Application of agonist in treatment of heatstroke brain injury and test method of agonist

Abstract

The invention belongs to the technical field of brain injury treatment medicines, and discloses application of an agonist in treatment of heatstroke brain injury and a test method of the agonist, cell culture of neuroblastoma SH-SY5Y uses a DMEM-F12 culture medium containing 10% of fetal calf serum and 1% of double antibodies; dividing the SH-SY5Y cells into a control group and a 0-hour group, a 3-hour group and a 6-hour group which are subjected to thermal strike rewarming, and culturing the control group in 5% CO2 at 37 DEG C all the time; dissolving an agonist TBHQ in DMSO, and adjusting the concentration of the agonist TBHQ to 40 [mu] M by using a culture medium; and various tests and analyses are carried out. On the basis of two damage mechanisms of oxidative stress, mitochondria and endoplasmic reticulum damage, an NRF2 signal channel is used as one of the most important action mechanisms of cells for resisting oxidative damage, and is activated under the oxidative stress condition to play an antioxidant role; the previous tests show that the NRF2 and HO-1 genes are obviously up-regulated in heatstroke patients and animal models respectively.

Description

Technical Field

[0001] The invention belongs to the technical field of brain injury therapeutic drugs, and in particular relates to the use of an agonist in treating heatstroke brain injury and a testing method thereof. Background Art

[0002] At present, severe heatstroke is an acute heat-related disease characterized by high fever and central nervous system disorders, which may be accompanied by systemic inflammatory response syndrome. Once it leads to multiple organ dysfunction syndrome (MODS), the mortality rate is as high as 30-60%. The central nervous system is one of the first organs to be hit by heatstroke. Previous tests have shown that the severity of brain damage after heatstroke is directly related to the patient's prognosis. The main known damage mechanisms of the central nervous system in severe heatstroke include direct damage from high fever, cerebral ischemia and hypoxia changes, systemic inflammatory response, oxidative stress, mitochondrial and endoplasmic reticulum damage, etc. At present, the main clinical treatment measures for severe heatstroke brain damage include reducing brain edema, anti-inflammatory, cell membrane protection and other treatment measures, but these traditional treatment methods have poor overall effects. 30% of survivors develop long-term sequelae of nervous system damage, which seriously affects the quality of life. Therefore, it is urgent to explore heatstroke-specific targets to reverse heatstroke-related brain damage.

[0003] At present, the treatment measures for severe heatstroke brain damage mainly include dehydration to reduce intracranial pressure, brain protection and neurotrophic drugs such as naloxone, and mild hypothermia therapy. As an emerging treatment for brain function damage in recent years, hyperbaric oxygen therapy has also been gradually applied to the clinical treatment of severe heatstroke. 1. Drug treatment: Neuroprotective drugs such as naloxone and some traditional Chinese medicine preparations such as Xingnaojing have been tested by scholars on their efficacy in severe heatstroke. They are believed to have the effects of promoting neurological function recovery, awakening, antipyretic and anticonvulsant, safe and effective, and fast efficacy, and have been widely used in clinical practice. It is generally believed that the efficacy of naloxone is mainly due to its effect on endogenous opioids, among which β-endorphins (β-EP) have the strongest activity. β-EP plays a certain role in the perception of pain, analgesia, secretion of pituitary hormones, cardiovascular activity and respiration. The test results show that the plasma β-EP content of patients with severe heatstroke is significantly higher than that of normal people, and it is clearly confirmed that heat stress is an important factor in promoting the secretion and release of β-EP. Xingnaojing injection is a traditional Chinese medicine preparation modified from Angong Niuhuang Pills, mainly composed of musk, gardenia, borneol and turmeric. Traditional Chinese medicine believes that it has a good function of opening the orifices and refreshing the mind, can open all the orifices and disperse evil fire, clear away heat and purge fire, cool blood and detoxify, and promote qi and blood circulation. Modern pharmacological tests also believe that Xingnaojing has a significant effect of scavenging oxygen free radicals and antagonizing cytokines. Animal model experiments have shown that it has a clear effect of reducing tissue cell pathological damage and effectively preventing and treating neuronal apoptosis. 2. Mild hypothermia therapy: Internationally, hypothermia is divided into mild hypothermia (33-35℃), moderate hypothermia (28-32℃), deep hypothermia (17-27℃) and ultra-deep hypothermia (<17℃), among which mild-moderate hypothermia is called mild hypothermia. In the mild hypothermia state, it can play the greatest protective role on important organs such as the heart and brain, and there are no obvious adverse reactions. Its main mechanism is: preventing the initiation of neuronal apoptosis; reducing the excessive release of excitatory amino acids and free radicals in the brain; preventing the loss of control of the body temperature regulation center due to the increase in intracranial temperature, which causes ischemic and hypoxic changes after the visceral blood flow flows outward. At present, there are few tests or case reports on the protective effect of hypothermia therapy on the brain in severe heatstroke at home and abroad, and its mechanism of action and exact efficacy remain to be elucidated. 3. Hyperbaric oxygen therapy: In recent years, hyperbaric oxygen therapy (HBO) has been widely used in the treatment of a variety of ischemic and hypoxic diseases in clinical practice as a non-invasive treatment measure. It has unique efficacy on brain damage, and has molecular biological effects such as improving tissue ischemia and hypoxia, improving circulatory disorders, reducing cerebral edema, and anti-inflammatory, anti-oxidative damage, and anti-apoptosis. Some tests believe that it can also promote awakening and neural repair for brain damage caused by heatstroke, and will be a new strategy for the treatment of severe heatstroke brain damage. In terms of efficacy, there are currently only a few clinical tests and case reports, but the role of hyperbaric oxygen in treating severe heatstroke brain damage is affirmed.Some tests also pointed out that the combined use of Xingnaojing and hyperbaric oxygen to treat patients with heat stroke coma can quickly relieve high fever symptoms, reduce ischemia-reperfusion-induced brain cell death, significantly reduce brain cell edema, effectively improve cerebral circulation, shorten the patient's coma time, and effectively improve the patient's prognosis. It is believed that the combined treatment of the two has a significant effect on the cranial nerve rehabilitation of patients with heat stroke coma.

[0004] Through the above analysis, the problems and defects of the prior art are as follows:

[0005] At present, the main clinical treatment measures for severe heatstroke brain damage include reducing brain edema, anti-inflammation, cell membrane protection and other treatment measures, but these traditional treatment methods are generally ineffective, and 30% of survivors may develop long-term sequelae of neurological damage, which seriously affects the quality of life. Therefore, it is urgent to explore heatstroke-specific targets to reverse heatstroke-related brain damage. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a use of an agonist in treating heatstroke brain damage and a testing method thereof.

[0007] The present invention is achieved by using an agonist in treating heat stroke brain damage, wherein the agonist is an NRF2 agonist.

[0008] Another object of the present invention is to provide a method for testing the use of the agonist in treating heat stroke brain damage, and the method for testing the use of the agonist in treating heat stroke brain damage comprises the following steps:

[0009] In the first step, neuroblastoma SH-SY5Y cells were cultured using DMEM-F12 medium containing 10% fetal bovine serum and 1% double antibody in a cell culture incubator at 5% CO2 and 37°C. The medium was changed every other day, and cells were passaged according to the cell growth conditions, and cells in the logarithmic growth phase were selected;

[0010] In the second step, SH-SY5Y cells were divided into a control group and a heat shock rewarming group at 0h, 3h, and 6h. The control group was cultured at 37°C and 5% CO2 all the time, and the experimental group was placed in a 43°C and 5% CO2 incubator for 2h for heat shock. After the heat shock, the cells were immediately transferred to a conventional incubator at 37°C and 5% CO2 for rewarming before various tests were performed.

[0011] In the third step, after dissolving the agonist TBHQ in DMSO, the concentration was adjusted to 40 μM with culture medium, and the DMSO concentration was kept below 0.01%; NRF2 siRNA was transfected into SH-SY5Y cells, and cell viability was detected, protein expression was detected by Western blot, mRNA expression was detected by RT-qPCR, ROS content was detected by flow cytometry, the morphological structure of SH-SY5Y cell MAMs was observed by transmission electron microscopy, and SH-SY5Y cell immunofluorescence analysis was performed.

[0012] Furthermore, in the third step, TBHQ was used to intervene in SH-SY5Y cells 24 hours before heat shock, and the intervention time was 24 hours; the experimental groups were: control group, TBHQ group only used agonist pretreatment, HS group only heat shock, and TBHQ and HS group agonist pretreatment followed by heat shock, and the heat shock treatment group selected the rewarming time with the highest NRF2 protein expression.

[0013] Further, in the third step, NRF2 siRNA transfects SH-SY5Y cells, and the NRF2 gene silencing sequence is NRF2-siRNA; by detecting the transfection efficiency of 5 to 100 nmol / L, the transfection effect with the least cytotoxicity is screened. The cells are plated one day in advance, and the cell density is 30% to 50% during transfection. NRF2 siRNA is dissolved in Opti-MEM serum-free medium, mixed, and placed for 5 minutes. At the same time, lipo2000 is dissolved in Opti-MEM serum-free medium, the two are mixed, and placed for 20 minutes. The above mixture is added to the culture well, mixed, and cultured in a 37°C, 5% CO2 incubator for 4 hours. After removing the supernatant, add a complete medium containing 10% fetal bovine serum and culture for 48 hours. After passing the PCR test, it is ready for use; the negative control NC NRF2-siRNA is transfected in the same way; the experimental groups are: control group, NRF2 siRNA group, HS group and NRF2 siRNA+HS group, and the heat shock treatment group selects the rewarming time with the highest NRF2 protein expression;

[0014] In the third step, cell viability detection, SH-SY5Y cells were inoculated in a 96-well cell culture plate, 5000 cells / well, 100 μL / well of culture medium, and cultured in a cell culture incubator for 24 hours. The experiment was divided into 4 groups: a control group, 0, 3, and 6 hours, and 3 replicates were set in each group. On the second day, the cells were heat-shocked at 43°C for 2 hours. After rewarming for 0, 3, and 6 hours, the culture medium was aspirated and discarded, and 100 μL of culture medium containing 10% CCK8 stock solution was added to each well. The cells were continued to be cultured in a cell culture incubator for 2 hours, and then the absorbance value (A450 value) of each group was detected using an ELISA reader at a wavelength of 450 nm; cell survival rate (%) = (A450 value of the experimental group - A450 value of the blank group) / (A450 value of the control group - A450 value of the blank group), and no cells were added to the blank group, only an equal amount of culture medium was added.

[0015] Furthermore, in the third step, Western blot is used to detect protein expression, including:

[0016] Cell protein extraction:

[0017] 1) Total protein extraction: Collect cells from the control group and experimental group, use 4°C pre-cooled protein lysis buffer RIPA: PMSF = 100:1 to fully lyse the cells on ice, centrifuge at 4°C, 12000r / min, 15min, and collect the supernatant, which is the total cell protein;

[0018] 2) Extraction of cytoplasmic and nuclear proteins:

[0019] Collect the cell pellet and add 200 μl of cytoplasmic protein extraction reagent A supplemented with PMSF for every 20 μl of cell pellet;

[0020] Vortex vigorously at the highest speed for 5 seconds to completely suspend and disperse the cell pellet, and place on ice for 10-15 minutes;

[0021] Add 10 μl of cytoplasmic protein extraction reagent B, vortex at the highest speed for 5 seconds, and place on ice for 1 minute;

[0022] Vortex at maximum speed for 5 seconds, centrifuge at 12,000-16,000 g for 5 minutes at 4°C;

[0023] Pipette the supernatant into a pre-cooled plastic tube, which is the extracted cytoplasmic protein;

[0024] For the precipitation, completely aspirate the remaining supernatant and add 50 μl of nuclear protein extraction reagent supplemented with PMSF;

[0025] Vortex at the highest speed for 15-30 seconds to completely suspend and disperse the cell pellet, then return to the ice bath and vortex at the highest speed for 15-30 seconds every 1-2 minutes for a total of 30 minutes.

[0026] Centrifuge at 12,000-16,000 g for 10 min at 4°C;

[0027] Pipette the supernatant into a pre-cooled plastic tube, which is the extracted nuclear protein;

[0028] Protein concentration determination:

[0029] 1) Draw a standard curve: add reagents to the ELISA plate in sequence;

[0030] 2) Take 2 μL of the sample to be tested and add it to the well, then add 18 μL of PBS to dilute the sample to be tested 10 times;

[0031] 3) Prepare BCA working solution at a ratio of 50:1, mix it thoroughly, and add 200 μL of BCA working solution to each well;

[0032] 4) Place the mixture in an incubator at 37°C for 30 minutes, measure the OD value at a wavelength of 562 nm, and then draw a standard curve with the protein content as the horizontal axis and the OD value as the vertical axis, and calculate the concentration according to the OD value of the sample to be tested;

[0033] Protein denaturation: Add 1 / 4 volume of 5× SDS-PAGE protein loading buffer to the collected protein sample and heat in a boiling water bath for 10 min to fully denature the protein;

[0034] Electrophoresis:

[0035] 1) Preparation of electrophoresis solution: 1g SDS, 3.03g Tris, 14.4g Glycine, add 1L ddH2O and stir to dissolve;

[0036] 2) According to the molecular weight of the sample protein to be tested, select SDA-PAGE gel, add 20 μg of protein to each SDS-PAGE gel well, run electrophoresis at 80V for 10 minutes, then switch to 120V and continue electrophoresis for 50 minutes;

[0037] Transfer:

[0038] 1) Preparation of transfer solution: 3.03g Tris, 14.4g Glycine, add 200mL methanol and 800mL ddH2O and stir to dissolve;

[0039] 2) Transfer conditions: Use PVDF membrane, transfer on ice at 250 mA for 90 min;

[0040] Blocking: 5% skim milk powder was blocked at room temperature for 2 h on a shaker (40 rpm);

[0041] Wash the membrane: discard skim milk powder, add appropriate amount of 1×TBST and wash the membrane 3 times on a shaker at 60 rpm for 5 min;

[0042] Primary antibody and secondary antibody incubation: add primary antibody and incubate at 4°C overnight, wash the membrane three times, add secondary antibody and incubate at room temperature for 1 hour on a shaker at 40 rpm, and wash the membrane three times;

[0043] Development: Add ECL chemiluminescent solution for development;

[0044] Analysis: Image Lab software was used for semi-quantitative analysis of protein bands.

[0045] Furthermore, in the third step, RT-qPCR detection of mRNA expression includes:

[0046] RNA extraction

[0047] 1) Sample lysis: cells were seeded on a 6-well plate. At the corresponding treatment time point, the 6-well plate was removed and the medium was discarded. The plate was washed 2-3 times with pre-cooled PBS, and then the PBS was discarded. 500 μl Buffer RL was added to each well.

[0048] 2) Removal of gDNA: transfer the lysate to FastPure gDNA-Filter Column III, 12,000 rpm (13,400 × g); centrifuge for 30 seconds and collect the filtrate;

[0049] 3) Adjust the upper column: add 0.5 times the volume of filtrate anhydrous ethanol to the filtrate and mix well;

[0050] 4) RNA adsorption: transfer the entire mixture to FastPure RNA Column III, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the filtrate;

[0051] 5) Remove impurities: add 700 μl Buffer RW1, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard the filtrate, add 700 μl Buffer RW2, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard the filtrate, add 500 μl Buffer RW2, centrifuge at 12,000 rpm (13,400 × g) for 2 minutes, and discard the collection tube;

[0052] 6) Elution of RNA: 50-200 μl RNase-free ddH2O, centrifugation at 12,000 rpm (13,400×g) for 1 min.

[0053] Furthermore, in the third step, the RNA concentration was determined by using Nanodrop1000 to detect the concentration and purity of the extracted RNA, using RNase-free ddH2O as a control, taking 2 μl of each sample for detection, and directly obtaining the concentration value; if the A260 / A280 ratio was in the range of 1.8-2.1, it indicated that the sample had a high purity; after the detection was completed, the reverse transcription reaction was immediately performed or temporarily stored in a -80°C refrigerator.

[0054] Further, in the third step, reverse transcription comprises:

[0055] 1) Genomic DNA removal

[0056] Prepare the mixture in an RNase-free centrifuge tube: gently pipette to mix, incubate at 42°C for 2 min;

[0057] 2) Prepare the reverse transcription reaction system and perform the reverse transcription reaction by directly adding 5×HiScript IIIqRT SuperMix into the reaction tube;

[0058] Mix well by gently pipetting, incubate at 37°C for 15 min, then at 85°C for 5 s. The product can be used for qPCR reaction or stored at -20°C.

[0059] Target gene primer design

[0060] Primer design is mainly obtained through two ways: method one: by searching for tests with the same gene, selecting primers with the same gene, and comparing them in NCBI's Blast; method two: using Primer5.0 software to design primers, then comparing the primer pairs in Blast, and finally sending the synthesized primer pairs to the company for synthesis;

[0061] PCR reaction

[0062] 1) Prepare the following mixture in a qPCR tube;

[0063] 2) Perform qPCR reaction according to the following conditions.

[0064] Furthermore, in the third step, the ROS content is detected by flow cytometry, including:

[0065] 1) After heat shock, discard the cell culture medium, wash once with pre-cooled PBS, add 0.25% trypsin, digest at 37°C for 5 minutes, and pipette 10-15 times until a cell suspension is formed;

[0066] 2) Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L;

[0067] 3) The cells were centrifuged at 500 rpm for 5 min and collected. The diluted DCFH-DA was added and placed in a 37°C cell culture incubator for 20 min. The cells were mixed once every 3 min to ensure full contact between the probe and the cells. The cells were washed once with serum-free medium and detected by flow cytometry as soon as possible within 1 h.

[0068] Furthermore, in the third step, the morphological structure of SH-SY5Y cell MAMs is observed by transmission electron microscopy, including:

[0069] 1) After heat shock, cells were collected and digested with 0.25% trypsin at 37°C for 5 min. Serum-containing culture medium was added to terminate the digestion reaction, and the cells were blown into a single cell suspension;

[0070] 2) After centrifugation at 500 rpm for 5 min, the supernatant was discarded and the volume of electron microscopy fixative was added at a ratio of 50:1 to the volume of cells;

[0071] 3) Send the fixed specimens to the electron microscope room for post-processing and observation;

[0072] In the third step, SH-SY5Y cell immunofluorescence analysis includes:

[0073] 1) Place a sterilized cover glass in a 6-well plate and coat it with poly-lysine, then plant SH-SY5Y cells in the wells and culture them, and perform heat shock treatment during the logarithmic growth phase;

[0074] 2) Discard the cell culture medium, rinse once with pre-cooled PBS, fix with 4% paraformaldehyde for 20 min, and then rinse three times with PBS, 10 min each time;

[0075] 3) Permeabilization: Dilute Triton 100 to 0.5% using sheep serum working solution, mix well and add to a 6-well plate, and place on ice for 10 min;

[0076] 4) Block the cells three times with sheep serum working solution, each time for 10 min;

[0077] 5) Add primary antibody: dilute the antibody with sheep serum working solution at a dilution ratio of 1:300, and then incubate at 4°C overnight;

[0078] 6) After discarding the primary antibody, wash the cells three times with sheep serum working solution, each time for 10 minutes;

[0079] 7) Add secondary antibody: Add fluorescent secondary antibody with wavelength of 594nm corresponding to the species of primary antibody, dilute at a ratio of 1:500, and incubate at room temperature in a dark place for 1 hour;

[0080] 8) After discarding the secondary antibody, rinse with PBS three times, 10 minutes each time; then remove the coverslip and air-dry it, and then turn the cell-seeded surface upside down on a slide with DAPI mounting medium added, air-dry it, store it at -20°C away from light, and observe it under a fluorescence microscope.

[0081] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0082] First, in view of the technical problems existing in the above-mentioned prior art and the difficulty of solving the problems, closely combined with the technical solutions to be protected by the present invention and the results and data in the research and development process, a detailed and in-depth analysis of how the technical solutions of the present invention solve the technical problems, and some creative technical effects brought about after solving the problems. The specific description is as follows: The present invention aims to clarify that the NRF2 signaling pathway reduces the level of oxidative stress, enhances the expression of mitochondrial-related endoplasmic reticulum membrane proteins, improves the morphological and functional abnormalities of mitochondrial-related endoplasmic reticulum membranes, and reshapes the crosstalk between mitochondria and endoplasmic reticulum, thereby exerting a protective mechanism against oxidative damage to the nervous system in severe heatstroke, and provides a theoretical basis for the treatment strategy of heatstroke-related brain injury targeting the NRF2 signaling pathway.

[0083] The preliminary experiments of the present invention found that the expression level of mitochondrial fusion protein 2 (MFN2), a key protein involved in the formation of MAMs, decreased in SH-SY5Y cells after heat shock, suggesting that abnormal changes in MAMs occurred after heat shock, thereby affecting normal mitochondrial and endoplasmic reticulum crosstalk. TBHQ is an NRF2-specific agonist that can activate the NRF2-KEAP1 pathway, reduce the production of ROS, maintain the function of the key protein MFN2 in MAMs of damaged primary hippocampal neurons, and protect damaged primary hippocampal neurons from oxidative stress and mitochondrial disorders. In addition, HO-1, a downstream protein of the NRF2 signaling pathway, can exert an endogenous protective effect on acute lung injury caused by sepsis by regulating NAD+-mediated mitochondrial communication. Therefore, the present invention speculates that NRF2 can promote the stability of the structure of MAMs by regulating oxidative stress, thereby exerting a protective effect on heatstroke-related brain damage.

[0084] Second, considering the technical solution as a whole or from the perspective of the product, the technical effects and advantages of the technical solution to be protected by the present invention are specifically described as follows: The present invention explores heatstroke-specific therapeutic targets based on two major damage mechanisms: oxidative stress, mitochondrial and endoplasmic reticulum damage. As one of the most important mechanisms of action of cells against oxidative damage, the NRF2 signaling pathway is activated under oxidative stress conditions to exert antioxidant effects. Previous tests have observed significant upregulation of NRF2 and HO-1 genes in heatstroke patients and animal models, respectively. The preliminary experiments of the present invention also confirmed that heatstroke can lead to the activation of the NRF2 signaling pathway from three aspects: cells, animals, and clinics. In recent years, NRF2 has received increasing attention as a molecular target for the treatment of sepsis patients in intensive care. According to a search on "ClinicalTrials.gov", there are about 800 clinical trials of sepsis focusing on the concept of NRF2-dependent treatment, among which the most tested are curcumin, stilbene derivative resveratrol, etc. Severe heatstroke is considered to be "sepsis-like", and the latest tests have also shown that shikonin can enhance the stress resistance of heatstroke mice by upregulating the expression of NRF2. Therefore, treatments that rely on NRF2 are feasible in severe heatstroke.

[0085] Mitochondria and endoplasmic reticulum (ER) are organelles closely related to oxidative stress. The two are connected by a tether composed of multiple proteins to form mitochondria-associated endoplasmic reticulum membranes (MAMs). MAMs are highly dynamic ER-mitochondria contact sites. As an important means of signal exchange between organelles, they are mainly involved in processes such as Ca2+ exchange, lipid transport, autophagy, inflammation and oxidative stress. The disorder of MAMs is one of the key factors leading to nervous system damage. Although there is currently no test of MAMs in severe heatstroke (sepsis-like), increased MAMs formation and increased mitochondrial fragmentation were observed in animal models of sepsis. By regulating the formation of MAMs and interfering with the crosstalk between ER and mitochondria, apoptosis and damage of cardiomyocytes in septic mice can be inhibited, thereby improving myocardial tissue damage.

[0086] Third, the present invention relates to a test method for the use of agonists in the treatment of heat stroke brain injury, especially the use of NRF2 agonists (such as TBHQ), which demonstrates significant technical progress in cell models. These advances are mainly reflected in the following aspects:

[0087] 1. Establishment and application of cell models:

[0088] Neuroblastoma SH-SY5Y cells were used as a model to simulate the environment of heat stroke brain damage. This cell model provides a relatively simple and controllable environment for research and can effectively simulate the biological response under the conditions of heat stroke brain damage.

[0089] 2. Heat shock simulation and treatment group settings:

[0090] By subjecting SH-SY5Y cells to heat shock at 43°C and 5% CO2, the environment of heat stroke brain damage was successfully simulated.

[0091] The experimental setup included different rewarming time groups (0h, 3h, 6h), which could help determine the dynamic changes of NRF2 protein expression after heat stroke.

[0092] 3. Application and pre-treatment of agonists:

[0093] TBHQ was used as an NRF2 agonist and dissolved and diluted to an appropriate concentration in DMSO for cell treatment. This pre-treatment strategy can evaluate the potential effect of NRF2 agonists on alleviating or preventing heat stroke brain injury.

[0094] Cells were treated with TBHQ 24 hours before heat shock, a step that allowed the assessment of the preventive effect of the agonist.

[0095] 4. Comprehensive biological detection methods:

[0096] The effects of NRF2 agonists on cells were comprehensively evaluated using a variety of biological techniques, such as cell viability assays, Western blot, RT-qPCR, flow cytometry, and transmission electron microscopy.

[0097] These methods not only evaluate the effects of agonists on cell survival, but also detect changes in protein and mRNA expression levels, ROS content, and cell structure, providing comprehensive biological information.

[0098] 5. Optimize treatment strategies:

[0099] Through the experimental results, the use of NRF2 agonists can be optimized to provide a more effective strategy for the treatment of heat stroke brain damage.

[0100] In summary, the application of this testing method not only provides a new strategy for treating heatstroke brain injury, but also enhances the understanding of the mechanism of heatstroke brain injury through comprehensive experimental design and meticulous biological testing, and lays the foundation for future clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 It is a flow chart of a testing method for the use of an agonist provided in an embodiment of the present invention in treating heatstroke brain damage.

[0102] Figure 2 It is a schematic diagram of a testing method for the use of an agonist provided in an embodiment of the present invention in treating heatstroke brain damage.

[0103] Figure 3 It is a schematic diagram of detecting the expression level of NRF2 gene provided by an embodiment of the present invention.

[0104] Figure 4 It is a schematic diagram of the effects of heat shock and the addition of NRF2-specific agonist TBHQ on SH-SY5Y cell viability provided in an embodiment of the present invention.

[0105] Figure 5 It is a schematic diagram of the effect of heat shock on the mRNA level of NRF2 signaling pathway in SH-SY5Y cells provided in an embodiment of the present invention.

[0106] Figure 6 Schematic diagram of the effect of heat shock on the protein level of NRF2 signaling pathway in SH-SY5Y cells provided in an embodiment of the present invention.

[0107] Figure 7 Schematic diagram of the effect of heat shock on MFN2 protein level in SH-SY5Y cells provided in an embodiment of the present invention.

[0108] Figure 8This is the effect of heat shock on SH-SY5Y cell viability provided by the embodiment of the present invention. Data are expressed as standard deviation ± mean value, n = 3. **P < 0.01, ***P < 0.001.

[0109] Fig. 9 The effect of heat shock on the gene expression profile of SH-SY5Y cells provided by the embodiment of the present invention. (A) The three volcano plots are the differentially expressed genes identified by comparing R0, R3, R6 with the control group. (B) Venn diagram of differentially expressed genes in the above three comparison combinations. (C) Cluster analysis results of differentially expressed genes in four groups of samples. (D) Enrichment analysis results of BP, CC, and MF with the top 10 most significant differences. (E) Enrichment analysis results of KEGG pathways with the top 20 most significant differences. BP: biological process; CC: cellular component; MF: molecular function.

[0110] Fig.10 The embodiments of the present invention provide that the oxidative stress level increases and the mitochondrial damage increases after heat shock. (A) Cluster analysis results of differentially expressed genes related to oxidative stress. (B) Effect of heat shock on ROS levels. (C) Cluster analysis results of differentially expressed genes related to MAMs. (D) Changes in the structure of MAMs after heat shock. (E) Changes in the contact distance and contact platform length of MAMs (contact length of Mito and ER <30nm) after heat shock. (F) Changes in MFN2 mRNA and protein expression levels after heat shock. Data are expressed as standard deviation ± mean, n = 3. *P < 0.05, **P < 0.01.

[0111] Fig.11 The NRF2 antioxidant signaling pathway is activated after heat shock provided by the embodiment of the present invention. (A) Cluster analysis diagram of genes related to the NRF2 signaling pathway after heat shock. (BC) Changes in the expression levels of mRNA and protein related to the NRF2 pathway after heat shock. (D) Changes in the localization of NRF2 protein after heat shock. Data are expressed as standard deviation ± mean, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0112] Fig.12The TBHQ pretreatment provided by the embodiment of the present invention can activate the NRF2 antioxidant signaling pathway and improve the viability of SH-SY5Y cells after heat shock. (A) Effect of heat shock on cell viability after TBHQ pretreatment. (BC) Effect of TBHQ pretreatment on the expression levels of mRNA and protein related to the NRF2 signaling pathway. (D) Changes in NRF2 protein localization after TBHQ pretreatment. Data are expressed as standard deviation ± mean, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

[0113] Fig.13 After the NRF2 signaling pathway was knocked down by NRF2 siRNA transfection provided in the embodiment of the present invention, the viability of SH-SY5Y cells after heat shock was significantly reduced. (A) Effect of heat shock on cell viability after NRF2 siRNA transfection. (BC) Effect of NRF2 siRNA transfection on the expression levels of mRNA and protein related to the NRF2 signaling pathway. (D) Changes in the localization of NRF2 protein after NRF2 siRNA transfection. Data are expressed as standard deviation ± mean, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0114] Fig.14 The NRF2 antioxidant signaling pathway provided in the embodiments of the present invention regulates the ROS level of SH-SY5Y cells. (A) Effect of activating the NRF2 signaling pathway with TBHQ on the ROS level of SH-SY5Y cells after heat shock. (B) Effect of knocking down NRF2 by NRF2 siRNA on the ROS level of SH-SY5Y cells after heat shock. Data are expressed as standard deviation ± mean, n = 3. *P < 0.05, **P < 0.01.

[0115] Fig.15 The effect of the NRF2 antioxidant signaling pathway provided by the embodiment of the present invention on the MAMs structure of SH-SY5Y cells after heat shock. (A) The effect of adding TBHQ to activate the NRF2 signaling pathway on the MAMs structure. (B) The effect of adding TBHQ on the contact distance and platform length of MAMs (platform with contact distance <30nm). (C) The effect of adding TBHQ on the expression of MFN2, a key molecule in the formation of MAMs structure. (D) The effect of knocking down the NRF2 signaling pathway by transfection of NRF2 siRNA on the structure of MAMs. (E) The effect of transfection of NRF2 siRNA on the contact distance and platform length of MAMs (platform with contact distance <30nm). (F) The effect of transfection of NRF2 siRNA on the expression of MFN2, a key molecule in the formation of MAMs structure. Data are expressed as standard deviation ± mean, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0116] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0117] The use of the agonist provided in the embodiment of the present invention in treating heat stroke brain damage is an NRF2 agonist.

[0118] like Figure 1 As shown, the testing method for the use of the agonist provided in the embodiment of the present invention in treating heat stroke brain injury comprises the following steps:

[0119] S101: Neuroblastoma SH-SY5Y cells were cultured in DMEM-F12 medium containing 10% fetal bovine serum and 1% double antibody in a cell culture incubator at 5% CO2 and 37°C. The medium was changed every other day, and cells were passaged according to their growth conditions, and cells in the logarithmic growth phase were selected.

[0120] S102: SH-SY5Y cells were divided into a control group and heat shock rewarming groups at 0h, 3h, and 6h. The control group was cultured at 37°C and 5% CO2 all the time, and the experimental group was placed in a 43°C, 5% CO2 incubator for 2h for heat shock. After the heat shock, the cells were immediately transferred to a conventional incubator (37°C, 5% CO2) for rewarming before various tests were performed.

[0121] S103: After dissolving the agonist TBHQ in DMSO, the concentration was adjusted to 40 μM with culture medium, and the DMSO concentration was kept below 0.01%; NRF2 siRNA was transfected into SH-SY5Y cells, and cell viability was detected, protein expression was detected by Western blot, mRNA expression was detected by RT-qPCR, ROS content was detected by flow cytometry, morphological structure of MAMs of SH-SY5Y cells was observed by transmission electron microscopy, and immunofluorescence analysis of SH-SY5Y cells was performed;

[0122] Embodiment 1:

[0123] 1. Test method: The present invention mainly includes: electron microscopy, HE staining, cell culture, Western Blot, RT-qPCR, immunofluorescence staining, PBMC separation and other testing methods.

[0124] 2. Technical route:

[0125] 1) Cell level test

[0126] The present invention attempts to demonstrate the effects of heat shock on the NRF2 antioxidant pathway of neurons and the morphological function of MAMs through in vitro experiments; and to clarify the positive regulatory effect of the NRF2 signaling pathway on MAMs.

[0127] 2) Animal level testing

[0128] This paper focuses on testing the effects of the NRF2 signaling pathway on the development time, vital signs and survival time, degree of brain damage and morphological function of MAMs in animal models of severe heatstroke, in order to prove the role of NRF2 in central nervous system damage in severe heatstroke in in vivo experiments, and to clarify the feasibility of NRF2-dependent treatment strategies in severe heatstroke. Figure 2 As shown;

[0129] 3) Clinical testing

[0130] This paper focuses on testing the changes in NRF2 gene levels in the development of severe heatstroke patients, and clarifies the mechanism of action of the NRF2 signaling pathway in severe heatstroke, such as Figure 3 As shown;

[0131] 3. Experimental test methods and steps:

[0132] 3.1 Cell experiments

[0133] 3.1.1 Cell culture and passaging

[0134] Neuroblastoma SH-SY5Y cells were purchased from Shanghai Biopharmaceuticals Co., Ltd. Cell culture was performed in DMEM-F12 medium containing 10% fetal bovine serum and 1% double antibody in a cell culture incubator at 5% CO2 and 37°C. The medium was changed every other day and the cells were passaged according to their growth. Cells in the logarithmic growth phase were used for experimental testing.

[0135] 3.1.2 Establishment of in vitro heat shock model

[0136] SH-SY5Y cells were divided into a control group and heat shock rewarming groups at 0h, 3h, and 6h. The control group was cultured at 37°C and 5% CO2 throughout, and the experimental group was placed in a 43°C, 5% CO2 incubator for 2h for heat shock. After the heat shock, they were immediately transferred to a conventional incubator (37°C, 5% CO2) for rewarming, and then various tests were performed.

[0137] 3.1.3 Intervention of SH-SY5Y cells with NRF2 antioxidant pathway-specific agonists

[0138] After the agonist TBHQ was dissolved in DMSO, its concentration was adjusted to 40 μM with culture medium, and the DMSO concentration was kept below 0.01%. TBHQ was used to intervene in SH-SY5Y cells 24 h before heat shock (intervention time was 24 h); experimental groups: control group, TBHQ group (pretreatment with agonist only), HS group (heat shock only) and TBHQ+HS group (heat shock after agonist pretreatment), and the heat shock treatment group selected the rewarming time with the highest expression of NRF2 protein.

[0139] 3.1.4 NRF2 siRNA transfection into SH-SY5Y cells

[0140] The sequence for silencing the NRF2 gene is NRF2-siRNA (SiNRF2-sense RNA, 5′-GGAUUAUUAUGACUGUUAAAU-3′; SiNRF2-antisense RNA, 5′-UUAACAGUCAUAAUAAUCCUU-3′). The transfection efficiency of 5-100 nmol / L was detected to screen the transfection effect with the lowest cytotoxicity. The cells were plated one day in advance, and the cell density was 30%-50% during transfection. NRF2 siRNA was dissolved in Opti-MEM serum-free medium, mixed, and placed for 5 minutes. At the same time, lipo2000 was dissolved in Opti-MEM serum-free medium, the two were mixed, and placed for 20 minutes. The above mixture was added to the culture well, mixed, and cultured in a 37°C, 5% CO2 incubator for 4 hours. The supernatant was removed, and a complete medium containing 10% fetal bovine serum was added to culture for 48 hours. After passing the PCR test, it was set aside. The negative control NC NRF2-siRNA was transfected in the same way. The experimental groups were control group, NRF2 siRNA group (only NRF2 siRNA transfection treatment), HS group (only heat shock) and NRF2 siRNA+HS group (NRF2 siRNA transfection followed by heat shock). The heat shock treatment group selected the rewarming time when NRF2 protein expression was the highest.

[0141] 3.1.5 Cell viability assay

[0142] SH-SY5Y cells were seeded in a 96-well cell culture plate, 5000 cells / well, 100 μL / well of culture medium. Cultured in a cell culture incubator for 24 hours. The experiment was divided into 4 groups: control group, 0, 3, 6 hours, and 3 replicates were set up in each group. On the second day, the cells were heat-shocked at 43°C for 2 hours, and after rewarming for 0, 3, and 6 hours, the culture medium was aspirated and 100 μL of culture medium containing 10% CCK8 stock solution was added to each well, and cultured in a cell culture incubator for 2 hours. Then, the absorbance value (A450 value) of each group was detected using an ELISA reader at a wavelength of 450 nm. Cell survival rate (%) = (A450 value of the experimental group - A450 value of the blank group) / (A450 value of the control group - A450 value of the blank group). No cells were added to the blank group, and only an equal amount of culture medium was added.

[0143] 3.1.6 Western blot detection of protein expression

[0144] 3.1.6.1 Cell protein extraction:

[0145] 1) Total protein extraction: Collect cells from the control group and the experimental group. Use sufficient 4°C pre-cooled protein lysis buffer (RIPA:PMSF=100:1) to fully lyse the cells on ice, centrifuge at 4°C (12000r / min, 15min) and collect the supernatant, which is the total cell protein.

[0146] 2) Extraction of cytoplasmic proteins and nuclear proteins:

[0147] 1. Collect the cell pellet and add 200 μl of cytoplasmic protein extraction reagent A supplemented with PMSF for every 20 μl of cell pellet.

[0148] 2. Vortex vigorously at the highest speed for 5 seconds to completely suspend and disperse the cell pellet. Incubate on ice for 10-15 minutes.

[0149] 3. Add 10 μl of cytoplasmic protein extraction reagent B. Vortex vigorously at the highest speed for 5 seconds and place on ice for 1 minute.

[0150] 4. Vortex at maximum speed for 5 seconds and centrifuge at 12,000-16,000g for 5 minutes at 4°C.

[0151] 5. Immediately transfer the supernatant to a pre-cooled plastic tube, which is the extracted cytoplasmic protein. It can be used immediately or frozen.

[0152] 6. For the precipitate, completely aspirate the remaining supernatant and add 50 μl of nuclear protein extraction reagent with PMSF added. (Failure to aspirate the supernatant will result in contamination of cytoplasmic proteins.)

[0153] 7. Vortex at the highest speed for 15-30 seconds to completely suspend and disperse the cell pellet. Then return to the ice bath and vortex at the highest speed for 15-30 seconds every 1-2 minutes for a total of 30 minutes.

[0154] 8. Centrifuge at 12,000-16,000g for 10 minutes at 4°C.

[0155] 9. Immediately transfer the supernatant to a pre-cooled plastic tube, which is the extracted nuclear protein. It can be used immediately or frozen at -70℃.

[0156] 3.1.6.2 Protein concentration determination:

[0157] 1) Draw a standard curve: Add reagents to the ELISA plate in sequence according to the table below;

[0158] Protein standard (μL) 0 1 2 4 8 12 16 20 PBS (μL) 20 19 18 16 12 8 4 0 Corresponding protein concentration (μg / μL) 0 0.025 0.05 0.1 0.2 0.3 0.4 0.5

[0159] 2) Take 2 μL of the sample to be tested and add it to the well, then add 18 μL of PBS to dilute the sample to be tested 10 times;

[0160] 3) Prepare BCA working solution at a ratio of 50:1, mix it thoroughly, and add 200 μL of BCA working solution to each well;

[0161] 4) Place the sample in an incubator at 37°C for 30 minutes, measure the OD value at a wavelength of 562 nm, and then draw a standard curve with the protein content as the horizontal axis and the OD value as the vertical axis, and calculate the concentration based on the OD value of the sample to be tested.

[0162] 3.1.6.3 Protein denaturation: Add 1 / 4 volume of 5×SDS-PAGE protein loading buffer to the collected protein samples and heat in a boiling water bath for 10 min to fully denature the protein.

[0163] 3.1.6.4 Electrophoresis:

[0164] 1) Preparation of electrophoresis solution: 1g SDS, 3.03g Tris, 14.4g Glycine, add 1L ddH2O and stir to dissolve.

[0165] 2) According to the molecular weight of the sample protein to be tested, select the appropriate SDA-PAGE gel, add 20 μg of protein to each SDS-PAGE gel loading hole, and run electrophoresis at 80V for 10 minutes, then switch to 120V and continue electrophoresis for 50 minutes.

[0166] 3.1.6.5 Transfer:

[0167] 1) Preparation of transfer solution: 3.03g Tris, 14.4g Glycine, add 200mL methanol and 800mL ddH2O and stir to dissolve.

[0168] 2) Transfer conditions: Use PVDF membrane, transfer on ice at 250 mA for 90 min.

[0169] 3.1.6.6 Blocking: Block with 5% skimmed milk powder on a shaker (40 rpm) at room temperature for 2 h.

[0170] 3.1.6.7 Washing the membrane: discard skimmed milk powder, add appropriate amount of 1×TBST and wash the membrane 3 times on a shaker (60 rpm, 5 min).

[0171] 3.1.6.8 Primary and secondary antibody incubation: Add primary antibody and incubate at 4°C overnight. Wash the membrane three times and then add secondary antibody and incubate at room temperature on a shaker (40 rpm) for 1 hour. Wash the membrane three times.

[0172] 3.1.6.9 Development: Add ECL chemiluminescent solution for development.

[0173] 3.1.6.10 Analysis: Image Lab software was used to perform semi-quantitative analysis of protein bands.

[0174] 3.1.7 RT-qPCR detection of mRNA expression

[0175] 3.1.7.1 RNA extraction

[0176] 1) Sample lysis: cells were seeded on a 6-well plate. At the corresponding treatment time point, the 6-well plate was removed and the medium was discarded. The plate was washed 2-3 times with pre-cooled PBS, and then the PBS was discarded. 500 μl Buffer RL was added to each well.

[0177] 2) Removal of gDNA: Transfer lysate to FastPure gDNA-Filter Column III, 12,000 rpm (13,400 × g)

[0178] Centrifuge for 30 seconds and collect the filtrate;

[0179] 3) Adjust the upper column: add 0.5 times the volume of filtrate anhydrous ethanol to the filtrate and mix thoroughly;

[0180] 4) RNA adsorption: transfer the entire mixture to FastPure RNA Column III, 12,000 rpm (13,400 × g)

[0181] Centrifuge for 30 seconds and discard the filtrate;

[0182] 5) Remove impurities: Add 700 μl Buffer RW1, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard the filtrate, add 700 μl Buffer RW2 (with anhydrous ethanol), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard the filtrate, add 500 μl Buffer RW2 (with anhydrous ethanol), centrifuge at 12,000 rpm (13,400 × g) for 2 minutes, and discard the collection tube.

[0183] 6) Elution of RNA: 50-200 μl RNase-free ddH2O, centrifugation at 12,000 rpm (13,400×g) for 1 min.

[0184] 3.1.7.2 RNA concentration determination

[0185] Use Nanodrop1000 to detect the concentration and purity of the extracted RNA, and use RNase-free ddH2O as a control. Take 2μl of each sample for testing to directly obtain the concentration value. If the A260 / A280 ratio is in the range of 1.8-2.1, it means that the sample purity is high. After the test is completed, reverse transcription reaction is immediately carried out or temporarily stored in a -80℃ refrigerator.

[0186] 3.1.7.3 Reverse transcription

[0187] 1) Genomic DNA removal

[0188] Prepare the following mixture in an RNase-free centrifuge tube:

[0189]

[0190] Mix by gently pipetting. Incubate at 42°C for 2 min.

[0191] 2) Prepare the reverse transcription reaction system and perform reverse transcription reaction

[0192] Add 5×HiScript III qRT SuperMix directly to the reaction tube in step 1.

[0193]

[0194] Mix well by gently pipetting. Incubate at 37℃ for 15min and then at 85℃ for 5s. The product can be used for qPCR reaction immediately or stored at -20℃.

[0195] 3.1.7.4 Target gene primer design

[0196] Primer design is mainly obtained through two ways: method one: by searching for tests with the same gene, selecting primers of the same gene, and performing gene comparison in NCBI's Blast; method two: using Primer5.0 software for primer design, then performing gene comparison of primer pairs in Blast, and finally sending the synthesized primer pairs to the company for synthesis.

[0197] 3.1.7.5 PCR reaction

[0198] 1) Prepare the following mixture in a qPCR tube

[0199]

[0200] 2) Perform qPCR reaction according to the following conditions

[0201]

[0202] 3.1.8 Detection of ROS content by flow cytometry

[0203] 1) After heat shock, discard the cell culture medium, wash once with pre-cooled PBS, add 0.25% trypsin, digest at 37°C for 5 minutes, and gently blow 10-15 times until a cell suspension is formed;

[0204] 2) Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L;

[0205] 3) After the cells were centrifuged (500 rpm, 5 min) and collected, diluted DCFH-DA was added and placed in a 37°C cell culture incubator for 20 min. The cells were mixed once every 3 min to ensure full contact between the probe and the cells. The cells were washed once with serum-free medium and detected by flow cytometry as soon as possible within 1 h.

[0206] 3.1.9 Transmission electron microscopy observation of SH-SY5Y cell MAMs morphology

[0207] 1) After heat shock, cells were collected and digested with 0.25% trypsin at 37°C for 5 min. Serum-containing culture medium was added to terminate the digestion reaction, and the cells were blown into a single cell suspension;

[0208] 2) After centrifugation (500 rpm) for 5 min, the supernatant was discarded and electron microscopy fixative (the ratio of its volume to cell volume was 50:1) was added;

[0209] 3) Send the fixed specimens to the electron microscope room for post-processing and observation.

[0210] 3.1.10 Immunofluorescence analysis of SH-SY5Y cells

[0211] 1) Place a sterilized cover glass in a 6-well plate and coat it with poly-lysine, then plant SH-SY5Y cells in the wells and culture them, and perform heat shock treatment during the logarithmic growth phase;

[0212] 2) Discard the cell culture medium, rinse once with pre-cooled PBS, fix with 4% paraformaldehyde for 20 min, and then rinse three times with PBS, 10 min each time;

[0213] 3) Permeabilization: Dilute Triton 100 to 0.5% using sheep serum working solution, mix thoroughly and add to a 6-well plate, and place on ice for 10 min;

[0214] 4) Block the cells three times with sheep serum working solution, each time for 10 min;

[0215] 5) Add primary antibody: dilute the antibody with sheep serum working solution at a dilution ratio of (1:300), and then incubate at 4°C overnight;

[0216] 6) After discarding the primary antibody, wash the cells three times with sheep serum working solution, each time for 10 minutes;

[0217] 7) Add secondary antibody: Add fluorescent secondary antibody (wavelength 594nm) corresponding to the species of the primary antibody, dilute at a ratio of (1:500), and incubate at room temperature in a dark place for 1 hour;

[0218] 8) After discarding the secondary antibody, rinse with PBS three times, 10 minutes each time. Then remove the coverslip and air-dry it, and then invert the cell-seeded surface on a slide with DAPI mounting medium added, air-dry it, store it at -20℃ away from light, and observe it under a fluorescence microscope.

[0219] The embodiments of the present invention have achieved some positive effects during the development or use process, and indeed have great advantages over the prior art. The following content is described in conjunction with data, charts, etc. of the test process.

[0220] 1. Preparation of severe heat stroke animal model

[0221] During the heat stress period, the changes of vital signs were continuously and dynamically monitored. Since estrogen has a protective effect against heat shock, male C57BL / 6 mice (6-8 weeks) were used in this experiment. The mice were placed in a constant temperature climate chamber, and the temperature in the chamber was set to 39±0.5℃ and the humidity was set to 60%±5%. The rectal temperature of the mice was measured with an electronic thermometer every 30 minutes. When the rectal temperature reached 41℃, the rectal temperature was measured every 15 minutes until the rectal temperature reached 42℃. The model was successfully established and immediately removed from the chamber and placed in an ambient temperature of 25±0.5℃ and a humidity of 35%±5% for different times (0h, 3h, 6h) as cooling treatment. After anesthesia, the mice were killed by dislocating the neck and the hippocampal tissue was taken.

[0222] 2. HE staining

[0223] Hippocampal tissues were immediately obtained after sacrifice, fixed with 4% paraformaldehyde, dehydrated and embedded into paraffin wax blocks, sliced ​​and stained with HE, and pathological changes of various tissues were observed under an upright optical microscope and photographed.

[0224] 3. Western blot detection of protein expression

[0225] 3.1. Protein extraction from hippocampal tissue:

[0226] 1) Cytoplasmic protein extraction: Weigh 50 mg of rat hippocampal tissue, grind it, add 900 μL Buffer A and 100 μL Buffer B, mix well on ice and let stand for 30 min. For every 1 mL of Buffer A, add 17 μL 100 mM PMSF and 1 μL protease inhibitor; centrifuge the sample at 4°C (3000 rpm, 10 min), collect the supernatant into a new centrifuge tube, and the cytoplasmic protein is obtained;

[0227] 2) Nuclear protein extraction: Add 200 μl of pre-cooled Buffer C to the above centrifuge tube (add 17 μl of 100 mM PMSF and 1 μl of protease inhibitor to every 1 mL of Buffer C before use), vortex at maximum speed for 15 seconds, place on ice for 30-60 minutes, and vortex at maximum speed for 15 seconds every 10 minutes; centrifuge the sample at 4°C in a centrifuge (14000g, 30 minutes), and transfer the supernatant to a new pre-cooled centrifuge tube as soon as possible to obtain the nuclear protein.

[0228] 3) Total protein extraction: Weigh 20 mg of rat hippocampal tissue, grind it, add 200 μL of lysis buffer (RIPA:PMSF=100:1), lyse on ice for 30 min, centrifuge at 4°C (12000 rpm, 20 min), and collect the supernatant into a new centrifuge tube, which is the total protein.

[0229] 3.2. Subsequent steps are the same as 3.1.6.2-10

[0230] 3.3. RT-qPCR detection of mRNA expression

[0231] RNA extraction

[0232] 1) Take fresh hippocampal tissue, add 500 μL Buffer RL for every 10-20 mg, and use a 1 mL syringe to repeatedly aspirate and blow to break it until there is no obvious tissue mass.

[0233] 3.4. Transmission electron microscopy observation of the morphology and structure of MAMs in hippocampal tissue

[0234] After dissecting the hippocampus on ice, cut the hippocampus into 1 mm pieces. 3 For large and small tissue blocks, three tissue blocks were selected from each rat and immersed in malondialdehyde fixative and sent to the electron microscope room for subsequent specimen processing and observation.

[0235] 3.5. Detection of MDA production in hippocampal tissue

[0236] 1) Rat hippocampal tissue was mixed with normal saline in a ratio of 1:9, and then ground with a homogenizer until a uniform tissue homogenate was formed;

[0237] 2) After the homogenate was centrifuged (3000 rpm, 10 min), most of the supernatant was taken for MDA content detection, and a small part was used for protein content determination;

[0238] 3) Prepare the solution and add the sample according to the table below;

[0239]

[0240] 4) After mixing the sample with a vortex mixer, tie the mouth of the test tube tightly with plastic wrap and pierce a small hole at the mouth of the test tube for ventilation. Heat in a 95°C water bath for 60 minutes, cool with running water and centrifuge (4000 rpm, 10 minutes);

[0241] 5) Take the supernatant and measure it with an enzyme-labeled instrument at a wavelength of 532 nm;

[0242] 6) MDA content calculation formula: ((OD value of the test tube - OD value of the control tube) / (OD value of the standard tube - OD value of the blank tube)) × standard concentration (10nmol / mL) / sample protein concentration (mgprot / mL).

[0243] 3.6. Detection of GSH production in hippocampal tissue

[0244] 1) The method for preparing rat hippocampal tissue homogenate is the same as above;

[0245] 2) Take 0.1 mL of homogenate supernatant, add 0.1 mL of reagent and mix well, centrifuge (3500 rpm, 10 min) and take the supernatant for testing;

[0246] 3) Perform the operations on the supernatant as shown in the following table;

[0247]

[0248] 4) After mixing and standing for 5 min, measure the OD value at a wavelength of 405 nm using an enzyme reader;

[0249] 5) The formula for calculating tissue GSH content is: ((OD value of the assay well - OD value of the blank well) / (OD value of the standard well - OD value of the blank well)) × standard tube concentration (20 μmol / L) × sample pre-treatment dilution factor / sample protein concentration (gprot / L).

[0250] 3.7. Immunofluorescence of hippocampal tissue

[0251] 1) After fixing the brain tissue, make wax blocks and slice them;

[0252] 2) Antigen repair: Place the tissue sections in a kit containing antigen repair buffer and perform repair in a microwave oven (8 minutes at medium heat, 8 minutes off heat, 7 minutes at low heat). Avoid excessive evaporation of the buffer during this process. Allow it to cool naturally and then wash it in PBS for 3 times, 10 minutes each time.

[0253] 3) Autofluorescence quenching: After the slices are slightly dried, use a histochemical pen to draw a circle on the tissue to prevent the loss of antibodies, then drop the autofluorescence quencher in the circle, incubate for 5 minutes, and rinse with tap water for 10 minutes;

[0254] 4) Add BSA to the circle and incubate for 30 minutes for blocking;

[0255] 5) After carefully shaking off the blocking solution, add a certain proportion of diluted primary antibody and place the slices in a humidified box at 4°C overnight;

[0256] 6) Wash the slides in PBS for 3 times, 10 min each time, spin dry, add secondary antibodies of the same species as the primary antibody, and then incubate at room temperature in the dark for 1 h;

[0257] 7) DAPI staining of cell nuclei: Wash the slides in PBS for 3 times, 10 min each time, spin dry the sections, add DAPI staining solution in the circle, and incubate at room temperature in the dark for 30 min;

[0258] 8) Sealing: Wash the slides in PBS for 3 times, 10 min each time, and then seal with a sealing agent that can prevent fluorescence quenching;

[0259] 9) Photographing and observation: Place the slices under a fluorescence microscope for observation and image collection.

[0260] 3.8 Clinical testing

[0261] 3.8.1 Isolation and extraction of peripheral blood mononuclear cells (PBMC)

[0262] Lymphocyte separation fluid (Ficoll) is used to separate mononuclear cells from fresh whole blood. Since the density of blood cells is different, after density gradient centrifugation of lymphocyte separation fluid, various blood cells will be distributed according to their specific gravity and appear in layers. In order to obtain the ideal separation effect, Ficoll separation fluid and PBS buffer should be balanced to room temperature before use.

[0263] Dilute peripheral blood: dilute peripheral blood with PBS at a ratio of 1:1 (the fresher the peripheral blood, the better), and mix thoroughly;

[0264] Aspirate Ficoll: Mix the Ficoll by inverting it, mix the solution thoroughly, open the lid, and inject some air with a sterile syringe to balance the pressure. Add an appropriate amount of Ficoll to the bottom of a new centrifuge tube (a 15mL centrifuge tube works better, avoid using a 50mL centrifuge tube);

[0265] Add diluted peripheral blood: slowly add the diluted peripheral blood to the surface of Ficoll liquid (Ficoll: diluted peripheral blood = 1:1);

[0266] Centrifugation: 400g, room temperature for 40 min, centrifuge speed adjusted to 0, speed adjusted to 2;

[0267] Aspirate PBMC: After centrifugation, the sample is divided into 4 layers, from bottom to top: red blood cell layer, Ficoll layer, PBMC layer, plasma and platelet layer. Use a pipette to carefully aspirate the PBMC layer;

[0268] Wash PBMC: Add 5 times the volume of PBS to wash the cells, centrifuge at 100g for 10 minutes, discard the supernatant, wash the cells again, discard the supernatant, and the cell pellet is PBMC. One part of PBMC is sent to the company for RNA sequencing, and the other part of PBMC is used for RT-qPCR.

[0269] 3.8.2 RT-qPCR detection of NRF2 mRNA expression

[0270] 3.8.2.1 RNA extraction

[0271] 1) Add 500 μL Buffer RL to the isolated PBMC precipitate and mix by pipetting until no precipitate remains;

[0272] 3.9 Statistical analysis

[0273] GraphPad Prism 8.0.2 software was used for statistical analysis. The measurement data were all in accordance with normal distribution and expressed as x±s. One-way analysis of variance was used for comparison among multiple groups, and Dunnett's multiple comparison test was used for further comparison between two groups. P<0.05 was considered statistically significant.

[0274] 4. Preliminary test results:

[0275] 4.1 Role of NRF2-mediated antioxidant signaling pathway in heat stroke-related brain damage

[0276] The research team previously confirmed from the cellular, animal, and clinical levels that heat shock can lead to the activation of the NRF2 signaling pathway, and that pretreatment with the NRF2-specific agonist TBHQ can effectively improve neuronal apoptosis caused by heat shock.

[0277] 1) Cell level: Based on the cell level, the pathophysiological process of severe heatstroke was simulated in vitro, and a heat shock neuroblastoma cell (SH-SY5Y cell) model was established. The CCK8 results showed that compared with the control group, the viability of neurons decreased significantly after heat shock, and as the rewarming time increased, the viability of neurons gradually weakened. The RT-qPCR results showed that the mRNA levels of NRF2, NQO1, and KEAP1 in each rewarming time group after heat shock were significantly lower than those in the control group, which was due to the increase in neuronal apoptosis after heat shock. As the rewarming time increased, neuronal apoptosis increased, and the mRNA expression levels of NRF2, HO-1, and P62 gradually increased, indicating that the NRF2 signaling pathway was activated in large quantities. Slightly different from the RT-qPCR results, the Western Blot results showed that the NRF2 protein expression levels in each rewarming time group after heat shock were not significantly different from those in the control group, but the protein expression level in the 6h rewarming group was significantly lower than that in the 3h rewarming group, and the KEAP1 protein expression level in the 6h rewarming group was significantly lower than that in the control group, which was related to the significant increase in neuronal apoptosis at 6h rewarming. The trend of RT-qPCR and Western Blot results is not synchronized because there is a time difference in the expression of genes and proteins at two different levels. If the rewarming time is further extended, consistency of the results at these two levels will be observed, but this requires more tests to verify.

[0278] The heat shock model of SH-SY5Y cells pretreated with NRF2-specific agonist TBHQ was established in vitro. Compared with the heat shock group, CCK8 results showed that the viability of neurons in the TBHQ pretreatment + heat shock group was significantly improved; RT-qPCR results showed that the mRNA expression levels of NRF2, HO-1, NQO1 and P62 in the TBHQ pretreatment + heat shock group were significantly increased; Western Blot results showed that the protein expression levels of NRF2 and P62 in the TBHQ pretreatment + heat shock group were significantly increased, and the protein expression level of KEAP1 was significantly decreased. The overall results showed that TBHQ pretreatment significantly activated the NRF2 signaling pathway and played a protective role in the apoptosis of neurons after heat shock.

[0279] like Figure 4 As shown in the figure, heat shock increased neuronal apoptosis, and the addition of NRF2-specific agonist TBHQ reduced neuronal apoptosis, suggesting that activation of the NRF2 signaling pathway has a protective effect on neurons.

[0280] like Figure 5 As shown in the figure, the NRF2 signaling pathway was activated as the rewarming time increased after heat shock.

[0281] like Figure 6 As shown in the figure, the NRF2 signaling pathway was activated as the rewarming time increased after heat shock.

[0282] 2) Animal level: Based on the animal level, the pathophysiological process of severe heatstroke was simulated in vivo, and a heat shock animal (C57BL / 6 mouse) model was established. After the modeling was completed, the hippocampal tissue was extracted for subsequent testing. Different from the results at the cell level, the RT-qPCR results of this test part showed that the expression level of NRF2 mRNA in the hippocampus of each rewarming time group after heat shock was significantly higher than that of the control group, among which the expression level of NRF2 mRNA had no significant difference at 0h and 3h of rewarming, and the expression level decreased significantly at 6h of rewarming, but it was still higher than that of the control group. The HO-1 mRNA level in the hippocampus of each rewarming time group after heat shock was also significantly higher than that of the control group, among which the highest expression was at 3h of rewarming, that is, as the rewarming time was prolonged, it generally showed a dynamic trend of first increasing and then decreasing. The overall results suggest that the NRF2 signaling pathway is activated in large quantities in the hippocampus tissue in the early stage after heat shock, but the continuous stress state will lead to the activation disorder of the NRF2 antioxidant pathway, resulting in reduced expression in the later stage, and the protective effect on the hippocampus tissue is also weakened.

[0283] The reason for the difference between the results at the cell level and the animal level is that the animal level is an in vivo test, and the extracted hippocampal tissue is composed of many neurons and glial cells, not a single cell group, so the activation of the NRF2 signaling pathway is affected by more factors than the cell level. However, both the results at the cell level and the animal level indicate that the NRF2 signaling pathway is activated in large quantities after heat shock.

[0284] 3) Clinical test: Peripheral blood samples were collected from patients with severe heat stroke on the 1st, 3rd, and 7th days after admission to the hospital, and then human peripheral blood mononuclear cells (PBMC) were isolated and extracted. PBMCs from healthy adults were also isolated and extracted for comparison. RT-qPCR was used to detect the difference in NRF2 mRNA expression levels between the two groups. The results showed that the NRF2 mRNA expression level in patients with severe heat stroke was significantly lower than that in the healthy control group, which was consistent with the RT-qPCR results at the cell level. There was no obvious pattern in the changes in NRF2 mRNA expression levels on the 1st, 3rd, and 7th days after admission, which was related to the changes in the condition of each patient.

[0285] Based on the preliminary test results of cells, animals and clinical practice, the NRF2 signaling pathway can be compensatorily activated after heat shock, exerting a protective effect against heatstroke brain damage, but continuous oxidative stress gradually weakens the expression of the NRF2 signaling pathway.

[0286] 4.2 Mechanism testing of mitochondrial-associated endoplasmic reticulum membrane MAMs structural disorder in heat stroke-related brain injury

[0287] The present invention detected the changes in protein and mRNA expression of MFN2, a key protein in the formation of MAMs after heat shock, at the cell and animal levels respectively:

[0288] 1) Cell level: Based on the cell level, the pathophysiological process of severe heatstroke was simulated in vitro, and a heat shock neuroblastoma cell (SH-SY5Y cell) model was established. Western Blot results showed that the expression of MFN2 at 0h and 3h after rewarming after heat shock was significantly lower than that of the normal control group. As the rewarming time increased, there was no significant difference in the change of MFN2 protein level, indicating that heat shock can cause the expression of key proteins in MAMs formation to decrease. After heat shock, cell apoptosis increased and the number of cell mitochondria decreased, which is also one of the reasons for the decrease in MFN2 protein expression level.

[0289] like Figure 7 As shown, heat shock can lead to a decrease in the expression of key proteins in the formation of MAMs.

[0290] 2) Animal level: Based on the animal model, the pathophysiological process of severe heatstroke was simulated in vivo, and a heat shock animal (C57BL / 6 mouse) model was established. After the modeling, the hippocampal tissue was extracted for subsequent testing. Western Blot and RT-qPCR results showed that the expression was highest at 0h after rewarming after heat shock, and there was no significant difference in the expression levels of the control group, the 3h rewarming group, and the 6h rewarming group. That is, the expression level of MFN2 increased significantly in the early stage after heat shock, and the expression gradually weakened with the extension of rewarming time. As a key protein in the formation of MAMs, the compensatory upregulation of MFN2 is a key means to block the structural disorder of MAMs, but as the injury time prolongs, the expression of MFN2 gradually decreases and enters an irreversible stage.

[0291] Based on the results of both cell and animal tests, heat shock can lead to a decrease in the expression of MFN2, a key protein in MAMs. However, the intuitive changes in MAMs still need to be further observed under a transmission electron microscope.

[0292] Combining the results of the two parts of the test, the present invention speculates that timely stimulating and maintaining the NRF2 signaling pathway in the early stages of heatstroke can reduce the level of oxidative stress, maintain the stability of the mitochondrial-related endoplasmic reticulum membrane structure and function, and reverse heatstroke-related brain damage, which is the key to treatment.

[0293] 5. Experimental research methods and steps:

[0294] 5.1 Cell culture and passaging

[0295] Neuroblastoma SH-SY5Y cells were purchased from Shanghai Biopharmaceuticals Co., Ltd. Cell culture was performed in DMEM-F12 medium containing 10% fetal bovine serum and 1% double antibody in a cell culture incubator at 5% CO2 and 37°C. The medium was changed every other day and the cells were passaged according to their growth. Cells in the logarithmic growth phase were used for experimental research.

[0296] 5.2 Establishment of in vitro heat shock model

[0297] SH-SY5Y cells were divided into a control group and heat shock rewarming groups at 0h, 3h, and 6h. The control group was cultured at 37°C and 5% CO2 throughout, and the experimental group was placed in a 43°C, 5% CO2 incubator for 2h for heat shock. After the heat shock, they were immediately transferred to a conventional incubator (37°C, 5% CO2) for rewarming, and then various tests were performed.

[0298] 5.3 Intervention of SH-SY5Y cells with NRF2 antioxidant pathway-specific agonists

[0299] After the agonist TBHQ was dissolved in DMSO, its concentration was adjusted to 40 μM with culture medium, and the DMSO concentration was kept below 0.01%. TBHQ was used to intervene in SH-SY5Y cells 24 h before heat shock (intervention time was 24 h); experimental groups: control group, TBHQ group (pretreatment with agonist only), HS group (heat shock only) and TBHQ+HS group (heat shock after agonist pretreatment), and the heat shock treatment group selected the rewarming time with the highest expression of NRF2 protein.

[0300] 5.4 NRF2 siRNA transfection into SH-SY5Y cells

[0301] The sequence for silencing the NRF2 gene is NRF2-siRNA (SiNRF2-sense RNA, 5′-GGAUUAUUAUGACUGUUAAAU-3′; SiNRF2-antisense RNA, 5′-UUAACAGUCAUAAUAAUCCUU-3′). The transfection efficiency of 5-100 nmol / L was detected to screen the transfection effect with the lowest cytotoxicity. The cells were plated one day in advance, and the cell density was 30%-50% during transfection. NRF2 siRNA was dissolved in Opti-MEM serum-free medium, mixed, and placed for 5 minutes. At the same time, lipo2000 was dissolved in Opti-MEM serum-free medium, the two were mixed, and placed for 20 minutes. The above mixture was added to the culture well, mixed, and cultured in a 37°C, 5% CO2 incubator for 4 hours. The supernatant was removed, and a complete medium containing 10% fetal bovine serum was added to culture for 48 hours. After passing the PCR test, it was set aside. The negative control NC NRF2-siRNA was transfected in the same way. The experimental groups were control group, NRF2 siRNA group (only NRF2 siRNA transfection treatment), HS group (only heat shock) and NRF2 siRNA+HS group (NRF2 siRNA transfection followed by heat shock). The heat shock treatment group selected the rewarming time when NRF2 protein expression was the highest.

[0302] 5.5 Cell viability assay

[0303] SH-SY5Y cells were seeded in a 96-well cell culture plate, 5000 cells / well, 100 μL / well of culture medium. Cultured in a cell culture incubator for 24 hours. The experiment was divided into 4 groups: control group, 0, 3, 6 hours, and 3 replicates were set up in each group. On the second day, the cells were heat-shocked at 43°C for 2 hours, and after rewarming for 0, 3, and 6 hours, the culture medium was aspirated and 100 μL of culture medium containing 10% CCK8 stock solution was added to each well, and cultured in a cell culture incubator for 2 hours. Then, the absorbance value (A450 value) of each group was detected using an ELISA reader at a wavelength of 450 nm. Cell survival rate (%) = (A450 value of the experimental group - A450 value of the blank group) / (A450 value of the control group - A450 value of the blank group). No cells were added to the blank group, and only an equal amount of culture medium was added.

[0304] 5.6 Western blot detection of protein expression

[0305] 5.1.6.1 Cell protein extraction:

[0306] 1) Total protein extraction: Collect cells from the control group and the experimental group. Use sufficient 4°C pre-cooled protein lysis buffer (RIPA:PMSF=100:1) to fully lyse the cells on ice, centrifuge at 4°C (12000r / min, 15min) and collect the supernatant, which is the total cell protein.

[0307] 2) Extraction of cytoplasmic proteins and nuclear proteins:

[0308] 1. Collect the cell pellet and add 200 μl of cytoplasmic protein extraction reagent A supplemented with PMSF for every 20 μl of cell pellet.

[0309] 2. Vortex vigorously at the highest speed for 5 seconds to completely suspend and disperse the cell pellet. Incubate on ice for 10-15 minutes.

[0310] 3. Add 10 μl of cytoplasmic protein extraction reagent B. Vortex vigorously at the highest speed for 5 seconds and place on ice for 1 minute.

[0311] 4. Vortex vigorously at maximum speed for 5 seconds and centrifuge at 12,000-16,000g for 5 minutes at 4°C.

[0312] 5. Immediately transfer the supernatant to a pre-cooled plastic tube, which is the extracted cytoplasmic protein. It can be used immediately or frozen.

[0313] 6. For the precipitate, completely aspirate the remaining supernatant and add 50 μl of nuclear protein extraction reagent with PMSF added. (Failure to aspirate the supernatant will result in contamination of cytoplasmic proteins.)

[0314] 7. Vortex at the highest speed for 15-30 seconds to completely suspend and disperse the cell pellet. Then return to the ice bath and vortex at the highest speed for 15-30 seconds every 1-2 minutes for a total of 30 minutes.

[0315] 8. Centrifuge at 12,000-16,000 g for 10 min at 4°C.

[0316] 9. Immediately transfer the supernatant to a pre-cooled plastic tube, which is the extracted nuclear protein. It can be used immediately or frozen at -70℃.

[0317] 3.1.6.2 Protein concentration determination:

[0318] 1) Draw a standard curve: Add reagents to the ELISA plate in sequence according to the table below;

[0319] Protein standard (μL) 0 1 2 4 8 12 16 20 PBS (μL) 20 19 18 16 12 8 4 0 Corresponding protein concentration (μg / μL) 0 0.025 0.05 0.1 0.2 0.3 0.4 0.5

[0320] 2) Take 2 μL of the sample to be tested and add it to the well, then add 18 μL of PBS to dilute the sample to be tested 10 times;

[0321] 3) Prepare BCA working solution at a ratio of 50:1, mix it thoroughly, and add 200 μL of BCA working solution to each well;

[0322] 4) Place the sample in an incubator at 37°C for 30 minutes, measure the OD value at a wavelength of 562 nm, and then draw a standard curve with the protein content as the horizontal axis and the OD value as the vertical axis, and calculate the concentration based on the OD value of the sample to be tested.

[0323] 3.1.6.3 Protein denaturation: Add 1 / 4 volume of 5×SDS-PAGE protein loading buffer to the collected protein samples and heat in a boiling water bath for 10 min to fully denature the protein.

[0324] 5.1.6.4 Electrophoresis:

[0325] 1) Preparation of electrophoresis solution: 1g SDS, 3.03g Tris, 14.4g Glycine, add 1L ddH2O and stir to dissolve.

[0326] 2) According to the molecular weight of the protein to be tested in the sample, select the appropriate SDA-PAGE gel, add 20 μg of protein to each SDS-PAGE gel loading hole, run electrophoresis at 80V for 10 minutes, then switch to 120V and continue electrophoresis for 50 minutes.

[0327] 5.1.6.5 Transfer:

[0328] 1) Preparation of transfer solution: 3.03g Tris, 14.4g Glycine, add 200mL methanol and 800mL ddH2O and stir to dissolve.

[0329] 2) Transfer conditions: Use PVDF membrane, transfer on ice at 250 mA for 90 min.

[0330] 5.1.6.6 Blocking: Block with 5% skimmed milk powder on a shaker (40 rpm) at room temperature for 2 h.

[0331] 5.1.6.7 Washing the membrane: discard the skimmed milk powder, add appropriate amount of 1×TBST and wash the membrane 3 times on a shaker (60 rpm, 5 min).

[0332] 5.1.6.8 Primary and secondary antibody incubation: Add primary antibody and incubate at 4°C overnight. Wash the membrane three times, then add secondary antibody and incubate at room temperature on a shaker (40 rpm) for 1 hour. Wash the membrane three times.

[0333] 5.1.6.9 Development: Add ECL chemiluminescent solution for development.

[0334] 5.1.6.10 Analysis: Image Lab software was used to perform semi-quantitative analysis of protein bands.

[0335] 5.7 RT-qPCR detection of mRNA expression

[0336] 5.1.7.1 RNA extraction

[0337] 1) Sample lysis: cells were seeded on a 6-well plate. At the corresponding treatment time point, the 6-well plate was removed and the medium was discarded. The plate was washed 2-3 times with pre-cooled PBS, and then the PBS was discarded. 500 μl Buffer RL was added to each well.

[0338] 2) Removal of gDNA: Transfer lysate to FastPure gDNA-Filter Column III, 12,000 rpm (13,400 × g)

[0339] Centrifuge for 30 seconds and collect the filtrate;

[0340] 3) Adjust the upper column: add 0.5 times the volume of filtrate anhydrous ethanol to the filtrate and mix thoroughly;

[0341] 4) RNA adsorption: transfer the entire mixture to FastPure RNA Column III, 12,000 rpm (13,400 × g)

[0342] Centrifuge for 30 seconds and discard the filtrate;

[0343] 5) Remove impurities: Add 700 μl Buffer RW1, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the filtrate.

[0344] Add 700 μl Buffer RW2 (with anhydrous ethanol), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard the filtrate, add 500 μl Buffer RW2 (with anhydrous ethanol), centrifuge at 12,000 rpm (13,400 × g) for 2 minutes, and discard the collection tube.

[0345] 6) Elution of RNA: 50-200 μl RNase-free ddH2O, centrifugation at 12,000 rpm (13,400 × g) for 1 min.

[0346] 5.1.7.2 RNA concentration determination

[0347] Use Nanodrop1000 to detect the concentration and purity of the extracted RNA, and use RNase-free ddH2O as a control. Take 2μl of each sample for testing to directly obtain the concentration value. If the A260 / A280 ratio is in the range of 1.8-2.1, it means that the sample purity is high. After the test is completed, reverse transcription reaction is immediately carried out or temporarily stored in a -80℃ refrigerator.

[0348] 5.1.7.3 Reverse transcription

[0349] 1) Genomic DNA removal

[0350] Prepare the following mixture in an RNase-free centrifuge tube:

[0351]

[0352] Mix by gently pipetting. Incubate at 42°C for 2 min.

[0353] 2) Prepare the reverse transcription reaction system and perform reverse transcription reaction

[0354] Add 5×HiScript III qRT SuperMix directly to the reaction tube in step 1.

[0355]

[0356] Mix well by gently pipetting. Incubate at 37℃ for 15min and then at 85℃ for 5s. The product can be used for qPCR reaction immediately or stored at -20℃.

[0357] 5.1.7.4 Target gene primer design

[0358] Primer design is mainly obtained through two ways: method one: by searching for studies with the same gene, selecting primers with the same gene, and performing gene comparison in NCBI's Blast; method two: using Primer5.0 software for primer design, then performing gene comparison of primer pairs in Blast, and finally sending the synthesized primer pairs to the company for synthesis.

[0359] 3.1.7.5 PCR reaction

[0360] 1) Prepare the following mixture in a qPCR tube

[0361]

[0362] 2) Perform qPCR reaction according to the following conditions

[0363]

[0364] 5.8 Flow cytometry detection of ROS content

[0365] 1) After heat shock, discard the cell culture medium, wash once with pre-cooled PBS, add 0.25% trypsin, digest at 37°C for 5 minutes, and gently blow 10-15 times until a cell suspension is formed;

[0366] 2) Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L;

[0367] 3) After the cells were centrifuged (500 rpm, 5 min) and collected, diluted DCFH-DA was added and placed in a 37°C cell culture incubator for 20 min. The cells were mixed once every 3 min to ensure full contact between the probe and the cells. The cells were washed once with serum-free medium and detected by flow cytometry as soon as possible within 1 h.

[0368] 5.9 Transmission electron microscopy observation of SH-SY5Y cell MAMs morphology

[0369] 1) After heat shock, cells were collected and digested with 0.25% trypsin at 37°C for 5 min. Serum-containing culture medium was added to terminate the digestion reaction, and the cells were blown into a single cell suspension;

[0370] 2) After centrifugation (500 rpm) for 5 min, the supernatant was discarded and electron microscopy fixative (the ratio of its volume to cell volume was 50:1) was added;

[0371] 3) Send the fixed specimens to the electron microscope room for post-processing and observation.

[0372] 5.10 Immunofluorescence analysis of SH-SY5Y cells

[0373] 1) Place a sterilized cover glass in a 6-well plate and coat it with poly-lysine, then plant SH-SY5Y cells in the wells and culture them, and perform heat shock treatment during the logarithmic growth phase;

[0374] 2) Discard the cell culture medium, rinse once with pre-cooled PBS, fix with 4% paraformaldehyde for 20 min, and then rinse three times with PBS, 10 min each time;

[0375] 3) Permeabilization: Dilute Triton 100 to 0.5% using sheep serum working solution, mix thoroughly and add to a 6-well plate, and place on ice for 10 min;

[0376] 4) Block the cells three times with sheep serum working solution, each time for 10 min;

[0377] 5) Add primary antibody: dilute the antibody with sheep serum working solution at a dilution ratio of (1:300), and then incubate at 4°C overnight;

[0378] 6) After discarding the primary antibody, wash the cells three times with sheep serum working solution, each time for 10 minutes;

[0379] 7) Add secondary antibody: Add fluorescent secondary antibody (wavelength 594nm) corresponding to the species of the primary antibody, dilute at a ratio of (1:500), and incubate at room temperature in a dark place for 1 hour;

[0380] 8) After discarding the secondary antibody, rinse with PBS three times, 10 minutes each time. Then remove the coverslip and air-dry it, and then invert the cell-seeded surface on a slide with DAPI mounting medium added, air-dry it, store it at -20℃ away from light, and observe it under a fluorescence microscope.

[0381] 5.11 Statistical analysis

[0382] GraphPad Prism 8.0.2 software was used for statistical analysis. The measurement data were all in accordance with normal distribution and expressed as x±s. One-way analysis of variance was used for comparison among multiple groups. P<0.05 was considered statistically significant.

[0383] 6. Preliminary research results:

[0384] 6.1 Decreased viability of SH-SY5Y cells after heat shock

[0385] We used the CCK8 method to evaluate the effect of heat shock on the viability of SH-SY5Y cells. Figure 8The results showed that compared with the control group, there was no significant difference in the survival rate of the rewarming group 0h after heat shock, while the cell survival rates of the rewarming groups 3h and 6h were significantly reduced (P<0.001, P<0.001). Moreover, as the rewarming time prolonged, the cell survival rates of the rewarming groups 3h and 6h were significantly lower than those of the rewarming group 0h (P<0.01, P<0.01).

[0386] 6.2 Effects of heat shock on gene expression profile of SH-SY5Y cells

[0387] In order to study the effect of heat shock on the gene expression profile of SH-SY5Y cells, we performed transcriptomic analysis on SH-SY5Y cell samples in the control group and heat shock rewarming 0h, 3h, and 6h groups. Fig. 9 C The results show that a large number of genes are differentially expressed. Fig. 9 As shown in A and B, compared with the control group, a total of 6106 differentially expressed genes were identified in the R0 group, of which 3890 genes were upregulated and 2216 genes were downregulated. A total of 10670 differentially expressed genes were identified in the R3 group, of which 7311 genes were upregulated and 3359 genes were downregulated. A total of 13199 differentially expressed genes were identified in the R6 group, of which 9086 genes were upregulated and 4113 genes were downregulated. There were 4347 common differentially expressed genes in these three comparison combinations. In order to understand the functions of all differentially expressed genes, we performed Go functional enrichment analysis on the differentially expressed genes. Fig. 9 D The results showed that the differentially expressed genes were mainly enriched in biological processes such as protein phosphorylation, microtubule movement, phosphorylation, etc., in cellular components such as dynein complex, microtubule-associated complex, myosin complex, etc., and in molecular functions such as DNA binding transcription factor activity, dynamin activity, and transcriptional regulator activity. The results of KEGG pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in MAPK signaling pathway, cell adhesion molecules, and neuroactive ligand-receptor interactions ( Fig. 9 E).

[0388] 6.3 Heat shock increased oxidative stress levels and aggravated mitochondrial damage in SH-SY5Y cells

[0389] In order to study the changes in oxidative stress levels in SH-SY5Y cells after heat shock, we performed differential analysis on 1031 oxidative stress-related genes and found that 153 oxidative stress-related genes were differentially expressed ( Fig.10 A). Fig.10 As shown in B, flow cytometry was used to detect ROS levels. We found that there was no significant difference between the control group and the heat shock rewarming group at 0h and 6h, but the ROS level at 3h after heat shock rewarming increased significantly (P < 0.05). During the rewarming period of 0h-6h after heat shock, the ROS level showed a trend of first increasing and then decreasing, and reached a peak at 3h.

[0390] Under oxidative stress conditions, mitochondria and ER are often damaged. MAMs are a platform for communication between mitochondria and ER. In order to understand whether MAMs in SH-SY5Y cells undergo abnormal changes as oxidative stress levels increase after heat shock, we analyzed the genes of MAMs-related proteins and found that 25 MAMs-related protein genes were differentially expressed ( Fig.10 C) Fig.10 DE, in order to observe this structure more intuitively, we used transmission electron microscopy to observe and found that compared with the control group, after heat shock, the mitochondria and endoplasmic reticulum showed different degrees of swelling, mitochondrial spines fell off, endoplasmic reticulum degranulation, and the contact between mitochondria and endoplasmic reticulum decreased and the distance increased. In order to study the specific molecular mechanism of abnormal changes in MAMs, we used RT-qPCR and Western Blot to detect the effect of heat shock on the mRNA and protein expression levels of MFN2, a key protein in the formation of MAMs. Fig.10 As shown in F, compared with the control group, the levels of MFN2 protein and mRNA at 0h and 6h after heat shock were significantly decreased (P<0.01, P<0.01; P<0.05, P<0.05).

[0391] The above results indicate that the oxidative stress level of SH-SY5Y cells increased significantly after heat shock, the structure of MAMs changed abnormally, and the normal communication between mitochondria and ER may be disturbed.

[0392] 6.4 Activation of NRF2 antioxidant signaling pathway in SH-SY5Y cells after heat shock

[0393] The NRF2 antioxidant signaling pathway is one of the most important antioxidant signaling pathways in the body and is usually activated when oxidative stress levels increase. In order to understand the effect of heat shock on the NRF2 signaling pathway, we found through transcriptomics analysis that NRF2, HO-1, NQO1, P62, and KEAP1 genes were differentially expressed genes ( Fig.11 A). In addition, we also detected the changes in the mRNA level of the NRF2 signaling pathway after heat shock by RT-qPCR. Fig.11As shown in B, compared with the control group, the mRNA levels of NRF2, NQO1, and KEAP1 were significantly decreased after heat shock (P < 0.0001, P < 0.0001, P < 0.001; P < 0.05, P < 0.01, P < 0.01; P < 0.0001, P < 0.0001, P < 0.0001), the mRNA level of HO-1 in the rewarming 3h and 6h groups was significantly increased (P < 0.05, P < 0.0001), and the mRNA level of P62 in the rewarming 0h group after heat shock was significantly decreased (P < 0.05). After heat shock, at 0-6h rewarming, the mRNA levels of NRF2, HO-1, and P62 were significantly increased (P < 0.001, P < 0.001, P < 0.001), the mRNA level of KEAP1 was significantly decreased (P < 0.05), and the mRNA level of NQO1 showed a downward trend, but the difference was not statistically significant. Fig.11 Western Blot results showed that compared with the control group, the NQO1 and KEAP1 protein levels in the heat shock group were significantly decreased (P < 0.05, P < 0.01, P < 0.05; P < 0.01, P < 0.001, P < 0.001), the nuclear NRF2 protein level at 0h and 3h of rewarming was significantly decreased (P < 0.01, P < 0.01), the P62 protein level at 3h and 6h of rewarming was significantly increased (P < 0.05, P < 0.01), and the cytoplasmic NRF2 protein level at 6h of rewarming was significantly decreased (P < 0.05). Compared with the 3h rewarming group, the total NRF2 protein level in the 6h rewarming group was significantly decreased (P < 0.05). During the rewarming period of 0h-6h, the levels of nuclear NRF2 protein, HO-1 and P62 protein were significantly increased (P<0.05, P<0.05, P<0.01), the levels of cytoplasmic NRF2 protein were significantly decreased (P<0.05), and there was no significant difference in the changes of NQO1 and KEAP1 proteins among the rewarming groups.

[0394] NRF2 is a nuclear transcription factor. Under stress, it dissociates from KEAP1 in the cytoplasm and enters the nucleus to bind to the promoter region for transcription and translation, thereby activating the downstream pathway. In order to more intuitively observe whether NRF2 protein undergoes nuclear translocation after heat shock, we localized NRF2 protein by immunofluorescence. The results are shown in Figure 2. Fig.11 D shows that in the control group, NRF2 protein was expressed in the nucleus and cytoplasm to varying degrees. After heat shock, NRF2 protein concentrated in the nuclear region, suggesting that after heat shock, NRF2 of SH-SY5Y cells entered the nucleus to exert its effect.

[0395] The above results show that the NRF2 signaling pathway is first inhibited after heat shock, and as the rewarming time increases, the expression of KEAP1 and cytoplasmic NRF2 gradually decreases, while the expression of nuclear NRF2, HO-1 and P62 gradually increases. That is, as the oxidative stress level increases, KEAP1 is inactivated, and NRF2 dissociates from KEAP1 and moves from the cytoplasm to the nucleus, activating downstream target genes such as HO-1 and P62.

[0396] 6.5 Protective effect of NRF2 antioxidant signaling pathway activation on SH-SY5Y cells after heat shock:

[0397] In order to understand whether this pathway plays a protective role against SH-SY5Y cell damage after heat shock, we explored this issue by activating the NRF2 signaling pathway through TBHQ pretreatment and knocking down the NRF2 gene through NRF2 siRNA.

[0398] 6.5.1 Activation of NRF2 antioxidant signaling pathway improves SH-SY5Y cell survival after heat shock

[0399] TBHQ is a NRF2-specific agonist. We added 40 μM TBHQ to the cell culture medium for 24 h pretreatment. In order to obtain sufficient cells, according to Figure 8 The CCK8 results selected the heat shock group after 3h of rewarming as the heat shock group for subsequent experiments. Fig.12 The results of ACCK8 showed that compared with the simple heat shock group, the cell viability of the heat shock group pretreated with TBHQ was significantly improved (P < 0.05). Fig.12 B RT-qPCR results showed that compared with the simple heat shock group, the mRNA levels of NRF2, NQO1, HO-1, and P62 were significantly increased after TBHQ pretreatment (P < 0.01, P < 0.001, P < 0.01, P < 0.05), and there was no statistically significant difference in the change of KEAP1 mRNA level. We used Western Blot to detect the expression level of NRF2 protein after TBHQ. Fig.12 The results showed that compared with the simple heat shock group, the expression levels of NRF2 total protein, nuclear NRF2, NQO1, and P62 proteins in the heat shock group pretreated with TBHQ were significantly increased (P < 0.05, P < 0.05, P < 0.05, P < 0.01), the expression level of KEAP1 protein was significantly decreased (P < 0.05), the level of cytoplasmic NRF2 protein did not change significantly, and the expression of HO-1 protein showed an upward trend, but the difference was not statistically significant. Fig.12D Immunofluorescence results showed that the NRF2 protein in the control group was expressed in both the cytoplasm and the nucleus, and the NRF2 protein in the control group after TBHQ was added was transferred to the nucleus. Compared with the control group, the NRF2 signaling pathway in the simple heat shock group had been activated, and the NRF2 protein was transferred from the cytoplasm to the nucleus, while more NRF2 protein was observed to be transferred to the nucleus in the heat shock group after TBHQ pretreatment. The above results indicate that TBHQ pretreatment can activate the NRF2 antioxidant signaling pathway of SH-SY5Y cells after heat shock and improve the survival rate of SH-SY5Y cells after heat shock.

[0400] After specifically knocking down NRF2 by cell transfection technology, cell viability was detected, such as Fig.13 As shown in A, compared with the simple heat shock group, the cell viability of the heat shock group after transfection was significantly decreased (P < 0.05). Fig.13 B. After knocking down NRF2, the mRNA levels of NRF2, NQO1, HO-1, and P62 were significantly decreased (P < 0.01, P < 0.001, P < 0.01, P < 0.001), and the mRNA expression level of KEAP1 was significantly increased (P < 0.05). Fig.13 Western Blot results showed that compared with the heat shock group, the expression levels of NRF2 total protein, nuclear NRF2, NQO1, HO-1 and P62 proteins in the heat shock group after NRF2 knockdown were significantly reduced (P < 0.001, P < 0.01, P < 0.01, P < 0.05, P < 0.01), the expression level of cytoplasmic NRF2 protein was significantly increased (P < 0.05), and the expression level of KEAP1 protein showed a downward trend, but there was no statistical significance. Fig.13 D. Immunofluorescence results also showed that the expression of nuclear NRF2 protein in the heat shock group after knocking down NRF2 was significantly reduced. The above results indicate that the activation of the NRF2 signaling pathway in SH-SY5Y cells was inhibited after transfection with NRF2 siRNA, and the cell viability was further reduced after heat shock.

[0401] 6.5.2 Activation of the NRF2 antioxidant signaling pathway reduces the increase in ROS levels in SH-SY5Y cells induced by heat shock

[0402] In order to understand the effect of NRF2 antioxidant signaling pathway on ROS level after heat shock, we used flow cytometry to detect ROS level in SH-SY5Y cells after heat shock after TBHQ pretreatment and NRF2 siRNA transfection. Fig.14 As shown in A, compared with the simple heat shock group, the ROS level of cells in the heat shock group after pretreatment with TBHQ was significantly reduced (P<0.05); Fig.14B. The results showed that the ROS level of heat-shocked SH-SY5Y cells increased significantly after knocking down NRF2 (P < 0.05). The NRF2 antioxidant signaling pathway plays an important role in regulating the ROS level after heat shock. By activating the NRF2 antioxidant signaling pathway, the oxidative damage of SH-SY5Y cells induced by heat shock can be significantly reduced.

[0403] 6.5.3 Activation of the NRF2 antioxidant signaling pathway can alleviate mitochondrial damage in SH-SY5Y cells induced by heat shock

[0404] In order to understand the role of NRF2 antioxidant signaling pathway in the structural disorder of MAMs in SH-SY5Y cells induced by heat shock, we used transmission electron microscopy to observe the MAMs structure of SH-SY5Y cells pretreated with TBHQ and transfected with NRF2 siRNA after heat shock. Fig.15 As shown in (AB), compared with the simple heat shock group, the contact area between ER and mitochondria in the heat shock group pretreated with TBHQ increased and the distance became smaller (P < 0.05, P < 0.05). Fig.15 C RT-qPCR and Western Blot results showed that the expression of MFN2 mRNA and protein increased significantly after the addition of TBHQ (P<0.05, P<0.05). Fig.15 (DE) The results showed that the contact distance between ER and mitochondria in SH-SY5Y cells transfected with NRF2 siRNA and heat-shocked cells increased (P < 0.05), mitochondrial swelling increased, and large-scale spine shedding occurred. Fig.15 The results of F also showed that after NRF2 siRNA transfection and knockdown of NRF2 in heat-shocked SH-SY5Y cells, the expression of MFN2 mRNA and protein was significantly reduced (P < 0.05, P < 0.05). In general, the NRF2 antioxidant signaling pathway regulates the structure of MAMs, and activation of this signaling pathway can significantly improve the structural disorder of MAMs in SH-SY5Y cells induced by heat shock, thereby alleviating mitochondrial damage caused by heat shock.

[0405] Two specific embodiments and corresponding implementation schemes are listed:

[0406] Example 1: Using TBHQ to prevent heat stroke brain damage

[0407] 1) Cell culture:

[0408] SH-SY5Y neuroblastoma cells were cultured in DMEM-F12 medium containing 10% fetal bovine serum and 1% double antibody.

[0409] The cells were cultured under the conditions of 5% CO2 and 37°C, and the medium was replaced regularly and passaged.

[0410] 2) Experimental groups:

[0411] The cells were divided into four groups: control group, TBHQ pretreatment group, heat shock group, and TBHQ pretreatment followed by heat shock group.

[0412] The control group was always cultured at 37°C and 5% CO2.

[0413] The cells in the heat shock group were exposed to 43°C and 5% CO2 for 2 hours and then returned to normal conditions for rewarming.

[0414] 3) TBHQ pretreatment:

[0415] 24 hours before heat shock, SH-SY5Y cells were treated with 40 μM TBHQ (dissolved in DMSO and diluted in culture medium) for 24 hours.

[0416] 4) Biological testing:

[0417] Cell viability was detected in all groups, protein expression was analyzed by Western blot, mRNA expression was detected by RT-qPCR, and ROS content was detected by flow cytometry.

[0418] Transmission electron microscopy was used to observe the morphological structure of mitochondria-endoplasmic reticulum contacts (MAMs) in SH-SY5Y cells.

[0419] Example 2: Evaluation of the effect of TBHQ on cell recovery after heat stroke brain injury

[0420] 1) Cell culture and grouping:

[0421] Similar to Example 1, SH-SY5Y cells were used and the cells were divided into the same four experimental groups.

[0422] 2) Heat shock treatment:

[0423] The cells in the experimental group were exposed to heat shock at 43°C and 5% CO2 for 2 hours, and then different groups were rewarmed at different time points (such as 0h, 3h, 6h) according to the protocol.

[0424] 3) TBHQ treatment:

[0425] For the TBHQ group and the TBHQ pretreatment followed by heat shock group, the mice were treated with 40 μM TBHQ for 24 hours 24 hours before heat shock.

[0426] 4) Functional and structural testing:

[0427] Cell viability detection, Western blot, RT-qPCR and ROS content detection were performed at different rewarming time points.

[0428] Immunofluorescence analysis was used to assess the expression and localization of key proteins within cells.

[0429] Transmission electron microscopy was used to observe changes in the internal structure of cells, especially the interaction between mitochondria and endoplasmic reticulum.

[0430] These two examples use TBHQ as an NRF2 agonist from the perspectives of prevention and treatment, respectively, and evaluate its effect on heatstroke brain damage through cell experimental methods, thereby providing basic data and theoretical basis for further animal experiments and clinical trials.

[0431] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. Use of an agonist in treating heat stroke brain damage, characterized in that: The agonist is an NRF2 agonist.

2. A method for testing the use of the agonist as claimed in claim 1 in treating heat stroke brain damage, characterized in that: Testing method for the use of the agonist in treating heat stroke brain damage The following steps are involved: In the first step, neuroblastoma SH-SY5Y cells were cultured using DMEM-F12 medium containing 10% fetal bovine serum and 1% double antibody in a cell culture incubator at 5% CO2 and 37°C. The medium was changed every other day, and cells were passaged according to the cell growth conditions, and cells in the logarithmic growth phase were selected; In the second step, SH-SY5Y cells were divided into a control group and a heat shock rewarming group at 0h, 3h, and 6h. The control group was cultured at 37°C and 5% CO2 all the time, and the experimental group was placed in a 43°C and 5% CO2 incubator for 2h for heat shock. After the heat shock, the cells were immediately transferred to a conventional incubator at 37°C and 5% CO2 for rewarming before various tests were performed. In the third step, after dissolving the agonist TBHQ in DMSO, the concentration was adjusted to 40 μM with culture medium, and the DMSO concentration was kept below 0.01%; NRF2 siRNA was transfected into SH-SY5Y cells, and cell viability was detected, protein expression was detected by Western blot, mRNA expression was detected by RT-qPCR, ROS content was detected by flow cytometry, the morphological structure of SH-SY5Y cell MAMs was observed by transmission electron microscopy, and SH-SY5Y cell immunofluorescence analysis was performed.

3. A method for testing the use of an agonist as claimed in claim 2 in treating heat stroke brain damage, characterized in that: In the third step, TBHQ was used to intervene in SH-SY5Y cells 24 hours before heat shock, and the intervention time was 24 hours; the experimental groups were: control group, TBHQ group only used agonist pretreatment, HS group only used heat shock, and TBHQ and HS groups were pretreated with agonists and then heat shocked. The heat shock treatment group selected the rewarming time with the highest NRF2 protein expression.

4. A method for testing the use of an agonist as claimed in claim 2 in treating heat stroke brain damage, characterized in that: In the third step, NRF2 siRNA is transfected into SH-SY5Y cells, and the NRF2 gene silencing sequence is NRF2-siRNA; by detecting the transfection efficiency of 5 to 100 nmol / L, the transfection effect with the minimum cytotoxicity is screened, the cells are plated one day in advance, the cell density is 30% to 50% during transfection, NRF2 siRNA is dissolved in Opti-MEM serum-free medium, mixed, and placed for 5 minutes, and lipo2000 is dissolved in Opti-MEM serum-free medium, the two are mixed, and placed for 20 minutes, the above mixture is added to the culture well, mixed, and cultured in a 37°C, 5% CO2 incubator for 4 hours, the supernatant is removed, and a complete medium containing 10% fetal bovine serum is added to culture for 48 hours, and after passing the PCR test, it is set aside; the negative control NC NRF2-siRNA is transfected in the same way; the experimental groups are: control group, NRF2 siRNA group, HS group and NRF2 siRNA + HS group, and the heat shock treatment group selects the rewarming time with the highest NRF2 protein expression; In the third step, cell viability detection, SH-SY5Y cells were inoculated in a 96-well cell culture plate, 5000 cells / well, 100 μL / well of culture medium, and cultured in a cell culture incubator for 24 hours. The experiment was divided into 4 groups: a control group, 0, 3, and 6 hours, and 3 replicates were set in each group. On the second day, the cells were heat-shocked at 43°C for 2 hours. After rewarming for 0, 3, and 6 hours, the culture medium was aspirated and discarded, and 100 μL of culture medium containing 10% CCK8 stock solution was added to each well. The cells were continued to be cultured in a cell culture incubator for 2 hours, and then the absorbance value (A450 value) of each group was detected using an ELISA reader at a wavelength of 450 nm; cell survival rate (%) = (A450 value of the experimental group - A450 value of the blank group) / (A450 value of the control group - A450 value of the blank group), and no cells were added to the blank group, only an equal amount of culture medium was added.

5. A method for testing the use of an agonist as claimed in claim 2 in treating heat stroke brain damage, characterized in that: In the third step, protein expression is detected by Western blot, including: Cell protein extraction: 1) Total protein extraction: Collect cells from the control group and experimental group, use 4°C pre-cooled protein lysis buffer RIPA: PMSF = 100:1 to fully lyse the cells on ice, centrifuge at 4°C, 12000r / min, 15min, and collect the supernatant, which is the total cell protein; 2) Extraction of cytoplasmic and nuclear proteins: Collect the cell pellet and add 200 μl of cytoplasmic protein extraction reagent A supplemented with PMSF for every 20 μl of cell pellet; Vortex vigorously at the highest speed for 5 seconds to completely suspend and disperse the cell pellet, and place on ice for 10-15 minutes; Add 10 μl of cytoplasmic protein extraction reagent B, vortex at the highest speed for 5 seconds, and place on ice for 1 minute; Vortex at maximum speed for 5 seconds, centrifuge at 12,000-16,000 g for 5 minutes at 4°C; Pipette the supernatant into a pre-cooled plastic tube, which is the extracted cytoplasmic protein; For the precipitation, completely aspirate the remaining supernatant and add 50 μl of nuclear protein extraction reagent supplemented with PMSF; Vortex at the highest speed for 15-30 seconds to completely suspend and disperse the cell pellet, then return to the ice bath and vortex at the highest speed for 15-30 seconds every 1-2 minutes for a total of 30 minutes. Centrifuge at 12,000-16,000 g for 10 min at 4°C; Pipette the supernatant into a pre-cooled plastic tube, which is the extracted nuclear protein; Protein concentration determination: 1) Draw a standard curve: add reagents to the ELISA plate in sequence; 2) Take 2 μL of the sample to be tested and add it to the well, then add 18 μL of PBS to dilute the sample to be tested 10 times; 3) Prepare BCA working solution at a ratio of 50:1, mix it thoroughly, and add 200 μL of BCA working solution to each well; 4) Place the mixture in an incubator at 37°C for 30 minutes, measure the OD value at a wavelength of 562 nm, and then draw a standard curve with the protein content as the horizontal axis and the OD value as the vertical axis, and calculate the concentration according to the OD value of the sample to be tested; Protein denaturation: Add 1 / 4 volume of 5× SDS-PAGE protein loading buffer to the collected protein sample and heat in a boiling water bath for 10 min to fully denature the protein; Electrophoresis: 1) Preparation of electrophoresis solution: 1g SDS, 3.03g Tris, 14.4g Glycine, add 1L ddH2O and stir to dissolve; 2) According to the molecular weight of the sample protein to be tested, select SDA-PAGE gel, add 20 μg of protein to each SDS-PAGE gel well, run electrophoresis at 80V for 10 minutes, then switch to 120V and continue electrophoresis for 50 minutes; Transfer: 1) Preparation of transfer solution: 3.03g Tris, 14.4g Glycine, add 200mL methanol and 800mL ddH2O and stir to dissolve; 2) Transfer conditions: Use PVDF membrane, transfer on ice at 250 mA for 90 min; Blocking: 5% skim milk powder was blocked at room temperature for 2 h on a shaker (40 rpm); Wash the membrane: discard skim milk powder, add appropriate amount of 1×TBST and wash the membrane 3 times on a shaker at 60 rpm for 5 min; Primary antibody and secondary antibody incubation: add primary antibody and incubate at 4°C overnight, wash the membrane three times, add secondary antibody and incubate at room temperature for 1 hour on a shaker at 40 rpm, and wash the membrane three times; Development: Add ECL chemiluminescent solution for development; Analysis: Image Lab software was used for semi-quantitative analysis of protein bands.

6. A method for testing the use of an agonist as claimed in claim 2 in treating heat stroke brain damage, characterized in that: In the third step, RT-qPCR detection of mRNA expression includes: RNA extraction 1) Sample lysis: cells were seeded on a 6-well plate. At the corresponding treatment time point, the 6-well plate was removed and the medium was discarded. The plate was washed 2-3 times with pre-cooled PBS, and then the PBS was discarded. 500 μl Buffer RL was added to each well. 2) Removal of gDNA: transfer the lysate to FastPure gDNA-Filter Column III, 12,000 rpm (13,400 × g); centrifuge for 30 seconds and collect the filtrate; 3) Adjust the upper column: add 0.5 times the volume of filtrate anhydrous ethanol to the filtrate and mix well; 4) RNA adsorption: transfer the entire mixture to FastPure RNA Column III, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the filtrate; 5) Remove impurities: add 700 μl Buffer RW1, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard the filtrate, add 700 μl Buffer RW2, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard the filtrate, add 500 μl Buffer RW2, centrifuge at 12,000 rpm (13,400 × g) for 2 minutes, and discard the collection tube; 6) Elution of RNA: 50-200 μl RNase-free ddH2O, centrifugation at 12,000 rpm (13,400×g) for 1 min.

7. A method for testing the use of an agonist in treating heat stroke brain damage as claimed in claim 2, characterized in that: In the third step, the RNA concentration is determined by using Nanodrop 1000 to detect the concentration and purity of the extracted RNA, using RNase-free ddH2O as a control, taking 2 μl of each sample for detection, and directly obtaining the concentration value; if the A260 / A280 ratio is in the range of 1.8-2.1, it indicates that the sample purity is high; after the detection is completed, the reverse transcription reaction is immediately performed or temporarily stored in a -80°C refrigerator.

8. A method for testing the use of an agonist in treating heat stroke brain damage as claimed in claim 2, characterized in that: In the third step, reverse transcription, comprises: 1) Genomic DNA removal Prepare the mixture in an RNase-free centrifuge tube: gently pipette to mix, incubate at 42°C for 2 min; 2) Prepare the reverse transcription reaction system, perform the reverse transcription reaction, and directly add 5×HiScript III qRTSuperMix into the reaction tube; Mix well by gently pipetting, incubate at 37°C for 15 min, then at 85°C for 5 s. The product can be used for qPCR reaction or stored at -20°C. Target gene primer design Primer design is mainly obtained through two ways: method one: by searching for tests with the same gene, selecting primers with the same gene, and comparing them in NCBI's Blast; method two: using Primer5.0 software to design primers, then comparing the primer pairs in Blast, and finally sending the synthesized primer pairs to the company for synthesis; PCR reaction 1) Prepare the following mixture in a qPCR tube; 2) Perform qPCR reaction according to the following conditions.

9. A method for testing the use of an agonist in treating heat stroke brain damage as claimed in claim 2, characterized in that: In the third step, the ROS content is detected by flow cytometry, including: 1) After heat shock, discard the cell culture medium, wash once with pre-cooled PBS, add 0.25% trypsin, digest at 37°C for 5 minutes, and then pipette 10-15 times until a cell suspension is formed; 2) Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L; 3) The cells were centrifuged at 500 rpm for 5 min and collected. The diluted DCFH-DA was added and placed in a 37°C cell culture incubator for 20 min. The cells were mixed once every 3 min to ensure full contact between the probe and the cells. The cells were washed once with serum-free medium and detected by flow cytometry as soon as possible within 1 h.

10. The testing method for the use of the agonist in treating heat stroke brain damage as claimed in claim 2, characterized in that: In the third step, the morphological structure of SH-SY5Y cell MAMs is observed by transmission electron microscopy, including: 1) After heat shock, cells were collected and digested with 0.25% trypsin at 37°C for 5 min. Serum-containing culture medium was added to terminate the digestion reaction, and the cells were blown into a single cell suspension; 2) After centrifugation at 500 rpm for 5 min, the supernatant was discarded and the volume of electron microscopy fixative was added at a ratio of 50:1 to the volume of cells; 3) Send the fixed specimens to the electron microscope room for post-processing and observation; In the third step, SH-SY5Y cell immunofluorescence analysis includes: 1) Place a sterilized cover glass in a 6-well plate and coat it with poly-lysine, then plant SH-SY5Y cells in the wells and culture them, and perform heat shock treatment during the logarithmic growth phase; 2) Discard the cell culture medium, rinse once with pre-cooled PBS, fix with 4% paraformaldehyde for 20 min, and then rinse three times with PBS, 10 min each time; 3) Permeabilization: Dilute Triton 100 to 0.5% using sheep serum working solution, mix well and add to a 6-well plate, and place on ice for 10 min; 4) Block the cells three times with sheep serum working solution, each time for 10 min; 5) Add primary antibody: dilute the antibody with sheep serum working solution at a dilution ratio of 1:300, and then incubate at 4°C overnight; 6) After discarding the primary antibody, wash the cells three times with sheep serum working solution, each time for 10 minutes; 7) Add secondary antibody: Add fluorescent secondary antibody with wavelength of 594nm corresponding to the species of primary antibody, dilute at a ratio of 1:500, and incubate at room temperature in a dark place for 1 hour; 8) After discarding the secondary antibody, rinse with PBS three times, 10 minutes each time; then remove the coverslip and air-dry it, and then turn the cell-seeded surface upside down on a slide with DAPI mounting medium added, air-dry it, store it at -20°C away from light, and observe it under a fluorescence microscope.

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