CN114432354A

CN114432354A - Helianthus annuus Linne disk for improving atherosclerosis

Info

Publication number: CN114432354A
Application number: CN202111174160.4A
Authority: CN
Inventors: 王健兵; 单士军
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-10-09
Filing date: 2021-10-09
Publication date: 2022-05-06

Abstract

The study aims to discuss the anti-atherosclerosis effect of sunflower discs and the related mechanism thereof. High fat diet for 24 weeks resulted in a C57BL background deficiency of apolipoprotein E (Apoe)^‑/‑) Severe atherosclerosis, H, throughout the aorta in mice&E and oil red O staining showed severe plaques in the aortic intima, reducing the extent of high fat diet-induced atherosclerotic lesions through sunflower disc intervention. The data show that HAL reduces atherosclerosis by modulating lipid, inhibiting oxidative stress, modulating gut flora, decreasing gut permeability, and inhibiting inflammatory response. The research finds that the sunflower discs reduce the inflammatory reaction of atherosclerosis by regulating the intestinal flora and reducing the intestinal permeability. Our findings provide new insights into the role and mechanism of the anti-atherosclerotic potential of sunflower discs.

Description

Helianthus annuus disk for improving atherosclerosis

Technical Field

The invention relates to a natural oral medicament for preventing and treating atherosclerosis. The study aims to discuss the anti-atherosclerosis effect of sunflower discs and the related mechanism thereof. High fat diet for 24 weeks resulted in a C57BL background deficiency of apolipoprotein E (Apoe)^-/-) Severe atherosclerosis, H, throughout the aorta in mice&E and oil red O staining showed severe plaques in the aortic intima, reducing the extent of high fat diet-induced atherosclerotic lesions through sunflower disc intervention. The data show that HAL reduces atherosclerosis by modulating lipid, inhibiting oxidative stress, modulating gut flora, decreasing gut permeability, and inhibiting inflammatory response. The research finds that the sunflower discs reduce the inflammatory reaction of atherosclerosis by regulating the intestinal flora and reducing the intestinal permeability. Our findings provide new insights into the role and mechanism of the anti-atherosclerotic potential of sunflower discs.

Background

Atherosclerosis (AS) is a lipid-driven inflammatory disease of the arterial wall and is the underlying cause of most cardiovascular diseases. One third of the worldwide deaths are caused by cardiovascular disease and are a huge economic burden. AS is the leading cause of cardiovascular disease-related morbidity and mortality, especially in the elderly population. Cardiovascular diseases are a major public health problem in China, and the burden is increasing sharply. Although primary and secondary prevention strategies significantly reduce the incidence of CVD, they are still a major cause of morbidity and mortality. Therefore, finding an effective treatment is crucial. At present, the market is lack of the AS treatment medicine which is reliable, safe and effective.

The most important pathogenic factor of AS is apolipoprotein B-containing low density lipoprotein cholesterol (LDL-C), and there is a clear causal relationship between LDL-C and cardiovascular disease. In addition to dyslipidemia, macrophages play a key role in AS, AS they are the primary cells that invade atherosclerotic lesions and form lipid-containing foam cells. These foam cells initiate the inflammatory cascade by releasing cytokines that accelerate lipoprotein retention and promote vascular inflammation. In early and middle stages of AS, including endothelial cell dysfunction, secretion of chemokines and cytokines, such AS Interleukin (IL) -1 β, IL-6, Tumor Necrosis Factor (TNF) - α, intercellular adhesion factor-1 (ICAM-1) and vascular cell adhesion factor-1 (VCAM-1) overexpression. While immune cells such as T lymphocytes, dendritic cells, and macrophages aggregate into plaques, migration and proliferation of Vascular Smooth Muscle Cells (VSMCs) into the plaques further exacerbates the inflammatory response. At the same time, VSMCs release a number of oxidation products, inactivating the vascular protective molecule Nitric Oxide (NO), leading to the development of endothelial dysfunction. Therefore, inhibition of inflammatory responses and oxidative stress may be critical for the treatment of early and intermediate AS.

In addition, there is increasing evidence that the gut microbiota is closely associated with the progression of atherosclerotic cardiovascular disease. Data show that the relationship between the intestinal microbiota and CVD in patients with coronary heart disease, a higher firmicutes to Bacteroides (F-B) ratio, a lower Bacteroides level, and a higher lactic acid bacteria level. Especially undigested dietary fiber, alters the microbial ecosystem of the gut and its composition, as the gut microbiota uses dietary fiber as a primary source of energy. The results of several human studies showed lower F-B ratios and higher bacteroidetes levels in humans on a high fiber diet. The results of large epidemiological studies have consistently supported the view that intake of dietary fiber can prevent AS.

Therefore, new therapeutic approaches should be based on reducing inflammation and oxidative stress early in the development of arterial plaque, eventually preventing the progression of the lesion to severe stenosis. At present, the medicines for improving atherosclerosis mainly comprise statin medicines and fibrate medicines, and the preventive medicines are blank. The long-term oral administration of statins and fibrates may have serious adverse effects including elevated liver enzymes, rhabdomyolysis, etc. Primary prevention of AS is of great concern once it occurs is essentially irreversible. At present, various AS-resistant health care products exist in the market, which are alleged to contain multiple active ingredients or plant extracts and the like, but the action mechanisms of the AS-resistant health care products are rejected or are not clear or effective at all, most AS-preventing products have the effects of regulating blood fat or promoting blood circulation to remove blood stasis rather than really resisting endothelial inflammation and oxidative stress, and therefore the conventional AS-resistant health care products cannot achieve the expected effect. Sunflower (A)Helianthus Annuus L.,HAL) contains flavonoids and polysaccharides as main ingredients, and may have antiinflammatory effect or antioxidant effect. Sunflower discs, AS a traditional Chinese herbal medicine, are mainly used for treating hypertension in the aspect of cardiovascular diseases, are not used for preventing and treating AS, and belong to the blank in the field. Therefore, the objective of this study was to observe whether sunflower discs have anti-AS effects and to investigate their possible mechanism of action.

Disclosure of Invention

The invention aims to clarify the function and related mechanism of sunflower discs in improving atherosclerosis.

The research shows that the sunflower discs can reduce high fat diet inductionFormation of atherosclerotic plaques;

the sunflower head can reduce the contents of serum LDL-C and Triglyceride (TG), increase high density lipoprotein (HDL-C) and reduce the ratio of LDL-C/HDL-C, thereby improving dyslipidemia;

the sunflower discs can obviously reduce the concentration of serum Malondialdehyde (MDA), obviously improve the concentrations of superoxide dismutase (SOD), NO and glutathione peroxidase (GSH-Px), and effectively reduce endothelial cell damage caused by oxidative stress;

the sunflower discs can obviously reduce the expression of serum IL-1 beta, IL-6, TNF-alpha and other inflammatory factors and reduce vascular endothelial inflammation;

the sunflower heads increase the relative abundance of bacteroidetes, reduce the relative abundance of firmicutes and obviously increase the intestinal Ackermanella (A) and (B)Akkermansiamuciniphila) And lactic acid bacteria (A), (B), (C)Lactobacillus) Abundance, and significantly reduced unidentified enterobacteriaceae: (unidentified_Enterobateriaceae) Sphingomonas(s) (ii) SSphingomonas) And Methylobacterium (meth) (meth)Methylobacterium) The results show that the sunflower extract can regulate the intestinal microbial composition of mice, increase the abundance of probiotics and reduce the abundance of harmful bacteria;

sixthly, the sunflower disc raises the level of colon occluding protein (occludin) and zon-1 (ZO-1), reduces the intestinal permeability and reduces the inflammatory mediator from entering the blood.

The technical scheme adopted by the invention

The invention is designed for animal experiments. This study will lack apolipoprotein E (Apoe) in the C57BL background^-/-) 30 female mice of 8 weeks of age were randomly divided intoAnd (3) groups. The control group (control) was fed normal diet, the model group (model) was given High Fat Diet (HFD), and the HAL group was given HFD doped with 5% HAL powder. After 24 weeks of feeding, by H&E and oil red O staining the aortic plaques were analyzed. Detecting serum lipid index, and determining activity of MDA, SOD, NO and GSH-Px in aorta. ELISA method detects IL-1 beta, IL-6, TNF-alpha concentration in serum, RT-qPCR method detects IL-1 beta, IL-6, TNF-alpha mRNA expression level in aorta, 16S rRNA method detects intestinal flora composition, immunohistochemical method is adopted to determine colon occludin and ZO-1 level.

Obtaining sunflower discs: the sunflower flower disc is prepared by crushing a flower disc, from which sunflower seeds are removed, after sunflower growing in saline-alkali soil is mature, and has the main nutritional ingredients: a mixture of neutral sugars (such as d-xylan) (59.3%), alpha-cellulose (52%), pectin (27.5%) and lignin (12.3%). Separating the sunflower disc extract by chromatography to obtain 11 diterpene compounds. Their structure is elucidated by spectroscopic data analysis. They include: -epoxy-17-al-19-oic acid, ent-kaurane-2-16-diol, ent-kaurane-15-16 ent-kaurane-16 β -ol, phenylloran-16 β -ol, ent-tisan-16-ol, grandifloric aicd, angeloylgrandifloric aicd, ent-kaurane-16-en-19-oic aicd, ent-kaurane-17-hydroxy-15-en-19-oic aicd, ent-kaurane-16 β -17-dihydroxy-19-oic aicd and ciliaric aicd.

Prevention and treatment effect of sunflower discs on atherosclerosis

The results of the study suggest that after 24 weeks of feeding, lipid plaque formation was found to be visible in the whole aorta of the model group mice (fig. 1). To assess the effect of sunflower discs on atherosclerotic plaques, we performed H & E staining (fig. 1.1) and ORO staining (fig. 1.2). We observed that high fat diet significantly increased the percentage of atherosclerotic plaques, while H & E positive and ORO positive decreased significantly after sunflower disc treatment, indicating a decrease in atherosclerotic plaque formation.

The results of the serum lipid analysis are shown in table 1. (iii) the serum total cholesterol, triglyceride, low density lipoprotein and LDL-C/HDL-C in the model group are found to be obviously increased compared with the control group: (P<0.01). Serum Total biliary fixation in HAL group compared to model groupSignificant decreases in alcohol, triglycerides, low density lipoproteins, and LDL-C/HDL-C (TG, TC:P<0.05, the restP<0.01). (Table 1)

TABLE 1 comparison of blood lipids for each group

The concentrations of MDA, SOD, NO, GSH-Px in aortic tissue were determined and are shown in Table 2. Compared with the control group, the concentration of the pro-oxidation factor MDA of the model group is obviously increased (P<0.05), the concentrations of antioxidant factors SOD, NO and GSH-Px are obviously reduced (P<0.01). HAL significantly reduced serum MDA concentrations compared to model groups (P<0.05), the concentrations of SOD, NO and GSH-Px were significantly increased (GSH-Px: P<0.05, the restP<0.01). (Table 2)

TABLE 2 comparison of the levels of oxidized cytokines in aortic tissues for each group

We found that the contents of IL-1, IL-6 and TNF-alpha in serum are obviously increased in the model group compared with the control group (see table 3) (the concentrations of proinflammatory factors IL-1 beta, IL-6 and TNF-alpha in the model group are obviously increased: (P<0.05). HAL significantly reduced serum IL-1 β levels compared to model groups (P<0.01), significantly reduce the content of IL-6 and TNF-alpha in serum (P<0.05). The mRNA expression levels of IL-1 beta, IL-6 and TNF-alpha in aorta were determined by RT-qPCR with primers as shown in Table 4. Expression of IL-1 β, IL-6, TNF-a in the aorta is shown in FIG. 2. Compared with the control group, the expression of IL-1 beta, IL-6 and TNF-TNF-in the model group is obviously increased (P<0.01). HAL significantly reduced its expression concentration compared to the model group (P<0.01). (FIG. 3 and tables 3 and 4)

TABLE 3 comparison of serum inflammatory cytokine levels in each group

TABLE 4 mouse target Gene primer sequences

Changes in the intestinal microbiota of AS model mice after HAL treatment were studied using high throughput sequencing of 16S rRNA. We obtained 77328 available reads and 1576 Operable Taxa (OTUs) from 18 samples. The Shannon diversity index of the model group was higher than that of the control and HAL groups (fig. 3.1). Venn plot analysis showed a total of 824 OTUs, 938 both in the control and model groups, 882 in the control and HAL groups, and 970 in the model and HAL groups (FIG. 3.2). The primary coordinate analysis (PCoA) showed that the intestinal flora of the model group and the HAL group were statistically different from the control group (fig. 3.3). Consistent with this result, phylogenetic trees showed significant differences between groups (fig. 3.4), and the species of intestinal flora and their relative abundance were further investigated. At the phylum level, all samples had 10 phyla, the most abundant of which was Bacteroides: (A) ((B))Bacteroidetes) And Thelluria firma: (Firmicutes) (FIG. 3.4). Of sets of modelsFirmicutesThe relative abundance is obviously higher than that of the control group,Bacteroidetesis significantly lower than the control group. HAL is increased compared with model groupBacteroidetesRelative abundance of, reduceFirmicutesRelative abundance of (c) (fig. 3.4). At the genus level, model groupAkkermansia muciniphila(FIG. 3.5a) andLactobacillusrelative abundance (fig. 3.5b) no difference compared to control group: (P>0.05), compared to the model group and the control group, and the HAL groupAkkermansia muciniphilaIs significantly increased (P)< 0.01)，LactobacillusAlso model increases in abundance of (a) ((ii))P<0.05). Model setunidentified_ Enterobateriaceae(P<0.01, FIG. 3.5c),Sphingomonas(P<0.01, FIG. 3.5d) andMethylobacterium(P<0.05, fig. 3.5e) was higher relative abundance than the control group. HAL is significantly reduced compared to the model groupunidentified_Enterobateriaceae（P<0.01）、Sphingomonas(P<0.05) andMethylobacterium(P<0.05) abundance in the intestinal tract. Taken together, these results indicate that HAL can modulate the gut microbiota composition, increase probiotic abundance, and decrease harmful bacterial abundance in AS model mice. (FIG. 3)

Intestinal permeability was analyzed by immunohistochemical staining. Through detection, compared with a control group, the levels of model colon occludin (occludin, figure 4.14.3) and tight junction protein 1 (ZO-1, figure 4.24.4) are obviously reduced (P<0.01). The level of occludin and ZO-1 in HAL group was significantly increased compared with that in model group (P<0.05), suggesting that HAL may elevate colonic occludin and ZO-1 levels, decrease colonic permeability, and reduce inflammatory responses by reducing the influx of inflammatory mediators into the blood. (FIG. 4)

In conclusion, the research result shows that the sunflower discs improve atherosclerosis by regulating lipid, inhibiting vascular endothelial oxidative stress and inflammatory reaction. The research finds that the sunflower discs reduce inflammatory reaction by regulating intestinal flora and reducing intestinal permeability so as to improve atherosclerosis. One of the highlights of this study is that we have conducted more intensive mechanistic studies in animal models and have obtained consistent results.

Drawings

FIG. 1.1: h & E staining (200 ×); 1.2: ORO staining (400X).

FIG. 2.1: IL-1. beta. mRNA aortic expression; 2.2:: IL-6 mRNA aortic expression; 2.3: TNF-alpha mRNA is expressed in the aorta.

FIG. 3.1: all samples are thinned to Shannon index calculated after reading equivalent sequence; 3.2: venn plot of the number of OTUs from each group; 3.3: PCoA score; 3.4: gut flora phylogenetic clustering trees, HAL treatment altered microbial communities at the phylum level (bar graph); 3.5: the relative abundances of each bacterium Akkermansia (a), Lactobacillus (b), unidentified _ Enterobacteriaceae (c), Sphingomonas (d), Methylobacterium (e).

FIG. 4.1: occludin; 4.2: ZO-1; 4.3: quantitative analysis of Occludin; 4.4: and (4) carrying out quantitative analysis on ZO-1.

Claims

1. The function of improving atherosclerosis of sunflower discs is characterized in that: the atherogenic effect of sunflower discs was shown by a reduction in high fat diet-induced aortic plaque formation.

2. The sunflower discs of claim 1 have effects in regulating lipid, relieving inflammation, resisting oxidative stress, regulating intestinal flora, and reducing intestinal permeability.