Study On Antioxidant Effect And Mechanism Of Recombinant Glutathione Reroxidase 1 And 4

Reactive oxygen species（ROS）are the normal metabolites in the process of cellular oxidative respiration.It usually exists in the form of free radicals or non-radicals,including superoxide anions（O2.-）,hydroxyl free radicals（OH●）,hydrogen peroxide（H₂O₂）and singlet oxygen and so on.At normal physiological concentrations,ROS can participate in a series of physiological processes such as immunity,activation of transcription factors,signal pathway conduction,and cell apoptosis and differentiation.It plays an important role in maintaining the stability of the human body environment.However,when the level of ROS produced in the cell is higher than the antioxidant defense ability of the cell,ROS can cause oxidative damage to DNA,protein,and membrane lipids in cells and lead to an imbalance between the body’s oxidation and antioxidant systems,leading to the occurrence of oxidative stress.Oxidative stress can affect the occurrence and development of a series of human diseases such as cardiovascular diseases,diabetes,skin diseases and neurodegenerative diseases.Therefore,effective elimination of ROS is the key to preventing and treating oxidative stress-related diseases.Glutathione peroxidase（GPx）is an important selenium-containing antioxidant enzyme in the body’s antioxidant defense system.Glutathione peroxidase 1（GPx1）is also called intracellular GPx.It has the highest content and the most widespread distribution in the human body,and it exists in the cytoplasm and mitochondria of almost all cells.In the mammalian cells,GPx1 can use Glutathione（GSH）as a reduction substrate,which can not only catalyze toxic H₂O₂ into non-toxic water but also other water-soluble hydroperoxidess,thereby protecting cell from oxidative stress injury.Glutathione peroxidase 4（GPx4）is also known as phospholipid hydroperoxidase GPx.It is the only GPx in monomeric form.Due to its structure,it can not only catalyze small molecular hydroperoxides but also directly reduce the phospholipid hydroperoxide on the biological membrane to inhibit cell lipid peroxidation.Studies have shown that GPx1 and GPx4 could directly or indirectly participate in a variety of normal physiological or pathological processes such as reproductive development,aging,diabetes,atherosclerosis,ischemia/reperfusion injury and inflammation.So they have good prospects for the development of drugs.Due to the limited source of natural GPx,the development of recombinant GPx is needed.However,since the active site of GPx is selenocysteine（Sec）,the insertion of Sec requires a special coding mechanism,which makes the development of recombinant GPx drugs more difficult.In a previous work,our group developed a new chimeric tRNA^UTuT6 method to express recombinant GPx in amber-less Escherichia coli.This method is low-cost,using ordinary LB medium,and per liter of medium can be obtained 4 to 8 mg of target protein;simple operation,convenient for large-scale production.In addition,recombinant human glutathione peroxidase 1（hGPx1）and recombinant human glutathione peroxidase 4（hGPx4）expressed by this method have a natural GPx structure and high catalytic activity,which is extremely promote the drug development of hGPx1 and hGPx4.However,the antioxidant effects of hGPx1 and hGPx4 at the cellular and animal levels have not been verified,and the antioxidant mechanism is still unclear.In this study,we used hGPx1 and hGPx4 to study their antioxidant effects at the cellular and animal levels.The specific study results are as follows:（1）Preparation of recombinant GPx1 and GPx4In this chapter,by comparing the amino acid sequence,structure,enzymatic properties and steady-state kinetics of hGPx1 and m GPx1,a mouse model was selected to study the antioxidant effect of hGPx1.At the same time,in order to accurately reflect the antioxidant effect of homologous recombination GPx1,recombinant mouse glutathione peroxidase 1（m GPx1）was used as a control of hGPx1 for the antioxidant study of mouse models.According to the classic method reported in our laboratory,hGPx1,hGPx4 and m GPx1 were successfully prepared in amber-type termination-deficient E.coli by using the new chimeric tRNA^UTuT6,laying the foundation for subsequent antioxidant experiments.（2）Study and mechanism on the antioxidant effect of recombinant hGPx1.In this chapter,the isoproterenol（ISO）-induced oxidative stress injury model of H9C2 cells and the BALB/c mouse heart oxidative stress injury model were established,and the antioxidant effects and mechanisms of hGPx1 at the cellular and animal levels were studied and observed the difference between hGPx1 and m GPx1 in antioxidant experiments.Cell experiment results showed that both hGPx1 and m GPx1 could significantly prevent ISO-induced oxidative stress injury in H9C2 cells by eliminating ROS in time and inhibit ISO-induced apoptosis in H9C2 cells through a mitochondria-dependent pathway,and both have the same antioxidant effect.Animal experiment results showed that both hGPx1 and m GPx1 could inhibit myocardial lipid peroxidation,repair pathological damage of heart oxidative stress,and significantly reduce ISO-induced the abnormal increase of specific myocardial enzymes in mouse serum,reduce the content of malondialdehyde（MDA）in mouse serum and heart tissue.Since hGPx1 and m GPx1 have the same antioxidant effect in cell models,the antioxidant effect of m GPx1 in animal models is stronger than hGPx1.In order to explore the reason,we conducted in vivo antibody detection of hGPx1 and m GPx1 and in vitro neutralizing antibody analysis.Study results showed that hGPx1 has immunogenicity in mice,and the antibodies it produces can inhibit its original antioxidant activity.Nevertheless,this study provided a reliable theoretical basis for the development of hGPx1 as an antioxidant drug.（3）Study and mechanism on anti-UV damage effect of recombinant hGPx1In this chapter,in order to further explore the medicinal value of hGPx1,a Ultraviolet B（UVB）induced Ha Ca T cell oxidative stress injury model was established to study the anti-UV effect of hGPx1 and its mechanism.Experimental results showed that hGPx1 could effectively prevent UVB-induced oxidative stress injury of skin epidermal cells by clearing ROS,and prevent skin epidermal cell apoptosis by indirectly inhibiting mitochondrial-dependent apoptosis.In addition,hGPx1 can indirectly inhibit the overexpression and secretion of MMP-1 and MMP-9 proteins in skin epidermal cells by clearing ROS in time,thereby reducing the excessive degradation of MMP-1 and MMP-9 proteins on collagen,preventing collagen loss caused by excessive ultraviolet radiation.The research in this chapter provides a reliable theoretical basis for the development and application of hGPx1 as a drug to prevent UV damage.（4）Study and mechanism on the antioxidant effect of recombinant hGPx4In this chapter,the monomeric hGPx4,namely phospholipid hydroperoxide GPx,is selected.The smaller molecular weight makes it possible to have lower immunogenicity and better tissue permeability.We have established male SD rat myocardial ischemia injury model and an in vitro model of cardiomyocytes to study the protective effect of hGPx4 on myocardial ischemia injury and its mechanism.Animal experiment results showed that hGPx4 can effectively prevent myocardial ischemic pathological damage in rats by clearing ROS in time,prevent arrhythmia in rats and inhibit myocardial hypertrophy in rats,and has a good preventive effect on myocardial ischemic damage in rats.Cell experiment results showed that hGPx4 could eliminate ROS in time and inhibit cell lipid peroxidation,thereby preventing cardiomyocytes oxidative stress injury,and inhibiting cardiomyocytes apoptosis by inhibiting mitochondrial-dependent apoptosis pathways.In addition,hGPx4 can also indirectly inhibit the excessive consumption of Nrf2 in the nucleus of cardiomyocytes and HO-1 in the cytoplasm of cardiomyocytes by clearing ROS in time,maintain the basic normal levels of Nrf2 and HO-1 in cardiomyocytes,and ensure that the Nrf2/HO-1 signaling pathway can perform its antioxidize effect normally,thereby preventing cardiomyocytes from oxidative stress damage.Although the catalytic activity of hGPx4 is not as good as that of hGPx1,it has a wide range of reaction substrates,can also reduce phospholipid hydroperoxide on the biological membrane,and also has a good antioxidant effect.Since the animal experiment is designed as a short-term prevention experiment,there is no need for immunological control.In short,hGPx4 can play an important role in the prevention of myocardial ischemia injury and has huge application potential.In conclusion,this study explored the medicinal value of hGPx1 and hGPx4 through the therapeutic effect of hGPx1 on myocardial oxidative stress injury,the preventive effect of UV damage and the preventive effect of hGPx4 on myocardial ischemia injury,and proved them have a good antioxidant effect at the cellular and animal levels,providing data support and laying a theoretical foundation for the development and application of hGPx1 and hGPx4 as anti-oxidative stress-related diseases.