Male C57BL/6J mice (6–8 weeks, 18–20 g) were sourced from SPF Biotechnology Co., Ltd. (Beijing, China) and housed for one week under a 12-h light/dark cycle in specific pathogen-free conditions before the experiment. To evaluate the effect of BBR (A75865, InnoChem, Beijing, China; purity $\ge$97%) on LPS-induced ALI in septic mice, the mice were randomly allocated into four groups: Control, LPS, LPS + BBR (25 mg/kg/day), and LPS + BBR (100 mg/kg/day), with six mice in each group. The LPS-induced ALI model was modified based on previously described methods [13, 14]. The mice were administered a single intraperitoneal injection of LPS at a dosage of 10 mg/kg of body weight without the use of analgesia, as previously used [15, 16, 17]. This minimally invasive procedure did not induce signs of distress and adhered to the 3R principles of animal welfare. Briefly, following intragastric administration of BBR dissolved in 0.5% carboxymethyl cellulose sodium (CMC-Na) or a control solvent twice daily for three days, LPS (L8880, Solarbio, Beijing, China) dissolved in normal saline was injected intraperitoneally at a dosage of 10 mg/kg body weight two hours following BBR treatment on day 4. At six hours post-LPS injection, the mice underwent a final treatment with either BBR or the control solvent. Eighteen hours after LPS injection, the mice were subjected to terminal anesthesia using 5% isoflurane (I8000, Solarbio, Beijing, China). After confirming that the mice were in a state of deep anesthesia and the absence of pain reflexes, their abdominal cavities were opened. Blood samples were obtained from the inferior vena cava with a 1-mL syringe, and the mice were euthanized via exsanguination, following which lung tissues were collected. The harvested lung tissues were either preserved at –80 °C for further analysis or fixed in 10% neutral formaldehyde for histological examination. All animal-related procedures received approval from the Ethics Committee of Beijing Friendship Hospital, Capital Medical University (Approval number #24-2034) and were performed in accordance with the National Guidelines for the Housing and Care of Laboratory Animals.

Blood samples underwent centrifugation at 2500 g for a duration of 10 min to obtain serum. The levels of HMGB1 in serum and cell culture supernatants were measured using a mouse HMGB1 enzyme-linked immunosorbent assay (ELISA) kit (SEKM-0145, Solarbio, Beijing, China). Additionally, mouse lung tissues were homogenized, and commercial assay kits were utilized to quantify malondialdehyde (MDA) (S0131, Beyotime Biotechnology, shanghai, China), superoxide dismutase (SOD) (S0101, Beyotime Biotechnology, shanghai, China), and myeloperoxidase (MPO) (A044-1-1, Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China) to assess oxidative stress in the lungs and infiltration of neutrophils.

Lung tissues that were fixed in paraffin, and sections measuring 5 µm were subjected to standard hematoxylin and eosin (H&E) staining to assess histopathological alterations using a light microscope. A semi-quantitative scoring system was utilized to evaluate the infiltration of neutrophils, the presence of pulmonary edema, disorganization within the lung parenchyma, and any hemorrhagic changes in the lung tissue. The scoring criteria were defined as follows: 0, no pathological changes observed in the visual field; 1, 2, 3, and 4 represent pathological changes present in less than 25%, 25%–50%, 51%–75%, and greater than 75% of the total visual field, respectively [18]. Apoptotic characteristics in lung tissues preserved with formaldehyde were identified using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, which employed the DAB (SA-HRP) (PR30010, Proteintech, Wuhan, China) TUNEL Cell Apoptosis Detection Kit (G1507, Servicebio, Wuhan, China). The proportion of TUNEL-positive cells was quantified using ImageJ software (NIH, Bethesda, MD, USA). Additionally, immunofluorescence was performed to detect HMGB1 in lung tissue, and its expression and intracellular distribution were evaluated.

Mitochondria were isolated from lung tissue using a mitochondrial isolation kit (C3606, Beyotime Biotechnology, Shanghai, China). In summary, the lung tissue underwent homogenization followed by centrifugation at 4 °C and 600 g for 10 min to eliminate cell nuclei. The resulting supernatant was centrifuged a second time at 11,000 g and 4 °C for 10 minutes to collect crude mitochondrial pellets. Next, the supernatant underwent centrifugation at 12,000 g for 10 minutes to separate cytoplasmic proteins. The concentration of proteins was assessed using a BCA protein assay kit (#23225, Thermo Fisher Scientific, Waltham, MA, USA), after which the proteins were heated and analyzed via Western blot.

RNA was extracted from the cells using the RaPure total RNA kit (R4011, Magen, Shanghai, China), in accordance with the instructions provided by the manufacturer. The mRNA expression levels of the inflammatory cytokine genes Tnf$\alpha$ and Il6, along with the internal control gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh), were amplified using specific primers: Gapdh (5^′-CTCTGGAAAGCTGTGGCGTGATG-3^′ and 5^′-ATGCCAGTGAGCTTCCCGTTCAG-3^′), Tnf$\alpha$ (5^′-CCAAAGGGATGAGAAGTTCC-3^′ and 5^′-CTCCACTTGGTGGTTTGCTA-3^′), and Il6 (5^′-CCATCCAGTTGCCTTCTTGG-3^′ and 5^′-TGCAAGTGCATCATCGTTGT-3^′) [14, 19]. Quantification was performed using one-step qRT-PCR with the HiScript II One Step qRT-PCR SYBR Green Kit (Q221-01, Vazyme). The comparative Ct method was employed to calculate relative mRNA levels, following normalization to Gapdh.

Lung homogenates or cell lysates were collected utilizing a protein extraction reagent (78510, Thermo Fisher Scientific, Waltham, MA, USA) combined with inhibitor cocktails for proteases and phosphatases (C0001 and C0004, TargetMol, Boston, MA, USA). The concentration of protein was assessed using a BCA protein assay kit. For Western blotting, protein samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% fat-free milk and incubated overnight at 4 °C with antibodies against ACTB (GB15001, Servicebio, Wuhan, China), HMGB1 (ET1601-2, HUABIO, Hangzhou, China), AMPK$\alpha$1 (ET1608-40, HUABIO, Hangzhou, China), p-AMPK$\alpha$1 (2535, CST, Danvers, MA, USA), NF-$\kappa$B p65 (4764, CST, Danvers, MA, USA), p-NF-$\kappa$B p65 (3033, CST, Danvers, MA, USA), cleaved Caspase-3 (WL02117, WanleiBio, Shenyang, China), Cleaved Caspase-9 (WL01838a, Wanleibio, Shenyang, China), Bax (WL01637, Wanleibio, Shenyang, China), Bcl-2 (WL015556, Wanleibio, Shenyang, China), and histone H3 (9715, CST, Danvers, MA, USA). The horseradish-peroxidase-conjugated secondary antibody was used to incubate the PVDF membrane, and the signals of the target proteins were assessed with Enhanced Chemiluminescence (ECL) chemiluminescence detection kits (E412, Vazyme, Nanjing, China) utilizing the ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). Signal intensity was measured using the Gel-Pro Analyzer (Thermo Fisher Scientific, Waltham, MA, USA), calculating the ratio of target proteins to the internal control protein ACTB, which was then normalized to 1.0 for the control group.

Mouse monocyte/macrophage cells RAW264.7 (CL-0190, Procell Life Science & Technology, Wuhan, China) were maintained in a 5% CO₂ atmosphere at 37 °C and cultured in DMEM supplemented with 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin. RAW264.7 cells were validated by STR profiling and tested negative for mycoplasma (D101, Vazyme, Nanjing, China). To investigate the critical role of HMGB1 in BBR-mediated NF-$\kappa$B inhibition, RAW264.7 cells were seeded in 6-well plates until they reached a confluence of 70%. After a pretreatment period of 2 hours with either 2 or 10 µM BBR, the cells were treated with 0.5 µg/mL LPS for 24 h. Subsequently, total protein and RNA were extracted to assess the levels of p-NF-$\kappa$B and NF-$\kappa$B proteins, as well as the mRNA expression of the inflammatory genes Tnf$\alpha$ and Il6. The supernatant HMGB1 level in cell culture was quantified using the ELISA method. Similarly, RAW264.7 cells were pretreated with 2 and 10 µM BBR and then simultaneously treated with 1 µg/mL recombinant mouse HMGB1 (rmHMGB1, 50913-M01H, Sino Biological, Beijing, China) protein for 24 h to evaluate NF-$\kappa$B activation and the inflammatory response.

To investigate whether AMPK activation by BBR contributes to the reduction of extracellular HMGB1 levels in LPS-stimulated RAW264.7 cells, we inhibited AMPK activation using RNA silencing or the inhibitor compound C (#T1977, TargetMol, Boston, MA, USA). Briefly, either 50 nM negative control siRNA or siRNA targeting AMPK$\alpha$ 1 (target sequence: 5^′-ATGATGTCAGATGGTGAATTT-3^′), synthesized by Sangon Biotech (Shanghai, China) [20, 21] was transiently transfected into RAW264.7 cells via Lipofectamine RNAiMAX reagent (13778, Invitrogen, Carlsbad, CA, USA) and incubated for an additional 24 h. Subsequently, 0.5 µg/mL LPS, with or without 10 µM BBR treatment, was added to the culture medium for another 24 h. The protein expressions of AMPK$\alpha$1 and p-AMPK$\alpha$1 were detected by Western blot, while the extracellular HMGB1 content was measured using the ELISA method. Additionally, RAW264.7 cells were pretreated with 10 µM BBR and BBR plus 5 µM Compound C for 2 hours, followed by induction with 0.5 µg/mL LPS for 24 hours, after which AMPK activation and extracellular HMGB1 levels were also assessed. To evaluate potential cytotoxicity in these experiments, the corresponding treatments were conducted in 96-well plates, and cell viability was assessed according to the cell counting kit-8 method with commercial kits (C0005, TargetMol, Boston, MA, USA).

The data in this article are presented as the mean $\pm$ SD or as representative figures. Statistical analyses were performed using SPSS 17.0 (IBM, Armonk, NY, USA) or GraphPad Prism 8 (GraphPad Prism Software Inc. La Jolla, CA, USA) and analyzed by Student’s t-test or One-way analysis of variance followed by Student-Newman-Keuls post hoc tests. The threshold for statistical significance was established at p 0.05 or 0.01.

To evaluate the effects of BBR (Fig. 1A) on LPS-induced ALI, mice were administered BBR twice daily (a total of 25 or 100 mg/kg/day) for 3 days before LPS induction, with an additional treatment at 6 hours post-LPS injection (Fig. 1B). 18 h following LPS stimulation, lung tissues stained with H&E from the group treated solely with LPS displayed marked infiltration of inflammatory cells, edema, increased interstitial congestion, and thickened alveolar walls. Conversely, BBR treatment effectively alleviated these pathological changes (Fig. 1C), with the lung injury score corroborating the protective effect of BBR in ALI (Fig. 1D). Inflammatory mediators play a vital role in the development of ALI triggered by LPS. Additionally, our findings indicated that BBR markedly reduced the overexpression of the inflammatory genes Tnf$\alpha$ and Il6 in the lungs of mice subjected to LPS (Fig. 1E,F). Neutrophil infiltration in lung tissues occurs during ALI, with MPO activity serving as a key indicator. As depicted in Fig. 1G, LPS significantly elevated MPO activity, whereas BBR treatment markedly reduced MPO activity, further confirming the protective effects of BBR against lung injury and inflammation. These findings suggest that BBR notably reduces the inflammatory response and alleviates LPS-induced ALI in mice.

ALI is characterized by an excessive inflammatory response that often exacerbates oxidative stress and apoptosis [22]. To investigate the effects of BBR on oxidative stress injury, we measured the activity of the antioxidant enzyme SOD and the levels of MDA in lung tissues. The results indicate that LPS significantly reduced SOD activity and increased MDA levels in lung tissues, whereas BBR treatment significantly reversed these alterations (Fig. 2A,B), emphasizing its potent antioxidant properties. To evaluate the effect of BBR on apoptosis, we conducted the TUNEL assay on lung tissues and analyzed the expression of apoptosis-related proteins. Our findings revealed a marked increase in the proportion of TUNEL-positive cells following LPS treatment, which was notably reduced by BBR (Fig. 2C,D). Additionally, BBR markedly suppressed the levels of apoptosis-related proteins, specifically cleaved caspase-3 and cleaved caspase-9, induced by LPS (Fig. 2E,F). Compared with the control group, the LPS group exhibited lower B cell lymphoma 2 (BCL2), levels and higher BCL2-Associated X protein (BAX) levels, and these abnormalities were also significantly attenuated by BBR treatment (Fig. 2E,F). The findings collectively affirm the antioxidative stress and anti-apoptotic properties of BBR in LPS-induced ALI, which correspond to its anti-inflammatory function.

Fig. 2.

BBR inhibits oxidative stress and apoptosis during LPS-induced ALI in mice. (A,B) MDA levels and SOD activity in lung tissues were assessed as oxidative stress indicators at 18 h after LPS and/or BBR treatment. (C,D) Representative staining (200$\times$, scale bar = 50 µm) and positive-cell quantification of the TUNEL assay in mouse lung tissues. (E,F) Western blot analysis and quantification of apoptosis-associated proteins in mouse lung tissues. The results are shown as means $\pm$ standard deviation (n = 6 for every group) or as representative figures. One-way analysis of variance (ANOVA) was conducted. *p 0.05 and **p 0.01 vs. the control group. ^#p 0.05 and ^#⁢#p 0.01 vs. the LPS group. MDA, malondialdehyde; SOD, superoxide dismutase; TUNEL, transferase dUTP nick end labeling; BCL2, B cell lymphoma 2; BAX, BCL2-Associated X protein.

BBR has been widely reported to activate AMPK, which in turn contributes to the maintenance of oxidant-antioxidant balance in various inflammatory conditions [12, 23]. Conversely, excessive HMGB1 release triggers inflammatory responses and subsequent apoptosis in sepsis [4, 5]. Consequently, we chose to examine the roles of AMPK and HMGB1 in mediating the protective effects of BBR against sepsis-induced inflammatory activation, a critical mechanism underpinning ALI. The results indicate that LPS significantly reduced phosphorylated AMPK (p-AMPK) levels and increased total HMGB1 protein levels, resulting in NF-$\kappa$B activation in mouse lung tissues (Fig. 3A,B). In contrast, BBR treatment partially reversed these effects without affecting the total AMPK levels (Fig. 3A,B). The regulatory effect of BBR on AMPK aligns with previous reports on its effects in other inflammation-related models [23, 24]. Furthermore, our results demonstrated that BBR inhibits the nuclear translocation of HMGB1, as evidenced by increased nuclear and decreased cytoplasmic HMGB1 protein levels compared with the LPS-induced ALI model (Fig. 3C,D). Immunofluorescence staining of HMGB1 in mouse lungs also showed increased total and cytoplasmic HMGB1 levels following LPS induction, which were mitigated by BBR treatment (Fig. 3E). The function of HMGB1 depends on its release into circulating fluids, where it acts as a DAMP by binding to cell surface receptors, ultimately triggering the inflammatory response. Consequently, we measured the serum levels of HMGB1 in the ALI model induced by LPS. As anticipated, LPS resulted in a marked increase in serum concentrations of HMGB1, while treatment with BBR significantly lowered these levels in a dose-dependent way (Fig. 3F). Consequently, BBR exerts inhibitory effects on HMGB1-mediated NF-$\kappa$B activation in LPS-induced ALI, and this mechanism may be related to the activation of AMPK during this process.

Fig. 3.

BBR activates AMPK and suppresses HMGB1-mediated NF-$\kappa$B activation in LPS-induced ALI in mice. (A,B) Western blot analysis and quantification of p-AMPK, AMPK, p-NF-$\kappa$B, NF-$\kappa$B, and HMGB1 in mouse lung tissue. (C,D) Western blot analysis and quantification of HMGB1 in the nuclei and cytoplasm. (E) Immunofluorescence analysis of HMGB1 protein expression in mouse lung tissue (scale bar = 25 µm; enlarge image scale bar = 10 µm). (F) ELISA analysis of serum HMGB1 levels. Data are presented as the mean $\pm$ SD (n = 6 for every group) or the representative figures. One-way analysis of variance (ANOVA), *p 0.05 and **p 0.01 vs. the control group. ^#p 0.05 and ^#⁢#p 0.01 vs. the LPS group. AMPK, adenosine-monophosphate-activated protein kinase; HMGB1, high mobility group box-1; NF-$\kappa$B, nuclear factor Kappa B; ELISA, enzyme-linked immunosorbent assay.

The primary mechanism underlying sepsis is immune dysfunction, with macrophages playing a pivotal role [25]. In this study, we utilized the mouse macrophage cell line RAW264.7 to elucidate the role of HMGB1 in BBR-mediated inhibition of NF-$\kappa$B activation during sepsis. After 24 hours of treatment with LPS and BBR, LPS significantly induced the activation of NF-$\kappa$B (Fig. 4A), increased the extracellular HMGB1 level (Fig. 4B), and was accompanied by excessive expression of Tnf$\alpha$ (Fig. 4C) and Il6 (Fig. 4D) mRNA. In contrast, BBR reversed the increased extracellular HMGB1 level and inflammatory activations induced by LPS (Fig. 4A–D). HMGB1 typically acts as a potent inflammation inducer by binding to multiple cell-surface receptors [26] or by binding with LPS to amplify its pro-inflammatory activity [27, 28]. To further verify the role of HMGB1 in the protective effect of BBR during sepsis, we evaluated the anti-inflammatory effect of BBR in HMGB1-stimulated RAW264.7 cells. The results indicated that HMGB1 significantly promoted NF-$\kappa$B activation and upregulated the inflammatory genes Tnf$\alpha$ and Il6 expression, whereas BBR partially mitigated this effect (Fig. 4E–G). Therefore, the evidence that BBR decreases extracellular HMGB1 in LPS-induced RAW264.7 cells and inhibits HMGB1-induced NF-$\kappa$B activation suggests that BBR alleviates sepsis-related inflammatory injury, at least partially, through decreasing extracellular HMGB1, which is consistent with the results derived from animal experiments (Fig. 3).

Fig. 4.

BBR decreases extracellular HMGB1 in LPS-induced RAW264.7 cells and inhibits HMGB1-stimulated NF-$\kappa$B activation. (A,B) The p-NF-$\kappa$B and NF-$\kappa$B expression (A), and the extracellular HMGB1 level (B) after treatment with BBR in LPS-induced RAW264.7 cells. (C,D,F,G) The mRNA expression of inflammatory gene Tnf$\alpha$ and Il6 detected by qRT-PCR. (E) The p-NF-$\kappa$B and NF-$\kappa$B expression after treatment by rmHMGB1 and/or BBR in RAW264.7 cells. Data are presented as mean $\pm$ SD (n = 3). One-way analysis of variance (ANOVA), *p 0.05, **p 0.01 vs. the control group; ^#p 0.05 and ^#⁢#p 0.01 vs. the LPS or rmHMGB1 treated group.

As extracellular HMGB1 serves as a final effector in the inflammatory activation process, we further assessed whether BBR-induced decreases in extracellular HMGB1 levels are mediated through AMPK activation in LPS-stimulated RAW264.7 cells. For this, we utilized AMPK$\alpha$1-specific small interfering RNA (siRNA) and the pharmacological p-AMPK inhibitor compound C to knock down or inhibit AMPK activation. Results indicate that, under non-cytotoxic conditions in RAW264.7 cells (Fig. 5A), AMPK protein expression was inhibited after transfection with siAMPK, leading to subsequently lower p-AMPK levels (Fig. 5B). Compared with the control group, LPS reduced p-AMPK levels, whereas BBR enhanced its level. However, by knockdown of AMPK$\alpha$1, the p-AMPK level remains lower even in the presence of BBR (Fig. 5B). Correspondingly, the inhibition of supernatant HMGB1 level by BBR in LPS-induced RAW264.7 cells was reversed by AMPK silencing (Fig. 5C). Similarly, at a non-cytotoxic concentration (Fig. 5D), Compound C also reversed BBR’s effects on p-AMPK activation and extracellular HMGB1 levels (Fig. 5E,F). The findings indicate that the reduction in extracellular HMGB1 levels caused by BBR is at least partially facilitated through the enhancement of p-AMPK level in RAW264.7 cells stimulated by LPS.

Fig. 5.

Silencing the activation of AMPK reverses BBR-reduced extracellular HMGB1 level in LPS-stimulated RAW264.7 cells. (A,D) Cytotoxicity detection in RAW264.7 cells after 24 h or 48 h treatment with LPS, BBR, siAMPK, or compound C. (B,E) Western blot analysis of p-AMPK and AMPK protein levels. (C,F) HMGB1 levels in cell culture supernatants detected via an ELISA method. Data are presented as the mean $\pm$ SD (n = 3). Student’s t-test and One-way analysis of variance (ANOVA), *p 0.05, **p 0.01 vs. the corresponding control group.