Mechanism of San Jie Tong Mai Fang in Atherosclerosis Attenuation by JAK/STAT-mediated Inhibition of Macrophage M1 Polarization

Summary

This study presents a protocol using an ApoE^-/- mouse atherosclerosis model. We examined JAK/STAT pathway activation and macrophage M1/M2 polarization in aortic tissues, revealing that San Jie Tong Mai Fang attenuates atherosclerosis by suppressing JAK/STAT signaling and balancing macrophage polarization, thus clarifying its anti-atherogenic mechanism.

Abstract

Macrophage polarization influences the atherosclerotic plaque microenvironment by driving inflammatory responses, with the JAK/STAT signaling pathway serving as a critical regulator of this process. This study investigated whether San Jie Tong Mai Fang (SJTMF), an herbal formulation reported to promote M2-type macrophage polarization, alleviates atherosclerosis (AS) by modulating the JAK/STAT signaling pathway. An AS model was established in ApoE^-/- mice via 12-week high-fat diet feeding, followed by 4-week treatment with SJTMF alone or combined with a JAK inhibitor or macrophage scavenger.

Our findings demonstrated that SJTMF significantly attenuated atherosclerotic plaque formation in ApoE^-/- mice, concomitant with improved blood lipid metabolism and inflammatory levels. We also observed that the expression of Arginase-1 (Arg-1) and interleukin-10 (Il-10) was upregulated by SJTMF, whereas the expression of inducible Nitric Oxide Synthase (iNos) and interleukin-1beta (Il-1β) was downregulated in the aortic tissues of ApoE^-/- mice. Notably, the effect of SJTMF increased with co-administration of JAK inhibitors (decreased p-JAK2 and p-STAT3 levels, p < 0.01), whereas it was significantly inhibited by the combination with macrophage scavengers. Our results demonstrated that SJTMF may contribute to inhibiting AS by modulating M1-type polarization of macrophages, thereby attenuating inflammation; this effect may occur through suppression of the JAK/STAT signaling pathway.

Introduction

Atherosclerosis (AS) is a vascular disease driven by both congenital and adaptive immune inflammation. According to the World Health Organization, cardiovascular diseases claim approximately 17.9 million lives annually, accounting for 31% of global mortality and representing a critical public health challenge requiring urgent intervention strategies¹. The 2019 global burden of cardiovascular diseases and risks report highlights atherosclerosis as an important pathological target for the progression of cardiovascular disease². Inflammation is involved in the initiation, development, and thrombotic complications of AS³.

Specifically, early-stage endothelial dysfunction facilitates the transendothelial accumulation of oxidized low-density lipoprotein (ox-LDL) within the subendothelial layer through impaired barrier function, a pivotal initiating event in atherogenesis⁴. These ox-LDL, synergizing with pro-inflammatory cytokines, activate endothelial cells and trigger the chemotactic recruitment of monocytes into the arterial intima⁵. The monocytes bind to the adhesion molecules expressed on the endothelium of the injured vessels and then transform into macrophages. The macrophages engulf the ox-LDL, which induces the release of inflammatory cytokines from inflamed tissues. This leads to the formation of lipid-rich, necrotic cores within the atherosclerotic lumen, causing the vessels to lose elasticity and eventually form atherosclerotic plaques⁶.

Macrophages, as very important leukocytes in the body, especially in plaques, are representative cells of AS. Macrophages can alter their phenotypic and functional characteristics in accordance with the local microenvironment in a reversible process called "macrophage polarization"^7,8. Polarized macrophages can be roughly classified into two major groups: classic M1-type macrophages and classic M2-type macrophages (M2a, M2b, M2c, M2d). M1-type macrophages induce pro-inflammatory effects via the production of Th1 cytokines such as Interleukin-1beta (IL-1β) and IL-12. In contrast, M2-type macrophages support the effector function of Th2-associated cytokines by producing anti-inflammatory cytokines such as iIL-10 and transforming growth factor-beta, which control inflammation and promote tissue repair⁹.

M1-type macrophages occur in the early stages of AS where they are mainly found in progressive plaques and the fatty necrosis core. M2-type macrophages appear in the late stages of AS where they predominate in stable plaques, where inflammation resolves, and in the vascular adventitia¹⁰. This indicates a correlation between the contents of M1 and M2 macrophages and the development trend of blood vessels in AS, which also partially influences the final course of the atherosclerotic plaque. Surface markers on M1-type macrophages, such as CD80 and CD86, are involved in initiating inflammatory responses or damaging endothelial tissue, while surface markers on M2-type macrophages, such as CD206 and CD163, are involved in the repair of inflammatory responses or controlling fibrosis^11,12,13. Thus, modulation of M1/M2 macrophage polarization, particularly promoting phenotypic transition from pro-inflammatory M1 to anti-inflammatory M2 phenotypes, represents a promising therapeutic strategy for mitigating inflammatory responses and attenuating atherosclerotic progression.

Numerous and complex pathways, such as Phosphatidylinositol 3-kinase/protein kinase B, Notch, Janus kinase/signal transducer and activator of transcription (JAK/STAT), and Toll-like receptor 4 pathways, are involved in the development of macrophage polarization¹⁴. In contrast, JAK/STAT, a classical inflammatory pathway, is involved in many important biological processes. It not only induces macrophages to maintain a proinflammatory phenotype but also promotes the development of AS^15,16. Wan et al. demonstrated that targeted inhibition of the JAK/STAT signaling pathway markedly suppressed M1-type macrophage polarization, thereby attenuating cadmium-exposure-induced AS progression¹⁷.

With the advancement of scientific modernization in Traditional Chinese Medicine (TCM), coupled with its cost-effectiveness and favorable safety profiles, the therapeutic potential of TCM in attenuating AS has been increasingly recognized. Notably, experimental studies have revealed that TCM compounds can reduce macrophage infiltration within atherosclerotic plaques and promote polarization toward the anti-inflammatory M2-type. This phenotypic shift contributes to the restoration of M1/M2 macrophage homeostasis in AS⁹. Of note, the JAK/STAT signaling pathway has been identified as a key target through which TCM affects the pathogenesis of AS^18,19,20. Meanwhile, our previous study had demonstrated that bioactive components in San Jie Tong Mai Fang (SJTMF)-including organic acids, flavonoids, phenylpropanoids, and terpenoids-selectively enhance M2-type macrophage polarization through the modulation of macrophage autophagy, thereby attenuating AS progression²¹. Therefore, we hypothesize that SJTMF can modulate macrophage polarization via the mediation of the JAK/STAT signaling pathway, thereby attenuating AS in ApoE^-/- mice.

Protocol

The protocol was approved by the Experimental Animals Committee of Changchun University of Chinese Medicine (Ethics Approval No. 2023521). Healthy male C57BL/6J and ApoE^-/- male mice (SPF grade; 18-22 g, 5 weeks old) were used in the study (Laboratory Animal Use License Nos. SCXK (JI) 2023-002 and SCXK (SU) 2021-0013). The details of the reagents and equipment used are listed in the Table of Materials.

1. Acclimatization

1. House all mice in specific pathogen-free (SPF) facilities under controlled conditions (22-26 °C, 50-60% humidity) with a 12 h light/dark cycle. Provide ad libitum access to food and water. Allow 7 days for acclimatization before initiating the experimental protocol.

2. Preparation of SJTMF

1. To prepare SJTMF, weigh the following 14 traditional Chinese medicines, including Salvia miltiorrhiza (Dan Shen) 30 g, Panax notoginseng (San Qi) 10 g, Artemisia scoparia (Yin Chen) 10 g, Poria cocos (Fu Ling) 10 g, Alisma plantago-aquatica L (Ze Xie) 30 g, Amomum villosum (Sha Ren) 10 g, Santalum albumL (Tan Xiang) 10 g, Citrus aurantium (Zhi Qiao) 10 g, Citrus reticulata peel (Chen Pi) 10 g, Whitmania pigra Whitman (Shui Zhi) 10 g, Bombyx batryticatus (Jiang Can) 10 g, Periostracum cicadae (Chan Tui) 10 g, Caulis Bambusae in Taeniam (Zhu Ru) 10 g, and Ophicalcitum (Hua Rui Shi) 30 g.
2. Decoct the 14 herbal materials (total weight: 1,800 g) 3x with 8-fold volumes of distilled water (1.5 h per extraction).
3. Combine the decoctions and filter through sterile gauze (100 mesh nylon filter; pore size: 149 µm).
4. Concentrate the filtrate at 60 °C to obtain a dense extract (relative density: 1.23-1.27). Dry the concentrated extract under reduced pressure at 70 °C.
5. Pulverize the resulting solid residue (370.55 g) through an 80 mesh sieve (20.586% yield).
6. Calculate the murine equivalent dose using body surface area normalization (3.56 g∙kg^-1∙day^-1).

3. Model establishment

1. Feed ApoE^-/- mice on a high-fat diet (HFD) (20% fat, 1.25% cholesterol) for 12 weeks to induce AS. Change bedding weekly and measure body weight at 7 day intervals to monitor metabolic status.
2. Assign C57BL/6J mice to the Control group (Control, normal diet + 0.025 mL∙g^-1∙day^-1 saline). Randomly assign successfully modeled mice into six groups: Model group (Model, saline 0.025 mL∙g^-1∙day^-1), Atorvastatin group (Atorvastatin, 0.0026 mg/g atorvastatin), SJTM-L group (SJTM-L, SJTMF 3.56 g∙kg^-1∙day^-1), SJTM-H group (SJTM-H, SJTMF 7.12 g∙kg^-1∙day^-1), SJTM-H+Clodronate group (SJTM-H + Clod, SJTMF 7.12 g∙kg^-1∙day^-1), SJTM-H+JAK-IN group (SJTM-H+JAK-IN ,SJTMF 7.12 g∙kg^-1∙day^-1). Administer all interventions (Atorvastatin and SJTMF formulations) via standardized intragastric gavage at a fixed volume of 0.4 mL per mouse daily in the morning for 4 weeks.
3. Prepare 100 mL of SJTMF stock solution for the SJTM-L group by dissolving 24.92 g in warm water. Prepare 100 mL of SJTMF stock solution for the SJTM-H group by dissolving 49.84 g in warm water. Store the solutions at 4 °C. Before intragastric gavage, warm the required stock solution in a 37 °C water bath for 5 min.
   NOTE: Begin experimental interventions in week 9. Align the administration protocol with clinical TCM decoction delivery by maintaining temporal rhythm (morning dosing) and applying a human-to-mouse dose conversion factor of 9.1.
4. For the SJTM-H+Clod group: Administer clodronate liposomes (5 mg/kg, 0.2 mL per mouse) intraperitoneally once weekly, beginning 1 week before modeling and continuing throughout the experiment²².
5. For the SJTM-H+JAK-IN group: Administer JAK-IN-1 (10 mg/kg, 0.1 mL per mouse) intraperitoneally once weekly for 4 weeks during the experimental intervention period²³.

4. Sample collection

1. Blood collection and serum preparation
   1. Collect blood from the orbital venous plexus via the eyeball removal method²⁴.
   2. Transfer blood samples into centrifuge tubes and allow them to rest at room temperature for 1-2 h. Centrifuge to isolate the serum (following a previously published report²¹).
2. Aorta harvesting and preservation
   1. Isolate the aorta from the aortic root to the common iliac artery bifurcation.
   2. Fix the aorta in paraformaldehyde or flash-freeze it in liquid nitrogen.
      NOTE: Collect the serum and store aortic tissues at -80 °C for future analysis.

5. Histopathological assessment

NOTE: The entire aortic segment from the aortic root to the abdominal aorta was harvested for gross Oil Red O staining to assess plaque distribution. A 1 cm proximal aortic segment (adjacent to the heart) was dissected; the tissues were rinsed with precooled PBS at 4 °C to remove blood stains, fixed in 4% paraformaldehyde for over 24 h, dehydrated through a graded ethanol series (75%, 85%, 90%, 95%), embedded in paraffin, sectioned into 4 µm slices, and stained with hematoxylin-eosin (HE) for analysis of plaque tissue structure and extracellular matrix lipid deposition²⁵.

1. Gross oil red O staining
   1. Fix the aorta in 4% paraformaldehyde for 24 h, then wash 2x with PBS.
   2. Dissect the vessels longitudinally, immerse them in Oil Red O stain, and incubate at 37 °C for 60 min.
   3. Destain luminal fatty plaques with 75% ethanol until reddish-orange coloration develops, ensuring nonfatty plaques remain nearly colorless.
   4. Fix the stained aortic tissue on a blue background, adjust the focus under optimal lighting, and photograph.
2. HE staining
   1. Deparaffinization and rehydration
      1. Immerse sections sequentially in xylene for 2 x 20 min, followed by absolute ethanol for 2 x 10 min. Hydrate sections sequentially in graded ethanol series (95%, 90%, 80%, 70%) for 5 min per concentration.
   2. Histochemical staining
      1. Stain sequentially with 0.4% hematoxylin for 3-8 min and 0.5% eosin solution for 1-3 min.
         NOTE: Rinse thoroughly with distilled water.
   3. Dehydration and mounting
      1. Dehydrate sections through an ascending ethanol series (95% to absolute ethanol) for 5 min per concentration. Clear sections in xylene for 2 x 5 min. Air-dry completely and mount with neutral resin.
   4. Observe under a microscope and photograph.

6. Enzyme-linked immunosorbent assay (ELISA)

NOTE: Hyperlipidemia is a well-established risk factor for AS. Inflammatory responses, mediated through the release of proinflammatory cytokines and recruitment of immune cells such as monocytes/macrophages, critically drive the formation and progression of atherosclerotic plaques. Consequently, measurement of lipid levels and inflammatory cytokines are essential for prevention and treatment of AS.

1. Total cholesterol and triglycerides (TC and TG)
   NOTE: Serum samples cryopreserved at -80 °C were thawed at 4 °C prior to experimental procedures.
   1. Prepare a 96-well plate and designate blank, calibration, and sample wells. Add 2.5 µL of serum, calibration standard, or blank solution to each respective well.
   2. Add 250 µL of working solution to each well and mix gently. Incubate for 10 min at 37 °C.
   3. Measure absorbance at 500 nm using a microplate reader according to the manufacturer's protocol.
2. Low-density lipoprotein and high-density lipoprotein (LDL-C and HDL-C)
   1. Prepare a 96-well plate and designate blank, calibration, and sample wells. Add 2.5 µL of serum, calibration standard solution, or blank solution to each respective well.
   2. Add reagents according to the kit instructions: 180 µL of Reagent 1 or 60 µL of Reagent 2 per well.
   3. Incubate the plate for 2 x 5 min at 37 °C, then set the measurement wavelength to 600 nm.
3. Inflammatory cytokines (IL-6, TNF-α, IL-1β)
   1. Prepare standard curves using serially diluted standards. Include blank wells and standard wells (50 µL per standard well).
   2. Add 10 µL of the test sample to each sample well, followed by 40 µL of sample diluent.
      NOTE: Leave blank wells untreated.
   3. Add 100 µL of enzyme-labeled reagent to all standard and sample wells (excluding blank wells). Seal the plate with adhesive film and incubate at a constant temperature for 60 min at 37 °C.
   4. Discard the liquid, gently pat the wells dry, wash with washing buffer for 1 min, shake off excess buffer, and repeat the wash cycle 5x.
   5. After adding 50 µL of substrate A and 50 µL of substrate B to each well, incubate at 37 °C in the dark for 15 min.
   6. After adding 50 µL of stop solution to each well, measure the optical density (OD) at 450 nm within 15 min.

7. Quantitative real-time polymerase chain reaction (qPCR)

NOTE: qPCR was used to analyze the expression levels of specific genes in mouse aortic tissues, aiming to assess the impact of experimental interventions on these genes.

1. RNA extraction from aortic tissue
   1. Precool a homogenization tube containing 1 mL of RNA extraction solution on ice. Transfer approximately 20 mg of tissue into the precooled homogenization tube.
   2. Homogenize the aortic tissues in RNA lysis buffer. Centrifuge the homogenate at 13,780 × g for 10 min, then collect the supernatant. Add 250 µL of chloroform, vortex vigorously, and centrifuge again to isolate the aqueous phase.
   3. Transfer the aqueous phase to a new tube and precipitate RNA by adding an equal volume of 70% ethanol. Add 52 µL of RNase-free water to the adsorption column to elute RNA, then centrifuge at 13,780 × g for 1 min.
   4. Determine RNA concentration and purity using a spectrophotometer.
      NOTE: Refer to Supplemental Table S1 for RNA purity assessment details.
2. RNA reverse transcription
   1. Incubate the first-step reaction mixture at 42 °C for 2 min, and the reaction by heating at 85 °C for 5 s. Store the cDNA products at -20 °C for further use.
      NOTE: Refer to Supplemental Table S2 and Supplemental Table S3 for detailed reverse transcription reaction conditions and qPCR primer sequences, respectively.
3. PCR amplification
   1. Predenature at 94 °C for 30 s, then perform 40 amplification cycles consisting of denaturation at 94 °C for 15 s, annealing at 61 °C for 35 s, and extension at 72 °C for 25 s per cycle, followed by melting curve analysis through sequential incubations at 95 °C for 10 s, 65 °C for 60 s, and 97 °C for 1 s.
      NOTE: Mus β-actin was used as an internal reference gene, and the 2^-ΔΔCT value of each group was the relative expression of the gene (refer to Supplemental Table S4 for PCR amplification conditions).

8. Immunofluorescence

NOTE: The spatial distribution of antigens in mouse aortic tissues was analyzed by immunofluorescence staining with fluorescently labeled specific antibodies.

1. Tissue preparation
   1. Process aortic tissues by paraffin embedding and dewaxing to water (refer to step 5.2.1). For antigen retrieval, heat sections in 0.01 M EDTA buffer (125 °C, 103 kPa). Rinse the heated sections for 3 x 3 min with PBS.
2. Endogenous peroxidase blocking
   1. Incubate sections in 3% hydrogen peroxide (10 min, humidified chamber), then wash for 3 x 5 min with PBS (pH 7.4) on a laboratory shaker.
3. Blocking
   1. Treat sections with 10% goat serum (30 min, humidified chamber), then rinse for 3 x 3 min with PBS.
4. Primary antibody incubation
   1. Apply diluted primary antibodies (e.g., CD206, 1:200; CD86, 1:150; p-STAT3, 1:100; see Table of Materials) to sections and incubate overnight at 4 °C. Wash sections for 3 x 3 min with PBS.
5. Secondary antibody incubation
   1. Incubate sections with fluorescence-conjugated secondary antibody (1:400 dilution) at 37 °C for 1 h. Rinse sections with PBST (3 x 3 min). Remove unbound antibody with PBST washes (4 x 3 min).
6. Nuclear counterstaining
   1. Stain nuclei with DAPI, then rinse with PBST (4 x 5 min).
7. Mounting and imaging
   1. Finalize with PBS (pH 7.4) washes (3 x 5 min), mount slides using mounting medium, and capture images under a fluorescence microscope.

9. Western blotting (WB)

NOTE: WB allows for the evaluation of activation status and interactions among key signaling molecules, as well as detection of protein phosphorylation. This technique thereby enables assessment of pharmacological interventions' effects on protein expression levels.

1. Tissue preparation
   1. Wash the aortic tissues with precooled PBS to remove blood contamination, cut them into small pieces, and transfer into a homogenization tube.
   2. Homogenize the aortic tissues in RIPA lysis buffer (containing protease inhibitors) at a 10:1 volume-to-tissue ratio for 30 min. Centrifuge the lysate at 13,780 × g for 10 min and collect the supernatant. Store protein extracts at -20 °C.
2. Protein quantification
   1. Determine total protein concentration using the BCA assay kit following the manufacturer's instructions (see Table of Materials).
3. Sample preparation
   1. Mix the protein solution with 5x loading buffer (containing β-mercaptoethanol and SDS) at a 4:1 ratio and denature the proteins by heating the mixture at 100 °C for 15 min. Cool the samples to room temperature and store at -20 °C for subsequent use.
4. SDS-PAGE gel preparation
   1. Prepare a 5% SDS-PAGE gel and assemble it into the electrophoresis apparatus (see Supplemental Table S5).
5. Electrophoresis
   1. Load 40 µg total protein per well and run the gel.
   2. Apply 75 V to the stacking gel and 120 V to the separating gel until the dye front reaches the bottom.
6. Membrane transfer
   1. Activate the PVDF membrane by placing it in methanol for 5 min.
   2. Place the sponge pad into the transfer cassette. Position the preequilibrated PVDF membrane on top. Carefully overlay the deionized water-rinsed gel onto the membrane. Complete the assembly by adding the second sponge pad.
7. Blocking and antibody incubation
   1. Block the PVDF membranes with 5% skim milk in TBST for non-phosphorylated proteins or 3% BSA for phosphorylated proteins, then incubate with primary antibodies (e.g., JAK2, 1:1,000; p-JAK2, 1:1,000; STAT3, 1:3,000; p-STAT3, 1:1,000; β-actin, 1:5,000) overnight at 4 °C on a shaker.
8. Washing and secondary antibody incubation
   1. Wash the membranes 3 x 5 min with TBST.
   2. Dilute the secondary antibody with TBST at a 1:10,000 ratio. Incubate on a shaker at room temperature for 60 min.
9. Detection and analysis
   1. Visualize protein bands using a chemiluminescence imaging system. Quantify band intensities with imaging software.

10. Statistics

1. Perform statistical analysis.
2. Express results as mean ± standard deviation (SD).
3. Compare differences between two groups with the t-test and among multiple groups with one-way ANOVA, considering p < 0.05 statistically significant.
4. Visualize data using software.

Results

Multi-group comparisons: One-way ANOVA was performed on data satisfying normality and homogeneity of variances. If ANOVA revealed significant intergroup differences, post hoc pairwise comparisons were conducted using Tukey’s Honest Significant Difference (HSD) test to control type I error in multiple comparisons. For heteroscedastic data, the Games-Howell test was employed for post hoc analysis.

Inhibition of atherosclerotic plaque formation
Aortic atherosclerotic plaque f...

Discussion

Since Ross first proposed in 1999 that AS is an inflammatory disease, local inflammatory responses trigger activation of diverse cell populations within atherosclerotic lesions²⁶. As central immune cells in the plaque microenvironment, macrophages exert dual regulatory roles during AS pathogenesis through phenotypic polarization. Notably, cholesterol crystals and lipopolysaccharide promote macrophage polarization toward the pro-inflammatory M1-type, consequently exacerbating the inflammatory casca...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% Paraformaldehyde Fix Solution	Sinopharm Chemical Reagent Co., Ltd.	80096618
30% Hydrogen Peroxide Solution	Sinopharm Chemical Reagent Co., Ltd.	10010018
0.5–10 µL Micropipette	eppendorf	4924000029
10 – 100 µL Micropipette	eppendorf	4924000053
20 – 200 µL Micropipette	eppendorf	4924000061
100 – 1,000 µL Micropipette	eppendorf	4924000088
1000 µL Pipette Tips	NEST Life Science Co., Ltd.	303216
10 µL Pipette Tips	NEST Life Science Co., Ltd.	301016
20 µL Pipette Tips	NEST Life Science Co., Ltd.	302106
15 mL Centrifuge Tube	NEST Life Science Co., Ltd.	601052
1.5 mL Centrifuge Tube	NEST Life Science Co., Ltd.	615601
Ammonia Water	Sinopharm Chemical Reagent Co., Ltd.	10011028
Anti - Fluorescence Quenching Mounting Medium	SouthernBiotech	0100 - 01
Autoclave	Shanghai Sanshen Medical Devices Co., Ltd.	YM75
BCA Protein Assay  Reagent A	GBCBIO	G3422
BCA Protein Assay Reagent B	GBCBIO	G3522
Benchtop High - Speed Refrigerated Centrifuge	Hunan Kecheng Instrument Equipment Co., Ltd.	H1 - 16KR
Bromophenol Blue	Sinopharm Chemical Reagent Co., Ltd.	71008060
Chloroform	Sinopharm Chemical Reagent Co., Ltd.	10006818
Chemiluminescence Imaging System	Hangzhou Shenhua Technology Co., Ltd.	SH - 523
Clean Bench	Suzhou Jinghua Equipment Co., Ltd.	SW-CJ-2D
Cover Glass	Jiangsu Shitai Experimental Equipment Co., Ltd.	10212450C
ChamQ SYBR qPCR Master Mix	VAZYME	Q311-02
DAPI	Beyotime	C1002
Dithiothreitol	Biofroxx	1111gr005
Digital Display Constant Temperature Water Bath	Changzhou Jinnan Instrument Manufacturing Co., Ltd.	HH - W600
Destaining Shaker	Wuhan Lingsi Biotechnology Co., Ltd.	TSY-B
DAB Concentrated Kit	Wuhan Lingsi Biotechnology Co., Ltd.	DA1010-2*3ml
Dewatering machine	Wuhan Junjie Electronics Co., Ltd.	JT-12J
DNase/RNase-Free Water	solarbio	R1600
Eosin	Wuhan Lingsi Biotechnology Co., Ltd.	E8090
Embedding Paraffin	Sinopharm Chemical Reagent Co., Ltd.	69019361
Ethanol Absolute	Sinopharm Chemical Reagent Co., Ltd.	10009218
eBlot L1 Rapid Wet Transfer System	Genscript Biotech Corporation	L00686C
Embedding Machine	Wuhan Junjie Electronics Co., Ltd.	JB-P5
ECL Substrate Solution	affinity	KF8003
Fluorescein (FITC) - labeled Goat Anti - Rabbit IgG	Thermo Fisher Scientific	F2765
Fluorescein (Cy3) - labeled Goat Anti - Mouse IgG	Thermo Fisher Scientific	A10521
Fluorescein (Cy3) - labeled Goat Anti - Rabbit IgG	Thermo Fisher Scientific	A10520
GraphPad Prism	/	8.0.2
Glacial Acetic Acid	Sinopharm Chemical Reagent Co., Ltd.	10000218
Glycerol	Sinopharm Chemical Reagent Co., Ltd.	10010618
Glycine	Biofroxx	1275GR500
Goat Blocking Serum	Wuhan Lingsi Biotechnology Co., Ltd.	SL038-10ml
HRP - conjugated Goat Anti - Mouse Secondary Antibody	Wuhan Lingsi Biotechnology Co., Ltd.	LJS - S - 0002
HRP - conjugated Goat Anti - Rabbit Secondary Antibody	Wuhan Lingsi Biotechnology Co., Ltd.	LJS - S - 0001
HDL-C Detection Kit	Nanjing Jiancheng Bioengineering Institute	A112 - 1 - 1
Hematoxylin	Wuhan Lingsi Biotechnology Co., Ltd.	G1140
Horizontal Shaker	Haimen Qilinbeier Instrument Manufacturing Co., Ltd., Jiangsu	TS - 1
High - Speed Refrigerated Centrifuge	Hunan Kecheng Instrument Equipment Co., Ltd.	H1 - 16K
Hydrophobic Barrier Pen	Servicebio	WG1066-1
HCI	Xinyang Chemical Reagent Factory	GB622-89
Isopropano	Sinopharm Chemical Reagent Co., Ltd.	80109218
Image-Pro Plus	Media Cybernetics	Version 6.0
Imaging System	Nikon	DS-Fi3
IRIS-Fine Micro Dissecting Scissors	RWD Life Science Co., Ltd.	S12004-09
JAK Inhibitor (JAK - IN - 1)	MCE	HY - 13827
KCI	Sinopharm Chemical Reagent Co., Ltd.	10016318
KH2PO4	Sinopharm Chemical Reagent Co., Ltd.	10017618
LDL-C Detection Kit	Nanjing Jiancheng Bioengineering Institute	A113 - 1 - 1
Macrophage Depleting Agent (Clodronate Liposomes)	Shanghai Shunna Biotechnology Co., Ltd.	SN - MLE03 - 5
Mouse TNF-α ELISA	Sinobestbio	YX-2014077M
Mouse IL-6 ELISA	Sinobestbio	YX-061206M
Mouse IL-1β ELISA	Sinobestbio	YX-091203M
Mouse Anti - CD86 Antibody	Novus	NBP2 - 25208
Multiskan FC Microplate Reader	Thermo Fisher Scientific	1410101
Microscope Slide	Nantong Mevid Life Science Co., Ltd.	PC2-301
Microwave Oven	Galanz Microwave Oven Electrical Appliances Co., Ltd.	P70D20TL-P4
Neutral Resin	Wuhan Lingsi Biotechnology Co., Ltd.	G8590
Nano Spectrophotometer	Hangzhou Aosheng Instrument Co., Ltd.	Nano-100
NaCl	Sinopharm Chemical Reagent Co., Ltd.	10019318
Na2HPO4.12H2O	Sinopharm Chemical Reagent Co., Ltd.	10020318
n-Butanol	Sinopharm Chemical Reagent Co., Ltd.	100052190
Oil Red O Staining Solution	Sinopharm Chemical Reagent Co., Ltd.	71029781
Oven	Shanghai Huitai Instrument Manufacturing Co., Ltd.	DHG-9140A
PVDF Membrane (0.45 μm)	Millipore	IPVH00010
PVDF Membrane Transfer Reagent A	Genscript	L00791C
PVDF Membrane Transfer Reagent B	Genscript	L00793C
Phosphatase Inhibitor	Meilunbio	MB12707
PBS Phosphate Buffer Powder	Wuhan Lingsi Biotechnology Co., Ltd.	P1010
Pathological Microtome	Leica Instruments Co., Ltd.(Shanghai)	RM2016
Portable Centrifuge	Wuhan Lingsi Biotechnology Co., Ltd.	D1008
PCR Machine	Hangzhou Bohang Technology Co., Ltd.	GE48527
PCR Tube	NEST Life Science Co., Ltd.	401001
Rabbit Monoclonal Anti - CD206 Antibody	CST	24595T
Rabbit Polyclonal Anti - β - actin Antibody (42 kDa)	Wuhan Sanying Biotechnology Co., Ltd.	66009 - 1 - Ig
Rabbit Monoclonal Anti - JAK2 Antibody (131 KD)	Boster	BM4165
Rabbit Polyclonal Anti - p - JAK2 Antibody (131 KD)	Affinity	AF3024
Rabbit Polyclonal Anti - STAT3 Antibody (88 KD)	Wuhan Sanying Biotechnology Co., Ltd.	10253 - 2 - AP
Rabbit Polyclonal Anti - P - STAT3 Antibody (86 KD)	Affinity	AF3293
RIPA Lysis Buffer (1.5 ML)	Meilunbio	MA0151
Real - Time PCR System	Roche	LC96
RNA Isolater Total RNA Extraction Reagent	VAZYME	R401-01
SPSS software	IBM SPSS statistics	version 22
SDS	Sinopharm Chemical Reagent Co., Ltd.	30166428
Suture Tying Forceps	RWD Life Science Co., Ltd.	FC11001R-11
Stereo Dissecting Microscope	RWD Life Science Co., Ltd.	77001S
Spin Columns RM	CWBIO	CW0597S
Trizol	Thermo Fisher	15596026CZ
Total Cholesterol Detection Kit	Nanjing Jiancheng Bioengineering Institute	A111 - 1 - 1
Triglyceride Detection Kit	Nanjing Jiancheng Bioengineering Institute	A110 - 1 - 1
Tissue Flotation Machine	Wuhan Junjie Electronics Co., Ltd.	JK-6
TEMED	Sinopharm Chemical Reagent Co., Ltd.	80125336
Trise-Base	Biofroxx	1115GR500
Tween-20	Sinopharm Chemical Reagent Co., Ltd.	30189328
Vortex Mixer	Wuhan Lingsi Biotechnology Co., Ltd.	MX-F
Vertical Electrophoresis Tank	Beijing Liuyi Instrument Factory	DYCZ-24DN
Vannas Spring-Loaded Scissors	RWD Life Science Co., Ltd.	S11001-08
Xylene	Sinopharm Chemical Reagent Co., Ltd.	10023418