Effect of Soufeng Qutan Chinese medicine on blood lipid and IL-1β in atherosclerotic mice with AopE gene knockout

Bing Zhao, Li-Hong Gong , Fu-Long Xiao

1. Liaoning University of Traditional Chinese Medicine

2. Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China

Keywords:Soufeng Qutan Chinese Medicine Atherosclerosis Blood Lipid IL-1β

ABSTRACT Objective: To explore the possible mechanism of Soufeng Qutan traditional Chinese medicine in the treatment of atherosclerosis. Methods: 6-8 weeks old ApoE knockout mice were fed with a high-fat diet for 13 weeks to establish an atherosclerotic unstable plaque model, and they were randomly divided into model groups, low, medium, and high-dose Shoufeng expectorant groups. Torvastatin group; another 6-8 week old C57BL/6 mice were set as blank control group. Both the blank control group and the model group were given 0.9% normal saline by gavage. The low, medium, and high-dose groups of Soufeng Expectorant Chinese Medicine and the atorvastatin group were given different doses of Soufeng Expectorant Chinese Medicine Decoction and Atorvastatin. intervention. After 13 weeks, the mice were sacrificed,and the abdominal aortic blood vessels were histopathologically observed by HE staining. The blood lipids, total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol(LDL-C) were detected by a biochemical automatic analyzer), high-density lipoprotein cholesterol (HDL-C) content and ELISA method to detect IL-1β levels. Results: Compared with the blank control group, a large number of foam cells can be seen in the model group, and the plaque area is obvious; serum TC, TG, LDL-C, IL-1β expression levels increased (P＜0.05),and HDL-C expression levels decreased (P＜0.05); Compared with the model group, each dose group of Soufeng expectorant Chinese medicine and atorvastatin group had a small amount of foam cells and reduced plaque area; serum TC, TG, LDL-C, IL-1β expression levels Both decreased (P＜0.05), and the expression level of HDL-C increased (P＜0.05). Compared with the atorvastatin group, there was no significant difference in the expression levels of TC, TG, LDL-C, HDL-C, and IL-1β in the high-dose Soufeng and expectorant Chinese medicine group (P＞0.05). Conclusion: Soufeng Expectorant Herbal Medicine can inhibit the progression of cellular inflammation, down-regulate the expression of IL-1β, reduce plaque lipid content, and improve blood lipids. This may be one of the molecular mechanisms of Soufeng Expectorant Herbal Medicine in the early treatment of atherosclerosis.

1. Introduction

The pathological basis of atherosclerosis (AS) is lipid metabolism disorder. In the early stage of AS, the vascular endothelium is damaged, a large amount of lipid accumulates, which induces inflammation, and releases various inflammatory factors such as IL-1β, etc., leading to plaques. Block formation [1]. Inflammation and lipids are jointly involved in the occurrence and development of AS[2]. Abnormal management of blood lipids is the cornerstone of primary and secondary prevention of atherosclerosis. For every 1 mmol/L low-density lipoprotein cholesterol (LDL-C)reduction, The relative risk of major cardiovascular events will be reduced by 20% [3]. Inflammatory response has been extensively studied and has been shown to play a key role in the progression of AS, coronary artery disease (CAD) and myocardial ischemia reperfusion (I/R) injury, and has an important role in acute coronary syndromes [4, 5 ], its downstream inflammatory factor IL-1β is a well-characterized pro-inflammatory cytokine that plays a central role in many inflammatory diseases. The content of IL-1β protein in the adventitia vessel wall of coronary arteries in patients with AS is significantly increased. This increase is proportional to the severity of AS. Inhibiting the production of IL-1β can improve cardiovascular structure [6] and reduce plaque production. Therefore,in the early stage of AS, inhibiting the release of the inflammatory factor IL-1β and improving blood lipids can interfere with the development of AS [7] and reduce the production of plaques. Soufeng expectorant method is an effective TCM treatment rule for clinical treatment of AS[8]. Early in vivo animal experiments have confirmed that Soufeng expectorant Chinese medicine has significant anti-AS and stabilizing plaque effects[9, 10]. On this basis, we Observe the intervention effect of Soufeng Qutan Chinese Medicine on blood lipids and IL-1β in atherosclerotic ApoE knockout mice, so as to further explore the mechanism of Soufeng Qutan Chinese Medicine in preventing and treating AS.

2. Materials and methods

2.1 Experimental animals

60 6-week-old male ApoE mice, purchased from Beijing Huafukang Biotechnology Co., Ltd., experimental animal license number: SCXK (Jing) 2020-0004. 20 6-week-old male C57BL/6J mice, purchased from Liaoning Changsheng Biotechnology Co.,Ltd., laboratory animal license number: SCXK (Liao) 2020-0001.The animal experiment conforms to the standards of the Laboratory Animal Ethics Committee of Liaoning University of Traditional Chinese Medicine, and the experimental animal ethics review number is 21000042020038.

2.2 Experimental drugs

Soufeng Expectorant Chinese Medicine: Scorpion 5 g, 1 centipede,15 g of earthworm, 15 g of tangerine peel, 15 g of Pinellia ternata,15 g of Atractylodes rhizome, 15 g of leech, processed into crude medicine 2 g/ml concentrated Chinese medicine Spare, purchased from the Chinese Medicine Bureau of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine; Atorvastatin calcium tablets: 10 mg/tablet (Pfizer Pharmaceutical Co., Ltd.,approval number EG6187).

2.3 Experimental reagents and instruments

Beijing Solebco); ELISA kit (batch number: 202104, Jiangsu enzyme standard); TC, TG, LDL-C, HDL-C test kits, (batch number:20210519 Nanjing built biology).

Refrigerated centrifuge, FC type microplate reader (Thermo);dehydrator, paraffin embedding machine, microtome (Leica); CHA type optical microscope (Olympus).

2.4 Building an AS model

60 ApoE knockout mice were given 1 week of adaptive general food and then changed to high-fat feeding. The feed was purchased from Shengwang Animal Husbandry Feed Factory in Yuhong District, Shenyang City. After 13 weeks, 4 animals were selected and sacrificed by the random number table method, and the abdominal aortic vessels were taken, and HE staining was used to determine whether the model was successful [11].

2.5 Grouping and processing

Fifty ApoE knockout mice that were successfully modeled were randomly divided into model groups, low, medium, and high dose groups of Soufeng expectorant Chinese medicine, and atorvastatin group, 10 mice in each group, continued to be fed with high-fat diet . The blank control group consisted of 10 C57BL/6J mice,which were fed daily with ordinary diet. According to the clinically recommended daily dose for adults and the equivalent dose conversion formula for mice and humans [12], the daily gavage dose for mice is obtained. The mice in the blank control group and the model group were given 0.3ml/d physiological saline; the mice in the atorvastatin group were given atorvastatin calcium tablets 25.4mg/(kg.d); the mice in each dose group of Soufeng expectorant Chinese medicine were given respectively They were given 6.175, 12.35, and 24.7g/(kg.d) gavage of Shoufeng and expectorant Chinese medicine once a day for 13 weeks. At the end of the experiment, blood was taken from the mouse eyeballs, stored in an anticoagulation tube,centrifuged at 3000 r/min for 15 minutes at 4°C, serum was separated and sterilized, and stored in a refrigerator at -80°C for later use. The mice were sacrificed by neck removal, and the heart and abdominal aorta were dissected and removed, and the heart and abdominal aorta were fixed with 10% formaldehyde.(Note: The dose of the Chinese medicine treatment group is based on the recommended daily clinical oral dose for adults, and the mouse gavage dose is calculated according to the equivalent dose conversion coefficient of humans and mice; the high-dose group is twice that of the middle-dose group. The dose group is twice that of the low-dose group.)

Hematoxylin and eosin staining solution (batch number: G1120,

2.6 Observation indicators and methods

2.6.1 HE staining to detect plaque area

After the animals were sacrificed, the abdominal aorta was immediately extracted, fixed with 4% paraformaldehyde for 24 h,and embedded in paraffin. The abdominal aorta sample was cut into 5 micron sections, stained with hematoxylin and eosin, and observed with an optical microscope.

2.6.2 The content of TC, TG, LDL-C, HDL-C detected by automatic biochemical analyzerAfter the experiment, blood was taken from the mouse eyeballs,the EP tubes were aliquoted, and the upper serum was collected by centrifugation, and the content of each index was detected according to the instructions.

2.6.3 ELISA method to detect the content of IL-1βTake the serum, and measure the index with the enzyme-linked immunosorbent assay kit.

2.7 Statistical methods

All results are expressed as±s. Through single factor difference analysis to determine the difference between multiple groups,P＜0.05 indicates a significant difference. Use SPSS software(version 21.0) for statistical analysis.

3. Results

3.1 HE staining results of mice in each group

Figure 1. Blank control group: There were no plaques in the blood vessels of the mice, and the intima of the abdominal aorta was smooth and intact. The abdominal aorta wall of the mice in the model group was damaged, and a large number of foam cells were seen to accumulate, which resulted in the thickening of the intima and the formation of atherosclerotic plaques. Soufeng expectorant Chinese medicine low, medium and high dose groups and atorvastatin group:abdominal aorta intima has plaques of varying degrees, but their area is smaller than the model group, of which Soufeng expectorant Chinese medicine middle dose group has plaques The block area is the smallest.

Figure 1 Plaque area of abdominal aorta of mice in each group(HE staining, ×100 times)

3.2 Test results of blood lipids in each group of mice

As shown in Table 1, compared with the blank control group,the content of TC, TG, and LDL-C in the model group increased(P＜0.05); the content of HDL-C decreased (P＜0.05); compared with the model group, search The contents of TG, T-CHO and LDL-C in each dose group of wind expelling phlegm Chinese medicine and atorvastatin group decreased (P＜0.05), and the content of HDL-C increased (P＜0.05).

3.3 Comparison of serum IL-1β detection in mice of each group

As shown in Table 2, compared with the blank control group,the IL-1β expression level of the model group was increased (P＜0.05); compared with the model group, the IL-1βexpression level of Soufeng Qutan Chinese medicine was compared with that of the atorvastatin group. The expression level of -1β decreased (P＜0.05).

Table 1 Comparison of TC, TG, LDL-C, HDL-C detection results of mice in each group (n=10, ±s)

Table 1 Comparison of TC, TG, LDL-C, HDL-C detection results of mice in each group (n=10, ±s)

Note: Compared with the blank control group,* P＜0.05;Compared with model group,△P＜0.05

GropeTC(mmol/L)TG(mmol/L)LDL-C(mmol/L)HDL-C(mmol/L)Blank control 2.845± 0.4641.178±0.2070.230±0.0892.360±0.190 Model group22.998± 4.771 *8.828±2.120 *6.500±1.701 *0.870±0.130 *Soufeng expectorant Chinese medicine low-dose group16.401± 3.584 *△6.977±1.047 *△3.570±1.384 *△1.260±0.210 *△Soufeng Expectorant Chinese Medicine Middle Dose Group15.348± 5.340 *△4.520±0.743 *△2.440±0.462 *△1.850±0.180 *△Soufeng expectorant Chinese medicine high-dose group16.515± 2.765 *△6.748±1.290 *△3.210±1.104 *△1.360±O.170 *△Atorvastatin group15.607± 2.160 *△4.638±0.773 *△2.600±1.714 *△1.710±0.100 *△F 13.5545.6262.5170.002 P＜0.001＜0.0010.0010.001

Table 2 Comparison of serum IL-1β levels of mice in each group (n=10, (±s))

Table 2 Comparison of serum IL-1β levels of mice in each group (n=10, (±s))

Note: Compared with the blank control group, * P＜0.05;Compared with model group,△ P＜0.05;

GropeIL-1β(pg/g-1)Blank control21.458±1.833 Model group23.465±1.177 *Soufeng expectorant Chinese medicine low-dose group 23.103±0.842 *△Soufeng Expectorant Chinese Medicine Middle Dose Group 22.630±1.055 *△Soufeng expectorant Chinese medicine high-dose group 23.158±1.099 *△Atorvastatin group22.649±1.074 *△F 3.02 P 0.006

4. Discussion

Abnormality of lipids and inflammatory factors is an important link in the occurrence and development of AS. Improving blood lipids and reducing related inflammatory factors can reduce the occurrence of cardiovascular events. Plaque formation caused by lipoprotein deposition is a common cause of AS. Reducing lipoprotein and plasma cholesterol levels can reverse the occurrence and development of AS to a certain extent [13]. IL-1β is one of the earliest inflammatory factors related to the pathogenesis of AS. The production of mature IL-1β induced by various inflammasome stimuli has been shown to contribute to the formation of AS plaques[14], LoDoCo, CANTOS and COLCOT, etc. Clinical trials have shown that it is feasible to treat AS with IL-1β as the target of Kanazumab[15], IL-1β inhibition has a powerful effect in improving AS[16], and clinical therapeutic interventions that interfere with IL-1β can Improving the cardiovascular structure has created a new era of AS anti-inflammatory therapy [17], showing its importance in preventing future cardiovascular events. The increase in IL-1β levels is related to dyslipidemia factors [18], which affects cholesterol homeostasis by promoting the uptake of Ox-LDL [19], so while inhibiting the production of IL-1β, it also plays a role in improving blood lipids. Will reduce the risk of cardiovascular disease.

The clinical benefits of statins for lowering blood lipids are still widely accepted, and are directly proportional to the reduction of LDL-C [20], and atorvastatin can reduce the formation of IL-1β and other related inflammatory factors, and inhibit cell pyrolysis [21] .Because atorvastatin has a certain effect on IL-1β and blood lipids,atorvastatin was selected as the western drug control drug in this experiment.

According to its clinical manifestations, AS can be classified into the categories of "chest pain" and "true heart pain". The treatment is mostly based on the treatment of promoting blood circulation to remove blood stasis, warming menstruation and dispelling cold,and promoting yang and eliminating phlegm [22]. According to the concept of "cardiovascular event chain" in western medicine,Professor Gong Lihong proposed the hypothesis of AS event chain in Chinese medicine: "deficiency-phlegm-blood stasis-poison(heat)-wind", and innovatively explained the occurrence of AS with the theory of wind phlegm. Development [23]. Elderly people with weak spleen and stomach, addicted to eating fat and sweet taste or emotional disorder can affect the function of spleen and stomach transportation and chemistry, stagnation of dampness and body fluid will produce turbid phlegm. Long-term turbid phlegm,kidney deficiency in old age, or internal emotional injury, etc., can cause poor circulation of qi and blood, endogenous blood stasis,and block the heart. Purple crimson or thick greasy tongue coating,slippery or tight pulse string. At this time, the pathogenesis is that phlegm turbidity and blood stasis interact with each other, which can’t be discharged, interlock and accumulate, and it can transform into poison (heat) over time. Toxin (heat) accumulates for a long time to produce "wind", and "wind" has "good deeds". The nature of "number change" is similar to the clinical manifestations of AS unstable plaques using the analogy method. Therefore, it is proposed that "deficiency" is the basis for the pathogenesis of AS, "phlegm-stasis-toxin (heat)" is the pathogenesis of AS, and"wind" For the "instability" of AS plaques, Professor Lihong of the Palace Museum summarized many years of clinical experience and believed that "wind phlegm" was the main cause of AS [24, 25].It is ruled that Chinese medicine for searching wind and expelling phlegm has a significant effect on the treatment of cardiovascular diseases. The scorpion and centipede in the prescription have the effects of searching for wind and relieving spasm, dredging collaterals and relieving pain. The combination of the two has the power to complement each other. Earthlong dredges the collaterals and activates the collaterals. The three are the monarch medicine;tangerine peel, pinellia and atractylodes are the minister medicines,which can invigorate the spleen. It can reduce dampness, reduce phlegm and dispel congestion, so that the movement can be restored,and the phlegm drinking can be reduced; leech is an adjuvant for removing blood stasis.

The results of this study showed that through HE staining, the blank control group had no obvious plaques in the abdominal aorta, and the model group showed more plaques. The plaque area of each dose group of Shoufeng Qutan Chinese medicine and the atorvastatin group was higher than that of the model group. The plaque area was measured to show that the Soufeng and expectorant Chinese medicine and atorvastatin have a certain effect on the treatment of atherosclerosis. At the same time, compared with the blank control group, the expression levels of TG, TC, LDL-C, and IL-1β in the model group, Soufeng expectorant Chinese medicine dosage groups,and atorvastatin group increased, while the expression of HDL-C decreased. It shows that the cell-related inflammatory factor IL-1β and blood lipids are at normal levels under normal conditions,so the expression level of IL-1β in the blank control group is low,and blood lipids are not abnormal. After the vascular endothelium is injured, lipids accumulate and IL-1β is activated, so the blood lipids of the model group appear abnormal, and the expression level of IL-1β increases. Both Soufeng Expectorant Herbal Medicine and Atorvastatin can improve blood lipids and reduce the production of IL-1β. Therefore, the expression levels of TG, TC, LDL-C, and IL-1β are reduced, and the expression level of HDL-C is increased.

In summary, Soufeng Expectorant Chinese Medicine can inhibit the progression of cellular inflammation, down-regulate the expression of IL-1β, reduce plaque lipid content, and improve blood lipids.This may be the early treatment of atherosclerosis by Soufeng Expectorant Chinese Medicine. One of the molecular mechanisms.

Authors’ contribution The experimental design is by the first author, Bing Zhao, and the corresponding author, Lihong Gong, with Bing Zhao as the author and Lihong Gong as the reviewer.