Use of xanthine dehydrogenase gene inhibitor in preparation of anti-hepatic fibrosis drugs

Abstract

This invention discloses the application of xanthine dehydrogenase gene inhibitors (Xdh gene) in the preparation of anti-liver fibrosis drugs. Using sgXdh, a guide RNA targeting the mouse Xdh gene, as an example, this invention investigated the application value of Xdh gene inhibitors in the preparation of anti-liver fibrosis drugs through in vivo mouse experiments. The results showed that in a mouse model of liver fibrosis, knocking out the Xdh gene with sgXdh reduced the expression of hepatic xanthine dehydrogenase and decreased the levels of liver fibrosis markers. This indicates that targeted inhibition of the Xdh gene can alleviate hepatic stellate cell activation and inhibit the progression of liver fibrosis, suggesting that targeted inhibition of the human Xdh gene can be used for the treatment of liver fibrosis. Xdh gene inhibitors can be used to prepare anti-liver fibrosis drugs and have promising clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the pharmaceutical use of xanthine dehydrogenase gene (Xdh gene) inhibitors. Background Technology

[0002] Liver fibrosis is an abnormal repair response of the liver under the continuous stimulation of various factors (such as hepatitis viruses and alcohol). It is characterized by excessive deposition and abnormal distribution of extracellular matrix (such as type I collagen, type III collagen, and fibronectin) in the liver, leading to changes in liver structure and function. If not intervened in time, liver fibrosis can further develop into cirrhosis, which can then lead to a series of decompensated events such as portal hypertension, esophageal and gastric variceal bleeding, ascites, and hepatic encephalopathy, and significantly increase the risk of hepatocellular carcinoma.

[0003] Current research demonstrates that early-stage liver fibrosis is reversible if the underlying cause is effectively controlled or eliminated. For example, patients with chronic hepatitis B (CHB) and liver fibrosis have a high reversal rate after effective antiviral therapy. Studies show that after 78 weeks of continuous and effective antiviral therapy, liver fibrosis can be reversed in 58% of CHB patients; after 5 years of antiviral therapy, the reversal rate in patients with chronic hepatitis B cirrhosis can reach 73%. Currently, there are no officially approved chemical drugs or biological agents specifically for treating liver fibrosis. Although some traditional Chinese medicine preparations used clinically (such as Compound Turtle Shell Softening Liver Tablets) have shown some efficacy, the evidence-based medicine and mechanistic studies are still insufficient. Therefore, there is an urgent need to find new intervention targets or anti-liver fibrosis drugs.

[0004] Xanthine dehydrogenase (XDH) is the rate-limiting enzyme in purine metabolism, converting hypoxanthine to xanthine, and then xanthine to uric acid. XDH protein levels are associated with the occurrence and development of various diseases, including hyperuricemia and gout. XDH inhibitors (such as allopurinol and febuxostat) have been used to treat hyperuricemia and gout, but their role in liver fibrosis remains unclear. Summary of the Invention

[0005] The purpose of this invention is to explore the anti-fibrotic mechanism with the Xdh gene as a direct target, in order to provide new treatment options and drugs for the treatment of liver fibrosis.

[0006] Based on research, the present invention provides the following technical solution:

[0007] 1. Application of Xdh gene inhibitors in the preparation of anti-hepatic fibrosis drugs.

[0008] Furthermore, the Xdh gene inhibitor is a guide RNA that targets the Xdh gene.

[0009] Furthermore, the Xdh gene inhibitor is a recombinant plasmid carrying a guide RNA targeting the Xdh gene.

[0010] Furthermore, the Xdh gene inhibitor is a recombinant lentivirus carrying a guide RNA targeting the Xdh gene.

[0011] As a specific technical solution, the Xdh gene inhibitor is a guide RNA targeting the mouse Xdh gene, namely sgXdh, whose nucleotide sequence is 5'-CACCGAGTTGGTCTTCTTTGTGAA-3' (SEQ ID No. 1); the anti-liver fibrosis drug is an anti-mouse liver fibrosis drug.

[0012] Furthermore, the Xdh gene inhibitor is a recombinant plasmid carrying sgXdh.

[0013] Specifically, the recombinant plasmid carrying sgXdh is obtained by cloning sgXdh into the pSECC vector.

[0014] Furthermore, the Xdh gene inhibitor is a recombinant lentivirus carrying sgXdh.

[0015] Specifically, the recombinant lentivirus carrying sgXdh is obtained by co-transfecting HEK293T cells with the recombinant plasmid carrying sgXdh and the lentiviral packaging plasmids pMD2.G and psPAX2. After the cells show obvious cytopathic effects, the culture supernatant containing the virus is collected to obtain the recombinant lentivirus carrying sgXdh.

[0016] The beneficial effects of this invention are as follows: Taking the guide RNA sgXdh targeting the mouse Xdh gene as an example, this invention investigated the application value of Xdh gene inhibitors in the preparation of anti-liver fibrosis drugs through in vivo mouse experiments. The results showed that in a mouse model of liver fibrosis, knocking out the Xdh gene with sgXdh reduced the expression of hepatic XDH protein, decreased the levels of liver fibrosis marker proteins (α-SMA, COL3A1, and COL1A1) and liver fibrosis marker genes (Acta2, Timp1, Col3a1, Col1a1, and Vim), indicating that targeted inhibition of the Xdh gene can alleviate hepatic stellate cell activation and inhibit the progression of liver fibrosis. This suggests that targeted inhibition of the human Xdh gene can be used for the treatment of liver fibrosis, and that Xdh gene inhibitors can be used to prepare anti-liver fibrosis drugs, showing promising clinical application prospects. Attached Figure Description

[0017] Figure 1 This study investigated the effect of sgXdh on XDH protein levels in mouse liver using Western blot analysis.

[0018] Figure 2 This study investigated the effect of sgXdh on the levels of liver fibrosis marker proteins in mice using Western blot analysis.

[0019] Figure 3 The effect of Real-time PCR on the gene levels of sgXdh, a marker of liver fibrosis in mice, is shown in the figure; ** indicates p < 0.01, indicating highly significant differences between groups.

[0020] Figure 4 The effect of sgXdh on the expression level of α-SMA in primary mouse hepatic stellate cells was detected using immunofluorescence technique. Detailed Implementation

[0021] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the preferred embodiments of this invention are described in detail below. Experimental methods in the preferred embodiments that do not specify specific conditions are generally performed under conventional conditions or conditions recommended by the reagent (kit) manufacturer.

[0022] Example 1. Synthesis and packaging of guide RNA targeting the mouse Xdh gene

[0023] First, a guide RNA targeting the mouse Xdh gene, namely sgXdh, was designed with the nucleotide sequence 5'-CACCGAGTTG- GTCTTCTTTGTGAA-3' (SEQ ID No. 1), and was commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0024] Subsequently, the sgXdh vector was cloned into the pSECC vector using the pSECC-sgRNA lentivirus construction method, and the successful construction was confirmed by DNA sequencing, resulting in the recombinant plasmid pSECC-sgXdh carrying sgXdh.

[0025] Finally, the recombinant plasmid pSECC-sgXdh and the lentiviral packaging plasmids pMD2.G and psPAX2 were co-transfected into HEK293T cells. After the cells showed obvious cytopathic effects, the culture supernatant containing the virus was collected to obtain the recombinant lentivirus carrying sgXdh.

[0026] Example 2. Guide RNA targeting the mouse Xdh gene reduces XDH protein levels in mouse liver.

[0027] The experiment was divided into three groups: AAV-Control+sgControl, AAV-HBV+sgControl, and AAV-HBV+sgXdh. The AAV-HBV+sgXdh group consisted of 6-8 week old C57BL / 6 mice injected with 2×10⁻⁶ sgXdh via the tail vein. 11A mouse model of HBV infection was constructed using AAV-HBV. One week after successful HBV infection, mice were injected via tail vein with a recombinant lentivirus carrying sgXdh (containing 1×10⁻⁶ sgXdh). 9 (One genome copy), and two weeks later, primary hepatocytes and primary hepatic stellate cells from mice were collected for examination. Mice in the AAV-Control+sgControl group were injected with neither AAV-HBV nor recombinant lentivirus carrying sgXdh; mice in the AAV-HBV+sgControl group were injected with only AAV-HBV and not recombinant lentivirus carrying sgXdh.

[0028] Primary mouse hepatocytes were collected, and XDH protein levels were detected by Western blot. Results are as follows: Figure 1 As shown, the XDH protein level in the AAV-HBV+ sgControl group (AAV-Control-, AAV-HBV+, sgControl+, sgXdh-) was significantly higher than that in the AAV-Control + sgControl group (AAV-Control+, AAV-HBV-, sgControl+, sgXdh-), while the XDH protein level in the AAV-HBV+ sgXdh group (AAV-Control-, AAV-HBV+, sgControl-, sgXdh+) was significantly lower than that in the AAV-HBV+ sgControl group. This indicates that HBV infection promotes XDH protein expression in mice, while guide RNA targeting the mouse Xdh gene inhibits XDH protein expression in mice.

[0029] Example 3. Guide RNA targeting the mouse Xdh gene reduces the level of liver fibrosis marker proteins in mice.

[0030] Primary mouse hepatic stellate cells collected in Example 2 were used, and the levels of liver fibrosis marker proteins α-SMA, COL3A1, and COL1A1 were detected by Western blot. The results are as follows: Figure 2As shown, the levels of liver fibrosis marker proteins α-SMA, COL3A1, and COL1A1 in the AAV-HBV+sgControl group (AAV-Control-, AAV-HBV+, sgControl+, sgXdh-) were significantly higher than those in the AAV-Control+sgControl group (AAV-Control+, AAV-HBV-, sgControl+, sgXdh-), while the levels of liver fibrosis marker proteins α-SMA, COL3A1, and COL1A1 in the AAV-HBV+sgXdh group (AAV-Control-, AAV-HBV+, sgControl-, sgXdh+) were significantly lower than those in the AAV-HBV+sgControl group. This indicates that HBV infection promotes the expression of these liver fibrosis marker proteins in mice, while guide RNA targeting the mouse Xdh gene inhibits the expression of these liver fibrosis marker proteins.

[0031] Example 4. Guide RNA targeting the mouse Xdh gene reduces the levels of liver fibrosis marker genes in mice.

[0032] Primary hepatic stellate cells from mice collected in Example 2 were used, and the levels of liver fibrosis marker genes Acta2, Timp1, Col3a1, Col1a1, and Vim were detected by real-time PCR. The results are as follows: Figure 3 As shown, the levels of liver fibrosis marker genes Acta2, Timp1, Col3a1, Col1a1, and Vim in the AAV-HBV+sgControl group were significantly higher than those in the AAV-Control+sgControl group, while the levels of these genes in the AAV-HBV+sgXdh group were significantly lower than those in the AAV-HBV+sgControl group. This indicates that HBV infection promotes the levels of these liver fibrosis marker genes in mice, while guide RNA targeting the mouse Xdh gene inhibits their levels.

[0033] Example 5. Guide RNA targeting the mouse Xdh gene reduces α-SMA expression levels in primary mouse hepatic stellate cells.

[0034] Hepatic stellate cells are normally in a quiescent state and do not express α-smooth muscle actin (α-SMA). Under pathological conditions, such as stimulation of the liver by physical, chemical, or viral infections, hepatic stellate cells are activated, transforming into myofibroblasts and expressing α-SMA. α-SMA expression is a marker of hepatic stellate cell activation. Sustained activation of hepatic stellate cells is a crucial step in the development and progression of liver fibrosis.

[0035] Primary mouse hepatic stellate cells collected in Example 2 were used to detect the expression level of α-SMA using immunofluorescence. The results are as follows: Figure 4 As shown, the α-SMA expression level in the AAV-HBV+sgControl group was significantly higher than that in the AAV-Control+sgControl group, while the α-SMA expression level in the AAV-HBV+sgXdh group was significantly lower than that in the AAV-HBV+sgControl group. This indicates that HBV infection promotes the expression of α-SMA in primary mouse hepatic stellate cells, while the guide RNA targeting the mouse Xdh gene inhibits the expression of α-SMA in primary mouse hepatic stellate cells.

[0036] The results of Examples 3-5 demonstrate that guide RNA targeting the mouse Xdh gene can inhibit the activation of mouse hepatic stellate cells and suppress the progression of liver fibrosis.

[0037] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. Application of xanthine dehydrogenase gene (Xdh gene) inhibitors in the preparation of anti-liver fibrosis drugs.

2. The application as described in claim 1, characterized in that: The Xdh gene inhibitor is a guide RNA that targets the Xdh gene.

3. The application as described in claim 1, characterized in that: The Xdh gene inhibitor is a recombinant plasmid carrying a guide RNA targeting the Xdh gene.

4. The application as described in claim 1, characterized in that: The Xdh gene inhibitor is a recombinant lentivirus carrying a guide RNA targeting the Xdh gene.

5. The application as described in claim 2, characterized in that: The Xdh gene inhibitor is a guide RNA targeting the mouse Xdh gene, namely sgXdh, with a nucleotide sequence of 5'-CACCGAGTTGGTCTTCTTTGTGAA-3'; the anti-liver fibrosis drug is an anti-mouse liver fibrosis drug.

6. The application as described in claim 5, characterized in that: The Xdh gene inhibitor is a recombinant plasmid carrying sgXdh.

7. The application as described in claim 6, characterized in that: The recombinant plasmid carrying sgXdh was obtained by cloning sgXdh into the pSECC vector.

8. The application as described in claim 5, characterized in that: The Xdh gene inhibitor is a recombinant lentivirus carrying sgXdh.

9. The application as described in claim 8, characterized in that: The recombinant lentivirus carrying sgXdh was obtained by co-transfecting HEK293T cells with the recombinant plasmid carrying sgXdh and the lentiviral packaging plasmids pMD2.G and psPAX2. After the cells showed obvious cytopathic effects, the culture supernatant containing the virus was collected to obtain the recombinant lentivirus carrying sgXdh.

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