Plant material was collected on November 29, 2019, from Sanfang Reservoir (Longxu, Cangwu, Guangxi, China). The voucher specimen (collection no. 450421191129020LY) was deposited in the Herbarium of Guangxi Institute of Botany (IBK) under voucher number IBK00430105. The specimen was identified by Yusong Huang on October 24, 2020, as Laggera alata (D. Don) Sch. Bip. ex Oliv, a species belonging to the genus Laggera of the family Asteraceae. The whole plant was used for extraction. The plant name was verified against The Plant List (http://www.theplantlist.org). TPLA was extracted according to previously published protocols (11). Dried Laggera alata herb (10 kg) was ground into coarse powder and extracted twice by reflux with 95% ethanol at ~84°C, using solvent-to-material ratios of 12:1 and 8:1 (L/kg) for 1.5 and 1 h, respectively. The combined extracts were filtered, concentrated under reduced pressure using a rotary evaporator, and subsequently dried in a 65°C water bath. The yield of TPLA (total phenolics: 13.52%) was 2.156 kg, with an extraction efficiency of 21.56%. The extract was sealed and stored in a dry environment. For experimental use, an accurately weighed amount of TPLA powder was dissolved in distilled water and diluted to the required concentration immediately before use.

The mouse macrophage cell line RAW264.7 (cat. no. CL-0190; Wuhan Pricella Biotechnology Co., Ltd.) was cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM; cat. no. E600003; Sangon Biotech Co., Ltd.) supplemented with 10% fetal bovine serum (FBS; Guangzhou Yunoer Biotechnology Co., Ltd.) and 1% penicillin-streptomycin (cat. no. P1400; Beijing Solarbio Science & Technology Co., Ltd.) at 37°C in a 5% CO₂ incubator. Cells were tested and confirmed to be mycoplasma-free and authenticated by short tandem repeat (STR) profiling (Wuhan Pricella Biotechnology Co., Ltd.). Cells at 80–90% confluence were used for experiments. To establish an in vitro LPS model group, cells were treated with 1 µg/ml lipopolysaccharide (LPS; cat. no. L2630; MilliporeSigma) for 24 h (22), followed by treatment with TPLA at low (10 µg/ml), medium (50 µg/ml) and high (100 µg/ml) concentrations or dexamethasone (DEX; 10 µM; cat. no. D4902; MilliporeSigma). The control group received no treatment. For mitophagy inhibition experiments, cells were additionally assigned to the LPS, LPS + TPLA-high, and LPS + TPLA-high + Mdivi-1 groups. Mdivi-1 (10 µM; cat. no. M0199; Sigma-Aldrich; Merck KGaA) was added 1 h before TPLA-high treatment. Mitophagy-related protein expression was subsequently evaluated by western blotting. In the Mdivi-1 inhibition experiment, macrophage polarization was further assessed by flow cytometry using CD80/CD86 as M1-associated markers and CD206/CD163 as M2-associated markers.

Levels of IL-1β (cat. no. MLB00C-1; R&D Systems, Inc.), IL-6 (cat. no. BMS603-2), TNF-α (cat. no. BMS607-3TEN), IL-1α (cat. no. 900-K82K), IL-4 (cat. no. 88-7044-88) and IL-10 (cat. no. 88-7105-88; all from Thermo Fisher Scientific, Inc.) were quantified using ELISA kits. Samples were added to 96-well plates pre-coated with capture antibodies and incubated for 2 h at 37°C. After washing, biotin-conjugated detection antibodies were added and incubated for 1 h at 37°C, after which the substrate solution (cat. no. A8043; Thermo Fisher Scientific, Inc.) was added for color development. Absorbance was measured at 450 nm using a microplate reader (Varioskan^™ LUX; Thermo Fisher Scientific, Inc.), and concentrations were calculated from standard curves.

MMP was assessed using JC-1 dye (cat. no. C2006; Beyotime Institute of Biotechnology). After 24 h of treatment, cells were washed with PBS and incubated with 5 µM JC-1 at 37°C for 30 min in the dark. After staining, cells were washed with JC-1 buffer and immediately analyzed by flow cytometry (Accuri C6; BD Biosciences). Fluorescence signals were collected in the FL1-H (green, JC-1 monomers, 530±15 nm) and FL2-H (red, JC-1 aggregates, 585±20 nm) channels under logarithmic scaling. At least 10,000 events were recorded per sample. The red/green fluorescence ratio was calculated to represent MMP, where red fluorescence indicates polarized mitochondria and green fluorescence indicates depolarization. Data were analyzed using FlowJo software (v10.8; BD Biosciences).

RAW264.7 cells were seeded at 6×10⁴ cell/ml (200 µl per well) in 96-well plates and treated according to group allocation. After 24 h of treatment, 20 µl of CCK-8 reagent (cat. no. CK04; Dojindo Laboratories, Inc.) was added to each well and incubated for 2–4 h at 37°C. Absorbance was then measured at 450 nm using a microplate reader (Synergy H1; BioTek; Agilent Technologies, Inc.).

Apoptosis was evaluated using the Annexin V-FITC/PI Apoptosis Detection Kit (cat. no. A211-01; Vazyme Biotech Co., Ltd.) following the manufacturer's instructions. After washing with PBS (cat. no. E600003; Sangon Biotech Co., Ltd.), cells were digested with 0.25% trypsin (cat. no. C0201; Beyotime Institute of Biotechnology) without EDTA, resuspended in 1X binding buffer, and incubated with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) for 15 min at room temperature in the dark. Samples were immediately analyzed by flow cytometry (Accuri C6; BD Biosciences). Fluorescence was detected using the FL1-H (FITC, 530±15 nm) and FL3-H (PI, >670 nm) channels under logarithmic scaling. Compensation was applied using single-stained controls. At least 10,000 events were acquired per sample, and debris and doublets were excluded by forward- and side-scatter gating. Early and late apoptotic cell percentages were calculated using FlowJo software (v10.8; BD Biosciences).

RAW264.7 cells were lysed in RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) after washing with PBS, and lysates were centrifuged at 12,000 × g for 15 min at 4°C to collect supernatants. Protein concentration was determined using a BCA protein assay kit (cat. no. P0012; Beyotime Biotechnology) according to the manufacturer's instructions. Equal protein amounts (30 µg) were resolved on 10% gels using SDS-PAGE together with a pre-stained protein marker (cat. no. G2086; Wuhan Servicebio Technology Co., Ltd.), followed by transfer onto PVDF membranes (MilliporeSigma). Membranes were blocked with 5% non-fat milk in TBST containing 0.1% Tween-20 for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C: LC3B rabbit monoclonal antibody (1:2,000; cat. no. ab192890; Abcam), p62 rabbit polyclonal antibody (1:10,000; cat. no. ab109012; Abcam), Parkin rabbit polyclonal antibody (1:1,000; cat. no. GB11596; Wuhan Servicebio Technology Co., Ltd.), PINK1 rabbit polyclonal antibody (1:500; cat. no. GB114934; Wuhan Servicebio Technology Co., Ltd.) and GAPDH rabbit polyclonal antibody (1:2,500; cat. no. ab9485; Abcam). After incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:10,000; cat. no. ab6721; Abcam), chemiluminescent signals were detected on X-ray films using ECL substrate (Thermo Fisher Scientific, Inc.). Band intensities were analyzed with ImageJ software (v1.80; National Institutes of Health).

Total RNA was extracted using RNAiso Plus reagent (cat. no. 9109; Takara Bio, Inc.), and RNA purity and concentration were determined using a NanoDrop spectrophotometer (Micro Drop; Bio-DL). cDNA synthesis was performed using PrimeScript RT Reagent Kit (cat. no. RR037A; Takara Bio, Inc.) under the following conditions: 37°C for 15 min, 85°C for 5 sec. qPCR was performed using PowerTrack^™ SYBR Green Master Mix (Thermo Fisher Scientific, Inc.) and specific primers as follows: inducible nitric oxide synthase (iNOS) forward, 5′-TCCTGGACATTACGACCCCT-3′ and reverse, 5′-AGGCCTCCAATCTCTGCCTA-3′; IL-12 forward, 5′-CTCCTGTGGGAGAAGCAGAC-3′ and reverse, 5′-CAGATAGCCCATCACCCTGT-3′; arginase 1 (Arg1) forward, 5′-AGATTATCGGAGCGCCTTTCT-3′ and reverse, 5′-CGTGGTCTCTCACGTCATACT-3′; IL-10 forward, 5′-CCAAGCCTTATCGGAAATGA-3′ and reverse, 5′-TTTTCACAGGGGAGAAATCG-3′; and GAPDH forward, 5′-GTGTTCCTACCCCCAATGTGT-3′ and reverse, 5′-ATTGTCATACCAGGAAATGAGCTT-3′. The PCR cycling conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. A melting curve analysis was performed at the end. Relative gene expression was calculated using the 2^−ΔΔCq method (23). GAPDH was used as the internal reference gene.

IF staining of CD80, CD86, CD206 and CD163 was performed to assess changes in macrophage polarization. After 24 h of cell culture, cells were washed twice with phosphate-buffered saline and fixed with 4% paraformaldehyde (cat. no. A0001; Sangon Biotech Co., Ltd.) for 20 min at room temperature. Cells were then permeabilized with 0.1% Triton X-100 (cat. no. A600413; Sangon Biotech Co., Ltd.) for 10 min, followed by blocking with 5% normal goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) for 1 h at room temperature. Subsequently, cells were incubated overnight at 4°C with the following primary antibodies: CD80 (1:200; cat. no. 66406-1-Ig), CD86 (1:200; cat. no. 13395-1-AP), CD206 (1:200; cat. no. 83485-1-RR) and CD163 (1:200; cat. no. 16646-1-AP; all from Proteintech Group, Inc.). The next day, cells were washed three times with PBS and incubated for 1 h at room temperature with Alexa Fluor 488-conjugated secondary antibody (1:1,000; cat. no. A11034) and Alexa Fluor 594-conjugated secondary antibody (1:1,000; cat. no. A11032; both from Thermo Fisher Scientific, Inc.). Nuclei were stained with 1 µg/ml DAPI (cat. no. C1002; Beyotime Institute of Biotechnology) for 10 min, and images were captured using a fluorescence microscope (IX71; Olympus Corporation).

Macrophage polarization was further quantified by flow cytometry. After the indicated treatments, RAW264.7 cells were collected, washed with PBS, and incubated with fluorochrome-conjugated anti-mouse antibodies against CD80-FITC (cat. no. 104705), CD86-PE (cat. no. 105007), CD206-PerCP/Cyanine5.5 (cat. no. 141715) and CD163-APC (cat. no. 155305; all from BioLegend, Inc.) according to the manufacturers' instructions. After incubation in the dark at 4°C for 30 min, cells were washed twice with PBS and analyzed using a BD Accuri C6 flow cytometer (BD Biosciences). Debris and doublets were excluded by forward- and side-scatter gating, and at least 10,000 events were acquired for each sample. The percentages of CD80/CD86-positive and CD206/CD163-positive cells were analyzed using FlowJo software (v10.8; BD Biosciences).

Male C57BL/6 mice (7–8 weeks old, 20–25 g) were obtained from Hunan Silaike Jingda Laboratory Animal Co., Ltd. [production license No. SCXK (Xiang) 2019-0004]. Only male mice were used in this initial in vivo experiment to reduce variability associated with sex-dependent immune and hormonal differences and because previous LPS/endotoxemia studies have reported sex-dependent inflammatory and organ-injury responses, with male mice often showing greater susceptibility or more pronounced injury than female mice (24). All animal experiments were conducted at the Guangxi University of Chinese Medicine, which holds the laboratory animal use license No. SYXK [Gui] 2019-0001. All procedures were approved by the Animal Ethics Committee of Guangxi University of Chinese Medicine (approval no. DW20240923-01; Nanning, China). Animals were housed in a controlled environment (25±2°C, 65% humidity, 12/12-h light/dark cycle) with free access to standard chow and purified water. All animal procedures complied with the ARRIVE guidelines (25) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Mice were randomly assigned to the following groups: control, LPS model, TPLA (low, medium and high dose) and DEX (n=6 per group). A total of 36 mice were used in the in vivo experiment, with six mice allocated to each group. All 36 animals completed the experimental protocol and were included in the final analysis. No animals were excluded, no animals were found dead, and no premature euthanasia was required during the study. At the scheduled endpoint, all 36 mice were euthanized for sample collection. TPLA was administered orally at 50, 100, or 200 mg/kg once daily for 27 consecutive days as a pretreatment regimen (11). The DEX group received DEX at 1 mg/kg (26). On the last two days of the experiment, mice were challenged with intraperitoneal LPS (20 mg/kg), twice daily for two consecutive days, to induce acute systemic inflammatory injury (27). Mice exhibiting lethargy, piloerection and labored breathing within 24 h were considered successfully modeled (28). Core body temperature was measured 6 h after the final LPS injection. The total duration of the in vivo experiment was 27 days, and mice were euthanized 6 h after the final LPS injection on day 27. At the scheduled endpoint, mice were deeply anesthetized and euthanized by intraperitoneal injection of 1% sodium pentobarbital solution (10 mg/ml; Sigma-Aldrich, P3761) at 150 mg/kg body weight (29,30), followed by cervical dislocation as a secondary physical method. Death was verified by the absence of spontaneous respiration and heartbeat, loss of corneal and pedal withdrawal reflexes, and absence of response to toe pinch before tissue collection. Humane endpoints were defined before the experiment to minimize animal suffering. Animals were monitored at least twice daily for signs of distress, including persistent lethargy, severe weight loss (>20% of body weight), hypothermia, hunched posture, tremors, unresponsiveness, or labored breathing. Mice showing any of these signs would have been immediately euthanized using the same sodium pentobarbital protocol.

Lung and liver tissues were collected and fixed in 10% neutral-buffered formalin (cat. no. HT501128; MilliporeSigma) for 24 h at room temperature. After dehydration, clearing and paraffin embedding, 4 µm-thick sections were cut using a microtome (cat. no. RM2255; Leica Biosystems). Sections were deparaffinized using xylene (X5; MilliporeSigma) and rehydrated through graded ethanol. Hematoxylin (cat. no. GHS132) and eosin (cat. no. E4003; both from MilliporeSigma) were used for nuclear and cytoplasmic staining, respectively. After dehydration and clearing, slides were mounted with neutral resin (cat. no. G8110; MilliporeSigma) and examined under a light microscope (BX53; Olympus Corporation). Semi-quantitative histopathological scoring was performed to evaluate lung and liver injury. For each animal, representative non-overlapping fields were observed, and the overall pathological injury was scored by two independent investigators blinded to the experimental groups. Lung injury was assessed according to alveolar structural destruction, alveolar septal thickening, inflammatory cell infiltration, congestion and alveolar collapse. Liver injury was assessed according to hepatocellular swelling, cytoplasmic rarefaction, inflammatory cell infiltration, nuclear pyknosis and disruption of hepatic cord architecture. The severity of pathological injury was graded using a 0–4 scale as follows: 0, no obvious injury; 1, mild injury; 2, moderate injury; 3, severe injury; and 4, very severe injury.

Statistical analyses and graph generation were performed using GraphPad Prism 10.0 (GraphPad Software; Dotmatics). Data are presented as the mean ± standard deviation. Semi-quantitative histopathological injury scores are presented as median with interquartile range. Data normality was assessed using the Shapiro-Wilk test. For normally distributed data, independent-samples t-tests were used for two-group comparisons, and one-way ANOVA with Tukey's post hoc test was used for multiple group comparisons. For non-normally distributed data, the Wilcoxon rank-sum test and Kruskal-Wallis test with Dunn's multiple comparisons were applied. P<0.05 was considered to indicate a statistically significant difference.

Flow cytometric analysis revealed a significant increase in both early and late apoptotic rates in macrophages treated with LPS, whereas treatment with low, medium and high doses of TPLA reduced apoptosis in a dose-dependent manner, with the high-dose group showing an effect similar to that of DEX (Fig. 1A). Quantitative analysis confirmed that the apoptotic rate in the LPS group increased to ~20%, while treatment with TPLA reduced this rate to ~15, 13 and 8%, respectively, and these reductions were statistically significant compared with the LPS group (Fig. 1B). Cell viability assessed by CCK-8 assay showed a significant reduction following LPS stimulation, which was restored in a dose-dependent manner by TPLA treatment to levels approaching those of the control group (Fig. 1C). ELISA results demonstrated that LPS significantly increased the levels of IL-1β, IL-6 and TNF-α, while TPLA treatment significantly suppressed the secretion of these cytokines, especially in the high-dose group, which exhibited cytokine levels comparable to the control (Fig. 1D).

Western blot analysis indicated that LPS treatment significantly reduced LC3-II expression and decreased the LC3-II/LC3-I ratio, suggesting suppression of autophagy-related activity. This impairment was reversed by medium and high doses of TPLA, which restored the LC3-II/LC3-I ratio to levels comparable to those in the DEX group. In addition, LPS elevated expression of the autophagy substrate p62 while downregulating the mitophagy-related proteins Parkin and PINK1. TPLA treatment dose-dependently reduced p62 expression and significantly increased Parkin and PINK1 levels, consistent with activation of PINK1/Parkin-associated mitophagy-related signaling (Fig. 2A). JC-1 staining revealed that LPS reduced MMP, as evidenced by increased green monomer fluorescence and decreased red aggregate fluorescence. This mitochondrial dysfunction was reversed by TPLA treatment, particularly at higher doses (Fig. 2B).

RT-qPCR analysis showed that LPS stimulation significantly increased M1-associated genes iNOS and IL-12, while decreasing M2-associated genes Arg1 and IL-10. TPLA treatment dose-dependently reversed this trend, decreasing iNOS and IL-12 expression and enhancing Arg1 and IL-10 expression (Fig. 3A). ELISA results demonstrated that LPS significantly elevated levels of M1-associated cytokines IL-1α and IL-1β and suppressed M2-associated cytokines IL-4 and IL-10. TPLA treatment decreased M1 cytokine levels and increased M2 cytokine levels in a dose-dependent manner (Fig. 3B). IF analysis further confirmed these results. CD80/CD86 expression (M1 markers) was increased in the LPS group, while CD206/CD163 expression (M2 markers) was reduced. TPLA treatment reversed these changes, with high-dose TPLA showing effects comparable to DEX (Fig. 3C). Flow cytometric analysis further confirmed the regulatory effect of TPLA on macrophage polarization. LPS stimulation significantly increased the proportion of CD80/CD86-positive M1 macrophages compared with the control group, whereas TPLA treatment reduced the CD80/CD86-positive population in a dose-dependent manner. In particular, high-dose TPLA decreased the proportion of CD80/CD86-positive macrophages to a level close to that observed in the DEX group (Fig. 4A). Conversely, LPS significantly decreased the proportion of CD206/CD163-positive M2 macrophages, while TPLA treatment dose-dependently restored the CD206/CD163-positive population. The effect of high-dose TPLA was comparable to that of DEX (Fig. 4B).

LPS stimulation significantly increased the proportion of CD80/CD86-positive M1 macrophages compared with the control group, whereas high-dose TPLA treatment reduced the CD80/CD86-positive population (Fig. 5A). Conversely, LPS significantly decreased the proportion of CD206/CD163-positive M2 macrophages, while high-dose TPLA treatment restored the CD206/CD163-positive population (Fig. 5B). However, co-treatment with Mdivi-1 partially reversed the effects of high-dose TPLA. To further examine whether the mitophagy-related effects of TPLA were associated with the PINK1/Parkin pathway, Mdivi-1 was used to inhibit mitochondrial fission- and mitophagy-related responses. Western blot analysis demonstrated that high-dose TPLA increased the expression levels of Parkin and PINK1, enhanced the LC3-II/LC3-I ratio, and reduced p62 accumulation compared with the LPS group. However, co-treatment with Mdivi-1 partially reversed these changes, as evidenced by decreased Parkin and PINK1 expression, reduced LC3-II/LC3-I ratio, and increased p62 expression (Fig. 5C). These results indicate that TPLA-induced activation of the PINK1/Parkin-associated mitophagy pathway was partially attenuated by Mdivi-1.

H&E staining of lung tissues revealed that the control group exhibited normal alveolar architecture with thin alveolar septa and minimal inflammatory cell infiltration. By contrast, the LPS group displayed marked pathological injury, characterized by alveolar collapse, interstitial thickening, congestion, and marked inflammatory cell infiltration. Semi-quantitative histopathological scoring further confirmed that the lung injury score was markedly increased in the LPS group compared with the control group. TPLA treatment reduced lung pathological injury in a dose-dependent manner, with the medium- and high-dose groups showing clear attenuation of alveolar structural disruption and inflammatory infiltration. The effect of high-dose TPLA was similar to that observed in the DEX group (Fig. 6A and B). In liver tissues, the control group demonstrated orderly hepatic cords, clear hepatic sinusoidal structures and intact hepatocyte morphology. The LPS group exhibited pronounced hepatocellular swelling, cytoplasmic rarefaction, inflammatory infiltration and nuclear pyknosis. Semi-quantitative scoring showed that LPS markedly increased the liver injury score, whereas TPLA treatment reduced hepatic pathological damage in a dose-dependent manner. In particular, medium- and high-dose TPLA significantly improved hepatocellular architecture and reduced inflammatory changes, with high-dose TPLA showing an effect comparable to DEX (Fig. 6C and D).

LPS administration markedly reduced rectal temperature in mice by ~2°C compared with the control group. Medium- and high-dose TPLA treatment significantly restored body temperature to levels comparable with the control and DEX groups, while the low-dose group showed no significant improvement (Fig. 7A). ELISA analysis revealed that LPS markedly increased serum levels of IL-1β, IL-6 and TNF-α, reaching ~400, 1,800 and 800 pg/ml, respectively. TPLA treatment reduced serum levels of these inflammatory cytokines in a dose-dependent manner, with the high-dose group showing the most pronounced effect, comparable to that of DEX (Fig. 7B).