All the experiments were performed as per the guidelines of the Institutional Animal Ethics Committee (IAEC-THSTI/163) of the Translational Health Science and Technology Institute (THSTI), Faridabad, India, and all the experiments were carried out following the control and supervision of experiments on animals (CPCSEA) guidelines (Govt. of India).

8–12 weeks old female BALB/c mice (18–25 g) were procured from the Small Animal Facility (SAF) of Translational Health Science and Technology Institute (THSTI), Faridabad, and housed in individually ventilated cages with controlled air flow as per the institute’s experimental animal guidelines. Access to normal chow and water was provided to them ad libitum. The animals were acclimatized for 1 week in the above conditions prior to initiating the experiments.

NCI-H460 cell line (human lung carcinoma cells) was procured from the National Centre of Cell Science, Pune, India, and was routinely maintained in RPMI-1640 medium containing 10% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen, United States). Cells were maintained in a humidified incubator with 5% CO₂ at 37°C. A549 cells were procured from ATCC and maintained in DMEM containing 10% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen, United States) and maintained in a humidified incubator with 5% CO₂ at 37°C, as above.

Aqueous extract of the W. somnifera (WS) roots was prepared in the good manufacturing practice (GMP) facility and was provided by NMPB, and its characterization has been reported previously (Kasarla et al., 2022).

Total RNA was isolated from cells treated with different concentrations of WS and lungs using Trizol Reagent (Thermo Fisher Scientific), according to the manufacturer’s protocol and used for the cDNA synthesis. cDNA was synthesized with an iScript cDNA synthesis kit (Applied Biosystems, United States) as per the manufacturer’s protocol. The gene-specific primers (Table 1) were designed using NCBI primer blast tool, and qPCR was performed on the QuantStudio 6 (Applied Biosystems, United States) real-time PCR systems. Reactions were carried out in 10 μl volumes containing: 5 μl Sybr green Master Mix (PowerUp SYBR Green mix, Applied Biosystems; Cat. No. A25742), 0.5 μl forward and 0.5 μl reverse primers, 3 μl water, and 1 μl cDNA. Cycling program used was: 7 min at 95°C and then 40 cycles with 10 s at 95°C and 20 s at 60°C. The specificity of the amplicons was analysed by a thermal dissociation curve. Data were normalized against the housekeeping gene, 18S, or GAPDH.

NCI-H460 cells were seeded at a density of 1 ×10⁴ cells/well in RPMI medium with 10% FBS in a 96-well plate and incubated at 37°C in a CO₂ incubator for 24 h followed by treatment with WS at 10, 3, 1, 0.3, 0.1, and 0.003 mg/ml. LPS (1 μg/ml) was added 30 min after the treatment with WS and the plate was incubated further in the CO₂ incubator for 24 h. Dexamethasone was used as a positive control (10 µM). For gene expression analysis, cells were grown at a density of 4 × 10⁵ cells/well in a 6 well plate. After 24 h, cells were treated with various concentrations of WS (10, 3, and 1 mg/ml) in RPMI 1640 medium with 10% FBS followed by stimulation with LPS (1 μg/ml), and the plate was incubated in the CO₂ incubator for another 24 h. Cells were lysed using the Trizol reagent and total RNA and cDNA was prepared followed by gene expression analysis as mentioned above. Similar experiments were carried out using A549 cells also. To determine the cytotoxicity of WS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. Cells treated as above in the inflammatory profiling setup were treated with MTT (100 μl of 0.5 mg/ml MTT stock in PBS) in an independent experiment, and the plate was incubated for 4 h at 37°C in a CO₂ incubator. The MTT mixture was aspirated and 100 μl of DMSO was added to each well to dissolve the formazan particles. The plate was shaken for 30 min on a plate shaker at 37°C to ensure complete dissolution of formazan crystals and read at 570 nm to measure the probable cytotoxicity of test substances if any.

Cell viability (%) = Total viable cells (unstained)/Total cells (unstained) + blue stained χ 100.

Intravenous blood was drawn from healthy individuals and collected in a heparinized tube. Heparinized blood was diluted with 1x PBS (1:1). After dilution, blood was layered on Histopaque-^®1077 (2:1) in a falcon tube and centrifuged at 1,600 rpm for 30 min. The middle layer of the buffy coat containing PBMCs was collected cautiously and washed twice with PBS. The pellet was dissolved in PBS for counting PBMCs using a hemocytometer.

The cells were plated at a cell density of 1 × 10⁶ cells/well in a 24-well plate. Cells were treated with WS (1 mg/ml) and incubated at 37°C in a CO₂ incubator for 30 min followed by treatment with LPS (1 μg/ml) and incubated again at 37°C in a CO₂ incubator for another 16 h. Cell culture supernatant was collected the next day and analyzed for the expression levels of different cytokines by ELISA as per the manufacturer’s instructions. Institutional ethics committee approval was obtained prior to initiating experiments with human blood obtained from healthy human volunteers (Institutional ethics approval number: THS1.8.1/100). No experiments were carried out in humans directly.

The effect of WS in LPS-induced neutrophilia was evaluated under two timepoints. In the first healthy mice were randomized based on their body weights and divided into five different groups: 1) Control; 2) LPS (25 µg/mice); 3) Dexamethasone (1 mg/kg); 4–6) WS, (10, 30, and 100 mg/kg). All the groups except the control group were injected intranasally with LPS in 200 µl PBS (pH 7.4). Mice in groups 4, 5, and 6 were treated orally with WS mixed in saline and challenged with LPS after 30 min of drug treatment. Mice in the control group were challenged with saline only and animals were euthanized 4 h after the WS or saline challenge. Broncho-alveolar lavage (BAL) fluid was collected from the lungs of mice in ice-cold Hank’s Balanced Salt Solution (HBSS) or 1X Phosphate buffer saline (PBS) and processed for the quantification of total and differential leukocyte counts. Lungs were isolated, homogenized, and lysed with Trizol reagent. Total RNA was isolated as per the manufacturer’s protocol. In the second study, the anti-inflammatory potential of WS was analysed in a 4-day LPS challenge model. Mice were treated with WS by oral route followed by intranasal administration of LPS (25 µg/mice) for 4 consecutive days. After 4 days mice were euthanized and analysed for the presence of leukocytes using BAL fluid as in the case of the 4 h study, mentioned above.

Smears were prepared using 50–100 µl of the freshly isolated BAL fluid on a frosted glass slide using a Cytospin centrifuge (Biotek, Agilent Technologies Inc. Santa Clara, CA United States) by spinning the slides at 1,000 rpm for 10 min. Slides were dried and fixed with 100% ethanol, further stained with Giemsa stain, and cell counts were taken by scientists who were blinded to the treatment groups.

Healthy mice were randomized based on their body weights and divided into four different groups: 1) Control; 2) LPS (25 µg/mice); 3) Dexamethasone (1 mg/kg); 4) WS (100 mg/kg). All the groups except the control group were injected intranasally with LPS in 200 µl PBS (pH 7.4). Mice were treated orally with WS mixed in saline after 30 min of LPS challenge. Mice in the control group were challenged with saline only and animals were euthanized 4 days after the WS or saline challenge.

Histological evaluation was done using the lung tissues of different groups of mice. Mice were sacrificed 4 days after the LPS administration and lower lobes from the left lung were dissected and fixed in 10% formalin, dehydrated with ethanol followed by embedding in paraffin and cutting into 5 μm sections. After deparaffinization, the tissues were stained with Hematoxylin and Eosin (H&E) as reported previously (Alwahaibi et al., 2015). Pathological changes among different groups were evaluated under a light microscope and lung injury scores were assessed using a semi-quantitative method.

Each experiment was performed three or more times independently with different cell passages. For animal work, each group consisted of n = 5 mice. Statistical analysis was performed on GraphPad PRISM 7 software (GraphPad, La Jolla, CA, United States) using one-way ANOVA. The p-values of <0.05 were considered statistically significant. The results were calculated as mean ± S.E.M.

To recapitulate the anti-inflammatory properties of WS in vitro, cell-based assays were conducted using a lung carcinoma cell line, NCI-H460, and lung epithelial cell line A549. WS extract was not found to be toxic in the MTT assay at the concentrations used in the in vitro experiments (Figure 1A). Treatment with LPS led to a significant increase in the secretion of TNF-α, IL-8, IL-6, chemokine (c-c) motif ligand 2 (CCL2), and IL-8 while pre-treatment of the cells with different concentrations of WS (10–0.03 mg/ml) showed a dose-dependent inhibition of pro-inflammatory cytokines by RT-PCR (NCI-H460: Figure 1A) and in A549 cell line for TNF-α, IL-8, IL-6 (Figure 1B) suggesting the anti-inflammatory properties of WS. Treatment with dexamethasone resulted in a decrease in the levels of pro-inflammatory cytokines.

Human PBMCs led to an increase in the expression levels of pro-inflammatory cytokines and chemokines such as TNF-α, IL-6, IL-1β, and IL-8, when induced with LPS (1 μg/ml). Pre-treatment of hPBMCs with different concentrations of WS (10, 3, 1, 0.3, 0.1, and 0.03 mg/ml) on the other hand, significantly reduced LPS-induced increase in the cytokines and chemokines (Figure 2).

To explore the beneficial effects of WS on endotoxin-induced airway inflammation in vivo, BALB/c mice were treated with three different doses of WS (10, 30, and 100 mg/kg for 30 min, followed by the LPS challenge by the intranasal route, and BAL fluid was collected after 4 h. Total number of leukocytes and differential neutrophil counts were analyzed in the BALF (Figure 3A). LPS challenge in mice resulted in a significant increase in the leukocytes influx compared to the unchallenged mice. Treatment with different doses of WS attenuated the number of total leukocytes as well as neutrophil counts in a dose-dependent manner. Dexamethasone was used as a positive control and led to a reduction in neutrophil counts.

LPS-induced leukocyte infiltration is associated with the levels of pro-inflammatory cytokines secreted into the lungs of mice (Gupta V et al., 2015). To test the effect of WS on LPS-induced inflammation in the lung tissue, mRNA expression studies were done using the lung tissues of mice challenged intranasally with LPS with or without WS by RT-PCR. LPS challenge in mice significantly increased the levels of IL-1β, IL-18, IL-4, IL-6, chemokine (c-x-c) motif ligand 1 (CXCL-1), IL-18, CCL2, and TNF-α (Figure 3B) which was found to be significantly reduced in mice pre-treated with WS in a dose-dependent manner. Treatment with dexamethasone resulted in a significant decrease in all tested cytokines except IL-4.

Intranasal administration of LPS has been reported to cause lung tissue damage in mice. Histological analysis of the lung tissues was carried out to assess the LPS-induced lung damage in a 4-day LPS challenge model using BALB/c mice (Figure 4). The lungs of mice in the control group, showed normal pulmonary architecture whereas the lungs of mice challenged with LPS showed notable inflammatory cell infiltration and alveolar haemorrhage. Treatment with WS significantly attenuated such pathological changes induced by LPS. The positive control dexamethasone also improved the histopathological conditions in LPS-induced mice. For semi-quantitative evaluation, the changes were also evaluated by calculating a lung injury score our data suggests that pre-treatment WS alleviates LPS-induced pathological changes in the mouse model.