New use of an orphan receptor GPR151

Abstract

The application discloses a new use of an orphan receptor GPR151, and particularly relates to application of the orphan receptor GPR151 in screening or preparation of a drug for preventing, diagnosing or treating colitis. The application finds that the GPR151 mRNA level is increased by about 2 times in a TNBS-induced colitis model maintenance stage through a TNBS-induced mouse colitis model, mRNA extraction of a colon and qPCR detection, and it is suggested that the GPR151 may play a role in the colitis inflammation maintenance stage. The colitis can be effectively relieved through inhibition of GPR151 expression or inhibition of the function, and it is indicated that the GPR151 participates in occurrence and development of the colitis. In conclusion, the application finds that the GPR151 can be used as a target point, and an inhibitor for inhibiting expression and function of the GPR151 can be used as an effective treatment drug for the colitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a new use of the orphan receptor GPR151, specifically the application of the orphan receptor GPR151 in screening or preparing drugs for the prevention, diagnosis or treatment of colitis. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the digestive tract, including Crohn's disease (CD) and ulcerative colitis (UC). Typical symptoms include diarrhea, rectal bleeding, abdominal pain, fatigue, and weight loss. Currently, the main factors contributing to IBD include genetic factors, abnormal immune system responses, and external environmental stimuli. Treatment for IBD typically includes medical and surgical approaches. Medical treatment generally uses medications such as glucocorticoids and TNF inhibitors, but these can lead to treatment ineffectiveness, drug resistance, or severe side effects. Therefore, in-depth research into the treatment of inflammatory bowel disease from a new perspective to identify effective drug targets has significant theoretical and practical implications.

[0003] The orphan receptor GPR151 is a member of the rhodopsin family A. GPR151 was initially identified as homologous to GPCR10, GPCR-2037, and GalRL-like glycopropeptide receptors. Phylogenetic analysis of the rhodopsin-like family sequence structure classifies GPR151 into "Class 14" along with GalR1 (glycopropeptide receptor 1), GalR2, GalR3, and Kisspeptin receptor (Kiss1R / Gpr54). Furthermore, comparable sequence analysis classifies Gpr151 into the broader "SOG" subfamily, which includes somatostatin, opioids, glycopropeptides, and Kisspeptin receptors. GPR151 expression is widespread, found in many tissues such as the gastrointestinal mucosa, small intestine, colon, spleen, and lungs. Current research reports significantly elevated GPR151 expression in the spinal cord, thereby mediating neuropathic pain. However, there are no reports of GPR151 directly involved in the development and progression of inflammation such as colitis. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a novel use for the orphan receptor GPR151. Experiments have shown that GPR151 can be used as a target, and inhibitors that suppress its expression and function can be used as effective treatments for colitis.

[0005] This invention is achieved through the following technical solution:

[0006] The application of an orphan receptor GPR151 in screening or preparing drugs for the prevention, diagnosis or treatment of colitis, using the orphan receptor GPR151 as a target.

[0007] The use of an inhibitor of the orphan receptor GPR151 in the preparation of drugs for the prevention, diagnosis or treatment of colitis.

[0008] Preferably, the inhibitor is a nucleic acid molecule that inhibits GPR151 mRNA, and the nucleic acid molecule is siRNA or shRNA;

[0009] The siRNA or shRNA has a sequence as shown in SEQ ID NO.5.

[0010] Preferably, the siRNA is modified with methoxy or cholesterol.

[0011] Preferably, the inhibitor is a recombinant vector that inhibits GPR151 mRNA, comprising a sequence of a nucleic acid molecule siRNA or shRNA capable of being transcribed to inhibit GPR151 mRNA and a vector, wherein the sequence is embedded in the vector;

[0012] The siRNA or shRNA has a sequence as shown in SEQ ID NO.5.

[0013] Preferably, the vector is a lentiviral vector or an adeno-associated virus vector.

[0014] A pharmaceutical composition for the prevention, diagnosis, or treatment of colitis, comprising an inhibitor of the orphan receptor GPR151.

[0015] Preferably, it also includes a pharmaceutically acceptable carrier.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention, through mining public GEO databases and using R language analysis, ultimately selected the gene GPR151, which had not been reported to have anti-inflammatory functions and could be experimentally verified. Subsequently, using a TNBS-induced mouse colitis model, colonic mRNA was extracted and analyzed by qPCR. The results showed that GPR151 mRNA levels increased approximately two-fold during the maintenance phase of the TNBS-induced colitis model, suggesting that GPR151 may play a role in the maintenance phase of colitis inflammation. Inhibiting GPR151 expression or function effectively alleviated colitis, indicating that GPR151 is involved in the development and progression of colitis. In summary, this invention experimentally demonstrates that GPR151 can be used as a target, and inhibitors that suppress its expression and function can be used as effective therapeutic drugs for colitis. Attached Figure Description

[0018] Figure 1The figures show the changes in various parameters in the TNBS-induced mouse colitis model of Example 1: A represents the change in mouse body weight after TNBS-induced colitis; B represents the change in mouse colon length after TNBS-induced colitis; and C represents the change in GPR151 gene expression in the mouse colon after TNBS-induced colitis. The data in the figures represent mean ± SEM (n = 3 or 4). Compared with the specified group, * P < 0.05 *** P < 0.001;

[0019] Figure 2 To illustrate the effect of GPR151-shRNA on the progression of TNBS-induced colitis in Example 2: A shows the effect of GPR151-shRNA on colon length in colitis-affected mice, and B shows the effect of GPR151-shRNA on body weight in colitis-affected mice; data in the figures represent means ± SEM (n = 3 or 4); compared to the specified group, ** P < 0.01;

[0020] Figure 3 To illustrate the progress of GPR151 gene knockout in suppressing TNBS-induced colitis in mice in Example 2: A shows colon tissue specimens from each group of mice; B shows the effect of GPR151 gene knockout on colon length in colitis-affected mice; C shows the effect of GPR151 gene knockout on body weight in colitis-affected mice; D shows HE staining results; data in the figure represent means ± SEM (n = 3 or 4); compared with the specified group, * P < 0.05 ** P < 0.01, *** P < 0.001. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0022] Example 1

[0023] A single enema of TNBS (2,4,6-trinitrobenzenesulfonic acid) with ethanol to induce inflammation is currently the most commonly used method for establishing colitis models. TNBS treatment in mice can establish a preclinical model of colitis that mimics clinical cases; the resulting immune response is Th1-mediated and characterized by CD4+. + Infiltration of T cells, neutrophils, and macrophages leads to transmural inflammation, resulting in transcolonial colitis. This model is prolonged, fully demonstrating the transition from acute to chronic inflammation, and exhibits a high degree of similarity to clinical colitis. Therefore, this embodiment uses a TNBS-induced mouse colitis model to extract colonic mRNA for qPCR detection to observe GPR151 expression during the development and progression of TNBS-induced colitis. The specific steps are as follows:

[0024] 1. Establishment of a TNBS-induced mouse colitis model

[0025] C57BL / 6J mice that were fasted for 24 hours but allowed free access to water were randomly divided into a 50% alcohol control group and a TNBS colitis model group, with 4 mice in each group.

[0026] (1) 50% alcohol control group: 100 μL of 50% alcohol was given as an enema once.

[0027] (2) TNBS model group: After one enema of 100 μL of 2.5% TNBS / 50% alcohol, the experiment ended on the 5th day.

[0028] The daily weight changes of mice in each group were monitored. After the mice were sacrificed, colon tissue specimens were collected and the length of the colon was recorded. The expression level of GPR151 mRNA in the mouse colon tissue was detected by RT-PCR.

[0029] 2. RNA extraction and real-time quantitative PCR

[0030] Total RNA was extracted using the Trizol method. After determining the concentration, 1 μg of total RNA was taken and reverse transcribed according to the instructions of the Vazyme HiScript II 1st Strand cDNA Synthesis Kit. The obtained cDNA product was diluted 8-fold and used as template for real-time quantitative PCR. 2 μL of template was added to each PCR system, and the PCR was performed according to the instructions of the Vazyme AceQ qPCR SYBR GreenMaster Mix. The primer sequences used are shown in Table 1 below. Results were analyzed using 2- ΔΔCT Legal analysis.

[0031] Table 1 Primer sequences

[0032] GPR151forward 1 5'-CCTAGTAGCTCCAAGTCAGACA-3' GPR151reverse 2 5'-ACCTTAGGTGGTCTTTGAGTCTC-3' GAPDH forward 3 5'-AAATGGTGAAGGTCGGTGTGAAC-3' GAPDH reverse 4 5'-CAACAATCTCCACTTTGCCACTG-3'

[0033] 3. Experimental Results

[0034] (1) TNBS induces colitis in mice

[0035] TNBS induced colitis in mice via enema, such as Figure 1 As shown in Figure A, compared with the 50% alcohol control group (Ctrl), the TNBS model group showed a significant decrease in body weight starting from day 3 after drug administration (n=4, * P < 0.05). At the end of the experiment, the mice were sacrificed and their colons were collected. The length of the mouse colon was measured. Figure 1 As shown in Figure B, the results indicate that, compared with the 50% alcohol control group, the colon length of mice in the TNBS model group was significantly shorter (n=4, *(P < 0.05). Weight loss and colon shortening indicate that TNBS induces inflammation in mice, and the colitis model was successfully established.

[0036] (2) Increased GPR151 gene expression in a TNBS-induced mouse colitis model

[0037] Colitis was induced in mice via TNBS enema. At the end of day 5, mouse colons were collected, and the expression of the GPR151 gene in the colon was analyzed by qPCR. Figure 1 As shown in Figure C, GPR151 mRNA expression in the colon of the TNBS model group was significantly increased (n=3, *** (P < 0.001) GPR151 mRNA levels increased approximately two-fold during the maintenance phase of the TNBS-induced colitis model. This indicates that GPR151 is involved in the development and progression of colitis.

[0038] Example 2: Effect of inhibiting GPR151 expression on colitis

[0039] 1. Design and synthesis of mouse GPR151 shRNA

[0040] The full-length sequence of mouse GPR151 mRNA (NM_181543.1) was obtained from the National Center for Biotechnology Information (NCBI) database. Based on the principles of RNAi and using design software, three candidate shRNAs targeting the mouse GPR151 gene transcript were designed. After BLAST alignment to ensure no homology with other genes, they were chemically synthesized.

[0041] 2. Screening for highly efficient GPR151-shRNA targets

[0042] With 2×10 5 Cells were seeded at a rate of (number of cells / well) into 24-well plates. When the cell confluence reached 70%–90%, a GPR151-shRNA interference efficiency screening experiment was performed. Cells were collected 48 h after transfection, and the interference effect of GPR151-shRNA was identified by qPCR.

[0043] The results showed that only one of the three candidate shRNAs had an interference efficiency greater than 60%, so this candidate shRNA with an interference efficiency greater than 60% was finally selected. The target sequence information of this GPR151-shRNA is shown in Table 2 below.

[0044] Table 2 Target sequence information for GPR151-shRNA and NC-shRNA (control)

[0045]

[0046] 3. Effects of GPR151 shRNA on the progression of colitis

[0047] C57BL / 6J mice were randomly divided into an NC-shRNA control group and a GPR151-shRNA group, with a normal control group receiving no treatment. Three mice were in each group. The specific steps are as follows:

[0048] (1) NC-shRNA control group: Mice were injected with NC-shRNA (as shown in SEQ ID NO.6);

[0049] (2) GPR151-shRNA group: Mice were injected with GPR151-shRNA (as shown in SEQ ID NO.5);

[0050] (3) Normal control group: Mice were not treated in any way.

[0051] C57BL / 6J mice were intraperitoneally injected with GPR151-shRNA or NC-shRNA for three consecutive weeks. One hour after the shRNA injection on day 3, the mice were given a single enema of 100 μL of 2.5% TNBS / 50% alcohol. The experiment concluded at week 4. Daily weight changes in the mice were monitored, colonic tissue specimens were collected, colonic length was recorded, and the degree of inflammation in the mouse colonic tissue was observed and scored using HE staining. The expression level of GPR151 mRNA in the mouse colonic tissue was detected by RT-PCR.

[0052] like Figure 2 As shown in Figures A and B, compared with the NC-shRNA group, injection of GPR151-shRNA in mice significantly inhibited TNBS-induced colitis. ** P < 0.01, n = 3 or 4).

[0053] 4. Effects of GPR151 gene knockout mice (GPR151KO mice) on the progression of colitis

[0054] WT C57BL / 6 mice and GPR151KO mice were given an enema of 2.5% TNBS once, and the weight changes of the mice were monitored until the end of the experiment on day 5.

[0055] like Figure 3 As shown in Figures A and B, compared with WT C57BL / 6J mice, GPR151KO mice significantly inhibited the shortening of colon length induced by colitis in mice. * P < 0.05, n = 3). For example... Figure 3 As shown in Figure C, compared with WT C57BL / 6J mice, GPR151KO mice significantly inhibited the weight loss induced by colitis in mice. ** P < 0.01 or ***P < 0.001, n = 4). Figure 3 HE staining results showed that, compared with WT C57BL / 6J mice, GPR151KO mice significantly suppressed TNBS-induced immune cell infiltration. These results indicate that inhibiting GPR151 expression can alleviate the progression of TNBS-induced colitis.

[0056] This study found that inhibiting GPR151 expression or function can effectively alleviate colitis, indicating that GPR151 is involved in the occurrence and development of colitis. Furthermore, shRNA targeting the GPR151 gene transcript was designed and recombined with an adeno-associated virus vector to construct a recombinant adeno-associated virus vector interfering with the GPR151 gene. This interfering virus was used to demonstrate that inhibiting GPR151 expression can alleviate the progression of colitis.

Claims

1. The application of a primer pair for detecting the expression level of the orphan receptor GPR151 gene in the preparation of drugs for diagnosing inflammatory bowel disease.

2. The use of an inhibitor of the orphan receptor GPR151 in the preparation of a medicament for treating inflammatory bowel disease, characterized in that, The inhibitor is a nucleic acid molecule that inhibits GPR151 mRNA, and the nucleic acid molecule is shRNA; the target sequence of the shRNA is shown in SEQ ID NO.

5.

3. The use of an inhibitor of the orphan receptor GPR151 in the preparation of a medicament for treating inflammatory bowel disease, characterized in that, The inhibitor is a recombinant vector that inhibits GPR151 mRNA, comprising a sequence of a nucleic acid molecule shRNA capable of being transcribed to inhibit GPR151 mRNA and a vector, wherein the sequence is embedded in the vector; the target sequence of the shRNA is shown in SEQ ID NO.5; and the vector is an adeno-associated virus vector.

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