The present study confirmed that the JPQT regulated the M1/M2 polarisation balance and restricted the inflammatory response, which further contributed to the attenuation of AS in ApoE−/− mice. The effects of the JPQT on M1/M2 polarisation balance were primarily achieved via increased STAT6 phosphorylation.
The immune-inflammatory response plays a crucial role in the development of atherosclerosis, and macrophages are one of the major players in the maintenance of immune homeostasis in the atherosclerotic plaque microenvironment. Due to their plasticity, macrophages are capable of undergoing different phenotypic transformations under different microenvironmental stimuli, with pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages predominating.
iNOS is a major marker protein for M1-type macrophages. Under inflammatory conditions, pro-inflammatory cytokines such as tumour necrosis factor α, interleukin-1, interferon-γ and LPS bind to cell surface receptors and activate kinases, leading to the phosphorylation of various intracellular proteins and the subsequent activation of specific transcription factors, such as NF-κB, STAT-1, etc., which induces the expression of iNOS(12). iNOS is virtually undetectable in the normal vascular system. However, it is abundantly expressed by smooth muscle cells and macrophages under pathological conditions such as atherosclerosis༈13༉. iNOS, in addition to being an M1-type macrophage marker, has an important role in the pathogenesis of atherosclerosis, which may be related to the large amount of rapid NO release, which induces endothelial dysfunction in the vasculature༈14༉. arg-1 is expressed predominantly in M2-type macrophages, which is the major marker protein of macrophages. Differential gene expression analysis of atherosclerosis-susceptible and drug-resistant rabbit macrophages showed that high macrophage Arg1 expression was associated with low atherosclerosis susceptibility and is expected to be a new candidate gene for atherosclerosis resistance༈15༉. Arginase I upregulation reduced macrophage infiltration and inflammation in rabbit atherosclerotic plaques༈16༉. Arg-1 was similarly found to be upregulated at admission in patients with ST-segment elevation myocardial infarction in clinical studies༈17༉.
STAT6 is a key regulator of the M2 transcriptional programme in macrophages. In response to IL-4 or IL-13 stimulation, the STAT6 dimer is activated by phosphorylation and translocates to the nucleus, where p-STAT6 induces the transcription of M2 marker genes such as Arg1, Ccl17, and Mrc1, and represses the activation of M1 or inflammatory genes. It was found that during macrophage activation, polarisation of M2-type macrophages is inhibited if Lys383 of Stat6 is acetylated by the acetyltransferase CREB-binding protein(18).Li et al. found that adipose-derived stem cells can inhibit inflammatory polarisation of macrophages through activation of the STAT6 pathway by exosomes secreted by them, exerting an antiatherosclerotic effect༈19༉. Chinese medicines and their monomers have also been found to be used to modulate phosphorylation of kinetic STAT6 to regulate macrophage polarisation; the Astragalus-Luteus medicinal pair was shown to improve lipid metabolism and attenuate atherosclerosis by increasing the regulation of M1/M2 and Th1/Th2 immune homeostasis through modulation of STAT6 phosphorylation ༈20༉; and ginseng saponin Rb1 was shown to reduce atherosclerosis through this being an inhibitory effect on M2/ M2 immunity through increasing levels of IL-4 and IL-13 as well as STAT6 phosphorylation to promote M2 macrophage polarisation and limit intraplaque inflammatory responses to prevent atherosclerosis༈21༉.
In Chinese medicine, spleen qi deficiency is one of the important symptoms of atherosclerosis(22), "spleen deficiency does not transport clear and turbid, stagnant fluid and phlegm", if the spleen and stomach dysfunction is easy to lead to qi stagnation and blood stasis, phlegm, including lipids in the blood, phlegm and turbid for a long time will become stasis, phlegm and stagnation of each other damage to the vein channel, the vein channel is unfavourable, the formation of plaques, the heart and vein paralysis and obstruction. Paralysis obstruction, increased permeability of the vessel wall lining, phlegm and stasis and other pathological products invade the lining, triggering inflammatory reactions and aggravating atherosclerosis.
"The heart receives qi from the spleen", "if the heart is suffering from labour disease, replenish the spleen qi to benefit it, and if the spleen is strong, it will be felt in the heart", so the spleen should be strengthened to strengthen the heart. The formula used in this study, Strengthening the Spleen and Dispelling Phlegm Formula, is an effective formula with clinical experience in coronary heart disease (thoracic paralysis), which is a key disease of the National Regional Chinese Medicine Diagnostic and Treatment Centre of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine. The formula is adapted from Sijunzi Tang, which consists of Codonopsis pilosulae, Radix Astragali, Rhizoma Atractylodis Macrocephalae, Poria, Semen Armeniacae, Piper betulae, Peach kernel, Safflower, and Radix et Rhizoma Glycyrrhizae. Radix et Rhizoma Ginseng and Radix Astragali are the monarchs, Radix et Rhizoma Ginseng strengthens the spleen, tonifies the middle Jiao Qi, Radix Astragali strengthens the spleen and tonifies the middle Jiao Qi; Atractylodes Macrocephalae, Poria, Semen Haematoxylae and Psoralea vulgaris are the ministers, Atractylodes Macrocephalae dries dampness and strengthens the spleen, Poria promotes water and seeps dampness, Semen Haematoxylae dries dampness and resolves phlegm, and Psoralea vulgaris disperses the phlegm; Roasted Glycyrrhiza glabra acts as the enabler to harmonise the medicines, which together can benefit Jiao Qi and tonify the spleen, and transport the water dampness.
In conclusions, we found that ApoE-/- mice showed a decrease in STAT6 phosphorylation in aortic tissue during the onset of atherosclerosis, an increase in pro-inflammatory M1 cells and a decrease in anti-inflammatory M2 cells, accompanied by an imbalance in the secretion of pro-inflammatory/anti-inflammatory factors, and an aggravation of inflammatory reactions. After administration of Spleen Strengthening and Expectorant Formula by gavage, it was found that Spleen Strengthening and Expectorant Formula activated the STAT6 pathway and enhanced the phosphorylation of STAT6. Immunofluorescence and WB results showed that the formula was able to regulate the balance of the number of M1/M2 macrophages in mice and promote the polarisation of M1 macrophages to M2 to balance the pro-inflammatory/anti-inflammatory responses. Meanwhile, it was found that the intervention of the formula alleviated the development of AS disease in ApoE-/- mice, improved the overall body weight and lipid level, and reduced the level of serum inflammatory factors, suggesting that the formula has a certain effect on improving AS. In conclusion, the results of the intervention study of the spleen-enhancing and expectorant formula showed that it could improve AS by activating STAT6 signalling and regulating the polarization balance of M1/M2 macrophages.
The limitations of this experiment are (1) the lack of evaluation and quantification of Chinese medicine symptoms for the spleen-deficiency syndrome during atherosclerosis mouse modelling and model evaluation; (2) the absence of a STAT6-specific inhibitor group for signaling pathway-related studies; and (3) the lack of exploration of upstream and downstream signaling factors in the signaling pathway.