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Treg Immunomodulation Contributes to the Anti-atherosclerotic Effects of Huxin Formula in ApoE-/- Mice

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Chinese Journal of Integrative Medicine Aims and scope Submit manuscript

Abstract

Objective

To explore the effects of Huxin formula (HXF) in curtailing atherosclerosis and its underlying mechanism.

Methods

According to random number table method, 24 specific pathogen free male ApoE-/- mice were randomly divided into model group, HXF low-dose (HXF-L) group (8.4 g/kg daily), HXF high-dose (HXF-H) group (16.8 g/kg daily), and pravastatin (8 mg/kg daily) group in Experiment I (n=6 per group). C57BL/6J mice served as the control group (n=6). ApoE-/- mice in HXF-L, HXF-H, pravastatin groups were fed a Western diet and administered continuously by gavage for 12 weeks, while C57BL/6J mice in the control group were fed conventional lab mouse chow for 12 weeks. Further, Tregs were depleted by weekly intraperitoneal injection of purified anti-mouse CD25 antibody (PC61, 250 µg per mouse) for 4 weeks in Experiment II (n=6 per group). Oil Red O and Masson staining were used to evaluate the plaque area and aortic root fibrosis. The CD4+CD25+Foxp3+Treg counts in the lymph nodes and spleen cells were detected using flow cytometric analysis. The transforming growth factor-β1 (TGF-β1), interleukin (IL)-10, and IL-6 serum levels were examined by MILLIPLEX® MAP technology. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot were utilized to assess the expression of TGF-β mRNA and protein in the aorta. The expression of CD4+T lymphocytes, macrophages and smooth muscle cells in the aortic root were detected by immunofluorescence staining.

Results

HXF reduced plaque area in ApoE-/- mice (P<0.01). HXF increased the Treg counts in the lymph nodes and spleen cells (P<0.05 or P<0.01). Moreover, HXF alleviated inflammatory response via elevating IL-10 and TGF-β 1 serum levels (P<0.05), while decreasing the IL-6 serum levels in ApoE-/- mice (P>0.05). Also, HXF upregulated the expression of TGF-β mRNA and protein in the aorta (P<0.05). Additionally, HXF attenuated CD4+T lymphocytes, macrophages and smooth muscle cells in aortic root plaque (P<0.01). Furthermore, the depletion of Tregs with CD25 antibody (PC61) curtailed the reduction in plaque area and aortic root fibrosis by HXF (P<0.01).

Conclusion

HXF relieved atherosclerosis, probably by restraining inflammatory response, reducing inflammatory cell infiltration and attenuating aortic root fibrosis by increasing Treg counts.

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Authors and Affiliations

  1. The Second Clinical College of Guangzhou University of Chinese Medicine, Guangzhou, 510405, China

    Xiao-min Ou, Jing Cai & Xiao-yue Hu

  2. Department of Cardiology, Guangdong Provincial Hospital of Chinese Medicine, the Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, 510020, China

    Xiao-min Ou, Jing Cai, Xiao-yue Hu, Qiao-huang Zeng, Tao-hua Lan &  Wei Jiang

  3. Guangdong Provincial Key Laboratory of Chinese Medicine for Prevention and Treatment of Refractory Chronic Diseases, Guangzhou, 510020, China

    Tao-hua Lan &  Wei Jiang

Authors
  1. Xiao-min Ou
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  2. Jing Cai
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  3. Xiao-yue Hu
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  4. Qiao-huang Zeng
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  5. Tao-hua Lan
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Contributions

Ou XM performed the experiments, analyzed the data and wrote the manuscript. Cai J performed the experiments and analyzed the data. Hu XY conducted the literature search and data collection. Zeng QH performed the experiments. Lan TH directed and revised the manuscript. Jiang W conceived and designed the experiments. All authors confirmed the final manuscript and approved it for publication.

Corresponding author

Correspondence to Wei Jiang.

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All authors declare no conflicts of interest.

Additional information

Supported by the National Natural Science Foundation of China (No. 81874432) and Traditional Chinese Medicine Bureau of Guangdong Province (No. 20221172)

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Ou, Xm., Cai, J., Hu, Xy. et al. Treg Immunomodulation Contributes to the Anti-atherosclerotic Effects of Huxin Formula in ApoE-/- Mice. Chin. J. Integr. Med. (2024). https://doi.org/10.1007/s11655-024-3663-2

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  • DOI: https://doi.org/10.1007/s11655-024-3663-2

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