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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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.
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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