Method Article

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

DOI:

10.3791/68546

⸱

July 18th, 2025

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1College of Traditional Chinese Medicine, Changchun University of Chinese Medicine, 2Department of Cardiology, the Third Affiliated Clinical Hospital of Changchun University of Chinese Medicine, 3Department of Respiratory, the Affiliated Hospital of Changchun University of Chinese Medicine, 4Department of Cardiology, the Affiliated Hospital of Changchun University of Chinese Medicine, 5Research Center of Traditional Chinese Medicine, the Affiliated Hospital of Changchun University of Chinese Medicine, 6Office of the Party Committee, the Affiliated Hospital of Changchun University of Chinese Medicine

In This Article

Summary

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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

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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

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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 AS

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Protocol

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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 li....

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Results

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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.......

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Discussion

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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.......

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Disclosures

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The authors declare that they have no competing interests.

Acknowledgements

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This study was supported by the Natural Science Foundation of Jilin Province (Nos. YDZJ202401644ZYTS).

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
4% Paraformaldehyde Fix SolutionSinopharm Chemical Reagent Co., Ltd.80096618
30% Hydrogen Peroxide SolutionSinopharm Chemical Reagent Co., Ltd.10010018
0.5–10 µL Micropipetteeppendorf4924000029
10 – 100 µL Micropipetteeppendorf4924000053
20 – 200 µL Micropipetteeppendorf4924000061
100 – 1,000 µL Micropipetteeppendorf4924000088
1000 µL Pipette TipsNEST Life Science Co., Ltd.303216
10 µL Pipette TipsNEST Life Science Co., Ltd.301016
20 µL Pipette TipsNEST Life Science Co., Ltd.302106
15 mL Centrifuge TubeNEST Life Science Co., Ltd.601052
1.5 mL Centrifuge TubeNEST Life Science Co., Ltd.615601
Ammonia WaterSinopharm Chemical Reagent Co., Ltd.10011028
Anti - Fluorescence Quenching Mounting MediumSouthernBiotech0100 - 01
AutoclaveShanghai Sanshen Medical Devices Co., Ltd.YM75
BCA Protein Assay  Reagent AGBCBIOG3422
BCA Protein Assay Reagent BGBCBIOG3522
Benchtop High - Speed Refrigerated CentrifugeHunan Kecheng Instrument Equipment Co., Ltd.H1 - 16KR
Bromophenol BlueSinopharm Chemical Reagent Co., Ltd.71008060
ChloroformSinopharm Chemical Reagent Co., Ltd.10006818
Chemiluminescence Imaging SystemHangzhou Shenhua Technology Co., Ltd.SH - 523
Clean BenchSuzhou Jinghua Equipment Co., Ltd.SW-CJ-2D
Cover GlassJiangsu Shitai Experimental Equipment Co., Ltd.10212450C
ChamQ SYBR qPCR Master MixVAZYMEQ311-02
DAPIBeyotimeC1002
DithiothreitolBiofroxx1111gr005
Digital Display Constant Temperature Water BathChangzhou Jinnan Instrument Manufacturing Co., Ltd.HH - W600
Destaining ShakerWuhan Lingsi Biotechnology Co., Ltd.TSY-B
DAB Concentrated KitWuhan Lingsi Biotechnology Co., Ltd.DA1010-2*3ml
Dewatering machineWuhan Junjie Electronics Co., Ltd.JT-12J
DNase/RNase-Free WatersolarbioR1600
EosinWuhan Lingsi Biotechnology Co., Ltd.E8090
Embedding ParaffinSinopharm Chemical Reagent Co., Ltd.69019361
Ethanol AbsoluteSinopharm Chemical Reagent Co., Ltd.10009218
eBlot L1 Rapid Wet Transfer SystemGenscript Biotech CorporationL00686C
Embedding MachineWuhan Junjie Electronics Co., Ltd.JB-P5
ECL Substrate SolutionaffinityKF8003
Fluorescein (FITC) - labeled Goat Anti - Rabbit IgGThermo Fisher ScientificF2765
Fluorescein (Cy3) - labeled Goat Anti - Mouse IgGThermo Fisher ScientificA10521
Fluorescein (Cy3) - labeled Goat Anti - Rabbit IgGThermo Fisher ScientificA10520
GraphPad Prism/8.0.2
Glacial Acetic AcidSinopharm Chemical Reagent Co., Ltd.10000218
GlycerolSinopharm Chemical Reagent Co., Ltd.10010618
GlycineBiofroxx1275GR500
Goat Blocking SerumWuhan Lingsi Biotechnology Co., Ltd.SL038-10ml
HRP - conjugated Goat Anti - Mouse Secondary AntibodyWuhan Lingsi Biotechnology Co., Ltd.LJS - S - 0002
HRP - conjugated Goat Anti - Rabbit Secondary AntibodyWuhan Lingsi Biotechnology Co., Ltd.LJS - S - 0001
HDL-C Detection KitNanjing Jiancheng Bioengineering InstituteA112 - 1 - 1
HematoxylinWuhan Lingsi Biotechnology Co., Ltd.G1140
Horizontal ShakerHaimen Qilinbeier Instrument Manufacturing Co., Ltd., JiangsuTS - 1
High - Speed Refrigerated CentrifugeHunan Kecheng Instrument Equipment Co., Ltd.H1 - 16K
Hydrophobic Barrier PenServicebioWG1066-1
HCIXinyang Chemical Reagent FactoryGB622-89
IsopropanoSinopharm Chemical Reagent Co., Ltd.80109218
Image-Pro PlusMedia CyberneticsVersion 6.0
Imaging SystemNikonDS-Fi3
IRIS-Fine Micro Dissecting ScissorsRWD Life Science Co., Ltd.S12004-09
JAK Inhibitor (JAK - IN - 1)MCEHY - 13827
KCISinopharm Chemical Reagent Co., Ltd.10016318
KH2PO4Sinopharm Chemical Reagent Co., Ltd.10017618
LDL-C Detection KitNanjing Jiancheng Bioengineering InstituteA113 - 1 - 1
Macrophage Depleting Agent (Clodronate Liposomes)Shanghai Shunna Biotechnology Co., Ltd.SN - MLE03 - 5
Mouse TNF-α ELISASinobestbioYX-2014077M
Mouse IL-6 ELISASinobestbioYX-061206M
Mouse IL-1β ELISASinobestbioYX-091203M
Mouse Anti - CD86 AntibodyNovusNBP2 - 25208
Multiskan FC Microplate ReaderThermo Fisher Scientific1410101
Microscope SlideNantong Mevid Life Science Co., Ltd.PC2-301
Microwave OvenGalanz Microwave Oven Electrical Appliances Co., Ltd.P70D20TL-P4
Neutral ResinWuhan Lingsi Biotechnology Co., Ltd.G8590
Nano SpectrophotometerHangzhou Aosheng Instrument Co., Ltd.Nano-100
NaClSinopharm Chemical Reagent Co., Ltd.10019318
Na2HPO4.12H2OSinopharm Chemical Reagent Co., Ltd.10020318
n-ButanolSinopharm Chemical Reagent Co., Ltd.100052190
Oil Red O Staining SolutionSinopharm Chemical Reagent Co., Ltd.71029781
OvenShanghai Huitai Instrument Manufacturing Co., Ltd.DHG-9140A
PVDF Membrane (0.45 μm)MilliporeIPVH00010
PVDF Membrane Transfer Reagent AGenscriptL00791C
PVDF Membrane Transfer Reagent BGenscriptL00793C
Phosphatase InhibitorMeilunbioMB12707
PBS Phosphate Buffer PowderWuhan Lingsi Biotechnology Co., Ltd.P1010
Pathological MicrotomeLeica Instruments Co., Ltd.(Shanghai)RM2016
Portable CentrifugeWuhan Lingsi Biotechnology Co., Ltd.D1008
PCR MachineHangzhou Bohang Technology Co., Ltd.GE48527
PCR TubeNEST Life Science Co., Ltd.401001
Rabbit Monoclonal Anti - CD206 AntibodyCST24595T
Rabbit Polyclonal Anti - β - actin Antibody (42 kDa)Wuhan Sanying Biotechnology Co., Ltd.66009 - 1 - Ig
Rabbit Monoclonal Anti - JAK2 Antibody (131 KD)BosterBM4165
Rabbit Polyclonal Anti - p - JAK2 Antibody (131 KD)AffinityAF3024
Rabbit Polyclonal Anti - STAT3 Antibody (88 KD)Wuhan Sanying Biotechnology Co., Ltd.10253 - 2 - AP
Rabbit Polyclonal Anti - P - STAT3 Antibody (86 KD)AffinityAF3293
RIPA Lysis Buffer (1.5 ML)MeilunbioMA0151
Real - Time PCR SystemRocheLC96
RNA Isolater Total RNA Extraction ReagentVAZYMER401-01
SPSS softwareIBM SPSS statisticsversion 22
SDSSinopharm Chemical Reagent Co., Ltd.30166428
Suture Tying ForcepsRWD Life Science Co., Ltd.FC11001R-11
Stereo Dissecting MicroscopeRWD Life Science Co., Ltd.77001S
Spin Columns RMCWBIOCW0597S
TrizolThermo Fisher15596026CZ
Total Cholesterol Detection KitNanjing Jiancheng Bioengineering InstituteA111 - 1 - 1
Triglyceride Detection KitNanjing Jiancheng Bioengineering InstituteA110 - 1 - 1
Tissue Flotation MachineWuhan Junjie Electronics Co., Ltd.JK-6
TEMEDSinopharm Chemical Reagent Co., Ltd.80125336
Trise-BaseBiofroxx1115GR500
Tween-20Sinopharm Chemical Reagent Co., Ltd.30189328
Vortex MixerWuhan Lingsi Biotechnology Co., Ltd.MX-F
Vertical Electrophoresis TankBeijing Liuyi Instrument FactoryDYCZ-24DN
Vannas Spring-Loaded ScissorsRWD Life Science Co., Ltd.S11001-08
XyleneSinopharm Chemical Reagent Co., Ltd.10023418

References

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  1. Shao, B. Z., Han, B. Z., Zeng, Y. X., Su, D. F., Liu, C. The roles of macrophage autophagy in atherosclerosis. Acta Pharmacol Sin. 37 (2), 150-156 (2016).
  2. Roth, G. A., Mensah, G. A., Fuster, V. The global burden of cardiovascular diseases and risks: A compass for globa....

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Tags

Atherosclerosis AttenuationJAK STAT PathwayMacrophage PolarizationM1 MacrophagesSan Jie Tong Mai FangApoE Knockout MiceInflammatory ResponsePlaque FormationM2 MacrophagesBlood Lipid Metabolism
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