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

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 CD4CD25Foxp3Treg 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 CD4T 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 CD4T 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.