Effects of Shenshao Decoction on the inflammatory response in the aorta of a rat atherosclerotic

Shenshao Decoction (参芍汤) is a traditional herbal medicine composed of Radix Salviae Miltiorrhizae , Astragalus Membranaceus , Radix Paeoniae Rubra , Radix Angelicae Sinensis , Rhizoma Chuanxiong , Semen Persicae , Flos Carthami , Pheretima Aspergillum , Hirudo , and Rhizoma Pinelliae . Shenshao Decoction was developed as an oral liquid a decade ago and has been registered with the State Food and Drug Administration of Hebei province (Z20050957, China). We have previously shown that Shenshao Decoction can effectively prevent and suppress left ventricular remodeling after acute myocardial infarction in rats.(1) We have also demonstrated that Shenshao Decoction has cardioprotective effects after acute ischemia-reperfusion in rats.(2,3) While similar results have also been observed in humans,(4) the underlying mechanisms remain unclear.

T h e i n f l a m m a t o r y r e s p o n s e i s a b a s i c pathological mechanism that stimulates the formation of atherosclerotic plaque. While macrophages are known for their contribution to the inflammatory element of atherosclerosis, the role of the T lymphocyte

is relatively understudied. The newly discovered interleukin (IL)-17A-producing T cell subset, known as the Th17 cell, plays a major role in chronic inflammatory and autoimmune diseases, such as rheumatoid arthritis, in ? ammatory bowel disease, and multiple sclerosis. Th17 cells have also been shown to contribute to atherogenesis.(5) Recently, the Th17-derived cytokine, IL-17A, was shown to be involved in the pro-in ? ammatory response of vascular smooth muscle cells. The IL-23 and IL-17A axis is emerging as a critical regulatory system that bridges the innate and adaptive arms of the immune system. IL-1β plays a crucial role in early Th17 cell differentiation,(6) and

ORIGINAL ARTICLE

Effects of Shenshao Decoction (参芍汤) on the In ? ammatory Response in the Aorta of A Rat Atherosclerotic Model

XUE Zhong-wen (薛忠文)1, SHANG Xiao-ming (尚小明)1, LV Song-hui (吕松桧)3,

XU Hong (徐 洪)4, ZHANG Qi (张 琦)2, and WANG Chen (王 晨)2

?The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2013

1. Department of Internal Medicine, Hebei Medical University, Shijiazhuang, Hebei Province (050000), China;

2. Department of Cardiology, the Tangshan Worker's Hospital of Hebei Medical University, Tangshan, Hebei Province (063000), China;

3. Department of Obstetrics, Beijing Fangshan District Maternal and Child Health Hospital, Beijing (102488), China;

4. Medical Research Center, Hebei United University, Tangshan, Hebei Province (063000), China

Correspondence to: Dr. SHANG Xiao-ming, Tel: 86-315-2814801, Fax: 86-315-3722242, E-mail: shangxiaoming0616@https://www.360docs.net/doc/c712901166.html, DOI: 10.1007/s11655-013-1457-z

ABSTRACT Objective : To investigate the effect of Shenshao Decoction (参芍汤) on the in ? ammatory status in the aorta in a rat model of atherosclerosis. Methods : Forty Sprague-Dawley rats were randomly divided into ? ve groups, 8 rats in each group: control untreated group, atherosclerosis group, atherosclerosis with Shenshao Decoction (low dose) group, atherosclerosis with Shenshao Decoction (high dose) group, atherosclerosis with simvastatin group. To stimulate atherosclerosis, the rats were fed vitamin D3 and a high-cholesterol diet. Four weeks later, treatments were maintained for eight weeks. Morphology changes were investigated by hematoxylin and eosin staining. Serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were obtained by enzymatic assays with use of an automated biochemical analyzer. The expression of malondialdehyde (MDA), glutathione peroxidase (GSH-PX) were detected by enzyme-linked immunosorbent assay. The expression levels of interleukin (IL)-1β, IL-17A, and IL-23 were detected by immunoblotting. Results : Shenshao Decoction treatment decreased TC, TG, LDL-C and MDA and increased GSH-PX levels (P <0.01). Compared with the control group, IL-1β, IL-17A, and IL-23 were lower in the high and low dose Shenshao Decoction groups and the simvastatin group (P <0.01). Conclusion : Shenshao Decoction could attenuate the progression of aortal atherosclerotic plaques by inhibiting the in ? ammatory response in a rat atherosclerotic model.

KEYWORDS Shenshao Decoction, atherosclerosis, malondialdehyde, glutathione peroxidase

is produced by activated macrophages. Considered a master switch of in?ammation, IL-1βhas been implicated in the pathogenesis of atherosclerosis.(7) Based on previous clinical and experimental trials, we hypothesized that the Chinese medicine Shenshao Decoction would protect against atherosclerosis by mediating the inflammatory and free radical pathogenic effects, as well as through stimulation of lipid-lowering and prevention of vascular smooth muscle migration.

METHODS

Shenshao Decoction Preparation

Radix Angelicae Sinensis and Ligusticum wallichii were re? uxed in 70% ethanol and extracted three times for 1.5 h each. The extraction was ? ltrated and preserved. The dregs and the remaining other eight herbs that make up Shenshao Decoction were boiled with water twice, the first time for 2 h and the second time for 1.5 h. The filtered liquors were amalgamated and incubated for 12 h. The supernatant was ? ltrated again and concentrated into an ointment with a density of 1.5-fold. The alcohol content of the ointment was regulated at 70% and was mixed with the alcohol extract of Radix Angelicae Sinensis and the others herbs. The mixture was obturated and kept in cold storage for 24 h, after which it was filtrated and concentrated into 800 mL. Then, Sirupus simplex (100 g) and an appropriate amount of pennyroyal was added into the mixture, and the pH was adjusted to 6.5 with sodium hydroxide. The new mixture was filtrated, and distilled water was added to 1000 mL final volume. Finally, the mixture was stirred and sterilized and designated as the working solution for experimental treatments.

Experimental Animals

Forty male Sprague-Dawley rats, weighing 180±10 g, were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (SCXK: 0175819, 2010-0038, Beijin, China). The animal experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Hebei United University.

A rat atherosclerosis model was produced by a regimen of intraperitoneal injection of 6×105 IU/(kg?d) vitamin D3 for 3 days and a high cholesterol diet for 4 weeks.(8) The high fat rat chow was purchased from the Experimental Animal Center of Hebei Union University, and was composed of 3% cholesterol, 0.5% sodium cholate, 0.2% propylthiouracil, 5% sugar, 10% lard, and 81.3% basic diet. After 4 weeks, the diet was replaced with a regular diet, and the experimental treatments were initiated and maintained for 8 weeks. Rats were randomly divided into 5 treatment groups with 8 rats in each group: (1) control group, fed a regular diet and administered physiological saline by oral gavage (8 mL/d); (2) atherosclerosis group, administered oral physiological saline (4 mL/d); (3) atherosclerosis group treated with low dose Shenshao Decoction [150 mg/(kg?d) by oral administration]; (4) atherosclerosis group treated with high dose Shenshao Decoction [300 mg/(kg?d) by oral administration]; and (5) atherosclerosis group treated with simvastatin which was produced by Merck Sharp & Dohme [5 mg/(kg?d) by oral administration]. Histopathology

A t t h e e n d o f t h e s t u d y,t h e r a t s w e r e anaesthetized with 10% chloral hydrate, and the aortas were removed. The resected tissues were then dissected from the root of the aorta to the branch of the common iliac artery. The upper half portion was fixed in formalin and embedded in paraffin, while the remaining portion was snap-frozen at –80℃ for subsequent immunoblotting analysis. Six μm-thick tissue sections were stained with hematoxylin and eosin (HE) for routine histopathology. Biochemical Assays

At the end of the 12-week protocol, blood was drawn from overnight fasted rats. Serum levels of malondialdehyde (MDA; HBT-0284R, Zhenjiang Hope Biotechnology Co., Ltd., China) and glutathione peroxidase (GSH-PX; HBT-0241R, Zhenjiang Hope Biotechnology Co., Ltd., China) were assayed by use of a highly sensitive enzyme-linked immunosorbent assay (ELISA) kit (Bio RAD, Imark, USA). Serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were obtained by enzymatic assays with use of an automated biochemical analyzer (Cobas Mira Sauto analyzer, Switzerland ). Immunohistochemistry for IL-1β, IL-17A, and IL-23

Paraf? n-embedded sections were deparaf? nized, rehydrated, and boiled in a microwave oven for 10 min for antigen retrieval. After incubation with

5% horse serum, sections were incubated with primary antibodies against IL-17A (6076, Santa Cruz Biotechnology, USA), followed by incubation with a biotinylated secondary antibody and the avidin-biotin complex (ABC) reagent (Wuhan Boshide Biological Engineering Co., Ltd., China). Immunoreactivity w a s v i s u a l i z e d w i t h t h e d i a m i n o b e n z i d i n e tetrahydrochloride (DAB) substrate. A brown-colored stain was considered as a positive result. Sections were counterstained with hematoxylin. Images were obtained from ? ve separate, randomly-selected ? elds (400×magni ? cation).

Immunoblotting Analysis of IL-1β, IL-17A, and IL-23

The thoracic aorta (100 mg) was lysed with lysis buffer (20 mmol/L Tris buffer, pH 7.4, containing 1 mmol/L sodium orthovanadate, 0.2 mmol/L EDTA, 0.2 mmol/L phenylmethylsulfonyl fluoride, 0.01 mmol/L NaCl, 50 mmol/L NaF, and 25 μg/mL aprotinin), and centrifuged at 14,000×g for 10 min. The supernatant was collected and protein content determined with a protein assay kit (Bio-Rad, USA). Total protein (90 μg/lane) was separated on a 8% gel by SDS-PAGE and electrotransferred onto a nitrocellulose membrane (Amervehicle Biosciences, Sweden). Membranes were blocked with 5% non-fat milk and incubated overnight at 4 ℃ with one of the following primary antibodies: IL-1β (SC-12742 Santa Cruz Biotechnology, USA), IL-17A (6076,

Santa Cruz Biotechnology), or IL-23 (271279, Santa

Cruz Biotechology). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (E030220, E020210, Earch Ox, USA). Target bands were visualized by incubation with 5-bromo-4-chloro-3-indolyl phosphate tetranitroblue tetrazolium chloride solution (E-116, Amresco, USA). Results were normalized with β-actin densitometry values and expressed as the fold-change of the corresponding actin control.

Statistical Analysis

All statistical analyses were performed with SPSS 13.0. The data were expressed as mean ±SEM. Multiple group comparisons were conducted using ANOVA. Group differences resulting in P -values of <0.05 were considered to be statistically signi ? cant.

RESULTS

Changes in Expression of TC, TG, LDL-C, MDA, and GSH-PX

We examined the expression levels of MDA, GSH-PX, TC, TG, and LDL-C by ELISA and automated biochemical analyzer. Compared with the control group, the expression of TC, TG, and LDL-C, MDA were higher and the expression of GSH-PX was lower in the atherosclerotic group. Shenshao Decoction and simvastatin treatments both signi ? cantly decreased the expression of TC, TG, and LDL-C (Figure 1).

Figure 1. Biochemical Measurements in the Five Treatment Groups of Rats

Notes: SSD: Shenshao Decoction; P <0.01, compared with the control group; △P <0.05, △△P <0.01, compared with the atherosclersosis group

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Morphological Observations

As shown in Figure 2, the control group showed intact structures of the tunica intima, tunica media, and tunica adventitia. In the atherosclerosis group, the aortas showed disorganized structure, coarse endangium, and large area of incrassation and apophysis. The endothelial cell layer was disrupted and plaque was formed. Foamy cells were present in the plaque, along with an amorphous area at the bottom of the plaque that contained cholesterol crystals, necrotic tissue, and fibrotic tissue. Smooth muscle cells were found to have migrated to the bottom of the endangium. The endochylema stained light, indicating lipid content. The hyperplastic smooth muscle cells in the tunica elastica were irregularly shaped, migrated towards the endangium, and integrated with the lipid.

In the low dose Shenshao Decoction group, the intima was coarse with a thick wall and hyperplasia. Overall, the aortas showed mild pathology. In the high dose Shenshao Decoction group, part of the intima was coarse and most of the endothelial layer was intact. The smooth muscle cells showed little hyperplasia. In the simvastatin group, the aortas were similar in appearance to those of the high dose Shenshao Decoction group. The intima was coarse and the

endothelial cell layer was largely intact and smooth muscle hyperplasia was mild.

Changes in Expression of IL-1β, IL-17A, and IL-23

We examined expression of IL-1β, IL-17A, and IL-23 by immunohistochemical staining (Figure 2). These proteins showed slight staining in the control group. The expression of IL-1β, IL-17A, and IL-23 was robustly up-regulated, however, in the atherosclerosis group, as evidenced by strong staining. Compared with the atherosclerosis group, the expression of IL-1β, IL-17A, and IL-23 was signi ? cantly reduced in the low and high dose Shenshao Decoction groups, as well as in the simvastatin-treated group (Figures 2 and 3).Immunoblotting analysis further revealed that the expression of IL-1β, IL-17A, and IL-23 increased 2.4-, 3.2-, and 2.3-fold, respectively, in the atherosclerosis group, as compared to the control group, all P <0.05). The expression of IL-1β, IL-17, and IL-23 were reduced by 65.56%, 59.5% and 50.43%, respectively, in the high dose Shenshao Decoction group, as compared to the atherosclerosis group (Figure 4).

DISCUSSION

The goal of this study was to evaluate the effect of Shenshao Decoction treatment on aortal plaque

Figure 3. Immunohistochemical Staining for IL-1β, IL-17A, and IL-23 (200×)

Figure 2. Morphological Changes in the Rat Aorta (HE staining, 200×)

Simvastatin

Control

Atherosclerosis

Low dose SSD

Simvastatin

Control

Atherosclerosis

Low dose SSD

High dose SSD

High dose SSD

IL-23

IL-1β

IL-17A

progression in a rat model of atherosclerosis. The signi ? cant results were that Shenshao Decoction was found to reduce in ? ammation and lipid accumulation in the aorta, resulting in reduced plaque formation. Our results suggest that Shenshao Decoction may serve as an effective treatment to prevent the development of aortal atherosclerosis.

Shenshao Decoction has shown promising clinical results since it was approved for use as a medical treatment. Coronary heart disease patients who were administered Shenshao Decoction for three months had significantly lower occurrence of angina pectoris and other cardiac adverse events than those who did not receive Shenshao Decoction.(4) In addition, coronary angiography demonstrated that the development of coronary vascular lesions in patients who took Shenshao Decoction was significantly delayed, compared with the control group.(4)

In an ischemia reperfusion animal model, Shenshao Decoction inhibited the production of collagen in a rabbit myocardial infarction model, decreased the level of ? brosis, postponed the effect of myocardial hypertrophy, and protected the ischemic cardiac muscle.(1-3) While these results indicated that Shenshao Decoction had a protective effect in cardiovascular disease, the specific underlying mechanisms remained unclear. Our study focused on whether Shenshao Decoction could significantly postpone the development of aortal atherosclerosis and/or alter lipid levels, inflammation, and oxidative stress.

Shenshao Decoction is derived from a group of herbal medicines that include Radix Salviae Miltiorrhizae , Astragalus Membranaceus , Radix Paeoniae Rubra , Radix Angelicae Sinensis , Rhizoma Chuanxiong , Semen Persicae , Flos Carthami , Pheretima Aspergillum , Hirudo , and Rhizoma

Pinelliae . Recent studies have demonstrated that Shenshao Decoction has pleiotropic effects, including improving endothelial function, reducing fibrosis, and inhibiting myocardial hypertrophy, which may be due to the cumulative or synergistic effects of the multiple compounds present in the herbal extract. For example, Radix Salviae Miltiorrhizae is the major ingredient of Shenshao Decoction, and has been successfully applied in Asian clinics as an independent therapeutic agent to prevent and treat coronary heart disease. It is possible that Radix Salviae Miltiorrhizae might regulate lipid metabolism, enhance antioxidant properties, and reduce the release of inflammatory factors.(9) Similarly, Astragalus Membranaceus has been shown to have cardiac protective and angiogenic effects.(10) Radix Angelicae Sinensis , Rhizoma Chuanxiong , Semen Persicae , and Flos Carthami are known in China as blood-activating and stasis-eliminating herbs. They elicit multiple physiologic effects, including inhibition of platelet coacervation, stimulation of blood circulation, and hematopoietic activation to induce the production of hemoglobin.(11-13) Pheretima Aspergillum , Hirudo , and Rhizoma Pinelliae have each been Demonstrated to promote blood circulation.(14) Our current study indicated that Shenshao decoction can decrease serum levels of TC, TG, and LDL in a rat atherosclerosis model, and can decrease the expression of MAD and increase GSH-PX.

The proliferation and migration of vascular smooth muscle cells play important roles in the formation and progression of intimal thickening that occurs in the early phase of atherosclerosis. While smooth muscle cell migration is stimulated and mediated by inflammatory factors, atherosclerosis is also a lipid-driven, chronic inflammatory disease of the vessel wall, in which both innate and adaptive immune responses play roles.(15) Immune cells and their mediators are integral components of the chronic arterial inflammation that is a hallmark of

Figure 4. The Relative Protein Expression in Thoracic Aorta from the Five Treatment Groups of Rats

Notes: P <0.01, compared with the control group; △P <0.05, △△P <0.01, compared with the atherosclerosis group

IL-1βIL-17A IL-23β-actin

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atherosclerosis. Here, we report that IL-1β, IL-23, and the innate mediator IL-17A are involved in the occurrence of atherosclerosis and further demonstrate an effect of Shenshao Decoction on these processes. IL-17A is a CD4 T cell-derived pro-in?ammatory cytokine, and is known to play an active role in the development of in? ammation, autoimmune diseases, and tumors.(16-18) More recently, IL-1β and IL-23 were shown to induce innate IL-17A production from CD T cells, leading to amplification of Th17 responses and autoimmunity.(19)However, the mechanisms underlying the pro-in? ammatory action of IL-17A are poorly understood. Our study showed that in an aortal atherosclerosis model, IL-1β, IL-17A, and IL-23 levels are increased in concert with endothelial cell damage and atherosclerosis plaque formation, and Shenshao Decoction inhibits all of these processes.

In conclusion, our study provided evidence that Shenshao Decoction can not only decrease the level of blood lipids in a rat aortal atherosclerosis model but also alleviate the pathological extent of the plaque and inhibit the expression of IL-1β, IL-17A, and IL-23. Collectively, these findings indicate that Shenshao Decoction may represent an effective anti-atherosclerotic agent by reducing in? ammatory and lipid pro? les.

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(Received November 30, 2012)

Edited by CHEN Yi-yu