Skate cartilage extracts containing chondroitin sulfate ameliorates hyperlipidemia-induced inflammation and oxidative stress in high cholesterol diet-fed LDL receptor knockout mice in comparison with shark chondroitin sulfate

Bo Gyeong Seol; Ji Hyun Kim; Minji Woo; Yeong Ok Song; Yung Hyun Choi; Jeong Sook Noh; Eun Ju Cho

doi:10.4162/nrp.2020.14.3.175

Journal List > Nutr Res Pract > v.14(3) > 1144994

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Seol, Kim, Woo, Song, Choi, Noh, and Cho: Skate cartilage extracts containing chondroitin sulfate ameliorates hyperlipidemia-induced inflammation and oxidative stress in high cholesterol diet-fed LDL receptor knockout mice in comparison with shark chondroitin sulfate

Original Research

Nutrition Research and Practice 2020; 14(3): 175-187.

Published online: 22 January 2020

DOI: https://doi.org/10.4162/nrp.2020.14.3.175

Skate cartilage extracts containing chondroitin sulfate ameliorates hyperlipidemia-induced inflammation and oxidative stress in high cholesterol diet-fed LDL receptor knockout mice in comparison with shark chondroitin sulfate

Bo Gyeong Seol¹

, Ji Hyun Kim¹

, Minji Woo^1,²

, Yeong Ok Song¹

, Yung Hyun Choi^3,⁴

, Jeong Sook Noh⁵

, Eun Ju Cho¹

¹Department of Food Science and Nutrition & Kimchi Research Institute, Pusan National University, Busan 46241, Korea.

²Busan Innovation Institute of Industry, Science & Technology Planning (BISTEP), Busan 48058, Korea.

³Anti-Aging Research Center, Dong-eui University, Busan 47227, Korea.

⁴Department of Biochemistry, Dong-eui University College of Korean Medicine, Busan 47227, Korea.

⁵Department of Food Science and Nutrition, Tongmyong University, Busan 48520, Korea.

Corresponding Author: Jeong Sook Noh. Department of Food Science and Nutrition & Kimchi Research Institute, Pusan National University, 2 Busandaehak-ro 63beon-gil, Geumjeong-gu, Busan 46241, Korea. Tel. +82-51-629-1716, Fax. +82-51-629-1709, jsnoh2013@tu.ac.kr

Corresponding Author: Eun Ju Cho. Department of Food Science and Nutrition, Tongmyong University, 428 Sinseon-ro, Nam-gu, Busan 48520, Korea. Tel. +82-51-510-2837, Fax. +82-51-583-3648, ejcho@pusan.ac.kr

Received 2 July 2019 Revised 6 September 2019 Accepted 4 December 2019

The Korean Nutrition Society and the Korean Society of Community Nutrition

(open-access, https://creativecommons.org/licenses/by-nc/4.0/):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/).

Abstract

BACKGROUND/OBJECTIVES

In this study, we investigated the beneficial effects of skate cartilage extracts containing chondroitin sulfate (SCS) on hyperlipidemia-induced inflammation and oxidative stress in high cholesterol diet (HCD)-fed mice in comparison with the effects of shark cartilage-derived chondroitin sulfate (CS).

MATERIALS/METHODS

Low-density lipoprotein receptor knockout (LDLR-KO) mice were fed HCD with an oral administration of CS (50 and 100 mg/kg BW/day), SCS (100 and 200 mg/kg BW/day), or water, respectively, for ten weeks.

RESULTS

The administration of CS or SCS reduced the levels of serum triglyceride (TG), total cholesterol (TC), and LDL cholesterol and elevated the levels of high-density lipoprotein cholesterol, compared with those of the control group (P < 0.05). Furthermore, CS or SCS significantly attenuated inflammation by reducing the serum levels of interleukin (IL)-1β and hepatic protein expression levels of nuclear factor kappa B, inducible nitric oxide synthase, cyclooxygenase-2, and IL-1beta (P < 0.05). In particular, the serum level of tumor necrosis factor-alpha was reduced only in the 100 mg/kg BW/day of SCS-fed group, whereas the IL-6 level was reduced in the 100 and 200 mg/kg BW/day of SCS-fed groups (P < 0.05). In addition, lipid peroxidation and nitric oxide production were attenuated in the livers of the CS and SCS groups mediated by the upregulation of hepatic proteins of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase (P < 0.05).

CONCLUSIONS

These results suggest that the biological effects of SCS, similar to those of CS, are attributed to improved lipid profiles as well as suppressed inflammation and oxidative stress induced by the intake of HCD.

Keywords: Chondroitin sulfates, cartilage, hyperlipidemias, inflammation, oxidative stress

INTRODUCTION

Hyperlipidemia is a common feature of dyslipidemia characterized by an increase of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) and a decrease of high-density lipoprotein cholesterol (HDL-C) in the blood. The high levels of plasma lipid are strongly associated with a typical pathological condition of numerous diseases, such as atherosclerosis, non-alcoholic fatty liver diseases (NAFLD), and inflammation-related disorder [1 2 3 4]. Dietary cholesterol is a well-known cause for hyperlipidemia [5]. Numerous in vivo studies demonstrated that a cholesterol-rich diet induces hyperlipidemia [5 6 7 8].

Under the condition of hyperlipidemia, the inflammation and oxidative stress are predominant [9]. Dietary cholesterol-induced hyperlipidemia leads to an inflammatory response and enhances oxidative stress in organs [5 6]. In particular, hepatic inflammation plays a vital role in the progression of steatohepatitis, fibrosis, and finally, cirrhosis [10]. The excessive intake of cholesterol provokes hepatic inflammation, which directly results in the development of hepatitis [4]. Inflammatory responses are promoted by the release of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6, and inflammatory enzymes, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), regulated by the nuclear factor-kappa B (NF-κB) activation [11]. In addition, elevated oxidative stress produces peroxynitrite and increases lipid peroxidation [12], which impairs the body's antioxidant status via downregulation of antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) [13].

Chondroitin sulfate is a glycosaminoglycan, a type of polysaccharide that is present in the cartilages, skin, blood vessels, ligaments, and tendons of the body [14]. Chondroitin sulfate is mainly used for the treatment of osteoarthritis due to its anti-inflammatory activities [14 15]. Besides, biological activities have established regarding the improvement of lipid/glucose metabolism, anti-atherosclerosis, antioxidant, and anti-apoptotic effects [16 17 18 19]. One of the major sources of chondroitin sulfate is shark cartilage. Recently, it has become necessary to replace shark cartilage-derived chondroitin sulfate (CS) because of the prohibition of the capture and killing of sharks [19 20]. Therefore, several studies have made attempts to extract chondroitin sulfate from various sources, including cattle, pigs, chickens, and sea cucumbers [19 21]. The skate (Raja Kenojei) is a commonly found fish species in the northwestern Pacific Ocean, Korea, Japan, China, and possibly elsewhere. Only the fillet of the skate is consumed due to its unique odor; the remaining parts (e.g. skin, cartilage, and bone) weighing about 30%, are discarded [22]. We previously investigated the protective effects of skate cartilage extracts containing chondroitin sulfate (SCS) in lipopolysaccharide (LPS)-induced liver damage [18]. However, it has not been studied whether SCS could alleviate hyperlipidemia-induced inflammation and oxidative stress in high cholesterol diet (HCD)-fed mice. Therefore, the aim of the present study is to investigate the beneficial effects of SCS on hyperlipidemia, inflammation, and oxidative stress in the LDL receptor knockout (LDLR-KO) mice fed an HCD in comparison with the effects of CS.

MATERIALS AND METHODS

Preparation of skate cartilage extracts containing chondroitin sulfate

The SCS was provided by Yeongsan Skate (Yeongsan Skate Co., Ltd., Busan, Korea). Skate cartilage was obtained during the skate processing as mentioned in the previous study [22]. Briefly, the byproduct of skate cartilage was heated at 95°C for 2 h to remove ribbon-type meat. Skate cartilage was dried for 24 h using a heated-air dryer and crushed. The powdered skate cartilage was added with 3 volumes of water and hydrolyzed by protease such as alcalase and protamex (Novozyme Co., Bagsværd, Denmark) at 50°C for 4 h, followed by heated at 95°C for 30 min. Afterward, it was deodorized, filtered, and concentrated at 65°C. The final sample was freeze-dried at −75°C. The composition of SCS was as follows; moisture 2.09%, protein 46.97%, ash 3.5%, and chondroitin sulfate 47.44% (Food Analysis Center, Pukyong National Univ., Busan, Korea). The positive control, CS was purchased from Sigma-Aldrich (C4384; Sigma-Aldrich, St. Louis, MO, USA).

Animal and experimental diets

Forty-five male LDLR-KO mice (6-week-old, each 20-25 g) were obtained from Jackson Laboratories (Bar Harbor, Me, USA). Mice were housed in a plastic cage with a 12 h light-dark cycle. The room had standard laboratory humidity (50 ± 10%) and temperature (20 ± 2°C). After 1 week of acclimatization, mice were randomly assigned into five groups on the basis of body weights (n = 8 per group). The experimental groups were shown in Table 1. The dose of CS and SCS was determined according to a previous report [18]. In particular, the concentration of SCS was considered by the content of chondroitin sulfate in SCS (47.44%) [18], thus the dose of SCS groups is a half of that of CS groups. CS and SCS were orally administered via a stomach tube every day for 10 consecutive weeks. During the experimental period, mice were provided with free access to water and HCD composed of 20 kcal% protein, 45 kcal% carbohydrate, 35 kcal% fat (D12336, Research Diets, New Brunswick, NJ, USA). The percentage of cholesterol in HCD was 1.25%. Body weight was recorded every week, and food intake was examined every day. The food efficiency ratio (%) was calculated as total body weight gain/total food intake × 100.

Table 1

The experimental groups in this study

Group	Treatment
Control	HCD + oral administration of water
CS50	HCD + oral administration of CS (50 mg/kg/day)
CS100	HCD + oral administration of CS (100 mg/kg/day)
SCS100	HCD + oral administration of SCS (100 mg/kg/day)
SCS200	HCD + oral administration of SCS (200 mg/kg/day)

HCD, high cholesterol diet; CS, shark cartilage-derived chondroitin sulfate; SCS, skate cartilage-derived chondroitin sulfate extract.

The mouse was fasted for 12 hours before sacrifice, and anesthetized with CO₂ gas. Immediately the blood was collected and centrifuged at 3,000 rpm for 20 min at 4°C to obtain serum. Liver tissues were perfused with 0.9% sodium chloride and removed. Serum and liver tissues were stored frozen in a deep freezer at −80°C. All experimental procedures were permitted (Approval No. PNU-2018-1887) using the guidelines established by the Pusan National University Institutional Animal Care and Use Committee (PNU-IACUC).

Serum lipid profiles, atherogenic index, and cardiovascular risk index

Serum triglyceride (TG, AM157S-K, Asan Pharm., Seoul, Korea), total cholesterol (TC, AM202-K, Asan Pharm, Seoul, Korea), and high-density lipoprotein cholesterol (HDL-C, AM203-K, Asan Pharm, Seoul, Korea) concentrations were measured using commercially available kits. Low-density lipoprotein cholesterol (LDL-C) concentration was calculated as TC - HDL-C / (TG / 5) [23]. The atherogenic index (AI) was calculated as (TC - HDL-C) / HDL-C, and the cardiac risk factor (CRF) was calculated as TC / HDL-C.

Serum inflammatory cytokine concentration

Serum inflammatory cytokine concentrations were measured using ELISA kits. The following cytokines were measured: TNF-α (#560478, BD, Franklin Lakes, NJ, USA), IL-1β (MLB00C, Minneapolis, MN, USA), IL-6 (#550950, BD, Franklin Lakes, NJ, USA), and prostaglandin E₂ (PGE₂, M6000B, Minneapolis, MN, USA).

Lipid peroxidation in the liver tissue

The content of malondialdehyde (MDA) in liver tissue was measured according to the method of Ohkawa et al. [24]. After centrifugation at 3,000 rpm for 10 min, the supernatant was mixed with 1% phosphoric acid and 0.67% thiobarbituric acid TBA), and the mixture was boiled for 30 min and then cooled. Seven milliliters of butanol was added and the mixture was centrifuged at 3,000 rpm for 10 min. The absorbance of the supernatant was measured at 540 nm. The standard curve was prepared using different concentrations of MDA, and the extent of lipid peroxidation was calculated.

Nitric oxide (NO) production in the liver tissue

The NO contents of liver tissue were measured according to the method of Schmidt et al. [25]. The liver tissues were homogenized with a homogenizer by adding a physiological saline solution (0.9% NaCl) and centrifuged at 3,000 rpm for 10 min. After, 150 µL of the supernatant and 130 µL of distilled water were mixed, and then 100 µL of the mixture was added to the Griess reagent (1:1 ratio) and incubated at room temperature for 15 min. The absorbance was measured at 540 nm. The standard curve was prepared by different concentrations of NaNO₂ and the inhibition of NO production was calculated.

Western blot analysis

The liver tissues were homogenized with lysis buffer containing a protease inhibitor cocktail. The homogenates were centrifuged at 12,000 rpm for 20 min at 4°C. After the supernatant was collected, the protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Irvine, CA, USA). An equal amount of proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membrane was incubated with 5% skim milk for 50 min at room temperature and then washed with PBS-T. The membrane was incubated with a primary antibody overnight at 4°C and then washed with PBS-T. The primary antibodies used were: β-actin (1:1,000; Cell Signaling, Beverly, MA, USA), NF-κB p65 (1:500, Abcam, Cambridge, UK), iNOS (1:500, Merk, Kenilworth, NJ, USA), COX-2 (1:1,000, Cell Signaling, Beverly, MA, USA), IL-1β (1:500; Bioss Antibodies, Boston, MA, USA), SOD (1:500; Santa Cruz, Dallas, TX, USA), catalase (1:500; Santa Cruz), and GSH-Px (1:500, Santa Cruz). The membranes were incubated with secondary antibodies for 1 h. The immuno-complexes were visualized by enhanced chemiluminescence solution (ELPIS Biotech, Daejeon, Korea) and bands were visualized with a chemiluminescent imaging system (Davinci Chemi, Seoul, Korea).

Statistical analysis

Data are presented as mean ± SD. Results were assessed using one-way analysis of variance (ANOVA) and Duncan's test for multiple comparisons. Statistical significance was considered as P < 0.05.

RESULTS

Body weight, food intake, and food efficiency ratio

Changes in body weight, the amount of daily food intake, and food efficiency ratio are presented in Table 2. These data were not significantly different among all experimental groups.

Table 2

Body weight and food intake in high cholesterol diet fed LDL receptor knockout mice for 10 weeks

Group¹⁾	Initial body weight (g)	Final body weight (g)	Body weight gain (g)	Food intake (g/day)	Food efficiency ratio (%)²⁾
Control	19.20 ± 1.23^NS	23.61 ± 1.64^NS	4.41 ± 1.01^NS	2.32 ± 0.41^NS	21.11 ± 4.84^NS
CS50	18.98 ± 1.39	23.86 ± 1.75	4.89 ± 1.19	2.36 ± 0.40	23.00 ± 5.58
CS100	18.68 ± 1.33	23.54 ± 1.69	4.86 ± 0.78	2.30 ± 0.39	23.47 ± 3.76
SCS100	18.79 ± 1.53	23.20 ± 1.53	4.41 ± 0.83	2.27 ± 0.42	23.32 ± 4.40
SCS200	18.80 ± 1.69	23.51 ± 1.63	4.71 ± 0.78	2.38 ± 0.41	22.03 ± 3.66

Values are mean ± SD (n = 8 each group). Statistical analyses were conducted by Duncan's multiple range test (P < 0.05). NS, Non-significance.

¹⁾Control, LDL receptor knockout (LDLR-KO) mice fed high cholesterol diet (HCD) and oral administration (OA) of purified water; CS50 and CS100, LDLR-KO mice fed HCD and OA of CS at a concentration of 50 and 100 mg/kg/day, respectively; SCS100 and SCS200, LDLR-KO mice fed HCD and OA of SCS at a concentration of 100 and 200 mg/kg/day, respectively.

²⁾Food efficiency ratio (%) : (total weight gain/total food intake) × 100.

Serum lipid profiles, atherogenic index, and cardiovascular risk index

As shown in Fig. 1, serum lipid profiles, atherogenic index (AI), and cardiovascular risk index (CRF) were determined. The administration of CS50, CS100, SCS100, and SCS200 significantly decreased the TG levels to 3.64 mmol/L, 3.72 mmol/L, 4.03 mmol/L, and 4.09 mmol/L, respectively, compared to those of the untreated control group (4.97 mmol/L). The concentration of TC was 71.45 mmol/L in the control group, whereas the CS50, CS100, SCS100, and SCS200 groups significantly reduced the TC level to 65.71 mmol/L, 64.20 mmol/L, 64.93 mmol/L, and 67.03 mmol/L, respectively. The LDL-C concentrations were significantly reduced in the CS50, CS100, SCS100, and SCS200 groups to 56.75 mmol/L, 54.67 mmol/L, 53.69 mmol/L, and 50.77 mmol/L, respectively, relative to that in the control group (63.96 mmol/L). The HDL-C concentrations were significantly increased in the CS50, CS100, SCS100, and SCS200 groups to 8.71 mmol/L, 9.74 mmol/L, 10.59 mmol/L, and 15.24 mmol/L, respectively, compared with the control group (4.07 mmol/L). Subsequently, the CS or SCS significantly decreased the AI and CRF. Compared with the control group, the CS50, CS100, SCS100 and SCS200 groups showed a decrease in the AI index by 6.98%, 6.22%, 5.35%, and 3.53%, and a decrease in the CRF index by 7.98%, 7.22%, 6.35%, and 4.53%, respectively.

Fig. 1

Effects of chondroitin sulfate from shark cartilage (CS) and chondroitin sulfate from skate cartilage extract (SCS) on serum lipid profiles in high cholesterol diet fed LDL receptor knockout mice for 10 weeks. (A) TG, triglyceride; (B) TC, total cholesterol; (C) LDL-C, low density lipoprotein cholesterol; (D) HDL-C, high density lipoprotein; (E) AI, atherogenic index; (F) CRF, cardiac risk factor. See the legend of Table 1 for experimental groups in detail. Values are means ± SD (n = 8 per group). ^a~cMeans with the different letters are significantly different (P < 0.05) by Duncan's multiple range test.

Serum inflammatory cytokine levels

The effects of CS and SCS on serum inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and PGE₂ are shown in Fig. 2. The concentrations of TNF-α were decreased to 20.38 pg/mL, 18.08 pg/mL, 16.54 pg/mL, and 21.62 pg/mL in the CS50, CS100, SCS100, and SCS200 groups, respectively, compared with control group (26.46 pg/mL). In particular, the SCS100 group showed a significantly decreased level of TNF-α. In CS50, CS100, SCS100, and SCS200 groups, the IL-1β levels were significantly reduced to 124.53 pg/mL, 121.16 pg/mL, 129.47 pg/mL, and 126.87 pg/mL, respectively, from those in the control group (145.05 pg/mL). In addition, the IL-6 level in the CS50, CS100, SCS100, and SCS200 groups were decreased by 112.69 pg/mL, 119.19 pg/mL, 116.94 pg/mL, and 111.94 pg/mL, respectively, compared with control group (123.69 pg/mL). However, no significant difference in PGE₂ levels was noted in any experimental group.

Fig. 2

Inflammatory cytokine concentrations in high cholesterol diet fed LDL receptor knockout mice for 10 weeks. (A) TNF-α, tumor necrosis factor-alpha; (B) IL-β, interleukin-1beta; (C) IL-6, interleukin-6; (D) PGE₂, prostaglandin E2. See the legend of Table 1 for experimental groups in detail. Values are means ± SD (n = 8 per group). ^a~cMeans with the different letters are significantly different (P < 0.05) by Duncan's multiple range test. NS, Non-significance.

Inflammation-related protein expression in the liver

The effects of CS and SCS on the hepatic inflammatory-related protein levels, such those of as NF-κB, iNOS, COX-2, and IL-1β, are shown in Fig. 3. The administration of CS or SCS lowered the protein expression levels of NF-κB, iNOS, COX-2, and IL-1β. In particular, compared to those of the control group, the protein expression levels of NF-κB and IL-1β in the CS100, SCS100, and SCS200 groups were significantly decreased by 31.17%, 33.50%, and 42.03%, respectively, and by 13.35%, 19.88%, and 16.97%, respectively (P < 0.05). Those levels of iNOS in the CS50, CS100, SCS100, and SCS200 groups were significantly reduced by 24.72%, 51.69%, 53.61%, and 38.46%, respectively (P < 0.05). Those levels of COX-2 in the CS100 and SCS200 groups were significantly decreased by 49.00% and 41.44%, compared to those of the control group (P < 0.05)

Fig. 3

Effects of chondroitin sulfate from shark cartilage (CS) and chondroitin sulfate from skate cartilage extract (SCS) on inflammation related protein expression in the liver of high cholesterol diet fed LDL receptor knockout mice. (A) Representative Western blot analysis; (B) NF-κB, nuclear factor kappa B; (C) iNOS, inducible nitric oxide synthase; (D) COX-2, cyclooxygenase-2; (E) IL-1β, interleukin-1beta. See the legend of Table 1 for experimental groups in detail. Values are means ± SD (n = 8 per group). β-actin was used as a loading control. ^a~eMeans with the different letters are significantly different (P < 0.05) by Duncan's multiple range test.

Lipid peroxidation and nitric oxide formation in the liver

The inhibitory effects of CS and SCS on lipid peroxidation and NO are shown in Fig. 4. The hepatic MDA levels were significantly decreased in the CS50, CS100, SCS100, and SCS200 groups, to 8.04 µmol/mg protein, 5.38 µmol/mg protein, 6.33 µmol/mg protein, and 8.39 µmol/mg protein, respectively, compared with the control group (12.04 µmol/mg protein). In particular, the CS100 and SCS100 groups showed the lowest MDA levels. The NO levels were significantly reduced in the CS100, SCS100, and SCS200 groups by 124.81 µmol/mg protein, 123.09 µmol/mg protein, and 123.94 µmol/mg protein, respectively, but there was no significant difference, compared with that of the control group (130.55 µmol/mg protein).

Fig. 4

Effects of chondroitin sulfate from shark cartilage (CS) and chondroitin sulfate from skate cartilage extract (SCS) on lipid peroxidation and nitric oxide formation in the liver of high cholesterol diet fed LDL receptor knockout mice. (A) MDA, malondealdehyde; (B) NaNO₂. See the legend of Table 1 for experimental groups in detail. Values are means ± SD (n = 8 per group). ^a~cMeans with the different letters are significantly different (P < 0.05) by Duncan's multiple range test.

The effects of CS and SCS on hepatic antioxidant enzymes, including SOD, catalase, and GSH-Px are shown in Fig. 5. The protein expression levels of SOD and catalase were significantly higher in the SCS100 (by 102.67% and 115.07%) and SCS200 groups (by 112.46% and 134.49%) than the control group. In addition, GSH-Px expression level was significantly increased in the CS50, CS100, SCS100, and SCS200 groups by 91.23%, 128.70%, 148.66%, and 174.12%, respectively, compared to that of the control group. In particular, SCS-administered groups showed a dose-dependent increase in the levels of hepatic antioxidant enzymes, including SOD, catalase, and GSH-Px.

Fig. 5

Effects of chondroitin sulfate from shark cartilage (CS) and chondroitin sulfate from skate cartilage extract (SCS) on antioxidant enzymes expression in the liver of high cholesterol diet fed LDL receptor knockout mice. (A) Representative Western blot analysis; (B) SOD, superoxide dismutase; (C) Catalase; (D) GSH-Px, glutathione peroxidase. See the legend of Table 1 for experimental groups in detail. Values are means ± SD (n = 9). ^a~eMeans with the different letters are significantly different (P < 0.05) by Duncan's multiple range test.

DISCUSSION

Blood TG and TC are essential components for maintaining the function of the human body [26]. However, it has been reported that excessive intake of HCD increases the fat concentration in the blood, which causes atherosclerosis and NAFLD [1 5 6 7 8 27 28]. Therefore, it is crucial to control the blood lipid level in order to prevent these chronic diseases. LDLR-KO mice have widely used in the study for hyperlipidemia and hepatic lipid metabolism [4 29 30]. Furthermore, the intake of HCD in LDLR-KO mice induces a rapid increase in plasma lipid levels as well as stimulates the TG synthesis in the liver. Chondroitin sulfate from salmon nasal cartilage has anti-hyperlipidemia via inhibition of absorption of dietary fat in high-fat diet fed mice [31]. In our previous study, SCS downregulated the protein expression levels of transcription factors, such as SREBP-1 and -2, thereby reducing serum TG and TC levels in LPS-injected mice [18]. In the present study, the CS and SCS administered groups had improved lipid profiles by decreasing the levels of TG, TC, and LDL-C, and increasing the levels of HDL-C compared with the control group. In particular, the AI and CRF indexes were the lowest in the SCS200 group among all experimental groups, which was probably due to the augmented HDL-C levels. These results might be associated with the reduced synthesis of fatty acid and cholesterol by SCS. These data indicate that SCS is expected to effectively alleviate hyperlipidemia in HCD-induced dyslipidemia.

Inflammatory responses are observed under the hyperlipidemia. Several inflammatory cytokines and mediators are involved in inflammation, such as TNF-α, IL-1β, IL-6, and PGE₂ [32]. TNF-α is a major pleiotropic cytokine that exerts proinflammatory effects in atherosclerosis [32]. In addition, IL-6 and IL-1β are multifunctional cytokines that regulate various aspects of the immune response, acute-phase reaction, and hematopoiesis [33]. In addition, NF-κB plays a key role in hepatic inflammation. NF-κB activated by IκB phosphorylation leads to release numerous pro-inflammatory genes such as iNOS and COX-2 in the nucleus [34 35]. iNOS leads to the production of excessive amounts of NO in the vascular smooth muscle cells, macrophages, and other cells [36]. COX-2 converts arachidonic acid to prostaglandin H2, which may trigger and maintain the inflammatory response in the various diseases, including atherosclerosis [37]. In the current study, the administration of SCS or CS decreased the serum levels of TNF-α and IL-1β and the expression levels of hepatic NF-κB, iNOS, and IL-1β, compared with those of the control group. In addition, the serum IL-6 level was reduced in the only SCS group. In particular, serum TNF-α and hepatic iNOS, COX-2, and IL-1β levels were the lowest in the SCS-administered group, with 100 mg/kg BW. In contrast, the serum IL-6 level was significantly lower only in the CS groups. These results were consistent with the previous study that chondroitin sulfate reduced serum inflammatory cytokines such as IL-1β and TNF-α in the obese mice and suppressed the activation of NF-κB in the inflamed human coronary artery endothelial cells [16]. In addition, chondroitin sulfate inhibits inflammatory reaction through downregulation of monocyte chemoattractant protein-1 (MCP-1) in the LPS-treated 3T3-L1 adipocytes [38]. Bovine-derived chondroitin sulfate attenuates IL-1β [39] or lipopolysaccharide [17] induced-inflammatory responses via suppression of NF-κB nuclear translocation. Similarly, our previous study demonstrated that SCS suppressed LPS-induced inflammation via downregulation of TNF-α, iNOS, and COX-2 in the liver tissue [18]. These results suggest that SCS could attenuate the inflammation induced by excessive intake of cholesterol in the serum and liver tissue, in a manner comparable to CS.

Oxidative stress elevated under hyperlipidemia is caused by free radicals produced by macrophages and directly damages lipid, protein, and other biological molecules. By contrast, oxidative stress could be accelerated due to hyperlipidemia and hypercholesterolemia [40 41]. The MDA, the final product of lipid peroxides, affects cell membrane function, modifies protein, induces DNA damage, and reduces their function [42]. The excessive production of NO easily reacts with reactive oxygen species, which generates peroxynitrite in the human body [43]. The production of peroxynitrite leads to lipid peroxidation as well as remarkable modifications in the antioxidant defense system [44]. Oxidative stress accompanies a lower level of antioxidant enzymes [45]. Hypercholesterolemia diminishes the antioxidant defense system and inhibits the activities of antioxidant enzymes such as SOD, GSH, and catalase [44]. In the present study, CS or SCS-administered groups showed a significant reduction of hepatic MDA levels, compared to the control group. In contrast, the expression level of GSH-Px was significantly higher in all CS and SCS groups, when compared to the CS-administered group. Moreover, those levels of SOD and catalase were elevated in SCS groups in which effects were dose-dependent. These results suggested that the reduction of MDA level in the liver was partially attributed to the elevation of antioxidant enzymes. These results are consistent that SCS exhibited the protective effects against hepatic oxidative stress via up-regulation of GPx in LPS-injected mice [18]. Similarly, the previous studies reported the protective effects of chondroitin sulfate on oxidative stress by downregulation of extracellular ROS and upregulation of antioxidant-related proteins such as heme oxygenase-1 in the hydrogen peroxide-treated cells [17]. In addition, chondroitin sulfate attenuated oxidative stress through the reduction of lipid peroxidation and activation of antioxidant enzymes in the CCl4-induced oxidative stress mouse model [46]. Therefore, the results of the present study indicate that CS or SCS may contribute to alleviating the oxidative stress induced by HCD through the enhanced expression of antioxidant enzymes in hypercholesteremic mice.

In conclusion, we investigated the beneficial effects of SCS on hyperlipidemia-induced inflammation and oxidative stress and compared them with those of CS in HCD-fed LDLR-KO mice. Administration of SCS improved the levels of serum lipid, reduced the levels of serum and hepatic inflammation-related proteins, and augmented the antioxidant status in dyslipidemic mice. These effects of SCS were comparable with those of the CS group. Our findings suggest that the biological effects of SCS are attributed to the alleviation of hyperlipidemia, inflammation, and oxidative stress like shark CS.

Notes

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A3B03034845).

Conflict of Interest: The authors declare no potential conflicts of interests.

Author Contributions:

Conceptualization: Woo M, Song YO.
Formal analysis: Woo M, Song YO.
Investigation: Seol BG, Kim JH.
Methodology: Choi YH, Noh JS, Cho EJ.
Supervision: Choi YH, Noh JS, Cho EJ.
Writing - original draft: Seol BG, Kim JH.

References

1. Tannock LR. Advances in the management of hyperlipidemia-induced atherosclerosis. Expert Rev Cardiovasc Ther. 2008; 6:369–383. PMID: 18327996.

2. Nelson RH. Hyperlipidemia as a risk factor for cardiovascular disease. Prim Care. 2013; 40:195–211. PMID: 23402469.

3. Tomizawa M, Kawanabe Y, Shinozaki F, Sato S, Motoyoshi Y, Sugiyama T, Yamamoto S, Sueishi M. Triglyceride is strongly associated with nonalcoholic fatty liver disease among markers of hyperlipidemia and diabetes. Biomed Rep. 2014; 2:633–636. PMID: 25054002.

4. Wouters K, van Gorp PJ, Bieghs V, Gijbels MJ, Duimel H, Lütjohann D, Kerksiek A, van Kruchten R, Maeda N, Staels B, van Bilsen M, Shiri-Sverdlov R, Hofker MH. Dietary cholesterol, rather than liver steatosis, leads to hepatic inflammation in hyperlipidemic mouse models of nonalcoholic steatohepatitis. Hepatology. 2008; 48:474–486. PMID: 18666236.

5. Onody A, Csonka C, Giricz Z, Ferdinandy P. Hyperlipidemia induced by a cholesterol-rich diet leads to enhanced peroxynitrite formation in rat hearts. Cardiovasc Res. 2003; 58:663–670. PMID: 12798440.

6. Czakó L, Szabolcs A, Vajda A, Csáti S, Venglovecz V, Rakonczay Z Jr, Hegyi P, Tiszlavicz L, Csont T, Pósa A, Berkó A, Varga C, Varga Ilona S, Boros I, Lonovics J. Hyperlipidemia induced by a cholesterolrich diet aggravates necrotizing pancreatitis in rats. Eur J Pharmacol. 2007; 572:74–81. PMID: 17628538.

7. Puskás LG, Nagy ZB, Giricz Z, Ónody A, Csonka C, Kitajka K, Hackler L Jr, Zvara A, Ferdinandy P. Cholesterol diet-induced hyperlipidemia influences gene expression pattern of rat hearts: a DNA microarray study. FEBS Lett. 2004; 562:99–104. PMID: 15044008.

8. Lee LC, Wei L, Huang WC, Hsu YJ, Chen YM, Huang CC. Hypolipidemic effect of tomato juice in hamsters in high cholesterol diet-induced hyperlipidemia. Nutrients. 2015; 7:10525–10537. PMID: 26694461.

9. Bondia-Pons I, Ryan L, Martinez JA. Oxidative stress and inflammation interactions in human obesity. J Physiol Biochem. 2012; 68:701–711. PMID: 22351038.

10. Harmon RC, Tiniakos DG, Argo CK. Inflammation in nonalcoholic steatohepatitis. Expert Rev Gastroenterol Hepatol. 2011; 5:189–200. PMID: 21476914.

11. Muniandy K, Gothai S, Badran KM, Suresh Kumar S, Esa NM, Arulselvan P. Suppression of proinflammatory cytokines and mediators in LPS-induced RAW 264.7 macrophages by stem extract of alternanthera sessilis via the inhibition of the NF-κB pathway. J Immunol Res. 2018; 2018:3430684. PMID: 30155492.

12. Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol. 2005; 25:29–38. PMID: 15539615.

13. Ho FM, Liu SH, Liau CS, Huang PJ, Lin-Shiau SY. High glucose-induced apoptosis in human endothelial cells is mediated by sequential activations of c-Jun NH(2)-terminal kinase and caspase-3. Circulation. 2000; 101:2618–2624. PMID: 10840014.

14. Abe S, Obata Y, Oka S, Koji T, Nishino T, Izumikawa K. Chondroitin sulfate prevents peritoneal fibrosis in mice by suppressing NF-κB activation. Med Mol Morphol. 2016; 49:144–153. PMID: 26880269.

15. Bauerova K, Ponist S, Kuncirova V, Mihalova D, Paulovicova E, Volpi N. Chondroitin sulfate effect on induced arthritis in rats. Osteoarthritis Cartilage. 2011; 19:1373–1379. PMID: 21884808.

16. Melgar-Lesmes P, Garcia-Polite F, Del-Rey-Puech P, Rosas E, Dreyfuss JL, Montell E, Vergés J, Edelman ER, Balcells M. Treatment with chondroitin sulfate to modulate inflammation and atherogenesis in obesity. Atherosclerosis. 2016; 245:82–87. PMID: 26714044.

17. Cañas N, Valero T, Villarroya M, Montell E, Vergés J, García AG, López MG. Chondroitin sulfate protects SH-SY5Y cells from oxidative stress by inducing heme oxygenase-1 via phosphatidylinositol 3-kinase/Akt. J Pharmacol Exp Ther. 2007; 323:946–953. PMID: 17885094.

18. Song YO, Kim M, Woo M, Baek JM, Kang KH, Kim SH, Roh SS, Park CH, Jeong KS, Noh JS. Chondroitin sulfate-rich extract of skate cartilage attenuates lipopolysaccharide-induced liver damage in mice. Mar Drugs. 2017; 15:E178. PMID: 28617322.

19. Hu SW, Tian YY, Chang YG, Li ZJ, Xue CH, Wang YM. Fucosylated chondroitin sulfate from sea cucumber improves glucose metabolism and activates insulin signaling in the liver of insulin-resistant mice. J Med Food. 2014; 17:749–757. PMID: 24949837.

20. Kim BH, Ahn SH, Choi BD, Kang SJ, Kim YL, Lee HJ, Oh MJ, Jung TS. In vivo evaluation of chondroitin sulfates from midduk (Styela clava) and munggae tunics (Halocynthia roretzi) as a cosmetic material. J Korean Soc Food Sci Nutr. 2004; 33:641–645.

21. da Cunha AL, Aguiar JA, Correa da Silva FS, Michelacci YM. Do chondroitin sulfates with different structures have different activities on chondrocytes and macrophages? Int J Biol Macromol. 2017; 103:1019–1031. PMID: 28536017.

22. Baek JM, Kang KH, Kim SH, Noh JS, Jeong KS. A study on development of high functional materials producing technique using by-products from skate processing (1)-development of chondroitin sulfate materials using skate cartilages. J Environ Sci Int. 2016; 25:645–654.

23. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972; 18:499–502. PMID: 4337382.

24. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979; 95:351–358. PMID: 36810.

25. Schmidt HH, Warner TD, Nakane M, Förstermann U, Murad F. Regulation and subcellular location of nitrogen oxide synthases in RAW264.7 macrophages. Mol Pharmacol. 1992; 41:615–624. PMID: 1373797.

26. Dietschy JM. Dietary fatty acids and the regulation of plasma low density lipoprotein cholesterol concentrations. J Nutr. 1998; 128(2 Suppl):444S–448S. PMID: 9478045.

27. Ichimura M, Kawase M, Masuzumi M, Sakaki M, Nagata Y, Tanaka K, Suruga K, Tamaru S, Kato S, Tsuneyama K, Omagari K. High-fat and high-cholesterol diet rapidly induces non-alcoholic steatohepatitis with advanced fibrosis in Sprague-Dawley rats. Hepatol Res. 2015; 45:458–469. PMID: 24827559.

28. Kainuma M, Fujimoto M, Sekiya N, Tsuneyama K, Cheng C, Takano Y, Terasawa K, Shimada Y. Cholesterol-fed rabbit as a unique model of nonalcoholic, nonobese, non-insulin-resistant fatty liver disease with characteristic fibrosis. J Gastroenterol. 2006; 41:971–980. PMID: 17096066.

29. Bieghs V, Van Gorp PJ, Wouters K, Hendrikx T, Gijbels MJ, van Bilsen M, Bakker J, Binder CJ, Lütjohann D, Staels B, Hofker MH, Shiri-Sverdlov R. LDL receptor knock-out mice are a physiological model particularly vulnerable to study the onset of inflammation in non-alcoholic fatty liver disease. PLoS One. 2012; 7:e30668. PMID: 22295101.

30. Subramanian S, Goodspeed L, Wang S, Kim J, Zeng L, Ioannou GN, Haigh WG, Yeh MM, Kowdley KV, O'Brien KD, Pennathur S, Chait A. Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice. J Lipid Res. 2011; 52:1626–1635. PMID: 21690266.

31. Han LK, Sumiyoshi M, Takeda T, Chihara H, Nishikiori T, Tsujita T, Kimura Y, Okuda H. Inhibitory effects of chondroitin sulfate prepared from salmon nasal cartilage on fat storage in mice fed a high-fat diet. Int J Obes Relat Metab Disord. 2000; 24:1131–1138. PMID: 11033981.

32. Wang J, Mazza G. Effects of anthocyanins and other phenolic compounds on the production of tumor necrosis factor α in LPS/IFN-γ-activated RAW 264.7 macrophages. J Agric Food Chem. 2002; 50:4183–4189. PMID: 12105943.

33. Kleemann R, Zadelaar S, Kooistra T. Cytokines and atherosclerosis: a comprehensive review of studies in mice. Cardiovasc Res. 2008; 79:360–376. PMID: 18487233.

34. Gil-Cardoso K, Ginés I, Pinent M, Ardévol A, Blay M, Terra X. Effects of flavonoids on intestinal inflammation, barrier integrity and changes in gut microbiota during diet-induced obesity. Nutr Res Rev. 2016; 29:234–248. PMID: 27841104.

35. Yuan Y, Naito H, Jia X, Kitamori K, Nakajima T. Combination of hypertension along with a high fat and cholesterol diet induces severe hepatic inflammation in rats via a signaling network comprising NF-κB, MAPK, and Nrf2 pathways. Nutrients. 2017; 9:E1018. PMID: 28906458.

36. Anavi S, Eisenberg-Bord M, Hahn-Obercyger M, Genin O, Pines M, Tirosh O. The role of iNOS in cholesterol-induced liver fibrosis. Lab Invest. 2015; 95:914–924. PMID: 26097999.

37. Angeli JK, Cruz Pereira CA, de Oliveira Faria T, Stefanon I, Padilha AS, Vassallo DV. Cadmium exposure induces vascular injury due to endothelial oxidative stress: the role of local angiotensin II and COX-2. Free Radic Biol Med. 2013; 65:838–848. PMID: 23973752.

38. Stabler TV, Montell E, Vergés J, Huebner JL, Kraus VB. Chondroitin sulfate inhibits monocyte chemoattractant protein-1 release from 3T3-L1 adipocytes: a new treatment opportunity for obesity-related inflammation? Biomark Insights. 2017; 12:1177271917726964. PMID: 28890654.

39. Jomphe C, Gabriac M, Hale TM, Héroux L, Trudeau LÉ, Deblois D, Montell E, Vergés J, du Souich P. Chondroitin sulfate inhibits the nuclear translocation of nuclear factor-kappaB in interleukin-1β-stimulated chondrocytes. Basic Clin Pharmacol Toxicol. 2008; 102:59–65. PMID: 17983423.

40. Kim HJ, Hwangbo MH, Lee JW, Im HG, Lee IS. Antioxidant effects of red ginseng powder on liver of benzo(α)pyrene-treated mice. Korean J Food Sci Technol. 2007; 39:217–221.

41. Lovlin R, Cottle W, Pyke I, Kavanagh M, Belcastro AN. Are indices of free radical damage related to exercise intensity. Eur J Appl Physiol Occup Physiol. 1987; 56:313–316. PMID: 3569239.

42. Niess AM, Dickhuth HH, Northoff H, Fehrenbach E. Free radicals and oxidative stress in exercise--immunological aspects. Exerc Immunol Rev. 1999; 5:22–56. PMID: 10519061.

43. Carr AC, McCall MR, Frei B. Oxidation of LDL by myeloperoxidase and reactive nitrogen species: reaction pathways and antioxidant protection. Arterioscler Thromb Vasc Biol. 2000; 20:1716–1723. PMID: 10894808.

44. Dahech I, Harrabi B, Hamden K, Feki A, Mejdoub H, Belghith H, Belghith KS. Antioxidant effect of nondigestible levan and its impact on cardiovascular disease and atherosclerosis. Int J Biol Macromol. 2013; 58:281–286. PMID: 23624165.

45. Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M, Matsuda M, Shimomura I. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest. 2004; 114:1752–1761. PMID: 15599400.

46. Lee JY, Lee SH, Kim HJ, Ha JM, Lee SH, Lee JH, Ha BJ. The preventive inhibition of chondroitin sulfate against the CCl4-induced oxidative stress of subcellular level. Arch Pharm Res. 2004; 27:340–345. PMID: 15089041.

TOOLS

Similar articles