The redox balance is essential to maintain the homeostasis of the organism and in it, ROS and enzymatic and non-enzymatic antioxidant systems participate. RS is a concept that is currently not well defined. However, in a similar way as OS, RS generates harmful effects32. In various studies, an increase in reducing equivalents and antioxidant systems has been observed; however, the mechanisms of action, the biological consequences, as well as the response of the cell to these changes remain unknown. A reducing environment is traditionally associated with beneficial effects on cellular functions and biological processes. However, the prolonged increase in these components disturbs the redox balance and generates harmful effects such as interference with the signaling and synthesis of ROS33. HSL is considered a "functional" food that provides various components with biological activity and can provide different minerals and essential amino acids that form part of the antioxidant system. However, these "beneficial" characteristics could become detrimental if consumed in excess or for a prolonged lapse of time, increasing antioxidant enzymes and excessively depleting ROS. This could lead to RS and to redox imbalance11, 34. Therefore, the objective of this study was to explore the effect of the ingestion of HSL infusions at different percentages on vascular reactivity and the possible RS generation due to the excess of antioxidant agents provided by the HSL infusion.
Our results show a significant increase in the elastic fibers, thickness of the tunica media and intima of the thoracic aorta. These changes suggest higher rigidity and hypertrophy, which was reflected in the increase in the SBP in the groups with HSL infusion at 3% and 6%. Structural alterations and hypertrophy in the conductive arteries which is associated with stiffness, inflammation, and increased blood pressure12. In this sense, a study carried out in male "Carworth" rat’s associated increased collagen and elastic fibers in the renal artery with the presence of renovascular hypertension35. The same effect was observed in another study when analyzing the structure of the aorta in hypertensive "Wistar" rats, where the hypertrophy caused in the wall decreased the elasticity of fibers and collagen biosynthesis leading to an increased blood pressure12. The hypertrophy of the aorta may stimulate the synthesis of angiotensin II and produce vasoconstriction favoring production of O2– through the activation of NAD/NADH+. This leads to inactivation of eNOS in the endothelium, causing, in turn, over-expression of iNOS that contributes to the inflammatory process17, 36. Furthermore, HSL contains tyrosine, and an excess of this amino acid could lead to higher availability of the substrate needed for the synthesis of NE by the sympathetic nerve endings, which could contribute to increase the SBP in the groups with HSL infusions at 3% and 6%37.
Our results show that vasoconstriction and vasodilation increased and decreased, respectively, in the rats that consumed the HSL infusion at 3% and 6%. This suggests that, in addition to the structural changes, the excess in the consumption of HSL infusion may alter vascular function, which is associated to the increase in antioxidants provided by the HSL infusion that modulate vascular reactivity through the increase in iNOS. This could influence the thickening of elastic fibers, the middle muscle zone, and the loss of elasticity in the thoracic aorta. In this sense, it is essential to maintain the redox balance in endothelial cells and a decrease in ROS can contribute to the alteration of vascular function and consequently lead to various vascular diseases2. The effects of ROS in the thoracic aorta are not uniform since they depend on which ROS molecule is acting and at what concentration38. In the thoracic aorta, the most important ROS are O2–, H2O2, NO and peroxynitrite (ONOO–)39.
The excess of antioxidants and precursors of the antioxidant systems provided by HSL infusions could decrease O2– and H2O2, which at physiological concentrations act as second messengers38. In our study, the vasoconstriction response in the experimental groups to infusions with different percentages of HSL eliminated the significant difference presented by the control group after being incubated with K2O. K2O reacts violently with water to form KOH and, in this process, O2– is formed. This suggests the participation of O2– in the vascular response, but also shows that an excess of antioxidants provided by HSL decreases the O2– concentration. This result can be associated with the alteration of the vascular reactivity and with increased SBP.
In contrast, H2O2 favors vasodilation and produces NO through the activation of different signaling pathways such as PI3K/Akt, Erk1/2, and p38MAPK39. H2O2 not only stimulates eNOS but can also increase its expression. Another protein that participates in the regulation of vasodilation is PKG1α. This protein kinase is sensitive to oxidation by H2O2 through the formation of a disulfide bond and the vasodilation that occurs is independent of cyclic guanosine monophosphate levels38. The results obtained in our study show that vasodilation increases after incubation with H2O2 in the group that consumed the HSL infusion at 6%, thus suggesting the participation of H2O2 in the vasodilatation response. They also suggest that the excess of antioxidants provided by the HSL infusion at 6% decreases the concentration of H2O2, which is reflected in the alteration of the arterial function, probably due to a decrease in the eNOS pathway. In this sense, the tendency, and the increase in the NO3–/NO2– ratio in the groups that consumed the HSL infusion at 3% and 6% could indirectly reflect the over-expression/activity of iNOS40. Some reactive nitrosative species such as ONOO– may participate in the inflammatory processes due to an increase in NO availability by iNOS pathway40–41. However, our results showed that NO3–/NO2– ratio present a tendency to increase, but the 3-NT was decreased. These results could be paradoxical because an increase in protein nitrosylation was expected. Through the iNOS metabolic pathway that probably would be increased in the aorta. However, the excess of antioxidants provided by the 6% HSL infusion could decrease the ONOO–, which participates in the nitrosylation process, thus explaining the decrease in 3-NT in the aortic homogenate. This decrease is also associated with the low concentration of thiol groups in the cysteines of proteins, by excessive consumption of HSL that decreases the concentration of thiol groups40. However, more studies are required to corroborate this hypothesis.
On the other hand, the enzyme G6PD is responsible for providing reducing equivalents such as NADPH and controlling ROS production through of the antioxidants enzymes that are NADPH dependent42. G6PD overexpression decreases the excess ROS in endothelial cells treated with H2O2 or TNFα. Moreover, the G6PD overexpression can increase the concentration of GSH through the glutathione reductase pathway42–43. Furthermore, in vascular cells, some processes that generate inflammation, such as the hypertrophy of the vascular wall in the metabolic syndrome and type 2 diabetes, could increase the G6PD expression or activity and favor high levels of NADPH as a compensatory mechanism43. Our results showed an increase in the activity of G6PD in the aorta homogenate. This could be attributed to the alteration of the vascular function and structural changes associated with the level of inflammation and hypertrophy of the aortic wall. The hypertrophy may cause NADPH oxidase activation and some interleukins that favor the pro-inflammatory state. These processes can stimulate the G6PD activation and favor the production NADPH that is a cofactor for some antioxidant enzymes such as GPx, TrxR, and iNOS. HSL could favor the G6PD overexpression or activity, which leads to an increase in NADPH44. However, more studies are necessary to corroborating this hypothesis.
In addition to the fact that G6PD overexpression can increase GSH concentration, HSL can by itself contribute to increase GSH since it contains cysteine, glycine, and glutamic acid, which are GSH precursors, which could increase the synthesis of this antioxidant19, 34. In this sense, our results showed a significant increase in the concentration of GSH in the groups that received the HSL infusion at 3% and 6%.
HSL in various concentrations also provides Se and the amount provided depends on the crop, type of soil, pH, type of plant species, and salinity45–46. Se is considered an essential micronutrient and its excess or depletion in the body favors the appearance of some diseases such as hypothyroidism, cardiovascular disorders, cognitive impairment, and weakening of the immune system46. Furthermore, Se is indispensable for 25 seleno enzymes such as GPx and TrxR, which contribute to maintaining the redox homeostasis47. Our results show that the Se concentration was increased in the groups of rats that received HSL infusion at 3% and 6%. This suggests that the excessive consumption the HSL could lead to selenosis, which is caused by excess intake of Se. However, more studies are required to corroborate this hypothesis.
The Se may be oxidized in the presence nitrogen species and GSH48. In this sense, Se compounds such as selenite and selenium dioxide can react with thiol groups present in GSH to produce O2–. This has been observed in experiments with isolated mitochondria treated with selenium-containing components, including selenite and selenocysteine47. This suggests that excess Se could lead to O2– and ONOO– generation and that the excessive antioxidants provided by the HSL infusion can decrease it.
A high level of Se concentration favors LPO, which causes DNA damage and favors oxidation of polyunsaturated fatty acids and the degradation of proteins47, and high Se concentration could cause thiol oxidation. Our results showed that the concentration of total thiol was decreased but that LPO was increased in the rats that received the HSL infusion at 3% and 6%. The low levels of thiols may contribute to the alteration of vascular function and structural changes in the thoracic aorta in these rats and are linked to cardiovascular events49. Another study associated the decrease in thiols and the increase in LPO with the severity of artery coronary disease lesions50.
To maintain thiol homeostasis in cysteines of proteins, mammalian cells depend on the TrxR system45, 51. This system also participates in the regulation of vascular tone, and when the TrxR is overexpressed, vascular reactivity is improved by increasing bioavailable NO52. A study in mice showed that the deletion of TrxR generated a lower response to vasodilation and a greater production of ONOO–53–54. Our results showed an increase in the activity of this enzyme in the group of rats that consumed the HSL infusion at 6%. These results suggest that an increase in the TrxR activity could be attributed to the excess Se and the increase of the G6PD activity52–54. This could be a compensatory mechanism that is aimed to increase the levels of thiols51 and could be associated with the consumption excessive antioxidants such as polyphenols, quercetin, flavonoids, Se, vitamin C, and gallic, protocatechuic, tannic acids, which may be provided by the HSL infusion.
Another enzyme that increased its activity in this study was GPx. This enzyme contains selenium-cysteine at its catalytic site and was overactive in the aorta homogenate from the rats of the group that consumed the HSL infusion at 6%. This increased activity could occur because of substrate surplus such as GSH and NADPH that were provided by the G6PD and to the elevation of Se. Furthermore, moderate ROS are required for keeping the formation of disulfide formation (thiols) in the cell and the overexpression of GPx can lead to a decrease of protein disulfides that is related to reduce signaling from growth factors and decreased mitochondrial function55.
On the other hand, GST is an antioxidant enzyme that participates in the detoxification process caused by oxidative damage, xenobiotic agents, and lipids oxidized, and may prevent the LPO effects56. This enzyme conjugates GSH with electrophilic and xenobiotic agents such as drugs, toxins, carcinogens, and detoxifies the organism57, our results showed a decrease in the GST activity in the group of rats that consumed the HSL infusion at 3% and 6%. The administration of tannic acid at doses of 80 mg/kg reduces the activity of the “mu” and “alpha” subunits of the GST in the liver56. It also decreases the “pi” subunit of the GST when protocatechuic acid is provided56. Another study demonstrated that high concentrations of flavonoids could inhibit GST activity and favor an increase in LPO57. This suggests that the excess consumption of flavonoids, tannic and protocatechuic acids, which are provided in the HSL infusion at 3% and 6% favored the decrease in the GST activity in the aorta and contributed to increased LPO. Figure 8 summaries the alteration in physiological and anatomical changes within the thoracic aorta in response to different percentages HSL infusions. In conclusion, the excessive consumption of antioxidants provided by the HSL infusion at 3% and 6% such as polyphenols, quercetin, flavonoids, Se, vitamin C, and gallic, protocatechuic, tannic acids may deplete the ROS thus deteriorating vascular function but not at low concentrations such as 1.5%. There was also an association with the increase in the thickness of the thoracic aorta wall that contributes to increasing the SBP. These changes are accompanied with the alteration of the redox homeostasis that could generate RS.