EAS has been shown to increase the expression of HSP70 mRNA and protein in HeLa cells, neuronal cell NG108-15, and hepatocyte cells 31, 32,33. Moreover, HSP70 overexpression increased the GSH/GSH disulfide ratio while reducing the ROS levels under hypoxia and glucose deprivation conditions 16. Therefore, the current study investigated whether EAS increases HSP70 expression. GSH is an antioxidant enzyme whose synthesis reduces heat shock-induced ROS generation with DNA damage to maintain the redox status in bovine cumulus-granulosa cells.
In the present study, EAS significantly increased the HSP70 mRNA and protein levels in bovine cumulus-granulosa cells at 5 mg/mL during 6 h of incubation. This result is consistent with a previous study by 33, who studied the dose and incubation time of EAS treatment in cultured hepatocyte cells. In addition, HeLa cells also increased HSP70 mRNA and protein expression when treated with a lower dose of EAS (4 mg/mL), but had a longer incubation time (24 h) than in the current study (6 h) 31. The variation in the dose of EAS in cells may be due to differences in cell type and culture conditions. HSP70 expression-enhancing activity by EAS may arise from asparagine, which has been found to elevate HSP70 in human monocyte HL-60 cells 18. On the other hand, resistance to anti-cancer drugs due to overexpression of HSP27 and HSP90 has been reported in several studies, but the expression of HSP27 and HSP90 in bovine cumulus-granulosa cells was not affected by EAS 34. Therefore, our results confirm the unique effect of EAS on HSP70 induction in bovine cumulus-granulosa cells under non-HS conditions.
In vivo and in vitro studies have shown a synergistic effect between HS and heat shock protein-inducing compounds on HSP70 expression 20, 35. In this study, HSP70 induction by EAS occurred in both non-HS and HS conditions, and it was highest under HS conditions. Paeoniflorin, an HSP70-inducing compound derived from peony (Paeonia lactiflora), increased HSP70 expression under HS conditions in HeLa cells 20. In addition, geranylgeranylacetone, a non-toxic HSP70 inducer, induced HSP70 levels in the brains of heatstroke rats 35. These data support that the combination of EAS with HS synergistically affects HSP70 induction in bovine cumulus-granulosa cells. The induction of HSF1, which is one of the main transcription factors of HSP70, by EAS has been reported in neuronal cells NG108-15 33. The significant increase in HSF1 gene expression by EAS in the present study supports the involvement of EAS in the HSP70 inducing pathway in bovine cumulus-granulosa cells. It is known that HSF1 is also available in non-stressed cells and binds to HSP70 as an inert monomer 36. Under cellular stress, HSF1 is activated and releases HSP70 to prevent the formation of misfolded polypeptides 36. In the present study, HSF1 gene expression was induced by EAS supplementation in both non-HS and HS conditions, and it was highest with EAS supplementation under HS conditions. In addition, the activator of HSF1 can also be stimulated by endogenous metabolites, synthetic compounds, and phytochemicals 37. Therefore, HSF1 enhancement by EAS was induced by the phytochemical compounds of asparagus, such as carotenoids, steroidal saponins, and flavonoids 29. These data suggest that EAS and HS have synergistic effects on the induction of HSF1 in bovine cumulus-granulosa cells. HSF2 is a heat shock transcription factor co-expressed with HSF1 that is activated in response to distinct developmental cues or differentiation stimuli 36. The interaction between HSF1 and HSF2 was discovered through their coiled-coil domains adjacent to their DNA-binding domains 37, however, HSF2 was not affected by EAS in the current study. In the present study, supplementation with EAS led to increased HSP70 expression under non-HS conditions. However, HSF1 expression was only induced under HS conditions with a synergistic effect by EAS. In addition, treatment with an HSP70 inhibitor reduced HSF1 gene expression. The inconsistency with results from previous studies may be due to the involvement of several factors. EAS may regulate the expression of HSP70 not only through HSF1 gene expression but also through other regulatory mechanisms. Additionally, research on different species and cell types has led to reports of different regulatory effects of EAS on HSF1 gene expression 33. Hence, it is conceivable that an increase in HSP70 expression by EAS is induced as a result of the asparagine content and enhancement of HSF1, with EAS exerting synergistic effects with HS on HSP70 induction.
Several studies have indicated that HSP70 has apoptosis-suppressive effects and anti-inflammatory activity, indicating that HSP70 exerts a cytoprotective function against various stresses 38. In addition, the overexpression of HSP70 reduces ROS induced by hypoxia and glucose deprivation 16. These results indicate that EAS with unique inducible HSP70 could protect cells against ROS under various stress conditions. Moreover, EAS was found to significantly reduce the amyloid beta peptide-induced production of ROS in differentiated PC12 cells 39. In the present study, EAS treatment significantly inhibited ROS expression in both normal and HS conditions in bovine cumulus-granulosa cells by activating GSH generation and antioxidant enzymes, such as SOD and PRDX. SOD is the first line of defense against ROS, and is comprised of three classes: Cu/Zn SOD (SOD1), Mn SOD/Fe SOD (SOD2), and Ni SOD 40. The reaction of the superoxide anion (O2•−) to form hydrogen peroxide (H2O2) is catalyzed by SOD 40. PRDX has six isoforms, PRDX1, 2, and 6 in the cytosol, PRDX3 in mitochondria, PRDX4 in the endoplasmic reticulum, and PRDX5 in both mitochondria and peroxisomes 41. PRDX contributes to the reduction of H2O2 to H2O 41. In addition, GSH concentration is used by PRDX6, which contributes to the elimination of H2O2 production 42. GSH has been shown to play an important role in antioxidant defense by interacting with ROS 43. Moreover, it is known that ROS activate c-Jun N-terminal kinase protein, which also increases under HS conditions 44. EAS clearly reduced the c-Jun N-terminal kinase protein induced by hydrogen peroxide in fibroblast cells 45. Quercetin, a flavonoid in asparagus, reduced the ROS levels while enhancing the antioxidant activity of bovine embryos under hyperoxic conditions 46. In conclusion, EAS reduced heat shock-related ROS generation in bovine cumulus-granulosa cells, which can be explained by the upregulation of GSH generation together with antioxidant enzymes and the downregulation of c-Jun N-terminal kinase protein.
ROS-induced damage can cause both single- and double-stranded DNA breaks 26. In the present study, EAS reduced γH2AX expression under non-HS conditions, however, the difference was not significant. Moreover, EAS significantly reduced the γH2AX levels under HS conditions. ROS generation from normal cellular metabolism and HS induces DNA damage in cells 47. In a previous study, EAS was shown to reduce cell damage induced by nitric oxide donor sodium nitroprusside or the hypoxia mimic reagent cobalt chloride of NG108-15 cells 32. HSP70 has the ability to repair DNA damage caused by HS 26. In the present study, HSP70 induction by the synergistic effect of EAS and HS contributed to reducing DNA damage in bovine cumulus-granulosa cells supplemented with EAS under HS conditions. Moreover, GSH also contributes to DNA repair activity, and the expression of GSH in the nucleus enhances protection against DNA damage 48. In the present study, GSH generation was increased by EAS in bovine cumulus-granulosa cells under non-stress and HS conditions; however, it was only significantly higher under non-stress conditions. These results indicate that EAS reduced DNA damage under non-HS conditions and was within the acceptable range of non-toxic levels of ROS production due to the enhancement of HSP70 and GSH levels.
GSH is a pivotal intracellular antioxidant that exerts potential cytoprotective ability by protecting cells against oxidants and electrophiles 49. In our study, EAS increased GSH in both non-HS and HS conditions, but only cells supplemented with EAS under non-HS conditions were statistically significantly different when compared with the control group. In agreement with this observation, previous reports have suggested that the overexpression of HSP70 enhances GSH expression 16. However, the GSH levels in the control group were similar to those in the HS group, which were exposed for 6 h at 41°C. In HeLa cells, HS treatment for 1, 2, or 3 h at 42°C resulted in the highest increase in GSH at 1 h and the lowest at 3 h 28. GSH biosynthesis is required by the action of two ATP-dependent enzymes: GCL, which assembles the formation of c-glutamyl-cysteine from glutamate-cysteine, and GS, which is involved in the ligation of c-glutamyl-cysteine to glycine in another ATP-dependent reaction to yield GSH 27. Our results showed that EAS increased the expression of GCL and GS under non-HS conditions. The transcription factor Nrf2, which has the potential to induce GCL and GS, is activated by ROS production 50. The transcription factor Nrf2 binds to Keap1 under normal conditions, known as the Nrf2/Keap1 signaling pathway, and is translocated to the nucleus in the presence of HS 51. In the nucleus, Nrf2 binds to antioxidant response elements (ARE) via the DNA-binding domain of small Mafs, thereby activating the transcription and translation of GCL and GS 51. Moreover, HSF1 may induce Nrf2 by activating p62, which can separate Nrf2 from Keap1 52. In this study, the GSH levels were not decreased by HS because of the compensatory effect of HSF1 with Nrf2, which can influence the GSH levels after 6 h of HS treatment. Therefore, there is a need to study the effect of HS treatment on GSH synthesis and the induction of genes. A previous study showed that reduced Keap1 expression is logically related to the induction of Nrf2 53. Together with the induction of Nrf2, EAS supplementation reduced Keap1 and induced HSF1 expression in the present study. In addition, supplementation of quercetin, a plant-derived flavonoid, comprised mainly of polyphenolic compounds from fruits and vegetables, induced Nrf2 expression in bovine embryos 46. EAS treatment increased the Nrf2 protein levels in NG108-15 neuronal cells 32. In conclusion, EAS increased GSH expression and γ-glutamyl cycle mRNA expression due to Nrf2, which was induced by the compensation effect of HSF1 with Nrf2 and the antioxidant content of EAS.
The higher expression of antioxidant enzyme genes in the HS group in the present study was similar to that reported in a previous study, in which HS induced SOD and PRDX expression in pig skeletal muscle 54 and HeLa cells 28, respectively. In another study, HS was reported to upregulate HSP70 and SOD2 in Chinese hamster lung fibroblast V79 cells 44. Interestingly, the heat shock protein family and antioxidant system reduce the harmful effects of ROS 55. In the present study, EAS increased HSP70 expression, antioxidant enzymes, and reduced ROS levels. Nrf2 expression has been shown to induce PRDX activity in HeLa cells 27 and SOD activity in mesenchymal stem cells 56. The binding of Nrf2 to ARE activates PRDX and SOD activity 27 56. In this study, Nrf2 expression was highest in bovine cumulus-granulosa cells treated with EAS under HS conditions, indicating that the induction of PRDX and SOD was due to Nrf2 expression. EAS extracted from asparagus (Asparagus officinalis L.) contains high levels of antioxidant, including carotenoids, steroidal saponins, and flavonoids 29. In addition, quercetin, which is one of the six subclasses of flavonoid compounds, increased the expression of Nrf2 and antioxidant genes, such as PRDX1 and SOD1 46. Therefore, the enhanced antioxidant activity of EAS treatment results from the induction of Nrf2 expression and the antioxidant content of asparagus (Asparagus officinalis L.).
PES has been shown to inhibit the function of HSP70 57. In the present study, PES reversed the effect of EAS on the GSH and ROS levels, indicating that HSP70 induction by EAS regulated the levels of GSH and ROS in bovine cumulus-granulosa cells. A previous study reported a correlation between the transcription factors Nrf2 and HSF1 for the protection of cells 58. There is evidence that Nrf2 and HSF1 compensate each other; the induction of HSP70 by methionine deprivation is dependent on Nrf2 but independent of HSF1 59. Both Nrf2 and HSF1 play important roles in cellular redox processes due to their ability to influence the levels of HSP70 and GSH. Therefore, distinct cell survival pathways, such as the HS response and Keap1/Nrf2/ARE pathway, are regulated by Nrf2 and HSF1 51. In the present study, PES also reversed the induction effect of both Nrf2 and HSF1. PES reduced the nuclear translocation of the nuclear factor-κB (NF-κB) pathway, which regulates the transcription of various gene families, including stress response, apoptosis, and receptor genes, and influences cell survival, differentiation, and proliferation 60. The nuclear translocation of NF-κB p65 enhances the ability of Nrf2 and plays a role in the antioxidant response in human kidney-2 cells 61. In addition, HSF1 activation in intestinal epithelial cells during HS is regulated by the NF-κB pathway 62. Moreover, PES reduced the levels of Keap1, which combined with Nrf2 to regulate the antioxidative protection system 51. These results indicate that HSP70 induction by EAS improved the redox balance by regulating the ROS and GSH levels via regulating the heat shock response through HSF1 and the antioxidant response through the Nrf2/Keap1 pathway in bovine cumulus-granulosa cells.
The beneficial effect of EAS on P4 synthesis demonstrated in the present study was similar to that reported in a previous study, in which the oral administration of asparagus root extract enhanced P4 levels in rats 63. P4 levels were enhanced by EAS was due to the presence of steroid saponins, such as sarsaponin, protodioscin, and diosgenin, in asparagus extract, which act as precursors of progesterone 63. This indicated that bovine cumulus-granulosa cells were important for reproductive function, especially steroidogenesis. As such, these findings indicate that EAS contributed to the improvement of P4 synthesis in bovine cumulus-granulosa cells.
In conclusion, EAS was shown to induce HSP70 under non-HS conditions, exerting a synergistic effect with HS on HSP70 induction in bovine cumulus-granulosa cells. Furthermore, EAS had beneficial effects, reducing the DNA damage induced by ROS, as well as increasing GSH synthesis and antioxidant enzyme levels to maintain the redox status, in addition to the P4 levels, in bovine cumulus-granulosa cells. HSP70 induced by EAS regulated the Nrf2/Keap1 pathway and HSF1 transcription factor levels, which contributed to the ROS and GSH levels in bovine cumulus-granulosa cells.
Taken together, the findings presented in this study demonstrate that EAS has potential uses in the regulation of reproductive functions by reducing physical stress and improving the properties of reproductive cells.