Broiler ascites syndrome is a disease caused by metabolic disorders that is common and poses a serious threat to broiler health while also limiting the economic benefits of broiler farming . Myocardial hypertrophy and failure are critical links in the development of broiler ascites syndrome. The onset of cardiac hypertrophy and failure is accompanied by a sustained increased oxygen radical response, such as the production of large amounts of ROS and Lipid Hydroperoxide, which can cause cell membrane damage, which in turn causes DNA damage and eventually induces cardiomyocyte death via apoptosis. Apoptosis of cardiomyocytes is a critical component of this pathogenesis . Excessive apoptosis of cardiomyocytes causes irreversible myocardial damage to the heart, resulting in cardiac hypertrophy and failure, ascites, and even death . Furthermore, endoplasmic reticulum stress is linked to a variety of cardiovascular diseases . Selenium (Se), an essential trace element in humans and animals, is involved in numerous important physiological processes and exerts biological effects in the form of selenoproteins . Selenoproteins are powerful antioxidants that play a role in immune enhancement and the prevention of cardiovascular disease. To maintain the body's normal functioning, selenium nanoparticles scavenge ROS by converting into selenoproteins with antioxidant functions and promoting the synthesis of antioxidant enzymes.
Apoptosis in cardiomyocytes was induced by molding with hydrogen peroxide (China, Sinopharm Chemical Reagent Co., Ltd.). A hydrogen peroxide concentration gradient of 50, 100, 200, 300, and 400 µmol/L was designed and tested for 4 hours. The hydrogen peroxide concentration close to the IC50 was discovered to be the optimal concentration for modeling by measuring CCK-8. The IC50 was calculated using the GraphPad Prism 7.0 software based on the CCK-8 assay results, and a concentration close the IC50 of 200 µmol/L hydrogen peroxide was used to replicate the apoptosis model by operating on the cells for 4 hours. The concentration of 200 µmol/L hydrogen peroxide applied to cells for 4 hours caused 23.2 percent early apoptosis and 16.3 percent late apoptosis, and the addition of Nano-selenium (1 µmol/L) reduced the early apoptosis rate by 9.2 percent and the late apoptosis rate by 12.16 percent. The findings showed that 1 µmol/L nano-selenium effectively reduced cardiomyocyte apoptosis caused by hydrogen peroxide simulation. In this experiment, the ROS assay was used to assess the effect of nano-selenium on hydrogen peroxide-induced oxidative stress in chicken cardiomyocytes. The fluorescence intensity of the Se + Y group was significantly lower than that of the Se + Y group, implying that nano-selenium inhibited hydrogen peroxide-induced oxidative stress in cells.
The biological effects of nano-selenium are dependent on the dose and duration of action. The CCK-8 results of different concentrations of nano-selenium on chicken cardiomyocytes for 24 hours revealed that the lower concentration of nano-selenium had no significant effect on cardiomyocyte activity, that when the concentration of nano-selenium was gradually increased to 5 µmol/L, nano-selenium showed a significant proliferative effect on chicken cardiomyocytes, and that when the concentration of nano-selenium reached 10 µmol/L, The effect on the survival rate of chicken cardiomyocytes tended to the blank control group when the nano-selenium concentration reached 100 µmol/L, and there was a steady trend to suppress cell activity. Although nano-selenium is biocompatible and has a low toxicity, a high concentration of nano-selenium acting on cells may cause a Trojan horse effect, and the nano-small selenium's particle size allows for uncontrolled passage across the cell barrier, which may have negative effects on cells.
It has been demonstrated that broiler antioxidant capacity is proportional to selenium levels within a certain range, and GSH-Px activity in broiler serum and tissues increases with increasing selenium levels in the diet[30-, and when GSH-Px activity in serum reaches a plateau, GSH-Px activity does not increase further with increasing selenium concentration in the diet . The activities of SOD, GSH-Px, and CAT in broiler serum were not significantly different between the Se and con groups in this experiment because the feed fed to the con group also had the basic selenium content of poultry. Meanwhile, the Se group added 1 mg/kg of nano-selenium to the basic diet. As stated above, when 0.15 mg/kg–1 mg/kg selenium was added to the diet, the activation response of broiler GSH-Px reached its peak . The GSH-Px activity in the serum of broilers after a certain dose of selenium intake did not increase with the increase of selenium level.
Furthermore, oxidative stress regulates many selenoproteins. For example, GPx1, GPx4, and TRx1 expression levels have been shown to be upregulated during oxidative stress. The expression of their associated cardioprotective selenoproteins and selenases has been shown to increase during cardiovascular oxidative stress , and mRNA and protein levels of MSRB1, TRx1, GPx3, and GPx4 were significantly increased in T3 or isoproterenol-induced myocardial hypertrophy.The results of this experiment revealed that there was no significant difference in serum GSH-Px activity between the Se + Y group and the Y group, and there was no significant difference in serum SOD activity between the Se + Y group and the Y group. SOD can scavenge oxygen radicals in the body, and its level in the organism can reflect the organism's ability to scavenge ROS indirectly. Serum CAT and T-AOC activities were significantly higher in the Se + Y group compared to the Y group, and MDA content was significantly lower in the Se + Y group. To sum up, nano-selenium can reduce oxidative stress in broiler chickens caused by brine modeling by increasing antioxidant enzyme activity.
The protein expression of ERS-related signaling pathways in broiler heart tissues was detected using Western Blot in this in vitro trial. The results of this experiment revealed that the expression levels of GRP-78, ATF-6, and CHOP proteins were significantly lower in the Se + Y group when compared to the Y group, indicating that Nano-selenium can reduce apoptosis in cardiomyocytes by inhibiting ERS. When ERS occurs, high levels of CHOP are induced, and high levels of CHOP induce the expression of apoptotic proteins such as GADD34 and DR5, where the DR5 receptor forms the death-inducing signaling complex (DISC), which activates the caspase cascade reaction. The results of this experiment showed that the Se + Y group had significantly lower caspase 3 and caspase 9 protein expression levels compared to the Y group, indicating that Nano-selenium could inhibit the activation of apoptotic proteins associated with the downstream of the ATF6-DR5 signaling pathway, thereby alleviating the occurrence of apoptosis in vivo. Overexpression of CHOP can also promote the production of ROS, which causes long-term endoplasmic reticulum stress and mediates apoptosis. When high levels of CHOP protein can cause a decrease in Bcl-2 expression, this can also result in Bax translocation from the cytoplasm to the mitochondria and the initiation of the mitochondrial apoptotic pathway . The current study found that the Se + Y group simultaneously decreased Bax protein expression while increasing Bcl-2 protein expression when compared to the Y group, and that Nano-selenium could inhibit caspase12 protein expression on the endoplasmic reticulum membrane.
In summary, this study shows that Nano-selenium can maintain endoplasmic reticulum homeostasis by inhibiting the expression of GRP-78, ATF-6, CHOP, caspase 12 and its downstream apoptosis-related proteins caspase 9, caspase 3, Bax, and increasing the expression level of Bcl-2, thereby alleviating cardiomyocyte apoptosis and reducing myocardial injury in broiler chickens. Broiler chicken myocardium was treated with nano-selenium. The ATF6-DR5 signaling pathway may be involved in the potential molecular mechanism.