1 Introduction

Atherosclerosis, characterized by lipid deposition, immune cell infiltration and plaque formation is a gradual process responsible for ischemic stroke and several heart conditions. Atherosclerosis majorly stems through chronic inflammation of the arterial wall, constituting the most common cause for stroke in middle aged population (Verde and De Pietro, 2019). Adaptive and innate originated immune cell infiltrates are observed in the arteries during the development of this disease. Moreover, in the adventitia artery, tertiary lymphoid organs could be observed as the disease advances (Hu et al., 2016). Change in the lifestyle and social environment are accompanying the rise in the incident rates of metabolic disorders and also an increase in the rate of cardiovascular disease due to progressing atherosclerosis. Progression and onset of atherosclerosis is closely related with dyslipidemia indicated by hypertriglyceridemia, hypocholesterolemia and hypo alpha lipoproteinemia (Hirayama, 2016).

Cholesterols’ such as low-density lipoprotein (LDL), high-density lipoprotein (HDL) and value of total cholesterol (TC) have been widely used as quantitative markers of lipids to evaluate cardiovascular risks (Hirayama, 2016). Induction of regression and limiting progression in atherosclerosis, with the aid of medical therapy has been associated extensively with marginalizing the risk of a cardiovascular events (Raggi et al., 2016). Development of insulin resistance, cardiovascular disease and atherosclerosis, can be attributed to hyperlipidemia (Li et al., 2016a,b). The progression of atherogenesis is followed majorly by the lipoperoxidation induced oxidative damage, therefore, in the atherosclerosis development, oxidized LDL is a major causative agent (Choi et al., 1862).

Flavonoids are a main class of phenolic compounds and secondary plant metabolites, having a wide array of reported pharmacological activities (Harborne and Williams, 2000). Vitexin (apigenin-8-C-glucoside), also known as ‘Mujingsu’ in China, is a c-glycosylated flavone found in various Chinese medicinal plants such as, hawthorn, pigeon pea, bamboo, chasteberry in seeds, fruits, flowers, etc. Vitexin has received greater attention, due to its wide spectrum of pharmacological interventions. Vitexin has been reported to downregulate lipogenesis and up regulate fatty acid oxidation and lipolysis in NAFLD (Non-alcoholic fatty liver disease) mice (Inamdar et al., 2019). Inhibition of the lipogenic genes were observed with vitexin administration in the diabetic mice (Kang et al., 2015a,b). Vitexin has also been reported in reducing the acetyl CoA carboxylase gene expression in adipose tissue of HFAD fed mice (Peng et al., 2019). Hepatoprotective effect of vitexin has been observed in colitis- induced liver injury by inhibiting the activation of NFκB (Duan et al., 2020). Vitexin prevents oxidative stress and improves histopathological structures in CCl4 induced liver damage (Manikya et al., 2012). Vitexin pharmacological interventions further includes its anti-cancer, anti-inflammatory, anti-oxidant, anti-Alzheimer's, anti-nociceptive, anti-hypertensive, anti-hypoxia and ischemia injury, anti-depressant-like actions and anti-viral, activities (He et al., 2016; Peng et al., 2019)

In the present work, effect of vitexin (in three different doses) as well as pravastatin (a selective 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor) administration both in combination and alone on lipid profile, AI, endothelial function, anti-atherogenic markers (paraoxonase 1 (PON1) and homocysteine thiolactonase (HT) activities), aortic nitrative-oxidative stress, adhesion molecules- inflammatory markers, liver HMG-CoA reductase activity, in high fat atherogenic diet fed (HFAD)animals, were studied.

2 Materials and methods

3 Results

3.5

3.5 Effect on aortic nitrosative-oxidative stress

As compared to normal control animals, the rats fed on HFAD for 45 days have showed significant increase in aortic nitro tyrosine level and significant decrease in T-SOD, GPx and CAT activities. This impairment was significantly corrected by the administration of vitexin (10 and 20 mg/kg) and pravastatin (10 mg/kg). Combination of vitexin (20 mg/kg) and pravastatin (10 mg/kg) have shown better effect on HFAD induced aortic nitrosative-oxidative stress as compared to both the treatments when administered alone. However, the effect of vitexin 5 mg/kg on aortic nitrosative-oxidative stress of HFAD animals was insignificant (Fig. 1a–d).

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Fig. 1

Effect on aortic nitrative-oxidative stress. a. Nitrotyrosine; b. Superoxide dismutase (SOD); c. Glutathione peroxidase (GPx); d. Catalase (CAT). Results are expressed as mean ± S.D. ^a p < 0.05 vs Normal control group; ^b p < 0.05 vs HFAD group; ^c p < 0.05 vs HFAD + treatment groups (Vitexin 10 or Vitexin 20 or Prava). HFAD-high fat atherogenic diet; Vitexin 10- Vitexin 10 mg/kg; Vitexin 20-Vitexin 20 mg/kg; Prava-Pravastatin 10 mg/kg.

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3.6

3.6 Effect on endothelial function

The HFAD rats have showed significant inhibition of acetylcholine-induced relaxation in thoracic aorta. The administration of vitexin (10 and 20 mg/kg) and pravastatin (10 mg/ kg) in combination or alone, significantly and dose dependently corrected the HFAD induced endothelial dysfunction induced in HFAD animals. There was no effect of any of the treatment on endothelial independent relaxation in aortic tissue (Fig. 2a and b).

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Fig. 2

Effect on endothelial function using organ chamber studies. a. Endothelium-dependent relaxation; b. Endothelium-independent relaxation. Results are expressed as mean ± S.D. ^a p < 0.05 vs Normal control group; ^b p < 0.05 vs HFAD group; ^c p < 0.05 vs HFAD + treatment groups (Vitexin 10 or Vitexin 20 or Prava); HFAD-high fat atherogenic diet; Vitexin 10- Vitexin 10 mg/kg; Vitexin 20-Vitexin 20 mg/kg; Prava-Pravastatin 10 mg/kg; ACh- acetylcholine; SNP- sodium nitro prusside.

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3.7

3.7 Effect on liver HMG-CoA reductase activity

As compared to normal-fat diet animals, the rats fed on HFAD for 45 days have showed significant increase in liver HMG-CoA reductase activities. This increase was significantly corrected by the administration of vitexin (10 and 20 mg/kg) and pravastatin (10 mg/kg). Combination of vitexin (20 mg/kg) and pravastatin (10 mg/kg) have shown better effect on HFAD increased liver HMG-CoA reductase activities as compared to both the treatments when administered alone. The effect of vitexin 5 mg/kg on liver HMG-CoA reductase activities of HFAD animals was insignificant (Fig. 3).

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Fig. 3

Effect on liver β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) Reductase Activity. Results are expressed as mean ± S.D. ^a p < 0.05 vs Normal control group; ^b p < 0.05 vs HFAD group; ^c p < 0.05 vs HFAD + treatment groups (Vitexin 10 or Vitexin 20 or Prava). HFAD-high fat atherogenic diet; Vitexin 10- Vitexin 10 mg/kg; Vitexin 20-Vitexin 20 mg/kg; Prava-Pravastatin 10 mg/kg.

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3.8

3.8 Histopathological results

H &E staining in the induction group showed microvascular depots of fat, with morphological alterations showing undefined contours, congestion and necrotic changes. Cell evidenced cellular ballooning were observed in the livers of high fat diet group with vitexin (5 mg/kg). Morphological alterations also showed alterations of peripheral nuclei, fragmented cells and characteristic of cellular necrosis. Treatment with vitexin (5, 10, 20 mg/kg) and pravastatin (10 mg/kg) have shown better effect in prevention of the morphological alterations caused by the HFAD (Fig. 4).

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Fig. 4

Effect on liver structure (H & E staining) 400×. a: Control group showing normal morphological characteristics. b: high fat atherogenic diet group animals showing microvascular depots of fat with morphological alterations of undefined contours along with congestion and necrotic changes. c: HFAD + vitexin (5 mg/kg) treated animal groups showing reduced fat depots with reduced congestion. d: HFAD + vitexin (10 mg/kg) treated animal groups, showing, reduction in the cytoplasmic ballooning and fragmented cells. e: HFAD + vitexin (20 mg/kg) treated animal group showing, largely reduced morphological alterations of peripheral nuclei, undefined contours, cellular ballooning and reduction in the fat depots. f: HFAD + vitexin(20 mg/kg) + pravastatin (10 mg/kg) treated animal groups showing near normal cells with reduction in morphological alterations induced by HFAD.

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4 Discussion

The results of this study showed that HFAD administration for 45 days has resulted in significant increase in serum TC, TG, LDL, AI, %HTR, MCP-1, VCAM-1, ICAM-1, IL-1β, IL6, TNF alpha, aortic nitro-tyrosine level and liver HMG-CoA reductase activity along with significant reduction in serum PON1, HT, aortic T-SOD, GPx, CAT activity and endothelial function. The results of the present study are in parallel to previously published reports (Blankenberg et al., 2003; Cho et al., 2012; Farid et al., 2010; Gu et al., 2015; Kosaka et al., 2005; Kruzliak et al., 2015; Li et al., 2016a,b; Misra et al., 2019; Paudel et al., 2016; Subramanian et al., 2009; Varatharajalu et al., 2010; Yang et al., 2015)

Exacerbation of obesity related conditions like endothelial dysfunction and atherosclerosis can be linked with increased fat in the diet, significantly altering cellular metabolism, leading to insulin resistance, dyslipidemia, increased cytokine expression and secretion from skeletal muscle and adipose tissue (Farid et al., 2010; Misra et al., 2019). Elevated serum cholesterol in itself is a developmental cause of atherosclerosis. Adhesion molecules and chemokines, including ICAM-1, VCAM-1 and MCP-1, are important atherosclerotic pathogenic marker, upregulated in smooth muscle cells, macrophages and endothelial cells during atherosclerosis. High fat diet is reported to increase macrophage accumulation in adipose tissue which produces cytokines causing inflammatory changes in the liver, blood vessels and other parts of the body, which play a role in formation of atherogenesis (Subramanian and Chait, 2009).

PON1 is an enzyme with potent antioxidant properties. It prevents LDL oxidation and detoxifies homocysteine thio-lactone, known as causative agents of atherosclerosis. Serum PON and HT activity decreases in atherosclerotic diseases and higher activities may provide an indication for anti-atherogenic effect (Varatharajalu et al., 2010). The HDL associated PON-1 enzyme protects serum lipids from oxidation (Farid et al., 2010). Activity of paraoxonase, lactonase protects against homocysteinylation, thus it is considered a potential contributing factor to prevent atherosclerosis (Locsey et al., 2013).

Peroxynitrite, a highly potent oxidant formed during the reaction of NO and superoxide anion, reacts with protein to form nitro tyrosine. Nitro tyrosine is used as a marker in in-vivo peroxynitrite mediated oxidative/nitrate stress and superoxide anion mediated NO inactivation. Excessive release of oxygen radicals, if not controlled by the body’s own defense anti-oxidant SOD, GPx and catalase enzymes, can lead to oxidant stress. Aortic nitrative and oxidative stress has been implicated and considered as an important risk factor in atherosclerosis (Misra et al., 2019; Wang et al., 2016)

At the onset of atherosclerosis, the damage to the endothelium leads to deposition of harmful cholesterol in the artery wall, thus compromising endothelial function. Thus, endothelial function is considered as an onset marker of atherosclerosis (Yang et al., 2015). As progressive damage to the endothelial system continues, it eventually leads to endothelial dysfunction. Genetic factors, factors involving diet such as high fat diet and high meat consumption, along with lifestyle factors such as tobacco use and increased oxidative stress are all implicated as promoters of atherosclerosis (Li et al., 2016a,b).

The main enzyme involved in the metabolism of TC is HMG-CoA reductase. Cholesterol synthesis is facilitated by HMG-CoA reductase by catalyzing mevalonate synthesis from HMG-CoA and further by generating TC from squalene. An overall decrease in the HMG-CoA can reduce generation of TC effectively (Gu et al., 2015). Inhibitors of HMG-CoA reductase like pravastatin are in main stay for the management of early risks and late life symptoms of atherosclerosis.

Administration of vitexin and pravastatin both alone and in combination, has significantly corrected HFAD induced impairments in levels of serum lipids, AI, adhesion molecules, chemoattractant-inflammatory mediators, anti-atherogenic markers, aortic nitroso-oxidative stress, endothelial function and liver HMG-CoA reductase activity. A very recent study has reported vitexin (10 mg/kg) have significant effect on serum lipids (TC, TG, LDL, HDL) and many other parameters of high fat diet induce obese mice (Tuso et al., 2015). In our study vitexin on two different doses (10 mg/kg and 20 mg/kg) have resulted into significant correction of serum lipids, and the effect of 20 mg/kg dose were almost similar to those observed by pravastatin 10 mg/kg. Further, administration of vitexin (20 mg/kg) in combination of pravastatin resulted in to highest lipid lowering effect.

Zhang colleagues (2017) has reported pre-treatment with vitexin may be responsible for inhibition of the ox-LDL-induced overexpression of IL-1β, IL-6, TNF-α, E-selectin, ICAM1 and VCAM1 in HUVECs. Vitexin has been shown to protect rat chondrocytes in ER stress-induced increase in the expression of inflammatory cytokines, IL-6 and TNF-α (Kang et al., 2015a,b). Vitexin promoted the expression of SOD, while reducing the ox-LDL-induced oxidative stress, along with inhibition of ROS and MDA (Xie et al., 2018). The harmful effects of HF diet on hepatic MDA, GSH, GPx, glutathione reductase and SOD activity were found to be reduced by the beta vulgaris L. extract rich in vitexin (Zhang et al., 2017). Also, vitexin administration alleviated oxidative stress and inflammatory parameters in brain and lungs’ insult (Bustos et al., 2018; Jiang et al., 2018; Lu et al., 2018). Vitexin exerted significant increase in endothelium dependent relaxations due to acetylcholine on isolated rat thoracic aortic ring preparation in chronic myocardial ischemia/reperfusion injury rats (Che et al., 2016). It is important to note here that, no previous study has been found which has studied the role of vitexin on the levels of serum anti-atherogenic factors (PON1, HT), aortic nitrative-oxidative stress, HMG-CoA reductase activity. Overall this is the first study which formally assess the effect of vitexin on serum lipid profile, AI, atherogenic markers, adhesion-inflammatory cytokines, chemokines, endothelial dysfunction, aortic nitrative-oxidative stress markers in HFAD rats.

Pravastatin has been used as positive control in the present study. Pravastatin, an HMG-CoA reductase inhibitor is already known for anti-atherosclerotic properties. Serum total cholesterol, low density lipoprotein-cholesterol, triglyceride in abdominal aorta were decreased. A 10 mg/kg dose of pravastatin was administered which has provided benefits on all the parameters studied in the present study (Wu et al., 2013). When given in combination, Vitexin has significantly enhanced the beneficial effect of pravastatin in all the parameters studied, may be due to HMG-CoA reductase inhibitory activity of vitexin, as observed in our study.

This study suggests that vitexin may be considered as potentially efficacious agent in HFAD induced risk of atherosclerosis due to its anti-hyperlipidemic, anti-atherogenic, anti-inflammatory, endothelium protectant and HMG-CoA reductase inhibitory activity.

5 Conclusion

Vitexin and pravastatin administration both alone and in combination has significantly prevented HFAD induced impairments in levels of serum lipids, AI, adhesion molecules, chemoattractant-inflammatory mediators, anti-atherogenic markers, aortic nitroso-oxidative stress, endothelial function and liver HMG-CoA reductase activity. This suggests that vitexin may be considered as a possible anti-atherogenic agent to minimize atherosclerosis risks, Thus, vitexin may be found useful in atherosclerosis and atherosclerosis associated conditions, such as Coronary Heart Disease, ischemic cerebrovascular disease and peripheral vascular disease. Further research is mandated to find the complete potential of vitexin in atherosclerosis condition.

6 Authors’ contributions

We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors.

XL and YY conceived and designed the study, YY collected and XL analyzed the data, both XL and YY wrote the manuscript. Both the authors read and approved the manuscript for publication.

7 Role of funding source

None.