Exploring the mechanism of atherosclerosis and the intervention of traditional Chinese medicine combined with mesenchymal stem cells based on inflammatory targets

Abstract

Atherosclerosis (AS) is a chronic inflammatory vascular disease, which is the common pathological basis of cardiovascular and cerebrovascular diseases. The immune inflammatory response throughout the course of AS has been evidenced by studies, in which a large number of immune cells and inflammatory factors play a crucial role in the pathogenesis of AS. The inflammation related to AS is mainly mediated by inflammatory cytokines (IL-1β, IL-6, IL-18, TNF-α, hs-CRP, SAA), inflammatory enzymes (Lp-PLA2, sPLA2-IIA, MMPs), and inflammatory signaling pathways (P38 MAPK signaling pathway, NF-κB signaling pathway, TLR2/4 signaling pathway). It is involved in the pathophysiological process of AS, and the degree of inflammation measured by it can be used to evaluate the risk of progression of AS plaque instability. In recent years, traditional Chinese medicine (TCM) has shown the advantage of minimal side effects in immune regulation and has made some progress in the prevention and treatment of AS. Mesenchymal stem cells (MSCs), as self-renewal, highly differentiated, and pluripotent stem cells with anti-inflammatory properties and immune regulation, have been widely used for AS treatment. They also play an important inflammation-immune regulatory function in AS. Notably, in terms of regulating immune cells and inflammatory factors, compared with TCM and its compound, the combination therapy has obvious anti-inflammatory advantages over the use of MSCs alone. It is an important means to further improve the efficacy of AS and provides a new way for the prevention and treatment of AS.

Graphical abstract

1. Introduction

Atherosclerosis (AS) is a chronic progressive disease with an extremely long incubation period and is the most common potential lesion of cerebrovascular disease, coronary artery disease, and peripheral artery disease, thus becoming a major global disease burden [1]. At present, the pathogenesis of AS mainly involves endothelial injury, immune inflammation, lipid infiltration, and other theories [2], among which the immune inflammation theory plays an important role in all stages of its occurrence and development, plaque rupture, and thrombosis [3]. Long-term clinical observation has found that the biological manifestations of AS are observed earlier than imaging. Therefore, inflammatory markers can be used as prominent indicators for predicting and evaluating the degree of inflammation or the risk of plaque progression in AS. The biological response of chronic inflammation starting from vascular endothelial dysfunction mainly causes the occurrence and development of AS [4], and a recent anti-inflammatory thrombosis outcome study on canakinumab (IL-1β monoclonal antibody, CANTOS) has confirmed the effectiveness of anti-inflammatory therapy for AS treatment [5]. Therefore, in addition to existing lipid-lowering therapies, targeting immune inflammatory pathways is a novel approach to prevent and treat AS [6].

Mesenchymal stem cells (MSCs), which are multipotent stem cells with anti-inflammatory and immunosuppressive properties, can be isolated from various tissues such as bone marrow, peripheral blood, fat, placenta, and other tissues, and play an important inflammation-immune regulatory function in the field of cardiovascular and cerebrovascular, especially in the process of AS [7]. Traditional Chinese medicine (TCM) can affect the occurrence and development of AS through multiple ways and targets and has been effective in the prevention and treatment of AS. TCM has the advantage of minimal side effects in immune regulation, which can promote the proliferation, differentiation, and other physiological activities of various stem cells in vivo and in vitro, regulate the homing and colonization of MSCs, and improve the survival rate of MSCs after transplantation [8,9]. Therefore, the incorporation of the unique advantages of TCM in MSC treatment will further improve the efficacy of AS. As a result, based on the theory of immune inflammation, the role of relevant inflammatory targets in the pathogenesis of AS was expounded, while the anti-inflammatory mechanism of the combination of TCM with MSCs in the treatment of atherosclerotic diseases (e.g., major atherosclerotic cerebral infarction and coronary atherosclerotic heart disease) was discussed through the regulation of immune cells and inflammatory factors. This paper aims to further study and summarize the existing research results in order to provide reference for future clinical and experimental research.

2. Immune inflammation theory

Immune response is a defense response that maintains the stability of the internal environment, including innate and adaptive immunity, while inflammatory response is an important part of biology, which has defects in function and regulation in addition to structural defects [10]. The theory of immune inflammation was first proposed by Professor Virchow in 1856, and Professor Peter Libby finally introduced this theory into the pathogenesis of AS through a large number of experimental studies and summaries. The cells involved in the formation of AS mainly include monocytes (MCs), macrophages (Mφs), smooth muscle cells (SMCs), endothelial cells (ECs), lymphocytes (LYs), and dendritic cells (DCs). Amid the development of AS, the innate immune response begins with the deposition of low-density lipoprotein (LDL) in the arterial wall through the binding of apolipoprotein B and proteoglycan to penetrate the endothelial barrier and deposit in the extracellular matrix of the subendothelial space, causing ECs damage and dysfunction, and MC migration, adhesion, and infiltration to ECs damage. After subendothelial LDL is oxidized by vascular cells, ECs and vascular SMCs can be stimulated to produce monocyte chemoattractant protein-1 (MCP-1) and macrophage colony stimulating factor (M-CSF). Under the induction of M-CSF, MCs are transformed into Mφs and activated to release many inflammatory factors [11]. (Fig. 1). At the same time, Mφ can phagocytose oxidized low-density lipoprotein (ox-LDL) to form foam cells, and the accumulation of foam cells forms lipid streaks and plaques, leading to the formation of atherosclerotic plaques. Subsequently, when DC presents a series of potential antigens to LY, adaptive immune response rapidly occurs rapidly, which further aggravate local inflammation and plaque formation. In addition, SMC in the middle layer proliferates and migrates to the intima, thus promoting monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase (MMP), and other pro-inflammatory factors were significantly increased. Moreover, Mφs recruitment and the uptake of lipids lead to plaque progression and rupture [12].

Fig. 1.

Immune-inflammatory microenvironment in atherosclerosis.

Mφs can be differentiated into two major functional phenotypes, M1 and M2, depending on the influence of the local microenvironment of blood vessels. M1 polarization produces tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, and other pro-inflammatory factors, which further aggravates the occurrence and development of AS. However, M2 polarization leads to the secretion of anti-inflammatory factors such as IL-10 and transforming growth factor-β (TGF-β), which have a protective effect on AS [13].

3. Inflammatory markers in the pathogenesis and clinical diagnosis of AS

Inflammation mainly causes the pathogenesis of AS. Therefore, the degree of inflammation measured by inflammatory markers can evaluate the risk of progression of AS plaque instability. AS-related inflammation is mainly mediated by inflammatory cytokines, enzymes, and signaling pathways and participates in the pathological process of AS.

3.1. IL-1β、IL-6 and IL-18

IL-1β has a high biological activity in the IL-1 family and is mainly produced by MC, Mφ, and DC [14]. The inflammatory characteristics of IL-1 family are dominated by innate immunity, but Il-1β and IL-6 can play a role in adaptive immunity [15]. IL-1β is dependent on the activation of NLRP3 inflammasome and requires processing to exert its biological effects, while local stimulation in plaques is conducive to the production of active IL-1β through the molecular assembly of NLRP3 [16,17]. In addition, IL-1β can be induced by TNF-α and then stimulate the secretion of varied cytokines and adhesion molecules through local paracrine and autocrine signaling, resulting in immune cell extravasation and persistent local inflammation [18]. Also, the proliferation and migration of vascular SMCs can be promoted and MMP can be induced to accelerate the degradation of the fibrous skeleton of AS plaques [19]. Studies have shown that the activation of IL-1β can increase the expression of its downstream factor IL-6, which in turn increases the production of CRP in the liver to promote the recruitment of white blood cells, leading to endothelial dysfunction and systemically inducing the inflammatory cascade [20,21]. However, IL-6 can make SMC in the media migrate to the subintima, proliferate, and carry out phenotypic transformation. It can also promote MC aggregation and infiltration, and the local expression of IL-6 increases in unstable plaques [22]. In the CANTOS study, the effect of canakinumab was significantly associated with a reduction in IL-6 levels [23], indicating a synergistic effect of IL-1β and IL-6, and its predicting AS function was also observed in another study [24]. IL-18 is a noticeable factor in the inflammatory network, which functions synergistically with many cytokines involved in the development of AS, such as IL-6, IL-12, and IFN-γ [25] and amplifies the inflammatory response in lesions. Several studies have shown that serum IL-18 level was elevated in patients with coronary artery diseases, while IL-18 and its receptors were overexpressed in different immune cells in AS plaques, such as Mφs, T cells, ECs, and SMCs [26], indicating a correlation between IL-18 and AS lesions. Moreover, IL-18 has also been found to be associated with the risk of substantial residual inflammation in canakinumab-treated patients [27]. Therefore, IL-1β, IL-6 and IL-18 can be used as potential markers for predicting the risk of AS and plaque instability.

3.2. TNF-α

TNF-α is an inflammatory and immunomodulatory factor that is mainly secreted by MC/Mφ. Clinical studies have found that TNF-α can recruit T cells and Mφ to the lesions of AS but not in normal tissues, thus direct confirming that TNF-α participates in the inflammatory response of AS [28]. Dudeck [29] has shown that TNF-α can increase the expression levels of the key genes involved in inflammation and cell proliferation by activating NF-κB, P38 MAPK, JAK, and other signaling pathways, leading to disease progression. In addition, TNF-α leads to increased leukocyte infiltration into blood vessels, which is also a key step in plaque formation. In TNF-α ApoE double knockout mice, the AS lesion area is reduced by more than 50 %, and plaque necrosis and apoptosis are increased, which have been evidenced by a study [30]. Also, a study has confirmed that TNF-α levels are significantly associated with early carotid AS [31]. In conclusion, TNF-α can activate multiple pathways and recruit various immune cells with multidirectional proinflammatory effects, is a potent modulator of inflammation in AS, and can be used as an effective clinical marker of early AS.

3.3. Hypersensitive C-reactive protein and serum amyloid A

Hypersensitive C-reactive protein (hs-CRP) and serum amyloid A (SAA) are both acute phase response proteins that are synthesized by the liver under the stimulation of pro-inflammatory factors. Both SAA and hs-CRP levels can be detected in AS lesions, and the association of SAA is higher than that of hs-CRP [32]. The short-term continuous increase and maintenance of high levels of hs-CRP can predict the presence of carotid plaque and the instability of plaque on MR imaging (low signal in T1-weighted image), and high levels of hs-CRP are significantly correlated with more new brain lesions detected by MR diffusion-weighted imaging during carotid artery stenting [33]. SAA can also stimulate the secretion of MMPs, thereby accelerating the degradation of matrix and matrix proteins [32]. Comprehensive analysis of relevant studies showed that hs-CRP and SAA, as sensitive inflammatory markers, can effectively evaluate AS and predict plaque rupture and potential cardiovascular and cerebrovascular diseases.

3.4. Lipoprotein-associated phospholipase A2 and secretory phospholipase A2 of group IIA

Lipoprotein-associated phospholipase A2 (Lp-PLA2), which mainly circulates a complex of LDL and HDL in the blood AS, can hydrolyze oxidized phospholipids in ox-LDL to produce pro-inflammatory lipids, and then produce various AS-causing effects. It also promotes the production of related cytokines [[34], [35], [36]]. However, Lp-PLA2 expression was upregulated in AS plaques, and its expression was enhanced in the Mφ of fibrous caps of vulnerable plaques [37]. Secretory phospholipase A2 of group IIA (sPLA2-IIA), an inflammatory protein, is activated through the PI3K/Akt pathway to increase ox-LDL-induced MCP-1 expression. It can also selectively activate the proinflammatory function in human aortic SMC, thereby exerting its AS effect [[38], [39], [40]]. In summary, PLA2 family members modify phospholipids to produce pro-inflammatory lipids that cause AS, and Lp-PLA2 and sPLA2-IIA are novel inflammatory markers for predicting and evaluating plaque instability in AS.

3.5. MMPs

MMPs are a superfamily of proteases secreted by Mφs, ECs, and LYs, which are responsible for tissue remodeling and extracellular matrix protein degradation and are involved in different stages of plaque progression in AS [41]. However, inflammatory cytokines are involved in ROS production and affect the expression and activity of MMPs. Tan C et al. [42] examined the association of MMP-9 and MCP-1 concentrations with the severity of carotid AS, as measured by carotid plaque and intima-media thickness (IMT), and found that an elevated serum MMP-9 concentration was independently associated with high total carotid plaque score, plaque instability, and increased IMT value. Nevertheless, MCP-1 concentration was independently associated with IMT rather than plaque morphology. Analysis of the corresponding changes in the APOE-deficient mouse model indicated that the increased expression of MMP-13 was due to the activation of the P38 mitogen-activated protein kinase (P38 MAPK) signaling pathway [43]. Increased MMP-7 activity may be associated with VSMC apoptosis and plaque instability within the fibrous cap [44]. Seifert et al. [45] confirmed that MMP-2 and MMP-9 activities were significantly higher in ApoE−/− models with unstable AS plaques compared to more stable plaque phenotypes downstream. In addition, MMPs, which were enhanced by the expression of MMP-9 under the stimulation of TNF-α, can promote VSMC proliferation and migration [46]. In the late stage of AS, MMPs can be regulated by TLR4/NF-κB and RAGE/NF-κB signaling, and the upregulation of MMPs mediated by NLRP3 inflammasomes is prone to plaque rupture [47,48]. In summary, MMPs play a key role in all stages of AS through vascular inflammation, VSMC migration, and plaque activation and rupture.

3.6. P38 MAPK signaling pathway

P38 mitogen activated protein kinase (P38 MAPK) is a member of the MAPK family, which includes extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs). The p38 MAPK signaling pathway can be activated in response to various intracellular and extracellular stimuli, including oxidative stress, cytokines, and growth factors, which are abundant in AS lesions, while p38 MAPKs can be activated by ox-LDL stimulation in Mφ [49,50]. MAPK phosphorylation in the blood can yield abundant ROS, promote the MAPK signaling pathway to induce the accumulation of MCs in the arterial wall, and reduce the secretion of collagen and other matrixes by vascular SMCs, finally triggering cytotoxicity [51] and causing foam cell necrosis in vascular plaques and AS plaque fragmentation. Activation of MAPK is driven by inflammation, and stimulation of intermittent hypoxia/reoxygenation (IHR) can activate the MAPK pathway in ECs and induce the expression of proinflammatory cytokines, such as TNF-α, IL-1, and IL-6 [52]. Therefore, p38 MAPK may facilitate the progression of AS by promoting oxidative stress and chronic inflammation. Furthermore, interventions in MAPK signaling can also modulate the degree of intimal proliferation, platelet activation, and apoptosis [53], all of which are key influencing factors in AS formation.

3.7. NF-κB signaling pathway

Nuclear factor-kappa B (NF-κB) is a critical nuclear transcription factor in the Rel family, including Rel (p65), NF-κB1 (p50), NF-κB2 (p52), RelB, and c-Rel. The activation of the IκB kinase (IKK) complex can initiate the NF-κB signaling pathway [54,55]. The NF-κB-mediated signaling pathway can not only accelerate the growth of lipid plaques and promote the inflammatory response by regulating the expression of proinflammatory factors, such as IL-1β, TNF-α, and INF-γ [56] but also regulate the invasion and colonization of inflammatory cells in the vascular wall, change the composition of the extracellular matrix, and promote the migration of SMCs [57]. Thus, it participates in the occurrence and development of AS. Endothelial dysfunction AS the initial event in the development of AS is induced by the production of NF-κB and downstream inflammatory mediators, and NF-κB-induced NLRP3 inflammasome contributes to the development of AS. Induced chemokines attract LY and trigger endothelial inflammation. It can also promote the recruitment and differentiation of MC by increasing the levels of adhesion factors and M-CSF in EC [58]. In conclusion, the activation of the NF-κB pathway can significantly increase the levels of its downstream proinflammatory factors and also increase the expression of its chemokines and adhesion factors, which directly leads to inflammatory and proapoptotic responses in vascular endothelial cells, and thereby motivate the progression of AS [59,60].

3.8. Toll-like receptor 2/4 signaling pathway

Toll-like receptors (TLR) are transmembrane proteins that are widely expressed in immune and vascular cells [61]. The signaling pathways mediated by TLR are mainly divided into myd88-and TRIF-dependent pathway. MyD88 can mediate the signaling of all TLR and IL-1 receptor family members, except TLR3, and activate the NF-κB and MAPK signaling pathways and the expression of inflammatory cytokines [62]. TLR2 induced VSMC migration in an IL-6-dependent manner. TLR2 also interacted with Nox 1 to induce ROS production, pro-inflammatory cytokine production, and MMP-2 secretion. TLR4 expression is necessary for Mφ to differentiate into foam cells, which can activate the MyD88/NF-κB signaling pathway, and ox-LDL can increase TLR4 expression without affecting TLR2 expression [63]. Lipopolysaccharide (LPS) can upregulate the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) through the TLR1 signaling pathway, which promotes Mφs to phagocytose oxidized lipids and converts them into foam cells, aggregating and increasing the plaque area [64]. In addition, ox-LDL can upregulate MMP-9 through the TLR9 signaling pathway, which promotes Mφs to express a series of inflammatory factors and accelerates plaque rupture [65]. Therefore, TLR2 and TLR4 are highly expressed in AS plaques of patients and play an important role in the initiation and progression of AS [66].

4. Study on the mechanism of Chinese medicine combined with MSCs in the treatment of as

AS is an inflammatory disease mediated by innate and adaptive immunity under pathological conditions, and it mainly causes ischemic stroke or myocardial infarction. Therefore, many anti-inflammatory strategies have become potential treatments for AS. The combination of TCM with MSCs has broad prospects and great potential in atherosclerotic diseases by targeting immune inflammatory pathways.

4.1. TCM combined with MSCs can regulate immune cells and inhibit inflammatory response

MC/Mφ is the main driver of the pathogenesis of AS and the main source of inflammatory cytokines in AS plaques, leading to innate immune response. T/B lymphocytes mainly recognize the presented antigen and release antibodies, leading to the occurrence of adaptive immune response in AS.

Arctium lappa root extract can reduce the vascular endothelial MC infiltration induced by TNF-α and the expression levels of IL-1β, IL-6, TNF-α, and MCP-1 in mouse aorta, indicating that it can directly or indirectly change the expression of inflammatory factors, reduce the infiltration of white blood cells into vascular endothelium, and participate in the treatment of AS [67]. Ginsenoside Rg3 can promote the transformation of Mφ into M2 type through peroxisome proliferator-activated receptor-γ-dependent mechanism, reduce inflammatory response, reduce the burden of AS plaque, and increase the stability of plaque [68,69]. In addition, Poria cocos polysaccharides [70] and icariin [71] can also induce the differentiation of M1-type Mφ into M2 type. Quercetin can inhibit the maturation of DCS by upregulating the domain antibody and then downregulate the Src/PI3K/Akt–NF–κB inflammatory pathway, thereby reducing the expression of DC surface markers CD80, CD86, MHC-Ⅱ, IL-6, and IL-12 and inhibiting the inflammatory response in AS lesions [72]. Buyang-huanwu decoction can increase the number of Treg cells in the spleen and peripheral blood of ApoE−/−AS mice, increase the proportion of Treg cells in CD4⁺ T cells, and regulate the levels of inflammatory factors [73]. Angong Niuhuang pill can reduce the expression level of Th17 cells and transcription factor RORγt and increase the expression level of Treg cells and transcription factor FOXP3, confirming that Angong Niuhuang Pill can inhibit chronic inflammation by regulating the balance of Th17/Treg [74]. Agarwood extract (phenethyl chromonone derivative GYF-21) can inhibit the survival, activation, proliferation, and differentiation of B cells mainly by blocking the signaling pathway activated by B cell activating factor and cause the apoptosis of B2 cells and a small increase in the proportion of B1 cells [75].

MSCs are natural immune privileged cells; hence, when the immune system is overactivated, MSCs can show anti-inflammatory effects to avoid excessive self-attack [76]. MSCs can regulate the polarization of Mφ by secreting prostaglandin E2 (PGE2), thereby reducing the secretion of inflammatory factors by changing the balance of M1/M2 [77]. Human amniotic MSCs can inhibit the inflammatory stimulation of Mφ induced by oxLDL by regulating the NF-кB pathway, inhibit the proliferation of Mφ, downregulate the levels of phosphorylated p65 and IκBα, remarkably reduce the expression of TNF-α, and increase the expression of IL-10 in order to achieve the anti-inflammatory effect on mice blood vessels and reduce the formation of plaque [78]. Human umbilical cord MSCs can participate in immune regulation and exert immunosuppressive effects by inhibiting the proliferation of T cells, downregulating Th1/Th17, and upregulating Treg cells [79]. BMSCs can inhibit the expression of p50 and p65 and affect the activation of NF-κB pathway through the overexpression of miR-23b, thereby inhibiting the maturation and differentiation of DC [80]. Tonsil derived MSCs inhibit the phytohemagglutinin-induced proliferation of peripheral blood MC, while the co-culture of tonsil derived MSCs significantly reduce the production of interferon-γ and IL-4, indicating their immunomodulatory effects on T cell proliferation and Th1 and Th2 specific cytokines [81].

The effect of the combination of asarone and MSCs on immune regulation has been evaluated by some studies [82] through in vitro and in vivo experiments. These research results have confirmed that asarone with umbilical cord MSCs can inhibit the proliferation of CD4⁺ or CD8⁺ T lymphocytes, downregulate Th1 cytokines, upregulate Th2 cytokines, and change the Th1/Th2 balance to a Th2 anti-inflammatory direction, thus inhibiting the inflammatory response. Zhao et al. [83] observed the immunogenicity and immunomodulation function of hMSCs after differentiation into neural-like cells induced by TCM herbs. Therefore, astragalus, gastrodiae, and ginseng, as TCM herbs, were selected to induce the differentiation of hMSCs into neural-like cells in vitro, and the one-way mixed lymphocyte reaction of differentiated cells was observed. The proliferation of lymphocytes in the experimental group was significantly lower than that in the control group. However, limited studies have focused on the regulation of immune cells by TCM combined with MSC therapy, and many studies are still needed to confirm this finding (Table 1).

Table 1.

Related studies on the mechanism of TCM/stem cells regulating immune cells to suppress inflammatory responses.

TCM Active Ingredients/MSCs	Mechanism of Action	Targets of Action	The Literature
Arctium lappa root extract	Inhibition of MC adhesion and aggregation	MC infiltration to the vascular endothelium ↓; IL-1β ↓、IL-6 ↓、TNF-α ↓、MCP-1 ↓	[67]
Ginsenoside Rg3	Regulation of Mφ polarization	Activity of M1 ↓，Activity of M2 ↑; Mφ of type M1 differentiated into Mφ of type M2	[68,69]
Poria cocos polysaccharides、icariin			[70] [71]
Quercetin	Inhibition of DC maturation	Domain specific antibody ↑、Src/PI3K/Akt–NF–κB ↓; CD80/86、MHC-Ⅱ、IL-6、IL-12 ↓	[72]
Buyang-huanwu decoction	Regulation of Th17/Treg balance	Number of Treg ↑, proportion of Treg in CD4+ T ↑	[73]
Angong Niuhuang pill		Th17 cells ↓、RORγt ↓; Treg cells ↑、FOXP3 ↑	[74]
Agarwood extracts (GYF-21)	Regulation of B cell function	B cell survival, activation, proliferation and differentiation ↓; The number of B2 cells ↓ and the proportion of B1 cells ↑	[75]
MSCs	Regulation of the M1/M2 balance	Activity of M1 ↓，Activity of M2 ↑	[77]
hAMSCs	Inhibition of Mφ proliferation	NF-κB signaling pathway ↓，The number of Mφ ↓; TNF-α、Phosphorylated p65 ↓、IκBα ↓，IL-10 ↑	[78]
hUC-MSCs	Regulation of Th1/Th17/Treg balance	Proliferation of T cells、Th1、Th17 ↓; Treg cell ↑	[79]
BMSCs	Inhibition of DC maturation and differentiation	Overexpression of miR-23b、NF-κB signaling pathway ↓	[80]
T-MSCs	Inhibition of peripheral blood MC and T cell proliferation	Interferon-γ in Th1 and IL-4 in Th2 ↓	[81]
Asarone combined with hUC-MSCs	Inhibition of proliferation of CD4+ or CD8+ T cells	Th1 cytokines ↓, Th2 cytokines ↑	[82]
Astragalus, gastrodin, and ginseng/hMSCs	Inhibition of T cell proliferation	The number of T cell proliferation ↓	[83]

Note: ↑ indicates increase, upregulation, activation; ↓ means decrease, down regulation, inhibition.

In conclusion, the activation of immune cells is characterized by the adhesion and aggregation of MCs, the formation of foam cells, the differentiation of M1/M2 Mφs, the maturation of DCs, and the differentiation of lymphocytes in the pathogenesis of AS. Among them, the main biological mechanisms of TCM herbs and MSCs in the treatment of AS by immune cells are comprised of inhibiting the adhesion, aggregation, infiltration and differentiation of MCs, inhibiting the polarization of M1 Mφs or promoting M2 Mφ polarization, inhibiting the maturation and differentiation of DCs as well as the proliferation of MCs and T cells, increasing the proportion of Treg cells, regulating the balance of Th1/Th17/Treg cells, and regulating the function of B cells, all of which can slow the process of AS. The combination of TCM with MSC has an obvious inhibitory effect on immune regulation, but limited studies have focused on this method. The combination of stem cells with immune cell therapy can effectively reduce the inflammatory response of AS, but MSCs do not have complete immune privileges in the allogeneic host with normal immune function, and how to reduce the immunogenicity of MSCs in allogeneic transplantation needs to be considered. Therefore, TCM can play a better role in the treatment of stem cells combined with immune cells, and the research of stem cell immunity combined with traditional Chinese and western medicine may also be the trend of future development.

4.2. TCM combined with MSCs can regulate inflammatory factors and inhibit inflammatory response

Cytokines are protein mediators of inflammation and immunity, which can act through autocrine, paracrine, or endocrine pathways. Inflammatory signaling pathways also play an important role in the inflammatory response.

Tanshinone ⅡA can inhibit the NF-κB or TLR4/NF-κB pathway, downregulate the levels of adhesion molecules ICAM-1 and VCAM-1, and inhibit the migration of Mφ, thus playing an immunomodulatory and anti-inflammatory role [84,85]. Baicalin inhibits the NF-κB and p38 MAPK signaling pathways in a dose-dependent manner, thereby reducing the increase of IL-6, TNF-α, and soluble vascular endothelial cadherin (SVE-cadherin) induced by AS [86]. Berberine can regulate the NLRP3/NF-κB pathway and reduce the expression of pro-IL-1β and other inflammatory factors [87]. Matrine can inhibit the NF-κB/MAPK signaling pathway and ROS production in hASMC, reduce the expression of inflammatory TNF-α, and inhibit the agES-induced M1 polarization of macrophages by inhibiting the RAGE-induced oxidative stress-mediated TLR4/STAT1 signaling pathway [88,89]. Andrographolide can inhibit the proliferation of VEC and relieve inflammation by inhibiting PI3K/Akt pathway and reducing TNF-α induced ICAM-1 expression [90]. Qingxin-jieyu decoction can reduce the levels of serum hs-CRP, IL-6, MMP-9, sCD40L, and other inflammatory factors in patients with CHD through the NLRP3/IL-1β pathway [91].

Human induced pluripotent stem cell-derived MSCs can inhibit the inflammatory response by reducing the expression levels of TNF-α and IL-6 or by reducing the expression of Notch1 gene in plaques [92]. The transplantation of skin-derived MSCs can significantly reduce the plaque area of AS mice, and skin-derived MSCS can inhibit the expression of TNF-α and enhance the expression of IL-10 in the plaque by regulating the NF-κB pathway in vivo and in vitro [93]. Human placenta-derived MSCs attenuate the NF-κB pathway by regulating the expression of TLR4 and the phosphorylation of IκBα and p65, thereby reducing the release of inflammatory factors and reducing the level of inflammation [94].

Zhang et al. [95] found that after 4 weeks of combined treatment with isoglycyrrhiza and BMSCs, the levels of TNF-α and IL-1β were and the level of IL-10 was higher than that those of the with monotherapy with isoglycyrrhiza and BMSCs, indicating the combined treatment can significantly inhibit inflammatory response and delay nerve tissue damage compared with the two alone treatment. Jiang [96] found that Xionggui prescription combined with BMSC-derived exosomes could reduce the mRNA expression levels of TNF-α and iNOS and the surface markers of M1-type MG compared with the control group. M2-type MG decreased the mRNA expression of pro-inflammatory factors IL-1β and IL-6, increased the mRNA expression of surface markers TGF-β and Arg-1, and increased the mRNA expression of anti-inflammatory factor IL-13. Liu [97] found that after lateral ventricle injection, neural-like cells co-cultured with Dendrobium officinale polysaccharide (DOP) and BMSCs could further reduce the levels of TNF-α, IL-1β, and IL-6 in the brain tissue of rats with cerebral ischemia, reduce neuroinflammation, and reduce cerebellar infarction volume compared with BMSCs alone. Li et al. [98] showed that the combination of Danshen Tongluo Jiedu decoction with BMSC transplantation could significantly improve myocardial injury in AMI model rats by regulating the NF-κB pathway, reduce the levels of TNF-α and IL-6, inhibit inflammatory response, and repair ischemic myocardium. Zhang et al. [99] found that the combination of Buyang-Huanwu decoction with BMSCs can synergize with TIMP-1 to reduce the expression of serum MMP-9 and MMP-2 in MCAO rats, inhibit the degradation of extracellular matrix caused by it, repair the blood–brain barrier, inhibit the inflammatory response, and thus reduce the formation of brain edema after ischemia (Table 2).

Table 2.

Related studies on the mechanism of TCM/stem cells regulating inflammatory factors and inhibiting inflammatory responses.

TCM Active Ingredients/MSCs	Pathways of action	Targets of Action	The Literature
Tanshinone ⅡA	NF-κB signaling pathway、TLR4/NF-κB signaling pathway	ICAM-1 ↓、VCAM-1 ↓	[84,85]
Baicalin	NF-κB signaling pathway、p38 MAPK signaling pathway	IL-6 ↓、TNF-α ↓、SVE-cadherin ↑	[86]
Berberine	NLRP3/NF-κB signaling pathway	pro-IL-1β ↓	[87]
Matrine	NF-κB/MAPK signaling pathway、TLR4/STAT1 signaling pathway	TNF-α ↓; Polarization of M1-type Mφ	[88,89]
Andrographolide	PI3K/Akt signaling pathway	TNF-α ↓、ICAM-1 ↓	[90]
Qingxin-jieyu Decoction	NLRP3/IL-1β signaling pathway	hs-CRP ↓、IL-6 ↓、MMP-9 ↓、sCD40L ↓	[91]
iPSC-MSC	–	IL-6 ↓、TNF-α ↓; Gene expression of Notch1↓	[92]
Skin-derived MSCs	NF-κB signaling pathway	TNF-α ↓、IL-10 ↑	[93]
HPMSCs	TLR4/NF-κB signaling pathway	Phosphorylated IκBα、p65 ↓	[94]
Isoglycyrrhiza combined with BMSCs	–	IL-1β ↓、TNF-α ↓	[95]
Xionggui prescription combined with BMSCs-exos	–	Expression of TNF-α and iNOS mRNA ↓、Expression of TGF-β and Arg-1 mRNA ↑; Expression of IL-1β and IL-6 mRNA ↓、Expression of IL-13 mRNA ↑	[96]
DOP combined with BMSCs	–	TNF-α ↓、IL-1β ↓、IL-6 ↓	[97]
Danshen Tongluo Jiedu decoction combined with BMSCs	NF-κB signaling pathway	TNF-α ↓、IL-6 ↓	[98]
Buyang-Huanwu decoction combined with BMSCs	–	MMP-2 ↓、MMP-9 ↓	[99]

Note: ↑ indicates increase, upregulation, activation; ↓ means decrease, down regulation, inhibition.

In conclusion, both TCM and MSC transplantation can increase the expression of anti-inflammatory cytokines (e.g., IL-10 and TGF-β) through the TLR4/NF-κB, p38 MAPK/NF-κB, and NLRP3/IL-1β signaling pathways. The expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, NF-κB, and MCP-1) decreased. In terms of regulating inflammatory factors, the effect of TCM combined with MSCs transplantation is more significant than that of single treatment. However, only animal experimental studies of TCM combined with MSCs transplantation have been conducted for the treatment of AS, and clinical trials of TCM combined with MSC transplantation remain lacking. In addition, AS is a chronic condition that is secondary to turbidness, phlegm, stasis, and toxicity in TCM based on the therapy including the stagnation of meridians or deficiency of meridians and the uncured disease for a long time, which leads to unfavorable channels and dysfunction of viscera organs. The main functions of the aforementioned TCM treatment are invigorating qi and activating blood circulation, removing blood stasis and dredging collaterals, and clearing heat and detoxifying, which are related to the pathological changes and clinical manifestations of AS. Meanwhile, TCM functions of nourishing qi and activating blood circulation, and removing blood stasis and dredging collaterals can make the body's qi carry blood with nutrition to reach all organs and tissues, which can improve pathological conditions, such as lipid deposition. TCM functions of heat-clearing and detoxifying can remove toxic factors, improve the vascular microenvironment, and inhibit the inflammatory response. Therefore, according to the mechanism of AS, the unique advantages of TCM, and the anti-inflammatory properties of stem cells, the combination of TCM and stem cells may effectively inhibit the inflammatory response of AS.

5. Summary and prospect

The concept of inflammation, as a driver of AS, has met considerable resistance in the past. However, inflammation provides a means through which traditional risk factors can exert their pathogenic effects at the level of arterial wall cells. According to this review, immune inflammatory response was observed throughout the course of AS, among which proinflammatory cytokines such as IL-6, TNF-α, and hs-CRP and inflammatory enzymes such as Lp-PLA2, sPLA2-IIA, and MMPs can be used as important indicators for the diagnosis of AS and the prediction and evaluation of the risk of plaque progression. P38 MAPK, NF-κB, and TLR2/4 signaling pathways play an important role in the pathogenesis of this disease. In terms of inhibiting inflammation by regulating immune cells and inflammatory factors, the effect of TCM combined with MSCs is more significant than that of single treatment.

In recent years, an increasing number of clinical studies has made new attempts into anti-AS treatment based on anti-inflammation and immune regulation. In addition to the success of the CANTOS clinical trial to establish anti-cytokine therapy, several large-scale RCTS are in progress, including ENTRACTE, COLCOT, and CIRT, which target IL-6, TNF-α, and neutrophils with methotrexate, etanercept, and colchicine, respectively. These studies aim to develop better therapeutic drugs for immune anti-inflammatory therapy of AS. A recent trial was conducted in China to evaluate the effect of optimal medical treatment of low-dose colchicine (0.5 mg/day) on cardiovascular outcomes in stroke patients with evidence of persistent coronary inflammation (based on hs-CRP). The theory of immune inflammation has become a new breakthrough in the prevention and treatment of AS, and inflammation in AS has developed from a theory to an established reality [100]. Aspirin is a classic nonsteroidal anti-inflammatory drug that can directly inhibit the formation of proinflammatory eicosanoids (PGI2 and PGE2) in endothelial cells. In contrast, the administration of aspirin will increase proresolved LXA4 formation, when COX-2 expression is induced in cells with Il-1β, thus supporting a beneficial anti-inflammatory and proresolved function of low-dose aspirin [101]. In addition, ox-LDL has significantly to do with the occurrence, development, and eventually thrombosis of AS. Therefore, the treatment of natural antioxidants, such as glutathione, is theoretically effective; Indeed this theoretical inference is not sufficient, and more relevant evidence-based medical evidence is needed. To date, in some international multicenter large-scale studies, natural antioxidants have shown negative results, which prove that natural antioxidants cannot achieve the ideal effect. However, some synthetic antioxidants, such as probucol, have shown positive effects. A series of clinical studies with small sample sizes in China and Japan have shown that probucol has preliminary results of reversing plaque, preventing vascular events, and changing the properties of plaque to a certain extent. In addition, the peripheral nervous system establishes a polysynaptic artery–brain–nerve circuit through the interaction of adventitial immune cells with atherosclerotic plaque, which is involved in the control of cardiovascular homeostasis and AS [102]. This study confirms for the first time that AS plaque is directly regulated by the nervous system. The results provide a new idea for the formation and development of AS.

Mediators produced and released by MSCs in AS lesions contribute to the regulation of inflammation and can improve impaired endothelial function. At present, many in vitro experiments and animal studies support the application of MSCs in the treatment of AS. Long-term beneficial effects of MSCs can be achieved through the engineering of MSCs to selectively deliver immunosuppressive and anti-angiogenic agents and thus reduce the development of AS lesions. Endothelial-to-mesenchymal transition (EndMT) may be a new direct source of MSCs, that dedifferentiation into MSCs by EC occurs in AS lesions, indicating that MSCs derived from EndMT are easily obtained, and MSCs need not be injected into the recipient as with traditional treatment methods [103,104]. Moreover, this method has a high safety and no graft rejection. However, many problems still need to be solved with MSCs. Among them, the two biggest challenges are the translation of in vitro and animal studies of MSCs to patients with atherosclerotic plaques and the use of MSCs to target the specific pathways involved in the development of AS and retention of MSCs at the site of action without affecting other regions and other mechanisms. Furthermore, the formation of AS is the result of the comprehensive action of many mechanisms, especially the immune inflammation always being involved in its pathological progression. TCM possess unique advantages in the prevention and treatment of AS; however, most researches on AS models are still in their infancy, and the upstream and downstream molecules in some drug target pathways have not been specifically studied. Therefore, the action mechanism of TCM and its compounds in the treatment of AS need to be discovered and verified by more researchers in the future. Considering that AS is a multi-target chronic vascular disease driven by inflammation, its treatment will require long-term administration. Therefore, AS treatment drugs require highly safe and multi-target anti-inflammatory drugs, and TCM will be a potential source of drug research in the future. Ultimately, and TCM combined with MSCs will also be an important direction of future research.

Funding

This work was supported by the National Natural Science Foundation of China（No. 81974564）; the National Natural Science Foundation of China（No. 82104730）; Zhongyuan talent plan--leading talent project for scientific and technological innovation（No. 224200510027）; and The Traditional Chinese Medicine Scientific Research Project of "Double first-class" establishment discipline in Henan Province（HSRP-DFCTCM-2023-1-04）. All the authors declare that financial support did not influence the opinion of the article or the statistical analysis and reporting of the objective results of the study data.

Author contribution statement

All authors listed have significantly contributed to the development and the writing of this article. Shibiao Sun conceived the content, drafted the manuscript, and approved the final version to be submitted. Feixiang Liu, Feiyan Fan, and Na Chen helped in writing the manuscript, revised it critically for important intellectual content, and approved the final version to be submitted. Xiaolong Pan and Zhihui Wei helped in writing the manuscript and approved the final version to be submitted. Yunke Zhang conceived the content, revised it critically for important intellectual content, and approved the final version to be submitted.

Data availability statement

No data was used for the research described in the article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Glossary

acronym

AS

atherosclerosis

MSCs

mesenchymal stem cells

MC

monocyte

Mφ

macrophage

SMC

smooth muscle cell

EC

endothelial cell

LY

lymphocyte

DC

dendritic cell

LDL

low-density lipoprotein

M-CSF

macrophage colony stimulating factor

ox-LDL

oxidized low density lipoprotein

MCP-1

monocyte chemoattractant protein-1

VCAM-1

vascular cell adhesion molecule-1

ICAM-1

intercellular adhesion molecule-1

MMP

matrix metalloproteinase

TNF-α

tumor necrosis factor-α

IL-1

interleukin-1

TGF-β

transforming growth factor-β

hs-CRP

hypersensitive C-reactive protein

SAA

serum amyloid A

Lp-PLA2

lipoprotein-associated phospholipase A2

sPLA2-IIA

secretory phospholipase A2 of group IIA

P38 MAPK

P38 mitogen activated protein kinase

NF-κB

nuclear factor-kappa B

TLR

Toll-like receptors

PGE2

prostaglandin E2

BMSCs

bone marrow mesenchymal stem cells

BMSCs-exos

BMSCs-derived exosomes

EndMT

endothelial-to-mesenchymal transition