Andrographolide Alleviates Mycoplasma pneumoniae Pneumonia in Children by Inhibiting Alveolar Epithelial Cell Pyroptosis Through the HMGB1/TLR4/NF-κB Pathway

3.1. Reagents and Compounds

Andrographolide (AG; purity ≥ 98%; Sigma) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution. The final DMSO concentration did not exceed 0.1% in any experimental group. Antibodies against NLRP3, HMGB1, phosphorylated NF-κB p65, NF-κB p65, TLR4, and β-actin were obtained from Thermo Fisher Scientific, Abcam, Santa Cruz Biotechnology, and Proteintech. All ELISA kits were obtained from MultiSciences Biotech Co., Ltd. The CCK-8 and LDH assay kits were supplied by Dojindo and Beyotime, respectively. Reagents for qPCR, including TRIzol, PrimeScript RT reagent kit, and SYBR Premix Ex Taq II, were obtained from Invitrogen and Takara, and primers were synthesized by Sangon Biotech (Shanghai).

3.2. Cell Culture

Mouse alveolar epithelial cells (MLE-12; ATCC) were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a 5% CO₂ atmosphere. Mycoplasma pneumoniae (Mp; FH strain; ATCC 15531) was cultured in PPLO broth medium to the logarithmic growth phase, collected by centrifugation, and resuspended in PBS. The bacterial concentration was adjusted to 1 × 10⁹ CCU/mL. For the infection model, cells were incubated with Mp at a multiplicity of infection (MOI) of 10 for 24 hours. Cells were divided into the following groups: control, Mp-infected, and Mp-infected plus AG at 10, 20, or 50 μM. All in vitro experiments were performed in triplicate and repeated independently at least 3 times.

3.3. Animals and In Vivo Model

Female Balb/c mice aged 4 - 6 weeks and weighing 18 - 22 g were housed in a standard animal center at 22 ± 2 °C, 50% ± 10% humidity, and under a 12-hour light/dark cycle. After 1 week of acclimatization, the mice were randomly divided into 4 groups (n = 10 per group): control group, Mp group, and Mp + AG groups at 25 and 50 mg/kg.

Mycoplasma pneumoniae (Mp) was collected, and the concentration was adjusted to 2 × 10⁸ CCU/mL. The bacterial suspension was slowly instilled into both nostrils of the mice (25 μL per nostril). Mice in the control group received 25 μL of PBS per nostril. Starting on the second day after infection, AG at 25 or 50 mg/kg, dissolved in 0.5% CMC-Na solution, was administered once daily for 7 consecutive days. The control and Mp model groups received an equivalent volume of 0.5% CMC-Na solution. Body weight was monitored daily during this period. At the end of the experiment, 1% pentobarbital sodium (50 mg/kg) was injected intraperitoneally to anesthetize the mice. Blood was collected from the eyeball, and serum was separated and stored for ELISA detection. Whole lung tissues were collected, rinsed with PBS, blotted dry with filter paper, and weighed. Some tissues were fixed in 4% paraformaldehyde for pathological examination. The remaining tissues were preserved in liquid nitrogen for subsequent Western blot and qPCR analyses.

3.4. Cell Viability and Cytotoxicity Assays

Cell viability was assessed using the CCK-8 method. MLE-12 cells were seeded in 96-well plates at 1 × 10⁴ cells/well. After 24 hours of culture, cells were treated according to the groups described above. After treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37 °C in the dark for 1.5 hours. Absorbance (OD) was measured at 450 nm using an enzyme-linked immunosorbent assay reader, and the cell survival rate and inhibition rate were calculated.

3.5. Detection of Lactate Dehydrogenase Release

Cell supernatants were collected and processed according to the instructions of the LDH kit (Beyotime, C0017). Absorbance was measured at 490 nm to assess the extent of cell membrane damage.

3.6. Protein Expression Analysis by Western Blotting

Total protein was extracted from each group of cells or lung tissues using RIPA lysis buffer containing protease and phosphatase inhibitors and was quantified using the BCA method. Protein samples (30 μg) were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skimmed milk for 2 hours at room temperature, incubated with primary antibodies overnight at 4 °C, washed with TBST, and then incubated with an HRP-labeled goat anti-rabbit/mouse secondary antibody for 1 hour at room temperature. Images were developed using an ECL chemiluminescence kit, and gray values were analyzed using Image Lab software (Bio-Rad). Protein expression levels were normalized to β-actin.

3.7. Cytokine Measurements

According to the kit instructions, levels of TNF-α, IL-6, and IL-1β in cell culture supernatants, mouse serum, and BALF were measured using ELISA kits. Absorbance was read at 450 nm, and cytokine concentrations were calculated based on standard curves.

3.8. Quantitative Real-Time PCR

Total RNA was extracted from lung tissue using TRIzol reagent, and purity and concentration were determined. Total RNA (1 μg) was reverse-transcribed according to the kit instructions. qPCR was performed using SYBR Premix Ex Taq II on the CFX96 Touch system. The 20-μL reaction system consisted of 10 μL of SYBR Premix, 2 μL of cDNA, 0.8 μL each of upstream and downstream primers, and 6.4 μL of ddH₂O. The reaction conditions were 95 °C for 30 seconds, followed by 40 cycles of 95 °C for 5 seconds and 60 °C for 30 seconds. Using GAPDH as the internal reference, relative mRNA expression was calculated using the 2^-ΔΔCt method. The primer sequences are listed in Table 1.

3.9. Histopathological Evaluation

Lung tissue was fixed in 4% paraformaldehyde and embedded in paraffin to prepare 4-μm-thick sections. The tissue was processed according to the instructions of the hematoxylin and eosin (H&E) staining kit, including xylene dewaxing, graded alcohol hydration, hematoxylin staining of nuclei, eosin staining of cytoplasm, and sealing with neutral gum. Pathological changes in lung tissue, including alveolar structure, inflammatory cell infiltration, hemorrhage, and edema, were observed under an optical microscope, and semi-quantitative analysis was conducted based on pre-established scoring criteria.

3.10. Statistical Analysis

All data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using SPSS 23.0 software. One-way ANOVA followed by the LSD post hoc test was used for multiple comparisons. A P value < 0.05 was considered statistically significant.

4.1. Andrographolide Restores Viability and Reduces Cytotoxicity in Mp-Infected Cells

We evaluated the effects of AG on MLE-12 cell function after Mycoplasma pneumoniae infection by assessing proliferation activity and membrane integrity. The results (Figure 1A) showed that, compared with the control group, Mp infection significantly inhibited MLE-12 cell proliferation, whereas treatment with different concentrations of AG (10, 20, and 50 μM) significantly increased cell survival rates, demonstrating a concentration-dependent restorative effect (P < 0.05).

Figure 1.

Effects of andrographolide (AG) on cell viability and LDH release in Mp-infected MLE-12 cells. A, Cell viability of MLE-12 cells; B, LDH release in MLE-12 cells [data are expressed as mean ± SEM (N = 3); *P < 0.05, **P < 0.01, ***P < 0.001].

The LDH release results shown in Figure 1B indicated that Mp infection significantly increased cytotoxicity, whereas AG intervention significantly reduced LDH release (P < 0.05), indicating that AG effectively protected cell membrane integrity (P < 0.05).

4.2. Andrographolide Suppresses NLRP3 Inflammasome Activation

NLRP3 is a core component of the inflammasome, and its activation can induce pyroptosis. We assessed the expression of NLRP3, a key signaling molecule for pyroptosis, by Western blotting. As shown in Figure 2A, Mp infection significantly upregulated NLRP3 expression (P < 0.001), whereas AG treatment at 10 - 50 μM dose-dependently inhibited this increase (P < 0.05), indicating that AG alleviated Mp-induced pyroptosis (Figure 2B).

Figure 2.

Effects of AG on NLRP3 and HMGB1/TLR4/NF-κB pathway protein expression in Mp-infected MLE-12 cells. A, Representative Western blot bands of NLRP3 protein; B, Quantitative analysis of NLRP3 protein expression; C, Representative Western blot bands of HMGB1, TLR4, phosphorylated p65 (p-p65), and total p65; D-F, Quantitative analysis of protein expression levels [data are expressed as mean ± SEM (N = 3); *P < 0.05, **P < 0.01, ***P < 0.001].

4.3. Andrographolide Inhibits HMGB1/TLR4/NF-κB Signaling Activation

We evaluated the effects of AG on the HMGB1/TLR4/NF-κB inflammatory signaling axis by Western blotting. The results showed (Figure 2C) that Mp infection significantly increased the protein levels of HMGB1 and TLR4 and the phosphorylation level of p65 (P < 0.001). AG treatment at 10 - 50 μM significantly downregulated the expression of HMGB1 and TLR4, and medium and high concentrations of AG (20 and 50 μM) significantly inhibited p65 phosphorylation (P < 0.01) (Figure 2D-F). These results suggest that AG may exert anti-inflammatory effects by inhibiting this signaling pathway through multiple targets.

4.4. Andrographolide Attenuates Inflammatory Cytokine Secretion

We also assessed the effects of AG on the levels of the inflammatory cytokines IL-6 and TNF-α in MLE-12 cells by ELISA. The results showed (Figure 3) that the concentrations of IL-6 and TNF-α significantly increased after Mp infection (P < 0.001). After AG intervention, the release of these two inflammatory factors was significantly reduced (P < 0.01), with the strongest inhibitory effect at 50 μM, further confirming the anti-inflammatory effect of AG.

Figure 3.

Effects of AG on the levels of inflammatory cytokines in the supernatant of Mp-infected MLE-12 cells. A, IL-6 level; B, TNF-α level [data are expressed as mean ± SEM (N = 3); *P < 0.05, **P < 0.01, ***P < 0.001].

4.5. Andrographolide Improves Systemic Manifestations and Lung Edema in Mice

The in vivo results showed that Mp infection led to a significant decrease in mouse body weight (Figure 4A) and a significant increase in lung index (lung weight/body weight) (Figure 4B), indicating disease activity and lung tissue edema (P < 0.001). However, AG treatment at 25 and 50 mg/kg effectively reversed weight loss and reduced the lung index (P < 0.05), indicating that AG has a protective effect against Mp infection.

Figure 4.

Effects of AG on general condition and lung histopathology in Mp-infected mice. A, Representative images of H&E-stained lung tissue sections (scale: 100 μm); B, Quantitative scoring results of pathological lung injury; C, Representative images of H&E-stained lung tissue sections (scale: 100 μm); D, Quantitative scoring results of pathological lung injury [data are expressed as mean ± SEM (A-B: N = 10; C-D: N = 3); *P < 0.05, **P < 0.01, ***P < 0.001].

4.6. Andrographolide Alleviates Lung Histopathological Injury

H&E staining of lung tissue (Figure 4C) showed that AG treatment significantly alleviated the severe destruction of alveolar structure, inflammatory cell infiltration, and interstitial thickening caused by Mp infection. The semi-quantitative lung injury scores (Figure 4D) indicated that the pathological changes caused by Mp infection were significantly alleviated after AG treatment, particularly at the higher dose of 50 mg/kg (P < 0.01). The H&E staining results further demonstrate that AG could serve as a potential therapeutic agent for Mycoplasma pneumoniae pneumonia.

4.7. Andrographolide Suppresses the Pulmonary HMGB1/TLR4/NF-κB Pathway In Vivo

We analyzed the expression of proteins in the inflammatory signaling pathway in lung tissue (Figure 5A). The results (Figure 5B-D) showed that Mp infection significantly upregulated the expression of HMGB1, TLR4, and p-p65 in lung tissue (P < 0.001), indicating that Mp activated the inflammatory response in lung tissue. Treatment with AG at 25 and 50 mg/kg reversed these changes. The expression levels of HMGB1, TLR4, and p-p65 proteins in the AG treatment groups were significantly inhibited, and the inhibitory effect of AG was dose-dependent, with the high dose of AG (50 mg/kg) showing the most pronounced effect (P < 0.05).

Figure 5.

Effects of AG on the HMGB1/TLR4/NF-κB pathway, inflammatory cytokines, and gene expression in Mp-infected mice. (A) Representative Western blot bands of HMGB1, TLR4, p-p65, and total p65 in lung tissues. (B-D) Quantitative analysis of protein expression levels. (E) IL-1β level in BALF detected by ELISA. (F) TNF-α level in BALF detected by ELISA. (G) Effects of AG on the mRNA expression of inflammation-related genes in lung tissues of Mp-infected mice. mRNA expression levels of TLR4, NF-κB p65, and NLRP3 were detected by qPCR [data are expressed as mean ± SEM (N = 3); *P < 0.05, **P < 0.01, ***P < 0.001].

4.8. Andrographolide Reduces Inflammatory Cytokines in BALF

As shown in Figure 5E-F, to further quantify the pulmonary inflammatory response, we measured the levels of the key proinflammatory cytokines IL-1β and TNF-α in BALF. Compared with the control group, the concentrations of IL-1β and TNF-α in the Mp infection model group were significantly increased (P < 0.001), indicating robust pulmonary inflammation triggered by Mp infection. After AG intervention, this abnormal inflammatory state was effectively controlled. All AG treatment groups (25 and 50 mg/kg) showed significantly reduced levels of IL-1β and TNF-α in BALF (P < 0.01), indicating that AG has a clear anti-inflammatory effect. These findings further confirmed, at the cytokine level, that AG can effectively inhibit the excessive inflammatory response driven by Mp infection, providing an important basis for its potential mechanism in treating Mycoplasma pneumoniae pneumonia.

4.9. Andrographolide Downregulates Inflammation-Related Gene Expression

To further explore the anti-inflammatory mechanism of AG at the transcriptional level, we used qPCR to measure the mRNA expression of key inflammatory signaling molecules in lung tissue. The results are shown in Figure 5G. Mp infection significantly upregulated the gene expression levels of TLR4, NF-κB p65, and NLRP3 (P < 0.001). After AG treatment, the abnormally high expression of these genes was significantly inhibited (P < 0.001). This result confirmed that AG can effectively inhibit the TLR4/NF-κB/NLRP3 inflammatory signaling pathway activated by Mp infection at the transcriptional level.