Epigallocatechin-3-Gallate Activates Nrf-2, NF-κB and TNF-α Protein Signaling Pathways in Rat Muscle Tissue Damage

Abstract

Cisplatin, used as an effective drug in cancer patients, has become a popular topic in studies conducted in recent years. It is known that cisplatin terminates the vital activities of healthy cells as well as cancer cells. Epigallocatechin-3-gallate (EGCG), a member of the catechin group, is a natural antioxidant known to promote and improve health. Catechins, due to their regulatory effect on apoptosis and autophagy, have attracted increasing interest in their positive health effects on various diseases. This study aims to investigate the effect of EGCG on Nrf-2, NF-κB, and TNF-α protein signaling pathways against muscle tissue damage caused by cisplatin. A total of 28 male rats were used in the study, creating 4 groups. The experimental period was 28 days (4 weeks). The protective and healing effects of EGCG against cisplatin-induced damage in muscle tissue were investigated. Catalase (CAT) activity, malonaldehyde (MDA), and glutathione (GSH) levels were measured in a spectrophotometer. Expression levels of Nrf-2, NF-κB, and TNF-α proteins were determined using the Western blot method. When compared to the cisplatin-administered group, MDA levels decreased in the EGCG+Cisplatin-administered group, while CAT activity and GSH levels increased. It was found that EGCG used for therapeutic purposes reduced the expression of Nrf-2, NF-κB, and TNF-α proteins. Histopathological examination showed that muscle tissue damage occurred in the cisplatin group, while the damage decreased in the EGCG+Cisplatin-administered group. Our findings show that EGCG can prevent toxicity due to its anti-inflammatory effect against tissue damage caused by cisplatin.

Keywords:
EGCG; Nrf-2; MDA; Muscle; TNF-α.

HIGHLIGHTS

EGCG reduced the expression of NF-κB and TNF-α proteins

EGCG reduced muscle tissue damage

GRAPHICAL ABSTRACT

INTRODUCTION

Camellia sinensis (green tea) is one of the oldest and most popular beverages in the world [¹]. Green tea is obtained by drying the leaves after the tea plant is harvested, rolling them, and subjecting them to a heat treatment, usually carried out with steam [²]. This process inactivates enzymes and prevents fermentation and oxidation, which polymerizes monomeric catechins into condensed polyphenols [³]. Since no fermentation method is applied to the green tea plant, the volatile oil components found in black tea are not formed [⁴]. As a result of the studies, it is claimed that angiogenesis, which is considered critical in tumor development, is suppressed by the polyphenols in green tea [⁵]. It has been suggested that Epigallocatechin-3-gallate (EGCG), a polyphenolic compound found in high concentrations in green tea, has activities that can be used to prevent or alleviate various chronic diseases such as cancer and heart [⁶]. In short, it has been explained that the EGCG compound regulates many disease-specific molecular targets [⁵]. Cisplatin (dichlorodiamino platinum) was first shown to be a suppressor of cell division in 1965. By 1969, cisplatin was found to have antitumor effects in animal models. The first report of nephrotoxicity in animal studies was made in 1971, and this report showed histopathological changes of acute tubular necrosis with azotemia [⁷]. Cisplatin is one of the most remarkable successes in the fight against cancer. Since its accidental discovery many years ago, cisplatin has been widely used in chemotherapy. Unlike most cancer treatment drugs, which are usually complex organic compounds, cisplatin is a simple inorganic molecule [⁸]. EGCG can be induced primarily by scavenging NO, peroxynitrite, and other reactive oxygen/nitrogen (ROS/RNS). It inhibits the transfection of NF-κB and oxidation potential-1 (AP-1) to downregulate the expression of cyclooxygenase-2 (COX-2) and NO synthase (iNOS) and reduces the production of inflammatory factors to exert anti-inflammatory effects. EGCG plays an antifibrosis role mainly by blocking the transfer of NF-κB from the cytoplasm to the nucleus to downregulate the expression of certain genes [⁹]. EGCG modulates gene expression by inhibiting various transcription factors, including the Sp1 gene, NF-κB, AP-1, and forkhead box protein O1 (FOXO1). EGCG-induced ROS production, which leads to the activation of NF-κB and nuclear factor erythroid-related factor-2 (Nrf-2), leads to increased expression of HO-1 and glutathione. Scavenging of ROS using various antioxidants abolished the heme oxygenase (HO-1) induction induced by EGCG, while pretreatment with EGCG had protective effects against hydrogen peroxide-induced cytotoxicity. Preincubation with EGCG protected against cell death induced by mitochondrial damage without changes in superoxide dismutase (SOD), glutathione peroxidase, Nrf-2, or B cell lymphoma-2 (Bcl-2) expression and oxidative stress. EGCG produces low levels of reactive oxygen species, including hydrogen peroxide, which can act as a second messenger for downstream signaling pathways [¹⁰]. EGCG increases the production of signaling molecules such as ROS, calcium ions (Ca2+), cyclic adenosine monophosphate (cAMP), or 3'-5'-cyclic guanosine monophosphate (cGMP) [¹¹].

MATERIAL AND METHODS

Chemical Substances Used

All chemical materials Epigallocatechin-3-gallate (A15722) were supplied by Alfa Aesar (Germany), BioRad, BioShop (Canada), Sigma-Aldrich (Germany), Merck (USA), Cisplatin (CAS-Nr: 15663-27-1) was supplied by Sigma-Aldrich (Germany). Primary antibodies used for Western Blot study, TNF-α (sc-52746, 1:1000), NF-ĸB (sc-8008, 1:1000), Nrf-2 (ab137550, 1:1000), Beta-actin (sc-8432, 1:500); secondary antibody (sc-516102,1:5000) was supplied by Santa Cruz Biotechnology (Germany).

Ethic and Experimental groups

Experimental Animals Used and Method of Creating Groups. The animal experiment phase of this thesis study was implemented at the Firat University Experimental Animals Research Center (FUDAM) with the protocol number 2019-141 and session number 2021/17 dated 27.10.2021 of the Firat University Animal Experiments Local Ethics Committee. All groups were provided with a lighting period of 12 hours per day, with light and dark periods. Four different groups were created: the control group, the EGCG group, the Cis group, and the Cis + EGCG group. Different diets were applied to each group. The initial live weights of the experimental animals were adjusted close to each other the groups were determined, and the live weights of the animals were recorded weekly during the study. Food and water were provided ad libitum during the study. The observation continued for 4 weeks, and as a result of this period, the muscle tissues of the rats were taken and the necessary examinations were performed.

Cisplatin (Cis) Preparation and Administration Method

On the 3rd day of the study, 2 mg/mL Cis was dissolved in 0.9% sodium chloride solution and administered by intraperitoneal injection as a single dose [¹²].

Epigallocatechin-3 Gallate (EGCG) Preparation and Administration Method

0.2 g Epigallocatechin Gallate was dissolved in 15 mL 0.9% sodium chloride and administered to experimental animals intraperitoneally at 50 mg/kg body weight three times a week starting from the 2nd week [¹³]. The application was continued until the 4 weeks were completed.

MDA Measurement

After the muscle tissue samples were cut into small pieces, 4.5 mL of 1.15% KCl was added to 0.5 grams of tissue and homogenized in a mechanical homogenizer. The determination of MDA (malondialdehyde), the final product of lipid peroxidation, was performed from this homogenate by modifying the Ohkawa method [¹⁴]. 750 µl of 8.1% sodium dodecyl sulfate (SDS) + 20% acetic acid solution (pH 3.5) and 750 µl of 0.8% (pH 3.5) TBA solution were added to 0.1 mL of homogenate. The final volume was made up of 4 mL of distilled water. Then it was incubated for 45 min in a 95℃ boiling water bath, after cooling, 5 mL of 15:1 (v/v) n-butanol-pyridine mixture and 1 mL of distilled water were added and vortexed. After centrifugation at 5000 rpm for 10 min, the upper phase was taken, and absorbance was measured and evaluated in a spectrophotometer at a wavelength of 532 nm. The results were recorded as nmol/g [¹⁵].

GSH Measurement

0.1 mL of cell homogenate was mixed with 0.4 mL of TCA (10% trichloroacetic acid) solution and vortexed for 20 seconds. Then, centrifugation was performed at 3000 rpm for 5 minutes. After the 0.1 mL of the supernatant obtained was transferred to another tube, 2 mL of Tris buffer (0.4M, pH 8.9), 0.1 mL of DTNB solution, and 0.9 mL of distilled water were added. The optical density of the resulting yellow color was read at 412 nm wavelength in the spectrophotometer [¹⁶].

AT Activity

For catalase analysis, a mixture containing 2 mL of phosphate buffer (pH 7), 0.95 mL of H₂O₂ (0.019 M), and 0.05 mL of supernatant (total 3 mL) was measured. The absorbance change of H₂O₂ was read every 10 seconds within 1 minute at 240 nm wavelength in the spectrophotometer. The number of enzyme units per mL was calculated from this optical density difference [¹⁷].

Muscle Tissue Homogenization for Western Blotting

Muscle tissue samples were cut into small pieces and digested in lysis buffer (EDTA, ß-Mercaptoethanol, 0.5 M Tris [pH 8], Phenyl methyl sulfonylfluoride [PMSF]) in a glass homogenizer. The samples belonging to the dissected tissues were centrifuged at 15000 rpm for 45 min. After centrifugation, the supernatant was taken and stored at -20 ℃ until further studies [¹⁸]. Muscle tissue protein samples were run on a 12% gel using the SDS-PAGE method and then transferred to a nitrocellulose membrane using the Western blotting method to determine the presence and density of proteins (Nrf-2, TNF-α, NF-κB) and their expression levels were examined [¹⁹, ²⁰] and protein levels were determined (Image J; National Institute of Health, Bethesda, USA).

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was provided by the BIO-RAD Mini-PROTEAN®3 Cell gel electrophoresis system. The separation of proteins was carried out with the following steps [²⁰]. For the 12% separation gel; 2.5 mL 1.5 M Tris hydrogen chloride (HCl) (pH; 8.8), 100 μl 10% SDS, 75 μl 10% ammonium persulfate, 3.35 mL distilled water, 2.5 mL sodium dodecyl sulfate (NaC1₂H₂5SO₄), 15 μl tetramethyl-enediamine and 4 mL 30% acrylamide/bis were used. For the 4% loading gel; 100 μl 10% SDS, 1.3 mL 30% acrylamide/bis, 2.5 mL 0.5 M Tris hydrogen chloride (pH; 6.8), 6.1 mL distilled water, 60 μl 10% ammonium persulfate ((NH₄)S₂O₈), 12 μl tetramethylendiamine (C₆H₁₆N₂) were used. SDS-PAGE SAB dye was added equally before loading the prepared samples into the wells. After boiling for 5 min, electrophoresis was performed by applying a 30 mA current [¹⁹]. After this application, the Western blotting technique was performed.

Determination of Protein Expression Levels of Nrf-2, NF-κB, and TNF-α by Western Blotting

Western blot was performed with the BIO-RAD Mini-PROTEAN®3 Cell gel electrophoresis system. Before using this technique, proteins were separated in the gel by the SDS-PAGE electrophoresis method. The same size as the gel, a nitrocellulose transfer membrane was cut and kept in the transfer buffer with the filter paper. After the electrophoresis process was carried out, the gel was kept in the transfer buffer, and then the cassette lid specific to Western blot was opened immediately after the filter paper was placed at the bottom, and the gel was placed on top. The nitrocellulose membrane and a new filter paper were placed so that no air bubbles remained, the cassette lid was closed, and placed in the electrophoresis tank. After the ice unit was placed in the tank, the transfer buffer was filled and connected to the power supply, and a current of 250 mA was applied for 90 minutes. For the regular distribution of heat, blotting was performed on a magnetic stirrer. Immediately after the blotting process, the membrane was washed in PBS Tween-20 for 15 minutes and then blocked in milk powder at 4 ℃ for 1 night. After this process, it was incubated at room temperature for 90 minutes, and the primary antibody (TNF-α, Nrf-2, NF-κB) was added. It was incubated again at room temperature (25 ℃) for 90 minutes and then washed for 15 minutes. A secondary antibody (sc-516102) was added and incubated for 90 minutes at room temperature. After washing again with PBS working solution for 15 minutes, the final volume was completed with 40 milliliters of distilled water, and it was kept in 40 μl of 30% H₂O₂, four tablets-20 mg 3.3’-diaminobenzidine (Amresco), 1.33 milliliters of 1 M Tris (pH 6.8 ) until the bands became visible. Then, the reaction was stopped by taking it into distilled water, [²¹, ²²].

Histopathological Analysis

The sections taken from the muscle tissues were fixed in 4% formaldehyde solution and observed under a light microscope [²¹, ²²].

Statistical Analysis

The obtained data were evaluated with variance analysis in the SPSS 22 package program. The differences in the 4 groups were determined by applying One-Way ANOVA Post Hoc Tukey, LSD, and Duncan tests. The measurements were evaluated by making three repetitions to ensure the reliability of the statistics.

RESULTS

Muscle Tissue MDA Level

The values given in Table 1 are calculated in accordance with the necessary procedures based on the standard MDA stocks of muscle tissue MDA. According to the results obtained, the MDA standard curve is shown in Figure 1. According to the graph given in Figure 1, the group with the highest muscle tissue MDA level is the Cis group, while the group with the lowest MDA level is the EGCG group. The MDA levels of the control and EGCG groups are close to each other, and the difference between them is not significant (p>0.05). However, the difference between the Cis and Cis+EGCG groups and the other two groups was found to be significant (p<0.05).

Table 1
Muscle tissue malondialdehyde (MDA) results

GROUPS	Muscle Tissue MDA Levels (nmol/g)
Control EGCG	52.65 ± 0.15c 48.55 ± 0.17c
Cis EGCG + Cis	93.55 ± 0.26a 82.95 ± 0.23b

Figure 1
Muscle tissue malondialdehyde (MDA) results in rats. a-c: Different letters indicate statistically significant difference between groups. Values are statistically significant at p<0.05 One-way analysis of variance (ANOVA) Post Hoc LSD Test.

Muscle Tissue GSH Level

When we look at the tables and graphs, it is understood that the group with the highest GSH level was EGCG, while the lowest was in the Cis group, and the GSH levels in the control and EGCG groups were close to each other and their was no significant difference between them (p>0.05). In the Cis and Cis+EGCG groups, GSH levels were found to be lower than in the other groups. It was determined that the difference between the EGCG group and Cis and EGCG + Cis was significant (p<0.05). It was observed that EGCG increased the low GSH level observed due to the damage caused by Cis. These measured values indicate that Cis causes damage to the antioxidant defense system and that EGCG provides a protective effect against this damage (Table 2 and Figure 2).

Table 2
Muscle tissue glutathione (GSH) results.

GROUPS	Muscle Tissue GSH Levels (µmol/mg protein)
Control EGCG	585.33 ± 0.34a 598.66 ± 0.31a
Cis EGCG + Cis	372.00 ± 0.23c 422.00 ± 0.26b

Figure 2
Muscle tissue glutathione (GSH) levels in rats. a-c: Different letters indicate statistically significant difference between groups. Values are statistically significant at p<0.05 One-way analysis of variance (ANOVA) Post Hoc LSD Test.

Muscle Tissue CAT Activity

According to Figure 3 and Table 3, it was observed that the CAT activity was the highest in the EGCG group and the lowest in the Cis group, and the CAT activities of the control and EGCG groups were close to each other, and there was no significant difference between them (p>0.05). In Cis and Cis+EGCG groups, CAT activities are lower than other groups, and at the same time, the difference between these two groups and the other groups is significant (p<0.05). The fact that the CAT level increased in the Cis+EGCG group compared to the Cis group and the difference between them is statistically significant (p<0.05) shows that EGCG has a protective effect.

Table 3
Muscle tissue catalase activity (CAT) results.

GROUPS	Muscle Tissue Catalase Activity (U/mg protein)
Control EGCG	92.00 ± 0.32a 98.66 ± 0.35a
Cis EGCG + Cis	65.33 ± 0.22c 82.00 ± 0.29b

Figure 3
Muscle tissue catalase enzyme activity (CAT) levels in rats. a-c: Different letters indicate statistically significant difference between groups. Values are statistically significant at p<0.05 One-way analysis of variance (ANOVA) Post Hoc LSD Test.

Expression Levels of Apoptotic Markers (Nrf-2, NF-κB, TNF-α proteins)

Nrf-2 Expression Levels in Groups

According to Figure 4A, the group with the highest Nrf-2 protein expression level is the EGCG-applied group. The expression level of the control group is close to the EGCG group but lower, and the difference between them is not significant (p>0.05). It was observed that the group with the lowest Nrf-2 expression level is the Cis-applied group. It was found that the Nrf-2 protein expression level was higher in the EGCG + Cis group than in the Cis group, and the difference between them was statistically significant (p<0.05). When the findings were evaluated, the increase in the Nrf-2 expression level with EGCG treatment compared to the Cis group revealed that it significantly corrected the muscle damage caused by Cis.

Figure 4
Muscle tissue western blotting mean protein expression results; A: Nrf-2, B: NF-κB, C: TNF-α, a-d: Different letters indicate statistically significant difference between groups. Values are statistically significant at p<0.05 One-way analysis of variance (ANOVA).

NF-κB Protein Expression Levels in Groups

When Figure 4B is examined, it is determined that the group with the highest NF-κB protein expression level compared to other groups is the cisplatin group, while the group with the lowest expression is the EGCG group. It was observed that the expression level in the EGCG+Cis group was lower than in the Cis group, and the difference between them was statistically significant (p<0.05). The difference in protein expression levels between the EGCG group and the control group is insignificant (p>0.05). The findings show that Cis, which causes damage in muscle tissue, increases NF-κB protein expression, while EGCG significantly treats the damage by reducing protein expression.

TNF-α Protein Expression Levels in Groups

When the values given in Figure 4C are examined, it is seen that the TNF-α expression level is the highest in the Cis group and the lowest in the EGCG group. It was determined that there was a significant decrease in expression levels in the EGCG+Cis group compared to the Cis group, and the difference between them was statistically significant (p<0.05). When the obtained data were examined, it was concluded that Cis caused damage in the muscle tissue by activating TNF-α expression, and that EGCG applied for treatment purposes also showed an effect by significantly reducing the damage.

Histopathological Findings

When the histopathological results of our study were examined, normal histology was observed in the control and EGCG groups of the muscle tissue. Inflammatory cell infiltration, edema, and significant loss of striation were observed in the Cis damage group.

It was determined that inflammatory cell infiltration and loss of striation significantly decreased in the EGCG + Cis treatment group compared to the Cis group. No edema formation was observed in the EGCG + Cis treatment group. In line with these findings, it was concluded that EGCG, which has many biological activities, significantly reduced the damage in the muscle tissue. The normal histology of the nuclei in the control group of muscle tissue examined under a microscope is shown in Figure 5A. In the group where EGCG was applied, the nuclei and transverse striations were observed to be normal as in the control group (Figure 5B). In Figure 5C, the parts indicated by black arrows in the Cis group show inflammatory cell infiltration and edema formation. There is a significant loss of striation in the part indicated by the double-headed arrow. In Figure 5D, it is observed that as a result of EGCG + Cis application, there is a decrease in inflammatory cell infiltration in the areas indicated by black arrows and a slight loss of striation in the areas indicated by double-headed arrows.

Figure 5
Muscle tissue histopathological results; A: Control, B: EGCG, C: Cis, D: EGCG+Cis

DISCUSSION

Green tea polyphenols reduce lymphocyte production of proinflammatory cytokines and also have the ability to reduce inflammation around trauma. Overexpression of inflammatory mediators in osteoarthritis can be inhibited by EGCG in polyphenols. Polyphenols also increase the production of vascular endothelial factors. In addition, polyphenols promote the regeneration of blood vessels around the injury due to the abundant capillaries around the nerve anastomosis [²³]. These findings are an indication of the healing effect of EGCG. Studies in animal models have shown the cancer-preventive activity of EGCG in different organs such as the liver, stomach, skin, lung, mammary glands, and colon. In studies with cell lines, EGCG has cancer-preventive activities such as inhibition of various protein kinases, DNA methyltransferase, and epidermal growth factor receptor signaling [⁶].

The biological effects of EGCG are plasma concentration dependent. Low or moderate EGCG concentrations (plasma levels ≤10 µM) may show an antioxidant effect mediated by EGCG-induced production of low amounts of ROS, which are primarily required for the stimulation of signal transduction pathways that promote cell protection. However, high EGCG concentrations (>10 µM) show a predominantly prooxidant effect, while the direct prooxidant effects of EGCG are due to its autoxidation, which leads to the production of hydrogen peroxide. When the values observed in this thesis study were examined, it was determined that there was an increase in the MDA level in the group in which muscle tissue damage was induced with cisplatin, while the MDA level decreased in the Cis+EGCG group, and the difference between them was found to be statistically significant (p<0.05). It can be said that EGCG prevents the damage caused by Cis in muscle tissue.

GSH levels decreased in the cisplatin-administered group and increased in the EGCG+Cis-administered group. When CAT activity was examined, it was found that CAT activity decreased in the cisplatin group and increased in the EGCG + Cis group. The difference between the two groups for both GSH level and CAT activity was found to be statistically significant (p<0.05). In other studies, EGCG, which inhibits NF-κB, was found to suppress inflammation in endothelial cells and cardiomyocytes. It was also found that EGCG reduced oxidative stress caused by cigarette smoke and weakened the expression of proinflammatory genes in bronchial epithelial cells. EGCG-induced NF-κB inhibition also led to anti-inflammatory effects related to carcinogenesis. For example, EGCG was observed to inhibit NF-κB activity in human colon, bladder, and lung cancer cells, which prevented cancer cell proliferation and migration. EGCG-induced suppression of NF-κB protein results in both anti-inflammatory and anti-tumor effects because NF-κB controls the synthesis of pro-inflammatory cytokines such as TNF-α or IL-1β and contributes to the regulation of cell growth [¹¹].

In our study, similar to the results of Mokra and coauthors [¹¹], it was found that EGCG inhibited NF-κB activity. In the study investigating the protective effect of royal jelly on pancreatic damage caused by fluoride, the levels of NF-κB, Bax, Bcl-2, TNF-α, and Caspase-3 protein expressions were examined, and it was found that TNF-α and NF-κB protein expression levels decreased compared to the control group. In addition, it was stated that royal jelly caused a decrease in MDA levels and an increase in GSH levels and catalase activity in pancreatic damage. According to our findings, it was determined that EGCG treatment against muscle tissue damage caused a decrease in NF-κB protein expression level. It was observed that the highest NF-κB protein expression level was in the Cis group and the lowest in the EGCG group (p<0.05). It was observed that the expression level in the EGCG+Cis group was lower than in the Cis group and the difference between them was also significant (p<0.05). In line with these results, it has been shown once again that EGCG has anti-inflammatory properties by affecting the NF-κB protein pathways [²³].

EGCG, the basic compound of green tea, has been found to have anti-inflammatory potential and inhibit proinflammatory cytokine activity. In some studies, there are findings that EGCG plays a leading role in TNF-α protein inhibition and can protect TNF-α-mediated lung inflammation through downregulation of oxidative stress and intercellular adhesion molecule-1 expression (ICAM-1). EGCG was found to inhibit TNF-α-induced monocyte chemoattractant protein-1 (MCP-1) production in human umbilical vein endothelial cells. As a result, it confirms that EGCG significantly reduces the TNF-α protein-induced protein and mRNA expression of MCP-1 [²⁴]. In our study, the highest TNF-α values were observed in the Cis group and the lowest in the EGCG group. The fact that the EGCG+Cis group showed an increase in expression levels compared to the Cis group and the difference between them was statistically significant (p<0.05) indicates that EGCG has an anti-inflammatory effect against muscle tissue damage [²⁴].

In a study conducted by Kim and coauthors [¹⁰], the researchers investigated whether EGCG (Epigallocatechin gallate) affected the expression of proinflammatory cytokines in microglial cells stimulated by hypoxia. The study found that CoCl₂, a hypoxia-mimicking agent, increased Nrf-2 levels in the cytosolic extract compared to the control group. However, when EGCG was introduced, it reversed the elevated cytosolic Nrf-2 levels and instead promoted an increase in nuclear Nrf-2 levels in comparison to the CoCl₂-treated group. These findings suggest that EGCG's anti-inflammatory effects may be linked to the upregulation of heme oxygenase-1 (HO-1) through the Nrf-2 pathway in BV2 microglial cells activated by hypoxia. This points to EGCG’s potential role in modulating inflammatory responses in neurological conditions associated with hypoxia.

As a result of our study, it was determined that the Nrf-2 protein expression level was the highest in the EGCG-applied group (p<0.05). In the EGCG + Cis group, it was observed that the Nrf-2 protein expression level was higher than in the Cis group (p<0.05), and it was concluded that EGCG treatment significantly prevented muscle damage caused by Cis. EGCG has the advantages of being natural and safe without any side effects. EGCG has many functions, such as anti-inflammatory, antioxidative, and antifibrotic effects in various tissues and cells, and has been shown to inhibit DNA methylation in tumor cell lines [²⁵].

Almatroodi and coauthors [²⁶] investigated the potential therapeutic targets of EGCG and its role in the treatment of various types of cancer. EGCG was found to inhibit TNF-alpha-induced monocyte chemoattractant protein-1 (MCP-1) production in human umbilical vein endothelial cells. As a result, it was confirmed that EGCG significantly reduced TNF-α-induced protein and mRNA expression of MCP-1. They also stated that EGCG suppresses TNF-alpha-induced MCP-1 expression in human umbilical vein endothelial cells and that this effect is provided by 67LR and occurs through inhibition of NF-κB activation [²⁵]. The effect of EGCG was investigated in a study where damage was induced in testicular tissue with cisplatin, and according to the obtained results, it was determined that TNF-α protein expression level was high in the cisplatin-administered groups and low in the groups aimed to be improved with EGCG (p>0.05). It has been observed that Cis increases the TNF-α protein expression level and causes damage to testicular tissue. It has been stated that EGCG application decreases the TNF-α protein expression level and shows antioxidant and anti-inflammatory activity against testicular tissue damage. When the biochemical analysis results are examined, it has been determined that GSH level and CAT activity increased in the EGCG group and decreased in the group where damage was created by Cis application (p<0.05) [²⁷].

Toprakoğlu and coauthors [²⁸] reported that EGCG decreased MDA levels in kidney tissue and plasma and caused an increase in catalase activity and GSH levels in cisplatin-induced kidney damage. They also observed that there was a significant decrease in IL-6 and TNF-α, p38α MAPK, and protein expression levels in the EGCG+Cis group compared to the cisplatin group. Arslan and coauthors [²⁹] investigated the effect of EGCG on cisplatin-induced damage in pancreatic tissue and observed that IL-6 protein expression levels were higher in the cisplatin-treated group compared to the other groups. When the Cis+EGCG group was compared with the Cis group, it was found to be highly increased in the Cis group (p<0.05). When the EGCG-treated group was examined, a significant decrease in IL-6 expression was observed in the pancreatic tissue. Cisplatin increased the IL-6 level and caused inflammation in the tissue. EGCG treatment proved its anti-inflammatory and healing effects by reducing the IL-6 level.

Recent developments support the potential role of EGCG in the chemoprevention and chemotherapy of various cancers by intervening in the initiation, development, and progression of cancer. Despite the frequently voiced criticisms regarding the stability, solubility, and bioavailability of polyphenols, it has been shown that EGCG can interact with DNA and RNA in the nucleus and possibly play a role in gene regulation. Furthermore, EGCG has been reported to bind to a wide variety of proteins such as kinases, Epidermal Growth Factor receptors, apoptotic proteins, and proteasomes, thus proving its ability to interfere with multiple signaling pathways [³⁰].

Singh and coauthors [³¹] conducted a study that suggested EGCG (Epigallocatechin-3-gallate) plays a significant role in inhibiting tumor formation across various organs. The study found that EGCG suppressed lipopolysaccharide-induced nitric oxide production and reduced the expression of inducible nitric oxide synthase (iNOS) genes in isolated peritoneal macrophages. This was achieved by inhibiting the activation of NF-κB, a key transcription factor involved in inflammation and immune response. Furthermore, EGCG was shown to inhibit platelet-derived growth factor (PDGF)-induced apoptosis and disrupt cell cycle regulation in vascular smooth muscle cells. These effects ultimately led to the inhibition of tumor growth, metastasis, and angiogenesis in vivo. Aslan and coauthors [³²], Gok and coauthors [³³], Beyaz and coauthors [³⁴] and Aslan [³⁵] indicated that kiwi fruit extract, ellagic acid, epigallocatechin-3 and Mulberry Juice reduced the oxidative stress. The findings suggest EGCG's potential as a therapeutic agent in cancer prevention and treatment. When the histopathological results of our study were examined, normal histology was observed in the control and EGCG groups of muscle tissue, while inflammatory cell infiltration, edema, and significant loss of striation were observed in the Cis injury group. EGCG directly stimulates cellular events and shows a healing effect. It was determined that inflammatory cell infiltration and loss of striation were significantly reduced in the EGCG + Cis treatment group compared to the Cis group. No edema formation was observed in the EGCG + Cis treatment group. In line with these results, it was concluded that EGCG, which has many biological activities, significantly reduced the damage in muscle tissue by exhibiting antioxidant properties.

CONCLUSION

The results indicated a decrease in MDA levels and an increase in CAT activity and GSH levels in the EGCG+Cis group compared to the Cis group. Additionally, it was found that EGCG treatment reduced the expression of NF-κB and TNF-α proteins while promoting the expression of Nrf-2 protein. Histopathological examination of muscle tissue revealed that the cisplatin group exhibited significant damage, including inflammatory cell infiltration, edema, and loss of striation. In contrast, the EGCG+Cis group showed reduced tissue damage due to the protective effects of EGCG. In the EGCG-treated groups, the increased Nrf-2 synthesis and decreased NF-κB protein expression indicate a reduction in oxidative damage compared to the Cis group, and specifically, the promotion of cell death in damaged cells. The decrease in TNF-α protein in the EGCG-treated groups suggests that damaged cells are directed towards apoptosis. In conclusion, the findings of this study are consistent with previous research on the healing and therapeutic benefits of EGCG, demonstrating that EGCG can not only exert anti-inflammatory effects but also prevent toxicity in cisplatin-induced tissue damage.

• Funding:
  This study was supported by Firat University Research Projects Unit (FUBAP) [Project Number: SYO.22.01].
• Institutional Review Board Statement:
  The animal study protocol was approved by the Institutional Ethics Committee of Firat University Experimental Animals Research Center (FUDAM) (protocol code 2021/17 and 27.10.2021 date of approval).
• Informed Consent Statement:
  Not applicable.

Acknowledgment:

This article was produced from the thesis titled “The Effect of Epigallocatechin-3-Gallate on Nrf-2, NF-κB and TNF-α Protein Signaling Pathways Against Muscle Tissue Damage Caused by Cisplatine.”

Use of Generative Artificial Intelligence

The authors declare that large language models and other generative artificial intelligence (AI) or AI-assisted technologies cannot be credited as authors and have not been listed as authors of this paper.

The author declare that did not use the artificial intelligence.

Data Availability Statement:

All data from the present study were inserted in this manuscript.

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• Editor-in-Chief:
  Paulo Vitor Farago

• Associate Editor:
  Jane Manfron

Publication Dates

• Publication in this collection
  03 Apr 2026
• Date of issue
  2026

History

• Received
  04 Aug 2025
• Accepted
  04 Dec 2025