Despite much advanced research, even with a decreasing death rate, acute lung injury (ALI) continues to be a major cause of morbidity and death for severely ill patients (Mowery et al., 2020). ALI or its extreme description, severe acute respiratory distress syndrome (ARDS), is a devastating lung disease that has an elevated rate of complications throughout the world (Mowery et al., 2020; Yu et al., 2021; Butt et al., 2016). ALI/ARDS is characterized by lung edema and hypercapnia caused by elevated alveolar-capillary membrane leakage (EACML) and the consequent reduction of arterial oxygenation (Ragaller and Richter, 2010; Mokra and Kosutova, 2015; An et al., 2019). The overall fatality rate of ALI is higher in the United States (Butt et al., 2016), at more than 55 per 100,000 cases annually, than in Australia, Europe, and other advanced nations, which have more than 27 per 100,000 cases (Goyal et al., 2012). ALI and ARDS account for almost 10% of all intensive care unit (ICU) cases and four percent of all hospital admissions. Two major causes of ALI are direct and indirect injuries. Microbial infections, lung contusions, pulmonary embolism, and near drowning are direct causes of ALI/ARDS, whereas sepsis, pancreatitis, trauma, and drug overdoses are categorized as indirect causes (Hussain et al., 2021). Understanding the pathophysiology of ALI has led to the identification of various biochemical indicators linked to non-satisfactory clinical outcomes. Events that either lead or predispose to ALI/ARDS often involve three interrelated pathophysiologic conditions: (i) increased microvascular permeability, (ii) alveolar instability reducing lung compliance, and (iii) overactive and disrupted lung inflammatory responses like interleukins (IL-1, IL-6), chemokines, tumor necrosis factor (TNF-α), oxidants (superoxide ions, hydrogen peroxide, nitric oxide), and proteases, which escalate inflammation by destroying alveolar architecture (Matuschak and Lechner, 2010).

Randomized clinical trials have found no particular drugs to be beneficial in treating ALI/ARDS (Calfee and Matthay, 2007). Although there is a paucity of data on outcomes, supportive care comprising mechanical respiration, positive end-expiratory pressure (PEEP), smaller tidal volumes, a conservative fluid approach, and lower inhalation pressures have slightly improved survival rates (Halter et al., 2007). Several intriguing novel treatments are being studied in current or planned clinical trials, such as nitric oxide, corticosteroids, heparin, surfactants, ibuprofen, azithromycin, activated protein C, β₂-agonists, lysofylline, phosphodiesterase inhibitors, neuromuscular blockers, aspirin, anti-TNF biologics, and antioxidants ((Mullen et al., 1993; Teke et al., 2008; Tanaka et al., 2008; Husari et al., 2014; Zhang et al., 2014; Kumari et al., 2015; Mokra and Kosutova, 2015; Kosutova et al., 2016; Meng, 2017; Dixon et al., 2021; Muhammad et al., 2023;; Rajasekar et al., 2024). Studies suggest that albumin and furosemide administration may only provide symptomatic relief in hypoproteinemic individuals suffering lung injury (Calfee and Matthay, 2007). However, no treatments have been demonstrated that enhance patient outcomes in therapeutic cost, quality of life, extent of morbidity, and mortality (Villar et al., 2020). There is thus great demand to develop new and efficient medications for the management of ALI/ARDS, considering its prevalence and poor prognosis (Alamgeer et al., 2018).

The intra-tracheal instillation of endotoxin (lipopolysaccharide) is extensively used to trigger ALI in test animals (Rojas et al., 2005). The pulmonary parenchymal cells are impaired by the discharge of reactive species, activated macrophages in the lung, and transmigrated neutrophils in the alveolar and interstitial sections. The endpoints are microvascular injury and diffuse alveolar damage (DAD) through hemorrhage within the lungs, swelling, neutrophil infiltration, deposition of fibrin, and complementary system activation (Triggianese et al., 2023; Shi et al., 2023). Pro-inflammatory markers were also released, including interleukins-1β, 6, and TNF-α, which enhance the level of transcription factor NF-ĸβ. ALI/ARDS has been induced using LPS, a fundamental element of Gram-negative bacteria. Animal models caused by LPS offer valuable insights into the mechanisms behind many diseases and can be used to identify new biomarkers and therapeutic targets (Chen et al., 2010).

Traditional medicinal plants contribute to combating a wide range of respiratory illnesses (Cock and Van Vuuren, 2020; Teka and Maryo, 2023). We can evaluate all traditional remedies that contain bioactive metabolites to create modern pharmaceuticals. Bioactive compounds such as polyphenols and flavonoids have anti-inflammatory, anti-oxidant, antibacterial, and anti-proliferative activities. Over 50% of all current drugs originate from natural sources, so researchers concentrate on drug discovery from botanical preparations (Singh et al., 2020). Considering the results of different biomedical and pharmacological studies, a variety of metabolites like flavonoids (Peng et al., 2019; Shokry et al., 2022), phenolics (Xing et al., 2019), alkaloids (Renushe et al., 2022), saponins (Lin et al., 2016), and terpenoids (Wang et al., 2022) have been suggested for the treatment of ALI (Georgiev et al., 2022). These botanical drugs have diverse anti-inflammatory and anti-oxidative potential and are a step toward novel drug discovery in pulmonology. Numerous studies on different plants have been shown to significantly reduce ALI manifestations, such as a decline in neutrophils infiltration and toll-like receptor pathways (TLR₄, NF-ĸβ, COX-2) and, consequently, TNF-α, Interleukins (IL-1, -6, -8, -10) and chemokines by reducing oxidative stress in experimental animals (Aboushanab et al., 2021; Liou et al., 2017; Baradaran Rahimi et al., 2019; Shokry et al., 2022; Feng et al., 2019; Tian et al., 2019a; Erdenechimeg et al., 2017; Feng et al., 2024; Tian et al., 2019b; Xing et al., 2019).

The botanical description of Polygonum bistorta L. is of a perennial herbaceous plant belonging to the family Polygonaceae. Its other scientific names are Bistorta officinalis Delarbre, Polygonon bistortum (L.), and Persicaria bistorta (L.). Its non-scientific names include adderwort, anjabar, asar-rai, bijband, bandak, ban-natia, dragon wort, hozar, meadow bistort, quanshen, snakeweed, stor-ormrot, wiesen-knoterich, serpentaire, schlangen-wiesenknoterich, and rhizomata bistortae. The plant is native to Pakistan, Turkey, China, and Afghanistan, as well as North America and Europe (Demiray et al., 2009; Klimczak et al., 2017). The botanical drug contains chlorogenic acid, gallic acid, catechin, procyanidin, caffeic acid, quercetin, luteolin, apigenin, and kaempferol. Among the identified physiologically active substances in the extract are various triterpenoids, phenolic acids, flavan-3-ols, flavonols, tannins, and fatty acids (Mehar and Tabarak, 2013; Klimczak et al., 2017). Their antimicrobial, antioxidant, hemostatic, immunostimulatory, anti-inflammatory, hepatoprotective, gastroprotective, and anticancer properties are all attributed to these metabolites (Liu et al., 2006). The roots of P. bistorta contain polysaccharides, polyphenols (anthraquinones, stilbenes, naphthalene), flavonoids (flavonols, 2, 5′,6′trihydroxy-4,2′-dimethoxy flavones, and 2, 3′,4′,4,6-pentahydroxy flavones, kaempferol, myricetin, arborinone, adeaninone, baicalin, arborinol, luteolin, rhamnetin), triterpenoids (24(E)-ethylidenecycloartanone and 24(E)-ethylidenecycloartan-3-ol), steroids, tannins (1-galloyl-β-d-glucose, castalagin, proanthocyanidin), catechins (epicatechins, protocatechuic, and gallocatechin), and glycosides (Manoharan et al., 2005; Orbán-Gyapai et al., 2015; Pillai et al., 2019; Pirvu et al., 2017). Another novel metabolite, 3-methyl-gallic acid 4-O-β-d-(6′-O-3″-methyl-galloyl)-glucopyranoside (isoquercetin), was elucidated from bistorts by Liu et al. (2006). Research has reported the presence of quercimeritrin, avicularin, rutin, hyperine, mequelianin, isoquercetin, spirozide, cyanidine, anthocyanidine, delphinidine, myristic, palmitic acid, furfural, linoleic acid, and oleic acid in bistorts (Sun et al., 2007; Intisar et al., 2013; Dhiman et al., 2023).

In folklore, phytomedicine (Javid et al., 2024) is enriched with a high content of polysaccharides and polyphenolics (Klimczak et al., 2017; Pirvu et al., 2017). Aerial parts of this plant are used in dock and savory puddings (Klimczak et al., 2017). According to British, European, Unani, and Chinese pharmacopoeias, the roots and rhizomes are consumed as fluid extract and dried in whole or fragmented forms. Traditionally, this medicinal plant is used to treat cough, bronchitis, severe respiratory infections (Javid et al., 2024), for other inflammatory diseases like acute gastritis, hemorrhoidal bleeding, and nasal bleeding (Munir et al., 2014; Karuppiah Pillai, 2021), as an expectorant, emetic, febrifugal (Mehar and Tabarak, 2013), and as an antidote for measles, smallpox, and snake bite. The reported pharmacological activities of this plant are anti-inflammatory (Pawłowska et al., 2020), antibacterial, (Intisar et al., 2012), antioxidant (Demiray et al., 2009), anti-microbial (Munir et al., 2014; Cecotti et al., 2012) anti-proliferative (Mittal et al., 2013), gastroprotective, anticancer (Khushtar et al., 2018), antipyretic (Kumar et al., 2012), and hepatoprotective (Mittal et al., 2013). The exploration of bistort extract has facilitated the identification of novel bioactive compounds, the assessment of their healing potential, and the assurance of their safety in the management of socially significant diseases. The object of this study was to examine the lung protective effects of an aqueous methanolic extract of the roots of P. bistorta L. against LPS-induced ALI in a murine model, as well as potential mechanism(s) of action, in context with the potential benefits of this traditionally used plant.

Botanical drugs have diverse anti-inflammatory and anti-oxidative potential and suggest a step toward novel drug discovery in pulmonological areas of science (Wanyo et al., 2024). This endeavor was intended to investigate the lung protective sound effects of Pb.Cr having bioactive metabolites against LPS-induced ALI in rats. In vitro, in vivo, and in silico studies were included to expand the broad range of evidence for a positive impact on health.

Research has shown that a variety of secondary metabolites may be employed in the management of ALI (Bae et al., 2010). The phenolics and flavonoids were quantified, indicating potential for anti-oxidative, anti-inflammatory, anti-bacterial, and anti-proliferative effects. The polyphenolic profile of Pb.Cr. obtained by this investigation was comparable to the results of Wang et al. (2016). The many phenolic metabolites, flavonoids in particular, have drawn interest because of their possible health advantages (Al-Khayri et al., 2022; Oluwole et al., 2022).

When there is acute lung injury, the lung cholinergic system plays a key role in reducing pulmonary inflammation (Pinheiro et al., 2021). Eserine (physostigmine) is a reversible inhibitor of ACHE and BChE. It is usually effective in the range of 0.1–10 µM. The degree of lung injury can be ascertained by measuring elevated levels of AChE and BChE in the lung lavage (Graham et al., 2006). The metabolites that decrease AChE potential can act as immunity boosters (Pohanka, 2014). Pb.Cr showed a marked inhibition of AChE. Pb.Cr has considerable effects on the immune system as well as anti-oxidation by enhancing the anti-inflammatory potential (Table 2).

GC-MS of Pb.Cr was conducted to identify and quantify metabolites. The data obtained showed the presence of 24 metabolites in Pb.Cr. Among these, 15 metabolites having a Qual factor of more than 80 are given in Table 3. Di (2-propylpentyl) ester and hexadecanoic acid are among the metabolites that have been found to have antioxidant and anti-inflammatory properties (Kala et al., 2011). Prior research has shown the anti-inflammatory properties of octadecadienoic acid while naphthalene and 1,2 benzene dicarboxylic acid have anti-microbial potential (Uma et al., 2009). Pyridine-3-carboxamide is a DNA gyrase inhibitor in bacteria and is considered antimicrobial (Yule et al., 2014). Octadecanoic, oleic, erucic, and vaccenic acids are all anti-oxidant and anti-inflammatory and (R)-(−)-14-methyl-8-hexadecyn-1-ol has the potential to treat injuries (Mohansrinivasan et al., 2015). Molecular docking was also incorporated as a step for drug design for ALI/ARDS treatment strategy.

An acute toxicity study was also conducted to estimate lethal dose (LD₅₀). Pb.Cr. was administered in mice at single doses of 0.3, 1, 3 and 7 g/kg according to the animal’s weight and was continuously observed for the initial 1, 2, and 12 hours, and then for a further 72 h to identify any harmful consequences that might have occurred after the treatment time. For 14 days, all animals were under constant observation. There were no discernible changes in any of the numerous metrics of the groups. Thus, at a maximum dosage of 7 g/kg, this plant seemed safe. A group of scientists reported no cytotoxic activity of chloroform and hexane extracts of bistorta at 100 μg/mL against cancer cell lines (Karuppiah Pillai et al., 2007). (Karuppiah Pillai , 2021) reported LD50 values of hexane and chloroform fractions of bistorta rhizomes extract in mice, inconsistent with our study.

The lung weight coefficient showed the grade of lung edema in broncho-alveolar areas. Alveolar edema, which is caused by a significant rise in the permeability of the pulmonary epithelial barrier, is one of the primary causes of hypoxemia in patients with acute ALI/ARDS. The impaired alveolar epithelium is believed to be the primary cause of amplified pulmonary permeability, which results in edema fluid having great levels of extravagated metabolites in the alveoli (Kushimoto et al., 2012). Pre- and post-treatment of Pb.Cr. at 100, 300, and 500 mg/kg doses expressed a significant decline in the edematous status of the pulmonary structure (Figure 2). This revealed the potential of P. bistorta roots against edema and inflamed states of pulmonary system by improving oxygen saturation and fluid balance.

BALF is recommended as an effective tool for describing inflammatory cellular metabolites and employs pulmonary scarring to illustrate cellular interactions in the lower respiratory system (Watters et al., 1987) and is more reliable than blood samples (Ye et al., 2021). Blood cell count was also assessed to evaluate the level of toxicity in systemic circulation. Neutrophils are an essential part of the inflammation that typifies ALI. When endotoxemia causes ALI, pro-inflammatory cytokines such as IL-1β and TNF-α are expressed by the neutrophils that migrate into the airways and infiltrate the lungs. These cytokines may also play a role in the oxidant-induced harm and degradation of epithelial strength that occur after endotoxemia (Abraham, 2003). Transmigrated neutrophils, activated macrophages, lymphocytes, and total cells were taken into account, and the findings showed an almost three- to four-fold elevation in the penetration of inflammatory cells, mainly neutrophils, in the LPS-treated animals. Pb.Cr. defended against LPS-induced ALI (Figure 3).

The histopathological findings described problems such as cell infiltration, pneumonia, atelectasis, bronchitis, pulmonary edema, and vascular degeneration in LPS-treated animals, while 300 and 500 mg/kg Pb.Cr. markedly reduced the severity of complications. No histological alterations were noted in the control group compared to the LPS-intoxicated group (Figure 4). The decision was made to produce inflammatory scores by applying Murray’s scoring system. A score of 0 indicated no lung injury, a grade of 1–2 indicated slight to moderate airway damage, a level of 3 or above suggested significant inflammation, and a level of 4–5 indicated serious harm (Raghavendran and Napolitano, 2011). Furthermore, all the doses of Pb.Cr declined MDA and TOS levels that were increased via LPS-induced oxidative damage in the airway. On the contrary, the levels of SOD and TAC were amplified with the administration of Pb.Cr (Figure 6). A lack of balance in the anti-oxidants and oxidative damage may lead to increased oxidative stress and pulmonary immune system activation (Tang et al., 2021).

PCR analysis showed increased levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the LPS-intoxicated group, while Pb.Cr (300 and 500 mg/kg) ameliorated the cytokines. IL-1β has become well-known for leading the inflammatory cytokine cascade (Goodman et al., 2003). The TNF-α pathway controls both cell proliferation and apoptosis at the same time, acting as a “sword with two blades”. During the phase of acute damage to tissue, irregular TNF-α signaling can set off cytokine storms that result in significant cell death. Conversely, tissue healing after the acute damage phase necessitates an ideal level of TNF-α signaling. Additionally, IL-6 is a crucial pro-inflammatory cytokine that potently induces acute inflammatory damage (Figures 6A–C). The level of COX-2 was increased in the LPS-treated group compared to control while significantly lowered in Pb.Cr.-treated animals (Figure 7). COX-2 inhibitors reduce proliferation as well as apoptosis in cellular levels (Mohseni et al., 2004).

NF-ĸβ stimulation was more enhanced in the LPS-intoxicated group than control, and declined levels of NF-ĸβ were observed in all the animals treated with Pb.Cr (Figure 8). The transcription of genes related to inflammation is started by the nuclear transcription factor NF-ĸβ. Toll-like receptors (TLR₄) initiate NF-ĸβ via many signaling pathways (Chen et al., 2019). According to earlier studies, TLR₄ that has been activated by LPS transmits downstream signaling, mainly through the NF-ĸβ and MAPK pathways, which in turn contributes to inflammatory reactions. (Chi et al., 2013). The five NF-ĸβ family members are NF-ĸβ₁ (p50), NF-ĸβ₂ (p52), Rel A (p65), Rel B, and c-Rel, which can control the transcription and translational activities of pro-inflammatory genes which are essential for controlling the inflammatory response (Alharbi et al., 2021). The suppressive protein IκBα typically sequesters the NF-ĸβ proteins in the cytoplasm. Following LPS stimulation, activated IKK causes a cascade of activations that leads to IκBα activation and depletion, which in turn releases p65. Then, phosphorylated p65 quickly moves into the nucleus and attaches to desired genes to encourage the transcription of subsequent genes (Tang et al., 2021). Here, octadecadienoic acid may decreased the amounts of phosphorylated IKKβ, IκBα, and p65 protein in a dose-dependent fashion, which may significantly inhibit p65 entering the nucleus.

Nowadays, a medication development process frequently uses computational molecular docking (Tabassum et al., 2022), which is valid for predict biochemical activities (Piccagli et al., 2008) to develop the blocker(s) of NF-ĸβ. Docking offers the benefit of detecting the type of contact between the study metabolites at the enzyme or receptor regions through particular significant relationships in addition to protein interacting structures—in this example, using chemical data from GCMS (Zhang et al., 2018). Molecular docking investigation was accomplished for AChE. A total of 23 metabolites were docked against AChE (PDB). The eight identified metabolites have the best binding affinities with this protein molecule (Supplementary Figure S1). These molecules thus have the potency to alter the molecular structures that may exert biological effects. The structural conformational changes indicated that 1,2-benzisothiazol-3-amine (−7.8) had pi-alkyl and alkyl type interactions for proline A:232 and A:529 and histidine 406, respectively, while H-bond-type interactions were observed for histidine A:390 and asparagine A:525. In interactions between proteins and ligands as well as other hydrophobic interactions like alkyl and pi alkyl, the hydrogen bond is crucial for maintaining the consistent binding of ligands to proteins (Borges et al., 2018). 10E,12Z-octadecadienoic acid (−8.0) showed the presence of H-bond and alkyl as well as Van der Waal’s force-type interactions between amino acids with different distances for each of them. For oleic acid (−7.4), H-bond-type interaction was noted for tyrosine A:121 and asparagine A:85. Pi-alkyl-type interactions were observed for five amino acids—tryptophan A:84, histidine A:440, and phenylalanine A:290, A:330, and A:331—with varying distances for each amino acid. Phthalates with binding affinity of −6.1 showed hydrophobic alkyl type interactions for valine A:323 distance of 3.23 Å, pi-sigma type interactions for valine A: 400, unfavorable Doner–Doner interactions for glutamic acid A:327 and histidine 326, and conventional H-bond type interaction for aspartic acid A:326, glycine A:441, and asparagine A:324. In CIS-vaccenic acid (_6.9), pi-alkyl-type interactions were observed for tryptophan A:85, phenylalanine A:330 and A:331, and histidine A:440 with different distance variations. Van der Waal’s force-type interactions were observed for the remaining amino acids. H-bond and alkyl type interactions were found for 7-pentadecyle (7.5). Cyclohexane carboxylic acid (−8.6) showed C-H bond, H-bond, Pi-Pi-T-shaped, and Pi-Pi-stacked type interactions between amino acids of this molecule. A major component of molecular docking is hydrogen bonding—important drug–target interactions that stabilize ligand–receptor protein complexes, leading to inhibition of microbial growth and exaggerated anti-inflammatory potential (Rozas, 2007). They can direct the ligand into the ideal binding shape and are among the strongest non-covalent interactions. One hydrogen atom can make a hydrogen bond with another electronegative atom on a different molecule if it is previously connected to a highly electronegative atom, such as oxygen or nitrogen. The binding force between a hydrogen atom and the single pair of electrons on the electronegative atom causes the hydrogen bond to form (Al-Malki, 2024). Alkyl, hydroxyl bond, Van der Waal’s forces, and covalent bonds are vital bonds that affect the short or permanent binding of ligand-receptor complexes. The binding energies are > −6.0 and the effectiveness of metabolites are strongly dependent on these binding affinities and the formation of bond type between ligand and receptor complexes (Rozas, 2007). The metabolites with binding affinities near the dimer’s DNA binding sites (Supplementary Figure S1) indicate that the compounds might have anti-inflammatory properties by preventing activated NF-ĸβ (p65) from binding to DNA (Chen et al., 1998). Thus, molecular docking results indicate that bonding interactions and their characterization are a step toward new NF-ĸβ (p65) blocker development in a medicinal approach to respiratory problems. However, further intensive studies are required to authenticate the inhibition of transcription factors.

GC-MS analysis expressed the presence of 24 different secondary constituents that belong to different classes, such as anti-oxidants and anti-inflammatories. In vivo and in vitro experimental outcomes showed a marked reduction in of neutrophil and macrophage infiltration, pulmonary edema, improved SO₂ levels, and restoration of anti-oxidant and anti-inflammatory markers. The AChE inhibitory profile indicates the use of Pb.Cr to treat inflammatory conditions. These findings offer a solid scientific basis for the anti-oxidant as well as anti-inflammatory properties of Pb.Cr by primarily inhibiting NF-ĸβ (p65) and shielding the lungs. Our research provides future insights into the potential of P. bistorta root for pharmacological breakthroughs to treat ALI/ARDS.