Heat Stress-induced Apoptosis Research Articles

Glutamate rescues heat stress-induced apoptosis of Sertoli cells by enhancing the activity of antioxidant enzymes and activating the Trx1-Akt pathway in vitro

Heat stress reduces the number of Sertoli cells, which is closely related to an imbalanced redox status. Glutamate functions to maintain the equilibrium of redox homeostasis. However, the role of glutamate in heat treated Sertoli cells remains unclear. Herein, Sertoli cells from 3-week-old piglets were treated at 44 °C for 30 min (heat stress). Glutamate levels increased significantly following heat stress treatment, followed by a gradual decrease during recovery, while glutathione (GSH) showed a gradual increase. The addition of exogenous glutamate (700 μM) to Sertoli cells before heat stress significantly reduced the heat stress-induced apoptosis rate, mediated by enhanced levels of antioxidant substances (superoxide dismutase (SOD), total antioxidant capacity (TAC), and GSH) and reduced levels of oxidative substances (reactive oxygen species (ROS) and malondialdehyde (MDA)). Glutamate addition to Sertoli cells before heat stress upregulated the levels of glutamate-cysteine ligase, modifier subunit (Gclm), glutathione synthetase (Gss), thioredoxin (Trx1) and B-cell leukemia/lymphoma 2 (Bcl-2), and the ratio of phosphorylated Akt (protein kinase B)/total Akt. However, it decreased the levels of Bcl2-associated X protein (Bax) and cleaved-caspase 3. Addition of the inhibitor of glutaminase (Gls1), Bptes (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, 30 μM)to Sertoli cells before heat stress reversed these effects. These results inferred that glutamate rescued heat stress-induced apoptosis in Sertoli cells by enhancing activity of antioxidant enzymes and activating the Trx1-Akt pathway. Thus, glutamate supplementation might represent a novel strategy to alleviate the negative effect of heat stress.

Hypoxia-inducible factor 1α inhibits heat stress-induced pig intestinal epithelial cell apoptosis through eif2α/ATF4/CHOP signaling

Unstoppable global warming and increased frequency of extreme heat leads to human and animals easier to suffer from heat stress (HS), with gastrointestinal abnormalities as one of the initial clinical symptoms. HS induces intestinal mucosal damage owing to intestinal hypoxia and hyperthermia. Hypoxia-inducible factor 1α (HIF-1α) activates numerous genes to mediate cell hypoxic responses; however, its role in HS-treated intestinal mucosa is unknown. This work aimed to explore HIF-1α function and regulatory mechanisms in HS-treated pig intestines. We assigned 10 pigs to control and moderate HS groups. Physical signs, stress, and antioxidant levels were detected, and the intestines were harvested after 72 h of HS treatment to study histological changes and HIF-1α, heat shock protein 90 (HSP90), and prolyl-4-hydroxylase 2 (PHD-2) expression. In addition, porcine intestinal columnar epithelial cells (IPEC-J2) underwent HS treatment (42 °C, 5 % O2) to further explore the functions and regulatory mechanism of HIF-1α. The results of histological examination revealed HS caused intestinal villi damage and increased apoptotic epithelial cell; the expression of HIF-1α and HSP90 increased while PHD-2 showed and opposite trend. Transcriptome sequencing analysis revealed that HS activated HIF-1 signaling. To further explore the role of HIF-1α on HS induced IPEC-J2 apoptosis, the HIF-1α was interfered and overexpression respectively, and the result confirmed that HIF-1α could inhibited cell apoptosis under HS. Furthermore, HS-induced apoptosis depends on eukaryotic initiation factor 2 alpha (eif2α)/activating transcription factor 4 (ATF4)/CCAAT-enhancer-binding protein homologous protein (CHOP) pathway, and HIF-1α can inhibit this pathway to alleviate IPEC-J2 cell apoptosis. In conclusion, this study suggests that HS can promote intestinal epithelial cell apoptosis and cause pig intestinal mucosal barrier damage; the HIF-1α can alleviate cell apoptosis by inhibiting eif2α/ATF4/CHOP signaling. These results indicate that HIF-1α plays a protective role in HS, and offers a potential target for HS prevention and mitigation.

Heat stress suppresses MnSOD expression via p53-Sp1 interaction and induces oxidative stress damage in endothelial cells: Protective effects of MitoQ10 and Pifithrin-α

AimTo investigate the mechanism of p53-mediated suppression of heat stress-induced oxidative stress damage by manganese superoxide dismutase (MnSOD) in endothelial cells (ECs). MethodsPrimary ECs isolated from mouse aortas were used to examine the effects of heat stress on vascular ECs viability and apoptosis. We measured MnSOD expression, reactive oxygen species (ROS) production, p53 expression, viability, and apoptosis of heat stress-induced ECs. We also tested the protective effects of MitoQ10, a mitochondrial-targeted antioxidant, and Pifithrin-α, a p53 inhibitor, in ECs from a mouse model of heat stroke. ResultsHeat stress increased cellular apoptosis, ROS production, and p53 expression, while reducing cellular viability and MnSOD expression in ECs. We also showed that the suppression of MnSOD expression by heat stress in ECs was mediated by interactions between p53 and Sp1. Furthermore, MitoQ10 and Pifithrin-α alleviated heat stress-induced oxidative stress and apoptosis in ECs. ConclusionOur results revealed that p53-mediated MnSOD downregulation is a key mechanism for heat stress-induced oxidative stress damage in ECs and indicated that MitoQ10 and Pifithrin-α could be potential therapeutic agents for heat stroke.

Viola yedoensis Makino alleviates heat stress-induced inflammation, oxidative stress, and cell apoptosis in the spleen and thymus of broilers

Ethnopharmacological relevanceViola yedoensis Makino (VYM) is a traditional Chinese herbal medicine widely distributed in China. It has many pharmacological effects such as anti-inflammatory, immune regulation and anti-oxidation. However, the protective effect of VYM on the spleen and thymus of broilers induced by heat stress has rarely been reported. Aim of the studyWe established a heat stress model of broilers to explore the protective effect of VYM on spleen and thymus of broilers. Materials and methodsIn this experiment, a heat stress model was made by adjusting the feeding temperature of broilers. The protective effect of VYM on the spleen and thymus of heat-stressed broilers were evaluated by detecting immune organ coefficient, histological observation, Enzyme-Linked Immunosorbent Assay, production of antioxidant enzymes and peroxides, TUNEL Staining, Quantitative Real-time PCR. ResultsIn this study, 60 healthy male AA broilers were divided into 6 groups: Control, 4.5% VYM, HS, HS + 0.5% VYM, HS + 1.5% VYM, HS + 4.5% VYM. After 42 days of feeding, serum, spleen and thymus were collected for detection and analysis. The study revealed that heat stress can lead to pathological damage in the spleen and thymus of broilers, reduce the content of immunoglobulin and newcastle disease (ND), infectious bursal disease (IBD) antibody levels, increase the expression of inflammatory factors IL-1β, INF-γ, heat shock 70 kDa protein (HSP70), heat shock 90 kDa protein (HSP90). Heat stress inhibits the activity of antioxidant enzymes CAT and SOD, promotes the production of MDA, and then lead to oxidative damage of the spleen and thymus. In addition, apoptotic cells and the ratio of Bax/Bcl-2 was increased. However, the addition of VYM to the feed can alleviate the adverse effects of heat stress on the spleen and thymus of broilers. ConclusionsThis study showed that the addition of VYM to the diet could inhibit oxidative stress and apoptosis, and reduce the inflammatory damage of heat stress on the spleen and thymus of broilers. This study provides a basis for further exploring the regulatory role of VYM in heat stress-induced immune imbalance in broilers. In addition, this study also provides a theoretical basis for the development of VYM as a feed additive with immunomodulatory effects.

Betaine ameliorates heat stress-induced apoptosis by affecting oxidative and endoplasmic reticulum stress in mouse Leydig cells.

In order to explore the potential protective role of betaine in heat stress (HS)-elicited apoptosis in mouse Leydig cells (mLCs). Betaine at 16mm exerted a greater inhibitory effect on HS-induced viability attenuation of cells, which also significantly suppressed the heat shock protein 70 level in HS-treated cells. Furthermore, betaine ameliorated certain negative effects, including increased cell apoptotic ratio, enhancement of apoptosis-related modulator caspase-3 activity, reduced activity levels of such antioxidant enzymes as SOD, CAT, GSH-Px, and MDA upregulation, and inhibited the protein levels of critical endoplasmic reticulum (ER) stress indices like CHOP and GRP78 in mLCs exposed to HS. Besides, treatment of cells with betaine significantly restored diminished testosterone production in response to HS. Correspondingly, betaine effectively rescued the reduced serum testosterone concentration in vivo. In summary, betaine ameliorated HS-induced apoptosis by affecting oxidative and ER stress, thereby providing benefits for the treatment of hyperthermia-related impairment in mLCs.

Chlorogenic acid ameliorates the heat stress-induced impairment of porcine Sertoli cells by suppressing oxidative stress and apoptosis

Sertoli cells are an important type of somatic cell in the testis that are in direct contact with spermatogonia in vivo and play an important role in the process of spermatogenesis. Chlorogenic acid (CGA) plays a pivotal role in the regulation of the mammalian cell heat stress response. For example, CGA treatment protects porcine oocytes from heat stress-induced apoptosis and prevents reduced embryo quality. However, the role of CGA treatment in protecting porcine testicular Sertoli cells against heat-induced damage has rarely been studied. This study aimed to identify the protective effects of CGA on oxidative stress and apoptosis in Sertoli cells under heat stress. The present results demonstrated that the addition of CGA significantly inhibited the accumulation of reactive oxygen species (ROS) and apoptosis in Sertoli cells induced by heat stress and decreased the expression of CASP3 protein and the BAX/BCL-2 protein ratio. CGA pretreatment also prevented the heat stress-induced reductions in the mitochondrial membrane potential, PCNA protein expression, and SOD and CAT activities. Moreover, CGA treatment reversed S phase cell cycle arrest and increased the HSP70 protein expression levels. Overall, these results suggest that oxidative damage participates in the inhibition of the proliferation of Sertoli cells and the increase in their apoptosis induced by heat stress, and the protective effects of CGA treatment on Sertoli cells under heat stress provide a theoretical basis for preventing heat stress injury in animals.

Alpha-lipoic acid attenuates heat stress-induced apoptosis via upregulating the heat shock response in porcine parthenotes

Heat stress (HS) is a long-standing hurdle that animals face in the living environment. Alpha-lipoic acid (ALA) is a strong antioxidant synthesized by plants and animals. The present study evaluated the mechanism of ALA action in HS-induced early porcine parthenotes development. Parthenogenetically activated porcine oocytes were divided into three groups: control, high temperature (HT) (42 °C for 10 h), and HT + ALA (with 10 µM ALA). The results show that HT treatment significantly reduced the blastocyst formation rate compared to the control. The addition of ALA partially restored the development and improved the quality of blastocysts. Moreover, supplementation with ALA not only induced lower levels of reactive oxygen species and higher glutathione levels but also markedly reduced the expression of glucose regulatory protein 78. The protein levels of heat shock factor 1 and heat shock protein 40 were higher in the HT + ALA group, which suggests activation of the heat shock response. The addition of ALA reduced the expression of caspase 3 and increased the expression of B-cell lymphoma-extra-large protein. Collectively, this study revealed that ALA supplementation ameliorated HS-induced apoptosis by suppressing oxidative and endoplasmic reticulum stresses via activating the heat shock response, which improved the quality of HS-exposed porcine parthenotes.

Nano-selenium Antagonizes Heat Stress-Induced Apoptosis of Rainbow Trout (Oncorhynchus mykiss) Hepatocytes by Activating the PI3K/AKT Pathway.

The cold-water fish rainbow trout (Oncorhynchus mykiss) shows poor resistance to heat, which is the main factor restricting their survival and yield. With the advancement of nanotechnology, nano-selenium (nano-Se) has emerged as a key nano-trace element, showing unique advantages, including high biological activity and low toxicity, for studying the response of animals to adverse environmental conditions. However, little is still known regarding the potential protective mechanisms of nano-Se against heat stress-induced cellular damage. Herein, we aimed to investigate the mechanism underlying the antagonistic effects of nano-Se on heat stress. Four groups were assessed: CG18 (0μg/mL nano-Se, 18°C), Se18 (5.0μg/mL nano-Se, 18°C), CG24 (0μg/mL nano-Se, incubated at 18°C for 24h and then transferred to 24°C culture), and Se24 (5.0μg/mL nano-Se, incubated at 18°C for 24h and then transferred to 24°C culture). We found that after heat treatment (CG24 group), T-AOC, GPx, and CAT activities in rainbow trout hepatocytes showed a decrease of 36%, 33%, and 19%, respectively, while ROS and MDA levels showed an increase of 67% and 93%, respectively (P < 0.05). Meanwhile, the mRNA levels of the apoptosis-related genes caspase3, caspase9, Cyt-c, Bax, and Bax/Bcl-2 in the CG24 group were 41%, 47%, 285%, 65%, and 151% higher than those in the CG18 group, respectively, while those of PI3K and AKT were 31% and 17% lower, respectively (P < 0.05). Besides, flow cytometry analysis showed an increase in the level of apoptotic cells after heat exposure. More importantly, we observed that nano-Se cotreatment (Se24 group) remarkably attenuated heat stress-induced effects (P < 0.05). We conclude that heat stress induces oxidative stress and apoptosis in rainbow trout hepatocytes. Nano-Se ameliorates heat stress-induced apoptosis by activating the PI3K/AKT pathway. Our results provide a new perspective to improve our understanding of the ability of nano-Se to confer heat stress resistance.

MiR-27a-3p relieves heat stress-induced mitochondrial damage and aberrant milk protein synthesis through MEK/ERK pathway in BMECs.

With global warming, heat stress has become a primary factor that compromises the health and milk quality of dairy cows. Here, we investigated the function and underlying regulatory mechanism of miR-27a-3p in bovine mammary epithelial cells (BMECs) under heat-stress conditions. The current study showed that miR-27a-3p could prevent heat stress-induced BMEC oxidative stress and mitochondrial damage by regulating the balance between mitochondrial fission and fusion processes. Importantly, we found that miR-27a-3p could increase cell proliferation under heat stress conditions by regulating the MEK/ERK pathway and cyclin D1/E1. Interestingly, miR-27a-3p is also involved in the regulation of milk protein synthesis-related protein expression, such as CSN2 and ELF5. Inhibition of the MEK/ERK signaling pathway by AZD6244 blocked the regulatory function of miR-27a-3p in cell proliferation and milk protein synthesis in BMECs under heat stress conditions. Our findings demonstrated that miR-27a-3p protects BMECs from heat stress-induced oxidative stress and mitochondrial damage through the MEK/ERK pathway, thereby promoting BMECs proliferation and lactation in dairy cows. The potential regulatory mechanism of miR-27a-3p in attenuating heat stress-induced apoptosis and lactation defect in BMECs.

Arbutin Inhibited Heat Stress-Induced Apoptosis and Promoted Proliferation and Migration of Heat-Injured Dermal Fibroblasts and Keratinocytes by Activating PI3K/AKT Signaling Pathway.

Objective Studies have shown that arbutin has antioxidant and anti-inflammatory activities, which makes it suitable for treating skin wounds. We designed this study to investigate the effect of arbutin on heat-induced apoptosis, proliferation, and migration of dermal fibroblasts and keratinocytes and to explore the molecular mechanism. Methods In vitro, HaCAT and dermal fibroblast (DFL) cells were cultured and used to establish a heat stress-injured skin cell model. We investigated the effects of arbutin on apoptosis, proliferation, and migration of HaCAT and DFL cells after heat stress injury. We then used immunoblotting to detect the expression of p-PI3K, PI3K, p-AKT, and AKT proteins for studying the underlying mechanisms and used a PI3K/AKT inhibitor (LY294002) to verify the efficacy of arbutin in HaCAT and DFL cells with heat stress injury. Results Arbutin strongly inhibited heat stress-induced apoptosis, proliferation inhibition, and migration inhibition of HaCAT and DFL cells in vitro. Our results also showed that arbutin strongly decreased the ratio of Bax/Bcl2 protein expression and PCNA protein expression in HaCAT and DFL cells after treatment with heat stress. Furthermore, we also found that arbutin significantly increased the ratio of p-PI3K/PI3K and p-AKT/AKT protein expression, and LY294002 markedly reversed the effect of arbutin on heat stress-induced apoptosis, proliferation inhibition, and migration inhibition of HaCAT and DFL cells. Conclusion Our finding indicated that arbutin inhibited heat stress-induced apoptosis and promoted proliferation and migration of heat-injured dermal fibroblasts and epidermal cells by activating the PI3K/AKT signaling pathway, suggesting that arbutin may provide an alternative therapeutic approach for the treatment of skin injury.

Melatonin alleviates the heat stress-induced impairment of Sertoli cells by reprogramming glucose metabolism.

Sertoli cells (SCs) provide structural and nutritional support for developing germ cells. Normal glucose metabolism of SCs is necessary for spermatogenesis. Melatonin could alleviate the effects of heat stress on spermatogenesis. However, the influences of heat stress on glucose metabolism in SCs remain unclear, and the potential protective mechanisms of melatonin on SCs need more exploration. In this study, boar SCs were treated at 43°C for 30 min, and different concentrations of melatonin were added to protect SCs from heat stress-induced impairment. These results showed that heat stress-induced oxidative stress caused cell apoptosis, inhibited the pentose phosphate pathway, and decreased the ATP content. Furthermore, heat stress increased the expressions of glucose intake-and glycolytic-related enzymes, which enhanced the glycolysis activity to compensate for the energy deficit. Melatonin relieved heat stress-induced oxidative stress and apoptosis by activating the Kelch-like ECH-associated protein 1 (KEAP1)/NF-E2-related factor 2signaling pathway to increase the capacity of antioxidants. In addition, melatonin enhanced heat-shock protein 90 (HSP90) expression through melatonin receptor 1B (MTNR1B), thereby stabilizing hypoxia-inducible factor-1α (HIF-1α). Activation of the HIF-1α signaling pathway enhanced glycolysis, promoted the pentose phosphate pathway, and increased cell viability. Our results suggest that melatonin reprograms glucose metabolism in SCs through the MTNR1B-HSP90-HIF-1α axis and provides a theoretical basis for preventing heat stress injury.

Overexpression of heat shock protein 70 induces apoptosis of intestinal epithelial cells in heat-stressed pigs: A proteomics approach

Heat stress (HS)-induced intestinal epithelial cell apoptosis may play a pivotal role in intestinal barrier dysfunction in animals. However, the underlying molecular mechanism by which HS induces apoptosis in intestinal epithelial cells is still poorly understood. Herein, a eukaryotic expression vector for an HSP70 gene was constructed and transfected into intestinal porcine epithelial cells (IPEC-J2). Afterward, functional proteomics approaches followed by liquid-chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify interacting proteins. Analysis of HSP70 transfected IPEC-J2 cells revealed 246 differentially expressed proteins (DEPs), and functional annotation indicated that most DEPs were primarily related to ECM-receptor interaction, focal adhesion, and apoptosis. Furtherly, the apoptosis rate and expression levels of apoptosis-related proteins in HSP70 transfected IPEC-J2 cells were detected, we found that the expression of caspase-3, PARP, and Bax were increased, but Bcl-2 were decreased in transfected cells. Lastly, an in vitro and in vivo heat stress model were established to explore the role of HSP70 in intestinal epithelia cell apoptosis. The results of in vitrol study showed that HS-induced cellular apoptosis and increases of caspase-3, PARP, and Bax, but decreased of Bcl-2 in IPEC-J2 cells. In vivo study, the cell apoptosis were induced significantly in the duodenum, cecum, and colon of heat stressed pigs, and upregulation of HSP70 was also detected in colon tissues. Therefore, it has been shown that HSP70 plays a crucial role in heat stress-induced apoptosis and may provide new insights into the molecular mechanisms of epithelial cell apoptosis induced by heat stress in pigs.

Nicotinamide mononucleotide alleviates heat stress-induced oxidative stress and apoptosis in BMECs through reducing mitochondrial damage and endoplasmic reticulum stress

Heat stress is directly correlated to mammary gland dysfunction in dairy cows, especially in summer. Abnormally high environmental temperature induces oxidative stress and apoptosis in bovine mammary epithelial cells (BMECs). Nicotinamide mononucleotide (NMN) has beneficial effects in maintaining the cellular physiological functions. In this study, we evaluate the protective effect of NMN on heat stress-induced apoptosis of BMECs and explore the potential underlying mechanisms. Our results showed that heat stress considerably decreased cell viability in BMECs, whereas pretreatment of BMECs with NMN (150 μM) for 24 h significantly alleviated the negative effects of heat stress on cells. NMN protected BMECs from heat stress-induced oxidative stress by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing the activity of antioxidant enzymes. It also inhibited apoptosis by reducing the ratio of Bax/Bcl2 and blocking proteolytic the cleavage of Caspase-3 in heat stressed-BMECs. Importantly, NMN treatment could reduce mitochondrial damage through mediating the expression of mitochondrial fission and fusion-related genes, including Dynamin related protein 1 (Drp1), Mitochondrial fission 1 protein (Fis1), and Mitofusin1, 2 (MFN1, 2); and suppress endoplasmic reticulum stress through unfolded protein response regulator Glucose regulated protein 78 (GRP78), and downstream elements Recombinant activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). Above all, our results demonstrate that NMN supplemention attenuates heat stress-induced oxidative stress and apoptosis in BMECs by maintaining mitochondrial fission and fusion, and regulating endoplasmic reticulum stress, which provides the convincing evidence that NMN has valuable potential in alleviating mammary gland injury of dairy cows caused by environmental heat stress.

The role of ALOX15B in heat stress-induced apoptosis of porcine sertoli cells

Heat stress reduces the number of Sertoli cells and impairs spermatogenesis. Mounting evidence indicates that lipid homeostasis is fundamental to cell survival. However, little is known about the role of lipid peroxides in the heat stress-induced apoptosis of Sertoli cells. In the present study, we used metabolomics to explore the changes of lipid peroxides in porcine Sertoli cells under heat stress using liquid chromatograph-mass spectrometry. These results showed a notable increase in the content of 8-hydroxyeicosatetraenoic acid (8-HETE), and 15-hydroxyeicosatetraenoic acid (15-HETE). Furthermore, we found that among arachidonate lipoxygenases, heat stress significantly increased the expression of arachidonate 15-lipoxygenase type B (ALOX15B). Moreover, baicalein, a specific inhibitor of ALOX15B, reduced the content of 8-HETE and 15-HETE, and decreased the apoptosis rate in heat stress-treated porcine Sertoli cells. In addition, baicalein and small interfering RNAs targeting ALOX15B increased the content of 8-HETE and 15-HETE, and activated the p38-p53 pathway, causing apoptosis in heat stress treated porcine Sertoli cells. Interestingly, a p38 inhibitor decreased the expression of ALOX15B, reduced the content of 8-HETE and 15-HETE, and decreased the expression of p53 and the apoptosis rate in heat stress treated porcine Sertoli cells. A p53 inhibitor had similar effect on Sertoli cells. These results indicated that heat stress enhanced the expression of ALOX15B, increased the content of 8-HETE and 15-HETE, and activated the p38-p53 pathway to cause apoptosis. ALOX15B and lipid peroxides obtained feedback from the p38-p53 pathway. Our findings will help to reveal the mechanism of lipid metabolism in Sertoli cells, and could provide a new targeted substrate for anti-heat stress strategies.

Protective Effect of Manganese on Apoptosis and Mitochondrial Function of Heat-Stressed Primary Chick Embryonic Myocardial Cells.

Heat stress, as a kind of oxidative stress, induces cell apoptosis. Apoptosis is a form of programmed cell death, and mitochondria play an important role in apoptosis. Manganese (Mn) has an antioxidant capacity by enhancing the activity of manganese superoxide dismutase (MnSOD). To investigate the potential effect of Mn on heat stress-induced apoptosis and mitochondrial function, we examined crucial related factors in the context of heat stress using primary chick embryonic myocardial cells pretreated with Mn for 24h. The results showed that Mn restored the heat stress-induced decrease in cell viability and reduced the activities of caspase-3 (P < 0.05). The repression of the Δψm and intracellular ATP content caused by heat stress was reversed dramatically in the Mn pretreatment group (P < 0.05). Additionally, Mn inhibited heat stress-induced mitochondrial fission, as shown by decreased mitochondrial fission-related protein dynamin-related protein 1 (Drp1) expression and increased mitochondrial fusion-related protein optic atrophy 1 (Opa1) and mitofusin 1 (Mfn1) (P < 0.05) in primary chick embryonic myocardial cells. It was concluded that Mn attenuates the mitochondrial-mediated apoptosis pathway and sustains mitochondrial structure and function under heat stress in primary chick embryonic myocardial cells.

Cold-inducible RNA-binding protein regulates cyclin B1 against spermatogenesis arrest caused by heat stress.

Spermatogenesis arrest and spermatogenic cell apoptosis occur in the testes of heat-stressed mice. Although heat stress-induced spermatogenic cell apoptosis is due to the decreased expression of cold-inducible RNA-binding protein (CIRBP), it remains unclear whether spermatogenesis arrest is also affected by CIRBP. Additionally, the specific mechanism by which CIRBP regulates spermatogenic cell apoptosis or inhibits spermatogenesis remains to be elucidated. To investigate the mechanism by which CIRBP contributes to heat stress-induced testicular spermatogenesis arrest. Target mRNAs downstream of CIRBP in testicular tissue of BALB/c mice, exposed or not to heat stress, were sequenced. Sequencing data were subjected to bioinformatics analysis to identify key mRNAs and pathways associated with heat stress-induced spermatogenic damage. The link between CIRBP and its target mRNA Ccnb1 (cyclin B1) was verified by western blotting, flow cytometry, and RNA pulldown assays, and the ability of CIRBP to inhibit germ cell cycle arrest by regulating cyclin B1 expression was investigated in a mouse spermatocyte cell line (GC-2spd). Changes in mRNA expression downstream of CIRBP were mainly associated with the cell cycle and RNA binding, transport and splicing. Cyclin B1 was found to regulate the G2/M transition during the first meiotic division of spermatogenic cells. Further, CIRBP was shown to bind directly to the 3'-untranslated region of Ccnb1 mRNA and was associated with cyclin B1-induced inhibition of spermatogenesis arrest. In conclusion, our results provide strong evidence that CIRBP may exert its key function in heat stress-induced testicular spermatogenic cell injury partly by regulating the expression of Ccnb1, the product of which inhibits spermatogenesis arrest.

Astaxanthin Protects Against Heat-induced Mitochondrial Alterations in Mouse Hypothalamus

The hypothalamus plays an essential role in regulating whole-body energy and temperature homeostasis when adapting to environmental changes. We previously reported that heat exposure causes mitochondrial dysfunction and apoptosis in mouse skeletal muscle, and pretreatment with astaxanthin (AST), an antioxidant, prevents this effect. How the hypothalamus responds to heat stress remains largely unexplored. In this study, we investigated the effects of heat exposure on hypothalamic mitochondria in mice with and without AST pretreatment. During heat exposure, both vehicle and AST-treated mice had a hyperthermic response though no significant differences in peak core body temperature were noted between the two groups. Heat exposure induced mitochondrial fission in the hypothalamus, as manifested by increased mitochondrial fragmentation and expression of both total and phosphorylated dynamin-related protein 1. In addition, transmission electron microscopy revealed damaged and degraded mitochondria in the hypothalamus of heat-exposed mice. Heat induced apoptosis and mitophagy were further confirmed by increased formation of reactive oxygen species, activation of caspase 3/7 and expression of LC3 proteins. Moreover, heat exposure increased the expression of PINK1 and Parkin in mouse hypothalamus. In contrast, pretreatment with AST reduced these effects. These results demonstrate that heat stress-induced hypothalamic apoptosis is associated with altered mitochondrial dynamics favoring fission and mitophagy. AST protects the hypothalamus against heat-induced injury by preserving redox homeostasis and mitochondrial integrity.

Manganese Mitigates Heat Stress–Induced Apoptosis by Alleviating Endoplasmic Reticulum Stress and Activating the NRF2/SOD2 Pathway in Primary Chick Embryonic Myocardial Cells

Heat stress leads to oxidative stress and induces apoptosis in various cells. Endoplasmic reticulum (ER) stress is an important apoptosis pathway. Manganese (Mn) has been shown to enhance the activity of manganese superoxide dismutase (MnSOD). To explore the potential effect of Mn on ER stress and apoptosis induced by heat stress, we examined crucial factors associated with heat stress, ER stress, and apoptosis in cultured primary chick embryonic myocardial cells that had been pretreated with 20μM Mn for 24h and then subjected to 4h of heat stress. The results showed that Mn decreased (P < 0.05) heat stress-induced reactive oxygen species (ROS) production and exerted antiapoptotic effects by increasing MnSOD enzymatic activity. The heat stress-induced accumulation of intracellular calcium was dramatically reduced (P < 0.05). Mn treatment significantly decreased (P < 0.05) the expression levels of the apoptosis-related gene Bax and ER stress markers glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) in primary chick embryonic myocardial cells. Additionally, Mn reduced oxidative stress by activating the nuclear factor E2-related factor 2 (NRF2)/SOD2 signaling pathway. Taken together, our findings indicate that Mn attenuates heat stress-induced apoptosis by inhibiting ROS generation, intracellular calcium accumulation, and the ER stress pathway and activating the NRF2/SOD2 signaling pathway to protect myocardial cells from oxidative stress during chick embryonic development.

Heat stress induces neuronal apoptosis by up-regulating endoplasmic reticulum stress pathway

To explore the role of endoplasmic reticulum stress in heat stress-induced apoptosis of human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were incubated at 43 ℃ for 2 h followed by further culture at 37 ℃ for 0, 3 h, or 6 h. With the cells cultured at 37 ℃ as the control, the cells exposed to heat stress were examined for morphological changes under optical microscope and changes in cell viability using CCK-8 assay. Flow cytometry was performed for detecting apoptosis of the cells following heat stress, and intracellular Ca2+ level in the cells was determined using flow cytometry and immunofluorescence confocal microscopy. The mRNA expression levels of caspase-12, BIP and XBP-1 in the cells were detected using qRT-PCR, and the protein expressions of caspase-12, BIP, P-JNK, JNK and XBP-1 were examined using Western blotting. The effect of pretreatment with 4-PBA on cell apoptosis following heat stress was analyzed with Western blotting. SH-SY5Y cells showed obvious cell shrinkage immediately after the exposure to heat stress, followed then by gradual cell stretching over time. The cell viability decreased significantly after heat stress (P=0.001), and the intracellular Ca2+ level increased significantly at 0 h and gradually recovered the normal level at 3 and 6 h. Heat stress induced significant increase in the protein expression of cleaved caspase-3 and time-dependent increase of caspase-12 (P=0.002) and BIP (P=0.008) expression at both the protein and mRNA levels. The expression of P-JNK/JNK protein increased significantly at 0 h (P=0.003) followed by gradual decrease; the expression levels of XBP-1 protein and mRNA gradually decreased after heat stress (P=0.005, P=0.002). Pretreatment with 4-PBA significantly reduced the expression level of cleaved caspase-3 in SH-SY5Y cells following heat stress. Heat stress induces apoptosis of SH-SY5Y cells by triggering endoplasmic reticulum stress and the imbalance of intracellular calcium ion homeostasis.

Choline attenuates heat stress-induced oxidative injury and apoptosis in bovine mammary epithelial cells by modulating PERK/Nrf-2 signaling pathway

Heat stress-induced decline in milk production and mammary glands dysfunction are economically important challenges facing the dairy industry, especially in summer. Choline is an organic water-soluble compound that can regulate a series of vital biological process, including cellular structural integrity and oxidative stress. However, it is unclear whether choline plays an anti-apoptosis and antioxidant effect in heat stress-induced mammary epithelial cells. This study aimed to determine the antioxidant effect of choline on heat stress-induced apoptosis and oxidative stress and its underlying molecular mechanism in bovine mammary epithelial cells (MAC-T cells). The MAC-T cells were divided into four treatment groups: control (37℃), choline (37℃), heat stress (HS, 42℃), and HS + choline. The results showed that heat stress up-regulated the HSP70 and HSP90 expression both in mRNA and protein, enhanced ROS accumulation, increased malondialdehyde (MDA) content, reduced the superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, significantly increased the expression of caspase-3 and upregulated the ratio of Bax/Bcl-2 and ultimately lead to oxidative stress and apoptosis in MAC-T cells. However, choline pretreatment reversed the above phenomenon compared with the HS group. The HS + choline group inhibited heat stress-induced phosphorylation of PERK, nuclear translocation of Nrf-2 and the protein expression of GRP78. In addition, the ratio of Bax/Bcl-2 and the expression of caspase-3 were significantly reduced in HS + choline group, thereby reduced the HS-induced oxidative stress and apoptosis in MAC-T cells. In conclusion, choline attenuates heat stress-induced oxidative stress and apoptosis of MAC-T cells by modulating PERK/Nrf-2 pathway.