2.4. Animals and in vivo study

The experimental protocols used for this animal study were approved by the Committee on Care and Use of Laboratory Animals of Kyungpook National University (KNU-2021-50). Four-week-old specific pathogen-free Sprague–Dawley rats were purchased from Orient Bio Inc. (Gyeonggi-do, Republic of Korea). They were acclimatized in a laboratory environment for 7 days under the following conditions: room temperature, 22 °C–26 °C; relative humidity, 55%–70%; and 12 h/12 h light and dark cycle. They were supplied with ad libitum filtered water and a standard pellet diet throughout the experiment. They were randomly distributed into eight groups with eight rats each (Table 1). Except for the normal control and levamisole groups, all the groups were intraperitoneally (i.p.) injected with cyclophosphamide (80 mg/kg/day) dissolved in sterile isotonic saline for the first 3 consecutive days to induce immunosuppression. Then, they were administered a daily oral dose of 1 mL of levamisole, LhLR, MhLR, HhLR, and LR dissolved in distilled water for 3 weeks through gavage feeding (Figure S1). Physical examination and body weight measurement were performed regularly for dosage adjustment. On day 21, the rats were terminated, and blood was collected and centrifuged (1000 × g, 10 min) to separate serum for enzyme-linked immunosorbent assay (ELISA); their organs were processed accordingly.

Description of treatment groups.

The weight of the rats was measured regularly throughout the experiment to adjust the dose. Before they were sacrificed, their body weight was taken, and their immune organs were excised and accurately weighed. The following formula was used to calculate the immune organ index:

(Fei et al. 2022).

Spleen tissues were fixed with 10% of buffered formalin solution. Then, the organs were processed and embedded in paraffin. The samples were cut (4–5 μm thick) using a frozen section machine and stained with hematoxylin and eosin (H&E). The histological characteristics of the tissues were assessed under a microscope.

After final drug administration, blood was collected from the sacrificed rats and placed in a heparinized tube. CBC analysis was performed following previously described procedures (Lee et al. 2018). The number of immune blood cells such as WBCs, granulocytes, lymphocytes, and mid-range absolute, which generally comprises basophils, eosinophils, and monocytes, were counted using URIT-300 Vet Plus (URIT Medical Electronic Co., Ltd., Guangxi, China).

Histopaque® (Sigma-Aldrich, USA) was used to separate neutrophils from the whole blood. Briefly, a 15 mL conical tube was filled with equal parts of whole blood and Histopque-1077; then, the specimen was centrifuged (400 × g, 30 min) at room temperature. After the upper layer was carefully aspirated within 0.5 cm of the opaque interface, the interface was collected, washed with 10 mL of PBS, and centrifuged at 250 × g for 10 min. The cells were counted using a hemocytometer by mixing with trypan blue after they were reconstituted in DMEM.

A neutrophil migration assay was conducted using a Cytoselect^TM 24-well migratory test kit (3 μm, fluorometric format) in accordance with the manufacturer’s instructions (Cell Biolabs, INC. CA, USA). Briefly, after neutrophils were isolated as previously described, 100 mL of 5 × 10⁶ cells/mL in serum-free DMEM was added to the insert, and 500 μL of the media containing 10% FBS was added to the lower well of the migratory plate. The cells were then incubated for 24 h, and the media were aspirated from the inside insert. They were further incubated at 37 °C for 30 min after the insert was transferred to a clean well containing 200 μL of a cell detachment solution. The cells were fully removed from the membrane’s underside by gently tilting the insert into the detachment solution that was then mixed with 400 μL of the media containing migrating cells. The mixture and 60 μL of a 4 × lysis buffer/CyQuant® dye solution (Thermo Fischer Scientific, Waltham, Massachusetts, USA) were poured into a 96-well plate and left to stand at room temperature for 20 min. The mixture (200 μL) was then placed in a 96-well plate to read the fluorescence at 480 nm and expressed as relative fluorescence unit (RFU).

CytoselectTM 96-well phagocytosis assay (zymosan substrate-based; Cell Biolabs, Inc., CA, USA) was performed in accordance with the manufacturer’s instructions to determine the phagocytic activity of neutrophils. Briefly, neutrophils (100 μL of 10⁶ cells/mL) was seeded in a 96-well plate, and 10 μL of zymosan was added to each well. The resulting sample was incubated at 37 °C for 2 h. After centrifugation, the culture medium was removed by aspiration and washed with cold PBS. The fixation solution (100 μL) was supplied to each well, and the wells were left to stand at room temperature for 10 min. Then, the fixation solution was eliminated by centrifugation and washed with PBS. The prepared blocking solution (100 μL) was added to each well and incubated at room temperature for 60 min. After the solution was centrifuged and washed, the prepared permeabilizing solution (100 μL) was added, and the mixture was left to stand at room temperature for 5 min. After the mixture was washed, a detection antibody solution (100 μL) was added to each well. After the solution was washed again with PBS, 50 μL of detection buffer was added to each well and incubated at room temperature for 10 min. A substrate solution (100 μL) was added to each well to initiate the reaction by incubating at 37 °C for 20 min. After 50 μL of stop reagent was added, absorbance was measured at 405 nm by using a microplate reader (BioTek Instruments, Inc., Winooski, USA).

Whole blood was collected into anticoagulant-coated tubes and mixed well. An equal volume of whole blood (3 mL) and Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA) was mixed in a 15 mL falcon tube to create a density gradient. The upper layer above the opaque interface containing the mononuclear cells was carefully removed after centrifugation at 400 × g and room temperature for 30 min. The opaque layer was collected and transferred into a conical tube. The cells were washed with 10 mL of PBS and then centrifuged at 250 × g for 10 min. After the supernatant was discarded, the cells were resuspended in 1 mL of FACS buffer. Finally, the cells were counted using a hemocytometer.

Splenocytes and thymocytes were prepared in accordance with previously described methods (Ahmad et al. 2015). Briefly, after the rats were terminated, the spleen and thymus were removed aseptically and kept in Hank’s balanced salt solution (HBSS; Gibco Life Technologies, New York, NY, USA). The organs were sieved through a 70 μm nylon cell strainer (BD Bioscience, CA, USA) by using a 3 mL syringe plunger. Then, the cells were centrifuged at 1800 rpm for 5 min, suspended in red blood cell (RBC) lysing buffer to lyse RBCs, and centrifuged again (1800 rpm, 5 min) to remove the lysed RBCs. The supernatant was discarded and washed thrice with PBS. The cell pellets were suspended in Roswell Park Memorial Institute (RPMI)-1640 media (Sigma-Aldrich, St. Louis, MO, USA), and the number of cells was counted using a hemocytometer via a trypan blue dye exclusion method.

A splenocyte proliferation assay was performed using mitogen concanavalin A (Con A) and lipopolysaccharide (LPS). In this procedure, 100 μL of 1 × 10⁶ cells/mL from different groups was seeded in a 96-well plate containing RPMI-1640 media supplemented with FBS (10%) and P/S (1%). The total volume was adjusted to 200 µL by adding mitogen Con A or LPS (5 μg/mL), and the cells were incubated at 37 °C for 72 h. The MTT reagents (20 μL) of 5 mg/mL were mixed in each well and incubated for 4 h. Then, 200 μL of dimethyl sulfoxide was added to each well, and absorbance was read at 570 nm.

The T lymphocyte phenotyping of the thymus, PBMCs, and spleen was performed at a concentration of 1 × 10⁶ cells/mL by harvesting them into a FACS tube. T cells were stained with the following anti-rat antibodies: anti-CD3⁺ (APC-conjugated), anti-CD4⁺ (PE-Cy7-conjugated), anti-CD8⁺ (FITC-conjugated), anti-CD45RA⁺ (PE-conjugated) and anti-CD28⁺ (BV711-conjugated). Then, 10 μL of 10⁶ cells/mL was mixed and incubated in a dark environment at 4 °C for 30 min. The cells were centrifuged (300× g, 5 min) with FACS buffer to remove unbound antibodies. For FACS analysis, the cells were suspended in 400 μL of FACS buffer. PBMCs, thymocytes, and splenocytes were analyzed in terms of CD3 T cells, CD4 Th, CD8 CTL, CD45RA, and CD28 regulatory T cells (Treg). Multicolor FACS with FACSDiva version 6.1.3 on BD FACSAria^TM III (BD Biosciences, San Diego, CA, USA) was used to acquire and analyze the data.

Cytokine concentrations were measured via ELISA in accordance with the manufacturer’s protocol (ELK Biotechnology, Wuhan, China). Briefly, 100 μL of the sample or standard was transferred to each well and incubated at 37 °C for 2 h. The wells were washed thrice with the wash buffer. Then, 100 μL of pre-diluted biotinylated antibody was mixed into each well and incubated at 37 °C for 1 h. The wells were washed three more times with the wash buffer before 100 μL of pre-diluted streptavidin-HRP was added to each well and incubated at 37 °C for 1 h. After three rounds of washing, 90 μL of TMB substrate was added to each well and incubated at 37 °C for 20 min. Lastly, 50 μL of the stop reagent was added to each well, and absorbance was read at 450 nm. Protein concentrations were determined by generating a standard curve, and results were expressed in picograms per milliliter.

Five groups (normal control, CTX, positive control, HhLR, and LR) were prepared to analyze the microbiota composition. The HhLR group was selected to represent the hLR treatment groups. Fecal microbial genomic DNA was extracted using a QIAamp DNA stool kit (Qiagen Inc., Hilden, Germany) following the manufacturer’s protocols. The rats’ fecal samples were microbially profiled on the basis of the 16S rRNA gene sequencing amplified by Pacific Biosciences performed in Chun Lab Inc. (Seoul, South Korea). The EzBioCloud Microbiome Taxonomic Profile (MTP) and EZBioCloud 16S database PKSSU4.0 were used to create the MTP from next-generation sequencing (Yoon et al. 2017). UCLUST was used to perform high-quality sequence clustering based on operational taxonomic units (OTUs) with a 97% similarity cutoff. The alpha-diversity (Chao 1, ACE, Jackknife, Shannon, and Simpson) and taxonomic profile were screened using EzBiocloud 16S-based MTP software (Yoon et al. 2017).