Application of RNF125 as new target in relieving cerebral ischemia-reperfusion injury

Abstract

The invention discloses application of RNF125 as a new target in relieving cerebral ischemia reperfusion injury, and relates to the technical field of biological medicine. Comprising one or more of the following items: application to reduction of cerebral infarction volume after middle cerebral artery embolism based on interference RNF125; based on RNF125 gene knock-down, the invention is applied to improvement of behavioral function defects after middle cerebral artery embolism; based on the interference RNF125, neuronal loss is reduced after middle cerebral artery embolism, and brain tissue damage is relieved; based on interference RNF125, the invention is applied to relieving inflammatory response after middle cerebral artery embolism. The application focuses on RNF125 as a new therapeutic target, and shows a remarkable effect in the aspect of relieving cerebral ischemia-reperfusion injury; by interfering with the expression of RNF125, the cerebral infarction volume in a mouse model can be effectively reduced, the neurological function can be improved, and brain tissue damage can be relieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to the application of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury. Background Art

[0002] Cerebral ischemia-reperfusion injury (CIRI) is one of the leading causes of death and disability in adults in China. Its high incidence, high disability rate, high mortality rate, and high recurrence rate, combined with the continuous increase in the global stroke disease burden, have made it a research focus that urgently needs to be broken through in the field of neuroscience. The pathological cascade reaction of CIRI involves multi-dimensional interaction mechanisms such as oxidative stress, calcium homeostasis imbalance, mitochondrial dysfunction, and activation of the neuroinflammatory network. Among them, the neuroinflammatory response, as the core driving factor, exacerbates neuronal apoptosis and neurological deficits through pathways such as microglia / astrocyte activation, cytokine storm (TNF-α, IL-1β, IL-6), and disruption of the blood-brain barrier integrity.

[0003] Recent studies have shown that the ubiquitin-proteasome system (UPS) plays a pivotal role in the regulation of neuroinflammation. The core functional unit of UPS - the E3 ubiquitin ligase regulates the ubiquitination modification status of key inflammatory signaling molecules (such as NF-κB, NLRP3 inflammasome) through substrate-specific recognition (such as the synergistic action of E1 activating enzyme and E2 conjugating enzyme). For example:

[0004] (1) Knockout of the E3 ubiquitin ligase c-Cbl in mice activates the PI3K / Akt / NF-κB pathway mediated by microglia and promotes the expression of inflammatory factors (TNF-α, IL-6, IL-1β, and MCP-1)6;

[0005] (2) Specific knockdown of TRIM45 in microglia significantly reduces the cerebral infarction volume in mice, decreases the neurological deficit score, and improves cognitive function by inhibiting NF-κB signal activation7;

[0006] (3) The E3 ubiquitin ligase FBXO3 plays a pro-inflammatory role in cerebral ischemia-reperfusion injury. Interfering with FBOX3 significantly reduces nerve injury in in vivo and in vitro models and inhibits the inflammatory response. These findings suggest the potential value of E3 ligases as "molecular switches" for inflammation regulation. Summary of the Invention

[0007] The purpose of the present invention is to solve the deficiencies in the prior art, and to propose the application of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury.

[0008] In the early stage of the present invention, through the integration of RNA-Seq sequencing technology and in-depth bioinformatics analysis, it was screened and found that the RING-type E3 ligase RNF125 showed a significant dynamic upregulation during the CIRI process (see the results section). In many studies on malignant tumors, it was found that the expression of RNF125 was significantly downregulated in tumor tissues, and overexpression of RNF125 could inhibit the proliferation and metastasis of tumor cells and play an anti-tumor role; in the endotoxemia model, RNF125 played an anti-inflammatory role by mediating NLRP3-K63 ubiquitination; in the study of osteoarthritis (OA) by Lu et al., it was found that the expression of RNF125 was downregulated in rat cartilage tissue and upregulated after physiological exercise. Overexpression of RNF125 could inhibit the degradation of the extracellular matrix of chondrocytes induced by IL-1β and alleviate the symptoms of OA; in Japanese encephalitis virus infection, miR-15b could promote the release of inflammatory factors by inhibiting RNF125 to reduce the inflammatory response. However, the functional study of RNF125 in cerebrovascular diseases has not been reported. Based on the mouse middle cerebral artery occlusion (MCAO) model, the results showed that:

[0009] (1) The protein expression of RNF125 was significantly upregulated after MCAO and reached the peak at 24 hours after reperfusion;

[0010] (2) Interference with RNF125 could improve the neurological score, reduce the cerebral infarction volume, and alleviate nerve injury;

[0011] (3) Interference with RNF125 significantly inhibited the expression of TNF-α, IL-1β, and IL-6. The above results suggest that interfering with RNF125 may reduce nerve injury through a pro-inflammatory mechanism in CIRI. Therefore, RNF125 may provide a new target for the treatment of cerebral ischemia-reperfusion injury.

[0012] To achieve the above object, the present invention adopts the following technical solutions:

[0013] The application of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury includes one or more of the following:

[0014] The application based on interfering with RNF125 in reducing the cerebral infarction volume after middle cerebral artery occlusion;

[0015] The application based on knocking down the RNF125 gene in improving the defects in behavioral function after middle cerebral artery occlusion;

[0016] The application based on interfering with RNF125 in reducing neuron loss and alleviating brain tissue injury after middle cerebral artery occlusion;

[0017] The application based on interfering with RNF125 in alleviating the inflammatory response after middle cerebral artery occlusion.

[0018] Preferably, the experimental verification is carried out in the following manner:

[0019] S1: Establishment of the MCAO model;

[0020] S2: Four-limb grip strength test;

[0021] S3: Rotarod test;

[0022] S4: Western Blot;

[0023] S5: Preparation of paraffin sections of brain tissue;

[0024] S6: HE staining;

[0025] S7: Nissl staining;

[0026] S8: Result analysis.

[0027] Preferably, in the above S1, the establishment of the MCAO model specifically includes:

[0028] S11: The mice are fasted 12 h before surgery and water-deprived 6 h before surgery. The surgical instruments are soaked in 75% alcohol for standby;

[0029] S12: After the mice are anesthetized, their four limbs and incisors are fixed with medical adhesive tape to keep the necks of the mice vertical, fully exposing the neck skin of the mice. The neck skin of the mice is disinfected with iodophor and deiodinated with 75% alcohol;

[0030] S13: A longitudinal incision is made directly above the trachea with a small surgical scissors to expose the gland directly above the trachea of the mice. Under a stereomicroscope, the fascia around the gland is gently bluntly dissected with forceps until the common carotid artery sheath wrapping the carotid artery, jugular vein and vagus nerve is exposed;

[0031] S14: Under a stereomicroscope, the common carotid artery sheath is gently bluntly dissected to isolate the common carotid artery, and the external carotid artery is searched distally. The proximal ends of the common carotid artery and the external carotid artery are ligated, and a thread is buried at the distal end of the common carotid artery;

[0032] S15: Under a stereomicroscope, the common carotid artery is gently lifted with forceps, and a small surgical scissors makes an incision on the common carotid artery. The thread embolism is inserted through the incision until the black dot marked on the thread embolism just passes through the bifurcation of the internal and external carotid arteries and the thread embolism can no longer pass forward;

[0033] S16: Under a stereomicroscope, the thread previously buried on the common carotid artery is tied with a slipknot and timed;

[0034] S17: After 60 min, the slipknot is loosened, the thread embolism is pulled out, the embolism removal time is recorded, and two surgical knots are made with forceps to prevent slippage;

[0035] S18: Suture the skin, place the modeled mice on an electric blanket until they wake up, and then keep them in an environment of 25°C;

[0036] S19: 24 hours after surgery, the mice were taken out for subsequent experiments. The mice in the Sham group only had their skin cut without plugging, and subsequent experiments were performed at the same time as the MCAO mice 24 hours later.

[0037] Preferably: In S1, mice are screened by neurological function scoring, as follows:

[0038] ① Take out the mice after MCAO surgery and score them using Zea-Longa's 5-level 4-point standard:

[0039] 0 points: no neurological deficit symptoms;

[0040] 1 point: When the mouse's tail is lifted, it can be observed that the mouse's contralateral forelimb cannot be fully extended;

[0041] 2 points: The mouse was placed on a level surface without obstacles, and the mouse turned its head to the opposite side while walking;

[0042] 3 points: The mouse was placed on a level surface without obstacles, and it fell to the opposite side while walking;

[0043] 4 points: The mouse loses its autonomous consciousness and has no autonomous activities;

[0044] 5 points: mouse died;

[0045] ②Mice with scores of 0 and 5 were not included in the experiment and were used as the criteria for inclusion in subsequent experiments.

[0046] Preferably: in S2, the limb gripping strength test specifically includes:

[0047] S21: Lift the mouse by its tail and let its limbs grasp the metal rod of the measuring instrument, keeping the mouse's tail parallel to the metal rod;

[0048] S22: Click the start button on the grip tester to start testing the grip;

[0049] S23: Gently lift the mouse's tail and gradually remove the mouse's limbs from the metal rod at a constant speed;

[0050] S24: Record the data displayed on the grip tester;

[0051] S25: Perform the test three times continuously, with an interval of 10 minutes between each test, and calculate the average value.

[0052] Preferably: in S3, the rotarod test specifically includes:

[0053] S31: Place the mouse on a slowly rotating rod for 1 min. After adaptation, start the rod, which accelerates uniformly from 5 r / min, and record the time when the mouse falls off the rod.

[0054] S32: Each mouse exercises for 3 days before modeling, with the number of times remaining the same. After modeling, measure 3 times and take the average, with a 10-min interval between each test.

[0055] Preferably, in the said S4, Western Blot specifically includes:

[0056] S41: Prepare an SDS-PAGE gel with a lower layer concentration of 12.5%. After solidifying for 20 min, prepare the upper gel. After the upper gel solidifies for 1 h, use it or store it in a 4°C refrigerator for later use.

[0057] S42: Mix the sample and 5×loading buffer in a ratio of 4:1, boil at 99°C for 5 min, let it return to room temperature before use, or store it in a -20°C refrigerator.

[0058] S43: Loading: Slowly add 20 - 40 μg of protein into the wells of the upper layer of the SDS-PAGE gel.

[0059] S44: Electrophoresis: Use a voltage of 50 V for electrophoresis for about 30 min, then adjust to 100 V and electrophorese until the bromophenol blue reaches the bottom.

[0060] S45: Electrotransfer: Transfer the protein on the SDS-PAGE gel to a 0.20-μm PVDF membrane, with a constant current of 250 mA, and transfer the membrane at 4°C for 40 min.

[0061] S46: Blocking: Use the blocking solution to block at room temperature for about 30 min.

[0062] S47: Primary antibody incubation: Dilute the antibody with the dilution solution, soak the PVDF membrane in the corresponding antibody, ensure that the membrane is completely covered, and gently shake overnight in a 4°C refrigerator; the next day, wash the membrane with TBST three times, 10 min each time, for a total of 30 min.

[0063] S48: Secondary antibody incubation: Use the secondary antibody of the corresponding species and incubate at room temperature for 1 h; wash the membrane three times again using the same method.

[0064] S49: Chemiluminescence: Prepare the luminescent solution in a 1:1 ratio, drop it on the PVDF membrane on the wax plate, and then automatically expose it with the instrument; analyze the relative optical density values of each well on the strip.

[0065] Preferably, in the said S5, preparing the paraffin section of the brain tissue specifically includes:

[0066] S51: After taking out the anesthetized mouse, open the chest cavity and perform cardiac perfusion.

[0067] S52: First, perfuse with PBS, and then slowly perfuse with 4% paraformaldehyde;

[0068] S53: Open the skull, carefully remove the brain tissue and place it in 4% paraformaldehyde for fixation for at least 48 hours

[0069] S54: Dehydration: Dehydrate successively with 75% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol, 2 hours for each step, and dry the brain tissue in the middle;

[0070] S55: Clearing: Prepare a 1:1 mixture of 100% alcohol and xylene, soak in it for 10 minutes, and then soak in xylene for 20 minutes;

[0071] S56: Impregnation with wax: Soak in a 1:1 solution of xylene and paraffin at 56 °C for 30 minutes, and then soak in paraffin for 3 hours;

[0072] S57: Embedding, sectioning, and baking the sections.

[0073] Preferably: In S6, the HE staining specifically includes:

[0074] S61: Dewaxing: Twice in xylene, 10 minutes each time;

[0075] S62: Deparaffinization: Wash successively in 100% alcohol, 95% alcohol, 80% alcohol, 70% alcohol for 3 minutes each, and wash in double-distilled water for 1 minute;

[0076] S63: Hematoxylin staining: Stain in hematoxylin for 8 minutes, wash in double-distilled water for 2 minutes, wash in 1% hydrochloric acid alcohol for 10 seconds, wash in double-distilled water for 1 minute, place in lithium carbonate for 2 minutes, and wash in double-distilled water for 1 minute;

[0077] S64: Eosin staining: Stain with eosin for 20 seconds, wash in double-distilled water for 1 minute, and dehydrate successively with 75% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol;

[0078] S65: Clearing: Clear in xylene;

[0079] S66: Sealing the coverslip: Seal the coverslip with neutral balsam;

[0080] S67: Observe the HE staining condition under the microscope and scan and collect the slices.

[0081] Preferably: In S7, the Nissl staining specifically includes:

[0082] S71: Dewaxing and deparaffinization;

[0083] S72: Staining: Stain with cresyl violet for 15 minutes, wash in double-distilled water for 1 minute;

[0084] S73: Color separation: Conducted in 95% ethanol;

[0085] S74: Dehydration: Dehydrated successively in 70% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol;

[0086] S75: Clearing: Conducted in xylene;

[0087] S76: Mounting: Mounted with neutral balsam;

[0088] S77: Observe the Nissl staining under a microscope, scan, and collect samples.

[0089] The beneficial effects of the present invention are as follows:

[0090] 1. The present invention focuses on RNF125 as a new therapeutic target and shows significant effects in reducing cerebral ischemia-reperfusion injury; by interfering with the expression of RNF125, it can effectively reduce the cerebral infarction volume in a mouse model, improve neurological function, and alleviate brain tissue damage.

[0091] 2. The present invention adopts a variety of experimental methods to evaluate the role of RNF125, including establishing a mouse middle cerebral artery occlusion (MCAO) model, neurological function scoring, Western Blot analysis, paraffin sectioning, and HE and Nissl staining, etc., ensuring the accuracy and reliability of the results.

[0092] 3. The results of the present invention show that after interfering with RNF125 using a specific siRNA fragment (si-1), the cerebral infarction volume after MCAO can be significantly reduced, the neurological function score can be improved, the retention time in the rotarod test can be prolonged, and the performance in the grip strength test can be restored, showing good therapeutic effects; in addition to the direct neuroprotective effect, interfering with RNF125 can also reduce the inflammatory response induced by MCAO and decrease the expression of inflammatory factors such as TNF-α, IL-1β, and IL-6, which may help further protect brain tissue from damage.

[0093] 4. In view of the important role of RNF125 in cerebral ischemia-reperfusion injury and its influence on the inflammatory response, the present invention provides new ideas and potential therapeutic targets for the development of new anti-cerebral ischemia drugs, and has important scientific research significance and potential clinical application prospects. Description of the Drawings

[0094] Figure 1 It is a statistical chart of the expression of RNF125 and its relative protein expression level after MCAO analyzed by Western blot of the present invention;

[0095] Figure 2This is a statistical chart of the expression of RNF125 and its relative protein expression level in the brain tissue of mice after MCAO by Western blot analysis of the present invention;

[0096] Figure 3 This is a statistical chart of the results of TTC staining and cerebral infarction volume statistics of the present invention;

[0097] Figure 4 This is a statistical chart of the neurological function score of the present invention;

[0098] Figure 5 This is a statistical chart of the rotarod test and the four-limb grip strength test of the present invention;

[0099] Figure 6 This is a HE staining map of the hippocampus and cerebral cortex of the present invention;

[0100] Figure 7 This is a Nissl staining map of the hippocampus and cerebral cortex of the present invention;

[0101] Figure 8 This is a schematic diagram of the protein expression levels of RNF125, IL-6, TNF-α, and IL-1β in the brain tissue of MCAO mice by Western blot analysis of the present invention;

[0102] Figure 9 This is a quantitative schematic diagram of the relative protein expression levels of RNF125, IL-6, TNF-α, and IL-1β in the brain tissue of mice of the present invention;

[0103] Figure 10 This is a statistical chart of the protein expression levels of IL-6, TNF-α, and IL-1β in the brain tissue of MCAO mice by ELISA analysis of the present invention. Detailed implementation manners

[0104] The technical solution of the present invention will be further described in detail below in conjunction with the specific implementation manners.

[0105] Example 1:

[0106] The application of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury was verified by the following experiment, specifically:

[0107] 1 Establish a mouse middle cerebral artery occlusion (MCAO) model

[0108] 1.1.1 Establishment of the MCAO model (suture method)

[0109] S11: Fast the C57 mice 12 h before surgery and withhold water 6 h before surgery. Soak the surgical instruments in 75% alcohol for standby;

[0110] S12: After anesthetizing the mice, fix the four limbs and incisors of the mice with medical adhesive tape to keep the necks of the mice vertical, fully expose the neck skin of the mice, disinfect the neck skin of the mice with iodophor, and deiodize with 75% alcohol;

[0111] S13: Make a longitudinal incision directly above the trachea with small surgical scissors, expose the gland directly above the trachea of the mice, and gently bluntly separate the fascia around the gland with forceps under a stereomicroscope until the common carotid artery sheath wrapping the carotid artery, jugular vein and vagus nerve is exposed;

[0112] S14: Gently bluntly separate the common carotid artery sheath under a stereomicroscope, isolate the common carotid artery, search for the external carotid artery towards the distal end, ligate the proximal ends of the common carotid artery and the external carotid artery, and bury a thread at the distal end on the common carotid artery;

[0113] S15: Gently lift the common carotid artery with forceps under a stereomicroscope, make an incision on the common carotid artery with small surgical scissors, insert the suture plug through the incision until the black dot marked on the suture plug just passes through the bifurcation of the internal carotid artery and the external carotid artery, and the suture plug can no longer pass forward;

[0114] S16: Tie a slipknot on the thread previously buried on the common carotid artery under a stereomicroscope and start timing;

[0115] S17: After 60 min, loosen the slipknot, pull out the suture plug, record the plug removal time, and make two surgical knots with forceps to prevent slippage;

[0116] S18: Suture the skin, place the modeled mice on an electric blanket until they wake up, and then raise them in an environment at 25 °C;

[0117] S19: 24 h after the operation, take out the C57 mice for subsequent experiments. The mice in the Sham group only have their skin cut without inserting a plug, and 24 h later, they conduct subsequent experiments simultaneously with the MCAO mice.

[0118] 1.1.2 Inclusion criteria - Neurological function score (Zea-Longa's score)

[0119] ① Take out the C57 mice after MCAO surgery and use the 5-grade 4-point method standard of Zea-Longa for scoring:

[0120] 0 point: No symptoms of neurological deficit;

[0121] 1 point: Lift the mouse's tail, and it can be observed that the contralateral forelimb of the mouse cannot be fully straightened;

[0122] 2 points: Place the mouse on a horizontal ground without obstacles. During the walking process of the mouse, the head turns to the contralateral side;

[0123] 3 points: Place the mouse on a horizontal ground without obstacles. During the walking process of the mouse, it tilts to the contralateral side;

[0124] Score 4: The mouse loses its autonomous consciousness and has no autonomous movement.

[0125] Score 5: The mouse dies.

[0126] ② C57 mice with scores of 0 and 5 are not included in the experiment, which is used as the standard for including subsequent experiments.

[0127] 1.2 Neurological function

[0128] 1.2.1 Neurological function scoring (see 1.1.2)

[0129] 1.2.2 Four-limb grip strength test

[0130] S21: Lift the mouse by its tail so that its four limbs grasp the metal rod of the measuring instrument, keeping the mouse's tail parallel to the metal rod.

[0131] S22: Click the start button on the grip strength tester to start detecting the grip strength.

[0132] S23: Gently lift the mouse's tail and gradually and uniformly disengage the mouse's four limbs from the metal rod.

[0133] S24: Record the data displayed on the grip strength tester.

[0134] S25: Detect continuously 3 times, with an interval of 10 min each time, and calculate the average value, which is used for statistical analysis.

[0135] 1.2.3 Rotarod test

[0136] S31: Place the mouse on the slowly rotating rotarod for 1 min. After adaptation, start the rotarod, which increases the speed uniformly from 5 r / min, and record the time when the mouse falls off the rotarod.

[0137] S32: Each mouse exercises for 3 days before modeling, with the number of times remaining the same. Measure 3 times after modeling and take the average value, with an interval of 10 min between each test.

[0138] 1.3 Western Blot

[0139] S41: Prepare an SDS-PAGE gel with a lower layer concentration of 12.5%. After solidifying for 20 min, prepare the upper layer gel. After the upper layer gel solidifies for 1 h, use it or store it in a refrigerator at 4 °C for later use.

[0140] S42: Mix the sample and 5×loading buffer in a ratio of 4:1, boil at 99 °C for 5 min, cool to room temperature before use, or store it in a refrigerator at -20 °C.

[0141] S43: Loading: Slowly add 20 - 40 μg of protein to the wells of the upper layer of the SDS-PAGE gel.

[0142] S44: Electrophoresis: Apply a voltage of 50 V and perform electrophoresis for approximately 30 min (until the protein sample reaches the boundary between the stacking gel and the separating gel), then adjust the voltage to 100 V and continue electrophoresis until the bromophenol blue reaches the bottom;

[0143] S45: Electrotransfer: Transfer the proteins on the SDS-PAGE gel to a 0.20-μm PVDF membrane at a constant current of 250 mA for 40 min at 4 °C (transfer conditions for proteins around 40 kDa);

[0144] S46: Blocking: Use a blocking solution and block at room temperature for about 30 min;

[0145] S47: Primary antibody incubation: Dilute the antibody with a dilution solution, soak the PVDF membrane in the corresponding antibody, ensure that the membrane is completely covered, and incubate slowly overnight in a 4 °C refrigerator; The next day, wash the membrane with TBST three times, 10 min each time, for a total of 30 min;

[0146] S48: Secondary antibody incubation: Use the secondary antibody of the corresponding species and incubate at room temperature for 1 h; Wash the membrane three times again using the same method;

[0147] S49: Chemiluminescence: Prepare the luminescence solution at a ratio of 1:1, drop it on the PVDF membrane on the wax plate, and then automatically expose it with an instrument; Analyze the relative light density values of each well on the strip.

[0148] 1.4 Preparation of paraffin sections and HE and Nissl staining

[0149] 1.4.1 Preparation of paraffin sections of brain tissue

[0150] S51: After taking out the anesthetized mouse, open the chest cavity and perform cardiac perfusion;

[0151] S52: First perfuse with PBS, and then slowly perfuse with 4% paraformaldehyde;

[0152] S53: Open the skull and carefully take out the brain tissue and place it in 4% paraformaldehyde for fixation for at least 48 h

[0153] S54: Dehydration: Dehydrate sequentially with 75% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol, 2 h for each step. Wipe the brain tissue dry midway and do not bring low-concentration alcohol into high-concentration alcohol;

[0154] S55: Clearing: Prepare a 1:1 mixture of 100% alcohol and xylene, soak in it for 10 min, and then soak in xylene for 20 min;

[0155] S56: Impregnation with wax: Soak in a 1:1 solution of xylene and paraffin at 56 °C for 30 min, and then soak in paraffin for 3 h;

[0156] S57: Embedding, sectioning, and baking the sections (at a temperature of 38 °C).

[0157] 1.4.2 HE staining

[0158] S61: Deparaffinization: Twice in xylene, 10 min each time;

[0159] S62: Dephenylation: Wash successively in 100% ethanol, 95% ethanol, 80% ethanol, 70% ethanol for 3 min each, and wash in double-distilled water for 1 min;

[0160] S63: Hematoxylin staining: Stain in hematoxylin for 8 min, wash in double-distilled water for 2 min, wash in 1% hydrochloric acid alcohol for 10 s, wash in double-distilled water for 1 min, soak in lithium carbonate for 2 min, and wash in double-distilled water for 1 min;

[0161] S64: Eosin staining: Stain in eosin for 20 s, wash in double-distilled water for 1 min, and dehydrate successively with 75% ethanol, 80% ethanol, 95% ethanol, and 100% ethanol;

[0162] S65: Clearing: Clear in xylene;

[0163] S66: Mounting: Mount with neutral balsam;

[0164] S67: Observe the HE staining under a microscope (white light) and scan and collect the sections.

[0165] 1.4.3 Nissl staining

[0166] S71: Deparaffinization and de-phenylation: The same as above;

[0167] S72: Staining: Stain in cresyl violet for 15 min, wash in double-distilled water for 1 min;

[0168] S73: Differentiation: Perform in 95% ethanol;

[0169] S74: Dehydration: Dehydrate successively in 70% ethanol, 80% ethanol, 95% ethanol, and 100% ethanol;

[0170] S75: Clearing: Perform in xylene;

[0171] S76: Mounting: Mount with neutral balsam;

[0172] S77: Observe the Nissl staining under a microscope (white light), scan, and collect the sections.

[0173] 1.5 Statistical analysis

[0174] All experiments were independently repeated at least three times. All statistical results were analyzed using Graphpad Prism 10.1.2. The data results were expressed as mean ± standard deviation (x ± s). Unpaired t-tests were used for differential analysis between two groups, and one-way ANOVA was used for differential analysis among more than two groups. Tukey's test was used for pairwise comparison among multiple samples after one-way ANOVA.

[0175] 2 Result Analysis

[0176] 2.1 Reduction of cerebral ischemia-reperfusion injury in mice after interfering with RNF125

[0177] 2.1.1 Expression of RNF125 after cerebral ischemia-reperfusion injury in mice

[0178] To study the changes in the protein level of RNF125 after cerebral ischemia-reperfusion in mice, the expression of RNF125 was measured at 12 h, 24 h, 48 h, and 72 h after ischemia-reperfusion. As Figure 1 shown, compared with the sham operation group (Sham), the expression level of RNF125 began to increase at 12 h of reperfusion, reached the peak at 24 h, and then decreased. This trend suggests that RNF125 may play a role in the injury caused by cerebral ischemia-reperfusion, especially the significant increase in expression at 24 h of reperfusion. Based on these findings, the time point for subsequent experiments was selected as 24 h after reperfusion.

[0179] 2.1.2 Screening of RNF125-siRNA with the best interference effect on mice

[0180] To interfere with the protein expression level of RNF125 in the brain tissue of C57 mice, three pairs of siRNA fragments with different sequences, namely si-1, si-2, and si-3, were used. At the same time, NC-siRNA was used as a negative control for the NC group. Each siRNA fragment was injected into the lateral ventricle of mice through a stereotaxic apparatus. After 24 h, the mice were subjected to MCAO modeling. After 24 h, the brain tissue was taken, and the interference efficiency of different fragments was detected by Western blot. As Figure 2 shown, the interference efficiency of si-1 on RNF125 was significantly higher than that of the NC group and other siRNA groups. Therefore, si-1 was used to interfere with the protein expression level of RNF125 in subsequent in vivo experiments.

[0181] 2.1.3 Reduction of the cerebral infarction volume in mice after interfering with RNF125

[0182] To clarify the role of RNF125 in cerebral ischemia-reperfusion injury, 24 h after establishing the MCAO model in mice, TTC staining combined with image acquisition and calculation analysis was used to detect the cerebral infarction volume in mice. The results of TTC staining showed that, asFigure 3 , the red part is the non-infarcted brain tissue, and the white brain tissue is the infarcted brain tissue. No obvious cerebral infarction was seen in the Sham group, while obvious cerebral infarction was observed in the MCAO group, MCAO+si-NC group, and MCAO+si-RNF125 group. As Figure 3 , the cerebral infarction volume was statistically analyzed. Compared with the MCAO group and MCAO+si-NC group, the infarction volume in the MCAO+si-RNF125 group was significantly reduced. The results showed that interfering with RNF125 could reduce the cerebral infarction volume in mice after MCAO.

[0183] 2.1.4 Reduction of neurological function in mice after MCAO by interfering with RNF125

[0184] To clarify the role of RNF125 in cerebral ischemia-reperfusion injury, 24 hours after establishing the MCAO model in mice, the Zea-Longa’s score was used to evaluate the neurological function injury of the mice. As Figure 4 , compared with the neurological function injury score of the Sham group, the neurological function injury scores of the MCAO group and MCAO+si-NC group were significantly increased (p < 0.0001), indicating severe neurological function injury in mice. Compared with the MCAO group and MCAO+si-NC group, the neurological function injury score of the MCAO+si-RNF125 group was increased (p < 0.01), suggesting that interfering with RNF125 could reduce the neurological function injury in mice after MCAO.

[0185] 2.1.5 Improvement of behavioral deficits in mice after MCAO by interfering with RNF125

[0186] As Figure 5 , clinically, patients with cerebral infarction will show cognitive and motor function disorders. Therefore, to explore the effect of RNF125 on the behavioral function of mice in MCAO, the rotarod test and the mouse grip strength test were used in this part to evaluate the changes in the behavioral function of mice. The experimental results showed that in the rotarod test, the time that mice in the MCAO, MCAO+si-NC group, and MCAO+si-RNF125 group stayed on the rotarod was significantly decreased compared with the Sham group. However, after interfering with RNF125, the staying time was prolonged compared with the MCAO and MCAO+si-NC groups; the results of the grip strength test showed that the grip strength of mice in the MCAO and MCAO+si-NC group was significantly weakened compared with the Sham group. However, after interfering with RNF125, the limb grip strength of mice was restored. The above results indicate that due to the aggravation of brain injury after the MCAO model, the behavioral function of mice is weakened, and knockdown of the RNF125 gene may improve the defects in behavioral function.

[0187] 2.1.6 Reduction of brain tissue injury in mice after MCAO by interfering with RNF125

[0188] AsFigure 6 HE staining showed that the cell morphology of the hippocampus and cerebral cortex in the Sham group was normal, the cell arrangement was regular, and the tissue structure was compact. However, in the MCAO group, MCAO+si-NC group, and MCAO+si-RNF125 group, the cells in the hippocampus and cerebral cortex were swollen, the cell arrangement was disordered, and the tissue structure was loose. Compared with the MCAO group and MCAO+si-NC group, the cell edema in the hippocampus and cerebral cortex of the MCAO+si-RNF125 group was reduced, the cell arrangement was relatively regular, and the tissue structure was relatively compact. This indicates that knocking down RNF125 can reduce the brain tissue damage in the hippocampus and cerebral cortex of mice after the MCAO model. As Figure 7 Nissl staining showed that the neurons in the hippocampus and cerebral cortex of the Sham group were arranged regularly, with distinct layers, and the surrounding tissues were arranged tightly. However, in the MCAO group, MCAO+si-NC group, and MCAO+si-RNF125 group, the neurons in the hippocampus and cortex were swollen, the arrangement was disordered, and the surrounding tissues were arranged loosely. Compared with the MCAO group and MCAO+si-NC group, the swelling of neurons in the MCAO+si-RNF125 group was reduced and the arrangement was relatively regular. These results indicate that interfering with RNF125 can reduce the loss of neurons and alleviate brain tissue damage in the mouse MCAO model.

[0189] 2.2 Interference with RNF125 alleviated the inflammatory response induced by cerebral ischemia-reperfusion injury in mice

[0190] To clarify the effect of RNF125 on the inflammatory response after MCAO in mice, 24 hours after establishing the mouse MCAO model, samples were taken and the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the mouse brain tissue were detected by western blot and ELISA. As shown, Figure 8 、 Figure 9 、 Figure 10 The WB and ELISA results showed that the expression levels of inflammatory factors IL-6, TNF-α, and IL-1β were increased in the MCAO group and MCAO+si-NC group, while the expression levels of inflammatory factors IL-6, TNF-α, and IL-1β were decreased in the MCAO+si-RNF125 group. The results indicate that interfering with RNF125 can alleviate the inflammatory response induced by MCAO in mice.

[0191] As described above, it is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and all should be covered by the protection scope of the present invention.

Claims

1. The application of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury, characterized in that: Include one or more of the following: Based on the interference of RNF125, it is used to reduce the volume of cerebral infarction after middle cerebral artery embolization; Application of RNF125 gene knockdown in improving behavioral deficits after middle cerebral artery embolism; Based on the interference of RNF125, it can reduce neuronal loss and alleviate brain tissue damage after middle cerebral artery embolism; Application of RNF125 interference in reducing inflammatory response after middle cerebral artery embolism.

2. The use of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury according to claim 1, characterized in that: The experimental verification is carried out in the following way: S1: Establishment of MCAO model; S2: limb grip test; S3: Rotarod test; S4: Western Blot; S5: Preparation of paraffin sections of brain tissue; S6: HE staining; S7: Nissl staining; S8: Result analysis.

3. The use of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury according to claim 2, characterized in that: In S1, the establishment of the MCAO model specifically includes: S11: The mice were fasted for 12 hours and deprived of water for 6 hours before surgery, and the surgical instruments were soaked in 75% alcohol for later use; S12: After the mouse is anesthetized, fix the mouse's limbs and incisors with medical tape to keep the mouse's neck upright, fully expose the mouse's neck skin, disinfect the mouse's neck skin with iodine, and deiodinate with 75% alcohol; S13: Use small surgical scissors to make a longitudinal incision just above the trachea to expose the gland just above the mouse trachea. Use forceps to gently and bluntly separate the fascia around the gland under a stereomicroscope until the common carotid sheath that wraps the carotid artery, jugular vein and vagus nerve is exposed. S14: Gently and bluntly separate the common carotid sheath under a stereo microscope, separate the common carotid artery, and search for the external carotid artery distally. Ligate the proximal ends of the common carotid artery and the external carotid artery, and bury the distal end of the thread on the common carotid artery. S15: Under a stereo microscope, forceps gently lift the common carotid artery, cut an incision on the common carotid artery with small surgical scissors, and insert the suture through the incision until the black dot marked by the suture just passes through the bifurcation of the internal and external carotid arteries, and the suture cannot pass forward any further; S16: Under a stereomicroscope, tie a slipknot on the thread pre-buried on the common carotid artery and start the timer; S17: After 60 minutes, loosen the slipknot, pull out the thread plug, record the time of pulling out the thread plug, and use forceps to make two surgical knots to prevent slippage; S18: Suture the skin, place the modeled mice on an electric blanket until they wake up, and then keep them in an environment of 25°C; S19: 24 hours after surgery, the mice were taken out for subsequent experiments. The mice in the Sham group only had their skin cut without plugging, and subsequent experiments were performed at the same time as the MCAO mice 24 hours later.

4. The use of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury according to claim 3, characterized in that: In S1, mice were screened by neurological function scoring, as follows: ① Take out the mice after MCAO surgery and score them using Zea-Longa's 5-level 4-point standard: 0 points: no neurological deficit symptoms; 1 point: When the mouse's tail is lifted, it can be observed that the mouse's contralateral forelimb cannot be fully extended; 2 points: The mouse was placed on a level surface without obstacles, and the mouse turned its head to the opposite side while walking; 3 points: The mouse was placed on a level surface without obstacles, and it fell to the opposite side during walking; 4 points: The mouse loses its autonomous consciousness and has no autonomous activities; 5 points: mouse death; ②Mice with scores of 0 and 5 were not included in the experiment and were used as the criteria for inclusion in subsequent experiments.

5. The use of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury according to claim 4, characterized in that: In S2, the limb grip test specifically includes: S21: Lift the mouse by its tail and let its limbs grasp the metal rod of the measuring instrument, keeping the mouse's tail parallel to the metal rod; S22: Click the start button on the grip tester to start testing the grip; S23: Gently lift the mouse's tail and gradually remove the mouse's limbs from the metal rod at a constant speed; S24: Record the data displayed on the grip tester; S25: Perform the test three times continuously, with an interval of 10 minutes between each test, and calculate the average value.

6. The use of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury according to claim 5, characterized in that: In S3, the rotarod experiment specifically includes: S31: Place the mouse on a slowly rotating rotarod for 1 min. After the mouse has adapted, start the rotarod. The speed of the rotarod increases uniformly from 5 r / min, and record the time it takes for the mouse to fall from the rotarod. S32: Each mouse was exercised for 3 days before modeling, and the number of times remained consistent. After modeling, it was measured 3 times and the average value was taken. The interval between each test was 10 minutes.

7. The use of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury according to claim 6, characterized in that: In said S4, Western Blot specifically includes: S41: Prepare a lower layer of SDS-PAGE gel with a concentration of 12.5%. After solidification for 20 minutes, prepare the upper layer of gel. After the upper layer of gel solidifies for 1 hour, use it or place it in a 4°C refrigerator for later use; S42: Mix the sample and 5× loading buffer in a ratio of 4:1, boil at 99°C for 5 min, return to room temperature before use, or freeze in a -20°C refrigerator; S43: Sample loading: slowly add 20-40ug protein into the wells of the upper gel of SDS-PAGE; S44: Electrophoresis: Use 50V voltage for about 30 minutes, then adjust to 100V voltage until bromophenol blue reaches the bottom; S45: Electrotransfer: Transfer the proteins on the SDS-PAGE gel to a 0.20 μm PVDF membrane at a constant current of 250 mA for 40 min at 4°C. S46: Blocking: Use blocking solution and block at room temperature for about 30 minutes; S47: Primary antibody incubation: dilute the antibody with diluent, soak the PVDF membrane in the corresponding antibody, make sure the membrane is completely covered, and slowly shake it in a 4°C refrigerator overnight; the next day, wash the membrane with TBST three times, 10 minutes each time, for a total of 30 minutes; S48: Secondary antibody incubation: Use the secondary antibody of the corresponding species, incubate at room temperature for 1 hour; wash the membrane three times using the same method; S49: Chemiluminescence: Prepare the luminescent liquid in a 1:1 ratio and drop it onto the PVDF membrane on the wax plate, then use the instrument to automatically expose the sample; analyze the relative optical density of each well on the strip.

8. The use of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury according to claim 7, characterized in that: In S5, making paraffin sections of brain tissue specifically includes: S51: After removing the mouse from anesthesia, the chest cavity was opened and the heart was perfused; S52: perfuse with PBS first, then slowly perfuse with 4% paraformaldehyde; S53: Open the skull, carefully remove the brain tissue and fix it in 4% paraformaldehyde for at least 48 hours S54: Dehydration: Dehydration was performed using 75% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol in sequence, with each step lasting 2 hours. The brain tissue was wiped dry during the dehydration process. S55: Transparent: 100% alcohol and xylene in a 1:1 ratio, soak in it for 10 minutes, and then soak in xylene for 20 minutes; S56: Wax immersion: immerse in a solution of xylene and paraffin in a ratio of 1:1 for 30 min at 56°C, and immerse in paraffin for 3 h; S57: embedding, slicing, and drying.

9. The use of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury according to claim 8, characterized in that: In said S6, HE staining specifically includes: S61: Dewaxing: twice in xylene, 10 min each time; S62: Debenzening: wash in 100% alcohol, 95% alcohol, 80% alcohol, 70% alcohol, 3 min each, and then wash in double distilled water for 1 min; S63: Hematoxylin staining: stain in hematoxylin for 8 min, wash in double distilled water for 2 min, 1% hydrochloric acid alcohol for 10 s, wash in double distilled water for 1 min, in lithium carbonate for 2 min, and wash in double distilled water for 1 min; S64: Eosin staining: Eosin staining for 20 seconds, washing with double distilled water for 1 minute, dehydration with 75% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol in sequence; S65: Transparent: Transparent in xylene; S66: Seal the slides with neutral gum; S67: Observe the HE staining under a microscope and scan the slides.

10. The use of RNF125 as a new target in alleviating cerebral ischemia-reperfusion injury according to claim 9, characterized in that: In said S7, Nissl staining specifically comprises: S71: dewaxing and debenzening; S72: Staining: stain with tar violet for 15 min, wash with double distilled water for 1 min; S73: Separation: carried out in 95% ethanol; S74: Dehydration: Dehydrate in 70% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol; S75: Transparent: carried out in xylene; S76: Seal the slides with neutral gum; S77: Observe the Nissl staining under a microscope, scan and take slides.

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