Abstract
Goishi tea, a traditional two-stage fermented tea is different from non-fermented green tea on the ingredient pattern. Although gallate-type catechins are reduced due to the assimilation of microorganisms, Goishi tea have antioxidant activity due to the increase of free-type catechins and other antioxidants. Since oxidative stress is greatly involved in the onset mechanism of atherosclerosis, ingestion of Goishi tea may be useful for the inhibition of atherosclerosis.
In this study, the effects of Goishi tea on atherosclerosis were evaluated by measuring plaque accumulation, inflammatory cytokines, and adiponectin in apolipoprotein E deficient mice. When, compared with the control group, the plaque area in the aortic root was significantly decreased in the Goishi tea group. In addition, TNF-α and IL-6 levels were significantly decreased and adiponectin was significantly increased in the Goishi tea group. These results suggest the possibility of inhibiting atherosclerosis with Goishi tea because of its antioxidant and anti-inflammatory properties.
Introduction
Atherosclerotic diseases such as myocardial infarction and cerebral infarction have become one of the major causes of death alongside cancer, thus necessitating the need to seek preventive measures to curb them (Hirata et al., 2017). While oxidative stress and inflammatory cytokines influence the onset and progression of this disease, oxidized low-density lipoprotein (LDL) formed during the oxidative modification of LDL by active oxygen plays a major role in the development of atherosclerosis. This oxidized LDL, when captured by macrophages leads to the formation of foam cells that accumulate large amounts of cholesterol. Agglomeration of these foam cells becomes fat streaks that trigger the initial stage of atherosclerosis (Yang et al., 2017). In addition to this, foam cells and activated endothelial cells release inflammatory cytokines that induce a chronic inflammatory response in the blood vessel. This, in turn, triggers smooth muscle cells to migrate to the vascular intima and an increase in the intima enlarges the plaque. This enlarged plaque causes stenosis and embolization due to stenosis of the blood vessel and thrombus formation following the rhexis (Marchio et al., 2019).
Conventional studies have identified the effectiveness of antioxidants in the inhibition of atherosclerosis (Malekmohammad et al., 2019). One of such natural sources of antioxidant is Goishi tea, a traditional two-stage fermented tea that has been handed down only to Otoyo-cho, Nagaoka-gun, Kochi prefecture. Goishi tea is made from leaves of Camellia sinensis and assimilated by microorganisms during aerobic fermentation (molding) and anaerobic fermentation (lactic acid fermentation), so the ingredient pattern is different from that of non-fermented green tea. Since gallate-type catechins are assimilated during the fermentation process, the total amount of catechins decreases, but free-type catechins such as (-)-catechin and (-)-gallocatechin increase (Shimamura et al., 2008, Moriyama and Takeda, 2008). In addition, since antioxidants such as pyrogallol (Shimamura et al., 2017) and cyclic dipeptides (Yamamoto, 2017) also increase during the fermentation process, Goishi tea is considered to have antioxidant activity. In fact, we reported that administration of Goishi tea to cholesterol-loaded rabbits reduced serum lipid peroxide amounts (Miyamura et al., 2008).
Furthermore, it has been clarified that the ingestion of Goishi tea to diet-induced obese model mice inhibits adipose cell enlargement, TNF-α, and IL-6 levels, and increases adiponectin levels (Yokota et al., 2013, Jobu et al., 2013). For these reasons, it can be suggested that Goishi tea inhibits atherosclerosis due to its antioxidant and anti-inflammatory properties.
Other types of teas like Green tea and Pu'er tea have also attracted interest due to their ability to reduce oxidative stress and prevent lifestyle diseases (Prasanth et al., 2019, Ahmed et al., 2010). Tea is broadly classified into non-fermented tea (Green tea), semi-fermented tea (Oolong tea), fermented tea (Black tea), and post-fermented tea (Goishi tea, Pu'er tea). Oolong tea and Black tea are fermented with the enzyme of tea leaves, whereas post-fermented tea stops the fermentation of the tea leaves by heat treatment and ferments them with microorganisms. Post-fermentation includes aerobic fermentation with aerobic bacteria like Aspergillus oryzae, anaerobic fermentation with anaerobic bacteria like lactic acid bacteria. It is also called two-stage fermented tea (Shimamura et al., 2008).
In this study, apolipoprotein E (ApoE) deficient mice were used as models to evaluate the inhibitory effects of Goishi tea on atherosclerosis. ApoE is one of the major apolipoproteins that make up lipoproteins such as VLDL, LDL, and HDL. It functions as a ligand for the LDL receptor family on the cell surface and is important for uptake of lipoproteins such as chylomicron and VLDL into cells and removal from plasma (Phillips, 2014). ApoE deficiency and abnormality have been reported to cause hyperlipoproteinemia type III, which is characterized by early atherosclerosis and blood cholesterol accumulation (Matsunaga and Saito, 2014). Many studies have administered antioxidants to ApoE deficient mice, and results from these studies have proven the effectiveness of antioxidants in inhibiting plaque accumulation by inhibiting LDL oxidation (Meng et al., 2019, Harauma et al., 2007, Koyama et al., 2006). Accordingly, this study evaluated the effects of Goishi tea on atherosclerosis in ApoE deficient mice (ApoE−/−) with a high cholesterol diet by measuring its levels of plaque accumulation, inflammatory cytokines, and adiponectin. Also, the antioxidant activity of Goishi tea was compared with other types of tea.
Materials and Methods
Preparation of Goishi tea and other teas Goishi tea was purchased from the Goishi Tea Producers Association (Kochi, Japan). Other types of tea such as Green tea (Sencha Uji, Kyoto, Japan), Oolong tea (Tikuanyin, Fujian, China), Black tea (Margaret's Hope, Darjeeling, India), Pu'er tea (Menghai Puer, Yunnan, China) were purchased from LUPICIA Co., Ltd (Tokyo, Japan). Following the general preparation method, the teas were prepared by adding 6 g of tea leaves to 0.4 L of boiling water for 5 min. Then, the tea extracts were used to measure the antioxidant activity and for the animal experiments.
Measurement of antioxidant activity of tea extracts A Superoxide Dismutase (SOD) Assay Kit-WST (Dojindo Molecular Technologies, Inc, Tokyo, Japan) was used to measure the Superoxide anion scavenging activity (SOSA) of the tea extracts.
Inhibitory effects of Goishi tea on atherosclerosis Materials and reagents MF containing 1 % cholesterol (Oriental Yeast Co., Ltd., Tokyo, Japan) was used for the high cholesterol diet (HCD), and CE-2 (CLEA Japan, Inc., Tokyo, Japan) was used for the normal diet (ND). The nutritional composition of these diets is shown in Table 1. Other reagents used were commercial guaranteed-grade products.
Table 1.
Feed composition
|
ND (CE-2) |
HCD (MF included 1% cholesterol) |
| Moisture (%) |
9.1 |
8.2 |
| Crude Protein (%) |
25.0 |
23.1 |
| Crude Fat (%) |
4.8 |
4.9 |
| Crude Fiber (%) |
4.2 |
5.7 |
| Crude Ash (%) |
6.7 |
2.9 |
| Cholesterol (%) |
0 |
1.0 |
| NFE (%) |
50.1 |
54.2 |
| Physiological fuel value (kcal) |
344.2 |
359.2 |
|
|
(contained in 100g) |
Experimental animals Male BALB/c.KOR/StmSlc-Apoeshl mice (5 weeks old) were purchased from Japan SLC, Inc. (Hamamatsu, Japan). After they were acclimated at a room temperature of 23 ± 2 °C for 7 d, they were divided into 3 groups as follows: ND group (tap water [ND]) and HCD groups (purified water [HCD], Goishi tea [HCD-G]). Each group consists of 6 mice. Respective diets for each of the groups were administered from the first day until the last day of the experiment which lasted 8 weeks. Sample food and water consumption were recorded every day. The body mass of each animal was also recorded soon after feeding for the entire period since the start of breeding until the end of the experiments. Approval for the animal experiments was obtained from the Animal Research Committee of Kochi University (Approval No. J-00064), and experiments were conducted in accordance with the guidelines on animal experimentation outlined by Kochi Medical School.
Biochemical blood constituents and measurements of inflammatory cytokines and adiponectin After 8 weeks of feeding, laparotomy was conducted under the mixture of three anesthetic agents (medetomidine hydrochloride, midazolam, butorphanol tartrate), and blood was collected from the abdominal vena cava after 12 h fasting period. Serum total cholesterol (T-CHO) and triglyceride (TG) were measured using Fuji DRI-CHEM slides (FUJIFILM Corporation, Tokyo, Japan). An ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) was used to measure the serum tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). A mouse/rat adiponectin ELISA kit (Otsuka Pharmaceutical, Tokyo, Japan) was used to determine the serum adiponectin.
Inhibitory effects of Goishi tea on plaque accumulation in the aorta The aorta and heart were removed after perfusion with PBS and fixed with 10 % formalin replaced with 4 % sucrose solution, and then frozen. To determine the cross-sectional lesion area, hearts containing the aortic root were cut into 10-µm sections for the quantitative atherosclerosis assay using the method described by Daugherty et al., (2017). Thereafter the sections were stained with Oil Red O and Hematoxylin. The percentage of the Oil Red O staining area to the cross-sectional vessel area was measured with a computer analysis software (WinROOF, Mitani Corporation, Tokyo, Japan).
Statistical analysis The obtained values are shown as mean ± standard deviation. A one-way ANOVA was used for dispersion analysis, followed by Dunnett's multiple comparison test. A significance level p < 0.05 was used to state the significant difference.
Results
Comparison of Goishi tea and other teas on SOSA Fig. 1 shows the SOSA of Goishi tea and other teas. There was no significant difference between Goishi tea and Green tea on SOSA. On the other hand, Black tea, Pu'er tea, and Oolong tea, which are fermented teas, showed significantly lower SOSA than Goishi tea.
Inhibitory effects of Goishi tea on atherosclerosis Effects of Goishi tea on body weight, food intake, and beverage intake The results of the body weight, food intake, beverage intake measured are shown in Table 2. There were no observed differences between the groups in terms of body weight, food intake, and beverage intake for 8 weeks.
Table 2.
Effects of Goishi tea on Body weight gain, Food intake and Beverage intake in apolipoprotein E deficient mice for 8 weeks.
|
ND |
HCD |
HCD-G |
| Body weight (g) |
|
|
|
| initial (g) |
24.5 ± 0.7 |
24.1 ± 0.6 |
25.2 ± 0.8 |
| Final (g) |
31.0 ± 0.9 |
31.7 ± 1.1 |
31.5 ± 1.1 |
| Gain (g) |
6.5 ± 0.9 |
7.5 ± 1.1 |
6.3 ± 1.0 |
| Food intake (g/d) |
4.2 ± 0.1 |
4.3 ± 0.2 |
4.2 ± 0.2 |
| Beverage intake (mL/d) |
8.8 ± 0.3 |
9.0 ± 0.4 |
8.9 ± 0.3 |
Result are expressed means±SD of 6 mice.
Effects of Goishi tea on blood lipid profiles, inflammatory cytokines, and adiponectin The effects of Goishi tea supplementation on serum blood lipid profiles, inflammatory cytokines, and adiponectin are shown in Table 3. T-CHO in the HCD group was significantly higher than that observed in the ND group, and there were no significant differences between the HCD group and the HCD-G group. TG in all groups had no significant difference. In the HCD group, TNF-α and IL-6 levels were significantly increased and adiponectin levels were significantly decreased compared to the ND group. While in the HCD-G group, TNF-α and IL-6 levels were significantly decreased and adiponectin level was significantly increased compared to the HCD group.
Table 3.
Effects of Goishi tea on blood lipid profiles, inflammatory cytokines and adiponectin in apolipoprotein E deficient mice for 8 weeks.
|
ND |
HCD |
HCD-G |
| T-CHO (mg/dL) |
973 ± 103.9** |
1828 ± 527.4 |
1848 ± 327.6 |
| TG (mg/dL) |
47.2 ± 14.13 |
52.2 ± 9.81 |
53.2 ± 7.26 |
| TNF-α (pg/mL) |
4.91 ± 0.47** |
6.30 ± 0.49 |
5.02 ± 0.80* |
| IL-6 (pg/mL) |
20.6 ± 0.99** |
23.8 ± 1.49 |
20.1 ± 0.75** |
| Adiponectin (µg/mL) |
14.5 ± 2.28** |
11.8 ± 1.13 |
13.1 ± 1.01** |
Result are expressed means±SD of 6 mice.
**
p < 0.01 compared with HCD group (analysis of variance followed by Dunnett's test).
Effects of Goishi tea on aortic plaque Fig 2. shows the effects of Goishi tea on an aortic plaque. The measured Oil Red O staining area (plaque area) was significantly increased in the HCD group as compared to the ND group. Also, in the HCD-G group, the plaque area ratio decreased significantly compared to the HCD group.
Discussion
The experimental results showed that the SOSA of Goishi tea was significantly higher than other fermented teas. In addition, administering Goishi tea to ApoE deficient mice significantly decreased the inflammatory cytokines, increased the adiponectin level, and decreased the plaque area in the aortic root compared to the control group. These results can be related to the antioxidant and anti-inflammatory effects of Goishi tea.
In atherosclerotic diseases, pathological accumulation of deposits such as lipids, smooth muscle cells, and extracellular matrix occurs in the vascular intima, forming atheromatous raised lesions. It progresses gradually or sometimes rapidly, resulting in narrowing of the blood vessel due to the thickening of plaques, stenosis, and embolization due to thrombus formation following the rhexis.
When the antioxidant activity of Goishi tea and other teas were compared, it was observed that the SOSA of Goishi tea was significantly higher than other fermented teas. This might be attributed to the antioxidant effect of Goishi tea which prevented the oxidative modification of LDL thereby inhibiting the foaming of macrophages and activation of endothelial cells which overall inhibits the progression of atherosclerosis.
Also, a HCD was administered to ApoE deficient mice as the atherosclerotic disease model for 8 weeks. This is because ApoE plays an important role in the cellular uptake and removal of lipoproteins such as chylomicron and VLDL from the plasma (Phillips, 2014), and ApoE deficiency or abnormality causes early atherosclerosis (Matsunaga, Saito, 2014). In this study, the result showed that plaque accumulation was confirmed in all groups, but administering Goishi tea significantly decreased the measured plaque area in the cross-sectional vessel area of the aortic root compared to control group.
In addition to LDL oxidation, inflammatory cytokines are greatly involved in plaque formation. Inflammatory cytokines released from activated endothelial cells and foam cells induce a chronic inflammatory in blood vessels. After which they cause the migration of smooth muscle cells to the intima of the blood vessel and increase the intima, which enlarges the plaque. In this study, results showed that the administration of Goishi tea significantly reduced TNF-α and IL-6 levels and significantly increased the adiponectin level compared to the control group. This inhibited the plaque enlargement thus exhibiting atherosclerosis inhibitory effects.
Also, in previous study, Goishi tea reduced Monocyte Chemoattractant Protein-1 (MCP-1) in diet-induced obese model mice (Yokota et al., 2013). MCP-1 promotes inflammatory response by migrating monocytes to lesions and develops atherosclerosis.
Therefore, we considered that Goishi tea suppressed the development of atherosclerosis by reducing not only inflammatory cytokines but also MCP-1 in this study.
These mechanisms might be involved with NF-κB, a transcription factor that plays the central role of the inflammatory response. It has been reported that NF-κB is activated by oxidative stress and increases the expression of inflammatory cytokines such as TNF-α and IL-6 (Zhang et al., 2016). Therefore, many studies have been conducted to evaluate the anti-inflammatory effects of antioxidants targeting NF-κB (Hsuan et al., 2015, Luo et al., 2015). Hence, Goishi tea might inhibit NF-κB activation by the antioxidant effects and inhibit the inflammatory cytokines released from the foam cells and activated endothelial cells.
Goishi tea has been shown in previous studies to contain functional components such as pyrogallol, gallocatechin (Shimamura et al., 2017), and cyclic dipeptides (Diketopiperazines: DKP) (Yamamoto, 2017, Otsuka et al., 2020). Therefore, we considered that Goishi tea showed antioxidant and anti-inflammatory effects by these functional components, and the aortic plaque area was reduced.
DKP is a group of cyclic organic compounds in which two amino acids form lactams by peptide bonds, and there are more than 200 types (Wang et al., 2017). DKP is known to show various physiological activities due to its diversity and inherent properties such as anti-cancer effects (Mollica et al., 2014), anti-influenza effects (Zhang et al., 2018), and hypoglycemic effects (Belkacem et al., 2017). DKP increases in content during aerobic and anaerobic fermentation stages of Goishi tea (Yamamoto, 2017). A lot of DKP found in Goishi tea contain proline (Otsuka et al., 2020) and it is known that cyclo (Leu-Pro) has antioxidant effects (hydroxy radical scavenging effects) (Furukawa et al., 2012), and cyclo (Phe-Pro) has anti-inflammatory effects (TNF-α inhibitory effects in RAW 264.7 macrophages) (Khan et al., 2015). Hence, it can be assumed that DKP is also involved in the atherosclerosis inhibitory effect shown in this study. Also, Shimamura reported that pyrogallol and gallocatechin contributed significantly to the antioxidant effects of Goishi tea (Shimamura et al., 2017). In particular, the contribution rate of SOSA of pyrogallol in Goishi tea was as high as 28.2 %. Thus, Goishi tea has various functional components that may be activated in combination to show its atherosclerosis inhibitory effects.
Overall, results from this study suggest that Goishi tea exhibits atherosclerosis inhibitory effects due to its antioxidative effects, anti-inflammatory effects by decreasing TNF-α and IL-6 levels and increasing adiponectin level. Future research would identify the functional components related to the atherosclerosis inhibitory effects of Goishi tea.
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