Weight change and biochemicals in serum and tissue
The body weight and heart index are shown in Table 1. The weight gain of the Sham (4.36 g), NC (4.20 g), and NS (4.50 g) groups showed no significant differences. The PM group (1.60 g) significantly decreased compared to the NC group. However, the GTE groups (GTE20, 4.38 g; GTE40, 3.95 g) significantly increased compared to the PM group. The heart index of the Sham (0.32%), NC (0.32%), and NS (0.33%) groups showed no significant differences. The PM group (0.43%) significantly increased compared to the NC group. However, the GTE groups (GTE20, 0.40%; GTE40, 0.39%) were significantly decreased compared to the PM group.
Table 1
Protective effect of aqueous extract of green tea extract (GTE) on body weight as 0 weeks and 12 weeks, weight gain, heart index, serum chemicals, and cardiac chemicals.
Group 1) | Sham | NC | NS | PM | GTE20 | GTE40 |
Body weight (0 weeks, g) | 22.75 ± 0.91 a | 23.30 ± 0.80 a | 22.80 ± 0.92 a | 23.65 ± 1.09 a | 23.15 ± 1.09 a | 23.35 ± 1.18 a |
Body weight (12 weeks, g) | 27.11 ± 1.78 a | 27.50 ± 1.36 a | 27.30 ± 2.63 a | 25.25 ± 2.36 b | 27.53 ± 1.02 a | 27.30 ± 1.84 a |
Weight gain (g) | 4.36 ± 0.87 a | 4.20 ± 0.56 a | 4.50 ± 1.71 a | 1.60 ± 1.27 b | 4.38 ± 0.07 a | 3.95 ± 0.66 a |
Heart index (%) 2) | 0.32 ± 0.01 c | 0.32 ± 0.01 c | 0.33 ± 0.01 c | 0.43 ± 0.05 a | 0.40 ± 0.06 ab | 0.39 ± 0.01 b |
CKMB (U/L) | 130.24 ± 10.22 c | 129.00 ± 11.52 c | 122.02 ± 13.00 c | 198.30 ± 10.16 a | 180.12 ± 13.02 b | 120.98 ± 12.01 c |
LDH (mg/dL) | 155.22 ± 12.01 d | 165.11 ± 20.16 d | 157.16 ± 22.02 d | 451.02 ± 30.16 a | 265.02 ± 22.01 c | 178.15 ± 19.02 c |
SOD activity (%) | 33.01 ± 1.03 a | 35.12 ± 2.65 a | 35.16 ± 1.51 a | 27.11 ± 2.41 c | 30.51 ± 2.56 bc | 32.02 ± 0.65 b |
Reduced GSH content (% of control) | 99.01 ± 5.17 c | 100.00 ± 3.05 c | 101.25 ± 2.08 c | 82.05 ± 4.78 a | 91.01 ± 6.08 ab | 95.08 ± 2.58 b |
MDA content (nnmole/mg of protein] | 3.02 ± 0.41 a | 3.12 ± 0.59 a | 3.26 ± 0.32 a | 5.08 ± 0.51 c | 4.56 ± 0.77 b | 3.92 ± 0.19 b |
Results shown are mean ± SD (body weight, heart index, CKMB content, and LDH content, n = 10; SOD activity, reduced GSH content, and MDA level, n = 5). Data were statistically considered at p < 0.05, and different small letters represent statistical difference. |
1) CKMB, creatine kinase MB isoenzyme; LDH, lactate dehydrogenas; SOD, superoxide dismutase; GSH, glutathione; MDA, malondialdehyde. |
2) Heart index was calculated according to the formula: (heart weight/body weight) × 100. |
The serum CKMB contents and LDH contents are shown in Table 1. The CKMB contents of the Sham (130.24 U/L), NC (129.00 U/L), and NS (122.02 U/L) groups showed no significant differences. The PM group (198.30 U/L) significantly increased compared to in the NC group. However, the GTE groups (GTE20, 180.12 U/L; GTE40, 120.98 U/L) were significantly suppressed compared to the PM group. The LDH contents of the Sham (155.22 mg/dL), NC (165.11 mg/dL) and NS (157.16 mg/dL) groups showed no significant differences. The PM group (451.02 mg/dL) significantly decreased compared to the NC group. However, the GTE groups (GTE20, 265.02 mg/dL; GTE40, 178.15 mg/dL) were significantly ameliorated compared to the PM group.
Cardiac SOD activity, reduced GSH contents, and MDA contents, related to antioxidant system, are shown in Table 1. The SOD activity in heart tissue of the Sham (33.01%), NC (35.12%), and NS (35.16%) groups showed no significant differences. The PM group (27.11%) significantly decreased compared to the NC group. However, that of the GTE groups (GTE20, 30.51%; GTE40, 32.02%) in the heart tissues significantly increased compared to the PM group. The reduced GSH contents in the heart tissues of the Sham (99.01% of the control), NC (100.00% of the control), and NS (101.25% of the control) groups showed no significant differences. The PM group (82.05% of the control) significantly decreased compared to the NC group. However, the GTE groups (GTE20, 91.01% of the control; GTE40, 95.08% of the control) in the heart tissues were significantly increased compared to the PM group. The MDA contents in the heart tissues of the Sham (3.02 mmole/mg of protein), NC (3.12 mmole/mg of protein), and NS (3.26 mmole/mg of protein) groups showed no significant differences. The PM group (5.08 mmole/mg of protein) significantly increased compared to in the NC group. However, the GTE groups (GTE20, 4.56 mmole/mg of protein; GTE40, 3.92 mmole/mg of protein) in the heart tissues were significantly attenuated compared to the PM group.
PM causes heart enlargement and damage to the antioxidant system [21]. Various proteins and factors are involved in this mechanism, among which SOD, GSH, and lipid peroxidation play an important role [22]. Antioxidant enzymes in the body play a key role in protecting cells from oxidative stress, but they can be oxidized by fine dust and toxic chemicals, leading to oxidative stress [23]. Exposure to PM2.5 reduces the activity of SOD, glutathione peroxidase (GPx), and GSH and can no longer alleviate oxidative stress [21]. It induces lipid peroxidation in cardiac tissue and causes cardiac hypertrophy by the oxidation of lipid components [24]. Moreover, the increased activation of NF-κB stimulated by PM2.5 reduces the antioxidant activity of SOD and GSH, increasing the production of lipid peroxides [25]. As a result, PM2.5 exposure might cause damage to the antioxidant system associated with the development and progression of heart disease, such as cardiac hypertrophy [26]. Therefore, to confirm the protective effect of GTE against PM2.5-induced antioxidant system damage, heart index, acute myocardial infarction index, such as CKMB, and LDH content in serum, SOD activity, GSH content, and MDA content were measured (Table 1). In a previous study, GTE suppressed antioxidant deficits by increasing antioxidant enzyme levels and suppressing lipid peroxidation in lung, skin, and brain tissues [17]. The administration of GTE reduced serum glutamic oxaloacetic transaminase, glutamine pyruvic transaminase, and LDH content in a metabolic imbalance mice model [14]. Catechin also restored reduced GSH and the activities of antioxidant enzymes, including catalase, SOD, and glutathione-S-transferase, and attenuated serum LDH, creatine kinase, and CKMB content in doxorubicin-induced cardiotoxicity [27]. Gallic acid ameliorated SOD, catalase, GSH, GPx, and glutathione reductase in colon carcinogenesis rats [28]. Therefore, this study suggests that GTE, shown to have significant antioxidant activity in a previous study [18], might be used as a considerable functional food material that can protect against antioxidant damage.
Protein expression of inflammation
Inflammatory protein expression levels are presented in Fig. 1. The TLR2 (206.51%), TLR4 (165.48%), phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (p-IκB-α, 132.05%), caspase-1 (Cas-1, 184.41%), cyclooxygenase-2 (COX-2, 145.78%), inducible nitric oxide synthase (iNOS, 205.78%), tumor necrosis factor-alpha (TNF-α, 240.57%), and interleukin 1beta (IL-1β, 123.57%) expression levels in the PM group were significantly upregulated compared to those in the NC group (100%). However, TLR2 (150.21%), TLR4 (121.57%), p-IκB-α (85.79%), Cas-1 (109.97%), COX-2 (105.87%), iNOS (148.17%), TNF-α (134.08%), and IL-1β (107.83%) expression levels in the GTE40 group were statistically downregulated compared to the PM group.
When exposed to air pollutants such as PM, TLRs are activated in cardiac cells [8]. Activated TLRs activate myeloid differentiation primary response 88 (MyD88), a downstream signaling protein, which in turn activates interleukin-1 receptor-associated kinases, TNF receptor-associated factor 6, and transforming growth factor-β-activated kinase 1 [29]. Activation of these downstream proteins activates NF-κB, which transcribes inflammatory genes to produce inflammatory factors such as IL-1, IL-6, and TNF-α, resulting in inflammation [30]. The activation of TLRs stimulated by PM exposure induces an inflammatory response by NF-κB [31]. In addition, activation of the TLRs-NF-κB pathway and inflammatory response due to exposure to PM was observed in the tissues of patients with arrhythmogenic right ventricular cardiomyopathy [32]. These studies showed that exposure to PM2.5 causes an inflammatory response in the heart by interaction with TLRs and NF-κB signal [30]. Moreover, in previous studies, it was confirmed that PM2.5 could produce various inflammatory factors [33–35]. Similar to these studies, expression of TLR2, TLR4, p-IκB-α, Cas-1, COX-2, iNOS, TNF-α, and IL-1β were evaluated to assess the anti-inflammatory effect of GTE in heart tissue (Fig. 1). Those protein expression levels were down-regulated with the administration of GTE. In a previous study, green tea suppressed pulmonary inflammation by down-regulating TNF- α, p-JNK, p-IκB-α, p-NF-κB, and IL-1β [17]. In addition, quercetin, a green tea flavonoid, reduced PM2.5-induced ROS production by regulating the abnormal structure and function damage of mitochondria in bronchial epithelial cells [36]. In addition, EGCG protected against the progression and inflammation of heart failure by regulating inflammatory cytokines such as IL-1β, IL-4, and TNF-α [37]. In conclusion, green tea, with compounds that have physiological activities, significantly inhibited PM2.5-induced inflammation by regulation of the TLR pathway, and it is suggested that green tea plays an important role in the regulation of inflammation.
Protein expression of Apoptosis
Apoptotic protein expression levels are presented in Fig. 2. The phosphorylated protein kinase B (p-Akt, 51.48%), heme oxygenase-1 (HO-1, 51.86%), and B-cell lymphoma 2 (BCl-2, 57.88%) expression levels in the PM group were significantly downregulated compared to those in the NC group (100%). However, p-Akt (119.72%), HO-1 (89.17%), and BCl-2 (73.18%) expression levels in the GTE40 group were statistically upregulated compared to the PM group. The phosphorylated c-Jun N-terminal kinases (p-JNK, 184.73%) and BCl-2-like protein 4 (BAX, 260.57%) expression levels and BAX/BCl-2 ratio (450.19%) in the PM group were significantly upregulated compared to those in the NC group (100%). However, p-JNK (108.73%) and BAX (157.92%) expression levels and BAX/BCl-2 ratio (215.80%) in the GTE40 group were statistically downregulated compared to the PM group.
PM2.5 induces apoptosis in cardiac tissue, which is achieved through the interaction of BAX, BCl-2, and caspase proteins [38]. When exposed to environmental pollutants, oxygen radicals and various inflammatory factors are produced within cells [8]. These environmental factors increase the expression of the BAX, which in turn increases the BAX/BCl-2 ratio and induces cell death [39]. BAX migrates into mitochondria and damages the mitochondrial membrane, causing cytochrome c to leak into cells [40]. This cytochrome c activates caspase-9 and caspase-3 initiating the caspase cascade, which degrades various proteins inside the cell and induces apoptosis [41]. These mechanisms cause damage to cardiac tissue and an inflammatory response that negatively affects heart health [30]. Thus, to estimate the protective effect of GTE against PM2.5-induced apoptosis, the protein expression levels of p-Akt, p-JNK, HO-1, BCl-2, and BAX were measured in this study, and the GTE significantly attenuated apoptotic signals (Fig. 2). Similar to this study, long-term treatment with green tea downregulated the expression of hypoxia-inducible factor-1α, insulin-like growth factor-binding protein-3, and BAX/BCl-2 ratio in aging rats [42]. Kaempferol, one of the green tea phenolic compounds, regulated the expression levels of BAX and BCl-2 in ischemia-reperfusion-induced cardiac damage by regulation of endoplasmic reticulum stress proteins [43]. Also, EGCG inhibited cigarette smoke-exposed cardiac apoptosis by regulating the protein expression levels of caspase-3, BAX, BCl-2, cytosolic cytochrome c, and caspase-9 [44]. Based on these results, the consumption of GTE with bioactivities significantly attenuated various toxicant-induced apoptosis and cytotoxicity, and it suggests that green tea also has significant activity in the PM2.5-induced apoptotic signaling pathway.