Baicalein inhibits human neutrophil myeloperoxidase and protects mice from LPS-induced lung inflammation

Abstract

Baicalein (BAI), a phenolic flavonoid widely used in East Asian countries, is protective in various fibrotic and inflammatory diseases. This research aimed to determine the function of BAI and the specific mechanism by which it contributes to lipopolysaccharide (LPS)-induced lung inflammation in mice. Intratracheal injection of LPS produced inflammation in the lungs of the mice. The lung injury was assessed pathologically. Bronchoalveolar lavage and lung tissue were obtained to cells, quantify inflammatory cell counts, and total protein content. The zymography approach measured metalloproteinase activity. After neutrophil depletion in mice, the DCFH fluorescence assay was used to measure reactive oxygen species (ROS) formation. The tetramethyl benzidine oxidation technique was used to evaluate the activity of myeloperoxidase (MPO). The lung damage and the number of positive cells in the BAI-treated groups showed considerable improvement compared to the LPS group in mice with LPS-induced lung inflammation. Our investigation revealed that BAI decreased the total cell count and neutrophil count in the bronchoalveolar lavage fluid (BALF) after LPS treatment. Additionally, it suppressed the excessive production of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in the bronchoalveolar lavage fluid (BALF). Moreover, BAI decreased MPO activity, and LPS activated ROS production in BALF cells via the TLR4/NF-κB pathway. The findings of our study demonstrate that BAI successfully reduced LPS-induced pulmonary edema, neutrophil infiltration, proinflammatory cytokine generation, and oxidative stress via the TLR4/NF-κB pathway.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-43806-z.

Introduction

Pneumonia is a common lower respiratory tract infection, primarily caused by viral, bacterial, and fungal pathogens, and has a high incidence and mortality worldwide^1,2. Pneumonia is widely occurring in children, mainly due to the low immunity of children and the defects of respiratory tract anatomy, and its high incidence and recurrence rate can cause various serious complications and poor prognosis, seriously affecting the development and life health of children^3–5. Therefore, it is very important to identify the underlying pathogenesis of pneumonia in children and develop new therapeutic strategies to inhibit the progression of pneumonia^6–8.

In recent years, the anti-inflammatory effect of Chinese herbs has been widely discussed^9,10. Baicalein (5,6,7-trihydroxyflavone) has anti-inflammatory and antioxidative properties; it was extracted from the root of the traditional Chinese herb Scutellaria baicalensis Georgi. Yan discovered that BAI inhibits oxidative stress and neuroinflammation in BV-2 cells triggered by LPS¹¹. In prior research, BAI was shown to reduce inflammation and macrophage dysfunction after acute lung damage by blocking excessive mitochondrial fission¹². Our previous study also found BAI exerts a protective effect on LPS-induced pulmonary inflammation by modulating macrophage polarization and inhibiting pyroptosis. However, the molecular mechanism of BAI in pulmonary remains poorly understood. In this report, we discuss the findings of our investigation into the impact of BAI on an animal model of pneumonia and the underlying processes responsible for these effects.

Materials and methods

LPS-induced pneumonia model and lung function test in mice

C57BL/6 mice (7–8 weeks, purchased from HuaFukang Biotechnology Company) were anaesthetized (4% isoflurane) and fixed, then LPS (10 µg LPS was dissolved in 50 µL PBS) was injected intratracheally through a micro syringe to induce the pneumonia model. The experiment ended 24 h after LPS induction, and the SCIREQ small animal lung function detector (Model: FLEXIVENT) tested the lung function indexes of mice. Mice were randomly divided into four groups (each group: n = 6): control (0.5% CMC-Na)-treated, BAI (5 mg/kg)-treated, LPS-treated, and BAI (5 mg/kg) + LPS-treated. Mice in the baicalein group were administered 5 mg/kg baicalein (baicalein was dissolved in 0.2 ml of 0.5% CMC-Na, the concentration of which was 1 µg/µl) via intraperitoneal injection. LPS (L2880, Escherichia coli 055:B5) and baicalein were purchased from Sigma-Aldrich. At the end of the experiments, mice were euthanized by cervical dislocation under deep anesthesia, confirmed by the absence of a pedal reflex. All experimental protocols were approved by the Experimental Animal Ethics Committee of Shenzhen Longhua District Maternal and Child care Health Hospital. All methods were performed in accordance with the relevant guidelines and regulations of this committee, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the ARRIVE guidelines.

Collection of bronchoalveolar lavage fluid (BALF)

BALF was collected as previously described. The lungs were lavaged twice with 1 mL of normal saline. Immediately after removing the lavage solution. The sample was put on ice and centrifuged at 2,500 rpm for 15 min at 4 ℃. The supernatant was stored at −80 ℃, and the precipitation consisted of cells in the lavage solution.

Protein content determination in BALF

The total protein concentration of the supernatant was determined using the BCA Protein Assay Kit (SolarBio).

BALF Smear staining of BALF cells

After BALF cell smear staining, the suspended cells were settled and fixed in methanol, and the images were stained and fixed with methanol after drying naturally. After fixation for 20 s, the cells were passed. Diff-QuikⅡ was stained for 10 s and removed immediately. After Diff-QuikⅡ staining for 15 s, it was taken out directly after washing with xylene transparent and sealed for preservation.

Enzyme-linked immunosorbent assay (ELISA)

The commercial ELISA kits measured the concentrations of IL-1α, IL-1β, and TNF-α in different groups (Nanjing SenBeiJia Biological Technology).

Gelatin zymography

According to the manufacturer’s instructions, MMP-2 and MMP-9 activity was measured by gelatin zymography assay kit (Shanghai Xinfan Biotechnology Co., Ltd).

Western blot assay

The total protein from each group was extracted and subjected to electrophoresis, membrane transfer, and closure, and the GAPDH (Cell Signaling), MPO, MMP2, and MMP9 (Abcam) primary antibody dilutions were added, respectively, incubated at 4℃ overnight, and the second antibody was added after membrane washing and incubated at room temperature for 1 h. For immunoblotting, membranes were cut horizontally according to the molecular weight markers to probe for proteins of different sizes. Therefore, the presented bands represent the relevant sections of the membrane.

Statistical analysis

All data are normally distributed, and the results are expressed as “mean ± standard difference.” One-way ANOVA was conducted between groups using GraphPad Prism 9. 0 software. P < 0.05 indicated a significant difference.

Results

Lung protective effects of BAI on LPS-induced pneumonia in mice

We examined the effects of BAI on LPS-induced pneumonia in mice. The results showed that mouse lung tissue was significantly damaged after LPS treatment, and the degree of damage was reduced considerably after BAI pretreatment (Fig. 1A). Functionally, indicators for lung capacity, such as pressure-volume (PV), inspiratory capacity (IC), and static compliance (Crs), were most improved in BAI-treated mice. BAI therapy was also associated with the greatest improvement in functional indicators for lung fibrosis, such as elastic resistance (Ers) and respiratory resistance (Rrs) (Fig. 1B). These results indicated that BAI pretreatment attenuated LPS-induced pneumonia in mice.

Fig. 1.

The effects of BAI on LPS-induced pneumonia. (A) Histological images of lung tissues. (B) Lung mechanical function was measured by FlexiVent, showing PV curves, IC, Rrs, Crs, and Ers for all the experimental groups. *p < 0.05, **p < 0.01, and ***p < 0.001.

Effect of BAI on inflammatory cell count and protein concentration in the BALF of LPS-induced pneumonia mice

Mice given LPS via the intratracheal method had a markedly elevated BALF protein concentration (Fig. 2A), total BALF cells (Fig. 2B), and BALF neutrophils (Fig. 2C and D) in contrast to animals given only the vehicle or BAI alone. However, BAI post-treatment suppressed BALF protein, the number of total cells, and neutrophils.

Fig. 2.

Effect of BAI on BALF protein content and cellularity in LPS-treated mice. (A) Protein content in the BALF was determined using a BCA assay. (B) The total number of cells in BALF (C) and (D), BALF neutrophil ratio, and Diff-QuikⅡ staining. *p < 0.05, **p < 0.01, and ***p < 0.001.

BAI attenuated LPS-induced inflammatory cytokine production

To learn more about how BAI reduces inflammation, we used Luminex assays and ELISA to measure its effects on IL-1α, IL-1β, and TNF-α, inflammatory cytokines produced in BALF and lung tissue. In the LPS model, BAI administration reduced IL-1α, IL-1β, and TNF-α levels in BALF (Fig. 3A). Additionally, BAI decreased IL-1α, IL-1β, and TNF-α levels in lung tissue (Fig. 3B).

Fig. 3.

BAI attenuated LPS-induced inflammatory cytokine production. (A) Inflammatory cytokines TNF-α, IL-6, and IL-1β in BALF were quantified by Luminex assay. (B) ELISA was used to quantify the inflammatory cytokines TNF-α, IL-6, and IL-1β in lung tissue. *p < 0.05, **p < 0.01 and ***p < 0.001.

Effects of BAI on LPS-induced oxidative stress in LPS–induced pneumonia mice

To evaluate the effects of BAI on LPS-induced oxidative stress, MPO activity, and ROS generation were assessed 24 h after LPS administration. When mice were treated with LPS alone, the MPO activity and ROS levels were significantly elevated, but they recovered to normal when BAI was administered (Fig. 4A and B). Inflammation and injury trigger the production of MMPs in large quantities, perhaps to repair the extracellular matrix that has been damaged [23]. As shown by zymography data, MMP-2 and MMP-9 activities were significantly elevated in the LPS-treated group compared to the control group (Fig. 4C). Protein expressions showed comparable outcomes. LPS-induced lung tissues enhanced MMP–2 and 9 protein expressions (Fig. 4D). Therefore, BAI may decrease the activity and protein levels of MMP-2 and 9 compared to the group stimulated by LPS. The results suggested that BAI might potentially inhibit the migration of inflammatory cells into lung tissues by decreasing the activities and protein expressions of MMP-2 and 9.

Fig. 4.

Effects of BAI on LPS-induced oxidative stress in LPS-induced pneumonia mice. (A) MPO activity. (B) ROS generation in BALF was determined using flow cytometry. (C) MMP–2 and MMP–9 activities in the BALF of LPS-induced pneumonia mice. (D) The protein expression of MMP–2 and MMP–9 was determined by Western blot. *p < 0.05, **p < 0.01 and ***p < 0.001.

Effects of BAI on the TLR4/NF-κB pathway in LPS–induced pneumonia mice

Lipopolysaccharide (LPS) is well recognized for its ability to stimulate Toll-like receptor 4 (TLR4), activating NF-κB. Figure 5A demonstrates that LPS substantially impacted the release of TLR4 and NF-κB p65, resulting in a large increase compared to the control group. However, BAI treatment reduced TLR4 and NF-κB p65, indicating that BAI inhibited inflammation in LPS-induced pneumonia mice. Moreover, the observations by IHC analyses showed similar trends as those in WB analyses (Fig. 5B).

Fig. 5.

Effects of BAI on the TLR4/NF-κB pathway in LPS-induced pneumonia mice (A) The protein expression of TLR4 and NF-κB was determined by western blot. (B) Immunofluorescence staining of TLR4. *p < 0.05, **p < 0.01 and ***p < 0.001.

Discussion

In this study, we used an LPS-induced mouse model of acute lung inflammation to evaluate the anti‑inflammatory and antioxidant effects of baicalein BAI. Treatment with BAI reduced pulmonary edema, neutrophil accumulation in bronchoalveolar lavage fluid and lung tissue, MPO activity, and the levels of proinflammatory cytokines (IL‑1α, IL‑1β, TNF‑α). BAI also attenuated oxidative stress by preserving antioxidant enzyme activities and small‑molecule scavengers.Together, these findings indicate that BAI limits both cellular and biochemical components of LPS‑driven lung injury.

The LPS, a prominent constituent of the outer membranes of Gram-negative bacteria, is now recognized as the primary pathogen responsible for pulmonary inflammation, which may potentially result in pneumonia¹³. It was postulated that LPS might boost MPO activity in lung tissues by increasing the amount of neutrophils in the BALF. Similarly, alveolar edema and significant neutrophil infiltration were seen in the lung tissues of the mice that had pneumonia. By reducing tissue leukocytes and pulmonary edema, BAI may be a useful treatment option. Based on our analysis of BAI’s effects on MPO, we can say that it inhibits the recruitment of leukocytes, especially neutrophils. Furthermore, neutrophil activation, capillary leakage, and tissue edema accompanied by the overproduction of proinflammatory cytokines in the lungs (mainly IL-1α, IL-1β, and TNF-α) are associated with the progression of pneumonia^14–16. Mice pretreated with BAI reduced the production of these cytokines in a dose-dependent manner after developing LPS-induced pneumonia. Based on these results, it seems that BAI significantly reduces inflammation in the animals. Furthermore, we investigated the potential role of NF-κB/TLR4 signalling pathways in the protective mechanism of glycoprotein against inflammation and oxidative stress in mice infected with LPS-induced pneumonia.

Finally, in mice that developed pneumonia due to LPS, we looked at how BAI affected inflammation and antioxidant levels. BAI reduced pulmonary edema, decreased neutrophil recruitment into lung tissue, and suppressed the synthesis of inflammatory mediators. In addition, BAI eased the oxidative load by enhancing the antioxidant enzyme activities and micromolecular scavenger content. The results show that BAI prevents infection-related damage to the lung tissue by acting as an antibiotic, suppressing the activation of the NF-κB/TLR4 signalling pathway, and maybe by reducing inflammation and free radicals. This study could provide a fresh perspective on how traditional Chinese medicine might be used to treat and prevent respiratory illnesses caused by bacteria.

While this study strongly supports the role of MPO inhibition and NF-κB/TLR4 suppression, we acknowledge certain limitations. The precise intracellular signaling upstream of NF-κB and the relative contribution of other cell types (e.g., macrophages) warrant further investigation. Nevertheless, our findings position BAI as a promising multi-target agent for inflammatory lung conditions. They offer a scientific rationale for the traditional use of its source, Scutellaria baicalensis, and suggest that targeting MPO could be a novel strategic component in developing therapies for bacterial pneumonia and other sterile inflammatory lung injuries.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

C.W., J.S., N.G., G.Z., and J.C. performed the research. C.W., and H.D. designed the research study. C.W., J.S., N.G., G.Z., J.C. and Q.B. contributed essential reagents or tools. C.W., Y.K., and Q.B. analysed the data. J.S. wrote the paper; all authors read and approved the final manuscript.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

The ethical standards of the study were approved by the Shenzhen Longhua District Maternal and Child Health care Hospital.