Application of Chchd10 overexpression reagent in preparation of medicine for treating diseases

Abstract

The invention discloses application of a Chchd10 overexpression reagent in preparation of a medicine for treating diseases, and belongs to the technical field of biological medicines. Researches find that Chchd10 is remarkably expressed in Ube2c + PC, especially is highly expressed in Ube2c + PC in an inflammation recovery stage (D9), ROS up-regulation can be effectively inhibited by up-regulating the expression quantity of Chchd10, and meanwhile, expression of pro-inflammatory molecules (IL-1beta and IL-6) of plasma cells can be reduced, so that recovery of ulcerative colitis is promoted, and a new scheme and thought are provided for treatment of ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of a Chchd10 overexpression reagent in preparing a drug for treating a disease. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory disease of the rectum and colon. However, the etiology and pathophysiology of ulcerative colitis remain unknown. Environmental influences, genetic factors, and imbalances in the microbiota are reported to drive immune responses and inflammatory processes. Activation of the intestinal mucosal immune response is a direct cause of the development and progression of intestinal inflammation in ulcerative colitis. In patients with ulcerative colitis, primary lesions show plasma cell (PC) infiltration in the lamina propria. The mucosal immune system consists of the innate immune system and the adaptive immune system, which play a major role in the pathophysiology of ulcerative colitis disease. T cells and B cells make up the majority of the adaptive immune system. The pathophysiology of ulcerative colitis is thought to be driven by a Th2 response, while little attention has been paid to the role of B lymphocytes in intestinal immune homeostasis in ulcerative colitis. Plasma cells in the intestinal mucosa mainly secrete immunoglobulin A (IgA), which is a barrier that protects the mucosal epithelium from pathogens, toxins, and food antigens. At the same time, IgA can help maintain the stability of the intestinal microbiota. Plasma cells play an important role in maintaining the stability of the intestinal immune microenvironment. Optimizing the intestinal microbial community can reduce the severity of intestinal inflammation. Therefore, changes in plasma cells in colitis may affect the process of inflammatory resolution to a certain extent. A study reported that plasma cells accounted for a large proportion of lymphocytes in the inflammatory infiltration of ulcerative colitis, and the expansion of these cells was associated with the risk of disease recurrence. Therefore, plasma cells may play a key role in the course of ulcerative colitis.

[0004] Treatment options for ulcerative colitis include non-targeted drugs (such as aminosalicylic acid and glucocorticoids) and targeted therapies (such as anti-tumor necrosis factor and anti-interleukin-12). Although targeted therapies are beneficial for many patients, a small number of patients will experience treatment failure or resistance. A deeper understanding of the mechanisms of the occurrence and resolution of colitis inflammation and the discovery of new therapeutic targets will provide new ideas for exploring more effective disease control methods. Summary of the invention

[0005] In view of this, the present invention provides an application of a Chchd10 overexpression reagent in preparing a drug for treating a disease, and the present invention provides a new idea for the treatment of calcific ulcerative colitis.

[0006] In a first aspect, the present invention provides a use of a Chchd10 overexpression reagent in the preparation of a medicament for treating ulcerative colitis.

[0007] Chchd10 is a coiled-coil domain protein, and its amino acid sequence may be substantially identical to the sequence shown in GenBank accession number 103172, or may be substantially identical to the sequence shown in other variant sequence numbers. The amino acid sequence of the Chchd10 protein formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. The Chchd10 protein or its biologically active fragment includes a replacement sequence of a portion of conservative amino acids, and the sequence replaced with amino acids does not affect its activity or retains part of its activity. Appropriate replacement of amino acids is a well-known technique in the art, which can be easily implemented and ensures that the biological activity of the resulting molecule is not changed. In general, changing a single amino acid in a non-essential region of a polypeptide will not substantially change the biological activity.

[0008] Preferably, the Chchd10 overexpression reagent is an overexpression vector containing Chchd10.

[0009] Furthermore, the overexpression vector includes an overexpression plasmid or a lentiviral vector.

[0010] Furthermore, the overexpression vector is an overexpression plasmid.

[0011] Preferably, the drug for treating ulcerative colitis is a pharmaceutical composition prepared from a Chchd10 overexpression agent and a pharmaceutically acceptable carrier.

[0012] Furthermore, the dosage form of the pharmaceutical composition is capsule, granule, tablet, pill, oral solution or injection.

[0013] Furthermore, the pharmaceutically acceptable carrier includes one or more of a filler, a binder, a lubricant, a surfactant, a coating agent, a capsule shell, a taste masking agent, a flavoring agent, a solvent, a co-solvent, a suspending agent, a pH adjuster or an antioxidant.

[0014] Preferably, the Chchd10 overexpression reagent has one or more of the following effects:

[0015] Inhibit ROS upregulation;

[0016] Reduce the expression of IL-1β;

[0017] Reduce the expression of IL-6.

[0018] In a second aspect, the present invention provides the use of Chchd10 as a target in screening drugs for treating ulcerative colitis.

[0019] Preferably, the drug for treating ulcerative colitis comprises a pharmaceutically acceptable carrier.

[0020] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0021] The present invention provides an application of a Chchd10 overexpression reagent in the preparation of a drug for treating ulcerative colitis. Through research, it is found that Chchd10 is significantly expressed in proliferating plasma cells (Ube2c+PC), especially highly expressed in Ube2c+PC in the inflammation recovery period (D9), and up-regulating the expression of Chchd10 can effectively inhibit the upregulation of ROS, and at the same time can reduce the expression of pro-inflammatory molecules (IL-1β and IL-6) of plasma cells, thereby promoting the recovery of ulcerative colitis, and providing a new solution and idea for the treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 The single-cell sequencing analysis of the expression of different genes in Ube2c+PCs at different stages of inflammation in Example 1 of the present invention;

[0024] Figure 2 The changes in Chchd10 protein expression in Ube2c+PC at different stages were determined by qPCR in Example 2 of the present invention;

[0025] Figure 3 The changes in Chchd10 protein expression in Ube2c+PC at different stages were determined by flow cytometry in Example 2 of the present invention;

[0026] Figure 4It is the efficiency of Chchd10 in plasma cells under siRNA transfection and the intracellular level of ROS in siNC and siChchd10 plasma cells verified in Example 3 of the present invention; wherein A is the efficiency of Chchd10 in plasma cells under siRNA transfection verified by qPCR; B is the efficiency of Chchd10 in plasma cells under siRNA transfection verified by western blot; C is the intracellular level of ROS in siNC and siChchd10 plasma cells measured by flow cytometry using DCFH-DA;

[0027] Figure 5 It is the efficiency of verifying Chchd10 in plasma cells by plasmid transfection in Example 4 of the present invention; wherein A is the efficiency of verifying Chchd10 in plasma cells by plasmid transfection through qPCR, and B is the efficiency of verifying Chchd10 in plasma cells by plasmid transfection through western blot;

[0028] Figure 6 The present invention relates to the effect of overexpressing Chchd10 on the mRNA expression of IL-1β and IL-6 in colon plasma cells and the effect of ROS levels in the DSS-induced colitis model of Example 4; wherein A is the effect on the mRNA expression of IL-1β, B is the effect on the mRNA expression of IL-6, and C is the intracellular level of ROS in Control and OE-Chchd10 plasma cells measured by flow cytometry using DCFH-DA. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0030] The technical solution of the present invention is further described below in conjunction with specific examples. The present invention has no particular limitation on the source of the reagents used in the following examples, and commercially available products known to those skilled in the art can be used.

[0031] Example 1

[0032] Single-cell sequencing revealed that Chchd10 expression was increased in plasma cells of the DSS-induced ulcerative colitis model.

[0033] 1. Experimental methods

[0034] (1) Establishment of DSS model of ulcerative colitis in mice:

[0035] Six-week-old male C57BL / 6 mice were housed in an animal room with an ambient temperature of 25°C and a 12-hour light cycle (12 hours of light and 12 hours of darkness), with free access to food and water. After one week of adaptive feeding, they were randomly divided into groups. Adding 2.2% dextran sulfate sodium (DSS) to drinking water for 5 days can induce colitis. Colon tissues were collected at 0 days (D5), 4 days (D9) and 9 days (D14) after drug withdrawal and normal controls (D0). Single-cell sequencing samples were obtained from the colorectal region of mice.

[0036] (2) Isolation of lamina propria cells from colonic mucosa

[0037] The colorectal cleansing reagent consists of HBSS, 5% FBS, and 2mM EDTA, which is preheated to 37°C. After euthanizing C57BL / 6 mice, the colorectum of the mice was dissected from the abdomen and placed in a culture dish filled with PBS. After dissection, the colon was cut into three sections for cleaning and placed in a preheated 50mL centrifuge tube containing 30mL of the cleaning reagent. Shake on a shaker at 37°C and 250rpm for 20 minutes. Repeat this operation once after changing the colorectal cleansing reagent.

[0038] The colorectal digestion reagent consists of 1.5 mg / mL type IV collagenase, 40 μg / mL DNaseI, 5% FBS, and HBSS. Cut the colorectal fragments into small pieces for digestion. Place the minced tissue in a 50 mL centrifuge tube containing 20 mL of colorectal digestion reagent. Oscillate the mixture at 37°C and 200 rpm for 35 minutes, and vortex the remaining intestinal tissue for 5-10 seconds. Then filter the cell suspension through a 100 μm cell strainer and centrifuge at 1500 rpm for 5 minutes at 4°C. Discard the supernatant, resuspend the cells in 50 mL PBS, and then centrifuge at 1500 rpm for 5 minutes at 4°C. Repeat this step once to clean the cells.

[0039] (3) Flow cytometry sorting of colon immune cells

[0040] FITC anti-CD45, PE-cy7 anti-CD11c, APC anti-LY6G, APC anti-FcεRI, APC anti-Siglec-F, APC anti-CD19, APC anti-TCRβ, APC anti-TCRγ / δ antibodies and Zombie NIR were added to the above cell suspension. TM The viability kit can be fixed and incubated in the dark for 30 minutes. Subsequently, 3 mL of PBS was added to the tube and then centrifuged at 1500 rpm for 5 minutes. The supernatant was removed and the mixture was washed with 10% PBS and 4% PBS. 7The cells were resuspended in PBS at a ratio of 10 cells / 1 mL. The CD45+ Zombie NIR-CD11c- APC- cells separated by flow cytometry were added to a 1.5 mL centrifuge tube containing PBS containing 1% FBS.

[0041] (4) scRNA-seq analysis

[0042] Singleron The platform was used for scRNA-seq. Trypan blue staining was used to determine cell viability and concentration. scRNA-seq experiments were performed when cell viability was >80% and concentration was between 700 and 1200 cells / μL (volume not less than 100 μL). The platform injects the cell suspension into the SCOPE-chip microfluidic chip to complete the isolation of single cells. Millions of magnetic beads with unique cell barcodes are added to the chip microwells to ensure that only one magnetic bead falls into each microwell. After cell lysis, magnetic beads with unique cell labels (barcodes) and molecular labels (UMIs) capture mRNA by binding to the poly (A) tail on the mRNA, thereby labeling the cells and mRNA. The magnetic beads are collected from the chip. The mRNA captured by the magnetic beads is reverse transcribed into cDNA and amplified. Library preparation is performed using the GEXSCOPE Library Suite and sequencing is performed using the Illumina NovaSeq 6000.

[0043] (5) Processing of scRNA-seq data

[0044] FASTQ data were aligned to the GRCm38 reference sequence of the mouse genome. Seurat v4, a tool set for scRNA-seq data analysis, was used to process barcode matrices. Low-quality cells (number of genes less than 200 or greater than 4000, percentage of mitochondrial genes greater than 10%) were first removed. Subsequently, logarithmic transformation was used to normalize the UMI count data. To aggregate the samples into a merged dataset, the top 2000 highly variable genes (HGVs) were selected. The “RunHarmony” function in the harmony package was used to eliminate batch effects between individuals. Cell clusters were then detected using the “FindClusters” function in Seurat and described using uniform manifold approximation and projection (UMAP). Differentially expressed genes (DEGs) were evaluated using the “FindMarkers” or “FindAllMarkers” function with default parameters. The same approach was applied to discover variable genes, dimensionality reduction, and clustering for subclustering analysis. Cell clustering and subclustering were performed using the FindClusters function of the Seurat software package (resolution = 0.14).

[0045] 2. Experimental results

[0046] Single-cell sequencing analysis identified nine major cell types based on the marker genes of each cell cluster, namely mature PCs, Hspa1a+PCs, Ighv7-3 PCs, Ube2c+PCs, Igkv6-15 PCs, Igkv17-121 PCs, monocytes, neutrophils, and fibroblasts. Figure 1 Single-cell sequencing was used to analyze the expression of different genes in Ube2c+PCs at different stages of inflammation. It can be seen that genes regulating apoptosis signaling pathways are highly expressed in D9, such as Pycard, Gpx1, Lgals3 and Chchd10. Among them, Pycard, Gpx1 and Lgals3 have been shown to be involved in the process of inflammation resolution, but Chchd10 has not been reported.

[0047] Example 2

[0048] Experiments verified that Chchd10 expression was increased in plasma cells of the DSS-induced ulcerative colitis model.

[0049] 1. Experimental methods

[0050] (1) RNA isolation and quantitative real-time polymerase chain reaction (qPCR)

[0051] Total RNA was extracted from cells using the TRIzol method. Reverse transcription was performed according to the instructions of Takara's PrimeScript II FirstStrand cDNA Synthesis Kit. Sample loading was performed according to the instructions of Takara's SYBR Green Gene Expression Detection Kit. qPCR was performed using the Roche Applied Science LightCycler480II Real-Time PCR System. Primer sequences used:

[0052] Chchd10 upstream primer: 5′-CCACCACTCAGAGCGACCTAAC-3′;

[0053] Chchd10 downstream primer: 5′-AGCTCAGACCGTGATTGTATTTGC-3′;

[0054] GAPDH upstream primer: 5′-ATTCAACGGCACAGTCAAGGC-3′;

[0055] GAPDH downstream primer: 5′-ACACCAGTAGACTCCACGACATAC-3′.

[0056] (2) Flow Cytometry

[0057] FITC anti-CD45, PE-cy7 anti-CD11c, APC anti-LY6G, APC anti-FcεRI, APC anti-Siglec-F, APC anti-CD19, APC anti-TCRβ, APC anti-TCRγ / δ antibodies and Zombie NIR were added to the colon cell suspension. TM The viability kit can be fixed and incubated in the dark for 30 minutes. Add 3 mL of PBS to the test tube and centrifuge at 1500 rpm for 5 minutes. Remove the supernatant and perform the experiment according to the instructions of the Lianke fixation and permeabilization kit. Then add antibodies for Ube2c and Chchd10 and incubate at 4°C overnight. On the second day, centrifuge at 1500 rpm for 5 minutes. Remove the supernatant, add the corresponding secondary antibodies for Ube2c and Chchd10, incubate at room temperature for 1 hour, and then centrifuge at 1500 rpm for 5 minutes. Remove the supernatant and centrifuge at 10 7 Resuspend the cells in PBS at a ratio of 10 cells / 1 mL.

[0058] 2. Experimental results

[0059] like Figure 2 The qPCR results showed that in the DSS-induced colitis model, the mRNA expression level of Chchd10 in colonic immune cells fluctuated at different stages and was highly expressed at D9. Figure 3 Flow cytometry analysis showed that Chchd10+Ube2c+PC was highly expressed at D9.

[0060] Example 3

[0061] Downregulation of Chchd10 increased oxidative stress (ROS) levels in plasma cells.

[0062] 1. Experimental methods

[0063] (1) Isolation and culture of plasma cells

[0064] According to EasySep TM Instructions for use of the Mouse CD138 Positive Selection Kit Collect colon mucosal lamina propria cells to isolate CD138+ plasma cells. CD138+ plasma cells were cultured in RPMI-1640 medium containing 10% FBS, 1% penicillin-streptomycin solution, 10 mM HEPES, 1 mM sodium pyruvate and 55 μM 2-ME.

[0065] (2) Transfection of siRNA

[0066] The cells were plated at 5×10 5Cells were seeded in 6-well plates at a density of 10 cells / well. siRNA transfection was performed using Lipofectamine 3000 according to the instructions. The downregulation efficiency of siRNA was evaluated by qPCR 24 h after transfection and western blot 48 h after transfection.

[0067] Small interfering RNA (siRNA) of Chchd10 and its negative control siRNA (siNC) were purchased from Hippobio (Huzhou, China).

[0068] The sequences of siRNA are listed below:

[0069] siChchd10-1: 5'-GGCCCUCAAACAGUGCAAAUATT-3';

[0070] siChchd10-2: 5'-GCAGACUAACAGGAGACAUAATT-3';

[0071] siNC: 5'-UUCUCCGAACGUGUCACGU-3'.

[0072] (3) Western blot

[0073] Cells were lysed in a protein extraction reagent containing protease inhibitors and phosphatase inhibitors, and proteins were separated by SDS-PAGE and transferred to a PVDF membrane after electrophoresis. Then, the membrane was blocked with 5% milk at room temperature for 1 hour, and after washing, the membrane was incubated in an antibody against Chchd10 (1:2000) at 4°C overnight. The next day, the secondary antibody (1:3000) was used for incubation at room temperature for 1 hour. The protein bands were developed using the Tanon5200 chemiluminescence imaging system (Shanghai, China).

[0074] (4) Intracellular oxidative stress (ROS) detection

[0075] The cells were resuspended in RPMI-1640 medium containing 10 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and incubated for 30 min at 37° C. Flow cytometry was used to assess intracellular ROS.

[0076] 2. Experimental results

[0077] To further investigate the functional role of Chchd10 in PC, PC were isolated from colonic tissues of DSS-induced colitis mice at D9. Flow cytometry was used to detect the purity of PC obtained by CD138 magnetic bead sorting. qPCR and western blot experiments verified the downregulation efficiency of siRNA in PC. Figure 4A in the figure is the efficiency of down-regulating Chchd10 in plasma cells verified by qPCR. It can be seen that Chchd10 is significantly down-regulated at the mRNA level; Figure 4 B in the figure verifies the efficiency of Chchd10 down-regulation in plasma cells by siRNA transfection through western blot. It can be seen that Chchd10 is significantly down-regulated at the protein level. Figure 4 C in the figure shows the intracellular level of ROS in siNC and siChchd10 plasma cells measured by flow cytometry using DCFH-DA. It can be seen that the level of ROS in siChchd10 plasma cells is higher than that in siNC plasma cells. From the above results, it can be seen that downregulation of Chchd10 leads to a significant increase in the level of ROS in PC, indicating that Chchd10 plays a protective role against oxidative stress.

[0078] Example 4

[0079] Upregulation of Chchd10 can alleviate the pro-inflammatory level of plasma cells.

[0080] 1. Experimental methods

[0081] (1) Plasmid transfection

[0082] The cells were plated at 5×10 5 Cells were seeded in 6-well plates at a density of 10 cells / well. Chchd10 overexpression plasmid transfection was performed using Lipofectamine 3000 according to the instructions. The overexpression efficiency of the plasmid was evaluated by qPCR 24 h after transfection and western blot 48 h after transfection.

[0083] (2) RNA isolation and quantitative real-time polymerase chain reaction (qPCR)

[0084] Total RNA was extracted from cells using the TRIzol method. Reverse transcription was performed according to the instructions of Takara's PrimeScript II FirstStrand cDNASynthesis Kit. Sample loading was performed according to the instructions of Takara's SYBR Green Gene Expression Detection Kit. qPCR was performed using the Roche Applied Science LightCycler 480II Real-Time PCR System.

[0085] (3) Western blot

[0086] Cells were lysed in a protein extraction reagent containing protease inhibitors and phosphatase inhibitors, and proteins were separated by SDS-PAGE and transferred to a PVDF membrane after electrophoresis. Then, the membrane was blocked with 5% milk at room temperature for 1 hour, and after washing, the membrane was incubated in an antibody against Chchd10 (1:2000) at 4°C overnight. The next day, the secondary antibody (1:3000) was used for incubation at room temperature for 1 hour. The protein bands were developed using the Tanon5200 chemiluminescence imaging system (Shanghai, China).

[0087] (4) Intracellular oxidative stress (ROS) detection

[0088] The cells were resuspended in RPMI-1640 medium containing 10 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and incubated for 30 min at 37° C. Flow cytometry was used to assess intracellular ROS.

[0089] 2. Experimental results

[0090] Figure 5 A in the figure shows the efficiency of up-regulating Chchd10 in plasma cells verified by plasmid transfection by qPCR. It can be seen that Chchd10 is significantly up-regulated at the mRNA level; Figure 5 B in the figure shows the efficiency of up-regulating Chchd10 in plasma cells verified by plasmid transfection by western blot. It can be seen that Chchd10 is significantly up-regulated at the protein level. Figure 6 Figures A and B show the effects of overexpression of Chchd10 on the mRNA expression of IL-1β and IL-6 in colonic plasma cells in the DSS-induced colitis model. It can be seen that upregulation of Chchd10 can reduce the expression of pro-inflammatory molecules (IL-1β and IL-6) in plasma cells. Figure 6 Figure C shows the effect of overexpression of Chchd10 on ROS in colon plasma cells in a DSS-induced colitis model. It can be seen that upregulation of Chchd10 can inhibit the level of ROS. The above results indicate that Chchd10 can promote inflammation repair.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a Chchd10 overexpression reagent in the preparation of a drug for treating ulcerative colitis.

2. The use according to claim 1, characterized in that The Chchd10 overexpression reagent is an overexpression vector containing Chchd10.

3. The use according to claim 2, characterized in that The overexpression vector includes an overexpression plasmid or a lentiviral vector.

4. The use according to claim 3, characterized in that The overexpression vector is an overexpression plasmid.

5. The use according to claim 1, characterized in that The medicine for treating ulcerative colitis is a pharmaceutical composition prepared from a Chchd10 overexpression reagent and a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that The dosage form of the pharmaceutical composition is capsule, granule, tablet, pill, oral solution or injection.

7. The use according to claim 5, characterized in that The pharmaceutically acceptable carrier includes one or more of a filler, a binder, a lubricant, a surfactant, a coating agent, a capsule shell, a taste masking agent, a flavoring agent, a solvent, a co-solvent, a suspending agent, a pH adjuster or an antioxidant.

8. The use according to claim 1, characterized in that The Chchd10 overexpression reagent has one or more of the following effects: Inhibit ROS upregulation; Reduce the expression of IL-1β; Reduce the expression of IL-6.

9. Application of Chchd10 as a target in screening drugs for the treatment of ulcerative colitis.

10. The use according to claim 9, characterized in that The drug for treating ulcerative colitis comprises a pharmaceutically acceptable carrier.

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