Experimental method for relieving ferroptosis of cerebral apoplexy rats by tVNS

Abstract

The invention discloses an experimental method for relieving ferroptosis of cerebral apoplexy rats through tVNS, and relates to the technical field of nerve regulation and control. Comprising the following steps: S1, constructing a rat MCAO model, and constructing an SD rat right cerebral ischemia reperfusion model by adopting a suture method; s2, experiment grouping: randomly dividing experimental animals into (1) a false operation group: an SHAM group; (2) a cerebral ischemia reperfusion group: an MCAO group; (3) an ear vagus nerve electrical stimulation group after cerebral ischemia reperfusion: a tVNS group; and S3, performing ear vagus nerve electrical stimulation intervention. Ferroptosis related proteins such as GPX4, ACSL4 and FTH1 are detected through Western blot, it is proved that tVNS can improve stroke rat brain tissue ferroptosis, and clear molecular mechanism support is provided for intervention; the content of reduced glutathione and the content of malondialdehyde are respectively detected by using a GSH and MDA content detection kit, and the effectiveness of tVNS in the aspect of relieving ferroptosis is further verified.

Description

Technical Field

[0001] The present invention relates to the field of neural regulation technology, and in particular to an experimental method for alleviating ferroptosis in stroke rats by using tVNS. Background Art

[0002] Stroke is an acute cerebrovascular disease with high morbidity, high disability and high mortality. Its pathological basis mainly includes cerebral ischemia and cerebral hemorrhage, of which cerebral ischemia accounts for the majority. After cerebral ischemia, due to interruption of blood flow, the brain tissue is not supplied with enough oxygen and nutrients, which in turn triggers a series of complex pathophysiological reactions, ultimately leading to brain cell death and neurological dysfunction.

[0003] At present, the treatments for stroke mainly include thrombolytic therapy, antiplatelet therapy, surgical thrombectomy, and rehabilitation therapy. However, these treatments often have problems such as time window limitations, risk of complications, and limited therapeutic effects. Therefore, it is of great significance to explore new strategies for the treatment of stroke.

[0004] In recent years, non-invasive neuromodulation technologies such as transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS) and transaural vagus nerve stimulation (tVNS) have gradually attracted attention. These technologies may provide new ways to restore neurological function after stroke by regulating the activity of the nervous system. Among them, tVNS, as a simple, safe and effective non-invasive neuromodulation method, has been widely used in the treatment of various diseases, including depression, anxiety, epilepsy and pain. Studies have shown that tVNS can activate the vagus nerve and promote the release of neurotransmitters, thereby regulating neuronal activity in areas such as the cerebral cortex and hippocampus, and exerting a neuroprotective effect.

[0005] Based on the above background, the present invention aims to deeply reveal the potential of tVNS in stroke treatment by constructing a rat MCAO model and performing tVNS intervention on it, combining a variety of biochemical and molecular biological detection methods. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose an experimental method for alleviating ferroptosis in stroke rats by tVNS.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] An experimental method for alleviating ferroptosis in stroke rats by tVNS, comprising:

[0009] S1: Construction of rat MCAO model. The right cerebral ischemia-reperfusion model of SD rats was established by suture embolism method.

[0010] S2: Experimental groups: The experimental animals were randomly divided into ① sham operation group: SHAM group; ② cerebral ischemia reperfusion group: MCAO group; ③ tVNS group after cerebral ischemia reperfusion;

[0011] S3: Auricular vagus nerve stimulation intervention;

[0012] S4: Neurological function score, to explore the improvement effect of tVNS on the neurological function of stroke rats;

[0013] S5: Brain tissue protein extraction;

[0014] S6: Protein concentration determination by BCA method;

[0015] S7: Western blot detection of ferroptosis-related proteins;

[0016] S8: Determination of cerebral infarction volume, detecting the area of ​​cerebral infarction in stroke rats;

[0017] S9: Tunnel staining of brain tissue to detect brain cell death in rats;

[0018] S10: Detection of reduced glutathione content in brain tissue, and detection of changes in ferroptosis indicators using ferroptosis-related kits;

[0019] S11: Detection of malondialdehyde content in brain tissue, and detection of changes in ferroptosis indicators using ferroptosis-related kits.

[0020] Preferably: in S1, the right cerebral ischemia-reperfusion model of SD rats is established by thread embolism method, which specifically includes: preoperative preparation, anesthesia and fixation, tissue separation and embolism and reperfusion;

[0021] Among them, tissue separation and embolization include:

[0022] ① After depilating the rat's neck, disinfect it with iodine, make a longitudinal incision along the middle of the neck, first use forceps to bluntly separate the right anterior cervical fascia layer by layer to expose the anterior cervical muscles, and use a retractor to pull the anterior cervical muscles to the right to fully expose the carotid sheath;

[0023] ② Use micro forceps to slowly and gently separate the vagus nerve, jugular vein and common carotid artery along the carotid sheath, and ligate the distal end of the common carotid artery with surgical thread; the common carotid artery is divided into the external carotid artery and the internal carotid artery after reaching the cervical cross in the direction of the head, ligate the external carotid artery, tie a loose knot at the cervical cross, and temporarily block the blood flow to the internal carotid artery with an artery clamp;

[0024] ③ Use vascular scissors to make an oblique incision between the common carotid artery knot and the cervical cross slipknot, insert the prepared thread plug from the incision through the slipknot to the internal carotid artery vascular clamp, tighten the loose knot to fix the thread plug, loosen the artery clamp, adjust the angle of the thread plug, and continue to insert the thread plug into the skull until the thread plug mark point passes through the cervical crossknot and the thread plug encounters resistance and cannot continue;

[0025] Reperfusion includes:

[0026] The rats were placed on an electric blanket to maintain their body temperature at 37±0.3℃. After 90 minutes, the suture was removed to restore blood flow and reperfusion was performed after vascular occlusion. The neck tissue was then sutured layer by layer, and the sutures were disinfected with iodine. After the rats woke up, they were housed in separate cages and allowed to eat and drink freely. Among them, the rats in the SHAM group were not treated with sutures inserted into the neck.

[0027] Preferably, in S1, the auricular vagus nerve electrical stimulation intervention specifically includes:

[0028] The rats were fixed in a prone position to ensure that their heads were completely exposed. The conchae on both sides of the rats were cleaned and the temperature-controlled rat plate was maintained at approximately 37°C. In the tVNS group, ear clips connected to the ear vagus nerve electrical stimulation instrument were clamped on both sides of the auricle area of ​​the rats, and the current intensity was set to 1 mA. Each intervention lasted 30 minutes, once a day, for a total of 7 days. The current intensity of the SHAM and MCAO groups was 0, and the rest of the steps were the same as the tVNS group. The stimulation was performed by the same researcher at the same time every day for 7 consecutive days.

[0029] Preferably: in said S4, the neurological function score specifically includes:

[0030] On the seventh day after MCAO, the neurological function of rats in each group was evaluated using the mNSS scale; the mNSS test included motor function, sensory function and reflex tests;

[0031] Rats were placed on a flat surface and examined for their ability to move their forelimbs and hindlimbs as an indicator of motor function;

[0032] Next, the rats’ forelimbs and hindlimbs were touched with a soft brush, and their responses were examined as an indicator of sensory function;

[0033] Finally, the rats' forelimbs and hindlimbs were tapped and their responses were examined as an indicator of reflexes.

[0034] Preferably: in S5, the brain tissue protein extraction specifically includes:

[0035] Transfer the extracted cerebral cortex tissue to a 1.5 ml EP tube, add the prepared tissue protein lysis solution, use ultrasound to lyse the tissue, and then use a grinder to fully grind the tissue, take it out and let it stand on ice for 15 minutes, then place it in a high-speed low-temperature centrifuge and centrifuge it at 12000rpm, 4℃, 20min; after centrifugation, use a sterile pipette tip to aspirate the tissue supernatant into the marked EP tube and record the volume of the extracted supernatant.

[0036] Preferably: in S6, the BCA method protein concentration determination specifically includes:

[0037] ① Add 0, 1, 2, 4, 8, 12, 16, 20 μl of 0.5 mg / ml protein standard to a 96-well plate, and then add PBS to make up to 20 μl per well to form a concentration gradient of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / ml; make 3 replicate wells for each concentration;

[0038] ② After the sample is diluted 50 times, add 20 μl to each sample and make 3 replicate wells for each sample;

[0039] ③ Add 200 μl BCA working solution to each well, solution A: solution B = 50:1, and place at 37°C for 20-30 minutes;

[0040] ④Measure the absorbance at 547nm using an enzyme-labeled instrument;

[0041] ⑤ Calculate the standard curve based on the absorbance and concentration of the standard, and then calculate the protein concentration of the sample based on the standard curve and the sample absorbance and balance;

[0042] ⑥ After balancing, add 5× Loading Buffer at a ratio of 4:1 according to the final volume, mix well and place in a 95℃ metal bath for 10 minutes until the protein is completely denatured; after cooling, dispense 200μl into each tube and store in a -80℃ refrigerator.

[0043] Preferably: in S7, Western blot detection of ferroptosis-related proteins specifically includes:

[0044] ① Glue preparation: add 50μl of coagulant to 2.5ml of the lower glue A and B liquids of each glue block, mix well and quickly add to the glass plate on the glue preparation rack, add anhydrous ethanol to the upper edge of the short plate; let it stand for 20 minutes, pour out the anhydrous ethanol, add 0.75ml of the upper glue A and B liquids and 12μl of coagulant to each plate, and insert a ten-hole comb; ensure that no bubbles are generated in the glue during the whole process;

[0045] ② Preparation of electrophoresis solution: weigh 37.6 g of glycine, 6.06 g of Tris-base, and 2.0 g of SDS, mix in 2 L of double distilled water, and mix thoroughly;

[0046] ③ Preparation of transfer solution: weigh 28.8 g of glycine, 6.06 g of Tris-base, and 400 mL of methanol, mix in 1600 mL of double distilled water, and mix thoroughly;

[0047] ④ Preparation of washing solution TBST: Add TBST dry powder buffer into 2L of double distilled water and mix thoroughly;

[0048] ⑤ Sample electrophoresis: Place the sample in a 95℃ metal bath for 5 minutes in advance. During this period, fix the prepared gel on the electrophoresis rack and place it in the electrophoresis tank. Add the newly prepared electrophoresis solution to the inner tank until the glass plate is submerged, and ensure that the inner tank is tightly sealed and does not leak. Add the electrophoresis solution to the outer tank until the scale line is reached. Then remove the comb, remove the bubbles in the lane, and add 3μl of marker and 30-40μg of protein to the channel. Perform electrophoresis at a constant voltage of 80V for 20 minutes on the upper concentrated gel and at a constant voltage of 120V on the lower separation gel until the buffer reaches the bottom.

[0049] ⑥ Transfer: Pre-cool the prepared electrotransfer solution, cut a PVDF membrane of appropriate size, activate it with methanol solution for 20 seconds in advance, and then place it in the electrotransfer solution; arrange it in the order of black plywood-1 layer of sponge-3 sheets of filter paper-gel-PVDF membrane-3 sheets of filter paper-1 layer of sponge-white plywood from top to bottom, remove bubbles, clamp the plywood, and transfer it to the electrotransfer tank, place an ice box in the tank, and fill it with pre-cooled electrotransfer solution; place the entire electrotransfer tank in an ice bath, and transfer the membrane at a constant current of 250mA. The transfer time is calculated according to the target molecule;

[0050] ⑦ Blocking: Prepare 0.5% skim milk with TBST half an hour in advance and shake well on a shaker; after electroporation, transfer the PVDF membrane to skim milk and shake at a low speed on a shaker for 2 hours; special target proteins are blocked with Biyuntian blocking solution for 1 hour;

[0051] ⑧ Incubate with primary antibody: After blocking, wash with TBST solution for 3 times, 5 minutes each time, then add the prepared primary antibody solution to ensure that the primary antibody can cover the strips, and place on a shaker in a 4°C refrigerator overnight;

[0052] ⑨ Incubate with secondary antibody: Recover the primary antibody, wash the strips 5 times with TBST solution by shaking rapidly on a shaker, 6 min / time; add the prepared secondary antibody solution, and incubate on a shaker at room temperature for 1 hour;

[0053] ⑩ Development: Recover the secondary antibody and wash 5 times with TBST solution by shaking rapidly on a shaker, 6 min / time; prepare ECL developer in a dark place and develop with Bio-Rad gel imager.

[0054] Preferably, in S8, the cerebral infarction volume determination specifically includes:

[0055] The rats were anesthetized and killed and their brains were removed immediately. The blood stains on the surface of the brain were rinsed with pre-cooled PBS solution. The surface moisture of the rat brain was wiped dry and placed in a -20℃ refrigerator for 10 minutes. The frozen brain was taken out and five consecutive sections were cut along the coronal plane. The thickness of each section was about 2 mm. The slices were placed in TTC solution and incubated in the dark at 37℃ for 30 minutes. The solution was gently shaken and the slices were turned over every 5 minutes to allow the brain slices to fully contact and react with the TTC solution. After the reaction was completed, the slices were fixed in 4% paraformaldehyde and stored in the dark. Finally, the brain slices were placed in order and photographed for storage. ImageJ was used to calculate the cerebral infarction volume of each rat.

[0056] Preferably, in said S9, the brain tissue Tunnel staining to detect the death of rat brain cells specifically includes:

[0057] ① Brain slice preparation: After the rats were anesthetized, they were perfused with the heart. After the heart was exposed, a needle was inserted into the left ventricle and the needle was fixed. The right auricle was cut and normal saline was injected through the needle until the liver turned completely white. Then the normal saline was replaced with 4% paraformaldehyde and injected slowly and evenly until the limbs and tail of the rats became stiff. Finally, the complete rat brain tissue was carefully removed and fixed in 4% paraformaldehyde for 24 hours. After 24 hours, the residual paraformaldehyde in the brain tissue was washed with PBS to remove the residual paraformaldehyde. After the surface moisture was wiped off, the brain tissue was immersed in 30% sucrose for dehydration until the brain tissue sank to the bottom. After the dehydrated brain tissue was removed, the surface moisture was wiped off and the brain tissue was embedded with OTC embedding agent and placed in a -20℃ refrigerator for freezing. Finally, the slices were sliced ​​on a slicer and the cut brain slices were stored in a 24-well plate filled with antifreeze at -20℃.

[0058] ②Tunnel staining: Take out the required slices from the antifreeze solution and place them in a new 24-well plate, add PBS solution and wash 3 times, 5 min / time; add proteinase K solution to the well plate and incubate at 37℃ for 25 min; then wash with PBS solution 3 times, 5 min / time; add 0.3% TritonX-100 solution, wait for 20 min at room temperature, and wash with PBS solution 3 times, 5 min / time; mix the Tunnel reaction solution evenly and add it to the well plate to completely cover the brain slices, incubate at 37℃ for 1 hour, and then wash with PBS solution 3 times, 5 min / time; finally, add DAPI, incubate at room temperature for 15 min, and then continue to wash with PBS solution, as before; add anti-fluorescence quencher after pasting, and photograph with a confocal microscope after sealing.

[0059] Preferably: In said S10, the detection of reduced glutathione content in brain tissue, and the detection of changes in ferroptosis indicators by a ferroptosis-related kit, specifically include:

[0060] The ischemic cerebral cortex was taken as a sample, and the GSH content of the ischemic brain tissue was detected using a GSH kit;

[0061] In S11, the detection of malondialdehyde content in brain tissue, and the detection of changes in ferroptosis indicators using a ferroptosis-related kit, specifically include:

[0062] The ischemic cerebral cortex was taken as a sample, and the MDA content of the ischemic brain tissue was detected using an MDA kit.

[0063] Preferred: Based on the experimental method of tVNS alleviating ferroptosis in stroke rats, the application of tVNS in stroke treatment is obtained.

[0064] The beneficial effects of the present invention are:

[0065] 1. The present invention detects ferroptosis-related proteins such as GPX4, ACSL4, FTH1, etc. by Western blot, proving that tVNS can improve ferroptosis in brain tissue of stroke rats, providing clear molecular mechanism support for intervention; GSH and MDA content detection kits were used to detect the contents of reduced glutathione and malondialdehyde, respectively, to further verify the effectiveness of tVNS in alleviating ferroptosis.

[0066] 2. The present invention adopts the suture embolism method to construct the right cerebral ischemia-reperfusion model in rats. Through detailed steps and strict operating procedures, the standardization and repeatability of model construction are ensured. The neurological function of each group of rats is evaluated using the modified neurological deficit score, and the improvement effect of tVNS on the neurological function of stroke rats is comprehensively evaluated from multiple aspects such as motor function, sensory function and reflex.

[0067] 3. The present invention detected the death of rat brain cells by Tunnel staining, which intuitively proved the significant effect of tVNS in saving brain tissue cell death in stroke rats; the cerebral infarction volume of each rat was calculated using TTC staining, which objectively reflected the positive effect of tVNS in reducing the area of ​​cerebral infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is a protein band diagram of actin internal reference in the experimental method of tVNS to alleviate ferroptosis in stroke rats proposed by the present invention;

[0069] Figure 2 This is a key protein band diagram of GPX4 ferroptosis in the experimental method of tVNS to alleviate ferroptosis in stroke rats proposed by the present invention. DETAILED DESCRIPTION

[0070] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0071] Embodiment 1:

[0072] An experimental method for alleviating ferroptosis in stroke rats by tVNS, as follows:

[0073] 1. Construction of rat MCAO model

[0074] The right cerebral ischemia-reperfusion model was established in SD rats using suture embolism.

[0075] (1) Preoperative preparation: Rats were fed adaptively for 5 days, fasted for 12 hours before surgery, and had free access to water. The surgical instruments required for the operation were sterilized by high-pressure steam, and the operating table was sterilized by ultraviolet light.

[0076] (2) Anesthesia and fixation: 1% sodium pentobarbital (45 mg / kg) was injected intraperitoneally into the rats. After waiting for about 5 minutes, the rats were fixed on the surgical splint in a supine position. The rats maintained even breathing and no other movements. The tongues were pulled to one side with forceps to prevent suffocation and to keep the airway open during the operation.

[0077] (3) Tissue separation and embolization process: ① After depilating the rat's neck, disinfect it with iodine, make a longitudinal incision along the middle of the neck, first use forceps to bluntly separate the right anterior cervical fascia layer by layer to expose the anterior cervical muscles, and use a retractor to pull the anterior cervical muscles to the right to fully expose the carotid sheath. ② Use micro forceps to slowly and gently separate the vagus nerve, jugular vein and common carotid artery along the carotid sheath, and ligate the distal end of the common carotid artery with 4-0 surgical thread. The common carotid artery is divided into the external carotid artery and the internal carotid artery after the cervical cross in the direction of the head. Ligate the external carotid artery and tie a loose knot at the cervical cross. Use an artery clamp to temporarily block the blood flow to the internal carotid artery. ③ Use vascular scissors to make an oblique incision between the common carotid artery knot and the cervical chiasm slipknot, insert the prepared wire plug from the incision through the slipknot to the internal carotid artery vascular clamp, tighten the loose knot to fix the wire plug, loosen the artery clamp, adjust the angle of the wire plug, and continue to insert the wire plug into the skull until the wire plug marking point passes through the cervical chiasm and the wire plug encounters resistance and cannot continue.

[0078] (4) Reperfusion: The rats were placed on an electric blanket to maintain body temperature at 37±0.3℃. After 90 minutes, the suture was removed to restore blood flow and complete reperfusion after vascular occlusion. The neck tissue was then sutured layer by layer, and the sutured area was disinfected with iodine. After the rats woke up, they were housed in separate cages and allowed to eat and drink freely. The rats in the SHAM group were not treated with sutures inserted into the neck, and the rest of the procedures were the same as those in the model group.

[0079] 2. Experimental Grouping

[0080] The experimental animals were randomly divided into ① sham operation group: SHAM group; ② cerebral ischemia reperfusion group: MCAO group; ③ auricular vagus nerve stimulation group after cerebral ischemia reperfusion: tVNS group.

[0081] 3. Auricular vagus nerve electrical stimulation intervention

[0082] The rats were fixed in a prone position, ensuring that the head of the rats was completely exposed. The conchae on both sides of the rats were cleaned and the temperature-controlled rat plate was maintained at approximately 37°C. In the tVNS group, ear clips connected to the ear vagus nerve electrical stimulation instrument were clamped on both sides of the auricle area of ​​the rats, and the current intensity was set to 1 mA. Each intervention lasted 30 minutes, once a day, for a total of 7 days. The sham stimulation group (SHAM and MCAO group) had no current, and the rest of the steps were the same as the stimulation group. The stimulation was performed by the same researcher at the same time every day for 7 consecutive days.

[0083] Fourth, neurological function scoring, to explore the effect of tVNS on the improvement of neurological function in stroke rats. On the seventh day after MCAO surgery, the mNSS scale was used to evaluate the neurological function of rats in each group. The mNSS test consists of several separate tests that assess different neurological functions, including motor function, sensory function, and reflexes. In brief, the rats were placed on a flat surface and their ability to move their forelimbs and hindlimbs was checked as an indicator of motor function. The score was divided into 0-6 points, where 0 points represented normal motor function and 6 points represented no movement. Next, the forelimbs and hindlimbs of the rats were touched with a soft brush, and their reactions were checked as an indicator of sensory function. The rats were scored on a scale of 0-2 points, where 0 points represented normal sensory function and 2 points represented no reaction. Finally, the forelimbs and hindlimbs of the rats were tapped and their reactions were checked as an indicator of reflexes. The rats were scored on a scale of 0-2 points, where 0 points represented normal reflexes and 2 points represented no reaction. The scores of each test were then added together to obtain the total mNSS score for each mouse. Higher scores indicate greater neurological deficits, while lower scores indicate better neurological function. The specific scores are as follows:

[0084] Table 1 Modified Neurological Deficit Score (mNSS)

[0085]

[0086] 5. Brain Tissue Protein Extraction

[0087] Transfer the extracted cerebral cortex tissue to a 1.5 ml EP tube, add the prepared tissue protein lysis solution (PIRA: protease inhibitor: phosphatase inhibitor = 50:1:1), use ultrasound to lyse the tissue, and then use a grinder to fully grind the tissue, take it out and let it stand on ice for 15 minutes, then place it in a high-speed low-temperature centrifuge and centrifuge it at 12000rpm, 4℃, 20min. After centrifugation, use a sterile pipette tip to draw the tissue supernatant into the marked EP tube and record the volume of the extracted supernatant.

[0088] VI. Protein concentration determination by BCA method

[0089] ① Add 0, 1, 2, 4, 8, 12, 16, 20 μl of 0.5 mg / ml protein standard to a 96-well plate, and then add PBS to 20 μl per well to form a concentration gradient of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / ml. Make 3 replicates for each concentration.

[0090] ② After the sample was diluted 50 times, 20 μl was added to each sample and 3 replicate wells were made for each sample.

[0091] ③ Add 200 μl BCA working solution (solution A: solution B = 50:1) to each well and place at 37°C for 20-30 minutes.

[0092] ④ Use an enzyme-labeled instrument to measure the absorbance at a wavelength of 547 nm.

[0093] ⑤ Calculate the standard curve based on the absorbance and concentration of the standard, and then calculate the protein concentration of the sample based on the standard curve and the sample absorbance and balance it.

[0094] ⑥After balancing, add 5× Loading Buffer at a ratio of 4:1 according to the final volume, mix well and place in a 95℃ metal bath for 10 minutes until the protein is completely denatured. After cooling, dispense 200μl into each tube and store in a -80℃ refrigerator.

[0095] 7. Western blot detection of ferroptosis-related proteins proves that tVNS can improve ferroptosis in brain tissue of stroke rats

[0096] ① Glue preparation: According to the instructions of the Biyuntian PAGE gel rapid preparation kit, add 50μl of coagulant to 2.5ml of the lower gel A solution and B solution of each gel, mix well and quickly add to the glass plate on the gel preparation rack, add anhydrous ethanol to the upper edge of the short plate; let it stand for 20 minutes, pour out the anhydrous ethanol, add 0.75ml of the upper gel A solution and B solution and 12μl of coagulant to each plate, and insert a ten-hole comb; ensure that no bubbles are generated in the gel during the whole process;

[0097] ② Preparation of electrophoresis solution: weigh 37.6 g of glycine, 6.06 g of Tris-base, and 2.0 g of SDS, mix in 2 L of double distilled water, and mix thoroughly;

[0098] ③ Preparation of transfer solution: weigh 28.8 g of glycine, 6.06 g of Tris-base, and 400 mL of methanol, mix in 1600 mL of double distilled water, and mix thoroughly;

[0099] ④ Preparation of washing solution TBST: Add TBST dry powder buffer (Seville catalog number: G0001-2L) into 2L of double distilled water and mix thoroughly;

[0100] ⑤ Sample electrophoresis: Place the sample in a 95℃ metal bath for 5 minutes in advance. During this period, fix the prepared gel on the electrophoresis rack and place it in the electrophoresis tank. Add the newly prepared electrophoresis solution to the inner tank until the glass plate is submerged, and ensure that the inner tank is tightly sealed and does not leak. Add the electrophoresis solution to the outer tank until the scale line is reached. Then remove the comb, remove the bubbles in the lane, and add 3μl of marker and 30-40μg of protein to the channel. Perform electrophoresis at a constant voltage of 80V for 20 minutes on the upper concentrated gel and at a constant voltage of 120V on the lower separation gel until the buffer reaches the bottom.

[0101] ⑥ Transfer: Pre-cool the prepared electrotransfer solution, cut a PVDF membrane of appropriate size, activate it with methanol solution for 20 seconds in advance, and then place it in the electrotransfer solution; arrange it in the order of black plywood-1 layer of sponge-3 sheets of filter paper-gel-PVDF membrane-3 sheets of filter paper-1 layer of sponge-white plywood from top to bottom, remove bubbles, clamp the plywood, and transfer it to the electrotransfer tank, place an ice box in the tank, and fill it with pre-cooled electrotransfer solution; place the entire electrotransfer tank in an ice bath, and transfer the membrane at a constant current of 250mA. The transfer time is calculated according to the target molecule;

[0102] ⑦ Blocking: Prepare 0.5% skim milk with TBST half an hour in advance and shake well on a shaker; after electroporation, transfer the PVDF membrane to skim milk and shake at a low speed on a shaker for 2 hours; special target proteins are blocked with Biyuntian blocking solution for 1 hour;

[0103] ⑧ Incubate with primary antibody: After blocking, wash with TBST solution for 3 times, 5 min each time, then add the prepared primary antibody solution to ensure that the primary antibody can cover the band, and place it on a shaker in a 4°C refrigerator overnight; primary antibody concentration: GPX4 (1:5000), ACSL4 (1:2000), FTH1 (1:2000), β-Actine (1:5000);

[0104] ⑨ Incubate with secondary antibody: Recover the primary antibody, wash the strips 5 times with TBST solution by shaking rapidly on a shaker, 6 min / time; add the prepared secondary antibody solution (1:5000), and incubate on a shaker at room temperature for 1 hour;

[0105] ⑩ Development: Recover the secondary antibody and wash 5 times with TBST solution by shaking rapidly on a shaker, 6 min / time; prepare ECL developer in a dark place and develop with Bio-Rad gel imager.

[0106] 8. Determination of cerebral infarction volume: Detection of the cerebral infarction area of ​​stroke rats, proving that tVNS can reduce the area of ​​cerebral infarction

[0107] The rats were anesthetized and killed and their brains were removed immediately. The blood stains on the surface of the brain were rinsed with pre-cooled PBS solution. The surface moisture of the rat brain was wiped dry and placed in a -20℃ refrigerator for 10 minutes. The frozen brain was taken out and five continuous sections were cut along the coronal plane. The thickness of each section was about 2mm. The slices were placed in TTC solution and incubated in the dark at 37℃ for 30 minutes. The solution was gently shaken and the slices were turned over every 5 minutes to allow the brain slices to fully contact and react with the TTC solution. After the reaction was completed, the slices were fixed in 4% paraformaldehyde and stored in the dark. Finally, the brain slices were placed in order and photographed for storage. ImageJ was used to calculate the volume of cerebral infarction in each rat.

[0108] 9. Tunnel staining of brain tissue was used to detect brain cell death in rats, proving that tVNS can save brain tissue cell death in stroke rats

[0109] ① Brain slice preparation: After the rat is anesthetized, the heart is perfused. After the heart is exposed, the needle is inserted into the left ventricle and the needle is fixed. The right atrial appendage is cut and saline is injected through the needle until the liver turns completely white. Then the saline is replaced with 4% paraformaldehyde and injected slowly and evenly until the rat's limbs and tail are stiff. Finally, the complete rat brain tissue is carefully removed and fixed in 4% paraformaldehyde for 24 hours. After 24 hours, the residual paraformaldehyde in the brain tissue is rinsed with PBS, the surface moisture is wiped off, and it is immersed in 30% sucrose for dehydration until the brain tissue sinks to the bottom. The brain tissue after dehydration is removed, the surface moisture is wiped off, and it is embedded with OTC embedding agent, and then placed in a -20℃ refrigerator for freezing. Finally, it is sliced ​​on a slicer, and the cut brain slices are stored in a 24-well plate filled with antifreeze and stored at -20℃.

[0110] ②Tunnel staining: Take out the required slices from the antifreeze solution and place them in a new 24-well plate. Add PBS solution and wash 3 times, 5 minutes each time. Add proteinase K solution to the well plate and incubate at 37°C for 25 minutes. Then wash with PBS solution 3 times, 5 minutes each time. Add 0.3% TritonX-100 solution, wait for 20 minutes at room temperature, and wash with PBS solution 3 times, 5 minutes each time. Mix the Tunnel reaction solution evenly and add it to the well plate to completely cover the brain slices. After incubating at 37°C for 1 hour, wash with PBS solution 3 times, 5 minutes each time. Finally, add DAPI, incubate at room temperature for 15 minutes, and then continue to wash with PBS solution, as before. After patching, add anti-fluorescence quencher and seal the slices and take pictures with a confocal microscope.

[0111] 10. Detection of reduced glutathione (GSH) content in brain tissue, and detection of changes in ferroptosis indicators using ferroptosis-related kits. tVNS can alleviate ferroptosis.

[0112] The above-mentioned ischemic cerebral cortex was taken as a sample, and the GSH content of ischemic brain tissue was detected using a GSH kit (Seville Product No. G4305). The operation steps and data processing were strictly carried out in accordance with the instructions attached to the kit:

[0113] 1.1 Sample pre-treatment:

[0114] Use appropriate buffer (PBS, physiological saline, etc.) for homogenization and lysis, in which the ratio of tissue to physiological saline is 1:9 (0.1g:0.9mL); after homogenization and lysis, centrifuge at 4°C, 10000g for 10-15min, take the supernatant and mix it with protein removal reagent in a ratio of 1:1, and mix thoroughly; centrifuge at 4°C, 10000g for 10-15min, and take the supernatant for subsequent reduced glutathione detection;

[0115] 1.2 Standard sample configuration and preparation:

[0116] 1.2.1. Centrifuge the glutathione standard (GSH powder) in a low-speed centrifuge to ensure that the powder is at the bottom of the tube; dissolve it in 500 μL ultrapure water to prepare the reduced glutathione standard (1 mmol / L). It is recommended to store it at -20℃ after aliquoting and use it up within 1 month.

[0117] 1.2.2. Use ultrapure water to perform gradient dilution of reduced glutathione standard (1mmol / L) (for example, take 50μL of the standard and add it to 950μL ultrapure water, dilute it to 50μmol / L, and further dilute it to make a standard curve, i.e. 50 / 25 / 12.5 / 6.25 / 3.125μmol / L), and use it for standard curve method data analysis after subsequent detection with the sample to be tested.

[0118] 2. Reduced glutathione detection:

[0119] 2.1. Mix the detection probe and ultrapure water in a ratio of 1:19 to prepare the detection probe working solution (it is recommended to be prepared before use);

[0120] 2.2. Refer to the table below for reduced glutathione detection;

[0121] Standard tube Blank tube Sample tube Reduced Glutathione Standard 100μL PBS or water 100μL Samples to be tested 100μL Reduced Glutathione Assay Buffer 100μL 100μL 100μL Detection probe working solution 20μL 20μL 20μL

[0122] The plate was shaken for 1 min, incubated at room temperature (25°C) for 5 min, and the absorbance at 412 nm was measured using an ELISA reader.

[0123] Data analysis

[0124] GSH content per unit tissue (μmol / gprot) = (absorbance of sample group - absorbance of blank group) / (absorbance of standard tube - absorbance of blank group) × concentration of standard tube (300μmol / l) × 2 * ÷ protein concentration

[0125] Note: 2* is the dilution multiple after mixing the protein removal reagent.

[0126] 11. Detection of malondialdehyde (MDA) content in brain tissue and detection of changes in ferroptosis indicators using ferroptosis-related kits. tVNS can alleviate ferroptosis.

[0127] The ischemic cerebral cortex was taken as a sample, and the MDA content of the ischemic brain tissue was detected using an MDA kit (Seville Product No. G4300). The operation steps and data processing were strictly carried out in accordance with the instructions attached to the kit.

[0128] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An experimental method for alleviating ferroptosis in stroke rats by tVNS, characterized in that: include: S1: Construction of rat MCAO model. The right cerebral ischemia-reperfusion model of SD rats was established by suture embolism method. S2: Experimental groups: The experimental animals were randomly divided into ① sham operation group: SHAM group; ② cerebral ischemia reperfusion group: MCAO group; ③ tVNS group after cerebral ischemia reperfusion; S3: Auricular vagus nerve stimulation intervention; S4: Neurological function score, to explore the improvement effect of tVNS on the neurological function of stroke rats; S5: Brain tissue protein extraction; S6: Protein concentration determination by BCA method; S7: Western blot detection of ferroptosis-related proteins; S8: Determination of cerebral infarction volume, detecting the area of ​​cerebral infarction in stroke rats; S9: Tunnel staining of brain tissue to detect brain cell death in rats; S10: Detection of reduced glutathione content in brain tissue, and detection of changes in ferroptosis indicators using ferroptosis-related kits; S11: Detection of malondialdehyde content in brain tissue, and detection of changes in ferroptosis indicators using ferroptosis-related kits.

2. The experimental method of tVNS for alleviating ferroptosis in stroke rats according to claim 1, characterized in that: In S1, the right cerebral ischemia-reperfusion model of SD rats was established by thread embolism method, which specifically included: preoperative preparation, anesthesia and fixation, tissue separation and embolism and reperfusion; Among them, tissue separation and embolization include: ① After depilating the rat's neck, disinfect it with iodine, make a longitudinal incision along the middle of the neck, first use forceps to bluntly separate the right anterior cervical fascia layer by layer to expose the anterior cervical muscles, and use a retractor to pull the anterior cervical muscles to the right to fully expose the carotid sheath; ② Use micro forceps to slowly and gently separate the vagus nerve, jugular vein and common carotid artery along the carotid sheath, and ligate the distal end of the common carotid artery with surgical thread; the common carotid artery is divided into the external carotid artery and the internal carotid artery after reaching the cervical cross in the direction of the head, ligate the external carotid artery, tie a loose knot at the cervical cross, and temporarily block the blood flow to the internal carotid artery with an artery clamp; ③ Use vascular scissors to make an oblique incision between the common carotid artery knot and the cervical cross slipknot, insert the prepared thread plug from the incision through the slipknot to the internal carotid artery vascular clamp, tighten the loose knot to fix the thread plug, loosen the artery clamp, adjust the angle of the thread plug, and continue to insert the thread plug into the skull until the thread plug mark point passes through the cervical crossknot and the thread plug encounters resistance and cannot continue; Reperfusion includes: The rats were placed on an electric blanket to maintain body temperature. After 90 minutes, the suture was removed to restore blood flow and reperfusion after vascular occlusion. The neck tissue was then sutured layer by layer and the sutured area was disinfected. After the rats woke up, they were housed in separate cages. Among them, the rats in the SHAM group were not treated with sutures inserted into the neck.

3. The experimental method of tVNS for alleviating ferroptosis in stroke rats according to claim 2, characterized in that: In S1, the auricular vagus nerve electrical stimulation intervention specifically includes: The rats were fixed in a prone position to ensure that their heads were completely exposed. The conchae on both sides of the rats were cleaned. In the tVNS group, ear clips connected to the ear vagus nerve electrical stimulation instrument were clamped on both sides of the auricle area of ​​the rats, and the current intensity was set to 1 mA. Each intervention lasted 30 minutes, once a day, for a total of 7 days. The current intensity of the SHAM and MCAO groups was 0, and the rest of the steps were the same as the tVNS group. The stimulation was performed by the same researcher at the same time every day for 7 consecutive days.

4. The experimental method of tVNS for alleviating ferroptosis in stroke rats according to claim 1, characterized in that: In S4, the neurological function score specifically includes: On the seventh day after MCAO, the neurological function of rats in each group was evaluated using the mNSS scale; the mNSS test included motor function, sensory function and reflex tests; Rats were placed on a flat surface and examined for their ability to move their forelimbs and hindlimbs as an indicator of motor function; Next, the rats’ forelimbs and hindlimbs were touched with a soft brush, and their responses were examined as an indicator of sensory function; Finally, the rats' forelimbs and hindlimbs were tapped and their responses were examined as an indicator of reflexes.

5. An experimental method for alleviating ferroptosis in stroke rats by tVNS according to any one of claims 1 to 4, characterized in that: In S5, the brain tissue protein extraction specifically includes: Transfer the extracted cerebral cortex tissue to an EP tube, add the prepared tissue protein lysis solution, use ultrasound to lyse the tissue, and then use a grinder to fully grind the tissue, take it out and let it stand on ice, then place it in a high-speed low-temperature centrifuge for centrifugation; after centrifugation, use a sterile pipette tip to aspirate the tissue supernatant into the marked EP tube, and record the volume of the extracted supernatant.

6. The experimental method of tVNS for alleviating ferroptosis in stroke rats according to claim 5, characterized in that: In S6, the BCA method protein concentration determination specifically includes: ① Add 0, 1, 2, 4, 8, 12, 16, 20 μl of 0.5 mg / ml protein standard to a 96-well plate, and then add PBS to make up to 20 μl per well to form a concentration gradient of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / ml; make 3 replicate wells for each concentration; ② After the sample is diluted 50 times, add 20 μl to each sample and make 3 replicate wells for each sample; ③ Add 200 μl BCA working solution to each well, solution A: solution B = 50:1, and place at 37°C for 20-30 minutes; ④Measure the absorbance at 547nm using an enzyme-labeled instrument; ⑤ Calculate the standard curve based on the absorbance and concentration of the standard, and then calculate the protein concentration of the sample based on the standard curve and the sample absorbance and balance; ⑥ After balancing, add 5× Loading Buffer at a ratio of 4:1 according to the final volume, mix well and place in a 95℃ metal bath for 10 minutes until the protein is completely denatured; after cooling, dispense 200μl into each tube and store in a -80℃ refrigerator.

7. The experimental method of tVNS for alleviating ferroptosis in stroke rats according to claim 6, characterized in that: In S7, Western blot was used to detect ferroptosis-related proteins, specifically including: ① Glue preparation: add 50μl of coagulant to 2.5ml of the lower glue A and B liquids of each glue block, mix well and quickly add to the glass plate on the glue preparation rack, add anhydrous ethanol to the upper edge of the short plate; let it stand for 20 minutes, pour out the anhydrous ethanol, add 0.75ml of the upper glue A and B liquids and 12μl of coagulant to each plate, and insert a ten-hole comb; ensure that no bubbles are generated in the glue during the whole process; ② Preparation of electrophoresis solution: weigh 37.6 g of glycine, 6.06 g of Tris-base, and 2.0 g of SDS, mix in 2 L of double distilled water, and mix thoroughly; ③ Preparation of transfer solution: weigh 28.8 g of glycine, 6.06 g of Tris-base, and 400 mL of methanol, mix in 1600 mL of double distilled water, and mix thoroughly; ④ Preparation of washing solution TBST: Add TBST dry powder buffer into 2L of double distilled water and mix thoroughly; ⑤ Sample electrophoresis: Place the sample in a 95℃ metal bath for 5 minutes in advance. During this period, fix the prepared gel on the electrophoresis rack and place it in the electrophoresis tank. Add the newly prepared electrophoresis solution to the inner tank until the glass plate is submerged, and ensure that the inner tank is tightly sealed and does not leak. Add the electrophoresis solution to the outer tank until the scale line is reached. Then remove the comb, remove the bubbles in the lane, and add 3μl of marker and 30-40μg of protein to the channel. Perform electrophoresis at a constant voltage of 80V for 20 minutes on the upper concentrated gel and at a constant voltage of 120V on the lower separation gel until the buffer reaches the bottom. ⑥ Transfer: Pre-cool the prepared electrotransfer solution, cut a PVDF membrane of appropriate size, activate it with methanol solution for 20 seconds in advance, and then place it in the electrotransfer solution; arrange it in the order of black plywood-1 layer of sponge-3 sheets of filter paper-gel-PVDF membrane-3 sheets of filter paper-1 layer of sponge-white plywood from top to bottom, remove bubbles, clamp the plywood, and transfer it to the electrotransfer tank, place an ice box in the tank, and fill it with pre-cooled electrotransfer solution; place the entire electrotransfer tank in an ice bath, and transfer the membrane at a constant current of 250mA. The transfer time is calculated according to the target molecule; ⑦ Blocking: Prepare 0.5% skim milk with TBST half an hour in advance and shake well on a shaker; after electroporation, transfer the PVDF membrane to skim milk and shake at a low speed on a shaker for 2 hours; special target proteins are blocked with Biyuntian blocking solution for 1 hour; ⑧ Incubate with primary antibody: After blocking, wash with TBST solution for 3 times, 5 minutes each time, then add the prepared primary antibody solution to ensure that the primary antibody can cover the strips, and place on a shaker in a 4°C refrigerator overnight; ⑨ Incubate with secondary antibody: Recover the primary antibody, wash the strips 5 times with TBST solution by shaking rapidly on a shaker, 6 min / time; add the prepared secondary antibody solution, and incubate on a shaker at room temperature for 1 hour; ⑩ Development: Recover the secondary antibody and wash 5 times with TBST solution by shaking rapidly on a shaker, 6 min / time; prepare ECL developer in a dark place and develop with Bio-Rad gel imager.

8. The experimental method of tVNS for alleviating ferroptosis in stroke rats according to claim 6, characterized in that: In S8, the cerebral infarction volume determination specifically includes: The rats were anesthetized and killed and their brains were removed immediately. The blood stains on the surface of the brain were rinsed with pre-cooled PBS solution. The surface moisture of the rat brain was wiped dry and placed in a -20℃ refrigerator for 10 minutes. The frozen brain was taken out and five consecutive sections were cut along the coronal plane. The slices were placed in TTC solution and incubated in the dark at 37℃ for 30 minutes. The solution was gently shaken and the slices were turned over every 5 minutes to allow the brain slices to fully contact and react with the TTC solution. After the reaction was completed, the slices were fixed in 4% paraformaldehyde and stored in the dark. Finally, the brain slices were placed in order and photographed for storage. ImageJ was used to calculate the cerebral infarction volume of each rat.

9. The experimental method of tVNS for alleviating ferroptosis in stroke rats according to claim 6, characterized in that: In S9, brain tissue Tunnel staining to detect rat brain cell death specifically includes: ① Brain slice preparation: After the rats were anesthetized, they were perfused with the heart. After the heart was exposed, a needle was inserted into the left ventricle and the needle was fixed. The right auricle was cut and normal saline was injected through the needle until the liver turned completely white. Then the normal saline was replaced with 4% paraformaldehyde and injected slowly and evenly until the limbs and tail of the rats became stiff. Finally, the complete rat brain tissue was carefully removed and fixed in 4% paraformaldehyde for 24 hours. After 24 hours, the residual paraformaldehyde in the brain tissue was washed with PBS to remove the residual paraformaldehyde. After the surface moisture was wiped off, the brain tissue was immersed in 30% sucrose for dehydration until the brain tissue sank to the bottom. After the dehydrated brain tissue was removed, the surface moisture was wiped off and the brain tissue was embedded with OTC embedding agent and placed in a -20℃ refrigerator for freezing. Finally, the slices were sliced ​​on a slicer and the cut brain slices were stored in a 24-well plate filled with antifreeze at -20℃. ②Tunnel staining: Take out the required slices from the antifreeze solution and place them in a new 24-well plate, add PBS solution and wash 3 times, 5 min / time; add proteinase K solution to the well plate and incubate at 37℃ for 25 min; then wash with PBS solution 3 times, 5 min / time; add 0.3% TritonX-100 solution, wait for 20 min at room temperature, and wash with PBS solution 3 times, 5 min / time; mix the Tunnel reaction solution evenly and add it to the well plate to completely cover the brain slices, incubate at 37℃ for 1 hour, and then wash with PBS solution 3 times, 5 min / time; finally, add DAPI, incubate at room temperature for 15 min, and then continue to wash with PBS solution, as before; add anti-fluorescence quencher after pasting, and photograph with a confocal microscope after sealing.

10. The experimental method of tVNS for alleviating ferroptosis in stroke rats according to claim 6, characterized in that: In S10, the reduced glutathione content in brain tissue is detected by using a ferroptosis-related kit to detect changes in ferroptosis indicators, specifically including: The ischemic cerebral cortex was taken as a sample, and the GSH content of the ischemic brain tissue was detected using a GSH kit; In S11, the detection of malondialdehyde content in brain tissue, and the detection of changes in ferroptosis indicators using a ferroptosis-related kit, specifically include: The ischemic cerebral cortex was taken as a sample, and the MDA content of the ischemic brain tissue was detected using an MDA kit.

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