CN112891542B - Pharmaceutical composition containing UCHs inhibitor and application thereof - Google Patents

Abstract

The invention provides a pharmaceutical composition for promoting neuron growth, which comprises a UCHs inhibitor. The invention clearly discloses the action and mechanism of UCHs in the process of neuron growth. Finding that UCHs and Spastin have interaction, particularly UCHL1, which can mediate the degradation of Spastin and inhibit the microtubule cutting function of Spastin; and after UCHL1 is interfered, the degradation of the Spastin can be obviously reduced, and the microtubule cutting function of the Spastin is recovered, so that the growth of neurons is promoted. Discloses a new mechanism for regulating the growth of the UCHs neurons, and provides practical scientific basis and a new direction for the clinical treatment of spinal cord injury.

Description

Pharmaceutical composition containing UCHs inhibitor and application thereof

Technical Field

The invention belongs to the field of biological medicines, relates to a pharmaceutical composition containing UCHs inhibitors and application thereof, and particularly relates to a pharmaceutical composition containing UCHs modification inhibitors and used for promoting neuron growth and spinal cord injury repair and application thereof.

Background

The central nervous system, which is composed of the brain and spinal cord, is the most important part of the human nervous system, with 250000 to 500000 new cases of spinal cord injury patients worldwide each year. The spinal cord injury has the characteristics of high incidence, high disability rate, high consumption and low death rate, and brings heavy burden to the patient, family and society. In the prior art, spinal cord injuries are usually treated by a neuroprotective therapy such as methylprednisolone, or by a surgical method to relieve the stress on the central nervous system, or by a hyperbaric oxygen therapy within a short time after the injury. However, limitations of the traditional methods have prompted people to explore new methods and mechanisms for nerve repair after central nervous system injury, stem cell transplantation; novel biomaterials, such as hydrogels; epidural electrical stimulation, etc.

Despite the endless number of new treatment regimens, the ability to repair a lesion by protuberant regeneration or compensatory fiber growth following a central nervous system injury is extremely limited. With the progress of research, it has been recognized that promotion of outgrowth, branching, and then re-establishment of neuron-to-neuron connections after spinal cord injury is a key process in post-spinal cord injury repair. The branching and growth of processes are influenced by multiple factors and are achieved by cytoskeleton, microtubule movement, and microfilament remodeling. When the protruding branches are formed, the microtubules invade and are cut, and the microtubules further grow so that the protruding branches form new branches to promote the growth of the protruding branches.

Neurons are the basis of nervous system structure and function, and in the development process of neurons, the processes of protrusions are responsible for collecting environmental changes and stimulation, and the cell bodies are responsible for analyzing and processing information. The development process of the neuron is divided into three main stages, firstly, cell proliferation is carried out, and the fate of divided daughter cells is mainly determined by the difference of gene expression; then, cell migration starts from a place close to a ventricular origin, newly developed neurons migrate towards the periphery of the nerve tube and are positioned at different levels; finally, cell differentiation, including changes in extrinsic morphology and intrinsic function, is affected by secretion from the target organ. The microtubule backbone assembly, architecture and dynamic remodeling of neurons are essential for completing different stages of neuronal development.

The processes of neurons include axons and dendrites, axons grow earlier than dendrites, axons grow, elongate along specific routes, and depend on extracellular targeting molecules such as extracellular matrix, cell adhesion molecules, growth factors, etc. to travel. Meanwhile, cytoskeleton exists in cells, tubulin is responsible for transportation, and actin and myosin are responsible for stretching and steering to promote growth of the protrusion. The growth cone at the end of the axon guides axon extension by amoebic movement, and the axon extension and stabilization essentially do not leave the microtubule skeleton. Dendrites grow as regulated by axonal retrograde transport of some chemical information, such as NTF, while relying largely on changes in microtubule stability. During the rapid growth process, microtubules are unstable in dynamic, but the protruding growth ends are covered with microtubule-associated proteins, microtubules become more stable, and the growth of dendrites is regulated by regulating the balance between unstable microtubules and stable microtubules. Thus, microtubules play a large role in the growth of the neurite outgrowth.

Ubiquitination modification refers to the process by which one or more Ubiquitin molecules (Ubiquitin) are covalently bound to a substrate protein under the action of a series of enzymes. The entire process involves Ubiquitin molecules, protein substrates, various enzyme systems and proteasomes, which together constitute the Ubiquitin-Proteasome System (UPS). The ubiquitination-protease system mediates more than 80% of protein degradation in eukaryotes and is an important post-translational modification of proteins. Ubiquitination modification is closely related to nerve development and axon regeneration, and abnormality of ubiquitination modification is a pathogenic factor of many neurological diseases. Ubiquitin C-terminal hydrolases (UCHs) are cysteine proteases which are widely existed in different organisms, and are deubiquitinating enzymes (DUBs) which can release ubiquitin molecules from substrate proteins, have reverse regulation effect on protein degradation, and can promote ubiquitin recycling.

The inventor finds out in research that UCHs are highly expressed in the nervous system and have double functions in ubiquitin proteasome degradation. However, the existing research shows that UCHs can mediate protein deubiquitination to play a role in regulating protein stability by playing the role of hydrolase, so that the UCHs can play a role in promoting or inhibiting cancer at different development stages of different types of tumors. However, the specific action and mechanism of UCHs in the processes of microtubule cutting, neuron growth, spinal cord injury repair and the like are rarely studied in the prior art. Therefore, it is necessary to research the mechanism of UCHs for regulating axon development and regeneration, and provide a new theoretical basis and a new direction for the clinical treatment of spinal cord injury.

Disclosure of Invention

The invention aims to solve the problems in the prior art, thereby carrying out deep research on the functions and mechanisms of UCHs in neuron growth and spinal cord injury repair, disclosing a new mechanism for regulating neurite growth and promoting neuron regeneration by inhibiting UCHs, and providing practical experimental evidence and scientific basis for clinical treatment of spinal cord injury.

In order to solve the above technical problems, the present invention is achieved by the following technical solutions.

In a first aspect, the present invention provides a pharmaceutical composition for promoting neuronal growth comprising an inhibitor of UCHs.

Preferably, the UCHs inhibitor is selected from one or more of a UCHL1 inhibitor, a UCHL3 inhibitor and a UCHL5 inhibitor; most preferably, the inhibitor of UCHs is selected from an inhibitor of UCHL 1.

Preferably, the UCHL1 inhibitor is selected from siRNA designed based on the UCHL1 gene (siUCHL 1).

Preferably, the siUCHL1 is selected from one or more of siUCHL1-1, siUCHL1-2 and siUCHL 1-3; the sequences of siUCHL1-1, siUCHL1-2 and siUCHL1-3 are respectively shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3, respectively.

In a second aspect, the present invention provides a pharmaceutical composition for promoting repair of spinal cord injury, comprising a UCHs inhibitor.

Preferably, the UCHs inhibitor is selected from one or more of a UCHL1 inhibitor, a UCHL3 inhibitor and a UCHL5 inhibitor; most preferably, the inhibitor of UCHs is selected from an inhibitor of UCHL 1.

Preferably, the UCHL1 inhibitor is selected from siRNA designed based on the UCHL1 gene (siUCHL 1).

Preferably, the siUCHL1 is selected from one or more of siUCHL1-1, siUCHL1-2 and siUCHL 1-3; the sequences of siUCHL1-1, siUCHL1-2 and siUCHL1-3 are respectively shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3, respectively.

In a third aspect, the invention provides the use of a UCHs inhibitor for the preparation of a medicament for promoting neuronal growth.

Preferably, the inhibitor of UCHs promotes neuronal growth by promoting the microtubule cleavage function of Spastin.

Preferably, the UCHs inhibitor is selected from one or more of a UCHL1 inhibitor, a UCHL3 inhibitor and a UCHL5 inhibitor; most preferably, the inhibitor of UCHs is selected from an inhibitor of UCHL 1.

Preferably, the UCHL1 inhibitor is selected from siRNA designed based on the UCHL1 gene (siUCHL 1).

Preferably, the siUCHL1 is selected from one or more of siUCHL1-1, siUCHL1-2 and siUCHL 1-3; the sequences of siUCHL1-1, siUCHL1-2 and siUCHL1-3 are respectively shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3, respectively.

The fourth aspect of the invention provides the application of UCHs inhibitors in preparing medicines for promoting spinal cord injury repair.

Preferably, the inhibitor of UCHs promotes spinal cord injury repair by promoting the Spastin microtubule cleavage function.

Preferably, the UCHs inhibitor is selected from one or more of a UCHL1 inhibitor, a UCHL3 inhibitor and a UCHL5 inhibitor; most preferably, the inhibitor of UCHs is selected from an inhibitor of UCHL 1.

Preferably, the UCHL1 inhibitor is selected from siRNA designed based on the UCHL1 gene (siUCHL 1).

Preferably, the siUCHL1 is selected from one or more of siUCHL1-1, siUCHL1-2 and siUCHL 1-3; the sequences of siUCHL1-1, siUCHL1-2 and siUCHL1-3 are respectively shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3, respectively.

Ubiquitin ligase and deubiquitinase mediated protein ubiquitination/deubiquitination balance regulation plays an important role in tumor malignant process. UCHL1, UCHL3, UCHL5 have recently attracted attention as important members of the ubiquitin C-terminal hydrolase (UCHs) family, particularly UCHL 1. The existing research finds that UCHs play the role of hydrolase to mediate the deubiquitination of protein to play the role of regulating and controlling the stability of protein, thereby playing the role of promoting or inhibiting cancer in different development stages of different types of tumors. However, the specific action and mechanism of UCHs in the processes of microtubule cutting, neuron growth, spinal cord injury repair and the like are rarely studied in the prior art.

Previous studies by the present inventors found that Spastin is one of important microtubule-cleaving proteins and is highly expressed in the central nervous system. Spastin can interact with ubiquitin molecules, and Spastin can be ubiquitinated in the nervous system. Spastin, a microtubule-cleaving protein, regulates microtubules and promotes axon branching and growth. The ubiquitination modification can influence the microtubule cutting function of the Spastin protein by regulating the stability of the Spastin protein, thereby influencing the growth of the neurite. During the development of neuron axons, after the function of the Spastin protein is exerted, ubiquitination marking is required and the Spastin protein is degraded, so that abnormal microtubule cutting caused by overactivity of Spastin is avoided; in order to promote the formation of lateral branches and the growth of processes during spinal cord injury, the stability of the Spastin protein can be increased by inhibiting the ubiquitination modification of Spastin so as to enhance microtubule cutting, promote axon regeneration and accelerate spinal cord injury repair.

On the basis, the inventor of the invention carries out further intensive research and finds that UCHs and Spastin have interaction, particularly UCHL1, which can mediate the degradation of Spastin and inhibit the microtubule cutting function of Spastin; and after UCHL1 is interfered, the degradation of the Spastin can be obviously reduced, and the microtubule cutting function of the Spastin is recovered, so that the growth of neurons is promoted.

Compared with the prior art, the invention has the following technical effects:

(1) the invention deeply researches the growth of UCHs and neuron, and finds the inhibition of UCHs on microtubule cutting and neuron growth;

(2) the invention defines the specific action mechanism of UCHs mediated microtubule cutting and neuron growth process, namely UCHs can interact with Spastin, thereby influencing the stability of Spastin and further influencing the microtubule cutting function;

(3) the invention discovers that the degradation of the Spastin can be obviously reduced by inhibiting the expression of UCHs, and the microtubule cutting function of the Spastin is recovered, so that the growth of neurons is promoted; discloses a new mechanism for regulating the growth of the UCHs neurons, and provides practical scientific basis and a new direction for the clinical treatment of spinal cord injury.

Drawings

FIG. 1 is a schematic diagram showing the result of detecting Spastin by IP in a rat brain lysate.

FIG. 2 is a schematic diagram showing the result of detecting UCHL1 by IP in the rat brain lysate.

FIG. 3 is a diagram showing the co-immunoprecipitation of Spastin and UCHL1 in 293T cells.

FIG. 4 is a schematic diagram showing the qualitative result of degradation of endogenous Spastin by overexpression of GFP-UCHL1 in COS7 cells.

FIG. 5 is a diagram showing the quantitative results of the degradation of endogenous Spastin by over-expression of GFP-UCHL1 in COS7 cells.

Figure 6 is a graph of the effect of varying doses of UCHL1 on the level of degradation of endogenous Spastin.

Fig. 7 is a graph showing the effect of different doses of UCHL1 on the level of degradation of exogenous Spastin.

Fig. 8 is a graph of the effect of UCHL1 treatment on the level of degradation of Spastin at various times.

FIG. 9 is a schematic representation of the inhibition of UCHL1 by different siUCHL 1.

FIG. 10 is a diagram showing the qualitative results of siUCHL1-2 inhibiting degradation of endogenous Spastin.

FIG. 11 is a diagram showing the quantitative results of siUCHL1-2 inhibiting the degradation of endogenous Spastin.

FIG. 12 is a graph showing the effect of different doses of siUCHL1-2 on the inhibition of the level of degradation of endogenous Spastin.

FIG. 13 is a graph showing the effect of siUCHL1-2 treatment on the level of degradation of endogenous Spastin at various times.

FIG. 14 is a diagram showing the qualitative result of the effect of UCHL1 on the cleavage function of Spastin microtubules.

FIG. 15 is a diagram showing the quantitative results of UCHL1 on the cleavage function of Spastin microtubules.

FIG. 16 is a diagram showing the qualitative results of the effect of siUCHL1-2 on the recovery of the cutting function of Spastin microtubules.

FIG. 17 is a diagram showing the quantitative results of siUCHL1-2 on the recovery of the cutting function of Spastin microtubules.

Fig. 18 is a schematic diagram of confocal laser images of hippocampal neuronal cell processes acquired under UCHL1 processing.

FIG. 19 is a statistical plot of the number of hippocampal processes in each group under UCHL1 treatment.

FIG. 20 is a statistical plot of the length of hippocampal processes in each group under UCHL1 treatment.

FIG. 21 is a schematic diagram of confocal laser collection of hippocampal cell processes under siUCHL1-2 treatment.

FIG. 22 is a statistical plot of the number of hippocampal processes in two groups under siUCHL1-2 treatment.

FIG. 23 is a statistical plot of the length of hippocampal processes in two groups under siUCHL1-2 treatment.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Immortalized cell tools including rat hippocampal neuronal cell line and COS7 monkey fibroblast cell line listed in the context of the present invention are commercially available and cultured according to conventional methods in cell biology, unless otherwise specified. All cell lines were identified by short tandem repeat analysis of the chinese typical culture collection (wuhan) and verified for the presence of mycoplasma contamination using a PCR assay kit (shanghai Biothrive Sci) while being cryopreserved in liquid nitrogen and used for subsequent experiments. The reagents used in the present invention are commercially available. The experimental methods and techniques used in the present invention, such as COS7 culture, 293T cell culture, rat hippocampal neuron culture, Western blot, molecular cloning, PCR, immunofluorescent staining, laser confocal, immunoprecipitation, co-immunoprecipitation, animal experiments, etc., are all conventional methods and techniques in the art.

Representative results from selection of biological experimental replicates are presented in the context figure, and data are presented as mean ± SD and mean ± SEM as specified in the figure. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 5.0 or SPSS 20.0 software. And comparing the difference of the mean values of two or more groups by using a t test or an analysis of variance. p < 0.05 was considered a significant difference.

Example 1 validation of the interaction of UCHL1 with Spastin

(1) Selecting SD rat within 1 day of birth, taking rat brain, adding cell lysate, wherein 10 μ L of cell lysate is added to 1mg of rat brain;

(2) adding PMSF, fully grinding on ice, and then cracking for 30 min;

(3) centrifuging at 4 deg.C and 12000rpm for 10min, and collecting supernatant to obtain rat brain lysate;

(4) performing immunoprecipitation experiment after incubating the rat brain lysate and anti-UCHL1 antibody, and detecting Spastin protein by using Western Blot (see figure 1);

(5) performing immunoprecipitation experiment after incubating the mouse brain lysate and anti-Spastin antibody, and detecting UCHL1 protein by Western Blot (see figure 2);

(6) and (3) carrying out mass spectrum identification on the precipitate obtained after the Spastin immunoprecipitation.

The results are shown in FIGS. 1-2, respectively. From the results, it can be seen that Spastin can be detected in the precipitate obtained from IP ucastin 1, and likewise, ucahl 1 can be detected in the precipitate obtained from IP Spastin, which means that there is indeed an interaction between Spastin and ucal 1, i.e. there is a binding between Spastin and ucal 1 in vivo. Subsequently, the mass spectrum analysis of the precipitate after the IP Spastin shows that seven peptide sequences belonging to UCHL1 molecules are extracted, namely QIEELKGQEVSPKETVEEQASTTER, QFLSETEK, LSPEDR, NEAIQAAHDSVAQEGQCR, MPFPVNHGASSEDSLLQDAAK, LGVAGQWR and FSAVALCK, and the interaction of UCHL1 and the Spastin is further confirmed

Since Spastin and UCHL1 were able to bind in vivo, further, co-immunoprecipitation experiments using 293T cells were performed to verify whether Spastin and UCHL1 could interact in vitro:

(1) GFP or GFP-UCHL1 and Flag-Spastin are transfected into 293T cells;

(2) after 24h of transfection, the original culture medium is discarded, and PBS is added for washing for 2 times;

(3) adding cell lysis solution (containing PMSF), and lysing on ice for 30 min;

(4) scraping off cells by using a cell scraper, transferring the cells into a sterilized centrifuge tube, centrifuging the cells at 4 ℃ and 12000rpm for 10min, and taking supernatant to obtain cell lysate;

(5) anti-GFP is used as a precipitation antibody to carry out co-immunoprecipitation with 293T cell lysate, and Western Blot is used for detecting Flag tags.

The results are shown in FIG. 3. The results show that Flag-Spastin can be detected in both GFP-UCHL1 and the supernatant group, while no Flag-Spastin can be detected in the GFP control group, which indicates that the Spastin and UCHL1 can be combined with each other in vitro.

Example 2 UCHL1 can mediate the degradation of Spastin

(1) Preheating MEM, PBS and DMEM containing 10% FBS, and preparing a plurality of sterilized 1.5 mL centrifuge tubes;

(2) discarding the culture medium in the original COS7 cell culture plate, adding equal amount of MEM starved cells, and returning to the incubator;

(3) adding a proper amount of MEM into centrifuge tubes, wherein the final volume of each well in a 6-well plate is usually 200 mu L, and the final volume of each well in a 24-well plate is 50 mu L, wherein 1 centrifuge tube is added with GFP-UCHL1 plasmid, usually 4 mu g plasmid is added into each well in the 6-well plate, 1 mu g plasmid is added into each well in the 24-well plate, and 1 centrifuge tube is added with liposome according to the ratio of 1:3 of plasmid to liposome;

(4) adding the liposome and MEM mixture into the plasmid and MEM mixture, gently mixing, and standing at room temperature for 15 min;

(5) uniformly dripping the liposome and plasmid mixture into a cell culture plate;

(6) removing the original MEM after 3h, washing with PBS for 2 times, adding newly configured DMEM containing 10% FBS, and placing the cells back into the incubator to continue culturing until the cells are treated; COS7 cells transfected with GFP-UCHL1 plasmid were obtained, and COS7 cells transfected with GFP empty plasmid were obtained in the same manner;

(7) western Blot was used to examine the effect on endogenous Spastin expression levels in cells.

The results are shown in FIG. 4. The results show that over-expression of UCHL1 resulted in a significant decrease in Spastin protein levels compared to the empty vector GFP. The relative protein expression level of Spastin in GFP-UCHL1 experiment group was then calculated by repeating the above experiment 3 times with the control group Spastin/GAPDH normalized to 1 in FIG. 4. The results are shown in FIG. 5, which shows that the difference between the two contents is statistically significant (.)p< 0.05). Therefore, the UCHL1 can obviously reduce the stability of the Spastin protein through over-expression, thereby causing the degradation of the endogenous Spastin of COS7 cells.

Then, different doses of GFP-UCHL1 (0, 1 and 4 mu g) are respectively overexpressed in COS7 cells, and the expression levels of endogenous Spastin and GFP-UCHL1 in the cells are detected by using Western Blot.

The results are shown in fig. 6, and show that UCHL1 is significantly dose-dependent on degradation of endogenous Spastin.

Furthermore, GFP-Spastin (2. mu.g) was overexpressed in 293T cells in the same manner as described above, and Flag-UCHL1 (0, 1, 4. mu.g) was overexpressed at different doses, respectively, and then the expression levels of exogenous GFP-Spastin and Flag-UCHL1 in 293T cells were detected by Western Blot.

The results are shown in FIG. 7. The results show that UCHL1 can generate degradation effect on exogenous Spastin in 293T cells, and the degradation is obviously dose-dependent.

Further, GFP or GFP-UCHL1 was overexpressed in COS7 cells; CHX (Cycloheximide ) is added after 12h of culture, which can inhibit protein translation synthesis; and cracking the cells after 0, 6 and 12h of adding CHX to collect protein, and detecting the expression level of endogenous Spastin in the cells by using Western Blot.

The results are shown in FIG. 8. The results show that the content of the Spastin protein gradually decreases with time after the addition of the CHX inhibitor protein for translation; whereas the degradation level of Spastin was significantly increased after addition of UCHL1, andthe time dependence is shown, namely UCHL1 obviously accelerates the degradation of endogenous Spastin. Quantitative analysis of the results showed that the differences were statistically significant (.)p＜0.05）。

Example 3 interference with UCHL1 can retard the degradation of Spastin

(1) Designing siRNAs (siUCHL 1-1, siUCHL1-2 and siUCHL 1-3) aiming at UCHL1 gene, wherein the sequences of the three siRNAs are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3);

(2) overexpression of GFP-UCHL1 in COS7 cells;

(3) over-expressing siUCHL1-1, siUCHL1-2 or siUCHL1-3 in COS7 cells over-expressing GFP-UCHL 1;

(4) western Blot was used to examine the effect on endogenous Spastin expression levels in cells.

The results are shown in FIG. 9. The result shows that siUCHL1-2 has higher interference efficiency on UCHL1, thereby obviously reducing the degradation effect on Spastin; subsequent experiments were conducted using siUCHL1-2 for related studies.

Subsequently, GFP-UCHL1 was overexpressed in COS7 cells, siUCHL1-2 or NC (negative control) was simultaneously overexpressed, and the effect on the expression level of endogenous Spastin in the cells was examined by Western Blot.

The results are shown in FIG. 10. The results show that siUCHL1-2 can indeed significantly interfere with UCHL1 and thereby inhibit the degradation of endogenous Spastin. The ratio of the control group of Spastin/GAPDH in fig. 10 was normalized to 1, and the above experiment was repeated 3 times to calculate the relative protein expression content of two groups of spastins. The results are shown in FIG. 11, which shows that the difference between the two contents is statistically significant (.)p＜0.05）。

Furthermore, GFP and siUCHL1-2 (1, 2,4 mu L) with different concentrations are over-expressed in COS7 cells, and then the expression level of endogenous Spastin and the expression level of GFP-UCHL1 in the cells are detected by using Western Blot.

The results are shown in FIG. 12. The result shows that, in a certain range, with the increase of the concentration of siUCHL1-2, the degradation of endogenous Spastin in COS7 is delayed, namely, the concentration dependence of UCHL1 on the inhibition of the degradation of the endogenous Spastin is interfered, the higher the concentration of siUCHL1-2 is, the stronger the inhibition effect on UCHL1 is, the more obvious the inhibition effect on the degradation of the Spastin is, and the reverse shows that UCHL1 can promote the degradation of the Spastin.

Further, 4 μ g of GFP-UCHL1 was overexpressed in COS7 cells, while 4 μ L of siUCHL1-2 or NC was overexpressed; adding CHX after culturing for 12 h; and cracking cells after 0, 6 and 12h after CHX is added to collect protein, and detecting the influence of siUCHL1-2 or NC on the expression level of endogenous Spastin in COS7 cells by using Western Blot.

The results are shown in fig. 13, and show that the content of Spastin protein gradually decreases with time after the addition of CHX inhibitor protein for translation; and after siUCHL1-2 is added, the function of UCHL1 is obviously inhibited, the protein level of the Spastin can be restored, the time dependence is realized, and the reverse demonstration proves that UCHL1 can promote the degradation of the Spastin. Quantitative analysis of the results showed that the differences were statistically significant (.)p＜0.05）。

Example 4 Effect of UCHL1 on the cleavage function of Spastin microtubules

(1) Over-expressing GFP or GFP-Spastin, mCherry or mCherry-UCHL1, NC or siUCHL1-2 in COS7 cells;

(2) adding CHX after 12h of transfection, and performing immunocytochemistry after 12h of culture;

(3) incubating anti-Tubulin to detect the condition of microtubules in COS7 cells, collecting images through a confocal microscope, and counting the change of fluorescence intensity of the microtubules in COS7 by ImageJ.

The results are shown in FIG. 14. Wherein, Tubulin: a microtube; GFP: green fluorescent protein; mCherry: a red fluorescent protein; GFP-Spastin: spastin green fluorescent fusion protein; mCherry-UCHL 1: UCHL1 red fluorescent fusion protein; dapi: and (4) cell nucleus.

The results show that in COS7, with over-expressed GFP and mCherry as control groups, Tubulin can be kept intact and cannot be cut; when GFP and mCherry-UCH1 are over-expressed, Tubulin can be kept intact and can not be cut, namely mChery-UCHL 1 has almost no influence on microtubules; when GFP-Spastin and mCherry are over-expressed, the Tubulin fluorescence intensity is obviously weakened, and microtubules are cut; when GFP-Spastin and mCherry-UCHL1 are over-expressed, although the Tubulin fluorescence intensity is weakened compared with the control group, the microtubule fluorescence intensity is enhanced compared with GFP-Spastin and mCherry, which indicates that the microtubule cutting ability of GFP-Spastin can be weakened due to the existence of mChery-UCHL 1, and the scale is 20 μm.

The fluorescence intensity of the microtubes was then counted using ImageJ, and the results are shown in fig. 15. 30 cells were counted in each group and the control group was normalized to 1 and tested using complete random analysis of variance, the results are expressed as Mean ± SEMp< 0.05 indicated that the difference was statistically significant.

Furthermore, GFP-Spastin, mCherry-UCHL1, NC or siUCHL1-2 are over-expressed in COS7 cells; changes in Tubulin were observed by immunofluorescence chemical staining.

The results are shown in FIG. 16. Wherein, Tubulin: a microtube; GFP: green fluorescent protein; mCherry: a red fluorescent protein; GFP-Spastin: spastin green fluorescent fusion protein; mCherry-UCHL 1: UCHL1 red fluorescent fusion protein; dapi: cell nucleus; NC: negative Control; siUCHL 1-2: the small molecule interferes with UCHL 1-2.

The result shows that mCherry-UCHL1 red fluorescence is obviously interfered and disappeared, meanwhile, the GFP-Spastin fluorescence intensity is increased compared with GFP-Spastin and mCherry, and the microtubule fluorescence intensity is greatly reduced. Thus, when Spastin is present but UCHL1 is absent (no exogenously introduced or co-transformed siUCHL), Spastin can normally perform microtubule cleavage; in the presence of Spastin and in the presence of UCHL1 (exogenously introduced or co-transfected NC), Spastin microtubule cleavage capacity was reduced, whereas overexpression of UCHL1 alone had no detectable effect on microtubule cleavage. The over-expression UCHL1 can inhibit the microtubule cutting function of Spastin, and the interference of UCHL1 can recover the microtubule cutting function of Spastin, with the scale of 20 μm.

And then, counting the fluorescence intensity of the microtubes by using ImageJ, standardizing the fluorescence intensity of the microtubes of the control group to be 1, counting 30 cells in each group, carrying out T test on two independent samples, and representing the result by Mean +/-SEM, wherein the displayed difference has statistical significance as shown in figure 17.

Therefore, the over-expression of UCHL1 can inhibit the microtubule cutting function of Spastin, and the interference of UCHL1 can obviously restore the microtubule cutting function of Spastin.

Example 5 Effect of UCHL1 on neuronal growth

(1) Culturing and observing hippocampal neuron cells, and paying attention to the state of the hippocampal neuron cells and whether the hippocampal neuron cells are polluted or not;

(2) transferring the original culture medium to auxiliary wells, and adding 500 mu L of Neurobasal culture medium into each well for starvation;

(3) using a calcium phosphate transfection kit, 1. mu.g of GFP, mCherry-UCHL1, GFP-Spastin plasmid and 25. mu.L of CaCl were added to the EP tube₂Adding another EP tube into BBS solution;

(4) mixing the solution in the step (3), and standing for 15min in a dark box;

(5) dripping 50 μ L of mixed solution into each well, dripping the mixed solution into the wells in different directions, gently shaking for several times, after 40min, cleaning the calcium-phosphorus particles with 1 × SA for 2 times, and adding the original culture medium;

(6) after 24h of transfection, cells were fixed with 4% PFA, immunofluorescent-chemical stained and visualized under laser confocal.

The results are shown in FIGS. 18-20. Wherein, GFP: green fluorescent protein; mCherry: a red fluorescent protein; GFP-Spastin: spastin green fluorescent fusion protein; mCherry-UCHL 1: UCHL1 red fluorescent fusion protein; dapi: and (4) cell nucleus. FIG. 18 shows confocal images of hippocampal cell processes; FIG. 19 is a statistical plot of the number of hippocampal processes in each group; FIG. 20 is a statistical plot of the length of hippocampal processes in each group. n = 30/group and results are expressed as Mean ± SEM, with scale 20 μm.

The results show that when GFP and mCherry-UCH1 were overexpressed in hippocampal neurons as controls, there was no significant change in total hippocampal neurite outgrowth and total length of outgrowth; when GFP-Spastin and mCherry are over-expressed, the total number of hippocampal neurites and total length of the processes are obviously increased; when GFP-Spastin and mCherry-UCHL1 were overexpressed, the total number of hippocampal processes and the total length of processes were significantly reduced compared to the GFP-Spastin and mCherry groups. It can be seen that Spastin can mediate the growth of the neurite, and UCHL1 can significantly inhibit the growth of the neurite mediated by Spastin, so that both the length of the neurite and the number of neurites are significantly increased, and the difference has statistical significance.

Furthermore, GFP-Spastin, mCherry-UCHL1, NC or siUCHL1-2 are over-expressed in primary hippocampal neurons, and after immunofluorescence chemical staining, observation is carried out under laser confocal.

The results are shown in FIGS. 21-23. Wherein, Tubulin: a microtube; GFP-Spastin: spastin green fluorescent fusion protein; mCherry-UCHL 1: UCHL1 red fluorescent fusion protein; dapi: cell nucleus; NC: negative Control; siUCHL 1-2: the small molecule interferes with UCHL 1-2. FIG. 21 shows confocal images of hippocampal cell processes; FIG. 22 is a statistical plot of the number of hippocampal processes in two groups; FIG. 23 is a statistical plot of the length of hippocampal processes in two groups. n = 30/group and results are expressed as Mean ± SEM, with scale 20 μm.

The results show that the total number of hippocampal processes and the total length of processes are significantly reduced compared to GFP-Spastin and mCherry; when GFP-Spastin, mCherry-UCHL1 and siUCHL1-2 are over-expressed, mCherry-UCHL1 red fluorescence is obviously interfered, and weak red fluorescence can be only detected in the nucleus of the hippocampal neuron under a confocal microscope, so that the total number of the hippocampal neuron processes and the total length of the processes are obviously increased. Therefore, when Spastin is present but UCHL1 is absent (no exogenously introduced or co-transformed siUCHL 1-2), Spastin can promote the increase of the number and the length of the hippocampal neurites; in the presence of Spastin and UCHL1 (exogenously introduced or co-transfected NC), the ability of Spastin to promote increased numbers and length of hippocampal processes was inhibited by UCHL1, whereas over-expression of UCHL1 alone did not detect the effect on total number and length of hippocampal processes. The over-expression of UCHL1 is proved to inhibit the function of Spastin for promoting the number and length of the hippocampal neurites, and the interference of UCHL1 can increase the total number and total length of the hippocampal neurites promoted by SpastinThe function is restored. That is, siUCHL1-2 can obviously promote the growth of the neuron processes mediated by the Spastin, so that the number and the length of the neurons can be obviously increasedpLess than 0.05, the difference is statistically significant.

By combining the above results, the invention clearly reveals the action and mechanism of UCHs in the process of neuron growth. Finding that UCHs and Spastin have interaction, particularly UCHL1, which can mediate the degradation of Spastin and inhibit the microtubule cutting function of Spastin, thereby inhibiting the growth of neuron processes; and after UCHL1 is interfered, the degradation of the Spastin can be obviously reduced, and the microtubule cutting function of the Spastin is recovered, so that the growth of the neurite is promoted. Discloses a new mechanism for regulating the growth of the UCHs neurons, and provides practical scientific basis and a new direction for the clinical treatment of spinal cord injury.

The above detailed description section specifically describes the analysis method according to the present invention. It should be noted that the above description is only for the purpose of helping those skilled in the art better understand the method and idea of the present invention, and not for the limitation of the related contents. The present invention may be appropriately adjusted or modified by those skilled in the art without departing from the principle of the present invention, and the adjustment and modification also fall within the scope of the present invention.

Sequence listing

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<120> a pharmaceutical composition comprising UCHs inhibitor and uses thereof

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<212> RNA

<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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Claims (2)

1. A pharmaceutical composition for promoting neuronal growth, comprising an inhibitor of UCHL1, wherein said inhibitor of UCHL1 is selected from the group consisting of siUCHL1 designed based on the UCHL1 gene, wherein the sequence of siUCHL1 is as set forth in SEQ ID NO: 2, respectively.

Use of an inhibitor of UCHL1 for the manufacture of a medicament for promoting neuronal growth, said inhibitor of UCHL1 being selected from the group consisting of siUCHL1 designed based on the UCHL1 gene, said siUCHL1 having the sequence as set forth in SEQ ID NO: 2, respectively.

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