Single-Cell RNA Sequencing Revealing Yiqi Huayu Jiedu Formula Mediated Alleviation of Acute Respiratory Distress Syndrome via Macrophage Chemotaxis

Summary

This article describes the protective effects of Yiqi Huayu Jiedu Formula (YQHYJDF) against lung injury through inhibition of macrophage chemotaxis and neutrophil recruitment by suppressing IL-6, IL-1β, TNF-α, PI3K/AKT, MAPK, and NF-κB signaling pathways, along with downregulation of the chemokines CCL2, CCL7, and CCL12.

Abstract

Acute Respiratory Distress Syndrome (ARDS) is a life-threatening form of respiratory failure with widespread inflammation in the lungs. Based on decades of clinical practice by Professor Du Huaitang, the Yiqi Huayu Jiedu Formula (YQHYJDF) has been used to alleviate symptoms in ARDS patients. Although formal clinical trials are lacking, preliminary observations indicate its therapeutic potential, justifying further investigation. To elucidate its protective mechanisms, network pharmacology, single-cell RNA sequencing, and validation experiments both in vivo and in vitro were integrated. An ARDS rat model was established by tail vein injection of LPS, after pre-prepared intervention with YQHYJDF. Histopathological analysis was conducted to evaluate the pathological alterations. Single-cell RNA sequencing was performed to explore differentially expressed genes between groups. Network pharmacology analysis was executed for the prediction of the potential protective mechanism of YQHYJDF against ARDS. Following this prediction, the mechanism was further validated through both in vivo and in vitro experiments via quantitative real-time PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Hematoxylin-eosin (HE) staining demonstrated that YQHYJDF significantly ameliorated lung histopathological alterations, reduced inflammatory cell infiltration, and lowered lung injury scores. Single-cell RNA sequencing revealed that YQHYJDF effectively regulated macrophage chemotaxis and significantly attenuated neutrophil recruitment in ARDS lung tissues. Network pharmacology analysis illustrated that the efficacy of YQHYJDF on ARDS may be realized by inhibiting PI3K/AKT, MAPK, and NF-κB signaling pathways. In vivo experiments demonstrated that YQHYJDF markedly suppressed the mRNA expression of IL-6, IL-1ß, and TNF-α in lung tissues, and reduced aberrant activation of the PI3K/AKT, MAPK, and NF-κB signaling pathways. In vitro experiments showed that YQHYJDF effectively inhibited the synthesis and secretion of Ccl2, Ccl7, and Ccl12 in LPS-induced RAW264.7 cells. The protective effect of YQHYJDF on ARDS is likely associated with the suppression of macrophage chemotaxis and inflammatory responses in lung tissues.

Introduction

Acute respiratory distress syndrome (ARDS) is a severe clinical condition manifesting as acute hypoxemic respiratory failure, diagnosed by the radiographic finding of bilateral pulmonary infiltrates¹. Despite advances in critical care, ARDS mortality remains 30%–50%, and no specific pharmacological therapies have been shown to significantly improve outcomes². The etiology of ARDS encompasses infection, trauma, aspiration, inhalation of toxic substances, and acute pancreatitis, all of which can lead to progressive impairment of lung function and potentially life-threatening respiratory failure^3,4,5. Although the pathogenesis of ARDS is multifactorial, dysregulated and uncontrolled inflammatory responses play an important role in disease onset and progression. Accordingly, targeting the inflammatory cascade constitutes a critical strategy for reducing ARDS-associated mortality.

As central mediators of innate immunity, macrophages play a pivotal role in ARDS pathogenesis. The recruitment of circulating monocytes to injured lung tissue and their subsequent differentiation into proinflammatory macrophages represent a key pathophysiological mechanism underlying disease progression^6,7. Once activated, macrophages release large quantities of inflammatory mediators, including cytokines and chemokines, thereby amplifying pulmonary injury and perpetuating the inflammatory cycle^8,9. Notably, macrophages contribute to ARDS pathogenesis through multiple functional states, including polarization, pyroptosis, phagocytosis, and chemotaxis, each of which may represent a distinct therapeutic target^10,11,12.

Traditional Chinese Medicine (TCM) has shown significant potential in treating ARDS via multi-target and multi-pathway mechanisms, including anti-inflammatory, antioxidant, and alveolar repair processes^13,14,15. Research on TCM-based interventions for ARDS is increasingly focused on their potential to modulate immune responses, such as mitigating cytokine storms and restoring immune homeostasis^16,17,18. The Yiqi Huayu Jiedu Formula (YQHYJDF), a prescription formulated based on TCM principles of supplementing qi, promoting blood circulation and removing blood stasis, and detoxification, consists of Astragalus membranaceus, Scutellaria baicalensis, Lonicera japonica, Panax notoginseng, Paeonia lactiflora, processed Citrus aurantium, Lepidium apetalum, and Morus alba bark, and has shown potential clinical efficacy against ARDS. Previous studies have demonstrated that the YQHYJDF can protect against lung injury in ARDS model rats by reducing neutrophil adhesion activity and inhibiting inflammation-related signaling pathways^19,20,21. However, the precise protective mechanisms by which it confers benefit in ARDS remain to be elucidated.

The emergence of single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of cellular heterogeneity and dynamic changes in disease contexts^22,23. This technology enables unprecedented resolution for analyzing cell type-specific transcriptional programs and identifying previously unknown cellular subpopulations involved in disease progression²⁴. In ARDS research, scRNA-seq has proven valuable in characterizing complex immune cell landscapes and revealing novel protective targets²⁵. The combination of single-cell sequencing technology with TCM research offers a powerful, high-resolution approach to decipher complex pathological mechanisms^26,27,28. To this end, scRNA-seq technology was employed to explore the mechanisms by which the YQHYJDF intervenes in LPS-induced ARDS models, providing evidence for further clinical applications.

Protocol

This study was reviewed and approved by the Animal Welfare and Ethics Committee of Dongzhimen Hospital, Beijing University of Chinese Medicine (Approval No. 24-54) and conducted following institutional guidelines for the ethical care and use of laboratory animals. A total of 18 male Sprague-Dawley (SD) rats, aged 6 weeks and weighing (220 ± 10) g, were purchased (animal license number: SCXK (jing) 2025-0008). The reagents and the equipment used are listed in the Table of Materials.

1. Animals

Animals were housed at the Laboratory Animal Center of Dongzhimen Hospital, Beijing University of Chinese Medicine, with 3 rats per cage under standard diet conditions at a temperature of 22–24 °C and humidity of 50%–70%. Experiments were conducted after a 7-day acclimatization period, during which rats were randomly divided into a blank control group, ARDS model group, and YQHYJDF group, resulting in 6 rats per group. Following randomization into three groups of six rats each, three rats per group were designated for single-cell RNA sequencing of the left lung, while all rats per group provided right lung tissue for histopathological examination, qPCR, ELISA, and Western blot analyses.

2. Preparation of YQHYJDF

The YQHYJDF is composed of Astragalus membranaceus (30g), Panax notoginseng (10 g), Paeonia lactiflora (15 g), processed Citrus aurantium (12 g), Lonicera japonica (15 g), Scutellaria baicalensis (10 g), Morus alba bark (15 g), and Lepidium apetalum (15 g). All herbs were decocted in distilled water (10x volume) and processed into powder extract by specialized equipment (batch number: 20250228). In clinical practice, YQHYJDF is prescribed at a dose of 122 g per formulation, with one formulation administered daily. Assuming an adult body weight of 60 kg, the human equivalent dosage of YQHYJDF is 2.03 g (raw drug)/kg/day. Based on body surface area–based dose conversion between rats and humans, the corresponding dose for rats is estimated to be 6.25 times that of humans. Thus, the dosage administered to rats was determined to be 12.2 g (raw drug)/kg/day. Given that the ratio of raw herbs to extract for YQHYJDF is 2:1, the equivalent extract dose for rats is 6.1 g/kg/day. The extract was dissolved in distilled water to prepare a stock solution with a concentration of 0.61g/ml, which was stored at 4 °C for subsequent use.

3. Pharmacological Intervention

The blank control group and the ARDS model group were administered distilled water via oral gavage at a dose of 1 mL per 100 g of body weight. The YQHYJDF group received a pre-prepared YQHYJDF solution via oral gavage at a dose of 1 mL per 100 g of body weight. Prior to administration, both the distilled water and the YQHYJDF solution were prewarmed in a 37 °C water bath. Gavage was performed daily at 8:00 for 7 consecutive days.

4. ARDS model establishment

6 h after the final intragastric administration on day 7, rats in the ARDS model group and the YQHYJDF group were subjected to a tail vein injection of LPS solution (2 mg/kg), while the blank control group received an equivalent volume of 0.9% sodium chloride injection (2 mg/kg) through the same route. 16 h later, all rats were anesthetized, and samples were collected.

5. Sample collection and processing

Rats were anesthetized with isoflurane using a gas anesthesia machine (following institutionally approved protocols). The thoracic cavity of the rat was opened with a scalpel, and the lungs were completely dissociated. The left lung tissue was placed in a specialized preservation solution for subsequent single-cell sequencing, whereas the right lung was divided into two portions: one portion was fixed in 4% paraformaldehyde for histopathological analysis, and the other was snap-frozen in liquid nitrogen for qPCR, ELISA, and Western blot analyses.

6. Pulmonary histopathological examination

The right lung tissue was fixed in 4% paraformaldehyde for 12 h and then rinsed several times with PBS. The tissue was progressively dehydrated using a graded ethanol series, cleared with xylene in a fume hood, infiltrated with molten paraffin, and embedded in paraffin blocks. Sections were cut at a thickness of 4 µm, floated on a 45 °C water bath to expansion, and mounted onto adhesive-coated glass slides. The slides were dried in a 60 °C oven and subsequently subjected to hematoxylin and eosin (HE) staining according to the manufacturer’s protocol. Following mounting with neutral balsam, the sections were further subjected to whole-slide imaging.

Lung injury was evaluated using a standardized semi-quantitative scoring system (0–4, where 0 = normal, 4 = severe) based on four parameters: neutrophil infiltration, interstitial inflammation, edema, and congestion. Each parameter was graded on a scale of 0 to 4 according to the extent of pathological involvement: 0, no pathological changes; 1, involvement of ≤25% of the examined field; 2, ≤50%; 3, ≤75%; and 4, diffuse involvement of the entire field. The final lung injury score for each animal was calculated as the sum of the scores for all 4 parameters^29,30.

7. Preparation of drug-containing serum

A separate cohort of six male SD rats (3 rats per group, same age and weight range as the main experiment, housed under identical conditions) was used exclusively for drug-containing serum preparation and was independent of the 18 rats used in the main in vivo experiment. They were orally administered YQHYJDF or an equivalent volume of distilled water continuously once daily for seven consecutive days. 12 h before the last administration, food was fasted with free access to water, during which two consecutive doses of YQHYJDF or distilled water were administered. 1 h after the last administration, the rats were anesthetized, and blood was collected from the abdominal aorta. Whole blood was centrifuged at 1,500 × g for 15 min at 4 °C to isolate serum. The obtained serum was inactivated at 56 °C in a water bath for 30 min and then filtered through a 0.22 µm filter for sterilization. After aliquoting, the serum was stored at -80 °C until use.

8. Culture and treatment of RAW264.7 cells

RAW264.7 murine macrophage cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM,4.5 g/L glucose) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. All in vitro experiments were performed with three independent biological replicates (n = 3), each using separately passaged RAW264.7 cell batches under identical experimental conditions.

Cells were treated with varying concentrations of YQHYJDF drug-containing serum or normal rat serum. The optimal intervention concentration was determined using the MTS proliferation assay. To determine the optimal intervention concentration, RAW264.7 cells were exposed to YQHYJDF-containing serum at concentrations of 2.5%, 5%, 10%, 15%, 20%, and 25% (v/v), with normal rat serum at equivalent concentrations serving as controls. Cell viability was assessed after 24 h of incubation using the MTS proliferation assay. Cell viability remained above 85% at concentrations up to 10%, while concentrations of 15% and above resulted in a progressive and significant decline in cell viability. Therefore, 10% (v/v) YQHYJDF-containing serum was selected as the optimal concentration for all subsequent experiments, as it represented the highest pharmacologically relevant concentration that preserved acceptable cell viability.

For the experiment, 1 × 10⁶ RAW264.7 cells were seeded in 6-well plates and treated with YQHYJDF drug-containing serum or normal rat serum. Polarization was induced by stimulation with 1 ug/mL LPS, and this induction was maintained for 72 h. This duration was selected based on preliminary experiments and established protocols for inducing a stable M1-like inflammatory phenotype in RAW264.7 cells, allowing for the full expression and secretion of target chemokines. After the intervention, the cells were detached by pipetting and centrifuged at 500 × g for 5 min at 4 °C. The supernatant was collected for enzyme-linked immunosorbent assay (ELISA) analysis, and cell pellets were lysed using TRIzol for subsequent qPCR detection.

9. Single-cell suspension preparation

Fresh rat lung tissues were minced into approximately 1 mm³ fragments, washed three times with ice-cold PBS, and enzymatically dissociated using the Tumor Dissociation Kit for 30 min at room temperature. The resulting cell suspension was filtered through 70 um and 40 um cell strainers, followed by centrifugation (4 °C, 300 × g, 10 min). After removal of the supernatant, red blood cells were lysed using red blood cell lysis buffer for 10 min at 4 °C. The remaining cells were washed twice with DPBS containing 0.04% BSA. Cell suspensions were adjusted to a final concentration of 1,000 cells/µL, and samples with cell viability > 80% were considered qualified for subsequent analysis.

10. Single-cell RNA library preparation and sequencing

Single-cell RNA libraries were constructed using the High-Throughput Single-Cell RNA Library Prep Kit. Cells were encapsulated with barcoded beads using DNBelab C-TaiM 4 droplet generation system, followed by reverse transcription at 42 °C for 90 min. After magnetic bead-based purification of reverse transcription products, cDNA was PCR-amplified and purified. Sequencing libraries were constructed through cDNA fragmentation, end repair, adapter ligation, and indexing PCR, then sequenced on a high-throughput next-generation sequencing (NGS) platform with paired-end sequencing.

11. Single-cell RNA sequencing data processing

Raw sequencing data were processed using the scRNA analysis pipeline for sample demultiplexing, barcode processing, and unique molecular identifier (UMI) counting. Processed reads were aligned to the rat reference genome using splice-aware RNA sequencing read alignment software. Valid cell barcodes were identified using “barcodeRanks” function from a software package for preprocessing droplet-based single-cell RNA sequencing data. Gene expression quantification was performed with gene expression quantification software, and downstream analysis was conducted in R using the Seurat package.

For quality control, the following metrics were calculated for each cell, including: (1) number of detected genes (nFeature_RNA); (2) total UMI counts (nCount_RNA); (3) percentage of mitochondrial gene expression (genes with “mt-” prefix); (4) percentage of hemoglobin gene expression to exclude red blood cell contamination. Cells were retained if they met the following criteria: 500–7,000 detected genes, mitochondrial gene percentage <20%, hemoglobin gene percentage <3%, and total UMI counts >1,000. Data were normalized using the “LogNormalize” method with a scaling factor of 10,000. Highly variable genes were identified using the “vst” method, and the top 2,000 genes were selected for downstream analysis. Cell cycle scoring was performed using Seurat’s built-in cell cycle marker genes, and cell cycle effects (G2M.Score and S.Score) were regressed out during data scaling. Potential doublets were identified and removed using a computational tool for doublet detection in single-cell RNA sequencing data, and cells classified as doublets were excluded from subsequent analyses³¹.

Principal component analysis (PCA) was performed on highly variable genes, and the first 30 principal components were selected based on elbow plot inspection³². Batch effects across samples were corrected using a software package for batch-effect correction and integration of single-cell RNA sequencing data³³. Cell clustering was performed using the Leiden algorithm implemented in Seurat’s FindClusters function with a resolution of 0.1 to identify major cell types. Uniform Manifold Approximation and Projection (UMAP) was applied for visualization based on the Harmony-corrected first 30 principal components. Cell type annotation was performed based on the expression of canonical marker genes identified using the FindAllMarkers function (Wilcoxon rank-sum test, logfc.threshold = 0.25, p < 0.05). Major cell types were identified including: macrophages/monocytes (Cd163, Aif1, C1qa), natural killer cells (Nkg7, Klrd1), B cells (Cd79a, Ms4a1), T cells (Cd3e, Cd3d), endothelial cells (Cav1, Pecam1), neutrophils (S100a9, S100a8), fibroblasts (Col1a1, Den), epithelial cells (Epcam, Ager, Sftpc), and other minor cell types based on established markers reported in the literature. Macrophages and monocytes were extracted for in-depth subset analysis³⁴. Differential gene expression analysis between experimental groups was performed using the MAST method for each cell type. Genes with adjusted p-value < 0.05 and |log2 fold change| > 0.25 were considered significantly differentially expressed. Functional enrichment analysis was conducted for Gene Ontology (GO) and Encyclopedia of Genes and Genomes (KEGG) pathway analysis using the clusterProfiler package.

12. Network pharmacology prediction

Ingredients of YQHYJDF were retrieved from the Traditional Chinese Medicine Pharmacology Platform (https://www.tcmsp-e.com/#/database)³⁵. The components were screened based on the criteria of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 to identify active ingredients^36,37. The corresponding gene targets of these active ingredients were further obtained from the TCMSP database. Using the keyword “Acute Respiratory Distress Syndrome” related target genes were retrieved from the GeneCards database (https://www.genecards.org/)³⁸. The overlapping targets between YQHYJDF-related targets and ARDS-related targets were identified as key targets of YQHYJDF for the treatment of ARDS. A regulatory network of active ingredients and key targets of YQHYJDF for ARDS treatment was constructed and visualized using Cytoscape. The key targets were analyzed for protein-protein interaction (PPI) using the STRING database, and the PPI network was visualized with Cytoscape^39,40. The top 20 targets ranked by degree of connectivity were presented in a bar chart using R software. The key targets of YQHYJDF for ARDS treatment were imported into the Bioconductor database (https://www.bioconductor.org/) via R software (https://www.r-project.org/) for KEGG pathway enrichment analysis⁴¹. Pathways with P< 0.05 were considered statistically significant, and those pathways directly related to human diseases or metabolism were excluded. The top 15 pathways and their associated genes were used to construct a “target-gene-signaling pathway” network, which was visualized using Cytoscape⁴².

13. Real-time quantitative PCR

Lung tissues were pulverized in liquid nitrogen. Approximately 100 mg of tissue sample or 5 × 10⁶ cells were lysed in 1 mL of TRIzol reagent and incubated at room temperature for 5 min. After adding 200 µL of chloroform, the mixture was vigorously vortexed for 15 s, incubated at room temperature for 3 min, and finally centrifuged at 12,000 × g for 15 min at 4 °C. The upper aqueous phase was carefully collected and mixed with an equal volume of isopropanol by gentle inversion, followed by incubation at -20 °C overnight. After centrifugation (4 °C, 12,000 x g, 15 min), the supernatant was discarded, and the RNA pellet was washed twice with 75% ethanol. The pellet was air-dried at room temperature for 10 min and dissolved in DEPC-treated RNase-free water. RNA concentration and purity were measured using a a microvolume UV–Vis spectrophotometer.

For complementary DNA (cDNA) synthesis, 1 µg of total RNA was reverse transcribed using a reverse transcription kit in a 20 µL reaction system (15 min at 42 °C followed by 95 °C for 5 s to terminate the reaction). qPCR was performed using SYBR Green Real-Time PCR Master Mix. Each 20 µL reaction mixture contained 10 µL of SYBR Green Master Mix, 2 µL of cDNA template, 0.8 µL each of forward and reverse primers (10 µM), and 7.4 uL of ddH₂O. Amplification was performed on a quantitative real-time PCR (qPCR) system under the following conditions: an initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s. The ß-actin gene was used as an internal reference, and relative expression of target genes was calculated using the 2^-ΔΔCt method. The primer sequences used are listed in Table 1.

14. Western blot analysis

RIPA lysis buffer supplemented with phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors was prepared and kept on ice. Lung tissue was ground in liquid nitrogen, and 20 mg of tissue powder was accurately weighed using a precision balance and homogenized in 250 µL of ice‑cold lysis buffer. The mixture was lysed on ice for 30 min, followed by centrifugation (4 °C, 12,000 x g, 15 min) to collect the supernatant. Protein concentration was determined using a BCA protein assay kit, and all samples were adjusted to the same concentration with RIPA lysis buffer. Protein samples were mixed with 5× SDS loading buffer and denatured by boiling at 95 °C for 5 min. Equal amounts of denatured samples were separated by SDS‑PAGE and then transferred onto PVDF membranes. The membranes were blocked with 5% (w/v) non‑fat milk in TBST buffer for 1 h at room temperature, followed by incubation with appropriately diluted primary antibodies at 4 °C overnight. The dilution ratios were 1:1000 for the specific primary antibody and 1:80,000 for the β‑actin primary antibody. After washing three times with TBST (10 min each), the membranes were incubated with an HRP‑conjugated secondary antibody at a dilution of 1:2500 for 1 h at room temperature, followed by three additional TBST washes (10 min per wash). Protein bands were detected with an ECL chemiluminescence substrate and imaged using a chemiluminescence imaging system. Band intensities were quantified using ImageJ. To assess the activity of signaling pathways, the relative levels of phosphorylated proteins were normalized to their corresponding total proteins (p-AKT/AKT, p-p38 MAPK/p38 MAPK, and p-NF-κB p65/NF-κB p65). β-actin was used as a loading control to confirm consistent protein loading across samples.

15. ELISA

Cell culture supernatant was obtained by centrifugation (10,000 × g for 10 min at 4 °C for subsequent detection. Prior to the assay, the ELISA kit was allowed to equilibrate at room temperature for 30 min. Standard solutions were prepared by two-fold serial dilution according to the manufacturer’s instructions. Both standards and samples were added to the pre-coated microplate wells. Subsequently, biotinylated detection antibody, enzyme conjugate, chromogenic substrate, and stop solution were added sequentially as directed in the kit protocol. Absorbance was measured at 450 nm using a microplate reader, with a reference wavelength set at 630 nm. The concentration of the target protein in each sample was determined based on the standard curve generated using a curve-fitting and regression analysis software.

16. Statistical analysis

All statistical analyses were performed using a statistical analysis software. Data for continuous variables are presented as mean ± standard deviation (SD). For comparisons among the three experimental groups (blank control group, ARDS model group, and YQHYJDF pre-treatment group), one-way analysis of variance (ANOVA) was first applied to test for overall group differences. When a statistically significant main effect was identified, post hoc pairwise comparisons were conducted using Tukey's Honestly Significant Difference (HSD) test to control for the familywise error rate under multiple comparisons. A two-tailed p-value of less than 0.05 was considered statistically significant.

Results

Protective effects of YQHYJDF on pulmonary pathology in ARDS rats

Histological examination of HE-stained lung sections from the blank control group revealed well-preserved alveolar structures with intact alveolar walls, absence of alveolar or interstitial congestion or edema, and only minimal inflammatory cell infiltration (Figure 1A). In contrast, lung tissues from the ARDS model group exhibited pronounced pathological alterations, including dist...

Discussion

ARDS is a critical respiratory condition characterized by an excessive and dysregulated inflammatory response^43,44,45. The pathological process begins with injury to alveolar epithelial cells and pulmonary capillary endothelial cells, triggering an inflammatory cascade that leads to increased permeability of the alveolar-capillary membrane, excessive exudation causing pulmonary edema, and dysfunction of pulmonary surfactant

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% Paraformaldehyde	Servicebio	G1101
5x Protein Loading Buffer	Solarbio	P1040
ABI 7500 Real-Time PCR System	Applied Biosystems	4351107
AKT Antibody	Cell Signaling Technology	4691S
Antibody Diluent Solution	Beyotime Biotechnology	P0268
BCA Protein Assay Kit	Beyotime Biotechnology	P0012S
Bovine Serum Albumin (BSA)	Sangon Biotech	A600332-0005
clusterProfiler R Package	Yu Lab (Guangchuang Yu)	4.0.5	RRID:SCR_016884
Cytoscape	Cytoscape Consortium	3.9.1	RRID:SCR_003032
DNBelab C-TaiM 4 System	MGI Tech Co., Ltd.	Not Applicable	scRNA-seq library generating system
DNBelab Single-Cell Library Prep Kit	MGI Tech Co., Ltd.	1000020432
DoubletFinder R Package	McGinnis, C.S. et al.	2.0.3
Dulbecco's Phosphate-Buffered Saline (DPBS)	Gibco	10010031
Escherichia coliLipopolysaccharide (LPS)	Sigma-Aldrich	L6511
Harmony R Package	Korsunsky, I. et al.	1.0	RRID:SCR_022206
Hematoxylin and Eosin (H&E) Staining Kit	Solarbio	G1120
HRP-conjugated Goat Anti-Mouse IgG	Zhongshan Jingqiao Biotechnology	ZB-5305
HRP-conjugated Goat Anti-Rabbit IgG	Zhongshan Jingqiao Biotechnology	ZB-2301
Isoflurane	RWD Life Science	R510-22
MACS Tumor Dissociation Kit	Miltenyi Biotec	130-095-929
MGISEQ-2000RS Sequencing Platform	MGI Tech Co., Ltd.	Not Applicable	scRNA-seq sequencing system
Mouse CCL12/MCP-5 ELISA Kit	Novus Biologicals	MCC120
Mouse CCL2/MCP-1 ELISA Kit	4A Biotech	CME0046
Mouse CCL7/MCP-3 ELISA Kit	Novus Biologicals	KA2199
MTS Assay Kit	Promega	G3580	CellTiter 96 AQueous One Solution
NanoDrop 2000 Spectrophotometer	Thermo Fisher Scientific	ND-2000
NF-κB p65 Antibody	Cell Signaling Technology	8242T
Non-fat Dry Milk	Solarbio	D8340
p38 MAPK Antibody	Abclonal	A14401
Phenylmethylsulfonyl Fluoride (PMSF)	Solarbio	P0010
Phosphatase Inhibitor Cocktail	Beyotime Biotechnology	P1081
Phospho-AKT (Ser473) Antibody	Cell Signaling Technology	4060S
Phospho-p38 MAPK Antibody	Abclonal	AP0526
PISA (Protein Interfaces, Surfaces and Assemblies)	EMBL-EBI	Not Applicable	https://www.ebi.ac.uk/pdbe/pisa/
Polyvinylidene Fluoride (PVDF) Membrane	Millipore	IPVH00010
R Project for Statistical Computing	The R Foundation	4.1.3	RRID:SCR_001905
RAW 264.7 Cell Line	ProcellLifeScience&TechnologyCo.,Ltd.	GCL-0190
Red Blood Cell Lysis Buffer	Tiangen Biotech	RT122-1
Reverse Transcription System	Promega	A3500
RIPA Lysis Buffer	Solarbio	R0010
RNase-free water	Ambion	AM9937
SDS-PAGE Electrophoresis Buffer	Servicebio	G2144
Seurat R Package	Satija Lab	4.1.1	RRID:SCR_016341
SPSS Statistics	IBM	26.0	RRID:SCR_002865
STAR (Spliced Transcripts Alignment to a Reference)	Dobin, A. et al.	2.7.10a
SYBR Green Realtime PCR Master Mix	TOYOBO	QPK-201
TBST Buffer (10X)	Solarbio	T1085
TE Buffer, pH 8.0	Ambion	AM9858
Tris-Glycine Transfer Buffer	Servicebio	G2017
Trypan Blue solution	Gibco	15250061
β-Actin Antibody	Zhongshan Jingqiao Biotechnology	TA-09