Application of selenium element in relieving hyperglycemia cerebral ischemia reperfusion injury

Abstract

The invention relates to the technical field of biological medicine, in particular to application of selenium element in relieving hyperglycemia cerebral ischemia reperfusion injury, provides application of selenium element in relieving hyperglycemia cerebral ischemia reperfusion injury, and provides application of selenium element in relieving hyperglycemia cerebral ischemia reperfusion injury in relieving hyperglycemia cerebral ischemia reperfusion injury in relieving hyperglycemia cerebral ischemia reperfusion injury in relieving hyperglycemia cerebral ischemia reperfusion injury in relieving hyperglycemia cerebral ischemia reperfusion injury. According to the application of the cerebral ischemia-reperfusion injury relieving medicine for treating the cerebral ischemia-reperfusion injury, the mitochondrial division and fusion process is kept relatively balanced, and the activation of a mitochondrial apoptosis pathway is inhibited, so that the cerebral ischemia-reperfusion injury of a patient in the hyperglycemia state is relieved. Therefore, the cerebral ischemia reperfusion injury is aggravated; the selenium element can regulate and control a mitochondrial regeneration signal channel to improve the functions of mitochondria, so that the mitochondrial division and fusion processes are kept relatively balanced, and the activation of a mitochondrial apoptosis channel is inhibited, so that the cerebral ischemia reperfusion injury of a patient in a hyperglycemia state is relieved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an application of selenium in alleviating cerebral ischemia-reperfusion injury caused by hyperglycemia. Background Art

[0002] Stroke is an acute cerebrovascular disease, which is divided into two types: ischemic stroke and hemorrhagic stroke. It develops rapidly and can cause cerebral infarction, leading to neurological dysfunction and even death in severe cases. It has the characteristics of high morbidity, high disability rate, high recurrence rate and high mortality rate, which seriously endangers human health. Stroke patients are usually accompanied by metabolic diseases, such as obesity, hyperglycemia, diabetes, hyperlipidemia, hypertension, atherosclerosis, etc. Among these many factors closely related to the occurrence and development of stroke, hyperglycemia is one of the most closely related risk factors. Compared with people with normal blood sugar, people with hyperglycemia are two to three times more likely to suffer from ischemic stroke.

[0003] At present, the main clinical treatments for ischemic stroke include thrombolysis, reducing intracranial pressure, using anticoagulants, protecting blood circulation between arterioles and venules, and using neuroprotective drugs. However, after the blood vessels are reopened, the rapid reperfusion of blood will induce cerebral ischemia-reperfusion injury, such as metabolic disorders of tissue cell function and cell structure damage, etc., and cerebral ischemia-reperfusion injury will be further aggravated when people with hyperglycemia have a stroke, such as increasing the degree of neurological dysfunction, expanding the volume of cerebral infarction, and increasing the number of degenerated and edematous neurons. Existing treatments for improving cerebral ischemia-reperfusion injury, such as antihypertensive drugs, antiplatelet drugs, and lipid-lowering drugs, have poor therapeutic effects on patients with hyperglycemia, and stroke will still occur after treatment. Summary of the invention

[0004] In view of this, it is necessary to provide an application of selenium in reducing cerebral ischemia-reperfusion injury in hyperglycemia, so as to improve the therapeutic effect of cerebral ischemia-reperfusion injury in patients with hyperglycemia, thereby reducing the risk of stroke in patients with hyperglycemia after treatment.

[0005] The present invention provides an application of selenium in alleviating cerebral ischemia-reperfusion injury caused by hyperglycemia. Selenium improves mitochondrial function by regulating the mitochondrial regeneration signal pathway, thereby maintaining a relative balance between mitochondrial fission and fusion processes and inhibiting the activation of the mitochondrial apoptosis pathway, thereby alleviating cerebral ischemia-reperfusion injury in patients with hyperglycemia.

[0006] Preferably, the mitochondrial regeneration signaling pathway is the AMPK / SIRT1 / PGC-1α signaling pathway.

[0007] Preferably, the selenium element enters the human body through selenium-containing substances that can be ingested by the human body.

[0008] Preferably, the selenium intake is 17 mg / d to 400 mg / d.

[0009] Preferably, the selenium-containing substance ingestible by the human body is SelH.

[0010] Preferably, the selenium-containing substance that can be ingested by the human body is sodium selenite, and the intake of sodium selenite is 37.2 μg / d to 875.9 μg / d.

[0011] The above-mentioned application of selenium in reducing cerebral ischemia-reperfusion injury caused by hyperglycemia provides an application of selenium to improve mitochondrial function by regulating the mitochondrial regeneration signal pathway, so that the mitochondrial fission and fusion process maintain a relative balance, and inhibit the activation of the mitochondrial apoptosis pathway, thereby reducing cerebral ischemia-reperfusion injury in patients with hyperglycemia. Specifically, hyperglycemia aggravates cerebral ischemia-reperfusion injury by increasing ROS production, changing the balance of mitochondrial fission and fusion, and activating the mitochondrial-mediated apoptosis pathway; and selenium can regulate the mitochondrial regeneration signal pathway to improve mitochondrial function, so that the mitochondrial fission and fusion process maintain a relative balance, and inhibit the activation of the mitochondrial apoptosis pathway, thereby reducing cerebral ischemia-reperfusion injury in patients with hyperglycemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a diagram of the rat body in Example 1 of the present application.

[0013] Figure 2 This is a graph of rat insulin levels in Example 1 of the present application.

[0014] Figure 3 This is a graph of blood glucose levels of rats in Example 1 of the present application.

[0015] Figure 4 This is a graph of rat weight levels in Example 1 of the present application.

[0016] Figure 5 This is a diagram of rat pancreatic immunofluorescence detection in Example 1 of the present application.

[0017] Figure 6 This is a statistical result diagram of Longa nerve dysfunction in rats in Example 1 of the present application.

[0018] Figure 7 This is a statistical result diagram of the rat survival rate in Example 1 of the present application.

[0019] Figure 8 This is a graph showing the measurement results of rat infarct volume in Example 1 of the present application.

[0020] Fig. 9 This is a diagram of the HE staining results in Example 1 of the present application.

[0021] Fig.10 This is a diagram of the Nissl staining result in Example 1 of the present application.

[0022] Fig.11 This is a diagram of the TUNEL staining results in Example 1 of the present application.

[0023] Fig.12 This is a graph of the neuronal nuclear antigen staining results in Example 1 of the present application.

[0024] Fig.13 This is a graph showing the expression changes of the antioxidant enzymes UCP2 and SOD2 in the cortical area of ​​the rat cerebral ischemic penumbra in Example 1 of the present application.

[0025] Fig.14 It is a diagram of the mitochondrial morphology and structure and a diagram of the number statistics in Example 1 of the present application.

[0026] Fig.15 This is the immunohistochemical result of SIRT1, a mitochondrial regeneration-related factor in the cortical area of ​​the ischemic penumbra in Example 1 of the present application. A. Light microscopic image of SIRT1 immunohistochemistry.

[0027] Fig.16 This is the immunohistochemical result of mitochondrial regeneration-related factor N RF1 in the cortical area of ​​cerebral ischemic penumbra in Example 1 of the present application. A is a light microscopic image of NRF1 immunohistochemistry.

[0028] Fig.17 This is the immunohistochemical result of TFAM, a factor related to mitochondrial regeneration in the cortical area of ​​the ischemic penumbra in Example 1 of the present application. A is a light microscopic image of TFAM immunohistochemistry.

[0029] Fig.18 This is a graph showing the expression changes of mitochondrial regeneration-related factors p-AMPK, AMPK, SIRT1, PGC-1a, NRF1 and TFAM in the cortical region of the rat cerebral ischemic penumbra in Example 1 of the present application.

[0030] Fig.19 This is a diagram of SIRT1 gene silencing in siSIRT1-HT22 cells in Example 2 of the present application.

[0031] Fig. 20 It is a graph showing the results of using the activator AICAR and the inhibitor EX-527 in Example 2 of the present application.

[0032] Fig.21 This is a diagram showing the ROS production in each group of HT22 cells in Example 2 of the present application.

[0033] Fig. 22 This is a graph showing the SOD activity of each group of HT22 cells and the MDA content of each group in Example 2 of the present application.

[0034] Fig.23 This is a graph showing the intracellular Ca2+ content and the LDH content in the culture medium of each group of HT22 cells in Example 2 of the present application.

[0035] Fig.24 This is a diagram of the mitochondrial inner membrane potential of each group of HT22 cells in Example 2 of the present application.

[0036] Fig.25 This is a graph showing the expression changes of granulosa regeneration-related factors p-AMP K, AMPK, SIRT1, PGC-1a, NRF1 and TFAM in each group of HT22 cells in Example 2 of the present application.

[0037] Fig.26 This is a graph showing the SOD activity and MDA content of each group of SelH-HT22 cells in Example 2 of the present application.

[0038] Fig. 27 This is a graph showing the intracellular Ca2+ content and the LDH content in the culture medium of each group of SelH-HT22 cells in Example 2 of the present application.

[0039] Fig.28 This is a diagram of the mitochondrial inner membrane potential of SelH-HT22 cells in Example 2 of the present application.

[0040] Fig.29 This is a diagram showing the expression of p-AMPK, a mitochondrial regeneration-related factor, in each group of SelH-HT22 cells in Example 2 of the present application.

[0041] Fig.30 This is a graph showing the expression of mitochondrial regeneration-related factors p-AMPK, AMPK, SIRT1, PGC-1α, NRF1 and TFAM in each group of SelH-HT22 cells in Example 2 of the present application. DETAILED DESCRIPTION

[0042] The technical solutions and technical effects of the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings of the present invention.

[0043] The present invention provides an application of selenium in alleviating cerebral ischemia-reperfusion injury caused by hyperglycemia. Selenium improves mitochondrial function by regulating the mitochondrial regeneration signal pathway, thereby maintaining a relative balance between mitochondrial fission and fusion processes and inhibiting the activation of the mitochondrial apoptosis pathway, thereby alleviating cerebral ischemia-reperfusion injury in patients with hyperglycemia.

[0044] Furthermore, the mitochondrial regeneration signaling pathway is the AMPK / SIRT1 / PGC-1α signaling pathway.

[0045] Furthermore, selenium enters the human body through selenium-containing substances that can be ingested by the human body, such as inorganic selenium compounds, artificially synthesized organic selenium compounds, and selenium-enriched yeast preparations transformed by bioengineering.

[0046] Furthermore, the intake of selenium is 17 mg / d to 400 mg / d.

[0047] Furthermore, the selenium-containing substance that can be ingested by the human body is SelH.

[0048] Furthermore, the selenium-containing substance that can be ingested by the human body is sodium selenite, and the intake of sodium selenite is 37.2 μg / d to 875.9 μg / d.

[0049] Example 1: Animal experiment on the effect of selenium on alleviating cerebral ischemia-reperfusion injury in patients with hyperglycemia

[0050] 1. Experimental Grouping

[0051] A total of 92 male SD rats were randomly divided into three groups: (1) normoglycemic ischemia group (NI), (2) hyperglycemic ischemia group (HI), and (3) hyperglycemic ischemia + sodium selenite treatment group (HI+Se). According to the different surgical treatments and reperfusion time after MCAO, each group was divided into sham operation group (Sham), 24h reperfusion group, and 72h reperfusion group.

[0052] 2. Preparation of Hyperglycemia Model

[0053] SD rats were fasted for more than 15 hours, but not water, and STZ was injected intraperitoneally at a dose of 60 mg / kg. In the figure, STZ was freshly prepared from 0.1 mol / L citric acid solution and 0.1 mol / L trisodium citrate solution in a fixed ratio. The concentration of the mixed buffer was 12 mg / mL and the pH value was 4.5. All steps were completed on ice and kept away from light throughout the process. Blood was collected from the tail vein on the 3rd day, and the fasting blood glucose level of the rats was measured by a portable blood glucose meter. Blood glucose values ​​> 16.7 mmol / L met the standard of hyperglycemia and were considered to be successful in modeling. The animals were raised for a total of 8 weeks, and the weight and blood glucose levels of the rats were monitored at fixed time points every week.

[0054] 3. Serum Insulin Determination

[0055] After the tip of the rat's tail was cut off, blood was immediately collected, placed at room temperature for 2 hours, and then centrifuged for 20 minutes. The supernatant was taken for testing. The insulin content was determined by Elisa enzyme-linked immunosorbent assay. The specific steps are as follows:

[0056] ① Add 100uL of standard working solution to the first two wells of the ELISA plate, and two duplicate wells. Add 100uL of the sample to be tested to the other wells. Cover the ELISA plate with film and incubate at 37℃ for 90min;

[0057] ②Discard the liquid, spin dry, add 100uL of biotinylated antibody working solution to each well of the ELISA plate, and incubate at 37℃ for 1h;

[0058] ③Discard the liquid, add 350uL washing solution to each well of the ELISA plate, after 1-2min, discard the liquid, pat dry on absorbent paper, and repeat this step 3 times;

[0059] ④ Add 100uL of enzyme conjugate working solution to each well of the ELISA plate and incubate at 37℃ for 30min.

[0060] ⑤Discard the liquid in each well, spin dry, and repeat this step 5 times.

[0061] ⑥ Add 90uL substrate solution to each well of the ELISA plate and incubate in a light-proof incubator at 37℃ for 15min.

[0062] ⑦ Add 50uL of stop solution to each well to terminate the reaction.

[0063] ⑧The ELISA reader was set at a wavelength of 450 nm to measure the optical density of each well.

[0064] 4. Blood sugar level monitoring

[0065] Monitor the blood glucose level of rats at fixed time for 8 consecutive weeks, debug the blood glucose meter, and install the test strips. Disinfect the rat's tail several times with iodine-disinfected cotton balls. Prick the tip of the rat's tail with a blood glucose needle, squeeze out an appropriate amount of blood from the tip of the rat's tail, drip it on the blood glucose meter test strip, and record the blood glucose value. Press the punctured part of the rat's tail with a cotton ball, and after stopping the bleeding, put the rat back into the cage to drink water and eat freely.

[0066] 5. Sodium Selenite Administration

[0067] Sodium selenite powder was dissolved in phosphate buffer solution (PBS), and the sodium selenite treatment group was intraperitoneally injected with sodium selenite daily. The sodium selenite treatment group was intraperitoneally injected with sodium selenite at 0.4 mg / kg / d for 4 consecutive weeks starting from 4 weeks after diabetes induction and before MCAO induction.

[0068] 6. General condition of rats

[0069] The rats in the normoglycemic ischemic group (NI) were round, with smooth and shiny hair, good nutrition and mental state, and normal daily food intake, water requirement, and urine output. The rats in the hyperglycemic ischemic group (HI) with type 1 diabetes successfully induced by STZ injection were thin, with yellow, frizzy, and dry hair, poor hygiene and mental state, and significantly increased daily food intake, water requirement, and urine output. The sodium selenite high-glycemic ischemic group (HI+Se), which was treated with sodium selenite after the hyperglycemic rat model was successfully established, showed improved body shape, smooth hair, improved hygiene and mental state, and decreased daily food intake, water requirement, and urine output compared with the hyperglycemic ischemic group (HI). Figure 1 shown.

[0070] 7. Rat serum insulin levels

[0071] The serum insulin levels of rats were detected 1-8 weeks after intraperitoneal injection of STZ. The serum insulin levels of rats in the NI group remained basically unchanged, while those of rats in the HI and HI+Se groups showed a downward trend as the time after injection prolonged. The serum insulin levels of the HI group decreased by 19.5% at 8 weeks after STZ injection compared with those at 1 week, and the serum insulin levels of the HI+Se group decreased by 8.77% at 8 weeks after STZ injection, but there was no statistical significance. At 1 week after intraperitoneal injection of STZ, the serum insulin levels of the HI (0.3933±240.2848ng / mL, P<0.01) and HI+Se (0.38±0.02082ng / mL, P<0.01) groups began to decrease, which were significantly lower than those of the NI group (1.09±0.6928ng / mL). The serum insulin levels of the HI and HI+Se groups were lower than those of the NI group at all time points from 2 to 8 weeks after STZ injection (P<0.01). However, there was no significant difference in serum insulin levels between the HI+Se group and the HI group at each time point (P>0.05). Figure 2 shown.

[0072] In the figure, the data are expressed as mean ± SD (n = 8). **P < 0.01, compared with the NI group.

[0073] 8. Blood glucose levels in rats

[0074] The blood glucose levels of rats were detected 1-8 weeks after intraperitoneal injection of STZ. The blood glucose levels of rats in the NI group remained basically unchanged over time, while the blood glucose levels of rats in the HI group and the HI+Se group showed a general upward trend over time. In the first week after STZ injection, the blood glucose levels of the HI group (22.45±3.436mmol / L, P<0.01) and the HI+Se group (19.2±1.334mmol / L, P<0.01) were higher than those of the NI group (5.2±0.6mmol / L), and there was no statistical significance between the HI+Se group (19.2±1.334mmol / L) and the HI group (22.45±3.436mmol / L) (P>0.05); the blood glucose values ​​of the HI group and the HI+Se group were higher than those of the NI group at all time points from 2 weeks to 8 weeks after STZ injection (P<0.01), and there was no statistical significance between the HI+Se group and the HI group. The experimental results are as follows Figure 3 shown.

[0075] In the figure, the data are expressed as mean ± SD (n = 10). **P < 0.01, compared with the NI group.

[0076] 9. Weight level of rats

[0077] The body weight levels of rats in the NI group showed a significant upward trend over time, the body weight levels of rats in the HI group basically maintained at the baseline level over time, and the body weight levels of rats in the HI+Se group showed a slight upward trend over time. There was no significant difference in body weight levels among the three groups of rats in the NI, HI, and HI+Se groups at the first and second weeks after intraperitoneal injection of STZ; at the third week after STZ injection, the body weight values ​​of the HI group (204.0±9.201g, P<0.01) and the HI+Se group (213.75±29.409g, P<0.05) were lower than those of the NI group (265.5±24.57g), and there was no significant difference between the HI+Se group (213.75±29.409g) and the HI group (204.0±9.201g) (P>0.05); at the fourth, fifth, sixth, seventh, and eighth weeks after STZ injection, the body weight values ​​of the HI group and the HI+Se group were lower than those of the NI group (P<0.01 or P<0.05), and there was no significant difference between the HI+Se group and the HI group (P>0.05). Statistical results are as follows Figure 4 shown.

[0078] In the figure, the data are expressed as mean ± SD (n = 10). **P < 0.01 and *P < 0.05, respectively compared with the NI group.

[0079] 10. Results of immunofluorescence detection of rat pancreas

[0080] Insulin represents the pancreatic β cells in the pancreatic tissue, glucagon represents the pancreatic α cells in the pancreatic tissue, and DAPI represents all cell nuclei. In the NI group, the number of cells in the pancreatic islets of pancreatic tissue was large, and they were uniform in size and neatly arranged, and the cell clusters were round. The pancreatic β cells accounted for the majority of the pancreatic islet tissue and were arranged in the central area of ​​the islets. The cell intervals were of uniform size. The islet tissue was surrounded by the α cells surrounding the β cells, which accounted for a small part of the total number of cells in the islet tissue. In the HI group, the pancreatic islet tissue showed atrophy, the islet morphology was irregular, the cells were disordered and sparsely distributed, the cell outlines were unclear, the number of pancreatic β cells decreased or even disappeared, and the number of pancreatic α cells increased and distributed in the center of the islets. The experimental results are as follows Figure 5 shown.

[0081] In the figure, DAPI (blue) represents all cell nuclei, insulin (green fluorescence) represents pancreatic β cells, and glucagon (red fluorescence) represents pancreatic α cells. Scale bar = 200 um.

[0082] 11. Results of rat neurological function scoring

[0083] The neurological dysfunction score (Feeney score) of the balance beam test showed that the behavioral changes of rats in the NI group were not obvious. The rats could pass the balance beam smoothly and rarely fell. The scores at 0h, 24h, and 72h were 1.7143±0.48795, 3.8571±0.69007, and 3.0±0.57735, respectively. The rats in the HI group could not walk stably on the balance beam after MCAO. Most of the rats fell off the balance beam within 30s. The scores at 0h, 24h, and 72h were 1.7143±0.48795, 3.8571±0.69007, and 3.0±0.57735, respectively. The scores at each time point were 2.8571±0.37796, 5.7143±0.48795, and 4.4286±0.7868, respectively. After MCAO, most rats in the HI+Se group could hold the balance beam tightly, and their limbs fell from the balance beam. The time they maintained on the balance beam was no more than 120s. The scores at 0h, 24h, and 72h were 2.2381±0.62488, 4.5714±0.95618, and 3.2857±0.48795, respectively. The balance beam test scores of the HI group were higher than those of the NI group (P<0.01), while those of the HI+Se group were lower than those of the HI group (P<0.01 or P<0.05). The neurological dysfunction scores of the balance beam test are as follows:

[0084] Table 1

[0085]

[0086] Longa neurological dysfunction score showed that neurological dysfunction was the most severe at 24h of ischemia-reperfusion, and the symptoms of neurological dysfunction showed a recovery trend at 72h of reperfusion. The scores of rats in NI group at 0h, 24h, and 72h after MCAO were 2.2±0.632, 2.7±0.483, and 1.9±0.738, respectively; the scores of rats in HI group at 0h, 24h, and 72h after MCAO were 3.2±0.422, 3.5±0.527, and 2.5±0.527, respectively; the scores of rats in HI+Se group at 0h, 24h, and 72h after MCAO were 2.4±0.516, 2.5±0.707, and 1.9±0.316, respectively. After MCAO model, at 0h, 24h, and 72h of reperfusion, the Longa scores of the HI group were higher than those of the NI group (P < 0.01 or P < 0.05), while those of the HI+Se group were lower than those of the HI group (P < 0.01 or P < 0.05). Figure 6 shown.

[0087] In the figure, the data are expressed as mean ± SD (n = 10). **P < 0.01 and *P < 0.05, respectively, compared with the NI group; ##P < 0.01 and #P < 0.05, respectively, compared with the HI group.

[0088] 12. Statistical results of rat survival rate

[0089] After MCAO in rats, 2 of 10 rats in the NI group died between 8 and 18 hours of reperfusion, and 8 rats survived to 72 hours; 3 of 10 rats in the HI group died at 20 hours of reperfusion, 2 died at 42 hours of reperfusion, 3 died at 69 hours of reperfusion, and only 2 rats survived to 72 hours. Therefore, the survival rates of rats in the HI group at 20 hours, 42 hours, and 72 hours were 70%, 50%, and 20%, respectively. In the HI+Se group, 3 of 10 rats died within 60 hours of reperfusion, and 7 rats survived to 72 hours. Therefore, the survival rate of rats in the NI group at 72 hours was 80%, and the survival rate of the HI group at 72 hours was 20%, which was statistically significant compared with the NI group (P < 0.01). The survival rate of the HI+Se group at 72 hours was 70%, which was statistically significant compared with the HI group (P < 0.01). The experimental results are shown in the following table. Figure 7 shown.

[0090] 13. Measurement results of rat infarct volume

[0091] TTC staining was used to detect the infarct volume of rats. The normal area was stained red, and the infarct area did not change and was pale. Image-J software was used to measure the infarct area, and the infarct volume percentage was calculated using the formula. The results are as follows: Figure 8As shown in the figure, the sham group of normoglycemic rats had no infarction. The percentage of infarction volume in the NI group after 24h of ischemia-reperfusion was 3.6%, and the percentage of infarction volume in the HI group after 24h of ischemia-reperfusion was 44.72%, which was significantly larger than that in the NI group (P<0.01), and the infarction volume extended to the cerebral cortex. The percentage of infarction volume in HI+Se was 3.2%, which was significantly lower than that in the HI group (P<0.01), but there was no statistical difference compared with the NI group (P>0.05).

[0092] Figure A. TTC staining, pale area is the infarct area; B is the statistical result of infarct volume. Data are expressed as mean ± SD (n = 3-7). ##P < 0.01, compared with NI group; &&P < 0.01, compared with HI group.

[0093] 14. Morphological observation results of rats

[0094] 14.1 HE staining results

[0095] In order to observe the difference between the injuries of the NI and HI groups of rats, we chose to take pictures of the cortical area of ​​the rat brain. The brain tissue structure of the sham group of the normal blood sugar group was normal, the nerve cell structure was densely arranged, the cell nucleus was round or oval, the nucleolus was located in the center, there was more chromatin, and the cytoplasm was red; 24 hours after MCA O reperfusion, the neuronal cell bodies of the brain tissue of the NI group were slightly edematous, a small number of karyopyknotic cells were visible, the cytoplasm was lightly stained, and the cytoplasmic space of some cells increased; compared with the NI group, the degree of brain tissue edema in the HI group was more serious, the cytoplasm was partially stained light pink, vacuolar tissue structures were seen everywhere, and a large number of karyopyknotic neurons were revealed; the HI+Se group was relieved compared with the HI group in the above morphological changes, the degree of brain tissue edema was reduced, and the number of karyopyknotic neurons was reduced. Results are as follows Fig. 9 As shown in Figures AD, the number of pyknotic cells in each group was counted, and the statistical results are as follows Fig. 9 As shown in E, the number of pyknotic cells in the NI group was higher than that in the Sham group (P < 0.01), the HI group was higher than that in the NI group (P < 0.01), and the HI+Se group was lower than that in the HI group (P < 0.01).

[0096] In the figure, AD is the light microscopic image of the cortical ischemic penumbra area in the normal blood sugar Sham group and each group at 24h of cerebral ischemia reperfusion. Normal neurons (arrows), nuclear pyknosis and swollen neurons (arrow heads). Scale bar = 50um; E is the statistical result of the number of nuclear pyknosis neurons in each group. Data are expressed as mean ± SD (n = 5-8). **P < 0.01, compared with the Sham group; ##P < 0.01, compared with the NI group; &&P < 0.01, compared with the HI group.

[0097] 14.2 Nissl staining results

[0098] The disintegration or loss of Nissl bodies (blue) in the neuronal cytoplasm symbolizes the destruction of neurons. In the sham group of the normal blood sugar group, the number of neurons was larger and arranged tightly. The neuronal nuclei were approximately round with clear and distinct outlines. The nucleoli were clearly visible and dark blue. The chromatin in the nucleus was light blue. The Nissl bodies were abundant and the Nissl bodies in the neuronal cytoplasm were clearly and evenly stained. At 24 hours of ischemia-reperfusion, the neuronal cell bodies in the NI group were swollen or deformed, the cells were dissolved, the morphology was irregular, the nuclei were condensed and darkly stained, the Nissl bodies were blurred, and there was a phenomenon of disintegration or loss. The neuronal density in the HI group decreased significantly, the intercellular space widened, a large number of vacuolar structures were visible, and the Nissl bodies decreased or disappeared. The neuronal density in the HI+Se group was slightly more than that in the HI group. The outlines of individual Nissl bodies were present, and the vacuolar structures still existed. The results are as follows Fig.10 As shown in AD, the number of "surviving neurons" in each group was counted, and the statistical results are as follows Fig.10 As shown in E, the number of surviving neurons in the NI group was significantly less than that in the Sham group (P < 0.01), the number of surviving neurons in the HI group was significantly reduced compared with the NI group (P < 0.01), and the number of surviving neurons in the HI+Se group was significantly increased compared with the HI group (P < 0.05).

[0099] In the figure, AD is the light microscopic image of the cortical ischemic penumbra area in the normal blood sugar Sham group and each group at 24h of cerebral ischemia reperfusion. Scale bar = 50um; E is the statistical result of the number of surviving neurons in each group. Data are expressed as mean ± SD (n = 5-8). **P < 0.01, compared with the Sham group; ##P < 0.01, compared with the NI group; &P < 0.05, compared with the HI group.

[0100] 14.3 TUNEL staining results

[0101] There were few TUNEL-positive cells in the sham group of the normal blood sugar group. At 24h of ischemia-reperfusion, the number of TUNEL-positive cells in the NI group increased significantly (P < 0.01), and the number of TUNEL-positive cells in the HI group increased further than that in the NI group (P < 0.01). There was a significant difference between the HI+Se group and the HI group (P < 0.01), almost down to the level of the NI group. Apoptotic cells in the cortical area of ​​the ischemic penumbra of rats, the results are as follows Fig.11 As shown in A. The statistical results of the ratio of apoptotic cells to total cells in each group are shown in Fig.11 As shown in B.

[0102] In the figure, A shows the results of apoptotic cells in the cortical ischemic penumbra of the normoglycemic Sham group and each group at 24h of cerebral ischemia-reperfusion. TUNEL-positive cell nuclei are marked in red, and all cell nuclei are marked in blue by DAPI. Scale bar = 50um; B shows the statistical results of the ratio of apoptotic cells to all cells. Data are expressed as mean ± SD (n = 5-8). **P < 0.01 compared with the Sham group; ##P < 0.01 compared with the NI group; &&P < 0.01 compared with the HI group.

[0103] 14.4 Neuronal nuclear antigen staining results

[0104] Immunofluorescence was used to detect the neuronal nuclear antigen (NeuN) in the ischemic penumbra cortex. Fig.12 As shown in A, NeuN is mainly expressed in the nucleus and perinuclear cytoplasm of neurons. The number of NeuN-positive neurons in the sham group of the normal blood sugar group is relatively large. At 24h of ischemia-reperfusion, the number of NeuN-positive neurons in the NI group was significantly reduced (P < 0.01), and the number of NeuN-positive neurons in the HI group was further reduced compared with the NI group (P < 0.01). There was a significant difference between the HI+Se group and the HI group (P < 0.01). The statistical results of the number of NeuN-positive neurons in each group are shown in Fig.12 As shown in B.

[0105] In the figure, A is the staining of neuronal nuclear antigen in the cortical ischemic penumbra of the normal blood sugar Sham group and each group at 24h of cerebral ischemia reperfusion. Green is NeuN-positive cells, and blue is DAPI cell nuclei. Scale bar = 200um; B is the statistical result of the number of NeuN-positive cells. Data are expressed as mean ± SD (n = 5-8). **P < 0.01 compared with the Sham group; ##P < 0.01 compared with the NI group; &&P < 0.01 compared with the HI group.

[0106] 15. Changes of Antioxidant Enzymes UCP2 and SOD2 Expression in the Cortical Area of ​​the Ischemic Penumbra in Rats

[0107] In order to examine whether sodium selenite reduces the oxidative stress level in MCAO rats and its possible mechanism, we detected the protein levels of antioxidant enzymes UCP2 and SOD2 in the cortical area of ​​the ischemic penumbra of rats by Western blot. Fig.13As shown in A. Semi-quantitative immunoblotting showed that the UCP2 protein level in the NI group decreased slightly with the extension of reperfusion time (P < 0.05, P < 0.01); the UCP2 protein level in the HI group was at a lower level at each time point of reperfusion, which was lower than that in the NI group (P < 0.01); in the HI+Se group, the UCP2 protein level at each time point of reperfusion was significantly higher than that in the HI group (P < 0.01). The gray value quantification results are shown in Fig.13 As shown in B. The changes in SOD2 protein levels were roughly the same as those in UCP2 protein levels. In the NI group, the SOD2 protein level decreased slightly to moderately with the extension of reperfusion time (P < 0.01); the SOD2 protein level in the HI group was lower than that in the NI group at each time point of reperfusion (P < 0.05, P < 0.01); in the HI+Se group, at 24h and 72h of reperfusion, the SOD2 protein level was significantly higher than that in the HI group (P < 0.01), while there was no significant change in the Sham group. The gray value quantification results are shown in Fig.13 As shown in C.

[0108] In the figure, A is the strip diagram of UCP2 and SOD2; B and C are the relative gray value ratios of UCP2 or SOD2 to β-actin. Data are expressed as mean ± SD (n = 3-4). **P < 0.01 and *P < 0.05 were compared with the ham group; ##P < 0.01 and #P < 0.05 were compared with the NI group at the same reperfusion time; &&P < 0.01 were compared with the HI group at the same reperfusion time.

[0109] 16. Transmission electron microscopy observation results

[0110] In the sham group of the normal blood sugar group, the nuclei of the neurons were large and round, with smooth nuclear membranes, nucleoli clustered into clusters with high electron density, sufficient organelles in the cytoplasm with intact structures, mitochondria in oval or round shapes, and numerous and clear cristae arranged in a regular pattern. At 24 hours of ischemia-reperfusion, the shape of the nuclei in the NI group changed, the volume decreased, the nuclear membrane was not smooth and invaginated, the nucleoli disappeared, the chromatin dissolved, the mitochondria swelled like balloons, the cristae decreased, and the shape was irregular. The nuclear condensation in the HI group was more serious, the chromatin dissolved or condensed into masses, the organelles around the nucleus decreased or disappeared, some mitochondria were vacuolated, the cristae were difficult to identify, and the original structure was lost. The cells in the HI+Se group still had nuclear condensation, the surface was jagged, the degree of chromatin dissolution was lighter than that in the HI group, the organelles in the cytoplasm were more abundant than those in the HI group, the morphological structure of some mitochondria was restored, and the mitochondrial cristae were visible. Results Fig.14 As shown in A.

[0111] The number of brain mitochondria in each TEM photo of each group was quantified. TEM analysis showed that the number of brain mitochondria in the NI group was reduced compared with the Sham group (P < 0.05), and the number of brain mitochondria in the HI group was further reduced compared with the NI group (P < 0.01), while the number of mitochondria in the HI+Se group increased compared with the HI group (P < 0.01). The statistical results of the number of mitochondria in each group are shown in Figure 2. Fig.14 As shown in B.

[0112] In the figure, A is the transmission electron microscopy image of the normal blood sugar Sham group and each group at 24h of cerebral ischemia reperfusion. The lower row is an enlargement of the black box content in the upper row. Arrows indicate normal mitochondria, and the arrow heads indicate damaged mitochondria. The upper row scale = 2um, the lower row scale = 1um. Nu represents neuronal nucleus; B is the statistical result of the number of mitochondria in each group. The data are expressed as mean ± SD (n = 3-4). *P < 0.05 compared with the Sh am group; ##P < 0.01 compared with the NI group; &&P < 0.01 compared with the HI group.

[0113] 17. Changes in the expression of mitochondrial regeneration-related factors in the cortical area of ​​the ischemic penumbra of rats

[0114] 17.1 Immunohistochemical detection of changes in the expression of mitochondrial regeneration-related factors SIRT1, NRF1 and TFAM in the cortical region of the ischemic penumbra of rats The sections were subjected to immunohistochemical staining of factors such as SIRT1, NRF1 and TFAM. Five different fields of view of the cortical region of the ischemic penumbra were selected under a 40x microscope, and the number of positive cells was counted and statistically analyzed. The results of SIRT1 immunohistochemical staining were shown in Figure 1. Fig.15 A in the figure shows 1-15A, and the statistical results are as follows Fig.15 As shown in B, see Figure 1-15 B. SIRT1 was mainly expressed in the cell nucleus. Positive cells were detected in the NI and HI groups. Compared with the Sham group, the number of positive cells increased at 24h and 72h after reperfusion (P < 0.01); the number of positive cells in the HI group was less than that in the NI group at 72h after reperfusion (P < 0.05); in the HI+Se group, compared with the Sham group, the number of positive cells increased at 24h and 72h after reperfusion (P < 0.01), and there was a statistically significant difference between the HI+Se group and the HI group (P < 0.05 or P < 0.01). Fig.16 As shown in A, the statistical results are as follows Fig.16 As shown in Figure B, NRF1 is mainly expressed in the cell nucleus. Compared with the Sham group, the positive cell counts in the NI and HI groups increased at 24h and 72h after reperfusion (P < 0.01); the number of positive cells in the HI+Se group increased further compared with the HI group (P < 0.01). Fig.17 As shown in A, the statistical results are as follows Fig.17 As shown in B, TFAM was mainly expressed in the cell cytoplasm. In the NI group, compared with the Sham group, the positive cell count increased at 24h and 72h after reperfusion (P < 0.01); in the HI group, compared with the Sham group, the positive cell count increased at 24h after reperfusion (P < 0.05); at 72h after reperfusion, the number of positive cells in the HI group was less than that in the NI group (P < 0.01); the number of positive cells in the HI+Se group further increased compared with the HI group (P < 0.01).

[0115] In the figure, Fig.15 Results of immunohistochemistry of SIRT1, a factor related to mitochondrial regeneration, in the cortical area of ​​cerebral ischemic penumbra. A. Light microscopic image of SIRT1 immunohistochemistry. Scale bar = 50um; B. Statistical results of the number of SIRT1-positive cells. Data are expressed as mean ± SD (n = 5-8). **P < 0.01 compared with the Sham group; #P < 0.05 compared with the NI group at the same reperfusion time; &&P < 0.01 and &P < 0.05 compared with the HI group at the same reperfusion time; Fig.16 The immunohistochemical results of NRF1, a mitochondrial regeneration-related factor in the cortical area of ​​cerebral ischemic penumbra. A is a light microscopic image of NRF1 immunohistochemistry. Scale bar = 50um; B is the statistical result of the number of NRF1 positive cells (n = 5-8). Data are expressed as mean ± SD. **P < 0.01 compared with the Sham group; &&P < 0.01 compared with the HI group at the same reperfusion time; Fig.17 Results of immunohistochemistry of TFAM, a factor related to mitochondrial regeneration, in the cortical area of ​​cerebral ischemic penumbra. A is a light microscopic image of TFAM immunohistochemistry. Scale bar = 50um; B is the statistical result of the number of TFAM positive cells. Data are expressed as mean ± SD (n = 5-8). **P < 0.01 and *P < 0.05 were compared with the Sham group; ##P < 0.05 was compared with the NI group at the same reperfusion time; &&P < 0.01 was compared with the HI group at the same reperfusion time.

[0116] 17.2 Western blot detection of the expression changes of mitochondrial regeneration-related factors p-AMP K, AMPK, SIRT1, PGC-1a, NRF1 and TFAM in the cortical area of ​​rat cerebral ischemic penumbra

[0117] In order to study whether the hyperglycemic cerebral ischemia state affects the mitochondrial regeneration signaling pathway, and whether selenium can alleviate cerebral ischemia-reperfusion injury by promoting the mitochondrial regeneration signaling pathway, we used Western blot to detect the expression changes of regeneration-related factors in the cortical area of ​​the rat cerebral ischemic penumbra. Fig.18 As shown in A.

[0118] At 24h of reperfusion, the p-AMPK protein level in the NI group increased (P < 0.01), and the level dropped below the control level at 72h; at 24h and 72h of reperfusion, the p-AMPK protein level in the HI group increased slightly (P < 0.01); at 24h of reperfusion, the p-AMPK protein level in the HI group was significantly lower than that in the NI group (P < 0.01); at 24h and 72h of reperfusion, the p-AMPK protein level in the HI+Se group was significantly higher than that in the HI group (P < 0.01, P < 0.05). Fig.18 As shown in B.

[0119] In the NI and HI groups, the SIRT1 protein levels were higher than those in the Sham group at 24h and 72h after reperfusion (P<0.01 or P<0.05); at 72h after reperfusion, the SIRT1 level in the HI group was lower than that in the NI group (P<0.05); in the HI+Se group, the SIRT1 level in the Sham group and at 72h after reperfusion was significantly higher than that in the HI group (P<0.01). Fig.18 As shown in C.

[0120] In the NI and HI groups, the PGC-1α protein level did not change significantly with the extension of reperfusion time (P>0.05); at each time point of reperfusion, the PGC-1α protein level between the HI group and the NI group did not reach a statistical difference (P>0.05); in the HI+Se group, at each time point of reperfusion, the PGC-1α protein level was significantly higher than that of the HI group (P<0.01). Fig.18 As shown in D.

[0121] In the NI group, the NRF1 protein level first decreased with the extension of reperfusion time (P < 0.01), and then increased (P < 0.01); in the HI group, the NRF1 protein level showed an increasing trend with the extension of reperfusion time (P < 0.01); in the HI+Se group, the NRF1 protein level in the Sham group and at 24h of reperfusion was significantly higher than that in the HI group (P < 0.01). Fig.18 As shown in E.

[0122] In the NI group, the TFAM protein level increased significantly at 24h after reperfusion (P<0.01), and slightly increased at 72h (P<0.01); in the HI group, the TFAM protein level increased at 24h after reperfusion (P<0.05); at 72h after reperfusion, the TFAM level in the HI group was lower than that in the NI group (P<0.01); in the HI+Se group, the TFAM level in the Sham group and at 24h and 72h after reperfusion was significantly higher than that in the HI group (P<0.01). Fig.18 As shown in F.

[0123] In the figure, A is the strip diagram of p-AMPK, AMPK, SIRT1, PGC-1a, NRF1, and TFAM; BF is the relative gray value ratio of p-AMPK, AMPK, SIRT1, PGC-1a, NRF1, TFAM and β-actin, respectively. Data are expressed as mean ± SD (n = 3-4). **P < 0.01 and *P < 0.05 were compared with the Sham group, respectively; #P < 0.05 and ##P < 0.01 were compared with the NI group at the same reperfusion time, respectively; &&P < 0.01 and &P < 0.05 were compared with the HI group at the same reperfusion time.

[0124] It can be seen from the comparison of various data in this example that sodium selenite can reduce cerebral ischemia-reperfusion injury in patients with hyperglycemia.

[0125] Example 2: Cell experiment on regulating AMPK / SIRT1 / PGC-1a signaling pathway by sodium selenite and SelH to alleviate cerebral ischemia-reperfusion injury in patients with hyperglycemia

[0126] 1. Experimental Grouping

[0127] 1.1 Part I Experimental Grouping

[0128] siControl-HT22 and siSIRT1-HT22 cells were divided into sodium selenite group (Se), sodium selenite + hypoxia and high glucose group (OD+HG+Se), and sodium selenite + hypoxia and high glucose + AICAR group (OD+HG+Se+AICAR), respectively.

[0129] 1.2 Part II Experimental Grouping

[0130] SelH-HT22 cells were divided into control group (Control), hypoxia and high glucose group (OD+HG), hypoxia and high glucose + AICAR group (OD+HG+AICAR), hypoxia and high glucose + AICAR + EX-527 group (OD+HG+AICAR+EX-527), and hypoxia and high glucose + EX-527 group (OD+HG+EX-527).

[0131] 2. Sodium selenite promotes mitochondrial regeneration through the AMPK / SIRT1 / PGC-1α signaling pathway

[0132] 2.1 Detection of SIRT1 gene silencing in siSIRT1-HT22 cells

[0133] Western blot was used to detect whether SIRT1 gene silencing in siSIRT1-HT22 cells was successful. The results showed that the band diagram results were as follows Fig.19As shown in A. Semi-quantitative immunoblotting showed that the SIRT1 protein level in the siSIRT1-HT22 cell group was low (P < 0.01), indicating that the SIRT1 gene silencing in siSIRT1-HT22 cells was successful and subsequent experiments could be performed. The gray value quantification results are shown in Fig.19 As shown in B.

[0134] In the figure, A is a strip diagram of SIRT1; B is the relative gray value ratio of SIRT1 to β-actin. Data are expressed as mean ± SD (n = 4). **P < 0.01, compared with siControl cells.

[0135] 2.2 Selection of timing for SIRT1 inhibitors and AMPK activators

[0136] AICAR, also known as AICARiboside or Acadesine, is currently a widely recognized AMPK activator. Western blot results showed that the p-AMPK protein level was low in the Control group. After using 1mmol / L AICAR, the p-AMPK protein level increased significantly at 1h and reached a peak, which was statistically significant compared with the Control group (P<0.01). At 2h, the p-AMPK protein level began to decline and continued until 12h. In subsequent experiments, we selected 1h as the use time of the activator AICAR. The results are as follows Fig. 20 As shown in A. EX-527 is a widely accepted SIRT1 inhibitor. Western blot results showed that SIRT1 protein levels were highly expressed in the Control group. After using 50umol / L EX-527, SIRT1 protein levels showed a gradual downward trend. At 1 hour, SIRT1 protein levels dropped to 0.8252±0.03045, which was statistically significant compared with the Control group (P<0.01). In subsequent experiments, we selected 1 hour as the use time of the inhibitor EX-527. The results are shown in Fig. 20 As shown in B.

[0137] In the figure, A is the bar graph of p-AMPK and the relative gray value ratio of p-AMPK to β-actin; B is the bar graph of SIRT1 and the relative gray value ratio of SIRT1 to β-actin (n=4). Data are expressed as mean ± SD (n=4). **P < 0.01, compared with Control cells.

[0138] 2.3 Oxidative stress detection

[0139] 2.3.1 Reactive oxygen generation

[0140] DCFH-DA probe was used to detect the production of reactive oxygen species (ROS) in each group of cells. Fig.21 As shown in A, green fluorescence represents ROS, and the amount of ROS produced is expressed by relative fluorescence intensity (RFI). Fig.21 As shown in Figure B, in siControl-HT22 cells and siSIRT1-HT 22 cells, compared with the Se group, the amount of ROS produced in the two groups increased significantly after OD+HG intervention (P<0.01), and the amount of ROS produced in siSIRT1-HT22 cells was more than that in siControl-HT22 cells (P<0.01). Compared with the OD+HG group, after the simultaneous use of the activator AICAR, the amount of ROS produced in the two groups of cells was significantly decreased (P<0.01), but the decrease in ROS production in siSIRT1-HT22 cells was less than that in siControl-HT22 cells (P<0.01).

[0141] In the figure, A is the ROS production in each group. Green is ROS. Scale bar = 50um; B is the statistical result of ROS relative fluorescence intensity (RFI). Data are expressed as mean ± SD (n = 4). **P < 0.01 compared with Se group; ##P < 0.01 compared with OD + HG group; &&P < 0.01 and &P < 0.05 compared with siControl cells.

[0142] 2.3.2 Oxidative stress markers SOD activity and MDA content

[0143] Oxidative stress was evaluated by detecting intracellular SOD activity and MDA content. Fig. 22 As shown in A, in siControl-HT22 cells and siSIRT1-HT22 cells, compared with Se group, the intracellular SOD activity of both groups decreased significantly after OD+HG intervention (P<0.01), and the SOD activity of siSIR T1-HT22 cells was significantly downregulated compared with siControl-HT22 cells (47.63±1.387, 8.412±0.3203U / mg, P<0.01); compared with OD+HG group, after the simultaneous use of activator AICAR, the intracellular SOD activity of both groups was significantly increased (38.2±1.443, 68.17±1.738U / mg, P<0.01), but the SOD activity of siSIRT1-HT22 cells was significantly upregulated compared with siControl-HT22 cells (P<0.01). The results of brain tissue MDA content detection are shown in Figure 2. Fig. 22As shown in B, in siControl-HT22 cells and siSIRT 1-HT22 cells, compared with Se group, the intracellular MDA content of both groups increased significantly after OD+HG intervention (9.384±0.145, 11.65±0.525umol / mg, P<0.01), and the MDA content of siSIRT1-HT22 cells was significantly higher than that of siControl-HT22 cells (P<0.05). Compared with OD+HG group, after the simultaneous use of activator AICAR, the intracellular MDA content of both groups was significantly decreased (7.056±0.3509, 8.412±0.3203umol / mg, P<0.01), but the MDA content of siSIRT1-HT22 cells was significantly lower than that of siControl-HT22 cells (P<0.05).

[0144] In the figure, A is the SOD activity of each group; B is the MDA content of each group. Data are expressed as mean ± SD (n = 4). **P < 0.01 compared with the Se group; ##P < 0.01 compared with the OD + HG group; &&P < 0.01 and &P < 0.05 compared with siControl cells.

[0145] 2.4HT22 cell injury markers: intracytoplasmic Ca2+ and LDH levels in culture medium

[0146] The degree of HT22 cell damage was evaluated by detecting the levels of Ca2+ in the cytoplasm and LDH in the culture medium. Fig.23 As shown in A, in siControl-HT22 cells and siSIRT1-HT22 cells, compared with the Se group, the intracellular Ca2+ content of both groups increased significantly after OD+HG intervention (216.3±3.712, 233±1.732nM, P<0.01), but the Ca2+ content of siSIRT1-HT22 cells increased more than that of siControl-HT22 cells (P<0.05); compared with the OD+HG group, after the simultaneous use of the activator AICAR, the intracellular Ca2+ content of both groups decreased significantly (178.3±4.333, 197.3±4.485nM, P<0.01), but the Ca2+ content of siSIRT1-HT22 cells decreased less than that of siControl-HT22 cells (P<0.01). The expression trend of LDH levels in the culture medium was basically consistent with the results of Ca2+ levels in the cytoplasm. The results of LDH content detection in the intracellular culture medium are shown in Figure 2. Fig.23As shown in Figure B, in siControl-HT22 cells and siSIRT1-HT22 cells, compared with Se group, LDH content in the cell culture fluid of both groups was significantly increased after OD+HG intervention (94.6667±6.02771, 109.6667±5.50757U / L, P<0.01), but LDH content in siSIRT1-HT22 cells was more upregulated than that in siControl-HT22 cells (P<0.05). Compared with OD+HG group, after simultaneous use of activator AICAR, LDH content in the cell culture fluid of both groups was significantly decreased (78.3333±3.05505, 91.3333±2.51661U / L, P<0.01), but LDH content in siSIRT1-HT22 cells was less downregulated than that in siControl-HT22 cells (P<0.01).

[0147] In the figure, A is the Ca2+ content of each group; B is the LDH content of each group. Data are expressed as mean ± SD (n = 4). **P < 0.01 compared with the Se group; ##P < 0.01 compared with the OD + HG group; &&P < 0.01 and &P < 0.05 compared with siControl cells.

[0148] 2.5 Mitochondrial membrane potential

[0149] The intracellular mitochondrial membrane potential (MMP) was detected by JC-1 staining. The change of JC-1 from red fluorescence to green fluorescence represents the decrease of cellular MMP. Therefore, the ratio of red / green fluorescence intensity represents MMP. Fig.24 As shown in A. In siControl-HT22 cells and siSIRT1-HT22 cells, compared with the Se group, the intracellular MMP of both groups decreased significantly after OD+HG intervention (P<0.01), and the MMP decrease of siSIR T1-HT22 cells was significantly greater than that of siControl-HT22 cells (P<0.01); compared with the OD+HG group, after the simultaneous use of the activator AICAR, the intracellular MMP of both groups increased significantly (P<0.05), but the MMP increase of siSIRT1-HT22 cells was significantly less than that of siControl-HT22 cells (P<0.01). The results are shown in Fig.24 As shown in B.

[0150] In the figure, A is the MMP situation of each group; B is the MMP statistical results of each group. Data are expressed as mean ± SD (n = 4). **P < 0.01 compared with Se group; #P < 0.05 compared with OD + HG group; &&P < 0.01 compared with siControl cells.

[0151] 2.6 Western blot detection of mitochondrial regeneration-related factors p-AMPK, AMPK, SIRT1, PGC-1a, NRF1 and TFAM expression changes

[0152] In order to study whether sodium selenite promotes mitochondrial regeneration through the AMPK / SIRT1 / PGC-1α signaling pathway and alleviates hypoxia and high glucose damage in neurons, we detected mitochondrial regeneration-related factors in HT22 cells by Western blot. Fig.25 As shown in A.

[0153] p-AMPK, AMPK, SIRT1, PGC-1α, NRF1 and TFAM are mitochondrial regeneration-related factors that directly reflect mitochondrial regeneration function. In siControl-HT22 cells and siSIRT1-HT22 cells, compared with Se group, p-AMPK, SIRT1, PGC-1α, NRF1 and TFAM protein levels were significantly upregulated after OD+HG intervention (P<0.05 or P<0.01), but the increase in p-AMPK, SIRT1, PGC-1α, NRF1 and TFAM protein levels in siSIRT1-HT22 cells was less than that in siControl-HT22 cells (P<0.01), indicating that mitochondrial regeneration function was inhibited when SIRT1 gene was silenced in siSIRT1-HT22 cells; compared with OD+HG group, after simultaneous use of activator AICAR, p-AMPK, SIRT1, PGC-1α, NRF1 and TFAM protein levels in both groups were further increased (P<0.05 or P<0.01), but p-AMPK, SIRT1, PGC-1α, NRF1 and TFAM protein levels in siSIRT1-HT22 cells were less than those in siControl-HT22 cells. The protein levels of F1 and TFAM increased less (P < 0.05 or P < 0.01). Fig.25 As shown in BF.

[0154] In the figure, A is the strip diagram of p-AMPK, AMPK, SIRT1, PGC-1a, NRF1, and TFAM; BF are the relative gray value ratios of p-AMPK, AMPK, SIRT1, PGC-1a, NRF1, TFAM and β-actin, respectively. Data are expressed as mean ± SD (n = 4). **P < 0.01 and *P < 0.05 were compared with the Se group, respectively; ##P < 0.01 and #P < 0.05 were compared with the OD + HG group, respectively; &&P < 0.01 and &P < 0.05 were compared with siControl cells, respectively.

[0155] 3. SelH promotes mitochondrial regeneration through the AMPK / SIRT1 / PGC-1α signaling pathway

[0156] 3.1 Oxidative stress markers: intracellular SOD activity and MDA content

[0157] Oxidative stress was evaluated by detecting the intracellular SOD activity and MDA content in SelH-HT22 cells. Fig.26 As shown in A, compared with the Control group, the intracellular SOD activity was significantly decreased after OD+HG intervention (39.63±4.495U / mg, P<0.01); compared with the OD+HG group, the intracellular SOD activity was significantly increased after the simultaneous use of the activator AICAR (88.3±1.185U / mg, P<0.01); on this basis, after the simultaneous use of the inhibitor EX-527, the SOD activity was significantly downregulated compared with the AICAR group (66.03±2.314U / mg, P<0.01); after the use of the inhibitor EX-527 alone, the SOD activity was further downregulated compared with the AICAR+EX-527 group (38.53±4.032U / mg, P<0.01).

[0158] The results of MDA content detection in SelH-HT22 cells are as follows Fig.26 As shown in B, compared with the Control group, the intracellular MDA content was significantly increased after OD+HG intervention (10.87±0.3529umol / mg, P<0.01); compared with the OD+HG group, the intracellular MDA content was significantly decreased after the simultaneous use of the activator AICAR (6.221±0.217umol / mg, P<0.01); on this basis, after the simultaneous use of the inhibitor EX-527, the MDA content was significantly increased compared with the AICAR group (7.432±0.1467umol / mg, P<0.01); after the use of the inhibitor EX-527 alone, the MDA content was more increased than that of the AICAR+EX-527 group (11.44±0.15umol / mg, P<0.01).

[0159] In the figure, A is the SOD activity of each group; B is the MDA content of each group. Data are expressed as mean ± SD (n = 4). **P < 0.01 compared with the Control group; ##P < 0.01 compared with the OD + HG group; &&P < 0.01 compared with the AICAR group; @@P < 0.01 compared with the AICAR + EX-527 group.

[0160] 3.2 Intracytoplasmic Ca2+ and LDH levels in culture medium, markers of HT22 cell injury

[0161] The degree of HT22 cell damage was evaluated by detecting the intracytoplasmic Ca2+ level of SelH-HT22 cells and the LDH level in the culture medium. Fig. 27 As shown in A, compared with the Control group, the intracellular Ca2+ content was significantly increased after OD+HG intervention (213±4.041nM, P<0.01); compared with the OD+HG group, the intracellular Ca2+ content was significantly decreased after the simultaneous use of the activator AICAR (178.7±1.856nM, P<0.01); on this basis, the Ca2+ content was significantly increased after the simultaneous use of the inhibitor EX-527 compared with the AICAR group (194.3±2.404nM, P<0.01); after the use of the inhibitor EX-527 alone, the Ca2+ content was more increased than that of the AICAR+EX-527 group (224.3±2.028nM, P<0.01).

[0162] The expression trend of LDH level in culture medium was basically consistent with the result of intracytoplasmic Ca2+ level. The results of LDH content detection in intracellular culture medium are shown in Figure 27B. Compared with the Control group, the LDH content in culture medium was significantly increased after OD+HG intervention (91.67±1.764U / L, P<0.01); compared with the OD+HG group, the LDH content in culture medium was significantly decreased after the simultaneous use of activator AICAR (57.33±1.453U / L, P<0.01); on this basis, the LDH content was significantly increased after the simultaneous use of inhibitor EX-527 compared with the AIC AR group (81.67±2.028U / L, P<0.01); the LDH content was more increased after the use of inhibitor EX-527 alone than that of AICAR+EX-527 group (101.7±1.764U / L, P<0.01).

[0163] In the figure, A is the Ca2+ content of each group; B is the LDH content of each group. Data are expressed as mean ± SD (n = 4). **P < 0.01 compared with the Control group; ##P < 0.01 compared with the OD + HG group; &&P < 0.01 compared with the AICAR group; @@P < 0.01 compared with the AICAR + EX-527 group.

[0164] 3.3 Mitochondrial membrane potential

[0165] The MMP in SelH-HT22 cells was detected by JC-1 staining. The polymer showed red fluorescence and the monomer showed green fluorescence. The change of JC-1 from red fluorescence to green fluorescence represented the decrease of MMP in the cells. Therefore, the ratio of red / green fluorescence intensity represented MMP. The results of flow cytometry observation were as follows Fig.28As shown in AE. The red / green fluorescence intensity ratio of SelH-HT22 cells in the Control group was 14.77±0.4667, and the red / green fluorescence intensity ratio in the OD+HG group was 1.433±0.1438, indicating a decrease in MMP, which was statistically different from the Control group (P<0.01); after the simultaneous use of the AMPK activator AI CAR, the red / green fluorescence intensity ratio increased significantly to 11.52±0.735, indicating an increase in MMP, which was statistically different from the OD+HG group (P<0.01); and after the simultaneous use of the SIRT1 inhibitor EX-527 under the condition of using the AMPK activator AICAR, the red / green fluorescence intensity ratio decreased significantly to 5.493±0.4748 compared with the simple application of AICAR, and MMP decreased (P<0.01); after the simple application of EX-527, the red / green fluorescence intensity ratio decreased more significantly to 3.01±0.5417, and MMP decreased (P<0.01). MMP statistical results are as follows Fig.28 As shown in F.

[0166] 3.4 Expression of mitochondrial regeneration-related factors

[0167] 3.4.1 Immunofluorescence observation of the expression of mitochondrial regeneration-related factor p-AMPK

[0168] Immunofluorescence was used to observe the expression changes of mitochondrial regeneration regulator p-AMPK. p-AMPK showed green fluorescence and was mainly expressed in the cell cytoplasm. Mitotracker red fluorescent probe labeled the mitochondria of SelH-HT22 cells. The results showed that in the Control group, weak positive red fluorescence was observed, indicating that the probe detected normal mitochondria, and the p-AMPK fluorescence staining intensity was weak; the p-AMPK fluorescence staining intensity in the mitochondria and cytoplasm of the OD+HG group was significantly enhanced compared with the Control group; compared with the OD+HG group, after the simultaneous use of the activator AICAR, the p-AMPK fluorescence staining intensity in the mitochondria and cytoplasm of the cells was further enhanced; under the condition of using the AMPK activator AICAR and the SIRT1 inhibitor EX-527, the p-AMPK fluorescence staining intensity in the mitochondria and cytoplasm was weakened compared with the AICAR group; after using the inhibitor EX-527 alone, the p-AMPK fluorescence staining intensity in the mitochondria and cytoplasm was further weakened compared with the AICAR+EX-527 group. The results are as follows Fig.29 shown.

[0169] In the figure, blue is DAPI-stained cell nucleus, red is Mitotracker-labeled mitochondria, green is p-AMPK-positive cells, and scale bar = 100 um.

[0170] 3.4.2 Western blot detection of mitochondrial regeneration-related factors p-AMPK, AMPK, SIRT1, PGC-1α, NRF1 and TFAM expression

[0171] We also used Western blot to detect the protein levels of mitochondrial regeneration-related factors in in vitro experiments. The band diagram is shown in Fig.30 As shown in Figure 5A, the results showed that the levels of p-AMPK, SIRT1, NRF1 and TFAM proteins in SelH-HT22 cells in the OD+HG group were significantly upregulated compared with those in the Control group (P<0.01 or P<0.05), while the level of PGC-1α protein was not statistically significant compared with that in the Control group (P>0.05). Compared with the OD+HG group, the levels of p-AMPK, SIRT1, PGC-1α, NRF1 and TFAM proteins in the cells were further increased after the simultaneous use of the activator AICAR (P<0.01 or P<0.05). When the AMPK activator AICAR was used simultaneously with the SIRT1 inhibitor EX-527, the p-AMPK The protein levels of MPK, SIRT1, PGC-1α, NRF1 and TFAM were significantly decreased compared with those in the AICAR group (P < 0.01); after using the inhibitor EX-527 alone, the protein levels of p-AMPK, SIRT1, PGC-1α, NRF1 and TFAM were further downregulated compared with those in the AICAR + EX-527 group (P < 0.01 or P < 0.05). Fig.30 As shown in BF.

[0172] In the figure, A is the strip diagram of p-AMPK, AMPK, SIRT1, PGC-1a, NRF1, and TFAM; BF is the relative gray value ratio of p-AMPK, AMPK, SIRT1, PGC-1a, NRF1, TFAM and β-actin, respectively. Data are expressed as mean ± SD (n = 4). **P < 0.01 and *P < 0.05 compared with the Control group; ##P < 0.01 and #P < 0.05 compared with the OD + HG group; &&P < 0.01 compared with the AICAR group; @@P < 0.01 and @P < 0.05 compared with the AICAR + EX-527 group.

[0173] Through the comparison of the above experimental data, it can be concluded that sodium selenite and SelH can promote mitochondrial regeneration by regulating the AM PK / SIRT1 / PGC-1a pathway, thereby reducing cerebral ischemia-reperfusion injury in patients with hyperglycemia.

[0174] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of selenium in alleviating hyperglycemic cerebral ischemia-reperfusion injury, characterized in that: Selenium improves mitochondrial function by regulating the mitochondrial regeneration signaling pathway, thereby maintaining a relative balance between mitochondrial fission and fusion processes and inhibiting the activation of the mitochondrial apoptosis pathway, thereby alleviating cerebral ischemia-reperfusion injury in patients with hyperglycemia.

2. An application of selenium as claimed in claim 1 in alleviating cerebral ischemia-reperfusion injury caused by hyperglycemia, characterized in that: The mitochondrial regeneration signaling pathway is the AMPK / SIRT1 / PGC-1α signaling pathway.

3. A use of selenium as claimed in claim 1 in alleviating hyperglycemic cerebral ischemia-reperfusion injury, characterized in that: The selenium element enters the human body through selenium-containing substances that can be ingested by the human body.

4. A use of selenium as claimed in claim 3 in alleviating hyperglycemic cerebral ischemia-reperfusion injury, characterized in that: The selenium intake is 17 mg / d to 400 mg / d.

5. A use of selenium as claimed in claim 3 in alleviating hyperglycemic cerebral ischemia-reperfusion injury, characterized in that: The selenium-containing substance that can be ingested by the human body is SelH.

6. A use of selenium as claimed in claim 3 in alleviating hyperglycemic cerebral ischemia-reperfusion injury, characterized in that: The selenium-containing substance that can be ingested by the human body is sodium selenite, and the intake of sodium selenite is 37.2 μg / d to 875.9 μg / d.

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