (P<0.001). The expression of the anti-apoptotic protein Bcl-2 in the LPS + OV-TLR9 group was downregulated (P<0.001) and that in the LPS + sh-TLR9 group was upregulated (P<0.001) compared to that in the LPS and LPS + EV groups. These results indicate that TLR9 promotes LPS-induced apoptosis and that interference of TLR9 expression plays a protective role against apoptosis.
Fig. 5.
Effect of TLR9 on LPS-induced apoptosis in A549 and Beas-2B cells. Flow cytometry was used to measure the percentage of apoptotic A549 (A) and Beas-2B (B) cells. Expression of apoptosis-related proteins caspase 8 and Bcl-2 in A549 (C) and Beas-2B (D) cells. Data represent mean ± SD (n = 3). ***P < 0.001.
Effect of TLR9 on the expression of proteins associated with the TLR9/MyD88/NF-κB and MyD88/MAPKs pathway in LPS-stimulated A549 and Beas-2B cells
As shown in Fig. 6, the tendency of relative protein expression was similar in A549 and Beas-2B cells. Cells transfected with OV-TLR9 exhibited higher expression of TLR9, MyD88, p-NF-κB, AP-1, p-JNK, and p-p38MAPK and lower expression of p-ERK1/2 compared to those in the CON and OV-TLR9-EV groups (P<0.001). Transfection with sh-TLR9 remarkably lowered the expression of TLR9, MyD88, NF-κB, AP-1, p-JNK, and p-p38MAPK and elevated the expression of p-ERK1/2 compared to those in the CON and sh-TLR9-EV groups (P<0.001). These findings implied that the regulation of apoptosis and inflammation by TLR9 in A549 and Beas-2B cells may be mediated by the MyD88 and MAPK signaling pathways.
Fig. 6.
Effect of TLR9 on the protein expression in LPS-challenged A549 and Beas-2B cells. The protein expression of MyD88, NF-κB, p-NF-κB, and AP-1 in A549 (A) and Beas-2B (B) cells. The protein expression of p-ERK1/2, p-JNK, and p-p38MAPK in A549 (C) and Beas-2B (D) cells. Data represent mean ± SD (n = 3). ***P < 0.001.
Discussion
As an important component of the cell wall of Gram-negative bacteria, LPS induces ALI characterized by the overproduction of pro-inflammatory and oxidative stress-related factors. LPS has been extensively applied in the induction and pathogenesis of ALI in vitro [17,18]. In the present study, LPS administered to A549 human alveolar epithelial cells and Beas-2B human pulmonary epithelial cells resulted in a remarkable reduction in cell viability and upregulation of TLR9 protein expression. Thus, it was hypothesized that LPS-induced inflammation and oxidative stress in ALI are associated with TLR9 expression. Next, cells were transfected with TLR9 overexpression and interference vectors before LPS treatment to investigate the regulatory effect of TLR9 on LPS-induced inflammation. The results showed that inhibition of TLR9 reduced inflammation and oxidative stress and suppressed LPS-induced apoptosis in A549 and Beas-2B cells, whereas TLR9 overexpression contributed to these processes.
Experimental and clinical research has demonstrated that ALI is an early and deadly complication of septic shock [19]. TLR9 plays an important role in causing hyperinflammation, and its inhibition reduces sepsis-induced mortality by preventing excessive inflammatory responses [20]. Previous studies have shown that TLR9 can be activated by cytosine-phosphate-guanine oligodeoxynucleotides and subse-quently recruit MyD88, followed by the activation of downstream NF-κB activation [21]. Activation of the MyD88/NF-κB pathway might result in the release of a large number of inflammatory mediators, such as IL-1β, IL-6, and TNF-α, which aggravate inflammation in the lung and cause ALI [22–24]. Shen et al. [10] found that antagonizing or silencing the TLR9 gene attenuated paraquat-induced ALI by suppressing the release of inflammatory cytokines IL-1β and TNF-α mediated by MyD88 and NF-κB, which is consistent with the findings of the present study.
Meanwhile, MyD88 enhances the phosphorylation of MAPKs [25–27], thus regulating the expression of AP-1 and leading to pro-inflammatory cytokine production [28–30]. A large amount of NO is considered an intracellular messenger in the development of oxidative stress [31]. It has been previously demonstrated that reducing iNOS and NO production decreases oxidative stress and cotton smoke-induced lung injury [32]. In recent years, evidence has shown that oxidative stress plays an important role in the development of inflammation [33]. The activation of iNOS induces the prominent production of NO, which causes airway inflammation and tissue damage. It has been reported that iNOS expression is inhibited in response to the downregulation of AP-1 [34], leading to the reduction of NO. However, the production of low concentrations of NO by eNOS protects against excessive bronchoconstriction, and regulates pulmonary blood flow and immune defense. In this study, compared to the LPS group, TLR9 inhibition decreased the levels of iNOS and NO and increased those of eNOS, and these phenomena were reversed by TLR9 overexpression.
AP-1 also regulates the expression of chemokines related to inflammatory responses. Previous research has shown that angiotensin III increases the expression of MCP-1 by activating the transcription factor AP-1, thus promoting the occurrence of cellular inflammation [35]. In addition, MyD88 directly regulates the levels of interferon to influence inflammation [36] and immune responses [37]. In this study, inhibition of TLR9 reduced the levels of the chemokines MCP-1 and MCP-5, while promoting those of IFN-α, IFN-β, and IFN-γ compared to those in the LPS group, and these phenomena were reversed by TLR9 overexpression.
Recent studies have revealed that lung epithelial cell apoptosis is an important pathological feature of LPS-induced ALI [6,17,38]. Arunkumar et al. found that TLR9 agonists induced apoptosis in mouse B-cell lymphoma cells by activating NF-κB [39]. The present experimental results indicated that the interference of TLR9 expression attenuated LPS-induced apoptosis in A549 and Beas-2B cells, which was reversed by TLR9 overexpression. The anti-apoptotic protein Bcl-2 is an important member of the Bcl-2 protein family that plays a vital role in the mitochondrial apoptosis pathway [40], while caspase 8 is an important pro-apoptotic protein of the caspase family. In this study, inhibition of TLR9 decreased the protein expression of caspase 8 and increased that of Bcl-2 compared to those in the LPS group, and these phenomena were reversed by TLR9 overexpression, and the trends of each physiological change were consistent in both Beas-2B cells and A549 cells. Yu et al. [41] also confirmed the correlation between various physiological changes in both cell types.
In conclusion, our experimental results verified the hypothesis that LPS-induced inflammation and oxidative stress in A549 and Beas-2B cells were associated with TLR9 expression. Inhibition of TLR9 exerted a protective function against LPS-induced inflammation and oxidative stress, which may be relevant to the MyD88/NF-κB and MyD88/MAPK/AP-1 pathways. The present work provides a potential preventive method for the treatment of LPS-induced ALI. Experiments will be performed in vivo to further validate these speculations.
Supplementary material
Table S1.
Results of replicate experiments for Biochemical testing in A549 cells
| iNOS (U/ml) | eNOS (U/ml) | NO (μmol/L) | |
|---|---|---|---|
| LPS+OV-TLR9 | 67.40 | 25.52 | 195.11 |
| 71.36 | 24.87 | 190.30 | |
| 65.58 | 29.77 | 193.91 | |
|
| |||
| LPS | 51.78 | 37.99 | 158.73 |
| 53.30 | 36.15 | 162.88 | |
| 50.77 | 36.90 | 159.47 | |
|
| |||
| LPS+EV | 54.22 | 37.44 | 161.48 |
| 50.92 | 36.23 | 155.02 | |
| 48.86 | 37.63 | 157.98 | |
|
| |||
| LPS+sh-TLR9 | 45.55 | 45.55 | 121.46 |
| 42.40 | 47.29 | 117.48 | |
| 46.21 | 51.64 | 115.06 | |
Table S2.
Results of replicate experiments for Biochemical testing in Beas-2B cells
| iNOS (U/ml) | eNOS (U/ml) | NO (μmol/l) | |
|---|---|---|---|
| LPS+OV-TLR9 | 67.37 | 25.19 | 194.37 |
| 71.32 | 24.83 | 189.52 | |
| 65.54 | 29.68 | 193.16 | |
|
| |||
| LPS | 51.73 | 37.52 | 158.35 |
| 53.29 | 35.81 | 162.16 | |
| 50.75 | 36.55 | 159.23 | |
|
| |||
| LPS+EV | 54.19 | 36.95 | 160.82 |
| 50.91 | 36.21 | 154.76 | |
| 48.85 | 37.19 | 157.33 | |
| LPS+sh-TLR9 | 45.51 | 45.18 | 121.39 |
| 42.37 | 46.75 | 116.75 | |
| 46.19 | 51.29 | 114.37 | |
Table S3.
Results of replicate experiments for IL-1β, IL-6, and TNF-α in A549 cells
| TNF-α (pg/ml) | IL-1β (pg/ml) | IL-6 (pg/ml) | ||||
|---|---|---|---|---|---|---|
| OD values | concentration | OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 1.374 | 511.302 | 1.159 | 433.437 | 1.274 | 191.930 |
| 1.312 | 483.977 | 1.303 | 495.791 | 1.185 | 176.515 | |
| 1.360 | 505.101 | 1.187 | 445.438 | 1.161 | 172.401 | |
|
| ||||||
| LPS | 0.976 | 342.031 | 0.998 | 365.591 | 0.959 | 138.493 |
| 0.924 | 320.989 | 1.018 | 373.912 | 0.930 | 133.731 | |
| 1.008 | 355.103 | 1.023 | 375.997 | 0.942 | 135.698 | |
|
| ||||||
| LPS+EV | 0.943 | 328.649 | 0.969 | 353.580 | 0.948 | 136.683 |
| 0.949 | 331.074 | 0.996 | 364.761 | 0.931 | 133.894 | |
| 0.957 | 334.314 | 0.989 | 361.857 | 0.951 | 137.177 | |
|
| ||||||
| LPS+sh-TLR9 | 0.741 | 248.913 | 0.731 | 257.427 | 0.799 | 112.548 |
| 0.779 | 263.627 | 0.715 | 251.117 | 0.775 | 108.725 | |
| 0.778 | 263.238 | 0.742 | 261.776 | 0.797 | 112.228 | |
Table S4.
Results of replicate experiments for IL-1β, IL-6, and TNF-α in Beas-2B cells
| TNF-α (pg/ml) | IL-1β (pg/ml) | IL-6 (pg/ml) | ||||
|---|---|---|---|---|---|---|
| OD values | concentration | OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 1.370 | 509.528 | 1.147 | 428.312 | 1.248 | 187.401 |
| 1.282 | 470.881 | 1.261 | 477.441 | 1.174 | 174.627 | |
| 1.345 | 498.477 | 1.182 | 443.290 | 1.159 | 172.059 | |
|
| ||||||
| LPS | 0.933 | 324.613 | 0.967 | 352.754 | 0.922 | 132.421 |
| 0.918 | 318.577 | 0.996 | 364.761 | 0.906 | 129.809 | |
| 0.961 | 335.936 | 0.983 | 359.371 | 0.939 | 135.206 | |
|
| ||||||
| LPS+EV | 0.926 | 321.794 | 0.926 | 335.889 | 0.942 | 135.698 |
| 0.937 | 326.226 | 0.949 | 345.334 | 0.926 | 133.076 | |
| 0.949 | 331.074 | 0.971 | 354.407 | 0.908 | 130.135 | |
|
| ||||||
| LPS+sh-TLR9 | 0.722 | 241.606 | 0.721 | 253.481 | 0.747 | 104.289 |
| 0.749 | 252.000 | 0.694 | 242.866 | 0.729 | 101.450 | |
| 0.731 | 245.063 | 0.708 | 248.363 | 0.762 | 106.663 | |
Table S5.
Results of replicate experiments for IFN-γ, IFN-α, and IFN-β in A549 cells
| IFN-γ (pg/ml) | IFN-α (pg/ml) | IFN-β (pg/ml) | ||||
|---|---|---|---|---|---|---|
| OD values | concentration | OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 0.517 | 135.817 | 0.409 | 130.261 | 0.443 | 84.712 |
| 0.462 | 119.662 | 0.394 | 124.756 | 0.490 | 95.250 | |
| 0.471 | 122.293 | 0.374 | 117.442 | 0.508 | 99.311 | |
|
| ||||||
| LPS | 0.554 | 146.790 | 0.475 | 154.683 | 0.694 | 142.095 |
| 0.535 | 141.144 | 0.461 | 149.475 | 0.694 | 142.095 | |
| 0.607 | 162.656 | 0.530 | 175.285 | 0.662 | 134.628 | |
|
| ||||||
| LPS+EV | 0.567 | 150.665 | 0.555 | 184.724 | 0.714 | 146.785 |
| 0.554 | 146.790 | 0.461 | 149.475 | 0.662 | 134.628 | |
| 0.616 | 165.367 | 0.454 | 146.876 | 0.648 | 131.375 | |
|
| ||||||
| LPS+sh-TLR9 | 0.792 | 219.401 | 0.742 | 256.825 | 0.823 | 172.646 |
| 0.771 | 212.853 | 0.688 | 235.734 | 0.710 | 145.845 | |
| 0.775 | 214.098 | 0.656 | 223.340 | 0.782 | 162.858 | |
Table S6.
Results of replicate experiments for IFN-γ, IFN-α, and IFN-β in Beas-2B cells
| IFN-γ (pg/ml) | IFN-α (pg/ml) | IFN-β (pg/ml) | ||||
|---|---|---|---|---|---|---|
| OD values | concentration | OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 0.473 | 122.878 | 0.384 | 121.095 | 0.431 | 82.036 |
| 0.433 | 111.220 | 0.337 | 103.992 | 0.476 | 92.101 | |
| 0.429 | 110.060 | 0.358 | 111.613 | 0.458 | 88.064 | |
|
| ||||||
| LPS | 0.552 | 146.195 | 0.462 | 149.846 | 0.669 | 136.258 |
| 0.518 | 136.112 | 0.427 | 136.889 | 0.638 | 129.057 | |
| 0.574 | 152.756 | 0.495 | 162.148 | 0.643 | 130.215 | |
|
| ||||||
| LPS+EV | 0.526 | 138.478 | 0.503 | 165.143 | 0.662 | 134.628 |
| 0.535 | 141.144 | 0.421 | 134.677 | 0.628 | 126.743 | |
| 0.569 | 151.263 | 0.446 | 143.911 | 0.641 | 129.752 | |
|
| ||||||
| LPS+sh-TLR9 | 0.761 | 209.744 | 0.716 | 246.643 | 0.793 | 165.477 |
| 0.726 | 198.913 | 0.672 | 229.527 | 0.702 | 143.969 | |
| 0.743 | 204.165 | 0.635 | 215.248 | 0.742 | 153.379 | |
Table S7.
Results of replicate experiments for MCP-1 and MCP-5 in A549 cells
| MCP-1 (pg/ml) | MCP-5 (pg/ml) | |||
|---|---|---|---|---|
| OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 1.500 | 693.502 | 1.172 | 521.112 |
| 1.415 | 648.597 | 1.302 | 588.202 | |
| 1.403 | 642.303 | 1.229 | 550.341 | |
|
| ||||
| LPS | 1.038 | 456.256 | 1.040 | 454.551 |
| 0.973 | 424.220 | 0.975 | 422.352 | |
| 1.029 | 451.801 | 1.010 | 439.642 | |
|
| ||||
| LPS+EV | 0.992 | 433.550 | 0.976 | 422.845 |
| 1.030 | 452.295 | 0.994 | 431.725 | |
| 0.957 | 416.385 | 1.017 | 443.114 | |
|
| ||||
| LPS+sh-TLR9 | 0.789 | 335.327 | 0.745 | 311.483 |
| 0.853 | 365.945 | 0.752 | 314.787 | |
| 0.797 | 339.137 | 0.802 | 338.514 | |
Table S8.
Results of replicate experiments for MCP-1 and MCP-5 in Beas-2B cells
| MCP-1 (pg/ml) | MCP-5 (pg/ml) | |||
|---|---|---|---|---|
| OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 1.457 | 670.715 | 1.146 | 507.877 |
| 1.413 | 647.547 | 1.273 | 573.104 | |
| 1.375 | 627.662 | 1.208 | 539.538 | |
|
| ||||
| LPS | 0.979 | 427.163 | 0.981 | 425.308 |
| 0.952 | 413.940 | 0.946 | 408.110 | |
| 0.975 | 425.201 | 0.952 | 411.050 | |
|
| ||||
| LPS+EV | 0.948 | 411.986 | 0.936 | 403.216 |
| 0.980 | 427.654 | 0.978 | 423.830 | |
| 0.935 | 405.644 | 0.983 | 426.294 | |
|
| ||||
| LPS+sh-TLR9 | 0.749 | 316.354 | 0.698 | 289.415 |
| 0.815 | 347.727 | 0.731 | 304.888 | |
| 0.786 | 333.900 | 0.752 | 314.787 | |
Acknowledgements
This research was mainly hosted by the institute of Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Foshan Hospital of Traditional Chinese Medicine, and Foshan Central Hospital.
Abbreviation
- ALI
Acute lung injury
- LPS
Lipopolysaccharide
- TLR9
Toll-like receptor 9
- MyD88
Myeloid differentiation protein 88
- AP-1
Activator protein 1
- NF-κB
Nuclear factor-κB
- JNK
c-Jun N-terminal kinase
- MAPK
Mitogen-activated protein kinase
- ERK1/2
Extracellular-regulated protein kinases 1/2
Footnotes
Authors’ contributions
ZQ and ZH were responsible for the design of this work. ZQ, JC, MM, MD and YZ performed the experiments. ZQ and TM were involved in analyzing data. ZQ drafted the manuscript. ZH revised the manuscript. All authors read and approved the final manuscript.
Conflict of Interest
There is no conflict of interest.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1.
Results of replicate experiments for Biochemical testing in A549 cells
| iNOS (U/ml) | eNOS (U/ml) | NO (μmol/L) | |
|---|---|---|---|
| LPS+OV-TLR9 | 67.40 | 25.52 | 195.11 |
| 71.36 | 24.87 | 190.30 | |
| 65.58 | 29.77 | 193.91 | |
|
| |||
| LPS | 51.78 | 37.99 | 158.73 |
| 53.30 | 36.15 | 162.88 | |
| 50.77 | 36.90 | 159.47 | |
|
| |||
| LPS+EV | 54.22 | 37.44 | 161.48 |
| 50.92 | 36.23 | 155.02 | |
| 48.86 | 37.63 | 157.98 | |
|
| |||
| LPS+sh-TLR9 | 45.55 | 45.55 | 121.46 |
| 42.40 | 47.29 | 117.48 | |
| 46.21 | 51.64 | 115.06 | |
Table S2.
Results of replicate experiments for Biochemical testing in Beas-2B cells
| iNOS (U/ml) | eNOS (U/ml) | NO (μmol/l) | |
|---|---|---|---|
| LPS+OV-TLR9 | 67.37 | 25.19 | 194.37 |
| 71.32 | 24.83 | 189.52 | |
| 65.54 | 29.68 | 193.16 | |
|
| |||
| LPS | 51.73 | 37.52 | 158.35 |
| 53.29 | 35.81 | 162.16 | |
| 50.75 | 36.55 | 159.23 | |
|
| |||
| LPS+EV | 54.19 | 36.95 | 160.82 |
| 50.91 | 36.21 | 154.76 | |
| 48.85 | 37.19 | 157.33 | |
| LPS+sh-TLR9 | 45.51 | 45.18 | 121.39 |
| 42.37 | 46.75 | 116.75 | |
| 46.19 | 51.29 | 114.37 | |
Table S3.
Results of replicate experiments for IL-1β, IL-6, and TNF-α in A549 cells
| TNF-α (pg/ml) | IL-1β (pg/ml) | IL-6 (pg/ml) | ||||
|---|---|---|---|---|---|---|
| OD values | concentration | OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 1.374 | 511.302 | 1.159 | 433.437 | 1.274 | 191.930 |
| 1.312 | 483.977 | 1.303 | 495.791 | 1.185 | 176.515 | |
| 1.360 | 505.101 | 1.187 | 445.438 | 1.161 | 172.401 | |
|
| ||||||
| LPS | 0.976 | 342.031 | 0.998 | 365.591 | 0.959 | 138.493 |
| 0.924 | 320.989 | 1.018 | 373.912 | 0.930 | 133.731 | |
| 1.008 | 355.103 | 1.023 | 375.997 | 0.942 | 135.698 | |
|
| ||||||
| LPS+EV | 0.943 | 328.649 | 0.969 | 353.580 | 0.948 | 136.683 |
| 0.949 | 331.074 | 0.996 | 364.761 | 0.931 | 133.894 | |
| 0.957 | 334.314 | 0.989 | 361.857 | 0.951 | 137.177 | |
|
| ||||||
| LPS+sh-TLR9 | 0.741 | 248.913 | 0.731 | 257.427 | 0.799 | 112.548 |
| 0.779 | 263.627 | 0.715 | 251.117 | 0.775 | 108.725 | |
| 0.778 | 263.238 | 0.742 | 261.776 | 0.797 | 112.228 | |
Table S4.
Results of replicate experiments for IL-1β, IL-6, and TNF-α in Beas-2B cells
| TNF-α (pg/ml) | IL-1β (pg/ml) | IL-6 (pg/ml) | ||||
|---|---|---|---|---|---|---|
| OD values | concentration | OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 1.370 | 509.528 | 1.147 | 428.312 | 1.248 | 187.401 |
| 1.282 | 470.881 | 1.261 | 477.441 | 1.174 | 174.627 | |
| 1.345 | 498.477 | 1.182 | 443.290 | 1.159 | 172.059 | |
|
| ||||||
| LPS | 0.933 | 324.613 | 0.967 | 352.754 | 0.922 | 132.421 |
| 0.918 | 318.577 | 0.996 | 364.761 | 0.906 | 129.809 | |
| 0.961 | 335.936 | 0.983 | 359.371 | 0.939 | 135.206 | |
|
| ||||||
| LPS+EV | 0.926 | 321.794 | 0.926 | 335.889 | 0.942 | 135.698 |
| 0.937 | 326.226 | 0.949 | 345.334 | 0.926 | 133.076 | |
| 0.949 | 331.074 | 0.971 | 354.407 | 0.908 | 130.135 | |
|
| ||||||
| LPS+sh-TLR9 | 0.722 | 241.606 | 0.721 | 253.481 | 0.747 | 104.289 |
| 0.749 | 252.000 | 0.694 | 242.866 | 0.729 | 101.450 | |
| 0.731 | 245.063 | 0.708 | 248.363 | 0.762 | 106.663 | |
Table S5.
Results of replicate experiments for IFN-γ, IFN-α, and IFN-β in A549 cells
| IFN-γ (pg/ml) | IFN-α (pg/ml) | IFN-β (pg/ml) | ||||
|---|---|---|---|---|---|---|
| OD values | concentration | OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 0.517 | 135.817 | 0.409 | 130.261 | 0.443 | 84.712 |
| 0.462 | 119.662 | 0.394 | 124.756 | 0.490 | 95.250 | |
| 0.471 | 122.293 | 0.374 | 117.442 | 0.508 | 99.311 | |
|
| ||||||
| LPS | 0.554 | 146.790 | 0.475 | 154.683 | 0.694 | 142.095 |
| 0.535 | 141.144 | 0.461 | 149.475 | 0.694 | 142.095 | |
| 0.607 | 162.656 | 0.530 | 175.285 | 0.662 | 134.628 | |
|
| ||||||
| LPS+EV | 0.567 | 150.665 | 0.555 | 184.724 | 0.714 | 146.785 |
| 0.554 | 146.790 | 0.461 | 149.475 | 0.662 | 134.628 | |
| 0.616 | 165.367 | 0.454 | 146.876 | 0.648 | 131.375 | |
|
| ||||||
| LPS+sh-TLR9 | 0.792 | 219.401 | 0.742 | 256.825 | 0.823 | 172.646 |
| 0.771 | 212.853 | 0.688 | 235.734 | 0.710 | 145.845 | |
| 0.775 | 214.098 | 0.656 | 223.340 | 0.782 | 162.858 | |
Table S6.
Results of replicate experiments for IFN-γ, IFN-α, and IFN-β in Beas-2B cells
| IFN-γ (pg/ml) | IFN-α (pg/ml) | IFN-β (pg/ml) | ||||
|---|---|---|---|---|---|---|
| OD values | concentration | OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 0.473 | 122.878 | 0.384 | 121.095 | 0.431 | 82.036 |
| 0.433 | 111.220 | 0.337 | 103.992 | 0.476 | 92.101 | |
| 0.429 | 110.060 | 0.358 | 111.613 | 0.458 | 88.064 | |
|
| ||||||
| LPS | 0.552 | 146.195 | 0.462 | 149.846 | 0.669 | 136.258 |
| 0.518 | 136.112 | 0.427 | 136.889 | 0.638 | 129.057 | |
| 0.574 | 152.756 | 0.495 | 162.148 | 0.643 | 130.215 | |
|
| ||||||
| LPS+EV | 0.526 | 138.478 | 0.503 | 165.143 | 0.662 | 134.628 |
| 0.535 | 141.144 | 0.421 | 134.677 | 0.628 | 126.743 | |
| 0.569 | 151.263 | 0.446 | 143.911 | 0.641 | 129.752 | |
|
| ||||||
| LPS+sh-TLR9 | 0.761 | 209.744 | 0.716 | 246.643 | 0.793 | 165.477 |
| 0.726 | 198.913 | 0.672 | 229.527 | 0.702 | 143.969 | |
| 0.743 | 204.165 | 0.635 | 215.248 | 0.742 | 153.379 | |
Table S7.
Results of replicate experiments for MCP-1 and MCP-5 in A549 cells
| MCP-1 (pg/ml) | MCP-5 (pg/ml) | |||
|---|---|---|---|---|
| OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 1.500 | 693.502 | 1.172 | 521.112 |
| 1.415 | 648.597 | 1.302 | 588.202 | |
| 1.403 | 642.303 | 1.229 | 550.341 | |
|
| ||||
| LPS | 1.038 | 456.256 | 1.040 | 454.551 |
| 0.973 | 424.220 | 0.975 | 422.352 | |
| 1.029 | 451.801 | 1.010 | 439.642 | |
|
| ||||
| LPS+EV | 0.992 | 433.550 | 0.976 | 422.845 |
| 1.030 | 452.295 | 0.994 | 431.725 | |
| 0.957 | 416.385 | 1.017 | 443.114 | |
|
| ||||
| LPS+sh-TLR9 | 0.789 | 335.327 | 0.745 | 311.483 |
| 0.853 | 365.945 | 0.752 | 314.787 | |
| 0.797 | 339.137 | 0.802 | 338.514 | |
Table S8.
Results of replicate experiments for MCP-1 and MCP-5 in Beas-2B cells
| MCP-1 (pg/ml) | MCP-5 (pg/ml) | |||
|---|---|---|---|---|
| OD values | concentration | OD values | concentration | |
| LPS+OV-TLR9 | 1.457 | 670.715 | 1.146 | 507.877 |
| 1.413 | 647.547 | 1.273 | 573.104 | |
| 1.375 | 627.662 | 1.208 | 539.538 | |
|
| ||||
| LPS | 0.979 | 427.163 | 0.981 | 425.308 |
| 0.952 | 413.940 | 0.946 | 408.110 | |
| 0.975 | 425.201 | 0.952 | 411.050 | |
|
| ||||
| LPS+EV | 0.948 | 411.986 | 0.936 | 403.216 |
| 0.980 | 427.654 | 0.978 | 423.830 | |
| 0.935 | 405.644 | 0.983 | 426.294 | |
|
| ||||
| LPS+sh-TLR9 | 0.749 | 316.354 | 0.698 | 289.415 |
| 0.815 | 347.727 | 0.731 | 304.888 | |
| 0.786 | 333.900 | 0.752 | 314.787 | |