Application of protein UCN2 in relieving and treating spinal cord injury

Abstract

The invention provides application of protein UCN2 in relieving and treating spinal cord injury. The invention provides a novel application of UCN2 as an active ingredient in treatment of spinal cord injury. Experiments prove that the UCN2 can activate a cAMP-PKA signal channel, inhibit neuronal apoptosis, promote astrocytes to secrete GDNF, enhance conversion from A1 type astrocytes to A2 type astrocytes, inhibit microglial cells to secrete NF-kappa B and relieve inflammatory response, so that the purposes of promoting protrusion regeneration, promoting apoptosis and the like are achieved. Therefore, the UCN2 can be used as a medicine for repairing the spinal cord injury, and the UCN2 can be used as a medicine for repairing the spinal cord injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of protein UCN2 in alleviating and treating spinal cord injury. Background Art

[0002] Spinal cord injury (SCI) refers to damage to the spinal cord or cauda equina caused by spinal fracture or dislocation. Spinal cord injury is a serious injury to the central nervous system that usually leads to permanent neurological loss and severe disability in patients. It has the characteristics of high morbidity, high treatment costs, high disability rate and low age of onset, which will cause huge physical, psychological and economic burdens on patients and their families. After spinal cord injury, it will go through acute, subacute and chronic phases in sequence. In the acute phase of spinal cord injury, primary injury will cause damage to the blood-spinal cord barrier, and a large number of inflammatory factors will flow into the spinal cord, aggravating spinal cord swelling. In the chronic phase of spinal cord injury, microglia gather around the injury and continue to secrete inflammatory factors, which also leads to the continuous transformation of astrocytes into neurotoxic A1 astrocytes, accelerating neuronal apoptosis.

[0003] Due to the complexity of the pathological process of spinal cord injury, there is currently no widely recognized treatment plan. The main clinical treatment methods currently include oral glucocorticoids, surgical treatment and stem cell transplantation. Methylprednisolone is the only drug approved by the FDA to reduce secondary injury, but a large amount of clinical evidence shows that its side effects are significant, and it is recommended to be used within 8 hours after injury; surgical treatment can cause secondary injury, while stem cell transplantation is not effective, and the risk of tumorigenesis and immune response related to the later stage cannot be avoided. This leads to a situation where no effective drugs are available when spinal cord injury reaches the chronic stage. Therefore, there is an urgent need to explore therapeutic drugs for the chronic stage of spinal cord injury. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a medicine for alleviating and treating the chronic stage of spinal cord injury.

[0005] In a first aspect, the present invention provides use of protein UCN2 or a derivative thereof in the preparation of a medicament for alleviating and / or treating spinal cord injury in a subject, wherein the amino acid sequence of the protein UCN2 is shown in SEQ ID NO:1.

[0006] Furthermore, the subject is in a chronic stage of spinal cord injury.

[0007] Furthermore, the subject is a mammal.

[0008] Furthermore, the protein UCN2 alleviates and / or treats spinal cord injury in a subject by promoting neuronal process regeneration and inhibiting neuronal apoptosis.

[0009] Furthermore, the derivative of the protein UCN2 is obtained by modifying the protein UCN2 by at least one of pegylation, glycosylation, lipidation, protein fusion, antibody coupling, site mutation, supercharging, and introduction of non-natural amino acids.

[0010] Furthermore, the drug has a pharmaceutically acceptable dosage form.

[0011] Furthermore, the medicine is an injection.

[0012] The second aspect of the present invention provides a pharmaceutical composition, comprising protein UCN2 or a derivative thereof and a pharmaceutically acceptable carrier, wherein the amino acid sequence of the protein UCN2 is shown in SEQ ID NO:1.

[0013] Furthermore, the pharmaceutical composition has a pharmaceutically acceptable dosage form.

[0014] Furthermore, the pharmaceutical composition is an injection.

[0015] The present invention provides a new use of protein UCN2 as an active ingredient in the treatment of spinal cord injury. Experiments have confirmed that UCN2 can activate the cAMP-PKA signaling pathway, inhibit neuronal apoptosis, promote astrocytes to secrete GDNF, enhance the transformation of A1 astrocytes to A2 astrocytes, inhibit microglia from secreting NF-κB, and relieve inflammatory responses, thereby promoting protrusion regeneration and accelerating the recovery of hind limb motor ability in rats with chronic spinal cord injury, indicating that UCN2 can be used as a chronic spinal cord injury repair drug. The present invention shows through animal experiments that UCN2 can accelerate the recovery of hind limb motor ability in rats with chronic spinal cord injury, and can be used as a recovery drug for the chronic spinal cord injury, which supplements the current situation where there are no effective drugs available for the chronic spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of neuronal drug administration test; A is the regeneration of peripheral neuronal processes in the injured area, B is the apoptosis of peripheral neuronal processes in the injured area, C is the mRNA expression of PKA, CREB, RhoA, Bcl-2, and Bax in the cAMP-PKA signaling pathway, and D is the protein expression and protein phosphorylation of PKA, CREB, RhoA, Bcl-2, and Bax in the cAMP-PKA signaling pathway;

[0017] Figure 2The results of the neuron-astrocyte co-culture drug administration test; A is the regeneration of peripheral neuronal processes in the injured area, B is the apoptosis of peripheral neurons in the injured area, C is the mRNA expression of PKA and GDNF, and D is the protein expression and protein phosphorylation of PKA and GDNF;

[0018] Figure 3 The results of the neuron-microglia co-culture drug administration test; A is the regeneration of peripheral neuronal processes in the injured area, B is the apoptosis of peripheral neurons in the injured area, C is the mRNA expression of PKA and NF-κB, and D is the protein expression and protein phosphorylation of PKA and NF-κB;

[0019] Figure 4 is the BBB score of rats in each group 14 days after injury;

[0020] Figure 5 The gait analysis results of rats in each group 14 days after injury;

[0021] Figure 6 The test results of cAMP-PKA signaling pathway in the spinal cord tissue of rats in each group; A is the mRNA expression of CRHR2, PKA, NF-κB, CREB, RhoA, Bcl-2, Bax, and GDNF in the cAMP-PKA signaling pathway; B is the protein expression and protein phosphorylation of CRHR2, PKA, NF-κB, CREB, RhoA, Bcl-2, Bax, and GDNF in the cAMP-PKA signaling pathway;

[0022] Figure 7 The effects of knocking down CRHR2 on the functions of various types of nerve cells around spinal cord injury; A is the co-staining of neurons and Tunel around the injured area of ​​rats in each group; B is the expression of neurofilaments around the injured area of ​​rats in each group; C is the expression of microglia around the injured area of ​​rats in each group; D is the number of A2 astrocytes expressed around the injured area of ​​rats in each group; E is the number of A1 astrocytes expressed around the injured area of ​​rats in each group. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The following examples do not limit the present invention in any form. The raw materials and equipment used in the specific embodiments of the present invention are all known products. Unless otherwise specified, the reagents and materials used in the following examples are obtained by purchasing commercial products.

[0025] The protein Recombinant Urocortin 2 (UCN2) used in the following examples was purchased from Abmart with the product number RKRC58551, and the amino acid sequence is shown in SEQ ID NO:1.

[0026] The nucleotide sequences of the primers used in the following examples are shown in Table 1.

[0027] Table 1 Nucleotide sequences of primers involved in the present invention

[0028]

[0029] The primary antibodies used in the following examples include Anti-Phospho-PKAC (1:1000, 4781S, Cell Signaling Technology), Anti-PKAC (1:1000, 4782S, Cell Signaling Technology), Anti-Phospho-NF-κB (1:1000, 3033S, Cell Signaling Technology), Anti-NF-κB (1:1000, 8242T, Cell Signaling Technology), Technology), Anti-Phospho-CREB(S133)(1:750,GB114684-100,Servicebio), Anti-CREB(1:750,GB11539-100,Servicebio), Anti-Bcl-2(1:750,GB154380-50,Servicebio), Anti-B ax(1:750,GB15690-50,Servicebio), Anti-RhoA(1:750,GB115176-50,Servicebio), Anti-GDNF(1:750,GB11403-50,Servicebio) and Anti-Actin(1:1000,GB113225-100,Servicebio). Secondary antibodies included Anti-rabbit IgG (1:2000, 7074S, Cell Signaling Technology) and Anti-mouse IgG (1:2000, 7076S, Cell Signaling Technology).

[0030] Example 1: In vitro cell experiment

[0031] In this embodiment, primary spinal neurons, primary astrocytes and primary microglia were extracted from SD fetal mice, and primary spinal neurons were cultured alone, primary spinal neurons-primary astrocytes, and primary spinal neurons-primary microglia were co-cultured. Mechanical injury was simulated by a scratch model, and UCN2 was added for treatment. Immunofluorescence, qPCR, western blot and other experiments were performed after 48 hours to analyze the effects of UCN2 on the apoptosis of neurons and the growth of processes, and to explore the effect of UCN2 on spinal cord injury.

[0032] 1. Primary spinal cord neuron culture

[0033] 1.1. Extraction and administration of primary spinal cord neurons

[0034] Spinal cords were dissected from embryos of 13-day-old SD rats (SD rats were purchased from the Experimental Animal Center of Shanghai First People's Hospital) and digested with 0.05% trypsin and DNase (0.1 mg / mL). An equal volume of complete medium was then added and centrifuged at 1000 rpm, 4°C for 5 min. Cells were collected and resuspended in Neurobasal medium containing 2% N2, 2% B27, 0.5 mM glutamine, and 1% Pen / Strep and cultured at 3 × 10 6 The cells were seeded at a density of 100 μg / well on poly-lysine-coated 6-well plates.

[0035] After the cells differentiated into primary spinal cord neurons, the primary spinal cord neurons were divided into the Control group, Scratch group, Scratch+UCN2 group and Scratch+UCN2+H89 group. The Control group was not treated after inoculation as required; the Scratch group was scratched with a 200μl gun tip to simulate mechanical damage, 3 times horizontally and 3 times vertically; 100nM UCN2 was added to the Scratch+UCN2 group after scratching; 30μM PKA inhibitor H89 was added to the Scratch+UCN2+H89 group after 48 hours of cell culture, and 1 hour after culture, the cells were scratched with a 200μl gun tip to simulate mechanical damage, 3 times horizontally and 3 times vertically, and fresh culture medium containing 100nMUCN2 and 30μM H89 was added again. After 48 hours of cell culture in each group, immunofluorescence, qPCR, and western blot experiments were performed for analysis.

[0036] 1.2 Immunofluorescence experiment:

[0037] The cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, permeabilized with 0.3% Triton 100X (Sevier, Wuhan, China) for 10 min, incubated with blocking solution (Biyuntian, Shanghai, China) for 1 h, and incubated with anti-β-tubulin III antibody (1:100, T2200, Sigma-Aldrich) at 4°C overnight; on the next day, the cells were incubated with Goat anti-Rabbit IgG 488 (1:500, A-11035, Invitrogen) at 37°C for 2 h, and images were observed using a fluorescence microscope (Olympus, Japan) and analyzed using ImageJ software.

[0038] The results are as follows Figure 1 As shown in A, it can be seen that compared with the control group, the number of neuronal processes around the injury in the Scratch group was significantly reduced, compared with the Scratch group, the number of neuronal processes around the injury in the Scratch+UCN2 group was significantly increased, and compared with Scratch+UCN2, the number of neuronal processes around the injury in the Scratch+UCN2+H89 group was significantly reduced, indicating that UCN2 can promote the growth of neuronal processes.

[0039] 1.3 Tunel:

[0040] Fix the cells with 4% paraformaldehyde for 10 minutes. Follow the protocol of Fluorescein (FITC) Tunel Cell Apoptosis Detection Kit (G1501-50T, Servicebio) and permeabilize with proteinase K solution at room temperature for 10 minutes. Wash with PBS solution for 5 minutes, repeat 3 times, block with immunostaining blocking solution at room temperature for 1 hour, then incubate with primary antibody Anti-β-Tubulin III antibody (1:100, T2200, Sigma-Aldrich) at 4°C overnight, and wash the cells with PBS the next day. Prepare TdT incubation buffer according to the kit requirements, add Goat anti-Rabbit IgG 488 (1:500, A-11010, Invitrogen), and evenly add 50 μL of the reaction mixture to each tissue section and incubate at 37°C for 1 hour. Wash the slides with PBS solution for 5 minutes, repeat 3 times. Stain with DAPI and seal the slides and observe under a microscope.

[0041] Staining results and data analysis results are shown in Figure 1As shown in B, the content of Tunel in the Scratch group (model) was significantly higher than that in the Control group, while that in the Scratch+UCN2 group was comparable to that in the Control group, and that in the Scratch+UCN2+H89 group was significantly higher.

[0042] 1.4. Analysis of gene mRNA expression in primary spinal cord neurons:

[0043] Total RNA was extracted from cells using Trizol reagent, 4 μl 5x RT SuperMix was added to 1000 ng RNA, ddH2O was added to 20 μl, and the mixture was heated at 45°C for 15 min, followed by heating at 85°C for 5 seconds for RNA reverse transcription. The PCR reaction system included 1 μl extracted RNA, 10 μl SYBR Green Master Mix, 0.4 μl each of the front primer and the rear primer, and the final reaction volume was 20 μl. The reverse transcription reaction was performed using the QuantStudio 6Flex real-time fluorescence PCR system, first pre-denaturing at 95°C for 30 s, then denaturing at 95°C for 10 s, annealing and extending for 30 s, using GAPDH as the internal reference, and using 2- ΔΔt The relative expression of mRNA of each gene was calculated by the primer sequence shown in Table 1. Figure 1 As shown in C, the mRNA expression levels of PKA, CREB and Bcl-2 in the Scratch+UCN2 group were increased, and the mRNA expression levels of RhoA and Bax were decreased compared with those in the Scratch group and the Scratch+UCN2+H89 group.

[0044] 1.5. Western blot analysis:

[0045] Remove the culture medium, use a cell scraper to hang the cells, add 1ml RIPA, 10μl protease inhibitors and 10μl phosphatase inhibitors to the cell sample, and sonicate on ice, then centrifuge at 4℃, 12000rpm for 15min, and collect the supernatant. Use the BCA method to determine the protein concentration, dissolve the protein sample in the sample buffer, and heat it at 100℃ for 5min, then perform SDS-PAGE electrophoresis on the sample and transfer it to a PVDF membrane. After blocking with 5% skim milk powder, the primary antibodies were added and incubated overnight at 4°C. The primary antibodies included Anti-Phospho-PKA C (1:1000, 4781S, Cell Signaling Technology), Anti-PKA C (1:1000, 4782S, Cell Signaling Technology), Anti-Phospho-CREB (S133) (1:750, GB114684-100, Servicebio), Anti-CREB (1:750, GB11539-100, Servicebio), Anti-Bcl-2 (1:750, GB154380-50, Servicebio), Anti-RhoA (1:750, GB115176-50, Servicebio) and Anti-Actin (1:1000, GB113225-100, Servicebio). After washing with TBST, the secondary antibody Anti-rabbit IgG (1:2000, 7074S, Cell Signaling Technology), Anti-mouse IgG (1:2000, 7076S, Cell Signaling Technology), followed by optical density analysis.

[0046] The results are as follows Figure 1 As shown in D, the protein expression and phosphorylation of PKA in the Scratch+UCN2 group were increased compared with those in the Scratch group. The protein expression and phosphorylation of CREB and Bcl-2 in the Scratch+UCN2 group were increased compared with those in the Scratch group and Scratch+UCN2+H89 group, and the protein expression and phosphorylation of RhoA and Bax were decreased.

[0047] The above results indicate that UCN2 can promote neurite outgrowth and reduce neuronal apoptosis by activating the cAMP-PKA signaling pathway in neurons.

[0048] 2. Co-culture of primary spinal cord neurons and primary astrocytes

[0049] 2.1. Extraction of primary astrocytes

[0050] Primary astrocytes were extracted from 15-day-old SD rat embryos, digested with 0.05% trypsin and DNase (0.1 mg / mL), and then added with an equal volume of complete medium and centrifuged at 1000 rpm and 4°C for 5 min. After centrifugation, the cells were resuspended in DMEM medium containing 10% FBS, 1% Pen / Strep, and 0.5 mM glutamine and cultured at 3 × 10 6 / well was inoculated in a poly-lysine-coated culture dish and cultured for 48h.

[0051] Primary spinal cord neurons were extracted according to the steps in 1.1, cultured on coverlips for 48 h, and then added to primary astrocytes and co-culture medium, which consisted of Neurobasal medium supplemented with 2% B27 supplement and 1x glutamine.

[0052] Treat the cells according to the method in 1.1, scratch the neurons and astrocytes with a 200 μl pipette tip, and the subsequent drug administration and result detection methods are the same as 1.1.

[0053] The cells were tested according to the method shown in 1.2-1.5. The test results were as follows Figure 2 As shown. Figure 2 As shown in A, compared with the control group, the number of neurons around the injury in the Scratch group was significantly reduced, compared with the Scratch group, the number of neurons around the injury in the Scratch+UCN2 group was significantly increased, and compared with the Scratch+UCN2 group, the number of neurons around the injury in the Scratch+UCN2+H89 group was significantly reduced. Figure 2 As shown in Figure B, the content of Tunel in the model group was significantly higher than that in the control group, while that in the model+UCN2 group was equivalent to that in the control group, and that in the model+UCN2+H89 group was significantly higher. Figure 2 As shown in C, the mRNA expression of PKA and GDNF in the Scratch+UCN2 group was higher than that in the Scratch group and the Scratch+UCN2+H89 group. Figure 2 As shown in D, the protein expression and protein phosphorylation of PKA and GDNF in the Scratch+UCN2 group were higher than those in the Scratch group.

[0054] In summary, through neuron-microglia co-culture, it was found that UCN2 promoted the release of GDNF by activating the cAMP-PKA signaling pathway in neurons and astrocytes, thereby promoting neuronal process regeneration.

[0055] 3. Neuron-microglia co-culture

[0056] 3.1. Extraction of primary microglia

[0057] The embryonic cortex of SD rats at 18 days of pregnancy was digested with 0.05% trypsin and DNase I (0.1 mg / mL), and then an equal volume of DMEM containing 10% fetal bovine serum (FBS) was added and centrifuged at 1000 rpm at 4°C for 5 minutes. The cells were resuspended in DMEM medium containing 10% FBS and 1% Pen / Strep and cultured at 3×10 6 / well were inoculated in a poly-lysine-coated T75 flask. After 5-7 days of in vitro culture, the culture dish was placed on a shaker and shaken at 200 rpm for 4 hours. The supernatant was then collected and centrifuged at 1000 rpm and 4°C for 5 minutes to collect the microglia, which were resuspended in new culture medium and inoculated according to experimental requirements.

[0058] Select neuron-microglia co-culture system. The co-culture medium consists of Neurobasal medium supplemented with 2% B27 supplement and 1x glutamine. Neurons need to be cultured on coverlips for 48 hours before adding microglia.

[0059] In the neuron-microglia co-culture experiment, the resuspended primary microglia were cultured for 48 h, and then the co-culture medium was replaced, and coverlips containing primary spinal cord neurons were added. Neurons and microglia were scratched with a 200 μl pipette tip. Subsequent drug administration and result detection methods were the same as 1.1.

[0060] 3.2. Analyze the cells using the same method as 1.2-1.5. The analysis results are as follows: Figure 3 As shown. Figure 3 As shown in A, compared with the control group, the number of neurons around the injury in the Scratch group was significantly reduced, compared with the Scratch group, the number of neurons around the injury in the Scratch+UCN2 group was significantly increased, and compared with the Scratch+UCN2 group, the number of neurons around the injury in the Scratch+UCN2+H89 group was significantly reduced; according to Figure 3 As shown in B, the content of Tunel in the model group was significantly higher than that in the control group, while that in the model+UCN2 group was comparable to that in the control group, and that in the model+UCN2+H89 group was significantly higher; Figure 3 As shown in C, the mRNA expression of PKA in the Scratch+UCN2 group was increased, and the mRNA expression of NF-κB was decreased compared with the Scratch group and the Scratch+UCN2+H89 group; Figure 3As shown in D, the protein expression and phosphorylation of PKA in the Scratch+UCN2 group were higher than those in the Scratch group, and the protein expression and phosphorylation of NF-κB in the Scratch+UCN2 group were lower than those in the Scratch group and Scratch+UCN2+H89 group.

[0061] In summary, through neuron-microglia co-culture, it was found that UCN2 inhibited NF-κB expression by activating the cAMP-PKA signaling pathway in neurons and microglia, thereby promoting neuronal process regeneration and inhibiting neuronal apoptosis.

[0062] Example 2: In vivo experiments

[0063] The experimental animals used in this example were 6-8 week old male SPF Sprague-Dawley rats weighing 180-230 g, purchased from the Experimental Animal Center of Shanghai First People's Hospital. All rats were housed in the Experimental Animal Center of Shanghai First People's Hospital with free access to food and water, and the room temperature was maintained at 24±3°C with a 12-hour day and night cycle. All experiments were approved by the Ethics Committee of Shanghai First People's Hospital.

[0064] 1. Establishment of spinal cord injury model and drug treatment

[0065] The main way UCN2 exerts its biological effects is by binding to the G protein-coupled receptor CRH receptor (CRHR). There are two subtypes of CRHR, CRHR1 and CRHR2, of which CRHR2 regulates stress response better. Compared with CRH1, CRHR2 has a better affinity for UCN2. After the two bind, they activate downstream signaling pathways such as cAMP-PKA or ERK-MAPK, thereby regulating neurite plasticity processes such as dendrite stabilization, ion channel transmission, cAMP response element binding protein (CREB) and transcription of other genes.

[0066] The rats were randomly divided into Sham group (only the lamina was removed, i.e., control group), spinal cord injury (SCI) group, SCI+UCN2 group (treatment group), and SCI+UCN2+siCRHR2 group (UCN2 receptor knockdown, causing UCN2 to lose its therapeutic effect), with 3 rats in each group.

[0067] The rat SCI model was established by full transection of the right spinal cord. Shutai 50 was diluted with normal saline at 1:10 and injected intraperitoneally at 1 ml / 100 g to anesthetize the rats. A 2 cm midline longitudinal incision was made on the back of the rats with the T10 spinous process as the center, and the T10 lamina was exposed and removed to serve as the Sham group. The rats were anesthetized and the T10 lamina was removed using the same method to expose about 5 mm of the spinal cord segment. The right spinal cord was cut along the midline of the spinal cord with microscissors to serve as the spinal cord injury (SCI) group. The rats in the SCI+UCN2 and SCI+UCN2+siCRHR2 groups were surgically modeled in the same way as the spinal cord injury (SCI) group. The SCI+UCN2+siCRHR2 group was injected with 10 μl of siRNA (provided by GenePharma, sequence S: 5'-CCUACAUGCUCUUCUUUGUTT-3'; AS: 5'-ACAAAGAAGAGCAUGUAGGTT-3') into the spinal cord before suture, and 200 μl of siRNA was injected into the tail vein twice a week after surgery. The SCI+UCN2 and SCI+UCN2+siCRHR2 groups were intraperitoneally injected with UCN2 (7 μg / kg) every day starting from the second day after surgery, and the SCI group was intraperitoneally injected with the same volume of saline every day. All rats were returned to individual warm cages after surgery and given sufficient food and water until they woke up naturally. Rats with BBB behavioral scores of 0-2 were included in the experiment. Manual bladder squeezing was used to assist urination every 8-12 hours until the spontaneous urination function was restored. All rats were intraperitoneally injected with 20 mg of gentamicin sulfate daily for 3 consecutive days. All rats underwent behavioral testing and samples were collected on the 14th day after surgery.

[0068] 2. Behavioral testing

[0069] 2.1. The Basso, Beattie and Bresnahan (BBB) ​​scale has a total of 22 points, which can systematically evaluate the overall motor ability and hind limb recovery through behaviors such as weight support, joint activity, and tail control, ranging from 0 points (indicating no hind limb movement) to 21 points (normal walking). Each rat was placed in an open area and evaluated by two professionals who were unaware of the experiment, and the average score was taken.

[0070] The results are as follows Figure 4 As shown, the score of the spinal cord injury (SCI) group was significantly lower than that of the Sham group (only lamina resection, control group), the score of the SCI+UCN2 group was significantly higher than that of the spinal cord injury (SCI) group, and the score of the SCI+UCN2+siCRHR2 group was significantly lower than that of the SCI+UCN2 group, indicating that UCN2 can accelerate the recovery of hind limb motor ability in rats in the chronic stage of spinal cord injury.

[0071] 2.2. Gait experiment: The front feet of rats were painted with green non-toxic and odorless dye, and the hind feet were painted with red non-toxic and odorless dye. The animals were asked to pass through a rectangular tunnel of 100cm*7cm*7cm, and the gait analysis was performed on the middle part of the tunnel with a uniform speed.

[0072] The results are as follows Figure 5 As shown, the red dye area in the SCI group was reduced compared with the Sham group (only the lamina was removed, the control group), that is, the rats had a significant loss of hind limb motor ability; the red dye area in the SCI+UCN2 group was increased compared with the SCI group, that is, the rats had a significant recovery of hind limb motor ability; the red dye area in the SCI+UCN2+siCRHR2 group was reduced compared with the SCI+UCN2 group, that is, the rats had a significant loss of hind limb motor ability, indicating that UCN2 can accelerate the recovery of hind limb motor ability of rats in the chronic stage of spinal cord injury.

[0073] 3. cAMP-PKA signaling pathway in spinal cord tissue

[0074] 3.1 mRNA expression

[0075] Total RNA was extracted from rat spinal cord tissue using Trizol reagent. 4 μl 5x RTSuperMix was added to 1000 ng RNA, and ddH2O was added to 20 μl. The mixture was heated at 45°C for 15 min, and then heated at 85°C for 5 seconds for RNA reverse transcription. The PCR reaction system included 1 μl extracted RNA, 10 μl SYBR Green Master Mix, 0.4 μl each of the front primer and the rear primer, and the final reaction volume was 20 μl. The reverse transcription reaction was performed using the QuantStudio 6Flex real-time fluorescence PCR system, first pre-denaturing at 95°C for 30 s, then denaturing at 95°C for 10 s, annealing and extending for 30 s, using GAPDH as the internal reference, and using 2 -ΔΔt The relative expression of mRNA of each gene was calculated by the primer sequence shown in Table 1. Figure 6 As shown in A, the mRNA expression levels of CRHR2, PKA, CREB, Bcl-2, and GDNF in the SCI+UCN2 group were increased, and the mRNA expression levels of NF-κB, RhoA, and Bax were decreased compared with those in the SCI group and the SCI+UCN2+siCRHR2 group.

[0076] 3.2 Western Blot Analysis

[0077] The prepared spinal cord tissue was removed, 1 ml RIPA, 10 μl protease inhibitors and 10 μl phosphatase inhibitors were added, and ultrasonic grinding was performed on ice, followed by centrifugation at 12000 rpm for 15 min at 4 °C, and the supernatant was collected. The protein concentration was determined by the BCA method, the protein sample was dissolved in the sample buffer, and heated at 100 °C for 5 min, and then the sample was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk powder, the primary antibody was added and incubated overnight at 4 °C, the secondary antibody was added after washing with TBST, and then optical density analysis was performed.

[0078] Results Figure 6 As shown in B, it can be seen that the protein expression and phosphorylation conditions are consistent with the mRNA expression conditions, indicating that UCN2 can activate the downstream cAMP-PKA signaling pathway by binding to CRHR2.

[0079] 4. Effects of spinal cord injury on the functions of various nerve cells around the spinal cord

[0080] 4.1 Tunel

[0081] Wash the frozen sections with PBS solution for 10 minutes. According to the protocol of Fluorescein (FITC) Tunel Cell Apoptosis Detection Kit (G1501-50T, Servicebio), permeabilize with proteinase K solution at room temperature for 10 minutes. Wash the tissue sections with PBS solution for 5 minutes, repeat 3 times, block with immunostaining blocking solution at room temperature for 1 hour, and then incubate with primary antibody Anti-β-Tubulin III antibody (1:100, T2200, Sigma-Aldrich) at 4°C overnight, and wash the sections with PBS the next day. Prepare TdT incubation buffer according to the kit requirements, add Goat anti-Rabbit IgG488 (1:500, A-11010, Invitrogen), and evenly add 50 μL of the reaction mixture to each tissue section and incubate at 37°C for 1 hour. Wash the slides with PBS solution for 5 minutes, repeat 3 times. Stain with DAPI and seal the slides and observe under a microscope.

[0082] Results Figure 7 As shown in middle A, it can be seen that compared with the Sham group, the SCI group had increased neuronal apoptosis, compared with the SCI group, the SCI+UCN2 group had reduced neuronal apoptosis, and compared with the SCI+UCN2 group, the SCI+UCN2+siCRHR2 group had increased neuronal apoptosis.

[0083] 4.2 Immunofluorescence

[0084] Transverse spinal cord specimens fixed in 4% paraformaldehyde were immersed in 15% and 30% sucrose solutions for dehydration. Then, the specimens were cut into 8-μm-thick longitudinal sections using a cryostat (Thermo, NX50, USA) and rinsed with PBS. The sections were incubated with blocking solution (Biyuntian, Shanghai, China) for 1 hour and incubated with primary antibodies overnight at 4°C: anti-neurofilament 200 antibody (1:100, N4142, Sigma-Aldrich), anti-Iba1 (1:100, 019-19741, Wako), anti-GFAP (1:100, GB12100-100, Sewell), anti-C3 (1:100, BA2298-1, Boster), and anti-S100A10 (1:100, A02787-2, Boster). The next day, the samples were incubated with secondary antibodies at 37°C for 2 h, and the secondary antibodies were goat anti-mouse IgG 546 (1:500, A-11030, Invitrogen) and goat anti-rabbit IgG 488 (1:500, A32731, Invitrogen). Cell nuclei were stained with DAPI. Images were observed using a fluorescence microscope (Olympus, Japan) and analyzed using ImageJ software.

[0085] The results are as follows Figure 7 As shown in B, it can be seen that compared with the Sham group, the neurofilament damage in the SCI group increased, compared with the SCI group, the neurofilament damage in the SCI+UCN2 group decreased, and compared with the SCI+UCN2 group, the neurofilament damage in the SCI+UCN2+siCRHR2 group increased.

[0086] The results are as follows Figure 7 As shown in C, it can be seen that compared with the Sham group, the number of activated microglia in the SCI group increased, compared with the SCI group, the number of activated microglia in the SCI+UCN2 group decreased, and compared with the SCI+UCN2 group, the number of activated microglia in the SCI+UCN2+siCRHR2 group increased.

[0087] The results are as follows Figure 7 As shown in D, it can be seen that compared with the Sham group, the number of A2 astrocytes in the activated astrocytes in the SCI group decreased, compared with the SCI group, the number of A2 astrocytes in the activated astrocytes in the SCI+UCN2 group increased, and compared with the SCI+UCN2 group, the number of A2 astrocytes in the activated astrocytes in the SCI+UCN2+siCRHR2 group decreased.

[0088] The results are as follows Figure 7As shown in E, it can be seen that compared with the Sham group, the number of type A1 astrocytes in the activated astrocytes in the SCI group increased, compared with the SCI group, the number of type A1 astrocytes in the activated astrocytes in the SCI+UCN2 group decreased, and compared with the SCI+UCN2 group, the number of type A1 astrocytes in the activated astrocytes in the SCI+UCN2+siCRHR2 group increased.

[0089] In summary, UCN2 promoted the recovery of hind limb motor function in rats with spinal cord injury 14 days after injury. After UCN2 treatment, rats with spinal cord injury had reduced neuronal apoptosis, reduced neurofilament damage, reduced expression of A1 astrocytes, and increased expression of A2 astrocytes (A1 astrocytes are mainly pro-inflammatory phenotypes, which aggravate injuries, and A2 astrocytes are mainly secreted anti-inflammatory factors and neurotrophic factors. In the chronic stage of spinal cord injury, activated astrocytes are mainly A1 type, and after adding UCN2 treatment, astrocytes are transformed into A2, thus playing a therapeutic role), and activated microglia are reduced (in spinal cord injury, activated microglia have been shown to be M1 microglia, whose main function is to release pro-inflammatory factors, which promote inflammatory reactions around the injury in the chronic stage of spinal cord injury, leading to prolonged spinal cord injury. After UCN2 treatment, activated microglia are reduced, improving the environment around the injury). The mRNA and protein expressions of PKA, CREB, and Bcl-2 in the cAMP-PKA signaling pathway of rats with spinal cord injury were upregulated, while the mRNA and protein expressions of NF-κB, RhoA, and Bax were decreased.

[0090] In summary, UCN2 can activate the cAMP-PKA signaling pathway, inhibit neuronal apoptosis, promote the secretion of GDNF by astrocytes, enhance the transformation of type A1 astrocytes to type A2 astrocytes, inhibit the secretion of NF-κB by microglia, and relieve the inflammatory response, thereby promoting neurite regeneration and accelerating the recovery of hind limb motor ability in rats with chronic spinal cord injury.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of protein UCN2 or its derivatives in the preparation of a drug for alleviating and / or treating spinal cord injury in a subject, characterized in that: The amino acid sequence of the protein UCN2 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The subject is in a chronic stage of spinal cord injury.

3. The use according to claim 1 or 2, characterized in that: The subject is a mammal.

4. The use according to any one of claims 1 to 3, characterized in that: The protein UCN2 alleviates and / or treats spinal cord injury in a subject by promoting neuronal process regeneration and inhibiting neuronal apoptosis.

5. The use according to claim 1, characterized in that: The protein UCN2 derivative is obtained by performing at least one modification of the protein UCN2 including pegylation, glycosylation, lipidation, protein fusion, antibody coupling, site mutation, supercharge, and introduction of non-natural amino acids.

6. The use according to claim 1, characterized in that: The drug has a pharmaceutically acceptable dosage form.

7. The use according to claim 1, characterized in that: The medicine is an injection.

8. A pharmaceutical composition, characterized in that It comprises protein UCN2 or its derivatives and a pharmaceutically acceptable carrier, wherein the amino acid sequence of the protein UCN2 is shown as SEQ ID NO:

1.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition has a pharmaceutically acceptable dosage form.

10. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition is an injection.

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