CN111729075A

CN111729075A - Application of recombinant glutathione peroxidase in preparation of antioxidant anti-aging health products and medicines or medicines for resisting myocardial infarction

Info

Publication number: CN111729075A
Application number: CN202010794088.4A
Authority: CN
Inventors: 魏景艳; 刘畅; 闫琦
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2020-08-10
Filing date: 2020-08-10
Publication date: 2020-10-02

Abstract

The application of the recombinant glutathione peroxidase in the preparation of antioxidant anti-aging health products and medicines or medicines for resisting myocardial infarction belongs to the field of biotechnology. Experimental results show that the recombinant human glutathione peroxidase (GPX 1-GPX 8) can inhibit cell damage caused by oxidative stress, reduce cell membrane damage caused by oxidative stress, block lipid peroxide chain reaction, reduce cell malondialdehyde content, inhibit intracellular ROS generation caused by oxidative stress, inhibit apoptosis caused by oxidative stress, change electrocardiogram abnormality caused by myocardial infarction, inhibit myocardial enzyme abnormal rise caused by myocardial infarction, reduce oxidative stress reaction caused by myocardial infarction, reduce myocardial infarction area and improve the pathological state of myocardial tissue morphology caused by myocardial infarction, thereby laying a foundation for the development of the recombinant human glutathione peroxidase in the preparation of antioxidant anti-aging health care products and medicines or anti-myocardial infarction medicines.

Description

Application of recombinant glutathione peroxidase in preparation of antioxidant anti-aging health products and medicines or medicines for resisting myocardial infarction

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of recombinant glutathione peroxidase in preparation of antioxidant anti-aging health products and medicines or medicines for resisting myocardial infarction.

Background

The balance of the oxidative and antioxidant systems is an important mechanism for the body to maintain homeostasis and self-protection. However, oxidative stress is induced when the level of Reactive Oxygen Species (ROS) produced in human cells is higher than the antioxidant defense capacity of the cells. Oxidative stress can affect the development and progression of many important diseases such as cardiovascular and cerebrovascular diseases, diabetes, neurodegenerative diseases.

Glutathione peroxidase is a selenase which plays an important role in the antioxidant defense system of the organism. Glutathione peroxidase can competitively catalyze hydrogen peroxide (H) by using Glutathione (GSH) as a substrate with catalase₂O₂) And can scavenge organic hydroperoxides in the body. Glutathione peroxidase 4 can also catalyze H with GSH and phospholipid hydroperoxide on biological membrane as substrates₂O₂And all kinds of lipid hydroperoxides. Thus, glutathione peroxidase plays an important role in regulating ROS levels in the body. Research shows that the glutathione peroxidase can directly or indirectly participate in various normal physiological/pathological processes such as aging, diabetes, hypertension, atherosclerosis, ischemia/reperfusion injury, inflammation and the like, and has good development prospects of medicines and health-care products. However, since the source of native glutathione peroxidase is limited and the active center of glutathione peroxidase is selenocysteine (Sec) which needs a special coding mechanism to be inserted, it has increased difficulty in drug development of recombinant glutathione peroxidase. Chinese patent 201510431360.1 of this subject group discloses a hybrid tRNA and successfully prepares a new recombinant glutathione peroxidase (GPX, including GPX 1-GPX 8), which has higher catalytic activity and better stability. The invention uses the method disclosed in the patent to prepare recombinantGlutathione Peroxidase (GPX) and researches the antioxidation of the GPX on the level of cells and animals so as to prepare antioxidant anti-aging health products and medicines or myocardial infarction medicines, and lays a foundation for the development of recombinant human glutathione peroxidase in medicines or health products for oxidative stress diseases.

Disclosure of Invention

The first purpose of the invention is to provide the application of GPX in the preparation of health products for preventing oxidative stress damage of cells, namely, in the preparation of antioxidant anti-aging health products. Specifically, see (1) to (8) and examples (2) to (10) of the advantageous effects of the present invention, and also see fig. 2 to 9.

The second purpose of the invention is to provide the application of GPX in preparing medicines for resisting cell oxidative stress damage, namely, resisting oxidation and resisting aging. See in particular (9) to (16) and examples (11) to (18) of the advantageous effects of the present disclosure, and also see fig. 10 to 17.

The third purpose of the invention is to provide the application of GPX in preparing the anti-myocardial infarction medicament. Specifically, see (17) to (23) and examples (19) to (25) of the advantageous effects of the present invention, and also see fig. 18 to 30.

The GPX and the medicinal carrier or the auxiliary material can be prepared into the medicinal composition by the conventional technology, the medicinal composition can adopt various preparation forms, and can be a liquid preparation, an injection, a granule, a tablet, a suppository, an ointment, an aerosol, an ophthalmic preparation, a sustained-release controlled-release preparation, a targeting preparation and the like, and also can be used as a health-care product.

The recombinant glutathione peroxidase GPX comprises recombinant glutathione peroxidase GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7 and GPX 8; wherein GPX1 represents recombinant human glutathione peroxidase 1, GPX2 represents recombinant human glutathione peroxidase 2, and so on. In this patent, GPX is prepared by the expression method of UAG engineering bacteria (see Chinese patent 201510431360.1), ISO represents isoproterenol, H₂O₂Represents hydrogen peroxide. Animal experiments were performed according to the care and use committee of experimental animals of the university of Jilin, with the experimental protocol approved by the ethical committee of the university of Jilin.

The invention takes GPX1 and GPX4 as implementation objects, and has the following beneficial effects:

(1) GPX1 is able to prevent cellular damage caused by oxidative stress:

at 100. mu. mol/L H₂O₂In an induced H9C2 cell oxidative stress model, the cell survival rate of a control group is 100.19 +/-8.06%; the survival rate of the GPX1 group cells is 98.93 +/-6.49%; h₂O₂The survival rate of the model group cells is only 49.52 +/-10.23%; and GPX1+ H containing 0.1U/mL GPX1₂O₂The survival rate of the group cells is 78.17 +/-4.59 percent; it can be seen that GPX1+ H₂O₂The survival rate of the cells of the group is 1.58 times that of the H2O2 model group, which shows that GPX1 can effectively prevent the cell damage caused by oxidative stress.

(2) GPX1 is able to prevent cell membrane damage caused by oxidative stress:

at 100. mu. mol/L H₂O₂In an induced H9C2 cell oxidative stress model, the release rate of the lactate dehydrogenase of the cells of the control group is 99.31 +/-11.06%; the release rate of the GPX1 group cell lactate dehydrogenase is 99.11 +/-9.38%; h₂O₂The release rate of the lactate dehydrogenase of the model group cells is 246.44 +/-17.27%; and GPX1+ H containing 0.1U/mL GPX1₂O₂The release rate of the group cell lactate dehydrogenase is 140 +/-13.33%; compared with H₂O₂Model group, GPX1+ H₂O₂The release rate of the group cell lactate dehydrogenase is obviously reduced, which shows that GPX1 can effectively prevent the cell membrane damage caused by oxidative stress.

(3) GPX1 is capable of preventing oxidative lipidation, reducing cellular malondialdehyde content:

at 100. mu. mol/L H₂O₂In the induced H9C2 cell oxidative stress model, the content of malondialdehyde in the cells of the control group is 1.26 +/-0.31 (nmol/mg pro); the malondialdehyde content of the cells of the GPX1 group is 1.31 +/-0.25 (nmol/mg pro); h₂O₂The malondialdehyde content of model group cells is 5.27 +/-0.27 (nmol/mg pro); and GPX1+ H containing 0.1U/mL GPX1₂O₂The malondialdehyde content of the group cells is 3.01 +/-0.23 (nmol/mg pro); compared with H₂O₂Model group, GPX1+ H₂O₂Group cell malondialdehydeThe content was significantly reduced, indicating that GPX1 was effective in preventing oxidative lipidation caused by oxidative stress.

(4) GPX1 is able to prevent intracellular ROS generation caused by oxidative stress:

at 100. mu. mol/L H₂O₂In the induced H9C2 cell oxidative stress model, the relative fluorescence intensity of ROS in the cells of the control group is 1.01 +/-0.11; the relative fluorescence intensity of ROS in GPX1 group cells is 1.12 +/-0.15; h₂O₂The relative fluorescence intensity of ROS in the model group cells is 4.27 +/-0.32; and GPX1+ H containing 0.1U/mL GPX1₂O₂The relative fluorescence intensity of ROS in the group cells is 1.79 plus or minus 0.18; compared with H₂O₂Model group, GPX1+ H₂O₂The relative fluorescence intensity of ROS in the group cells was significantly reduced, indicating that GPX1 is effective in preventing intracellular ROS production caused by oxidative stress.

(5) GPX1 is able to prevent apoptosis caused by oxidative stress:

at 100. mu. mol/L H₂O₂In an induced H9C2 cell oxidative stress model, the apoptosis rate of a control group cell is 4.89 +/-0.76%; the apoptosis rate of GPX1 group cells is 6.79 +/-0.86%; h₂O₂The apoptosis rate of model group cells is 53.45 +/-5.19%; and GPX1+ H containing 0.1U/mL GPX1₂O₂The apoptosis rate of the group cells is 21.37 +/-1.99 percent; compared with H₂O₂Model group, GPX1+ H₂O₂The apoptosis rate of the group cells is obviously reduced, which shows that GPX1 can effectively prevent the apoptosis caused by oxidative stress.

(6) GPX4 is capable of preventing oxidative lipidation, reducing cellular malondialdehyde content:

at 100. mu. mol/L H₂O₂In the induced H9C2 cell oxidative stress model, the content of malondialdehyde in the cells of the control group is 1.23 +/-0.18 (nmol/mg pro); the malondialdehyde content of the cells of the GPX4 group is 1.29 +/-0.22 (nmol/mg pro); h₂O₂The malondialdehyde content of the model group cells is 4.79 +/-0.21 (nmol/mg pro); and GPX4+ H containing 0.01U/mL GPX4₂O₂The malondialdehyde content of the group cells was 2.49. + -. 0.28(nmol/mg pro); compared with H₂O₂Model group, GPX4+ H₂O₂Group cell CThe dialdehyde content was significantly reduced, indicating that GPX4 was effective in preventing the occurrence of lipid peroxide chain reactions caused by oxidative stress.

(7) GPX4 is able to prevent intracellular ROS generation caused by oxidative stress:

at 100. mu. mol/L H₂O₂In the induced H9C2 cell oxidative stress model, the relative fluorescence intensity of ROS in the cells of the control group is 1.23 +/-0.41; the relative fluorescence intensity of ROS in GPX4 group cells is 1.33 +/-0.17; h₂O₂The relative fluorescence intensity of ROS in the model group cells is 3.86 +/-0.19; and GPX4+ H containing 0.01U/mL GPX4₂O₂The relative fluorescence intensity of ROS in the group cells is 1.95 plus or minus 0.14; compared with H₂O₂Model group, GPX4+ H₂O₂The relative fluorescence intensity of ROS in the group cells was significantly reduced, indicating that GPX4 is effective in preventing intracellular ROS production caused by oxidative stress.

(8) GPX4 is able to prevent apoptosis caused by oxidative stress:

at 100. mu. mol/L H₂O₂In an induced H9C2 cell oxidative stress model, the apoptosis rate of a control group cell is 5.02 +/-0.81%; the apoptosis rate of GPX4 group cells is 6.64 +/-0.92%; h₂O₂The apoptosis rate of model group cells is 45.96 +/-4.59%; and GPX4+ H containing 0.01U/mL GPX4₂O₂The apoptosis rate of the group cells was 23.06 + -4.59% compared with H₂O₂Model group, GPX4+ H₂O₂The apoptosis rate of the group cells is obviously reduced, which shows that GPX4 can effectively prevent the apoptosis caused by oxidative stress.

(9) GPX1 is capable of treating cellular damage caused by oxidative stress:

the cell survival rate of the control group in a 50 mu mol/L ISO-induced H9C2 cell oxidative stress model is 101.05 +/-5.49%; the survival rate of the GPX1 group cells is 99.16 +/-8.66%; the cell survival rate of the ISO model group is only 50.16 +/-8.26%; the cell survival rate of GPX1+ ISO group containing 0.1U/mL GPX1 is 80.17 +/-9.56%; the cell survival rate of the GPX1+ ISO group is 1.6 times that of the ISO model group, which shows that GPX1 can effectively treat cell damage caused by oxidative stress.

(10) GPX1 is capable of treating cell membrane damage caused by oxidative stress:

in a 50 mu mol/L ISO-induced H9C2 cell oxidative stress model, the release rate of the lactate dehydrogenase of the cells in the control group is 98.75 +/-9.19%; the release rate of the GPX1 group cell lactate dehydrogenase is 100.29 +/-10.01%; the release rate of the ISO model group cell lactate dehydrogenase is 226.31 +/-7.27%; the release rate of the GPX1+ ISO group cell lactate dehydrogenase containing 0.1U/mL GPX1 is 131 +/-11.29%; compared with the ISO model group, the release rate of the GPX1+ ISO group cell lactate dehydrogenase is remarkably reduced, which shows that GPX1 can effectively treat cell membrane injury caused by oxidative stress.

(11) GPX1 is capable of treating oxidative lipidation, reducing cellular malondialdehyde content:

in a 50 mu mol/L ISO-induced H9C2 cell oxidative stress model, the content of malondialdehyde in control cells is 1.18 +/-0.29 (nmol/mg pro); the malondialdehyde content of the cells of the GPX1 group is 1.27 +/-0.33 (nmol/mg pro); the content of malondialdehyde in the cells of the ISO model group is 6.14 +/-0.31 (nmol/mg pro); while the content of malondialdehyde in GPX1+ ISO group cells containing 0.1U/mL GPX1 is 3.35 +/-0.43 (nmol/mg pro); compared with the ISO model group, the cell malondialdehyde content of GPX1+ ISO group is remarkably reduced, which indicates that GPX1 can treat oxidative lipidization caused by oxidative stress.

(12) GPX1 is capable of treating intracellular ROS production caused by oxidative stress:

the relative fluorescence intensity of ROS in the control group cells in a 50 mu mol/L ISO-induced H9C2 cell oxidative stress model is 1.02 +/-0.21; the relative fluorescence intensity of ROS in GPX1 group cells is 1.22 +/-0.13; the relative fluorescence intensity of ROS in the ISO model group cells is 5.43 +/-0.41; and GPX1+ H containing 0.1U/mL GPX1₂O₂The relative fluorescence intensity of ROS in the group cells is 2.28 +/-0.37; the relative fluorescence intensity of intracellular ROS was significantly reduced in the GPX1+ ISO group compared to the ISO model group, indicating that GPX1 was effective in treating intracellular ROS generation caused by oxidative stress.

(13) GPX1 is capable of treating apoptosis caused by oxidative stress:

in a 50 mu mol/L ISO-induced H9C2 cell oxidative stress model, the apoptosis rate of a control group cell is 5.76 +/-0.94%; the apoptosis rate of GPX1 group cells is 6.09 +/-0.58%; the apoptosis rate of ISO model group cells is 62.79 +/-4.09%; the apoptosis rate of GPX1+ ISO group containing GPX1 of 0.1U/mL is 32.13 +/-4.29%; compared with the ISO model group, the apoptosis rate of GPX1+ ISO group cells is remarkably reduced, which shows that GPX1 can effectively treat apoptosis caused by oxidative stress.

(14) GPX4 is capable of treating oxidative lipidation, reducing cellular malondialdehyde content:

in a 50 mu mol/L ISO-induced H9C2 cell oxidative stress model, the content of malondialdehyde in control cells is 1.28 +/-0.3 (nmol/mg pro); the content of malondialdehyde in GPX4 group cells is 1.34 +/-0.42 (nmol/mg pro); the content of malondialdehyde in the cells of the ISO model group is 5.79 +/-0.49 (nmol/mg pro); while the content of malondialdehyde in GPX4+ ISO group cells containing 0.01U/mL GPX4 is 3.26 +/-0.52 (nmol/mg pro); compared with the ISO model group, the cell malondialdehyde content of the GPX4+ ISO group is obviously reduced, which indicates that GPX4 can effectively treat the occurrence of lipid peroxide chain reaction caused by oxidative stress.

(15) GPX4 is capable of treating intracellular ROS production caused by oxidative stress:

the relative fluorescence intensity of ROS in the control group cells in a 50 mu mol/L ISO-induced H9C2 cell oxidative stress model is 1.19 +/-0.18; the relative fluorescence intensity of ROS in GPX4 group cells is 1.27 +/-0.2; the relative fluorescence intensity of ROS in the ISO model group cells was 4.89. + -. 0.23, while GPX4+ H containing 0.01U/mL GPX4₂O₂The relative fluorescence intensity of ROS in the group cells is 2.01 +/-0.34, compared with that in an ISO model group, the relative fluorescence intensity of ROS in GPX4+ ISO group cells is obviously reduced, and the fact that GPX4 can effectively treat the generation of ROS in the cells caused by oxidative stress is shown.

(16) GPX4 is capable of treating apoptosis caused by oxidative stress:

in a 50 mu mol/L ISO-induced H9C2 cell oxidative stress model, the apoptosis rate of a control group cell is 6.06 +/-0.83%; the apoptosis rate of GPX4 group cells is 7.19 +/-0.93%; the apoptosis rate of the ISO model group cells is 58.36 +/-3.09%; and the apoptosis rate of GPX4+ ISO group containing 0.01U/mL GPX4 is 28.56 +/-3.33%, and compared with the apoptosis rate of GPX4+ ISO group, the apoptosis rate of GPX4+ ISO group is obviously reduced, which shows that GPX4 can effectively treat apoptosis caused by oxidative stress.

(17) GPX1 can be used for treating electrocardiogram abnormality caused by myocardial infarction:

in a rat infarction model induced by 100mg/kg ISO, the mV of ST section of electrocardiogram of a control group is 0.05 +/-0.01 mV; the mV of ST section of the electrocardiogram ST of the GPX1 group is 0.06 +/-0.02 mV; the electrocardiogram ST section mV of the myocardial infarction model group is 0.5 +/-0.08 mV; while the mV of ST segment of the electrocardiogram of GPX1+ ISO group containing 4 mu g/kg/day GPX1 is 0.3 +/-0.08 mV, so that GPX1 can obviously repair the abnormality of the ST segment of the electrocardiogram, which shows that GPX1 can effectively treat the electrocardiogram abnormality caused by myocardial infarction.

(18) GPX1 can be used for treating abnormal increase of myocardial enzyme caused by myocardial infarction:

in a rat myocardial infarction model induced by 100mg/kg ISO, the activity of lactate dehydrogenase in the serum of a control group is 1002.26 +/-59.79U/L; the activity of lactate dehydrogenase in the serum of GPX1 group is 1013.29 +/-79.56U/L. The activity of the lactate dehydrogenase in the serum of the myocardial infarction model group is 2516.29 +/-75.09U/L, while the activity of the lactate dehydrogenase in the serum of the GPX1+ ISO group containing 4 mu g/kg/day GPX1 is 1501.21 +/-77.05U/L, and compared with the serum of the myocardial infarction model group, the activity of the lactate dehydrogenase in the serum of the GPX1+ ISO group is obviously reduced.

The activity of creatine kinase in the serum of a control group in a rat myocardial infarction model induced by 100mg/kg ISO is 1001.97 +/-49.29U/L; the activity of creatine kinase in the serum of GPX1 group is 1005.69 +/-62.09U/L; the activity of creatine kinase in the serum of the myocardial infarction model group is 2249.51 +/-87.29U/L; and the creatine kinase activity in GPX1+ ISO group serum containing 4 mu g/kg/day GPX1 is 1426.59 +/-89.59U/L, and compared with that in the myocardial infarction model group, the creatine kinase activity in GPX1+ ISO group serum is obviously reduced.

In a rat myocardial infarction model induced by 100mg/kgISO, the activity of creatine kinase isoenzyme in the serum of a control group is 1000.59 +/-59.49U/L; the activity of creatine kinase isoenzyme in the serum of the GPX1 group is 1003.26 +/-79.89U/L; the activity of creatine kinase isoenzyme in the serum of the myocardial infarction model group is 2355.99 +/-81.07U/L; the activity of creatine kinase isoenzyme in GPX1+ ISO group serum containing 4 mu g/kg/day GPX1 is 1463.3 +/-87.09U/L; compared with the myocardial infarction model group, the activity of creatine kinase isoenzyme in serum of the GPX1+ ISO group is obviously reduced, which shows that GPX1 can effectively treat abnormal increase of myocardial enzyme caused by myocardial infarction.

(19) GPX1 is capable of treating oxidative stress caused by myocardial infarction:

in a 100mg/kg ISO-induced myocardial infarction model of rats, the content of malondialdehyde in serum of a control group is 8.59 +/-0.59 (nmol/mL pro); the content of malondialdehyde in the serum of GPX1 group is 8.26 +/-0.75 (nmol/mL pro); the content of malondialdehyde in the serum of the myocardial infarction model group is 23.69 +/-1.56 (nmol/mL pro); while the content of malondialdehyde in the serum of GPX1+ ISO group containing 4. mu.g/kg/day GPX1 was 14.26. + -. 1.09(nmol/mL pro); compared with the myocardial infarction model group, the content of malondialdehyde in serum of GPX1+ ISO group is obviously reduced.

Malondialdehyde in heart tissue of control group was 2.34 + -0.21 (nmol/mg pro) in 100mg/kg ISO-induced myocardial infarction model in rats; the malondialdehyde content in heart tissue of GPX1 group was 2.43. + -. 0.35(nmol/mg pro); the content of malondialdehyde in the myocardial infarction model group tissue is 6.29 +/-0.43 (nmol/mg pro); while the content of malondialdehyde in the tissue of GPX1+ ISO group containing 4 mug/kg/day GPX1 is 4.12 +/-0.33 (nmol/mg pro); compared with the myocardial infarction model group, the content of malondialdehyde in the tissue of the GPX1+ ISO group is obviously reduced, which indicates that GPX1 can effectively treat the occurrence of oxidative stress caused by myocardial infarction.

(20) GPX1 was able to reduce myocardial infarct size:

in a rat myocardial infarction model induced by 100mg/kg ISO, the ratio of the myocardial infarction area of a control group to the whole myocardial area is 2.59 +/-0.29 percent; the ratio of the myocardial infarction area of the GPX1 group to the whole myocardial area is 3.59 +/-0.46 percent; the ratio of the myocardial infarction area of the myocardial infarction model group to the whole myocardial area is 33.21 +/-2.06%, and the ratio of the GPX1+ ISO myocardial infarction area of the GPX1 group containing 4 mu g/kg/dayGPX1 to the whole myocardial area is 18.65 +/-1.26%; compared with the myocardial infarction model group, the myocardial infarction area of the GPX1+ ISO group is obviously reduced, which shows that GPX1 can effectively relieve the symptoms of myocardial infarction and reduce the myocardial infarction area.

(21) GPX1 is capable of ameliorating the pathological state of myocardial histomorphology following myocardial infarction:

in the 100mg/kg ISO-induced myocardial infarction model in rats, the myocardial tissue structures of the control group and the GPX1 group were normal. The myocardial cells of the myocardial infarction model group are arranged disorderly, and edema and more inflammatory cell infiltration appear. In the GPX1+ ISO group containing 4 mug/kg/dayGPX 1, a small amount of interstitial muscle is present, edema is present, and the degree of pathological changes is obviously weakened. This shows that GPX1 can effectively and obviously improve pathological changes caused by myocardial infarction.

(22) GPX4 can be used for treating abnormal increase of myocardial enzyme caused by myocardial infarction:

in a rat myocardial infarction model induced by 100mg/kg ISO, the activity of lactate dehydrogenase in the serum of a control group is 1000.8 +/-71.64U/L; the activity of lactate dehydrogenase in the serum of GPX4 group is 1002.9 +/-76.46U/L. The activity of lactate dehydrogenase in the serum of the myocardial infarction model group is 2643.16 +/-80.44U/L; the activity of lactate dehydrogenase in GPX4+ ISO group serum containing 4 mu g/kg/day GPX4 is 1743.71 +/-79.41U/L; the activity of lactate dehydrogenase was significantly reduced in GPX1+ ISO group serum compared to the myocardial infarction model group.

In a rat myocardial infarction model induced by 100mg/kg ISO, the activity of creatine kinase in the serum of a control group is 1000.4 +/-59.49U/L respectively; the activity of creatine kinase in the serum of the GPX4 group is 1001.49 +/-72.16U/L; the activity of creatine kinase in the serum of the myocardial infarction model group is 2230.2 +/-64.79U/L; the activity of creatine kinase in GPX4+ ISO group serum containing 4 mu g/kg/day GPX4 is 1522.56 +/-74.26U/L; compared with the myocardial infarction model group, the activity of creatine kinase in the serum of the GPX1+ ISO group is obviously reduced.

In a rat myocardial infarction model induced by 100mg/kg ISO, the activity of creatine kinase isoenzyme in the serum of a control group is 1005.26 +/-64.19U/L; the activity of creatine kinase isoenzyme in the serum of the GPX4 group is 1007.16 +/-59.46U/L; the activity of creatine kinase isozyme in the serum of the myocardial infarction model group is 2477.99 +/-82.13U/L, while the activity of creatine kinase isozyme in the serum of GPX4+ ISO group containing 4 mu g/kg/day GPX4 is 1566.26 +/-90.56U/L; compared with the myocardial infarction model group, the activity of creatine kinase isoenzyme in the serum of the GPX1+ ISO group is obviously reduced. This indicates that GPX4 is effective in treating abnormal elevation of myocardial enzymes caused by myocardial infarction.

(23) GPX4 is capable of ameliorating the pathological state of myocardial histomorphology following myocardial infarction:

in the 100mg/kg ISO-induced myocardial infarction model in rats, the myocardial tissue structures of the control group and the GPX4 group were normal. Compared with the control group and the GPX4 group, the myocardial cells of the myocardial infarction model group are disorderly arranged, and edema and more inflammatory cell infiltration appear. Compared with the myocardial infarction model group, the GPX4+ ISO group containing 4 mug/kg/day GPX4 has a small amount of interstitial tissues with edema, and the lesion degree is obviously weakened. This shows that GPX4 can effectively and obviously improve pathological changes caused by myocardial infarction.

Drawings

FIG. 1 is a SDS-PAGE electrophoresis and Western Blot of GPX1 and GPX4 applied in the present invention. Wherein FIG (a) is an SDS-PAGE electrophoresis of GPX 1; FIG. (b) is a Western Blot of GPX 1; FIG. (c) is an SDS-PAGE electrophoresis of GPX 4; FIG. (d) is a Western Blot of GPX 4; in an SDS-PAGE electrophoresis picture, M is a standard protein Marker, and the molecular weights are respectively 14.3kDa, 20.1kDa, 29.0kDa, 44.3kDa, 66.4kDa and 97.2 kDa; in the Western Blot image, M is a rainbow protein Marker, and the molecular weights are respectively 14kDa, 25kDa, 30kDa, 40kDa, 50kDa and 70 kDa;

FIG. 2 shows that GPX1 of the present invention prevents H₂O₂Cell viability map of induced H9C2 cell injury (note: compared to control,^###P<0.001; and H₂O₂Compared with the model group, the model group is compared,^***P<0.001；n＝6)；

FIG. 3 shows that GPX1 of the present invention prevents H₂O₂Graph of the lactate dehydrogenase release rate of induced H9C2 cell membrane damage (note: compared to control,^###P<0.001; and H₂O₂Compared with the model group, the model group is compared,^***P<0.001；n＝6)；

FIG. 4 shows that GPX1 of the present invention prevents H₂O₂Graph of malondialdehyde content in induced oxidative lipidation of H9C2 cells (note: compared to control,^###P<0.001; and H₂O₂Compared with the model group, the model group is compared,^***P<0.001；n＝6)；

FIG. 5 shows that GPX1 of the present invention prevents H₂O₂Graph of intracellular ROS levels of induced oxidative stress of H9C2 cells (note: compared to control,^###P<0.001; and H₂O₂Compared with the model group, the model group is compared,^***P<0.001；n＝3)；

FIG. 6 shows that GPX1 of the present invention prevents H₂O₂Apoptosis rate profile of induced apoptosis of H9C2 cells (note: compared to control,^###P<0.001; and H₂O₂Compared with the model group, the model group is compared,^***P<0.001；n＝3)；

FIG. 7 shows that GPX4 of the present invention prevents H₂O₂Graph of malondialdehyde content in induced oxidative lipidation of H9C2 cells (note: compared to control,^###P<0.001; and H₂O₂Compared with the model group, the model group is compared,^***P<0.001；n＝6)；

FIG. 8 shows that GPX4 of the present invention prevents H₂O₂Graph of intracellular ROS levels of induced oxidative stress of H9C2 cells (note: compared to control,^###P<0.001; and H₂O₂Compared with the model group, the model group is compared,^***P<0.001；n＝3)；

FIG. 9 shows that GPX4 of the present invention prevents H₂O₂Apoptosis rate profile of induced apoptosis of H9C2 cells (note: compared to control,^###P<0.001; and H₂O₂Compared with the model group, the model group is compared,^***P<0.001；n＝3)；

figure 10 is a graph of cell viability of GPX1 of the present invention for treatment of ISO-induced H9C2 cell injury (note: compared to control,^###P<0.001; in contrast to the ISO model set,^***P<0.001；n＝6)；

FIG. 11 is a graph showing the release rate of lactate dehydrogenase in GPX1 of the present invention for treating ISO-induced H9C2 cell membrane damage (note: compared to control,^###P<0.001; in contrast to the ISO model set,^***P<0.001；n＝6)；

figure 12 is a graph of malondialdehyde content of GPX1 of the present invention in treating ISO-induced oxidative lipidation of H9C2 cells (note: compared to control,^###P<0.001; in contrast to the ISO model set,^**P<0.01；n＝6)；

figure 13 is a graph of intracellular ROS levels of GPX1 of the present invention in treating ISO-induced oxidative stress of H9C2 cells (note: compared to control,^###P<0.001; in contrast to the ISO model set,^**P<0.01；n＝3)；

figure 14 is a graph of the apoptosis rate of GPX1 of the present invention for treatment of ISO-induced apoptosis of H9C2 (note: compared to control,^###P<0.001; in contrast to the ISO model set,^***P<0.001；n＝3)；

figure 15 is a graph of malondialdehyde content of GPX4 of the present invention in treating ISO-induced oxidative lipidation of H9C2 cells (note: compared to control,^###P<0.001; in contrast to the ISO model set,^**P<0.01；n＝6)；

figure 16 is a graph of intracellular ROS levels of GPX4 of the present invention in treating ISO-induced oxidative stress of H9C2 cells (note: compared to control,^###P<0.001; in contrast to the ISO model set,^***P<0.001；n＝3)；

figure 17 is a graph of the apoptosis rate of GPX4 of the present invention for treatment of ISO-induced apoptosis of H9C2 (note: compared to control,^###P<0.001; in contrast to the ISO model set,^***P<0.001；n＝3)；

FIG. 18 is a II-lead electrocardiogram of GPX1 of the present invention for treating ISO-induced myocardial infarction in rats;

figure 19 is an electrocardiogram of ST-mV from GPX1 of the invention for treating ISO-induced myocardial infarction in rats (note: compared to control,^###P<0.001; compared with the myocardial infarction model group,^**P<0.01；n＝6)；

figure 20 is a graph of lactate dehydrogenase in serum of GPX1 of the present invention in treating ISO-induced myocardial infarction in rats (note: compared to control,^###P<0.001; compared with the myocardial infarction model group,^***P<0.001；n＝6)；

figure 21 is a graph of creatine kinase in serum of GPX1 of the present invention treated for ISO-induced myocardial infarction in rats (note: compared to control,^###P<0.001; compared with the myocardial infarction model group,^***P<0.001；n＝6)；

figure 22 is a graph of creatine kinase isozyme in serum of GPX1 of the present invention in treating ISO-induced myocardial infarction in rats (note: compared to control,^###P<0.001; compared with the myocardial infarction model group,^***P<0.001；n＝6)；

figure 23 is a graph of the malondialdehyde content in serum of GPX1 of the present invention in the treatment of ISO-induced myocardial infarction in rats (note: compared to control,^###P<0.001; compared with the myocardial infarction model group,^**P<0.01；n＝6)；

figure 24 is a graph of the malondialdehyde content in heart tissue of GPX1 treated with ISO-induced myocardial infarction in rats of the invention (note: compared to control,^###P<0.001; compared with the myocardial infarction model group,^**P<0.01；n＝6)；

figure 25 is a graph of infarct size ratio of GPX1 treated with ISO-induced myocardial infarction in rats of the present invention (note: compared to control,^###P<0.001; compared with the myocardial infarction model group,^**P<0.01；n＝3)；；

FIG. 26 is a myocardial histomorphogram of GPX1 of the present invention for treating ISO-induced myocardial infarction in rats; the grade is 1, 2, 3 and 4, and the higher the grade is, the more serious the myocardial damage is;

figure 27 is a graph of lactate dehydrogenase in serum of GPX4 of the present invention in treating ISO-induced myocardial infarction in rats (note: compared to control,^###P<0.001; compared with the myocardial infarction model group,^***P<0.001；n＝6)；

figure 28 is a graph of creatine kinase in serum of GPX4 of the present invention treated ISO-induced myocardial infarction in rats (note: compared to control,^###P<0.001; compared with the myocardial infarction model group,^***P<0.001；n＝6)；

figure 29 is a graph of creatine kinase isozyme in serum of GPX4 of the present invention in treating ISO-induced myocardial infarction in rats (note: compared to control,^###P<0.001; compared with the myocardial infarction model group,^***P<0.001；n＝6)；

FIG. 30 is a cardiac histomorphogram of GPX4 of the present invention for treating ISO-induced myocardial infarction in rats; the grade is 1, 2, 3 and 4, and the higher the grade is, the more serious the myocardial damage is;

Detailed Description

Example 1 preparation of recombinant glutathione peroxidase 1(GPX1)

GPX1 was prepared according to the method of patent CN105018482A granted to this group of subjects, and the amino acid sequence, preparation and characterization method of GPX1 are described in detail in this patent. The invention identifies the target protein by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (Western blot), and the result is shown in figures 1(a) and (b), the molecular weight is 21.9kDa, and the protein is specifically combined with an anti-GPX 1 antibody, thereby proving that the human GPX1 protein is successfully obtained. The GPX activity is 27291U/mu mol protein, which is higher than that of GPX mutant reported previously.

Example 2 preparation of recombinant glutathione peroxidase 4(GPX4)

Preparing GPX4 according to the method of patent CN105018482A granted to the subject group, designing primers according to the gene sequence (see NCBI, NM-002085.3) of human GPX4 disclosed in a gene library to amplify the coding gene, ensuring that the 5 'end of the gene contains an initiation codon (ATG) and a Nde I enzyme cutting site, and the 3' end of the gene contains a termination codon and a Hind III enzyme cutting site; specifically, the 5 'primer was 5'-CCCATATGTGCGCGTCC-3'and the 3' primer was 5'-CCCAAGCTTTTAGAAATAGTGGGGCA-3'. After double digestion with restriction endonucleases Nde I and Hind III, the fragments were ligated with DNA ligase into the same digested pCold III (TAKARA, Cat. #3369) vector, which was pCold III-tRNA-GPx 4. Under the premise of keeping other amino acid sequences unchanged, two completely isometric complementary site-directed mutation primers are designed according to the gene sequence of the 46 Sec adjacent amino acid in GPX4, the length of the primers is 25-50bp, and the codon (TGA) of the 46 Sec is taken as the center. With these two perfectly complementary primers and a rapid site-directed mutagenesis kit (Invitrogen, operating according to the kit instructions), the coding sequence TGA of Sec No. 46 of the GPX4 gene constructed on the prokaryotic expression vector pCold III was mutated to amber codon (TGA), and the success of the mutation was confirmed by DNA sequencing without the occurrence of other accidental gene mutations. Then, the vector (pCold III-tRNA-GPX4) containing the hybrid tRNA and GPX4 gene is used to transform the competent cells of the UAG readonly engineering bacterium C321. delta. A.exp, and a nutrient agar plate containing 100. mu.g/mL Amp is coated to screen positive strains. Positive transformants were inoculated into 1.2L of nutrient agar medium (containing 100. mu.g/mL ampicillin and 50uM sodium selenite) and shake-cultured at 160rpm in an air bath at 37 ℃ to OD600 of 0.6. Placing the bacterial solution in a low-temperature shaking table at 30 ℃ and shaking at 80rpm for 5h, then adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 0.1mmol/L, and shaking and culturing at 30 ℃ for 5h to express GPX4 protein in a soluble form in a periplasm cavity of the bacteria. The cells were collected (6000rpm, 10min) and washed twice with equal volumes of buffer T (50mM Tris buffer, pH7.5, containing 1mM EDTA). The cell pellet was resuspended in buffer T, PMSF (phenylmethylsulfonyl fluoride) was added to a final concentration of 1mM, the cells were sonicated, centrifuged at 12000rpm at 4 ℃ for 30min, and the supernatant was collected. Treating GSH affinity column according to the instruction, using buffer (50mmol/L Tris, pH7.5) for equilibration, adding the supernatant to GSH affinity column, eluting the hetero-protein with equilibration buffer, and eluting the target protein with 10mmol/L GSH. The eluate was dialyzed and lyophilized to give human GPX4 protein. The invention identifies the target protein by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (Western blot), and the result is shown in figures 1(c) and (d), the molecular weight is 21.1kDa, and the protein is specifically combined with an anti-GPX 4 antibody, thereby proving that the human GPX4 protein is successfully obtained. The GPX activity is 805.6U/mu mol protein, which is higher than that of GPX mutant reported previously.

Example 3 GPX1 vs H₂O₂Prevention of induced H9C2 cell damage

H9C2 cells at 1 × 10⁵Inoculating each/mL into 96-well plate containing DMEM medium containing 100mL/L fetal calf serum, culturing in cell incubator, and dividing into 4 groups (control group, GPX1 group, H) when cells grow to 85% fusion₂O₂Model set and GPX1+ H₂O₂And (4) grouping. Control group was added with H₂O₂An equivalent amount of physiological saline; GPX1 of 0.1U/mL is added into the GPX1 group; h₂O₂Model group was added to a final concentration of 100. mu. mol/LH₂O₂Acting for 6 hours; GPX1+ H₂O₂The group was first treated with 0.1U/mL GPX1 for 1 hour, then with 100. mu. mol/LH₂O₂The reaction was carried out for 6 hours. Then adding 20 μ L of 5mg/mL MTT solution into each well, culturing for 4 hr, removing culture supernatant from each well after terminating the culture, adding 150 μ L dimethyl sulfoxide (DMSO) into each well to terminate the reaction, and shaking for 10min to make the water insolubleThe 490nm wavelength is selected, the absorbance value of each well is measured on an enzyme labeling instrument, and the cell survival rate of each group is calculated, namely the cell survival rate (%) (the OD value of the experimental group/the OD value of the control group) is × 100%.

As shown in FIG. 2, at 100. mu. mol/L H₂O₂In an induced H9C2 cell oxidative stress model, the cell survival rate of a control group is 100.19 +/-8.06%; the survival rate of the GPX1 group cells is 98.93 +/-6.49%; h₂O₂The survival rate of the model group cells is only 49.52 +/-10.23%; and GPX1+ H containing 0.1U/mL GPX1₂O₂The survival rate of the group cells is 78.17 +/-4.59 percent; it can be seen that GPX1+ H₂O₂The survival rate of the cells of the group is 1.58 times that of the H2O2 model group, which shows that GPX1 can effectively prevent the cell damage caused by oxidative stress.

Example 4 GPX1 vs. H₂O₂Prevention of induced H9C2 cell membrane damage

H9C2 cells at 1 × 10⁵Inoculating each/mL into 96-well plate containing DMEM medium containing 100mL/L fetal calf serum, culturing in cell incubator, and dividing into 4 groups (control group, GPX1 group, H) when cells grow to 85% fusion₂O₂Model set and GPX1+ H₂O₂And (4) grouping. Control group was added with H₂O₂An equivalent amount of physiological saline; GPX1 of 0.1U/mL is added into the GPX1 group; h₂O₂Model group was added to a final concentration of 100. mu. mol/L H₂O₂Acting for 6 hours; GPX1+ H₂O₂Group was first treated with 0.1U/mL GPX1 for 1 hour, then with 100. mu. mol/L H₂O₂The reaction was carried out for 6 hours. Then, using a lactate dehydrogenase kit (A020-2-2) of Nanjing Kangji corporation, the lactate dehydrogenase indicator in the cell supernatant was measured and expressed as the lactate dehydrogenase release rate according to the procedure described in the specification.

As shown in FIG. 3, at 100. mu. mol/L H₂O₂In an induced H9C2 cell oxidative stress model, the release rate of the lactate dehydrogenase of the cells of the control group is 99.31 +/-11.06%; the release rate of the GPX1 group cell lactate dehydrogenase is 99.11 +/-9.38%; h₂O₂The release rate of the lactate dehydrogenase of the model group cells is246.44 +/-17.27%; and GPX1+ H containing 0.1U/mLGPX1₂O₂The release rate of the group cell lactate dehydrogenase is 140 +/-13.33%; compared with H₂O₂Model group, GPX1+ H₂O₂The release rate of the histiocyte lactate dehydrogenase is obviously reduced, which shows that GPX1 can effectively prevent H₂O₂Inducing cell membrane damage caused by cellular oxidative stress.

Example 5 GPX1 vs. H₂O₂Prevention of oxidative lipidation of induced H9C2 cells

H9C2 cells at 1 × 10⁵Inoculating each/mL into 96-well plate containing DMEM medium containing 100mL/L fetal calf serum, culturing in cell incubator, and dividing into 4 groups (control group, GPX1 group, H) when cells grow to 85% fusion₂O₂Model set and GPX1+ H₂O₂And (4) grouping. Control group was added with H₂O₂An equivalent amount of physiological saline; GPX1 of 0.1U/mL is added into the GPX1 group; h₂O₂Model group was added to a final concentration of 100. mu. mol/L H₂O₂Acting for 6 hours; GPX1+ H₂O₂Group was first treated with 0.1U/mL GPX1 for 1 hour, then with 100. mu. mol/L H₂O₂The reaction was carried out for 6 hours. The malondialdehyde in the cells was then measured using the malondialdehyde assay kit (a003-4-1) from buika corporation as described in the specification and expressed as malondialdehyde content.

As shown in FIG. 4, at 100. mu. mol/L H₂O₂In the induced H9C2 cell oxidative stress model, the content of malondialdehyde in the cells of the control group is 1.26 +/-0.31 (nmol/mg pro); the malondialdehyde content of GPX1 group cells is 1.31 +/-0.25 (nmol/mgpro); h₂O₂The malondialdehyde content of model group cells is 5.27 +/-0.27 (nmol/mg pro); and GPX1+ H containing 0.1U/mLGPX1₂O₂The malondialdehyde content of the group cells is 3.01 +/-0.23 (nmol/mg pro); compared with H₂O₂Model group, GPX1+ H₂O₂The malondialdehyde content of the group cells is obviously reduced, which shows that GPX1 can effectively prevent H₂O₂Induce oxidative lipidation of cells.

Example 6 GPX1 vs. H₂O₂Induction ofPreventing effect of oxidative stress of H9C2 cells

H9C2 cells at 1 × 10⁵Inoculating each/mL into 6-well plate containing DMEM medium containing 100mL/L fetal calf serum, culturing in cell incubator, and dividing into 4 groups (control group, GPX1 group, H) when cells grow to 85% fusion₂O₂Model set and GPX1+ H₂O₂And (4) grouping. Control group was added with H₂O₂An equivalent amount of physiological saline; GPX1 of 0.1U/mL is added into the GPX1 group; h₂O₂Model group was added to a final concentration of 100. mu. mol/L H₂O₂Acting for 6 hours; GPX1+ H₂O₂Group was first treated with 0.1U/mL GPX1 for 1 hour, then with 100. mu. mol/L H₂O₂The reaction was carried out for 6 hours. Then, the procedure was followed using Biyuntian ROS detection kit (S0033S), and 10. mu.ml of DCFH-DA was added, and after incubation at room temperature for 10min, intracellular ROS production was observed under a fluorescent microscope and photographed. Relative fluorescence intensity was measured for each set of pictures using ImageJ 1.52 software.

As shown in FIG. 5, at 100. mu. mol/L H₂O₂In the induced H9C2 cell oxidative stress model, the relative fluorescence intensity of ROS in the cells of the control group is 1.01 +/-0.11; the relative fluorescence intensity of ROS in GPX1 group cells is 1.12 +/-0.15; h₂O₂The relative fluorescence intensity of ROS in the model group cells is 4.27 +/-0.32; and GPX1+ H containing 0.1U/mLGPX1₂O₂The relative fluorescence intensity of ROS in the group cells is 1.79 plus or minus 0.18; compared with H₂O₂Model group, GPX1+ H₂O₂The ROS in the group cells is obviously reduced relative to the fluorescence intensity, which shows that GPX1 can effectively prevent the ROS in the group cells from being combined with the H₂O₂Induces intracellular ROS production caused by cellular oxidative stress.

Example 7 GPX1 vs. H₂O₂Prevention of induced apoptosis of H9C2 cells

H9C2 cells at 1 × 10⁵Inoculating each/mL into 6-well plate containing DMEM medium containing 100mL/L fetal calf serum, culturing in cell incubator, and dividing into 4 groups (control group, GPX1 group, H) when cells grow to 85% fusion₂O₂Model set and GPX1+ H₂O₂And (4) grouping. Control group was added with H₂O₂An equivalent amount of physiological saline; GPX1 of 0.1U/mL is added into the GPX1 group; h₂O₂Model group was added to a final concentration of 100. mu. mol/L H₂O₂Acting for 6 hours; GPX1+ H₂O₂Group was first treated with 0.1U/mL GPX1 for 1 hour, then with 100. mu. mol/L H₂O₂The reaction was carried out for 6 hours. Then, H9C2 cells were collected and washed with a binding buffer by using Annexin V/PI apoptosis assay kit (556547) from BD company according to the procedures described in the specification. Then stained with 5. mu.L Annexin V-FITC and 10. mu.L PI staining solution and incubated for 15 min at room temperature in the absence of light. Finally, apoptotic cells were analyzed using flow cytometry. Apoptosis is expressed as the rate of apoptosis.

As shown in FIG. 6, at 100. mu. mol/L H₂O₂In an induced H9C2 cell oxidative stress model, the apoptosis rate of a control group cell is 4.89 +/-0.76%; the apoptosis rate of GPX1 group cells is 6.79 +/-0.86%; h₂O₂The apoptosis rate of model group cells is 53.45 +/-5.19%; and GPX1+ H containing 0.1U/mLGPX1₂O₂The apoptosis rate of the group cells is 21.37 +/-1.99 percent; compared with H₂O₂Model group, GPX1+ H₂O₂The apoptosis rate of the group cells is obviously reduced, which shows that GPX1 can effectively prevent H₂O₂Inducing apoptosis caused by cellular oxidative stress.

Example 8 GPX4 vs H₂O₂Prevention of oxidative lipidation of induced H9C2 cells

H9C2 cells at 1 × 10⁵Inoculating each/mL into 96-well plate containing DMEM medium containing 100mL/L fetal calf serum, culturing in cell incubator, and dividing into 4 groups (control group, GPX4 group, H) when cells grow to 85% fusion₂O₂Model set and GPX4+ H₂O₂And (4) grouping. Control group was added with H₂O₂An equivalent amount of physiological saline; GPX4 of 0.01U/mL is added into the GPX4 group; h₂O₂Model group was added to a final concentration of 100. mu. mol/L H₂O₂Acting for 6 hours; GPX4+ H₂O₂0.01U is added into the groupmL of GPX4 was allowed to act for 1 hour, then 100. mu. mol/L H was added₂O₂The reaction was carried out for 6 hours. Then, using Nanjing to build a malondialdehyde assay kit (A003-4-1), malondialdehyde in the cells was determined according to the procedures described in the specification and expressed as malondialdehyde content.

As shown in FIG. 7, at 100. mu. mol/L H₂O₂In the induced H9C2 cell oxidative stress model, the content of malondialdehyde in the cells of the control group is 1.23 +/-0.18 (nmol/mg pro); the content of malondialdehyde in GPX4 group cells is 1.29 +/-0.22 (nmol/mgpro); h₂O₂The malondialdehyde content of the model group cells is 4.79 +/-0.21 (nmol/mg pro); and GPX4+ H containing 0.01U/mLGPX4₂O₂The malondialdehyde content of the group cells was 2.49. + -. 0.28(nmol/mg pro); compared with H₂O₂Model group, GPX4+ H₂O₂The malondialdehyde content of the group cells is obviously reduced, which shows that GPX4 can effectively prevent H₂O₂Induce oxidative lipidation of cells.

Example 9 GPX4 vs. H₂O₂Prevention of oxidative stress induced in H9C2 cells

H9C2 cells at 1 × 10⁵Inoculating each/mL into 6-well plate containing DMEM medium containing 100mL/L fetal calf serum, culturing in cell incubator, and dividing into 4 groups (control group, GPX4 group, H) when cells grow to 85% fusion₂O₂Model set and GPX4+ H₂O₂And (4) grouping. Control group was added with H₂O₂An equivalent amount of physiological saline; GPX4 of 0.01U/mL is added into the GPX4 group; h₂O₂Model group was added to a final concentration of 100. mu. mol/L H₂O₂Acting for 6 hours; GPX4+ H₂O₂Group was first treated with 0.01U/mL GPX4 for 1 hour, then with 100. mu. mol/L H₂O₂The reaction was carried out for 6 hours. Then, the procedure was followed using Biyuntian ROS detection kit (S0033S), and 10. mu.ml of DCFH-DA was added, and after incubation at room temperature for 10min, intracellular ROS production was observed under a fluorescent microscope and photographed. Relative fluorescence intensity was measured for each set of pictures using ImageJ 1.52 software.

As shown in FIG. 8, at 100. mu. mol/L H₂O₂In the induced H9C2 cell oxidative stress model, the relative fluorescence intensity of ROS in the cells of the control group is 1.23 +/-0.41; the relative fluorescence intensity of ROS in GPX4 group cells is 1.33 +/-0.17; h₂O₂The relative fluorescence intensity of ROS in the model group cells is 3.86 +/-0.19; and GPX4+ H containing 0.01U/mL GPX4₂O₂The relative fluorescence intensity of ROS in the group cells is 1.95 plus or minus 0.14; compared with H₂O₂Model group, GPX4+ H₂O₂The ROS in the group cells is obviously reduced relative to the fluorescence intensity, which shows that GPX4 can effectively prevent the ROS in the group cells from being combined with the H₂O₂Induces intracellular ROS production caused by cellular oxidative stress.

Example 10 GPX4 vs H₂O₂Prevention of induced apoptosis of H9C2 cells

H9C2 cells at 1 × 10⁵Inoculating each/mL into 6-well plate containing DMEM medium containing 100mL/L fetal calf serum, culturing in cell incubator, and dividing into 4 groups (control group, GPX4 group, H) when cells grow to 85% fusion₂O₂Model set and GPX4+ H₂O₂And (4) grouping. Control group was added with H₂O₂An equivalent amount of physiological saline; GPX4 of 0.1U/mL is added into the GPX4 group; h₂O₂Model group was added to a final concentration of 100. mu. mol/L H₂O₂Acting for 6 hours; GPX4+ H₂O₂Group was first treated with 0.01U/mL GPX4 for 1 hour, then with 100. mu. mol/L H₂O₂The reaction was carried out for 6 hours. Then, H9C2 cells were collected and washed with a binding buffer by using Annexin V/PI apoptosis assay kit (556547) from BD company according to the procedures described in the specification. Then stained with 5. mu.L Annexin V-FITC and 10. mu.L PI staining solution and incubated for 15 min at room temperature in the absence of light. Finally, apoptotic cells were analyzed using flow cytometry. Apoptosis is expressed as the rate of apoptosis.

As shown in FIG. 9, at 100. mu. mol/L H₂O₂In an induced H9C2 cell oxidative stress model, the apoptosis rate of a control group cell is 5.02 +/-0.81%; the apoptosis rate of GPX4 group cells is 6.64 +/-0.92%; h₂O₂The apoptosis rate of model group cells is 45.96 +/-4.59%; and GPX4+ H containing 0.01U/mL GPX4₂O₂The apoptosis rate of the group cells was 23.06 + -4.59% compared with H₂O₂Model group, GPX4+ H₂O₂The apoptosis rate of the group cells is obviously reduced, which shows that GPX4 can effectively prevent H₂O₂Inducing apoptosis caused by cellular oxidative stress.

Example 11 therapeutic Effect of GPX1 on ISO-induced H9C2 cell injury

H9C2 cells at 1 × 10⁵The method comprises the steps of inoculating each cell/mL into a 96-well plate containing DMEM medium containing 100mL/L fetal calf serum, placing the 96-well plate into a cell constant temperature incubator for culture, dividing the 96-well plate into 4 groups, namely a control group, a GPX1 group, an ISO model group and a GPX1+ ISO group when the cells grow to 85% fusion, adding normal saline with the same amount as ISO into the control group, adding 50 mu mol/L ISO of the final concentration into the ISO model group for 24 hours, adding 50 mu mol/L ISO into the GPX1+ ISO group for 1 hour, adding 0.1U/mL GPX1 for 24 hours, adding 20 mu L of MTT solution into each well, continuing to culture for 4 hours, removing culture supernatant in each well after the culture is stopped, adding 150 mu L of dimethyl sulfoxide (DMSO) into each well for stopping reaction, shaking for 10 minutes, fully dissolving water-insoluble formazan crystals, selecting a wavelength of 490nm, measuring the absorbance of each well on an enzyme reader, and calculating the cell survival rate of each group (the cell survival rate) is 100 percent (OD × 100).

As shown in fig. 10, the cell survival rate of the control group was 101.05 ± 5.49% in the 50 μmol/L ISO-induced oxidative stress model of H9C2 cells; the survival rate of the GPX1 group cells is 99.16 +/-8.66%; the cell survival rate of the ISO model group is only 50.16 +/-8.26%; the cell survival rate of GPX1+ ISO group containing 0.1U/mL GPX1 is 80.17 +/-9.56%; it can be seen that the cell survival rate of the GPX1+ ISO group is 1.6 times that of the ISO model group, which indicates that GPX1 can effectively treat the oxidative stress injury of the cells induced by ISO.

Example 12 therapeutic Effect of GPX1 on ISO-induced cell membrane damage of H9C2 cells

H9C2 cells at 1 × 10⁵Inoculating each/mL into 96-well plate containing DMEM medium containing 100mL/L fetal calf serum, culturing in cell incubator, and dividing into 4 groups (control group, GPX1 group, and ISO model group) when cells grow to 85% fusionAnd GPX1+ ISO group. Adding physiological saline with the same quantity as ISO into a control group; adding ISO with a final concentration of 50 mu mol/L to the ISO model group for 24 hours; the GPX1+ ISO group was exposed to 50. mu. mol/L ISO for 1 hour and then to 0.1U/mL GPX1 for 24 hours. Then, using Nanjing as a built lactate dehydrogenase kit (A020-2-2), the lactate dehydrogenase indicator in the cell supernatant was measured and expressed by the lactate dehydrogenase release rate according to the procedures described in the specification.

As shown in FIG. 11, in the H9C2 cell oxidative stress model induced by 50 μmol/L ISO, the release rate of lactate dehydrogenase in the control cells was 98.75. + -. 9.19%; the release rate of the GPX1 group cell lactate dehydrogenase is 100.29 +/-10.01%; the release rate of the ISO model group cell lactate dehydrogenase is 226.31 +/-7.27%; the release rate of GPX1+ ISO group cell lactate dehydrogenase containing 0.1U/mLGPX1 is 131 +/-11.29%; compared with the ISO model group, the release rate of the GPX1+ ISO group cell lactate dehydrogenase is remarkably reduced, which shows that GPX1 can effectively treat cell membrane damage caused by ISO-induced cell oxidative stress.

Example 13 therapeutic Effect of GPX1 on ISO-induced oxidative lipidation of H9C2 cells

H9C2 cells at 1 × 10⁵The cells/mL are inoculated into a 96-well plate containing a DMEM medium containing 100mL/L fetal bovine serum, placed in a cell incubator for culture, and divided into 4 groups when the cells grow to 85% confluence, namely a control group, a GPX1 group, an ISO model group and a GPX1+ ISO group. Adding physiological saline with the same quantity as ISO into a control group; adding ISO with a final concentration of 50 mu mol/L to the ISO model group for 24 hours; the GPX1+ ISO group was exposed to 50. mu. mol/L ISO for 1 hour and then to 0.1U/mL GPX1 for 24 hours. Then, using Nanjing to build a malondialdehyde assay kit (A003-4-1), malondialdehyde in the cells was determined according to the procedures described in the specification and expressed as malondialdehyde content.

As shown in FIG. 12, in the 50. mu. mol/L ISO-induced oxidative stress model of H9C2 cells, the malondialdehyde content in the control cells was 1.18. + -. 0.29(nmol/mg pro); the malondialdehyde content of the GPX1 group cells is 1.27 +/-0.33 (nmol/mgpro); the content of malondialdehyde in the cells of the ISO model group is 6.14 +/-0.31 (nmol/mg pro); and the content of malondialdehyde in GPX1+ ISO group cells containing 0.1U/mLGPX1 is 3.35 +/-0.43 (nmol/mg pro); compared with the ISO model group, the content of malondialdehyde in the cells of the GPX1+ ISO group is remarkably reduced, which shows that GPX1 can effectively treat oxidative lipidation of the cells induced by ISO.

Example 14 therapeutic Effect of GPX1 on ISO-induced oxidative stress of H9C2 cells

H9C2 cells at 1 × 10⁵each/mL of the cells were inoculated into 6-well plates of DMEM medium containing 100mL/L fetal bovine serum, cultured in a cell incubator, and when the cells grew to 85% confluence, they were divided into 4 groups, i.e., a control group, a GPX1 group, an ISO model group, and a GPX1+ ISO group. Adding physiological saline with the same quantity as ISO into a control group; adding ISO with a final concentration of 50 mu mol/L to the ISO model group for 24 hours; the GPX1+ ISO group was exposed to 50. mu. mol/L ISO for 1 hour and then to 0.1U/mL GPX1 for 24 hours. Then using Biyuntian ROS detection kit (S0033S), the procedure was as described in the specification, 10. mu.ml of DCFH-DA was added, and after incubation for 10 minutes at room temperature, intracellular ROS production was observed under a fluorescent microscope and photographed. Relative fluorescence intensity was measured for each set of pictures using ImageJ 1.52 software.

As shown in FIG. 13, the relative fluorescence intensity of ROS in control cells was 1.02. + -. 0.21 in the 50. mu. mol/L ISO-induced oxidative stress model of H9C2 cells; the relative fluorescence intensity of ROS in GPX1 group cells is 1.22 +/-0.13; the relative fluorescence intensity of ROS in the ISO model group cells is 5.43 +/-0.41; and GPX1+ H containing 0.1U/mLGPX1₂O₂The relative fluorescence intensity of ROS in the group cells is 2.28 +/-0.37; the relative fluorescence intensity of intracellular ROS was significantly reduced in the GPX1+ ISO group compared to the ISO model group, indicating that GPX1 was effective in treating intracellular ROS generation caused by ISO-induced cellular oxidative stress.

Example 15 therapeutic Effect of GPX1 on ISO-induced apoptosis of H9C2 cells

H9C2 cells at 1 × 10⁵each/mL of the cells were inoculated into 6-well plates of DMEM medium containing 100mL/L fetal bovine serum, cultured in a cell incubator, and when the cells grew to 85% confluence, they were divided into 4 groups, i.e., a control group, a GPX1 group, an ISO model group, and a GPX1+ ISO group. Adding physiological saline with the same quantity as ISO into a control group; adding ISO with a final concentration of 50 mu mol/L to the ISO model group for 24 hours; GPX1+ ISO group is added with 50 mu mol/L ISOAfter 1 hour of action, 0.1U/mL GPX1 was added for 24 hours of action. The H9C2 cells were then harvested using the Annexin V/PI apoptosis detection kit from BD (556547) following the procedure described in the specification, and the cells were washed with binding buffer. Then stained with 5. mu.L Annexin V-FITC and 10. mu.L PI staining solution and incubated for 15 min at room temperature in the absence of light. Finally, apoptotic cells were analyzed using flow cytometry. Apoptosis of H9C2 was expressed as the rate of apoptosis.

As shown in fig. 14, the apoptosis rate of the control group was 5.76 ± 0.94% in the H9C2 cell oxidative stress model induced by 50 μmol/L ISO; the apoptosis rate of GPX1 group cells is 6.09 +/-0.58%; the apoptosis rate of ISO model group cells is 62.79 +/-4.09%; the apoptosis rate of GPX1+ ISO group containing 0.1U/mLGPX1 is 32.13 +/-4.29%; compared with the ISO model group, the apoptosis rate of GPX1+ ISO group cells is remarkably reduced, which shows that GPX1 can effectively treat apoptosis caused by ISO-induced oxidative stress of cells.

Example 16 therapeutic Effect of GPX4 on ISO-induced oxidative lipidation of H9C2 cells

H9C2 cells at 1 × 10⁵The cells/mL are inoculated into a 96-well plate containing a DMEM medium containing 100mL/L fetal bovine serum, placed in a cell incubator for culture, and divided into 4 groups when the cells grow to 85% confluence, namely a control group, a GPX4 group, an ISO model group and a GPX4+ ISO group. Adding physiological saline with the same quantity as ISO into a control group; adding ISO with a final concentration of 50 mu mol/L to the ISO model group for 24 hours; the GPX4+ ISO group was exposed to 50. mu. mol/L ISO for 1 hour and then to 0.01U/mL GPX4 for 24 hours. Then, using Nanjing to build a malondialdehyde assay kit (A003-4-1), malondialdehyde in the cells was determined according to the procedures described in the specification and expressed as malondialdehyde content.

As shown in FIG. 15, in the 50. mu. mol/L ISO-induced oxidative stress model of H9C2 cells, the malondialdehyde content in the control cells was 1.28. + -. 0.3(nmol/mg pro); the content of malondialdehyde in GPX4 group cells is 1.34 +/-0.42 (nmol/mg pro); the content of malondialdehyde in the cells of the ISO model group is 5.79 +/-0.49 (nmol/mg pro); while the content of malondialdehyde in GPX4+ ISO group cells containing 0.01U/mL GPX4 is 3.26 +/-0.52 (nmol/mg pro); compared with the ISO model group, the content of malondialdehyde in the cells of the GPX4+ ISO group is remarkably reduced, which shows that GPX4 can effectively treat oxidative lipidation of the cells induced by ISO.

Example 17 therapeutic Effect of GPX4 on ISO-induced oxidative stress of H9C2 cells

H9C2 cells at 1 × 10⁵each/mL of the cells were inoculated into 6-well plates of DMEM medium containing 100mL/L fetal bovine serum, cultured in a cell incubator, and when the cells grew to 85% confluence, they were divided into 4 groups, i.e., a control group, a GPX4 group, an ISO model group, and a GPX4+ ISO group. Adding physiological saline with the same quantity as ISO into a control group; adding ISO with a final concentration of 50 mu mol/L to the ISO model group for 24 hours; the GPX4+ ISO group was exposed to 50. mu. mol/L ISO for 1 hour and then to 0.01U/mL GPX4 for 24 hours. Then using Biyuntian ROS detection kit (S0033S), the procedure was as described in the specification, 10. mu.ml of DCFH-DA was added, and after incubation for 10 minutes at room temperature, intracellular ROS production was observed under a fluorescent microscope and photographed. Relative fluorescence intensity was measured for each set of pictures using ImageJ 1.52 software.

As shown in FIG. 16, the relative fluorescence intensity of ROS in control group cells was 1.19. + -. 0.18 in the 50. mu. mol/L ISO-induced oxidative stress model of H9C2 cells; the relative fluorescence intensity of ROS in GPX4 group cells is 1.27 +/-0.2; the relative fluorescence intensity of ROS in the ISO model group cells was 4.89. + -. 0.23, while GPX4+ H containing 0.01U/mL GPX4₂O₂The relative fluorescence intensity of ROS in the group cells is 2.01 +/-0.34, compared with that in an ISO model group, the relative fluorescence intensity of ROS in GPX4+ ISO group cells is obviously reduced, and the fact that GPX4 can effectively treat the generation of ROS in the cells caused by ISO-induced cellular oxidative stress is shown.

Example 18 therapeutic Effect of GPX4 on ISO-induced apoptosis of H9C2 cells

H9C2 cells at 1 × 10⁵each/mL of the cells were inoculated into 6-well plates of DMEM medium containing 100mL/L fetal bovine serum, cultured in a cell incubator, and when the cells grew to 85% confluence, they were divided into 4 groups, i.e., a control group, a GPX4 group, an ISO model group, and a GPX4+ ISO group. Adding physiological saline with the same quantity as ISO into a control group; adding ISO with a final concentration of 50 mu mol/L to the ISO model group for 24 hours; the GPX4+ ISO group was exposed to 50. mu. mol/L ISO for 1 hour and then to 0.01U/mL GPX4 for 24 hours. Then use BD CoAnnexin V/PI apoptosis detection kit (556547), which was performed according to the procedures described in the specification, H9C2 cells were harvested and then washed with binding buffer. Then stained with 5. mu.L Annexin V-FITC and 10. mu.L PI staining solution and incubated for 15 min at room temperature in the absence of light. Finally, apoptotic cells were analyzed using flow cytometry. Apoptosis of H9C2 was expressed as the rate of apoptosis.

As shown in fig. 17, the apoptosis rate of the control group was 6.06 ± 0.83% in the H9C2 cell oxidative stress model induced by 50 μmol/L ISO; the apoptosis rate of GPX4 group cells is 7.19 +/-0.93%; the apoptosis rate of the ISO model group cells is 58.36 +/-3.09%; and the apoptosis rate of GPX4+ ISO group containing 0.01U/mL GPX4 is 28.56 +/-3.33%, and compared with the apoptosis rate of GPX4+ ISO group, the apoptosis rate of GPX4+ ISO group is obviously reduced, which shows that GPX4 can effectively treat apoptosis caused by ISO-induced oxidative stress of cells.

Example 19 therapeutic Effect of GPX1 on ISO-induced abnormalities in the electrocardiogram of myocardial infarction in rats

200-250g male (Sprague-Dawley) SD rats were randomly housed in groups in a temperature-suitable environment with a 12h light/dark cycle, and the rats were allowed free access to food and water and acclimatized for 7 days. Rats were divided into 4 groups, i.e., control group, GPX1 group, myocardial infarction model group, and GPX1+ ISO group. The control group was rats injected with physiological saline subcutaneously for 2 consecutive days; the GPX1 group was rats subcutaneously injected with 4 μ g/kg/day of GPX1 (1 μ g/kg per dose, 6 hours apart, 4 times a day, corresponding to a daily intake of approximately 280 μ g of adult selenium) for 2 consecutive days; the myocardial infarction model group is that rats are injected with 100mg/kg ISO subcutaneously for 2 consecutive days; the GPX1+ ISO group was administered continuously for 2 days, with rats being subcutaneously injected with 100mg/kg of ISO for 1 hour each day followed by 4 μ g/kg/day of GPX1 (1 μ g/kg for each dose administered, 4 times a day at 6 hour intervals, corresponding to a daily intake of selenium of approximately 280 μ g for adults), and after the last subcutaneous injection of ISO24h, rats were anesthetized with 50mg/kg of sodium pentobarbital, and the standard limb leads were used for testing and recording II-lead electrocardiograms of the rats.

As shown in FIGS. 18 and 19, in the 100mg/kg ISO-induced rat infarction model, the ST-segment mV of the electrocardiogram of the control group is 0.05 + -0.01 mV; the mV of ST section of the electrocardiogram ST of the GPX1 group is 0.06 +/-0.02 mV; the electrocardiogram ST section mV of the myocardial infarction model group is 0.5 +/-0.08 mV; while the mV of ST segment of the electrocardiogram of GPX1+ ISO group containing 4 mu g/kg/day GPX1 is 0.3 +/-0.08 mV, so that GPX1 can obviously repair the abnormality of the ST segment of the electrocardiogram, which shows that GPX1 can effectively treat the electrocardiogram abnormality caused by myocardial infarction.

Example 20 therapeutic Effect of GPX1 on serum Biochemical indicators of ISO-induced myocardial infarction in rats

200-250g male (Sprague-Dawley) SD rats were randomly housed in groups in a temperature-suitable environment with a 12h light/dark cycle, and the rats were allowed free access to food and water and acclimatized for 7 days. Rats were divided into 4 groups, i.e., control group, GPX1 group, myocardial infarction model group, and GPX1+ ISO group. The control group was rats injected with physiological saline subcutaneously for 2 consecutive days; the GPX1 group was rats subcutaneously injected with 4 μ g/kg/day of GPX1 (1 μ g/kg per dose, 6 hours apart, 4 times a day, corresponding to a daily intake of approximately 280 μ g of adult selenium) for 2 consecutive days; the myocardial infarction model group is that rats are injected with 100mg/kg ISO subcutaneously for 2 consecutive days; the GPX1+ ISO group was administered continuously for 2 days, after injecting 100mg/kg of ISO subcutaneously into rats every day for 1 hour, 4. mu.g/kg/day of GPX1 was injected subcutaneously (each dose was 1. mu.g/kg, administered every 6 hours, 4 times a day, corresponding to about 280. mu.g of selenium daily intake for adults), fresh blood was obtained from rats by abdominal aortic hemorrhaging after the last subcutaneous injection of ISO24h, and after the blood was left at 37 ℃ for 1 hour, serum was obtained from rats by centrifugation and used for the measurement of serum biochemical indicators, including lactate dehydrogenase, creatine kinase and creatine kinase isoenzymes. Serum biochemical indexes are detected by establishing a lactate dehydrogenase kit (A020-2-2), a creatine kinase kit (A032-1-1) and a creatine kinase isozyme kit (E006-1-1) through Nanjing according to the steps described in the specification.

As shown in FIG. 20, the lactate dehydrogenase activity in the serum of the control group was 1002.26. + -. 59.79U/L in the 100mg/kg ISO-induced myocardial infarction model in rats; the activity of lactate dehydrogenase in the serum of GPX1 group is 1013.29 +/-79.56U/L. The activity of lactate dehydrogenase in the serum of the myocardial infarction model group is 2516.29 +/-75.09U/L, while the activity of lactate dehydrogenase in the serum of GPX1+ ISO group containing 4 mu g/kg/dayGPX1 is 1501.21 +/-77.05U/L, and compared with the serum of the myocardial infarction model group, the activity of lactate dehydrogenase in the serum of GPX1+ ISO group is obviously reduced.

As shown in FIG. 21, the creatine kinase activity in the serum of the control group was 1001.97 + -49.29U/L in the 100mg/kg ISO-induced myocardial infarction model of rats; the activity of creatine kinase in the serum of GPX1 group is 1005.69 +/-62.09U/L; the activity of creatine kinase in the serum of the myocardial infarction model group is 2249.51 +/-87.29U/L; and the creatine kinase activity in GPX1+ ISO group serum containing 4 mu g/kg/dayGPX1 is 1426.59 +/-89.59U/L, and compared with that in the myocardial infarction model group, the creatine kinase activity in GPX1+ ISO group serum is obviously reduced.

As shown in FIG. 22, the creatine kinase isoenzyme activity in the serum of the control group was 1000.59 + -59.49U/L in the 100mg/kg ISO-induced myocardial infarction model of rats; the activity of creatine kinase isoenzyme in the serum of the GPX1 group is 1003.26 +/-79.89U/L; the activity of creatine kinase isoenzyme in the serum of the myocardial infarction model group is 2355.99 +/-81.07U/L; the activity of creatine kinase isoenzyme in GPX1+ ISO group serum containing 4 mu g/kg/day GPX1 is 1463.3 +/-87.09U/L; compared with the myocardial infarction model group, the activity of creatine kinase isoenzyme in the serum of the GPX1+ ISO group is obviously reduced.

These results indicate that GPX1 can effectively treat abnormal increase of lactate dehydrogenase, creatine kinase and creatine kinase isoenzyme caused by myocardial infarction, and protect myocardial cells.

Example 21 therapeutic effect of GPX1 on ISO-induced oxidative stress in myocardial infarction in rats.

200-250g male (Sprague-Dawley) SD rats were randomly housed in groups in a temperature-suitable environment with a 12h light/dark cycle, and the rats were allowed free access to food and water and acclimatized for 7 days. Rats were divided into 4 groups, i.e., control group, GPX1 group, myocardial infarction model group, and GPX1+ ISO group. The control group was rats injected with physiological saline subcutaneously for 2 consecutive days; the GPX1 group was rats subcutaneously injected with 4 μ g/kg/day of GPX1 (1 μ g/kg per dose, 6 hours apart, 4 times a day, corresponding to a daily intake of approximately 280 μ g of adult selenium) for 2 consecutive days; the myocardial infarction model group is that rats are injected with 100mg/kg ISO subcutaneously for 2 consecutive days; the GPX1+ ISO group was administered for 2 consecutive days, with rats being given 100mg/kg of ISO at 1 hour per day followed by 4 μ g/kg/day of GPX1 subcutaneously (1 μ g/kg for each dose given, 4 times per day with 6 hour intervals, corresponding to a daily intake of approximately 280 μ g of adult selenium), and after the last subcutaneous injection of ISO24h, rat serum and heart tissue were obtained and prepared as a tissue homogenate. The malondialdehyde in serum and heart tissue was measured by the malondialdehyde assay kit (A003-4-1) from Nanjing construction company according to the procedures described in the specification and expressed as malondialdehyde content.

As shown in FIG. 23, in the 100mg/kg ISO-induced myocardial infarction model in rats, the content of malondialdehyde in the serum of the control group was 8.59. + -. 0.59(nmol/mL pro); the content of malondialdehyde in the serum of GPX1 group is 8.26 +/-0.75 (nmol/mL pro); the content of malondialdehyde in the serum of the myocardial infarction model group is 23.69 +/-1.56 (nmol/mL pro); while the content of malondialdehyde in the serum of GPX1+ ISO group containing 4. mu.g/kg/day GPX1 was 14.26. + -. 1.09(nmol/mL pro); compared with the myocardial infarction model group, the content of malondialdehyde in serum of GPX1+ ISO group is obviously reduced.

As shown in FIG. 24, in the 100mg/kg ISO-induced myocardial infarction model in rats, the malondialdehyde content in the heart tissue of the control group was 2.34. + -. 0.21(nmol/mg pro); the malondialdehyde content in heart tissue of GPX1 group was 2.43. + -. 0.35(nmol/mg pro); the content of malondialdehyde in the myocardial infarction model group tissue is 6.29 +/-0.43 (nmol/mg pro); while the content of malondialdehyde in the tissue of GPX1+ ISO group containing 4 mug/kg/day GPX1 is 4.12 +/-0.33 (nmol/mg pro); compared with the myocardial infarction model group, the content of malondialdehyde in the tissue of the GPX1+ ISO group is obviously reduced, which indicates that GPX1 can effectively treat oxidative stress caused by myocardial infarction.

Example 22 therapeutic Effect of GPX1 on ISO-induced myocardial infarct size in rats

200-250g male (Sprague-Dawley) SD rats were randomly housed in groups in a temperature-suitable environment with a 12h light/dark cycle, and the rats were allowed free access to food and water and acclimatized for 7 days. Rats were divided into 4 groups, i.e., control group, GPX1 group, myocardial infarction model group, and GPX1+ ISO group. The control group was rats injected with physiological saline subcutaneously for 2 consecutive days; the GPX1 group was rats subcutaneously injected with 4 μ g/kg/day of GPX1 (1 μ g/kg per dose, 6 hours apart, 4 times a day, corresponding to a daily intake of approximately 280 μ g of adult selenium) for 2 consecutive days; the myocardial infarction model group is that rats are injected with 100mg/kg ISO subcutaneously for 2 consecutive days; the GPX1+ ISO group was administered for 2 consecutive days, rats were injected subcutaneously with 100mg/kg of ISO for 1 hour each day followed by 4 μ g/kg/day of GPX1 (each dose was 1 μ g/kg, administered 4 times a day at 6 hour intervals, corresponding to a daily intake of selenium of approximately 280 μ g for adults), rat heart tissue was obtained after the last subcutaneous injection of ISO24h, fresh rat heart tissue was first placed in a-20 ℃ freezer, frozen for 30 minutes, after which rat heart was cross-sectioned to a thickness of 2mm and stained with 1% TTC for 20 minutes at 37 ℃ and then fixed with 4% paraformaldehyde. Heart sections were taken using a camera and infarct size was measured using ImageJ 1.52 software.

As shown in fig. 25, the ratio of the myocardial infarction area of the control group to the entire myocardial area was 2.59 ± 0.29% in the 100 mg/kgISO-induced myocardial infarction model of rats; the ratio of the myocardial infarction area of the GPX1 group to the whole myocardial area is 3.59 +/-0.46 percent; the ratio of the myocardial infarction area of the myocardial infarction model group to the whole myocardial area is 33.21 +/-2.06%, and the ratio of the GPX1+ ISO myocardial infarction area of the GPX1 containing 4 mu g/kg/day GPX1 to the whole myocardial area is 18.65 +/-1.26%; compared with the myocardial infarction model group, the myocardial infarction area of the GPX1+ ISO group is obviously reduced, which shows that GPX1 can effectively relieve the symptoms of myocardial infarction and reduce the myocardial infarction area.

Example 23 therapeutic Effect of GPX1 on ISO-induced myocardial tissue morphology in myocardial infarcted rats

200-250g male (Sprague-Dawley) SD rats were randomly housed in groups in a temperature-suitable environment with a 12h light/dark cycle, and the rats were allowed free access to food and water and acclimatized for 7 days. Rats were divided into 4 groups, i.e., control group, GPX1 group, myocardial infarction model group, and GPX1+ ISO group. The control group was rats injected with physiological saline subcutaneously for 2 consecutive days; the GPX1 group was rats subcutaneously injected with 4 μ g/kg/day of GPX1 (1 μ g/kg per dose, 6 hours apart, 4 times a day, corresponding to a daily intake of approximately 280 μ g of adult selenium) for 2 consecutive days; the myocardial infarction model group is that rats are injected with 100mg/kg ISO subcutaneously for 2 consecutive days; the GPX1+ ISO group was administered for 2 consecutive days, with rats being subcutaneously injected with 100mg/kg of ISO for 1 hour each day followed by 4 μ g/kg/day of GPX1 (1 μ g/kg for each dose administered 4 times a day, 6 hour apart, corresponding to a daily intake of approximately 280 μ g of selenium for adults), with rat heart tissue obtained after the last subcutaneous injection of ISO24h and soaked overnight in 4% paraformaldehyde before being embedded in paraffin to form tissue blocks. It was then cut into 5 μm tissue sections using a paraffin microtome. Finally, tissue sections were stained with hematoxylin-eosin (HE) and observed under a light microscope and evaluated for myocardial severity as necrosis, inflammatory cell infiltration and edema.

As shown in FIG. 26, in the 100mg/kg ISO-induced myocardial infarction model in rats, the myocardial tissue structures of the control group and the GPX1 group were normal. The myocardial cells of the myocardial infarction model group are arranged disorderly, and edema and more inflammatory cell infiltration appear. In the GPX1+ ISO group containing 4 mug/kg/dayGPX 1, a small amount of interstitial muscle is present, edema is present, and the degree of pathological changes is obviously weakened. This indicates that GPX1 can effectively treat pathological changes caused by myocardial infarction.

Example 24 therapeutic Effect of GPX4 on serum Biochemical indicators of ISO-induced myocardial infarction in rats

200-250g male (Sprague-Dawley) SD rats were randomly housed in groups in a temperature-suitable environment with a 12h light/dark cycle, and the rats were allowed free access to food and water and acclimatized for 7 days. Rats were divided into 4 groups, i.e., control group, GPX4 group, myocardial infarction model group, and GPX4+ ISO group. The control group was rats injected with physiological saline subcutaneously for 2 consecutive days; the GPX4 group was rats subcutaneously injected with 4 μ g/kg/day of GPX4 (1 μ g/kg per dose, 6 hours apart, 4 times a day, corresponding to a daily intake of approximately 280 μ g of adult selenium) for 2 consecutive days; the myocardial infarction model group is that rats are injected with 100mg/kg ISO subcutaneously for 2 consecutive days; the GPX4+ ISO group was administered continuously for 2 days, after injecting 100mg/kg of ISO subcutaneously into rats every day for 1 hour, 4. mu.g/kg/day of GPX4 was injected subcutaneously (each dose was 1. mu.g/kg, administered every 6 hours, 4 times a day, corresponding to about 280. mu.g of selenium daily intake for adults), fresh blood was obtained from rats by abdominal aortic hemorrhaging after the last subcutaneous injection of ISO24h, and after the blood was left at 37 ℃ for 1 hour, serum was obtained from rats by centrifugation and used for the measurement of serum biochemical indicators, including lactate dehydrogenase, creatine kinase and creatine kinase isoenzymes. Serum biochemical indexes are detected by establishing a lactate dehydrogenase kit (A020-2-2), a creatine kinase kit (A032-1-1) and a creatine kinase isozyme kit (E006-1-1) through Nanjing according to the steps described in the specification.

As shown in FIG. 27, the lactate dehydrogenase activity in the serum of the control group was 1000.8. + -. 71.64U/L in the 100mg/kg ISO-induced myocardial infarction model in rats; the activity of lactate dehydrogenase in the serum of GPX4 group is 1002.9 +/-76.46U/L. The activity of lactate dehydrogenase in the serum of the myocardial infarction model group is 2643.16 +/-80.44U/L; the activity of lactate dehydrogenase in GPX4+ ISO group serum containing 4 mu g/kg/dayGPX4 is 1743.71 +/-79.41U/L; the activity of lactate dehydrogenase was significantly reduced in GPX1+ ISO group serum compared to the myocardial infarction model group.

As shown in FIG. 28, the creatine kinase activity in the serum of the control group was 1000.4 + -59.49U/L, respectively, in the 100mg/kg ISO-induced myocardial infarction model in rats; the activity of creatine kinase in the serum of the GPX4 group is 1001.49 +/-72.16U/L; the activity of creatine kinase in the serum of the myocardial infarction model group is 2230.2 +/-64.79U/L; the activity of creatine kinase in GPX4+ ISO group serum containing 4 mu g/kg/day GPX4 is 1522.56 +/-74.26U/L; compared with the myocardial infarction model group, the activity of creatine kinase in the serum of the GPX1+ ISO group is obviously reduced.

As shown in FIG. 29, the creatine kinase isoenzyme activity in the serum of the control group was 1005.26. + -. 64.19U/L in the 100mg/kg ISO-induced myocardial infarction model in rats; the activity of creatine kinase isoenzyme in the serum of the GPX4 group is 1007.16 +/-59.46U/L; the activity of creatine kinase isozyme in the serum of the myocardial infarction model group is 2477.99 +/-82.13U/L, while the activity of creatine kinase isozyme in the serum of GPX4+ ISO group containing 4 mu g/kg/dayGPX4 is 1566.26 +/-90.56U/L; compared with the myocardial infarction model group, the activity of creatine kinase isoenzyme in the serum of the GPX1+ ISO group is obviously reduced.

These results indicate that GPX4 can effectively treat abnormal increase of lactate dehydrogenase, creatine kinase and creatine kinase isoenzyme caused by myocardial infarction, and protect myocardial cells.

Example 25 therapeutic Effect of GPX4 on ISO-induced myocardial tissue morphology in myocardial infarcted rats

200-250g male (Sprague-Dawley) SD rats were randomly housed in groups in a temperature-suitable environment with a 12h light/dark cycle, and the rats were allowed free access to food and water and acclimatized for 7 days. Rats were divided into 4 groups, i.e., control group, GPX4 group, myocardial infarction model group, and GPX4+ ISO group. The control group was rats injected with physiological saline subcutaneously for 2 consecutive days; the GPX4 group was rats subcutaneously injected with 4 μ g/kg/day of GPX4 (1 μ g/kg per dose, 6 hours apart, 4 times a day, corresponding to a daily intake of approximately 280 μ g of adult selenium) for 2 consecutive days; the myocardial infarction model group is that rats are injected with 100mg/kg ISO subcutaneously for 2 consecutive days; the GPX4+ ISO group was administered for 2 consecutive days, with rats being subcutaneously injected with 100mg/kg of ISO for 1 hour each day followed by 4 μ g/kg/day of GPX4 (1 μ g/kg for each dose, administered 4 times a day at 6 hour intervals, corresponding to a daily intake of approximately 280 μ g of selenium for adults), after the last subcutaneous injection of ISO24h heart tissue was obtained and soaked overnight in 4% paraformaldehyde before being embedded in paraffin to form tissue blocks. It was then cut into 5 μm tissue sections using a paraffin microtome. Finally, tissue sections were stained with hematoxylin-eosin (HE) and observed under a light microscope and evaluated for myocardial severity as necrosis, inflammatory cell infiltration and edema.

As shown in FIG. 30, the myocardial tissue structures of the control group and the GPX4 group were normal in the 100mg/kg ISO-induced myocardial infarction model in rats. The myocardial cells of the myocardial infarction model group are arranged disorderly, and edema and more inflammatory cell infiltration appear. Compared with the myocardial infarction model group, the GPX4+ ISO group containing 4 mug/kg/dayGPX 4 has a small amount of interstitial tissues with edema, and the lesion degree is obviously weakened. This indicates that GPX4 can effectively treat pathological changes caused by myocardial infarction.

Claims

1. The application of the recombinant human glutathione peroxidase GPX in preparing the antioxidant and anti-aging health care product.

2. The application of the recombinant human glutathione peroxidase GPX in preparing the anti-oxidation and anti-aging drugs.

3. Application of recombinant human glutathione peroxidase GPX in preparation of anti-myocardial infarction medicaments.