CN115925997B - Polypeptide for intervening in treating cerebral hemorrhage and application thereof - Google Patents

Abstract

The invention discloses a polypeptide for intervening and treating cerebral hemorrhage and application thereof, wherein the polypeptide is Tat-CIRP, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 3; wherein Tat is a membrane penetrating structure sequence of 11 amino acids, and is specifically shown as SEQ ID NO. 1; CIRP is a 15 amino acid sequence on a CIRP protein sequence with highest affinity with IL-6R, and is specifically shown as SEQ ID NO. 2. The polypeptide can not only specifically target IL-6R and specifically bind IL-6R so as to competitively inhibit the formation of CIRP-IL-6R complex, but also further inhibit JAK/STAT3 mediated inflammatory reaction and inhibit the function of M1 microglial cells, thereby improving the secondary inflammatory injury after cerebral hemorrhage. The polypeptide has definite inflammation inhibition effect of cerebral hemorrhage, and provides a relatively complete experimental basis for clinical application.

Description

A polypeptide for intervention and treatment of cerebral hemorrhage and its application

Technical field

The invention belongs to the technical field of biomedicine, and specifically relates to a polypeptide for intervention and treatment of cerebral hemorrhage and its application.

Background technique

The mortality rate of patients with cerebral hemorrhage is very high. Most of the survivors have neurological dysfunction, which also brings huge material and human costs. The key mechanism of cerebral hemorrhage is still unclear. In addition to supportive treatment, there is currently a lack of accurate and effective treatment. treatment methods. Therefore, cerebral hemorrhage is not only a major clinical problem that needs to be overcome, but also a major public health problem.

Brain damage after cerebral hemorrhage is a complex pathophysiological process involving multiple genes and signaling pathways. The basic pathological changes after cerebral hemorrhage include primary damage and secondary damage (Feigin, V.L., Lawes, C.M., Bennett, D.A. and Anderson, C.S. ( 2003). Stroke epidemiology: a review of population-based studies of incidence, prevalence, and case-fatality in the late 20th century. Lancet Neurol 2, 43-53.).

Primary injuries are mainly mechanical injuries caused by tissue destruction during bleeding, hematoma expansion, and increased intracranial pressure. Secondary injury mechanisms include inflammatory response, apoptosis, necrosis, red blood cell lysis, and brain edema formation (Xi, G., Keep, R.F. and Hoff, J.T. (2006). Mechanisms of brain injury after intracerebralhaemorrhage. Lancet Neurol 5, 53-63.).

Studies have found that inflammation plays an important role in secondary damage after cerebral hemorrhage. The inflammatory mechanism mainly involves the activation of microglia, the infiltration of inflammatory cells and the release of cytokines and inflammatory chemokines, which ultimately leads to cell death and aggravates the brain's damage. The mechanisms of cerebral hemorrhage are complex and diverse, and their interrelationships are still poorly understood at this stage. Currently, effective control and reduction of inflammatory secondary damage after cerebral hemorrhage is the key to neuroprotective treatment of cerebral hemorrhage. Therefore, finding biomarkers and therapeutic targets related to inflammation in cerebral hemorrhage is the focus of research related to cerebral hemorrhage. This can not only provide more theoretical basis for the diagnosis of the severity and prognosis of cerebral hemorrhage, but also provide new information for patients with cerebral hemorrhage. diagnostic and treatment methods.

Apoptosis and necrosis are present after cerebral hemorrhage. After cell apoptosis or necrosis, a large number of cell signaling molecules are released, which can promote inflammation and are called danger-related molecules, including high mobility group box 1 (HMGB1), heat shock proteins, cold shock proteins, etc. , and increased HMGB1 and CIRP can stimulate IL-6R-like receptors to induce inflammatory responses. CIRP (cold shock protein) is a stress response protein that can be activated by various cellular stress sources and plays a role according to the specific cellular environment, that is, the specific presence of other stress response molecules. It is widely involved in various processes in the body. Physiological and pathological processes. CIRP is a new type of inflammatory mediator that can be secreted and released from the heart and liver into the circulation system in hemorrhagic shock and sepsis. It mediates the formation of IL-6R receptor complex (CIRP-IL-6R) and activates downstream JAK. -STAT3 signaling pathway upregulates the expression of TNF-α and HMGB1, plays the role of damage-associated molecular protein (DAMP), and triggers inflammatory response. Currently, CIRP has been identified as an important mediator during severe inflammation or ischemia and has the harmful function of aggravating cell damage. At the same time, studies have proven that IL-6R has become one of the most promising targets for treating inflammatory reactions. Targeted inhibition of IL-6R can effectively inhibit the inflammatory reactions induced by the IL-6R/JAK/STAT3 signaling pathway. However, there are currently no research reports on the use of peptide analogs to treat inflammatory reactions in cerebral hemorrhage.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides a polypeptide for intervention and treatment of cerebral hemorrhage and its application, providing a strategy for the clinical treatment of cerebral hemorrhage.

The present invention is achieved through the following technical solutions:

A polypeptide for intervention and treatment of cerebral hemorrhage, the polypeptide is Tat-CIRP, and its amino acid sequence is shown in SEQ ID NO.3; wherein, Tat is a transmembrane structural sequence of 11 amino acids, specifically shown in SEQ ID NO.1 ; CIRP is the 15 amino acid sequence of the CIRP protein sequence with the highest affinity for IL-6R, as shown in SEQ ID NO. 2.

Application of a polypeptide for intervention and treatment of cerebral hemorrhage in the preparation of medicine for intervention and treatment of cerebral hemorrhage.

A pharmaceutical composition for intervention and treatment of cerebral hemorrhage, including the above-mentioned polypeptide.

Preferably, a pharmaceutically acceptable carrier is also included.

The beneficial effects of the present invention are as follows:

(1) The present invention discovered for the first time that IL-6R specifically binds to one segment of CIRP, and the equilibrium dissociation constant (K _d ) of rhCIRP and rhIL-6R previously reported in the literature is 9.81×10 ^-8 M, indicating that both Has strong binding affinity. And it was confirmed that CIRP can combine with IL-6R to promote inflammatory response. This suggests that we can inhibit the pro-inflammatory response mediated by CIRP and IL-6R in microglia under inflammatory conditions. Based on the above findings, the present invention provides a new synthetic polypeptide Tat-CIRP as a drug for the intervention and treatment of cerebral hemorrhage. The amino acid fragment in the transcription transactivator protein Tat in the polypeptide drug drives the polypeptide to penetrate and enter cells. The partial sequence of CIRP The amino acid fragment can recognize IL-6R and bind to IL-6R. This polypeptide can not only competitively inhibit the formation of the CIRP-IL-6R complex by specifically targeting IL-6R and specifically binding to IL-6R, but can also further inhibit the inflammatory response mediated by JAK/STAT3 and inhibit M1 type Microglia function, thereby improving secondary inflammatory damage after intracerebral hemorrhage.

(2) The experimental results of the present invention show that treatment with the polypeptide drug can improve the spatial learning and memory abilities of mice and relieve anxiety. It is confirmed that the polypeptide Tat-CIRP has a clear inhibitory effect on inflammation in cerebral hemorrhage, and its clinical application is Provides a more complete experimental basis.

Description of drawings

Figure 1 shows the effects of polypeptide TCC on the body weight and neurobehavior of rats after intracerebral hemorrhage after collagenase IV injection into the striatum in Example 2: A is body weight, B is mNSS score, C is staggered step test, D is corner turning experiment;

Figure 2 shows the effect of the water maze detection polypeptide TCC on the learning and memory ability of rats after cerebral hemorrhage in Example 2: A is the latency to find the platform, B is the walking speed, C is the time to stay in the target quadrant, and D is the walking trajectory;

Figure 3 shows the ELISA in Example 2 to detect the TNF-α content secreted by the striatal area after intracerebral hemorrhage for 7 days after polypeptide TCC administration;

Figure 4 shows the effect of polypeptide TCC on microglia in rats after cerebral hemorrhage in Example 3: A is an immunofluorescent coronal section of the brain tissue 3 days after cerebral hemorrhage, and B is the presence of M1 type microglia/macrophages in the striatum of the brain. The distribution situation, C is the distribution situation of M2 type microglia/macrophages in the striatum of the brain;

Figure 5 shows the SPR analysis in Example 3 to detect that C22 inhibits the binding of rhCIRP and rhIL-6R: A is the binding ability of rhCIRP and rhIL-6R at different concentrations, and B is the binding ability of rhCIRP and rhIL-6R under 0 nM and 50 nM TCC.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are only used to illustrate the present invention and do not constitute a limitation on the scope of the claims. Other alternative means that those skilled in the art can think of are within the scope of the claims of the present invention.

Example 1

A polypeptide for intervention and treatment of cerebral hemorrhage, the polypeptide is Tat-CIRP (TCC); wherein Tat is a transmembrane structural sequence of 11 amino acids; CIRP is 15 amino acids on the CIRP protein sequence with the highest affinity for IL-6R sequence.

The transcription transactivator Tat can cross the membrane and enter the cell interior, and has a strong internalization effect. Tat protein generally consists of 86-102 amino acid residues, and the 47-57th amino acid fragment in the Tat protein is selected. The 47th-57th amino acid fragment is shown in SEQ ID NO.1, abbreviated as YGRKK RRQRR R, Specifically, it is Try-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg. The 47-57 amino acid fragment of the transcription transactivator Tat is located at the N-terminus of the polypeptide Tat-CIRP.

CIRP is the 15 amino acid sequence on the CIRP protein sequence with the highest affinity for IL-6R, that is, the 128-142 amino acid fragment of the CIRP protein, as shown in SEQ ID NO.2, its abbreviation is FESRS GGYGG SRDYY, specifically Phe- Glu-Ser-Arg-Ser-Gly-Gly-Tyr-Gly-Gly-Ser-Arg-Asp-Tyr-Tyr.

That is, the amino acid sequence of the polypeptide Tat-CIRP is shown in SEQ ID NO.3, abbreviated as YGRKK RRQRR RFESRSGGYG GSRDY Y, specifically Try-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Phe -Glu-Ser-Arg-Ser-Gly-Gly-Tyr-Gly-Gly-Ser-Arg-Asp-Tyr-Tyr.

In order to study whether CIRP aggravates the inflammatory response after cerebral hemorrhage by directly regulating the transformation of microglia into the "harmful" M1 type, based on the possible relationship between the CIRP content in serum and the degree of inflammation after cerebral hemorrhage, experimental studies will be carried out in vivo. (Example 2) and ex vivo (Example 3) two horizontal expansions.

Example 2

1. In vivo experiments to construct a rat cerebral hemorrhage model by injecting collagenase IV into the striatum.

The sham operation group (Sham), intracerebral hemorrhage group (ICH), and intracerebral hemorrhage plus peptide intervention group (ICH+TCC, hereinafter referred to as the peptide intervention group) were used. The peptide intervention group took Tat-CIRP (10 mg/kg) after intracerebral hemorrhage. After 2 hours of intravenous injection, the collagenase injection method was used in the cerebral hemorrhage model. The specific grouping steps are shown in Table 1 below.

Table 1 Experimental grouping

The surgical operation is as follows: After the rats are weighed, the rats are anesthetized by intraperitoneal injection of 10% chloral hydrate solution (400 mg/kg), and the stereotaxic instrument is adjusted to ensure that the working surface is level. Fix the anesthetized rat in the prone position on the stereotaxic instrument. Adjust the stereotaxic instrument so that the anterior and posterior fontanelles are basically on the same plane. Remove the hair on the top of the head and disinfect it with 75% alcohol. Make a cut with a length of about 1cm in the center of the skull. A longitudinal incision is made to expose the skull, anterior fontanelle, and posterior fontanel, and confirm again that the anterior and posterior fontanelles are in the same plane. At 0.2 mm in front of the bregma and 3.5 mm to the right of the midline, drill a circular hole with a diameter of about 1.0 mm in the skull using a micro electric drill, taking care to avoid damaging the dura mater. Quickly absorb 0.25mU/kg collagenase IV with a microsyringe and fix it on the stereotaxic instrument. Insert the needle vertically along the drilling direction from the surface of the dura mater to a depth of 5.5mm. The microsyringe was connected to the automatic injector and injected at a speed of 0.4 μL/min. After the injection, leave the needle for 10 minutes, slowly withdraw the microsyringe, seal the hole with bone wax, and suture the skin incision. The sham operation group was injected with normal saline. Wait for the animals to wake up in a warm and ventilated environment, then send them to a constant temperature (22°C) SPF room, provide normal food and water, and maintain 12h/d light. Check the body movements, such as turning in circles to the right, which is considered as the model preparation is successful.

2. Observation indicators

(1) General status observation: Observation time and frequency: Observe once a day at a fixed time after administration; Observation results: Overall status of the subject animal.

(2) Modified Neurological Severity Score (mNSS)

Neurological function measurements were performed using the mNSS test. The rats in each group were tested before hemorrhage and 1, 3, 5, 7, 14, 21, and 28 days after hemorrhage. mNSS is a combination of motor (muscle status, abnormal movements), sensory (visual, tactile and proprioceptive) and reflex tests. Neurologic function was graded on a scale of 0 to 18 (normal score 0; maximum deficit score 18). In the impairment severity score, 1 point is awarded for each abnormal behavior or lack of test reflex; 13-18 points indicate severe impairment; 7-12 points indicate moderate impairment; 1-6 points indicate mild impairment; therefore, The higher the score, the more severe the injury. The neurological impairment severity score is scored on an 18-point scale as shown in Table 2 below.

Table 2 mNSS scoring criteria

(3) Misstep experiment

To assess sensorimotor function, a staggered test was performed before hemorrhage and 1, 3, 5, 7, 14, and 28 days after hemorrhage. Let the rat walk on the grid. With each step, the forelimb or hindlimb may fall or slide between the grids, and if this occurs, it is recorded as a wrong step. Choose a large grid of 50L×40W and place it at a height of about 100cm. The rats were pre-trained for 3 days. During the formal test, the total number of steps the rats walked within 1 minute and the number of empty steps of the forelimbs and hindlimbs were recorded.

Wrong step rate = number of wrong steps/total number of steps × 100%

(4) Corner turning experiment

The installation consists of two walls connected at a sharp angle of 30°, where there is a small opening so that the curiosity of the animals drives them into the corner. The corner turning device consists of two walls forming a 30° angle and a 5mm gap at the apex of the triangular tip. Once the animal entered the corner, attempts to turn to the damaged or unimpaired side of the body were recorded. Place the open edge of the device near the edge of the table and allow the rodent to advance into the corner. A complete turn means that the body position is 180° from the starting point. Let the rodent advance into the corner. When the animal moves its body from side to side, rotation to the left or right should be achieved by placing one or both front paws against the wall. Allow 30 s between trials to avoid the animal developing an aversion to corners. Repeat the test 10 to 15 times and record the number of right (R) or left (L) turns.

CT score=[(R)/(R+L)]×100%

(5)Experimental results

After collagenase IV is injected into the striatum, it can degrade the proteolytic enzymes of the basement membrane and interstitial collagen, thereby inducing cerebral hemorrhage. After cerebral hemorrhage, the hematoma will compress the surrounding nerve tissue and affect the weight of the rat (A in Figure 1) and associated sensorimotor behaviors. The mNSS score (B in Figure 1), staggered step test (C in Figure 1) and corner turning test (D in Figure 1) can detect the sensorimotor function of rats.

As shown in Figure 1, the peptide Tat-CIRP (TCC) was treated by intraperitoneal injection 2 hours after cerebral hemorrhage. The staggered test and mNSS score indicators were improved on the 5th and 7th days, and the difference was statistically significant (P<0.05).

Therefore, this experiment concluded that peptide administration can significantly improve the sensorimotor function of rats after cerebral hemorrhage in the short term.

3. Water maze experiment

The water maze experiment mainly tests the cognitive functions of rats, including spatial learning ability, memory ability and spatial exploration ability. Therefore, a water maze was used to detect whether the learning and memory abilities of rats were improved after peptide administration.

Before the start of the experiment, adjust the water temperature in the maze to 25±1°C and maintain it. The room temperature is kept at 25°C. The water surface is 1cm higher than the platform. The environment is kept quiet. The animals are brought to the test room to adapt to the environment. Training started three days before the operation, and the animals were placed from each quadrant, and the time and activity track when they found the platform were recorded. If the platform was not found within 60 s, the incubation period was recorded as 60 s. At the end of each swimming session, the animals were placed on the platform for 10 s. Training began for three consecutive days before surgery, and the latency to find the platform was recorded. On the last day, remove the platform, place the animal from the opposite corner of the platform, and record the time the animal stays in the target quadrant within 1 minute. After each test, dry the animal with a towel and keep it warm.

As shown in Figure 2, on days 21 and 22, the latency of rats in the peptide intervention group to find the platform was different from that in the cerebral hemorrhage group (A in Figure 2), and the difference was statistically significant (P<0.05); in the water maze There was no difference in the mid-middle swimming speed between the peptide intervention group and the cerebral hemorrhage group (B in Figure 2); however, the peptide intervention group stayed longer in the target quadrant than the cerebral hemorrhage group (C in Figure 2). The difference is statistically significant (P<0.05); and it can be seen from the diagram of the rats swimming in the water (D in Figure 2) that after the platform was removed, the peptide intervention group had more trajectories in the target quadrant, indicating that the peptide intervention group had more trajectories in the target quadrant. Can significantly improve the memory function of rats. It can be seen that TCC intervention can effectively improve learning and memory dysfunction caused by cerebral hemorrhage.

4. ELISA quantitative detection

(1) Sample collection and storage

The striatum on the side of rat cerebral hemorrhage was washed in pre-cooled PBS (0.02mol/L, pH=7.0~7.2) to remove blood, weighed and used for later use (larger tissue pieces need to be chopped first and then homogenized). Multiple homogenization methods can be used at the same time to achieve better crushing effects: First, move the tissue block into the glass homogenizer, add 5 to 10 mL of pre-cooled PBS (the mass-volume ratio of tissue to PBS is recommended to be 1:5, that is, 1g of sample is added 5mL PBS) for full grinding (machine homogenization can be used in laboratories with conditions). This process needs to be carried out on ice; the obtained homogenate can be further processed by ultrasonic crushing or repeated freezing and thawing (pay attention to the cooling of the ice bath during the ultrasonic crushing process. ; Repeated freezing and thawing method can be repeated 2 times). Centrifuge the prepared homogenate at 5000 × g for 5 minutes, and keep the supernatant for detection.

(2) Reagent preparation

All reagents and samples need to be returned to room temperature (25±3°C) before testing.

If crystals appear in the concentrated reagent, warm the solution at 37°C until all crystals are dissolved.

①1×Lotion

Pipette 50 mL of 20× concentrated lotion into a 1L graduated cylinder, add distilled water to 1000 mL, and mix gently to avoid foaming. Transfer to a clean bottle. Store at 2～25℃, 1× lotion can be stored stably for 30 days.

②1×detection buffer

Pipette 5 mL of 10× concentrated detection buffer into a 100 mL graduated cylinder, add distilled water to 50 mL, and mix gently to avoid foaming. Store at 2-8℃, 1× detection buffer can be stored stably for 30 days.

③Detection of antibodies

Mix thoroughly before diluting. Depending on the number of standards and samples to be tested, dilute the concentrated detection antibody 1:100 with 1× detection buffer.

Note: The diluted detection antibody needs to be used within 30 minutes.

④ Horseradish peroxidase-labeled streptavidin

Mix thoroughly before diluting. Depending on the number of standards and samples to be tested, dilute the concentrated horseradish peroxidase-labeled streptavidin 1:100 with 1× detection buffer.

Note: Diluted horseradish peroxidase-labeled streptavidin needs to be used within 30 minutes.

⑤Signal enhancer concentrate

Mix thoroughly before diluting. Dilute the concentrated signal enhancer 1:100 with signal enhancer diluent according to the quantity of standards and samples to be tested.

Note: The diluted signal enhancer needs to be used within 30 minutes.

⑥Rat TNF-α standard

Centrifuge briefly before opening the cap, and redissolve the rat TNF-α standard in distilled water. The re-dissolved volume is marked on the label of the rat TNF-α standard. Vortex gently to ensure thorough mixing. The concentration of the standard after redissolution is 1000pg/mL. After reconstitution, let it sit for 10 to 30 minutes. Mix thoroughly before diluting.

⑦ Preparation of tissue standard curve

Take 230 μL of concentrated rat TNF-α standard and add 230 μL of standard diluent as the highest concentration of the standard curve (500 pg/mL). Add 230 μL of standard diluent to each test tube. Use high concentration standards to make 1:1 serial dilutions. Make sure to mix thoroughly each time you pipet. Use the standard dilution as the zero concentration of the standard curve.

(3)Detection steps

All reagents and samples need to be equilibrated to room temperature (25±3°C) before detection.

① Prepare all required reagents and working concentration standards.

② Remove the unnecessary slats, put them back into the aluminum foil bag containing the desiccant, and reseal them.

③Soak the enzyme plate: add 300 μL of 1× washing solution and let soak for 30 seconds. After discarding the wash solution, pat the microplate dry on absorbent paper. After washing the plate, use the microplate immediately and do not let the microplate dry.

④Add standard: Add 100 μL of 2-fold diluted standard to the standard hole. Add 100 μL of standard diluent (serum/plasma sample) to the blank well.

⑤Add sample: serum/plasma: Add 80μL 1× detection buffer and 20μL sample to the sample hole.

Ensure that steps ④ and ⑤ are added continuously without interruption. The sample addition process is completed within 15 minutes.

⑥Incubation: Use sealing film to seal the plate. Shake at 100~300r/min and incubate at room temperature (25±3℃) for 1.5h.

⑦ Washing: Discard the liquid, add 300 μL washing solution to each well to wash the plate, and wash 6 times. After each wash, pat dry on absorbent paper. To obtain ideal experimental performance, residual liquid must be completely removed.

⑧Add detection antibody: Add 100 μL of diluted detection antibody to each well (1:100 dilution). Seal the plate with sealing film. Shake at 100~300r/min and incubate at room temperature (25±3℃) for 30min.

⑨ Washing: Repeat step ⑦.

⑩Enzyme addition and incubation: Add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well. Seal the plate with new sealing film. Shake at 100~300r/min and incubate at room temperature (25±3℃) for 30min.

Washing: Repeat step ⑦.

Add signal enhancer for incubation: add 100 μL of diluted signal enhancer (1:100 dilution) to each well. Seal the plate with new sealing film. Shake at 100~300r/min and incubate at room temperature (25±3°C) for 15 minutes.

Washing: Repeat step ⑦.

Add enzyme and incubate again: add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well. Seal the plate with new sealing film. Shake at 100~300r/min and incubate at room temperature (25±3°C) for 15 minutes.

Washing: Repeat step ⑦.

Add substrate for color development: Add 100 μL of color development substrate TMB to each well, protect from light, and incubate at room temperature (25±3°C) for 5 to 30 minutes.

Add stop solution: Add 100 μL of stop solution to each well. The color changes from blue to yellow. If the color appears green or the color change is obviously uneven, tap the frame lightly to mix thoroughly.

Detection reading: Within 30 minutes, use a microplate reader to perform dual-wavelength detection and measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 570nm or 630nm. The calibrated OD value is the measured value at 450nm minus the measured value at 570nm or 630nm. Using only 450nm measurement will result in high OD values and reduced accuracy.

(4) Result calculation

Calculate the average OD value of the standards and samples, then subtract the OD value of the zero concentration standard.

Taking the standard concentration as the abscissa and the OD value as the ordinate, use computer software to perform regression fitting to generate a standard curve. Regression analysis determines the best-fit curve.

The standard curve can be linearized by taking a logarithmic fit to the concentration and OD values. This process may be able to obtain the concentrations of more samples, but the accuracy of the data will be reduced.

As shown in Figure 3, ELISA quantitatively detected an increase in the expression of TNF-α in the striatum area on the bleeding side, and peptide administration could improve the inflammatory response in the cerebral hemorrhage hemisphere and reduce the expression of TNF-α, and the difference was statistically significant (P <0.05). ELISA testing showed that peptide administration exerted a neuroprotective effect on rats with cerebral hemorrhage and reduced their inflammatory response.

5. Tat-CIRP-CMA can regulate the transformation of rat microglia to M1/M2 type after cerebral hemorrhage.

After the intracerebral hemorrhage model was established in rats, the rats were intraperitoneally injected with peptides for treatment. The intracerebral hemorrhage group was injected with an equal amount of normal saline as a control. The brains were harvested by perfusion 3 days after intracerebral hemorrhage, dehydrated with sucrose, frozen sections, and subjected to immunohistofluorescence.

Tissue immunofluorescence: After the tissue is frozen and sectioned, float it onto a glass slide and leave it at room temperature overnight. The next day, rupture the membrane with PBST (PBS+Trition), wash 3 times for 15 minutes each time, block with 5% DS for 2 hours, and use primary antibody 4 Incubate at ℃ for 15 hours or overnight. The next day, rewarm the slices incubated with primary antibodies at room temperature for 0.5 hours, PBST (PBS+Tween-20), wash 15 minutes each time, wash 3 times, incubate with secondary antibodies in the dark for 2 hours, PBST (PBS+ Tween-20) wash for 15 minutes each time, wash 3 times, mount the slide with DAPI-containing mounting medium, fix it with nail polish, confirm the fluorescence signal under a fluorescence microscope and take pictures.

As shown in Figure 4, the phagocytic ability of M1 type microglia is significantly lower than that of M0 type (unstimulated normal microglia), while the phagocytic ability of M2 type is 2-3 times higher than that of M1 type (A in Figure 4 ). Three days after cerebral ischemia in rats, it was observed that treatment with Tat-CIRP (TCC) could significantly reduce the production of M1 type microglia in the brain compared with the cerebral hemorrhage group (B in Figure 4). This experiment demonstrates that the peptide exerts a neuroprotective effect.

6 Conclusion

Behavioral testing The impact of the polypeptide intervention group on the neurobehavioral prognosis of rats was mainly reflected in the improvement of limb movement disorders and the improvement of learning and memory abilities in rats. The expression of TNF-α in the striatum area on the bleeding side was quantitatively detected by ELISA, and peptide administration could improve the inflammatory response in the cerebral hemorrhage hemisphere.

Example 3

This example studies whether interfering or reducing the biological function of CIRP/IL-6R after cerebral hemorrhage can effectively inhibit the IL-6RJAK/STAT3 signaling pathway and thereby inhibit the cascade effect of inflammatory mediators and reduce secondary damage to the central nervous system. of destruction.

1. SPR analysis

This experiment uses surface plasmon resonance (SPR) analysis to determine whether different concentrations of TCC will destroy the binding of extracellular CIRP to IL-6Rα, as well as to detect rhCIRP (human CIRP, equivalent to extracellular CIRP) and Whether IL-6R and the synthetic peptide TCC can block the binding ability of CIRP and IL-6R in the body.

Biacore analysis of IL-6Rα-CIRP and C22-IL-6Rα-CIRP interactions was performed on a Biacore 3000 instrument (GE Healthcare) using SPR technology. Binding reactions were performed in 1×HBS EP (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% P20, pH=7.4). The CM5 dextran chip (flow cell 2) was first injected with 89 μL of 0.1 M N-ethyl-N'-[3-diethylamino-propyl]-carbodiimide and 0.1 M N-hydroxysuccinimide. activation. For ligand immobilization, recombinant human (rh) IL-6Rα protein (R&D) diluted at 5 μg/mL in 10 mM sodium acetate (pH=4.5) was injected into flow cell 2 of the CM5 chip in a volume of 200 μL. Next, the remaining active sites were blocked by injecting 135 μL of 1 M ethanolamine (pH=8.2). To assess non-specific binding, flow cell 1 not coated with rhIL-6Rα protein was used as a control. Binding assays were performed using a flow rate of 30 μL/min at 25°C. To assess binding, analyte rhCIRP protein (OriGene) (500 nM rhCIRP for yes or no binding assay, or range 62.5 to 500 nM rhCIRP with or without 50 μM C22 for kinetic analysis) was injected into flow cells-1 and Flow cell-2 and record the association of analyte and ligand in the presence or absence of C22. The blank channel (flow cell-1) signal was subtracted from the ligand-coated channel (flow cell-2). Data were analyzed by Biacore 3000 evaluation software. Data were globally fit to a Langmuir model to achieve 1:1 binding.

As shown in Figure 5, rhCIRP binds to IL-6Rα with high affinity, and the equilibrium dissociation constant K _d value is 8.08×10 ^-8 M (A in Figure 5). At a concentration of 50 μM, TCC completely abolished the binding of eCIRP to IL-6Rα (B in Figure 5 ). This shows that TCC can inhibit the direct binding of CIRP to IL-6Rα.

2. Conclusion

In vitro experiments first used SPR analysis to detect rhCIRP (human CIRP) and IL-6R as well as the synthetic peptide TCC. It was found that the peptide TCC can block the binding of CIRP and IL-6R in the body. Then, in the rat cerebral hemorrhage model, Tat-CIRP (TCC) peptide treatment was given (specifically down-regulates the endogenous CIRP effect), and the impact of the peptide intervention group on the neurobehavioral prognosis of rats was tested behaviorally, which was mainly manifested in rats. Improvement of physical mobility impairment and improvement of learning and memory abilities. The expression of TNF-α in the striatum area on the bleeding side was quantitatively detected by ELISA, and peptide administration could improve the inflammatory response in the cerebral hemorrhage hemisphere. By detecting the ratio of M1 type microglia to M2 type microglia in rat coronal brain slices by immunohistofluorescence, it was confirmed that peptide intervention in the content of endogenous CIRP can effectively inhibit the inflammatory response and promote M1 type microglia. Transform to M2 type and improve the prognosis of cerebral hemorrhage.

Claims (4)

1. A polypeptide for the intervention and treatment of cerebral hemorrhage, characterized in that the polypeptide is Tat-CIRP, and its amino acid sequence is as shown in SEQ ID NO. 3; wherein, Tat is a transmembrane structural sequence of 11 amino acids, specifically as follows As shown in SEQ ID NO.1; CIRP is the 15 amino acid sequence of the CIRP protein sequence with the highest affinity to IL-6R, specifically as shown in SEQ ID NO.2. 2. Application of a polypeptide for the intervention and treatment of cerebral hemorrhage as claimed in claim 1 in the preparation of a drug for the intervention and treatment of cerebral hemorrhage. 3. A pharmaceutical composition for intervention and treatment of cerebral hemorrhage, characterized by comprising the polypeptide of claim 1. 4. A pharmaceutical composition for intervention and treatment of cerebral hemorrhage according to claim 3, further comprising a pharmaceutically acceptable carrier.