Research Progress On The Mechanism Of Anti Atherosclerosis Of Cistanche Glycoside

Abstract: Atherosclerosis is a chronic vasculitis disease, and endothelial dysfunction is a key early stage of atherosclerosis. Cistanche glycoside is a flavonoid component isolated from Cistanche deserticola. It has a variety of pharmacological effects. It can reduce the oxidative damage and apoptosis of endothelial cells, inhibit the process of endothelial mesenchymal transformation, inhibit the proliferation and migration of smooth muscle cells, prevent the formation of foam cells, inhibit inflammation, regulate the expression of multiple miRNAs, and play an anti-atherosclerosis role in a variety of ways. This paper summarized the mechanism of anti-atherosclerosis of cistanche glycoside, hoping to provide a reference for the clinical application of cistanche glycoside.

Keywords: Cistanche glycoside; Atherosclerosis; Endothelial cells; Smooth muscle cells; Foam cell; inflammatory reaction

Atherosclerosis is a chronic vascular inflammatory disease, which is characterized by persistent inflammatory reactions, activation of inflammatory bodies, and damage of cellular inflammation. Endothelial cells, smooth muscle cells, monocytes, macrophages, foam cells, and platelets participate in the inflammatory reaction of blood vessel walls caused by atherosclerosis [1]. Endothelial dysfunction is a key early stage of atherosclerosis. After the vascular endothelial injury, low-density lipoprotein enters the subendothelial layer and is oxidized, mediating the secretion of various cell adhesion molecules and inflammatory mediators, promoting the migration and adhesion of monocytes, promoting the formation of foam cells, and accelerating the process of atherosclerosis [2]. 

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Cistanche glycoside is a flavonoid component isolated from Cistanche deserticola, which has various pharmacological effects such as anti-inflammatory, antioxidant, anti-osteoporosis, anti-tumor, and immune regulation. Zhou Yufei et al. [4] elaborated the anti-atherosclerosis mechanism of Cistanche deserticola extract from the aspects of lipid metabolism, macrophage-derived foam cells, vascular smooth muscle cells, vascular endothelial cells, inflammatory reaction, etc., providing a way for the research of Cistanche deserticola glycoside on anti-atherosclerosis. Guo Yingjie et al. [5] summarized the possible targets and core genes of cistanche glycoside against atherosclerosis based on network pharmacology methods and found that cistanche glycoside can play an anti-atherosclerosis role by inhibiting cell proliferation, reducing oxidative stress, and improving vascular remodeling. Cistanche glycoside has been proven to be beneficial to cardiovascular diseases such as atherosclerosis [6], but the mechanism of action of Cistanche glycoside on atherosclerosis is still not completely clear. Cistanche glycoside can reduce the oxidative damage and apoptosis of endothelial cells, inhibit the process of endothelial mesenchymal transformation (EndMT), inhibit the proliferation and migration of smooth muscle cells, prevent the formation of foam cells, inhibit the inflammatory response, regulate the expression of multiple miRNAs, and play an anti-atherosclerosis role in a variety of ways. This article summarized the anti-atherosclerosis mechanism of cistanche glycoside, hoping to provide a reference for the clinical application of cistanche glycoside.

Cistanche deserticola experiment

1. Reduce endothelial cell oxidative damage and apoptosis

1.1 Reduce monocyte cell adhesion

Atherosclerosis is a chronic vasculitis disease. Its pathological feature is that cholesterol and lipid accumulation lead to narrowing and rigidity of the lumen. Oxidized low-density lipoprotein (ox LDL) plays a key role in the initiation of atherosclerotic lesions. It can damage endothelial function, induce vascular smooth muscle contraction, and upregulate intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) The expression of many vascular adhesion molecules such as E-neneneba selectin promotes monocytes to recruit into the subendothelial space, changes the secretion activity of the endothelium, reduces the antioxidant capacity, enhances endothelial cell apoptosis, and speeds up the formation of atherosclerosis [8]. Hu et al. [9] treated human umbilical vein endothelial cells (HUVECs) with icariin and found that 10, 20, and 40 μ Mol/L cistanche glycoside significantly reduces the cell viability of HUVECs, monocyte cell adhesion induced by ox LDL, and the secretion of ICAM-1, VCAM-1, and E-nenenebb selection in a concentration-dependent manner. The results show that cistanche glycoside can reduce monocyte cell adhesion and play a role in preventing and treating atherosclerosis by reducing the oxidative damage of vascular endothelial cells.

1.2 Inhibition of endothelial cell apoptosis

Ox LDL participates in the pathogenesis of atherosclerosis by damaging vascular endothelial cells, directly targeting vascular endothelial cells, promoting the mitochondrial apoptosis pathway, and also promoting the upregulation of reactive oxygen species (ROS) level, inducing endothelial cell apoptosis [10]. Hu et al. [11] intervened with cistanche glycoside in human vascular endothelial cells and found that 10, 20, and 40 μ Mol/L cistanche glycoside can reduce the damage of endothelial cells, reduce the apoptosis of endothelial cells, and significantly inhibit the decrease of caspase in endothelial cells and the increase of B-cell lymphoma 2 (Bcl-2). The results show that cistanche glycoside can inhibit the regulation of caspase and Bcl-2 expression by increasing the secretion of ROS, and inhibit the apoptosis of vascular endothelial cells to delay the process of atherosclerosis.

1.3 Inhibition of nuclear factor E2 related factor 2 (Nrf2)/antioxidant response element (ARE) pathway

The oxidative stress reaction is one of the important reasons leading to atherosclerosis. A large number of oxidative free radicals can aggravate the oxidative stress injury of endothelial cells and also regulate downstream nuclear transcription factors (NF- κ B) The signaling pathway promotes cell apoptosis, and the activation of the Nrf2/ARE signaling pathway not only promotes antioxidant stress response but also inhibits cell apoptosis [12]. Xu Yongyan et al. [13] found in the experiment of using cistanche glycoside for atherosclerosis in ApoE deficient mice that 100 mg/kg cistanche glycoside can effectively reduce pathological changes such as fat deposition and plaque instability in the arteries of mice, and can also reduce the volume of sclerotic plaque, help reduce the apoptosis rate of vascular smooth muscle cells, increase the level of total antioxidant capacity in vascular smooth muscle, and reduce 8-hydroxydeoxyguanosine The levels of malondialdehyde increase the expression of Nrf2 and ARE, indicating that Cistanche glycosides can inhibit cell apoptosis and alleviate oxidative stress response by inhibiting the Nrf2/ARE pathway to prevent the formation of vulnerable plaques.  

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1.4 Inhibition of phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway

The PI3K/Akt signaling pathway is closely related to oxidative stress response and cell apoptosis. PI3K can promote Akt activation, and Akt phosphorylation can promote the secretion of pro-apoptotic proteins, inhibit the release of apoptosis-inducing factors and cytochrome C from mitochondria, and regulate cell apoptosis [14]. Luo Yunmei et al. [15] also confirmed that 0.01, 0.1, 1 μ Mol/L cistanche glycoside can effectively reduce the oxidative damage of human umbilical vein endothelial cells induced by H2O2. Its mechanism is to inhibit the PI3K/Akt signaling pathway, effectively reduce the apoptosis caused by oxidative stress, reduce the cell vitality and ROS secretion in a dose and time-dependent manner, and play an anti-atherosclerosis role. Zhang Li et al. [16] applied cistanche glycoside to human umbilical vein endothelial cells and found that 12.5, 25, and 50 μ G/mL cistanche glycoside can reduce the oxidative damage of HUVECs induced by H2O2 in a dose-dependent manner, reduce the morphological changes of endothelial cells, increase the number of endothelial cells, increase the level of SOD, and reduce the expression of ICAM-1 and human macrophage chemoattractant protein-1 (MCP-1). The results show that cistanche glycoside can reduce the expression of related cytokines and improve atherosclerosis by reducing the oxidative stress damage of endothelial cells.

1.5 Inhibition of eukaryotic cell translation initiation factor-2 α （eIF-2 α）  

Eukaryotic cell eIF-2 α It is a marker of cellular endoplasmic reticulum stress, can significantly promote the expression of H2O2 in cells, and induce oxidative stress injury of vascular endothelial cells [17]. Under the stimulation of H2O2, vascular smooth muscle cells can also promote the activity of alkaline phosphatase (ALP), increase the expression of osteocalcin (OC), collagen I (Col Ia), osteopontin (OPN), and other calcification-related factors, accelerate the process of vascular calcification, and aggravate the process of atherosclerosis [18]. Bai Xiaojun et al. [19] used icariin in aortic vascular smooth muscle cells (VASMCs) and found that 1, 0.1, 0.01 μ Mol/L Cistanche glycoside can enhance the activity of VASMCs, significantly reduce the degree of calcification of VASMCs, significantly reduce the activity of ALP and calcium content in cells, and downregulate the expression of Runx2 protein, ATF4, and eIF2 α Phosphorylation of, upregulated α- The expression of SMA protein significantly reduced the expression of OC, Col Ia, OPN and Runx2 mRNA. The results showed that cistanche glycoside could inhibit the expression of eIF-2 in eukaryotic cells α Its expression inhibits the calcification of smooth muscle cells by an anti-oxidative stress reaction to interfere with the process of atherosclerosis.  

1.6 Regulatory Protein Arginine Methyltransferase (PRMT)/Asymmetric Dimethylarginine (ADMA)/Dimethylarginine Dimethylaminohydrolase (DDAH) Pathway

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Endothelial dysfunction is the key pathological feature of atherosclerosis. ADMA is the main marker of endothelial dysfunction. DDAH can catalyze ADMA to produce dimethylamine and l-citrulline. PRMT is an enzyme that catalyzes ADMA production. PRMT/ADMA/DDAH pathway may become a new drug target for treating endothelial dysfunction. This signal pathway is regulated by oxidative stress [20]. Xiao et al. [21] used Cistanche glycoside in high-fat fed ApoE deficient mice and human umbilical vein endothelial cells, and found that 10 and 30 mg/kg of Cistanche glycoside significantly inhibited the generation of ROS in the mouse aorta, upregulated the expression of DDAH II protein and gene in ApoE cells, and downregulated the expression of PRMT I protein and gene, 1,3,10 μ Mol/L cistanche glycoside can up-regulate the activity of DDAH in human umbilical vein endothelial cells, reduce the level of serum ADMA in mice, improve the endothelium-related vasodilation of ApoE aortic ring, increase the Emax value, and reduce the EC50 value. The results show that cistanche glycoside can inhibit oxidative stress by regulating the PRMT/ADMA/DDAH pathway to improve endothelial function, so as to prevent the formation of atherosclerosis.

1.7 Upregulation of endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) pathway

Endothelial damage caused by atherosclerosis leads to the decrease of eNOS biological activity, followed by the impairment of NO release, further promoting the production of ROS in the upper artery wall, leading to the increase of local NO degradation, and triggering the cascade reaction of oxidative sensitivity mechanism. Oxidative stress reaction can inhibit eNOS/NO signaling pathway, aggravate vascular endothelial damage, and cause a vicious cycle [22]. Xiao et al. [23] used cistanche glycoside in apoE-deficient atherosclerotic mice fed with a high-fat diet and found that 10 and 30 mg/kg cistanche glycoside could significantly reduce the body mass and levels of total cholesterol (TC) and triacylglycerol (TG) in mice, effectively inhibit the production of ROS in aortic cells, up-regulate the expression of eNOS, and increase the level of NO in plasma, The results showed that cistanche glycoside alleviated the oxidative stress injury of endothelial cells by up-regulating eNOS/NO pathway to inhibit the development of atherosclerosis caused by ApoE deficiency.

2. Suppress the EndMT process

EndMT refers to the phenotype and characteristics of mesenchymal cell proliferation, migration, and collagen synthesis after endothelial cells gradually lose their original shape and function, which can cause plaque calcification, fibrous cap thinning, and increased plaque instability, and participate in the process of atherosclerosis [24]. H19 is the first discovered long noncoding RNA (lncRNA) with paternal imprinting characteristics. It can selectively express maternal alleles, competitively regulate microRNA (miRNA), regulate the expression of miRNA target genes, and regulate the expression of ELF5 by directly interacting with miR-148b-3p. ELF5 can inhibit SMAD3 activation and effectively inhibit transforming growth factor- β （TGF- β） EndMT driven [25]. Liu et al. [26] applied cistanche glycoside to human umbilical vein endothelial cells and found that 10 μ Mol/L icariin can significantly inhibit endothelial cells α- The expression of SMA and S100A4 specific proteins, upregulation of VE-cadherin and CD31 expression, promotion of ox-LDL induced H19 expression in endothelial cells, effectively inhibiting the process of EndMT, upregulation of miR-148b-3p and ELF5 expression, further confirming that Cistanche glycosides can inhibit EndMT and further inhibit TGF through the H19/miR-148b-3p/ELF5 axis- β The results showed that cistanche glycoside could regulate EndMT by regulating the expression of H19, and play an anti-atherosclerosis role.

3. Inhibition of smooth muscle cell proliferation and migration

3.1 Prevention β Subtype PKC I (PKC β I) Expression of

Abnormal proliferation, apoptosis, and migration of vascular smooth muscle can accelerate arterial restenosis and atherosclerosis, PKC β As a member of the protein kinase C family, I participate in the metabolism, proliferation, migration, differentiation and other cell type-specific processes of vascular smooth muscle, especially plays a key role in cell migration and proliferation, and helps regulate the expression of cyclin D1 and matrix metalloproteinase-9 (MMP-9) [27]. Zhang et al. [28] used cistanche glycoside in atherosclerotic mice fed with high-fat cholesterol, and found that 20, 40, and 80 mg/kg cistanche glycoside significantly reduced the mortality of mice, significantly reduced TC, low-density lipoprotein cholesterol (LDL-C) levels, decreased the total area of atherosclerotic plates in the aortic lumen in a dose-dependent manner, and alleviated the proliferation and disorder of aortic smooth muscle cells The distribution of elastic fibers and the accumulation of foam cells and other pathological changes reduce PKC in aortic tissue in a dose-dependent manner β I expression, prevent ox LDL from stimulating the proliferation and cell migration of vascular smooth muscle cells, effectively reduce the expression of cyclin D1 and MMP-9 in vascular smooth muscle cells, and block vascular smooth muscle cells in G1/S phase. The results show that cistanche glycoside can prevent PKC β The expression of I inhibits the proliferation and migration of vascular smooth muscle cells and plays an anti-atherosclerotic role.

3.2 Deactivating the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway

ERK is a widely expressed protein kinase and participates in the proliferation of intracellular signaling molecules of cells. ox LDL can stimulate the activation of the ERK1/2 signaling pathway in vascular smooth muscle cells, thus further promoting the proliferation of vascular smooth muscle cells, promoting the thickening of the tunica intima, and accelerating or enhancing the process of Congee [29]. Hu et al. [30] used cistanche glycoside in human aortic vascular smooth muscle cells and found that 10, 20, 40 μ Mol/L Cistanche glycoside can inhibit ox-LDL-induced proliferation of vascular smooth muscle cells in a concentration-dependent manner, effectively increasing the proportion of G0/G1 phase cells, inhibiting the expression of proliferating cell nuclear antigen (PCNA) protein, and inhibiting ox-LDL induced ERK1/2 phosphorylation. The results showed that Cistanche glycoside can inactivate the ERK1/2 signaling pathway and inhibit the expression of PCNA, thereby inhibiting ox-LDL-induced proliferation of vascular smooth muscle cells, thereby reducing the progression of atherosclerosis.

3.3 Adjusting the miR-205-5p/ERBB4/Akt signaling pathway

MiR-205-5p is an anti-tumor gene that participates in cell proliferation and migration, can target and regulate the expression of ANGPT2, inhibit the activation of the ERK/Akt signaling pathway, ERBB4 belongs to the tyrosine kinase receptor family, and is highly expressed in atherosclerotic tissues. ox LDL can reduce the expression of miR-205-5p in vascular endothelial cells, and increase the expression of ERBB4 mRNA and p-ERBB4, p-Akt protein, Thus promoting the formation of atherosclerosis [31]. Huang et al. [32] studied the use of cistanche glycoside in human aortic vascular smooth muscle cells and found that 10 μ Mol/L cistanche glycoside can significantly reduce the viability of vascular smooth muscle cells, block the cell cycle process and cell migration ability induced by ox LDL, up-regulate the expression of miR-205-5p, down-regulate the expression of ERBB4 gene, reduce the p-AKT/Akt protein ratio and p-ERBB4/ERBB4 protein level, significantly reduce the expression of proliferation-related protein Cyclin D1, and increase the levels of Caspase-3, BAX/BCL-2 protein. The results confirm that Cistanche glycoside can inhibit the proliferation and migration of vascular smooth muscle cells induced by ox LDL by regulating the miR-205-5p/ERBB4/Akt signaling pathway and playing an anti-atherosclerosis role.

4.  Prevent the formation of foam cells

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High-density lipoprotein receptor (SR-BI) is a kind of high-density lipoprotein cholesterol (HDL-C) receptor, which mediates the selective uptake of HDL-C by cells. SR-BI is a protective signal of high-density lipoprotein-induced endothelial cells and macrophages to mediate atherosclerosis. SR-BI deficiency can significantly increase HDL-C levels, thereby promoting the formation of atherosclerosis, SR-BI expression was negatively correlated with plasma HDL-C level and atherosclerosis [33]. CD36 is a member of the scavenger receptor family of cell surface protein class B, which is related to ox LDL uptake and foam cell formation. It regulates the expression of CD36 by inhibiting p38 MAPK phosphorylation [34]. Yang et al. [35] studied the use of Cistanche glycoside in macrophages and found that 0.8, 4, and 20 μ Mol/L cistanche glycoside can significantly inhibit the production of foam cells, inhibit the lipid load level of foam cells, reduce the differentiation of lipopolysaccharide activated macrophages into foam cells, and reduce the synthesis of CD36 mRNA and protein in cells. Overexpression of the CD36 gene can restore the inhibitory effect of cistanche glycoside on the formation of foam cells, which proves the direct relationship between cistanche glycoside and the formation of foam cells, The results showed that cistanche glycoside could inhibit the formation of foam cells and atherosclerosis by down-regulating the expression of CD36 and up-regulating the expression of SR-BI.

5. Inhibition of inflammatory response

5.1 Inhibition of NF- κ B signal pathway

NF- κ Activation of B can trigger an inflammatory cascade reaction, promote the secretion of various inflammatory factors, and enter the nucleus to bind with corresponding sequences, inducing gene transcription and promoting macrophage apoptosis κ B as NF- κ B's inhibitory protein, which blocks NF by binding to the latter- κ B enters the nucleus to block NF- κ B activity [36]. Li Tengteng et al. [37] used icariin in mononuclear macrophage leukemia cells (RAW264.7) and found that 10, 20, 40 μ Mol/L Cistanche glycoside can increase the activity of RAW264.7, significantly reduce the apoptosis of RAW264.7 cells, help reduce cell damage induced by ox-LDL, and inhibit cell NF in a dose-dependent manner- κ The expression of B p65, Bax, and Caspase-3 proteins is upregulated κ B α、 The expression of Bcl-2 protein reduces IL-6 and IL-1 in cells β、 TNF- α The results indicate that Cistanche glycosides can inhibit NF by- κ B signaling pathway reduces the inflammatory response and inhibits endogenous cell apoptosis to play an anti-atherosclerotic role.

5.2 Inhibition of p38 MAPK signaling pathway

Activation of the p38 MAPK pathway can promote TNF- α And IL-6 expression, aggravate the inflammatory damage of vascular endothelial cells, further aggravate the vascular oxidation reaction, and promote the formation of atherosclerosis [38]. Hu et al. [39] used cistanche glycoside in atherosclerotic rats induced by a high cholesterol diet, and found that 30 and 60 mg/kg cistanche glycoside could significantly reduce the levels of TC, TG, LDL-C, increase the level of HDL-C, and significantly reduce serum IL-6, TNF- α It alleviates pathological changes such as thickening of arterial capsule media, endothelial cell dislocation with proliferation, and accumulation of foam cells, significantly reduces the intensity of fat stripes on aortic intima, and significantly reduces IL-6 and TNF in concentration correlation- α MRNA level, the results confirmed that cistanche glycoside could inhibit the activation of p38 MAPK signaling pathway to reduce inflammatory reaction and oxidative reaction and delay the process of atherosclerosis.

6.  regulates the expression of multiple miRNAs

MiRNA also plays an important role in the pathogenesis of atherosclerosis. From risk factors to the occurrence and progression of plaque to the formation of atherosclerosis neovascularization, miRNA can mediate the regulation of cistanche glycoside on the core signal pathways PI3K/Akt, Ras, ErbB, and VEGF [40]. Zhang et al. [41] used cistanche glycoside in apoE-deficient atherosclerotic mice induced by a high-fat diet. As a result, 40 mg/kg cistanche glycoside could significantly reduce the pathological changes of the atherosclerotic plate of the mouse aorta. A total of 46 miRNAs were obtained through TargetScan target gene technology, further establishing the miRNA GO network, and identifying mmu-miR-6931-5p, mmu-miR-3547-5p, mmu-miR-6368, mmu-miR-705 Mmu-miR - 7118-5p is the first five target genes, and the possible mechanism is related to angiogenesis, cell proliferation, and migration. Through the use of Gene Ontology (GO) analysis and miRNA GO Network verification, it can be seen that cistanche glycoside can play an anti-atherosclerosis role by regulating gene expression.

Noncoding RNA (ncRNA) contains RNA molecules with long ncRNAs (lncRNAs) and microRNAs (miRNAs), which have become powerful regulators of cardiovascular disease. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis can be used to predict the potential function of DE mRNA and corresponding lncRNAs, by establishing lncRNA mRNA co-expression networks, It is helpful to understand the hub lncRNA related to the anti-atherosclerosis effect of cistanche glycoside [42]. Zhang et al. [43] used cistanche glycoside in apoE-deficient atherosclerotic mice established by high-fat feeding. The results showed that 20 mg/kg cistanche glycoside could significantly reduce the serum TC and LDL-C levels of mice, significantly reducing the chief editor of atherosclerosis plate. DE mRNA enriched the GO function that was up-regulated, and key DE mRNA was enriched through up-regulated pathways, including ECM receptor interaction, GnRH signal pathway, axon guidance Focal adhesion, estrogen signaling pathway, Ras signaling pathway, etc., downregulated pathways include T cell receptors, NF- κ B. Apoptosis, B cell receptor signaling pathway, and Th1 and Th2 cell differentiation were confirmed by qPCR. The expression of lncRNA Gm5327, NONMMUT005483, Gm2904, NONMMUT031625, Gm031859, and NONMMUT000659 was upregulated in the aorta, while the expression of NONMMUT8, NONMMUT14, and Ighv6-2 was downregulated. The results showed that Cistanche glycosides can trigger differentially expressed lncRNAs, Playing the role of anti-atherosclerosis.

7.  Conclusion

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Cistanche glycoside can inhibit the EndMT process, inhibit the proliferation and migration of smooth muscle cells, prevent the formation of foam cells, reduce inflammation, regulate the expression of multiple miRNAs, and play an anti-atherosclerosis role in a variety of ways by alleviating the oxidative damage of endothelial cells and apoptosis. Cistanche glycoside is an active component extracted from Cistanche deserticola, which is more convenient to extract and has good drug safety and has good clinical application prospects. However, at present, the research of cistanche glycoside for atherosclerosis is still in the basic research, and has not yet been carried out in clinical trials for the human body, and its pharmacological effect and pharmacokinetics level need further discussion. And there is still a lack of direct evidence regarding the dose-effect-toxicity relationship of Cistanche glycosides. Clinically, there is no drug with cistanche glycoside as the main component, and we call on pharmaceutical companies to combine the existing pharmaceutical technology to develop a new drug of cistanche glycoside as soon as possible, so as to provide new ideas for the treatment of cardiovascular and cerebrovascular diseases such as coronary heart disease, cerebral infarction, carotid plaque, etc. through its anti-atherosclerosis effect in multiple ways and targets.

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