Anapc11 gene / protein for screening drugs for treating hyperuricemia-related multi-organ dysfunction

Abstract

The present application belongs to the field of biotechnology and medical technology, and particularly relates to Anapc11 gene / protein for screening drugs for treating hyperuricemia related multi-organ dysfunction. The present application uses Anapc11 gene as a molecular target or a drug action target for screening or preparing drugs for treating multi-organ dysfunction caused by hyperuricemia (including liver injury, testicular dysfunction and intestinal barrier damage). The present application provides a new use of cell cycle promoting complex subunit E3 ubiquitin ligase Anapc11 gene, provides a new molecular target for treating hyperuricemia related multi-organ dysfunction, breaks the limitation of existing uric acid lowering drugs only in the metabolic link, and provides a new direction and good application prospect for developing innovative drugs with organ protection effect.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and pharmaceutical technology, specifically relating to the use of the Anapc11 gene / protein for screening drugs to treat hyperuricemia-related multiple organ dysfunction. Background Technology

[0002] Hyperuricemia (HUA) is caused by purine metabolism disorders or impaired uric acid excretion, and its global prevalence continues to rise and is showing a trend towards affecting younger people. Besides forming urate crystals and inducing gout, high uric acid can also cause damage to multiple organs, including the liver, kidneys, and heart, through mechanisms such as oxidative stress, inflammasome activation, and mitochondrial dysfunction. While existing drugs such as allopurinol and febuxostat can lower uric acid levels, their efficacy is limited and they have significant side effects, making it difficult to block the tissue damage caused by high uric acid. Therefore, there is an urgent need to establish a drug screening system based on molecular mechanisms to discover safe and effective drugs to combat organ damage caused by hyperuricemia.

[0003] The Anapc11 gene encodes the catalytic subunit of the key E3 ubiquitin ligase in the Anaphase-Promoting Complex / Cyclosome (APC / C), containing a typical RING-finger zinc finger domain, and is an important component of the ubiquitin-proteasome system (UPS). APC / C maintains cell cycle progression and metabolic homeostasis by mediating the ubiquitination and degradation of cyclins, DNA replication, and metabolic regulatory proteins. Studies have shown that Anapc11 plays a central role in stem cell self-renewal and differentiation balance, and its abnormal expression is closely related to tumorigenesis, metabolic disorders, and cellular senescence. Mechanistically, Anapc11 can regulate signaling pathways such as p53, NF-κB, and mTOR, thereby affecting oxidative stress, inflammatory responses, and cell survival. Existing research suggests that hyperuricemia can induce oxidative damage, mitochondrial dysfunction, and inflammatory activation, all of which are closely related to the disruption of protein homeostasis. It is speculated that Anapc11 may play a key regulatory role in hyperuricemia-related organ damage. However, a lack of systematic research has revealed its biological function and molecular mechanism in hyperuricemia, limiting drug screening and mechanism validation targeting Anapc11. Research in this area is expected to fill the knowledge gap between hyperuricemia-induced organ damage and protein degradation regulation, providing a theoretical and technological foundation for establishing an Anapc11-based drug screening system.

[0004] Hyperuricemia-induced multi-organ damage is closely related to protein homeostasis imbalance. The ubiquitin-proteasome system is a key mechanism for maintaining intracellular protein mass control, with the APC / C complex being one of the major E3 ligase complexes. Anapc11, as its catalytic subunit, plays a central role in regulating protein degradation and cellular stress. Abnormal function of Anapc11 may lead to protein accumulation and persistent activation of inflammatory signals, contributing to hyperuricemia-induced tissue damage. Therefore, Anapc11 can serve as a potential molecular target for screening drugs against hyperuricemia-induced organ damage. Summary of the Invention

[0005] The purpose of this invention is to address the existing problems by providing an Anapc11 gene / protein for screening drugs to treat hyperuricemia-related multiple organ dysfunction.

[0006] This invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention relates to the use of the Anapc11 gene and its protein in screening drugs for the treatment of hyperuricemia-related multiple organ dysfunction.

[0008] Secondly, the present invention relates to the use of the Anapc11 gene and its protein in the preparation of drugs for treating hyperuricemia-related multiple organ dysfunction.

[0009] Thirdly, the present invention relates to the use of the Anapc11 gene and its protein in screening drugs that inhibit liver damage induced by hyperuricemia.

[0010] Fourthly, the present invention relates to the use of the Anapc11 gene and its protein in screening drugs that inhibit intestinal damage induced by hyperuricemia.

[0011] Fifthly, the present invention relates to the use of the Anapc11 gene and its protein in screening drugs to inhibit testicular damage induced by hyperuricemia.

[0012] Preferably, the Anapc11 gene is the Anapc11 gene of a human or mouse.

[0013] b. The Anapc11 gene is used as a molecular target or drug target.

[0014] Preferably, the nucleotide sequence of the Anapc11 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0015] SEQ ID NO.1:

[0016] atgaaggtgaaaattaaatgttggaatggtgtggccacttggctctgggtagccaatgatgagaactgcggcatctgcaggatggcgtttaatggctgctgtccagactgtaaggtgcctggtgatg actgccccctcgtgtggggacagtgctcccactgcttccacatgcactgcatcctcaagtggctgaatgcgcagcaggtgcagcagcactgccccatgtgtcgccaggagtggaagttcaaagagtga

[0017] SEQ ID NO.2:

[0018] MKVKIKCWNGVATWLWVANDENCGICRMAFNGCCPDCKVPGDDCPLVWGQCSHCFHMHCILKWLNAQQVQQHCPMCRQEWKFKE

[0019] Preferably, the drug comprises plant extracts developed with Anapc11 gene as a molecular target or drug target, compounds with well-defined chemical structures, peptides, nucleic acids, polysaccharides, viral vectors, liposomes, or nanoparticles.

[0020] The present invention has the following advantages over the prior art:

[0021] 1. This invention is the first to use Anapc11 in the study of hyperuricemia-related organ damage, constructs a drug screening system and explores its regulatory mechanism, providing new ideas for the development of highly effective and low-toxicity novel targeted drugs.

[0022] 2. This invention uses the Anapc11 gene as a molecular target and drug development target to screen and prepare drugs for treating hyperuricemia-related multi-organ dysfunction. These drugs can improve the structure and function of multiple organs such as the liver, testes, and intestines by activating Anapc11 function.

[0023] 3. This invention uses the Anapc11 gene as a molecular target and drug development target to screen and prepare drugs that inhibit uric acid-induced multiple organ dysfunction. These drugs can significantly reduce liver inflammation, improve the intestinal barrier, and restore testicular function.

[0024] 4. This invention provides a new target for the treatment of multiple organ dysfunction induced by hyperuricemia, breaking through the limitations of existing uric acid-lowering drugs that are limited to the metabolic process, and providing a new direction and good application prospects for the development of innovative drugs with organ-protective effects. Attached Figure Description

[0025] Figure 1 Targeting Anapc11 can inhibit liver dysfunction induced by hyperuricemia;

[0026] Figure 2 Targeting Anapc11 can inhibit hyperuricemia-induced intestinal dysfunction;

[0027] Figure 3 Targeting Anapc11 can inhibit testicular dysfunction induced by hyperuricemia. Detailed Implementation

[0028] To further explain the present invention, the following specific embodiments are described.

[0029] Example 1: Construction of a mouse model of hyperuricemia

[0030] Eight-week-old male C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used. The hyperuricemia model group was administered a diet containing 0.125% purine combined with adenine 200 mg / kg + potassium oxonate 300 mg / kg via gavage every 2 days. The normal control group received no treatment. AAV8 intervention was initiated on day 28. After 28 days of intervention, liver, intestine, and testicular tissues of the mice were collected for experiments.

[0031] Example 2: Overexpression of Anapc11 for the treatment of liver dysfunction in hyperuricemic mice

[0032] 2.1 Adeno-associated virus 8 (AAV8) treatment

[0033] Adeno-associated virus serum type 8 (AAV8-Anapc11) overexpressing Anapc11 under the CAG promoter was purchased from Shandong Weizhen Biotechnology Co., Ltd. (Shandong, China). Twenty-four 8-week-old C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into three groups. After successful model establishment, the mice were treated with the following regimens: normal control group (n = 6), hyperuricemia group (n = 6), hyperuricemia group + AAV8-Anapc11 group (n = 6), and hyperuricemia group + AAV8-Vector group (n = 6). After deep anesthesia, the C57BL / 6 mice were injected with a titer of 8 × 10⁻⁶... 10 The viral vector was injected via tail vein, and samples were collected for experiments on the 28th day of treatment.

[0034] The AAV8-Anapc11 target sequence used for overexpression of Anapc11 is as follows:

[0035] 5' sequencing primer: CCTCTGCTAACCATGTTCAT

[0036] 3' sequencing primers: AGGATGTCCCAGGCGAAGG

[0037] 2.2 Detection of key indicators of liver function in mice

[0038] After anesthetizing mice with 10% chloral hydrate, blood samples were extracted from the inferior vena cava, and serum was obtained by sedimentation and centrifugation. The main liver function indicators (ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; ALP, Alkaline phosphatase; TBIL, Total bilirubin) were then detected.

[0039] The results are as follows Figure 1 Figure A shows that, compared with the high uric acid group, the high uric acid + AAV8-Anapc11 treatment group of mice showed reduced liver damage.

[0040] 2.3 Histopathological evaluation of liver dysfunction in mice

[0041] After euthanasia, mouse liver tissue was perfused with 4% paraformaldehyde. Tissue sections were prepared and the liver tissue structure was observed using PSR.

[0042] The results are as follows Figure 1 As shown in Figures B and C, compared with the high uric acid group, the degree of liver fibrosis was reduced in mice treated with high uric acid + AAV8-Anapc11.

[0043] 2.4 Detection of liver injury-related genes in hyperuricemic mice overexpressing Anapc11 by quantitative real-time PCR

[0044] After homogenizing and extracting mRNA from mouse liver, cDNA was obtained by reverse transcription. The expression of Anapc11 gene and liver injury-related genes TNF-α, IL-6, TGF-β1, Gsdmd and Col1a1 was detected by real-time PCR using a Roche LC480 system.

[0045] The relevant data were analyzed using the ΔΔCt method and obtained as follows: Figure 1 Figure D shows that the expression levels of liver injury-related genes TNF-α, IL-6, TGF-β1, Gsdmd, and Col1a1 were decreased in the livers of mice in the hyperuricemia group.

[0046] The primers used for real-time PCR are shown in Table 1:

[0047] Table 1

[0048]

[0049] The above experimental results indicate that in vivo administration of AAV8-Anapc11 significantly slowed the progression of liver fibrosis in mice.

[0050] Example 3: Overexpression of Anapc11 to treat intestinal dysfunction in hyperuricemic mice

[0051] 3.1 Adeno-associated virus 8 (AAV8) treatment

[0052] Adeno-associated virus serum type 8 (AAV8-Anapc11) overexpressing Anapc11 under the CAG promoter was purchased from Shandong Weizhen Biotechnology Co., Ltd. (Shandong, China). Twenty-four 8-week-old C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into three groups. After successful model establishment, the mice were treated with the following regimens: control group (n = 6), model group (n = 6), model group + AAV8-Anapc11 treatment group (n = 6), and model group + AAV8 empty vector group (n = 6). After deep anesthesia, the C57BL / 6 mice were injected with a titer of 8 × 10⁻⁶... 10 The viral vector was locally injected into the intestinal lumen, and samples were taken for experiments on the 28th day of treatment.

[0053] The AAV8-Anapc11 target sequence used for overexpression of Anapc11 is as follows:

[0054] 5' sequencing primer: CCTCTGCTAACCATGTTCAT

[0055] 3' sequencing primers: AGGATGTCCCAGGCGAAGG

[0056] 3.2 Pathological evaluation of mouse intestinal tissue

[0057] After euthanasia, the mouse intestinal tissue was fixed with 4% paraformaldehyde, and the intestinal tissue structure was observed by IHC after supporting tissue sections.

[0058] The results are as follows Figure 2 As shown in Figure A, compared with the hyperuricemia model group, the intestinal dysfunction of mice in the hyperuricemia + AAV8-Anapc11 treatment group was significantly improved.

[0059] 3.3 Detection of genes related to intestinal dysfunction in mice by real-time quantitative PCR

[0060] After homogenizing and extracting mouse intestinal mRNA, cDNA was obtained by reverse transcription. The expression of intestinal dysfunction-related genes CLDN1 and TJP1 was detected by quantitative real-time PCR using a Roche LC480 system. The relevant data were analyzed using the ΔΔCt method. Figure 2 As shown in Figure B, compared with the hyperuricemia model group, the intestinal function of mice in the hyperuricemia model + AAV8-Anapc11 group was improved, and the expression levels of intestinal dysfunction-related genes CLDN1 and TJP1 were observed to increase.

[0061] The primers used for real-time PCR are shown in Table 2 below:

[0062] Table 2

[0063]

[0064] 3.4 Western Bolt detection of proteins related to intestinal dysfunction in mice

[0065] Mouse intestinal proteins were extracted from homogenized mice using RIPA lysis buffer, followed by SDS-PAGE electrophoresis, PVDF membrane transfer under constant current, blocking with 5% skim milk powder, three TBS / T washes, incubation overnight at 4°C with antibodies against intestinal dysfunction-related proteins CLDN1 (Abcam, ab307692) and TJP1 (Abcam, ab276131), and washing followed by incubation with secondary antibodies (Cell Signaling Technology, 7076, 7074) for development. Figure 2 Figure C shows that, compared with the hyperuricemia model group, the expression levels of CLDN1 and TJP1, proteins related to intestinal dysfunction, were significantly increased in the model group + AAV8-Anapc11 group mice. Figure 2 Figure D in the figure shows the grayscale statistics of CLDN1 and TJP1 proteins.

[0066] The above experimental results indicate that in vivo administration of AAV8-Anapc11 significantly improved intestinal dysfunction in mice.

[0067] Example 4: Overexpression of Anapc11 for the treatment of testicular dysfunction in hyperuricemic mice

[0068] 4.1 Adeno-associated virus 8 (AAV8) treatment

[0069] Adeno-associated virus serum type 8 (AAV8-Anapc11) overexpressing Anapc11 under the CAG promoter was purchased from Shandong Weizhen Biotechnology Co., Ltd. (Shandong, China). Twenty-four 8-week-old C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into four groups. After successful model establishment, the mice were treated with the following regimens: control group (Control, n = 6), model group (HUA, n = 6), model group + AAV8-Anapc11 treatment group (HUA + AAV8-Anapc11, n = 6), and model group + AAV8-vector group (HUA + AAV8-Vector, n = 6). After model establishment, the C57BL / 6 mice were deeply anesthetized and treated with a titer of 8 × 10⁻⁶. 10 The viral vector was locally injected into the testicle, and samples were taken for experiments on the 28th day of treatment.

[0070] The AAV8-Anapc11 target sequence used for overexpression of Anapc11 is as follows:

[0071] 5' sequencing primer: CCTCTGCTAACCATGTTCAT

[0072] 3' sequencing primers: AGGATGTCCCAGGCGAAGG

[0073] 4.2 Detection of key functional indicators of mouse testicular tissue

[0074] Mice were anesthetized with 10% chloral hydrate, and blood samples were extracted from the inferior vena cava. After sedimentation and centrifugation, serum testosterone was obtained and the reproductive function of the mice was detected.

[0075] The results are as follows Figure 3 Figure A shows that, compared with the hyperuricemia model group, the hyperuricemia + AAV8-Anapc11 group of mice had increased serum testosterone, while the hyperuricemia + AAV8-Vector group had no effect on serum testosterone.

[0076] 4.3 Pathological evaluation of mouse testicular tissue

[0077] After euthanasia, mouse testicular tissue was fixed with 4% paraformaldehyde. Testicular tissue sections were then analyzed for structure using IHC and immunofluorescence.

[0078] The results are as follows Figure 3 As shown in Figure B, compared with the model group, immunohistochemistry and fluorescence showed that the expression of StAR and CYP11A1, key enzymes in testosterone synthesis, was increased in the hyperuricemia model group + AAV8-Anapc11 group, and the testicular function of mice was significantly improved.

[0079] 4.4 Detection of genes related to testicular dysfunction in mice by quantitative real-time PCR

[0080] After homogenizing and extracting mouse intestinal mRNA, cDNA was obtained by reverse transcription. The expression of intestinal dysfunction-related genes StAR and CYP11A1 was detected by quantitative real-time PCR using a Roche LC480 system. The relevant data were analyzed using the ΔΔCt method. Figure 3 As shown in Figure C, compared with the model group, the testicular function of mice in the model group + AAV8-Anapc11 group was improved, and the expression levels of testosterone synthesis-related genes CYP11A1 and StAR were observed to increase.

[0081] The primers used for real-time PCR are shown in Table 3 below:

[0082] Table 3

[0083]

[0084] 4.5 Western Bolt detection of proteins related to testicular dysfunction in mice

[0085] Mouse intestinal proteins were extracted from homogenized mice using RIPA lysis buffer, followed by SDS-PAGE electrophoresis, PVDF membrane transfer under constant current, blocking with 5% skim milk powder, three TBS / T washes, incubation overnight at 4°C with antibodies against StAR (GeneTex, GTX636800) and CYP11A1 (GeneTex, GTX56293) antibodies related to testicular dysfunction at 4°C, washing, and then incubation with secondary antibodies (Cell Signaling Technology, 7076, 7074) for development. Figure 3 Figure D shows that, compared with the hyperuricemia model group, the expression levels of StAR and CYP11A1, proteins related to testicular dysfunction, were significantly reduced in the hyperuricemia model group + AAV8-Anapc11 group mice. Figure 3 Figure E in the figure is Figure 3 The grayscale statistics of StAR and CYP11A1 proteins in Figure D.

[0086] The above experimental results indicate that in vivo administration of AAV8-Anapc11 significantly slowed the progression of testicular dysfunction in mice.

[0087] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of the Anapc11 gene overexpression vector in the preparation of drugs for treating hyperuricemia-related multiple organ dysfunction, characterized in that, The overexpression vector is adeno-associated virus serotype 8 carrying the Anapc11 gene under the CAG promoter; The aforementioned multi-organ dysfunction refers to liver damage, intestinal damage, or testicular damage induced by hyperuricemia.

2. The use according to claim 1, characterized in that, The nucleotide sequence of the Anapc11 gene is shown in SEQ ID NO.

1.

3. The use of Anapc11 protein in the preparation of drugs for the treatment of hyperuricemia-related multiple organ dysfunction; The aforementioned multi-organ dysfunction refers to liver damage, intestinal damage, or testicular damage induced by hyperuricemia.

4. The use according to claim 3, characterized in that, The Anapc11 protein is encoded by the Anapc11 gene as shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

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