Tea Polyphenols Enhanced the Antioxidant Capacity and Induced Hsps to Relieve Heat Stress Injury (2025)

2.1. Cell Culture

H9C2 cells, derived from embryonic rat hearts, were purchased from the American Type Culture Collection. The cells were seeded in plastic tissue culture flasks (Corning, China) at a density of 1 × 10⁵/mL and cultured in Dulbecco's Modified Eagle Medium (Gibco, USA) supplemented with 10% fetal bovine serum (Biological Industries, BI), 100 U penicillin, and 100 μg/mL streptomycin (Gibco). Thereafter, the cells were incubated in a 5% CO₂ humidified atmosphere (Thermo, USA) at 37°C. When the cells formed a complete monolayer, they were passaged within 30 passages for the subsequent experiments.

2.2. Screening the Optimal Concentration of Tea Polyphenols

Concentration screening was performed according to the cell viability measured by cell counting kit-8 (CCK-8, Beyotime, China). The cells were seeded in 96-well plates. When the cell fusion degree reached 70%–80%, the medium was replaced with a fresh one containing tea polyphenols at 0, 5, 10, 20, 40, 60, or 80 μg/mL. The tea polyphenols was purchased from Jiangsu Dehe Biological Technology Co., Ltd., with polyphenol content > 98%, catechins > 80%, and epigallocatechin gallate (EGCG) > 59%. The plate was incubated at 37°C for 16 h, and the medium was replaced with a fresh medium containing 10% CCK-8 for incubation for 2 h at 37°C. Absorbance was measured at 450 nm with a microplate reader (NanoQuant, USA) to obtain the maximum safe concentration of tea polyphenols. After the cells were incubated with different concentrations of tea polyphenols for 16 h, they were transferred to a 42°C 5% CO₂ cell incubator for a 5 h heat stress to discriminate the protection of tea polyphenols under heat stress. The medium was replaced with a fresh one containing 10% CCK-8 for incubation for 2 h at 37°C, and the absorbance was detected. GraphPad Prism 6.01 was used for data analysis.

2.3. In Vitro Myocardial Cell Heat Stress Model

The cells were randomly divided into the control and tea polyphenol groups to evaluate the heat stress protection of tea polyphenols. When the cells grew to a fusion degree of 70–80%, the control group medium was changed to a fresh one. Meanwhile, the tea polyphenol group medium was changed to a fresh one containing 10 μg/mL tea polyphenols. After a continued culturing for 16 h, the cells were transferred into a 42°C 5% CO₂ cell incubator for 0, 2, 3, and 5 h heat stress (Figure 1(a)). Thereafter, the cells and supernatants were immediately harvested for analysis.

2.4. Histological Analysis of the H9C2 Cells

The H9C2 cells were seeded on coverslips and treated as the vitro heat stress model. The cells were collected, fixed in 4% paraformaldehyde for 30 min, and stained with haematoxylin for 5 min and with eosin for 3 min. Then, the cells were immediately passed through the gradient ethanol consisting of 75, 85, 95, 95, 100, and 100% for 2 min. After the cells were passed through xylene for 2 min, the coverslips were mounted using neutral resin and analyzed via light microscopy with an Axio Imager.A2 instrument (Zeiss, German).

2.5. Oxidative Damage-Related Indicator Analysis of H9C2

The H9C2 cells, seeded in 30 mm dishes, were treated as the vitro heat stress model, and the cells were collected for the detection of antioxidant indicators Hemeoxygenase-1 (HO-1) mRNA, total antioxidant capacity (T-AOC), and superoxide dismutase (SOD). T-AOC was performed with commercial kits in accordance with the manufacturer's instructions (A012-5, Nanjing Jiancheng). SOD was measured by Kingmed Diagnostics. The HO-1 mRNA level was analyzed by RT-PCR. The total RNA was extracted using an RNAiso Plus reagent (TaKaRa, Japan) and quantified with a NanoDrop 2000 (Thermo, USA) by the absorbance at 260 nm and A260/A280 ratio. Reverse transcription was then carried out with a real-time quantitative PCR (RT-PCR kit) (Vazyme, China) to synthesize the cDNA, which was used for RT-PCR with a Power SYBR Green Master Mix (Vazyme) according to the manufacturer's instructions. The relative transcription level of HO-1 was normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and quantified using the comparative Ct (2^−ΔΔCt) method. The primers for HO-1 were forward: 5′-CTTTCAGAAGGGTCAGGTGTC-3′, and reverse: 5′-TGCTTGTTTCGCTCTATCTCC-3′. Meanwhile, the primers for GAPDH were forward: 5′-GCAAGTTCAACGGCACAG-3′, and reverse: 5′-GCCAGTAGACTCCACGACAT-3′.

2.6. Dynamic Expression Levels of Nrf2, Hsps, and Cleaved-Caspase 3 in the H9C2 Cells

The dynamic expression levels were analyzed using western blot. The treated cells were washed with PBS three times and lysed with a RIPA lysis buffer (R0010, Solarbio) containing a 1% protease inhibitor (Nanjing Jiancheng Biochemical Reagent) in 4°C for 15 min. The supernatants were collected after centrifugation at 12,000 g for 10 min, and the protein concentration was measured with a BCA Protein Assay Kit (P0009; Beyotime). A 5x sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was added to the protein samples. The samples were then boiled for 15 min and stored at −20°C until needed. Approximately 15 μg of each protein sample was separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. After the membranes were blocked with 5% nonfat dry milk in tris-buffered saline and Tween 20 (TBST) buffer for 2 h, they were incubated with anti-Nrf2 (1 : 1000, Abcam), anti-Hsp70 (1 : 1000, Enzo), anti-Hsp27 (1 : 1000, Santa), anti-CRYAB (1 : 1000, CST), and anticleaved-caspase 3 (1 : 500, CST) at 4°C overnight. The membranes were washed with TBST and then incubated with anti-mouse IgG HRP conjugated (1 : 3000, CST) or anti-rabbit IgG HRP conjugated (1 : 3000, CST) secondary antibodies at room temperature for 2 h. After the membranes were washed with TBST, they were added with an enhanced chemiluminescence detection regent ECL (Thermo, USA) and detected by the ImageQuant LAS 500 digital imaging system (GE Healthcare, Japan). The intensity of the scanned bands was determined using Quantity One.

2.7. In Vivo Myocardial Cell Heat Stress Model

Eighty 300-day-old specific pathogen-free chickens were purchased from Shandong Health-Tec Laboratory Animal Breeding Co., Ltd., Jinan, China. The chickens were randomly divided into control and tea polyphenol groups. The experimental design is shown in Figure 1(b). All chickens were fed for adaptive growth for 7 d in the normal environment (23 ± 2°C, approximately 60% humidity) with free diet and drinking. Followed by a 7 d dosing period, the tea polyphenol group was fed with water supplemented with 0.2 g/L tea polyphenols. Meanwhile, the control group was fed with normal water. Then, heat stress (37 ± 1°C) was triggered for 0, 0.5, 2, and 5 d with the same drinking with dosing period. After the heat stress, the serum and heart tissues were immediately collected for analysis. All experiments were performed in accordance with the guidelines of the Animal Ethics Committee of Shandong Province (China) and approved by the Institutional Animal Care and Use Committee of Shandong Academy of Agricultural Sciences (SAAS-2019-032), China.

2.8. Pathological Examination of the Myocardial Tissues

The myocardial tissues fixed in 10% formalin were resized to 5 mm × 5 mm × 2 mm and then refixed in 10% formalin for 12 h. After 75%, 85%, 95%, 95%, 100%, and 100% ethanol gradient dehydration of each gradient for 1 h, the myocardial tissues were immersed in xylene for 20 min for transparency. Transparent tissues were embedded in paraffin wax and sliced into 5 μm thick serial sections. The sections were deparaffinized in xylene, rehydrated with reduced alcohol series, and stained with haematoxylin for 5 min and eosin for 2 min. The slices were sealed with coverslips using a neutral resin for light microscopic analysis with an Axio Imager.A2 instrument (Zeiss, German).

2.9. Injury and Oxidation-Related Index Analysis in the Chicken Serum

The lactate dehydrogenase (LDH), creatine kinase (CK), CK isoenzyme MB (CK-MB), and SOD in the chicken serum were sent to Kingmed Diagnostics for detection. The levels of TNF-α (JYM0033Ch, Wuhan Colorful Gene Biotechnology Co., Ltd.), malondialdehyde (MDA, A003-1-2), total antioxidant capacity (T-AOC, A012-5), and glutathione peroxidase (GSH-PX, A005-1-2) in the chicken serum were determined with kits in accordance with the manufacturer's instructions purchased from Nanjing Jiancheng Bioengineering Institute.

2.10. Dynamic Expression Levels of Nrf2, Hsps, and Cleaved-Caspase 3 in the Myocardial Tissues

Approximately 20 mg of myocardial tissue for each chicken was homogenized in a 200 μL RIPA buffer (R0010, Solarbio) containing 1% PMSF and lysed at 4°C for 30 min. The supernatant was collected after centrifugation at 12,000 g for 10 min. The protein concentration was measured with a BCA Protein Assay Kit (P0009; Beyotime). The 5x SDS-PAGE loading buffer was added to the protein samples. The samples were then boiled for 15 min and stored at −20°C until needed. Approximately 20 μg of each protein sample was used for western blot analysis as the method in H9C2 cells.

2.11. Statistical Analysis

Data differences between the experimental groups were analyzed by SPSS software v.20.0 by one-way analysis of variance and least significant difference multiple comparison test methods. Statistically significant differences were set at P < 0.05 (^∗ or ^#). Extremely significant differences were set at P < 0.01 (^∗∗ or ^##).

3.1. Optimal Concentration of Tea Polyphenols

The optimal concentration screening was performed according to the cell viability. Under a normal condition, the concentration of tea polyphenols in the range of 0–20 μg/mL was safe for the H9C2 cells. However, a dose of 40 μg/mL or higher could significantly lower the cell viability (P < 0.01); the cell viability decreased by 38.86% when the concentration reached 80 μg/mL (Figure 2(a)). Under the condition of heat stress for 5 h, the protection of tea polyphenols first increased and then decreased with the increasing concentration; the 10 μg/mL concentration presented the best, which could increase cell viability by 28.52% (Figure 2(b)). In summary, 10 μg/mL of tea polyphenols was the optimal concentration and used in the subsequent protection experiments.

3.2. Histological Analysis of the H9C2 Cells

Pathological lesions occurred when the H9C2 cells were exposed to heat stress (Figure 3). In the control group, the H9C2 cells demonstrated granular degeneration when they were exposed to heat stress for 2 h. Meanwhile, the supplement with 10 μg/mL tea polyphenols could significantly weaken granular degeneration. When the heat stress continued for 3 h, the granular degeneration in the control group still existed, and large vacuolar degeneration was observed. By contrast, the cells in the tea polyphenol group presented granular degeneration and relatively small vacuolar degeneration. When the heat stress lasted for 5 h, nucleus deep staining was observed in the control group, while the tea polyphenol group was relatively light.

3.3. Oxidative Damage-Related Indicator Analysis of H9C2

The levels of oxidative damage-related indicators T-AOC, HO-1 mRNA, and SOD in the H9C2 cells are shown in Figure 4. The heat stress could upregulate the T-AOC levels, which showed a significant difference at 5 h (P < 0.01). Such stress could upregulate the HO-1 mRNA levels and showed significant differences at 2, 3, and 5 h (P < 0.01). However, the level of SOD presented a downward trend and a significant difference at 5 h (P < 0.01). The supplement with 10 μg/mL tea polyphenols could increase the total antioxidant capacity (T-AOC) of the H9C2 cells, and a significant difference was observed at 5 h (P < 0.05). Heat stress could upregulate the HO-1 mRNA levels and showed a significant difference at 2 h (P < 0.01). Moreover, heat stress could enhance the activity of the SOD level and presented differences at 2, 3, and 5 h (P < 0.05).

3.4. Dynamic Expression Levels of Nrf2, Hsps, and Cleaved-Caspase 3 in the H9C2 Cells

The dynamic expression levels of Nrf2, Hsps, and cleaved-caspase 3 in the H9C2 cells are shown in Figure 5. Heat stress could increase the expression level of Nrf2 in the H9C2 cells, which showed a significant upregulation at 2, 3, and 5 h (P < 0.01). The supplement with 10 μg/mL tea polyphenols could induce the expression of Nrf2 under normal condition (0 h of heat stress) and at 5 h of heat stress (P < 0.01). Heat stress could downregulate the level of CRYAB and showed a significant decrease at 3 and 5 h (P < 0.01). The initial stage of heat stress for Hsp27 had a slight effect on its expression until at 5 h of heat stress which showed a significant decrease (P < 0.01). For Hsp70, heat stress induced extremely significant increases at 2 and 3 h (P < 0.01). The supplement with 10 μg/mL tea polyphenols increased the expression of CRYAB at 3 h of heat stress and could induce the expression of Hsp27 at 0, 2, and 3 h (P < 0.01) and Hsp70 at 3 and 5 h (P < 0.01). Heat stress increased the expression of cleaved-caspase 3, and a supplement with 10 μg/mL tea polyphenols had a very significant relief effect at 3 and 5 h (P < 0.01).

3.5. Pathological Examination of Myocardial Tissue

The pathological changes of the laying hen myocardial tissue are shown in Figure 6. Heat stress damaged the myocardial tissue. Such damage became severe with the prolongation of heat stress. The cardiomyocytes demonstrated vacuolar degeneration (→) when exposed to heat stress at 38°C for 0.5 d. When heat stress continued to 2 d, vacuole degeneration gradually became obvious, and capillary vasodilation (↑) could be seen. Cardiomyocyte necrosis (↓) characterized by nuclear hyperchromatism and karyopyknosis was observed when the heat stress continued to 5 d. The supplement with 0.2 g/L tea polyphenols in drinking water could effectively alleviate the cardiomyocyte damage caused by heat stress, thereby showing no obvious vacuolar degeneration and capillary vasodilation at 2 d and necrosis at 5 d.

3.6. Injury-Related Index Analysis in the Chicken Serum

LDH, CK, CK-MB, and TNF-α, which are indexes related to myocardial injury, were detected in the layer hen serum (Figure 7). Heat stress leads to the increases in LDH, CK, CK-MB, and TNF-α. Such stress showed significant differences at 2 and 5 d for LDH (P < 0.01); at 0.5, 2, and 5 d for CK and CK-MB (P < 0.01); and at 5 d for TNF-α (P < 0.01). The supplement with 0.2 g/L tea polyphenols in drinking water could effectively relieve the upregulation of LDH, CK, CK-MB, and TNF-α caused by heat stress. In comparison with the control group under the same conditions, the tea polyphenol group showed a significant downregulation of CK-MB on 0.5 d (P < 0.05) and extremely significant downregulation of CK and CK-MB on 2 d (P < 0.01). This group also showed a downward trend of CK (P < 0.05) and TNF-α (P < 0.01) at 5 d.

3.7. Oxidative Damage-Related Index Analysis in the Chicken Serum

Heat stress activated the body's antioxidant level and upregulated the levels of T-AOC, GSH-PX, and SOD (Figure 8). T-AOC and GSH-PX showed a significant upregulation at 2 and 5 d (P < 0.01). SOD presented an upward trend at 0.5 d, but it showed a downward trend with the duration of the heat stress and significantly decreased at 5 d (P < 0.01). The heat stress upregulated the level of MDA (Figure 8), and its upward trend was dependent on the time of heat stress. Significant differences were observed at 2 d and 5 d (P < 0.01). The supplement with 0.2 g/L tea polyphenols in drinking water increased the level of T-AOC in the cardiomyocytes under normal condition (P < 0.05) and GSH-PX (P < 0.01) and SOD at 0.5 d heat stress. However, such supplement decreased the level of MDA at 5 d heat stress (P < 0.01).

3.8. Dynamic Expression Levels of Nrf2, Hsps, and Cleaved-Caspase 3 in the Myocardial Tissues

The dynamic expression levels of Nrf2, Hsps, and cleaved-caspase 3 in the myocardial cells fare shown in Figure 9. In the early stage of heat stress, the laying hens activated the body's redox regulation pathway, which was shown by the increase of the Nrf2 level (P < 0.01) at 0.5 d. The expression level of Nrf2 showed a downward trend with the continuous increase in the heat stress time, thereby presenting significant difference at 2 d (P < 0.01). When the heat stress continued to 5 d, the level of Nrf2 basically recovered. The supplement with 0.2 g/L tea polyphenols in drinking water induced the expression level of Nrf2 under the condition of heat stress for 0 d (P < 0.01) and 2 d (P < 0.05). Heat stress increased the levels of CRYAB, Hsp27, and Hsp70 at the early stage of 0.5 d and presented a significant increase in Hsp27 (P < 0.01). Moreover, the heat stress showed an upward trend but no statistical difference for CRYAB and Hsp70. CRYAB and Hsp27 showed an extremely significant downregulation at 2 d (P < 0.01) with the duration of heat stress. The expression of CRYAB, Hsp27, and Hsp70 presented an increase when the heat stress lasted for 5 d compared with 2 d. The supplement with 0.2 g/L tea polyphenols in drinking water induced the expression levels of Hsp27 and Hsp70 under normal condition (heat 0 d, P < 0.01) and increased CRYAB, Hsp27, and Hsp70 under heat stress. With regard to the increases under heat stress, significant differences were observed at 0.5 d (P < 0.01) and 2 d (P < 0.05) for CRYAB, at 0.5 d (P < 0.01) and 5 d (P < 0.05) for Hsp27, and at 0.5 d (P < 0.01) for Hsp70. Heat stress induced the expression of cleaved-caspase 3 and showed an extremely significant increase at 0.5 d (P < 0.01). The supplement with 0.2 g/L tea polyphenols in drinking water could effectively inhibit the upregulation of cleaved-caspase 3 caused by heat stress. The inhibition effect was significant at 0.5 d (P < 0.01).

• 1.Lara L. J., Rostagno M. H. Impact of heat stress on poultry production. Animals. 2013;3(2):356–369. doi: 10.3390/ani3020356. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 2.Quinteiro-Filho W. M., Ribeiro A., Ferraz-de-Paula V., et al. Heat stress impairs performance parameters, induces intestinal injury, and decreases macrophage activity in broiler chickens. Poultry Science. 2010;89(9):1905–1914. doi: 10.3382/ps.2010-00812. [DOI] [PubMed] [Google Scholar]
• 3.Sahin N., Hayirli A., Orhan C., Tuzcu M., Komorowski J. R., Sahin K. Effects of the supplemental chromium form on performance and metabolic profile in laying hens exposed to heat stress. Poultry Science. 2018;97(4):1298–1305. doi: 10.3382/ps/pex435. [DOI] [PubMed] [Google Scholar]
• 4.Yin B., Tang S., Sun J. R., et al. Vitamin C and sodium bicarbonate enhance the antioxidant ability of H9C2 cells and induce HSPs to relieve heat stress. Cell Stress & Chaperones. 2018;23(4):735–748. doi: 10.1007/s12192-018-0885-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 5.Patinen T., Adinolfi S., Cortés C. C., Härkönen J., Jawahar Deen A., Levonen A. L. Regulation of stress signaling pathways by protein lipoxidation. Redox Biology. 2019;23, article 101114 doi: 10.1016/j.redox.2019.101114. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 6.Xu J., Tang S., Yin B., Sun J. R., Bao E. D. Co-enzyme Q10 upregulates Hsp70 and protects chicken primary myocardial cells under in vitro heat stress via PKC/MAPK. Molecular and Cellular Biochemistry. 2018;449(1-2):195–206. doi: 10.1007/s11010-018-3356-2. [DOI] [PubMed] [Google Scholar]
• 7.Elwan H. A., Elnesr S. S., Mohany M., Al-Rejaie S. S. The effects of dietary tomato powder (Solanum lycopersicum L.) supplementation on the haematological, immunological, serum biochemical and antioxidant parameters of growing rabbits. Journal of Animal Physiology and Animal Nutrition. 2019;103(2):534–546. doi: 10.1111/jpn.13054. [DOI] [PubMed] [Google Scholar]
• 8.Abo Ghanima M. M., Alagawany M., Abd El-Hack M. E., et al. Consequences of various housing systems and dietary supplementation of thymol, carvacrol, and euganol on performance, egg quality, blood chemistry, and antioxidant parameters. Poultry Science. 2020;99(9):4384–4397. doi: 10.1016/j.psj.2020.05.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 9.Abd El-Hack M. E., Elnesr S. S., Alagawany M., Gado A., Noreldin A. E., Gabr A. A. Impact of green tea (Camellia sinensis) and epigallocatechin gallate on poultry. World's Poultry Science Journal. 2020;76(1):49–63. doi: 10.1080/00439339.2020.1729672. [DOI] [Google Scholar]
• 10.Roh E., Kim J. E., Kwon J. Y., et al. Molecular mechanisms of green tea polyphenols with protective effects against skin photoaging. Critical Reviews in Food Science and Nutrition. 2017;57(8):1631–1637. doi: 10.1080/10408398.2014.1003365. [DOI] [PubMed] [Google Scholar]
• 11.Hu R., He Y., Arowolo M. A., Wu S., He J. Polyphenols as potential attenuators of heat stress in poultry production. Antioxidants. 2019;8(3, article 8030067) doi: 10.3390/antiox8030067. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 12.Wang J., Jia R., Celi P., et al. Green tea polyphenol epigallocatechin-3-gallate improves the antioxidant capacity of eggs. Food & Function. 2020;11(1):534–543. doi: 10.1039/C9FO02157D. [DOI] [PubMed] [Google Scholar]
• 13.Basiricò L., Morera P., Dipasquale D., et al. (-)-Epigallocatechin-3-gallate and hydroxytyrosol improved antioxidative and anti-inflammatory responses in bovine mammary epithelial cells. Animal. 2019;13(12):2847–2856. doi: 10.1017/S1751731119001356. [DOI] [PubMed] [Google Scholar]
• 14.Lai X., Chen J., Zheng J., Sun C., Xu G. Meta analysis of the effects of adding tea polyphenols in diets on the production performance and egg quality of laying hens. China Poultry. 2019;41(16):58–64. [Google Scholar]
• 15.Srikanth K., Kumar H., Park W., et al. Cardiac and skeletal muscle transcriptome response to heat stress in Kenyan chicken ecotypes adapted to low and high altitudes reveal differences in thermal tolerance and stress response. Frontiers in Genetics. 2019;10, article 00993 doi: 10.3389/fgene.2019.00993. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 16.Kim K. M., Im A. R., Lee S., Chae S. Dual protective effects of flavonoids from Petasites japonicus against UVB-induced apoptosis mediated via HSF-1 activated heat shock proteins and Nrf2-activated heme oxygenase-1 pathways. Biological & Pharmaceutical Bulletin. 2017;40(6):765–773. doi: 10.1248/bpb.b16-00691. [DOI] [PubMed] [Google Scholar]
• 17.Yao X., Bai Q., Yan D., Li G., Lu C., Xu H. Solanesol protects human hepatic L02 cells from ethanol-induced oxidative injury via upregulation of HO-1 and Hsp70. Toxicology in Vitro. 2015;29(3):600–608. doi: 10.1016/j.tiv.2015.01.009. [DOI] [PubMed] [Google Scholar]
• 18.Satoh T., Rezaie T., Seki M., et al. Dual neuroprotective pathways of a pro-electrophilic compound via HSF-1-activated heat-shock proteins and Nrf2-activated phase 2 antioxidant response enzymes. Journal of Neurochemistry. 2011;119(3):569–578. doi: 10.1111/j.1471-4159.2011.07449.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 19.Xu J., Tang S., Yin B., Sun J. R., Song E. B., Bao E. D. Co-enzyme Q10 and acetyl salicylic acid enhance Hsp70 expression in primary chicken myocardial cells to protect the cells during heat stress. Molecular and Cellular Biochemistry. 2017;435(1-2):73–86. doi: 10.1007/s11010-017-3058-1. [DOI] [PubMed] [Google Scholar]
• 20.Tang S., Yin B., Xu J., Bao E. D. Rosemary Reduces Heat Stress by Inducing CRYAB and HSP70 Expression in Broiler Chickens. Oxidative Medicine and Cellular Longevity. 2018;2018:10. doi: 10.1155/2018/7014126.7014126 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 21.Xu J., Tang S., Song E. B., Yin B., Bao E. D. Inhibition of heat shock protein 70 intensifies heat-stressed damage and apoptosis of chicken primary myocardial cells in vitro. Molecular Medicine Reports. 2017;15(5):2881–2889. doi: 10.3892/mmr.2017.6337. [DOI] [PubMed] [Google Scholar]
• 22.Yin B., Tang S., Xu J., et al. CRYAB protects cardiomyocytes against heat stress by preventing caspase-mediated apoptosis and reducing F-actin aggregation. Cell Stress & Chaperones. 2019;24(1):59–68. doi: 10.1007/s12192-018-0941-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 23.Zhang M., Zhu L., Zhang Y., et al. Effect of different short-term high ambient temperature on chicken meat quality and ultra-structure. Asian-Australasian Journal of Animal Sciences. 2019;32(5):701–710. doi: 10.5713/ajas.18.0232. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 24.Elnesr S. S., Elwan H. A. M., Xu Q. Q., Xie C., Dong X. Y., Zou X. T. Effects of in ovo injection of sulfur-containing amino acids on heat shock protein 70, corticosterone hormone, antioxidant indices, and lipid profile of newly hatched broiler chicks exposed to heat stress during incubation. Poultry Science. 2019;98(5):2290–2298. doi: 10.3382/ps/pey609. [DOI] [PubMed] [Google Scholar]
• 25.Elwan H. A., Elnesr S. S., Xu Q. Q., Xie C., Dong X. Y., Zou X. T. Effects of in ovo methionine-cysteine injection on embryonic development, antioxidant status, IGF-I and TLR4 gene expression, and jejunum histomorphometry in newly hatched broiler chicks exposed to heat stress during incubation. Animals. 2019;9(1):p. 25. doi: 10.3390/ani9010025. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 26.Ohtsuka K., Suzuki T. Roles of molecular chaperones in the nervous system. Brain Research Bulletin. 2000;53(2):141–146. doi: 10.1016/S0361-9230(00)00325-7. [DOI] [PubMed] [Google Scholar]
• 27.Kumar S., Stokes J., Singh U. P., et al. Targeting Hsp70: a possible therapy for cancer. Cancer Letters. 2016;374(1):156–166. doi: 10.1016/j.canlet.2016.01.056. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 28.Mounier N., Arrigo A. P. Actin cytoskeleton and small heat shock proteins: how do they interact? Cell Stress and Chaperones. 2002;7(2):167–176. doi: 10.1379/1466-1268(2002)007&#x0003c;0167:ACASHS&#x0003e;2.0.CO;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 29.Laskowska E., Matuszewska E., Kuczyska-Winik D. Small heat shock proteins and protein-misfolding diseases. Current Pharmaceutical Biotechnology. 2010;11(2):146–157. doi: 10.2174/138920110790909669. [DOI] [PubMed] [Google Scholar]
• 30.Luo J., Song J., Liu L., Xue B., Tian G., Yang Y. Effect of epigallocatechin gallate on growth performance and serum biochemical metabolites in heat-stressed broilers. Poultry Science. 2017;97:599–606. doi: 10.3382/ps/pex353. [DOI] [PubMed] [Google Scholar]
• 31.Elwan H., Xie C., Miao L. P., et al. Methionine alleviates aflatoxinb1-induced broiler chicks embryotoxicity through inhibition of caspase-dependent apoptosis and enhancement of cellular antioxidant status. Poultry Science. 2021;100(8, article 101103) doi: 10.1016/j.psj.2021.101103. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 32.Pinner K. D., Wales C. T., Gristock R. A., Vo H. T., So N., Jacobs A. T. Flavokawains A and B from kava (Piper methysticum) activate heat shock and antioxidant responses and protect against hydrogen peroxide-induced cell death in HepG2 hepatocytes. Pharmaceutical Biology. 2016;54(9):1503–1512. doi: 10.3109/13880209.2015.1107104. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 33.Sahin K., Orhan C., Tuzcu Z., Tuzcu M., Sahin N. Curcumin ameloriates heat stress via inhibition of oxidative stress and modulation of Nrf2/HO-1 pathway in quail. Food and Chemical Toxicology. 2012;50(11):4035–4041. doi: 10.1016/j.fct.2012.08.029. [DOI] [PubMed] [Google Scholar]
• 34.Nawab A., Li G., Liu W., et al. Effect of dietary curcumin on the antioxidant status of laying hens under high- temperature condition. Journal of Thermal Biology. 2019;86, article 102449 doi: 10.1016/j.jtherbio.2019.102449. [DOI] [PubMed] [Google Scholar]
• 35.Paul S., Ghosh S., Mandal S., Sau S., Pal M. NRF2 transcriptionally activates the heat shock factor 1 promoter under oxidative stress and affects survival and migration potential of MCF7 cells. The Journal of Biological Chemistry. 2018;293(50):19303–19316. doi: 10.1074/jbc.RA118.003376. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 36.Dayalan N. S., Kostov R. V., Dinkova-Kostova A. T. Transcription factors Hsf1 and Nrf2 engage in crosstalk for cytoprotection. Trends in Pharmacological Sciences. 2015;36(1):6–14. doi: 10.1016/j.tips.2014.10.011. [DOI] [PubMed] [Google Scholar]

Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.