Application of ADAMTS4 metalloproteinase in the preparation of drugs for early liver fibrosis

Abstract

This invention discloses the application of the metalloproteinase ADAMTS4 in the preparation of drugs for treating early liver fibrosis, belonging to the field of biopharmaceuticals. This invention discloses the application of the metalloproteinase ADAMTS4 as a target in the preparation of drugs for treating early liver fibrosis. The drug exerts its therapeutic effect on early liver fibrosis by knocking out or inhibiting ADAMTS4 expression. Experimental results show that ADAMTS4 deficiency can activate TGF-β1 expression, thereby promoting collagenase synthesis and inhibiting hepatic stellate cell activity to slow down liver fibrosis. This invention demonstrates the feasibility of delaying, stopping, and reversing the development of liver fibrosis by inhibiting ADAMTS4 protein activity, providing a theoretical basis and molecular approach for the treatment of related diseases and offering new ideas for controlling early liver fibrosis in liver cancer.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and in particular to the application of a metalloproteinase ADAMTS4 in the preparation of drugs for early liver fibrosis. Background Technology

[0002] Chronic liver damage caused by different factors shares a common pathological change: liver fibrosis, which is also an essential step in the progression of chronic liver disease to cirrhosis (O'Hara SP. et al., Hepatology, 2017; Zhang CY. et al., World J Gastroenterol, 2016). It is noteworthy that liver fibrosis is reversible in its early stages; however, if the underlying cause of liver damage persists, long-term liver fibrosis can evolve into cirrhosis, affecting the liver's metabolic function and causing portal hypertension, leading to permanent and irreversible liver damage, and potentially further progressing to liver cancer (Lai JC. et al., Am J Transplant, 2014; Tandon P. et al., Am J Gastroenterol, 2016). Therefore, exploring the mechanisms and key molecules involved in the development and progression of liver fibrosis is of great significance for delaying, terminating, and reversing liver fibrosis, and for the prevention and treatment of cirrhosis.

[0003] Key challenges in liver fibrosis research include understanding its mechanisms and identifying therapeutic targets. Over the past few decades, researchers have made significant progress in understanding the mechanisms of liver fibrosis. Studies have shown that hepatic stellate cell (HSC) activation plays a crucial role in the occurrence, development, and reversal of liver fibrosis (Yuji Iimuro et al., Pharmaceutical Research, 2008; Kisseleva T et al., Best Pract ResClin Gastroenterol, 2011). Activated HSCs exhibit increased proliferation frequency, significantly upregulated expression of the activation marker α-smooth muscle actin α-SMA, increased synthesis of pre-type I collagen α1 chains (COL1A1), and increased secretion of extracellular matrix (ECM) (Hitoshi Yoshiji et al., Hepatology, 2002). In vivo experiments in mice have shown that while hepatic fibrosis is induced by hepatic sclerosis, hepatic sclerosis cells (HSCs) also produce enzymes such as matrix metalloproteinase-13 (MMP-13) to mitigate the progression of liver fibrosis (Hiroyuki Abe et al., Mol Ther Nucleic Acids, 2016). Furthermore, HSC activation is regulated by cytokines secreted by inflammatory cells, with hepatic macrophages (Kupffer cells) and T cell subsets also playing significant roles (Tacke F. et al., Journal of Hepatology, 2014; Pradere J. et al., Hepatology, 2013; Murray PJ. et al., Nature Reviews Immunology, 2011). The severity of liver fibrosis depends on the balance between matrix metalloproteinases (MMPs) released by HSCs and tissue inhibitors of metalloproteinases (TIMPs) released by Kupffer cells or other interstitial cells. How these two factors regulate each other, and whether there is a key regulatory molecule in the process of liver fibrosis, remains unclear. Therefore, this invention seeks a key factor that can regulate early liver fibrosis and investigates the regulatory role and molecular mechanism of this substance in the process of liver fibrosis, providing new insights for controlling early liver fibrosis in liver cancer. Summary of the Invention

[0004] The purpose of this invention is to provide an application of the metalloproteinase ADAMTS4 in the preparation of drugs for early liver fibrosis, so as to solve the problems existing in the prior art and achieve the effect of slowing down early liver fibrosis by inhibiting ADAMTS4 gene expression.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] This invention provides the application of the metalloproteinase ADAMTS4 as a target in the preparation of drugs for treating early liver fibrosis.

[0007] Furthermore, the treatment of early liver fibrosis can be achieved by knocking out or inhibiting ADAMTS4 expression.

[0008] Furthermore, the drug treats early liver fibrosis through one or more of the following pathways:

[0009] (1) Reduces liver cell damage;

[0010] (2) Reduce the formation of liver fibrosis;

[0011] (3) Reduce the expression of matrix metalloproteinases;

[0012] (4) Inhibits the activity of hepatic stellate cells;

[0013] (5) Promotes the synthesis of collagenase.

[0014] Furthermore, the reduction in liver fibrosis is achieved by decreasing the expression of TIMP-1 and TIMP-3.

[0015] Furthermore, the matrix metalloproteinase includes MMP14.

[0016] Furthermore, the collagenase includes collagenase I.

[0017] Furthermore, the drug is in a pharmaceutically acceptable dosage form.

[0018] Furthermore, the dosage form of the drug is tablets, capsules, granules, injections, or sprays.

[0019] Furthermore, the method of administration of the drug is oral or non-gastrointestinal.

[0020] The present invention discloses the following technical effects:

[0021] This invention targets the metalloproteinase ADAMTS4 to investigate its regulatory role in liver fibrosis. By comparing the expression of liver fibrosis-related genes in ADAMTS4 knockout mice and wild-type control mice in a carbon tetrachloride-induced chronic liver fibrosis model, the study found that ADAMTS4 deficiency inhibits fibrosis formation. Immunohistochemistry, RT-PCR, and Western blotting experiments confirmed that ADAMTS4 deficiency activates TGF-β1 expression, thereby promoting collagenase synthesis and inhibiting hepatic stellate cell activity, thus affecting liver fibrosis. Therefore, this study clarifies the regulatory role and molecular mechanism of ADAMTS4 in liver fibrosis, and verifies the feasibility of inhibiting ADAMTS4 protein activity in delaying, stopping, and reversing the development of liver fibrosis. This provides a theoretical basis and molecular approach for the treatment of related diseases and offers new insights into controlling early-stage liver fibrosis in hepatocellular carcinoma. Attached Figure Description

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The images show the establishment of a mouse model of liver fibrosis and the staining results of liver tissue. A shows the process of constructing a carbon tetrachloride-induced mouse liver fibrosis model; B shows the results of HE staining, Masson staining, Sirius red staining, and α-SMA staining of liver tissue from model mice and control mice; and C shows the statistical results of collagen volume fraction after Masson staining, Sirius red staining, and α-SMA staining of liver tissue from model mice and control mice.

[0024] Figure 2 The table shows the expression of ADAMTS4 in liver fibrosis samples. A represents the protein expression of ADAMTS4 in a mouse liver fibrosis model as detected by Western blotting; B represents the statistical results of ADAMTS4 protein expression in a mouse liver fibrosis model; C represents the protein expression of ADAMTS4 in a mouse liver fibrosis model as detected by RT-PCR; D represents the statistical results of ADAMTS4 protein expression in serum samples from patients with liver fibrosis as determined by enzyme-linked immunosorbent assay (ELISA); E represents the fluorescence microscopy results of ADAMTS4 expression in serum samples; and F represents the statistical results of ADAMTS4 expression in serum samples.

[0025] Figure 3The results of serological marker detection in ADAMTS4-deficient mice are shown, where A: AST; B: ALT; C: HA; D: PIIIP; E: IVC; F: LN;

[0026] Figure 4 HE staining results of liver tissues from model group and control group mice;

[0027] Figure 5 The results show the staining of liver tissue from mice in the model group and the control group; where A is the result of Masson staining; B is the statistical result of collagen volume fraction from Masson staining; C is the result of Sirius red staining; and D is the statistical result of collagen volume fraction from Sirius red staining.

[0028] Figure 6 The expression of metalloproteinases and protease inhibitors in mouse liver tissue; where A: MMP2; B: MMP9; C: MMP14; D: TIMP1; E: TIMP2; F: TIMP3;

[0029] Figure 7 The images show the α-SMA staining results in mouse liver tissue; the left image shows the staining results, and the right image shows the statistical results.

[0030] Figure 8 The expression of CollagenⅠ, TGF-b1, α-SMA, and ADAMTS4 proteins in the liver tissues of mice in the model group and control group is shown in Figure A, where A is a Western blot electrophoresis image and B is a statistical graph of protein expression.

[0031] Figure 9 Microscopic images of collagen fibers in the liver tissue of mice in the model group and control group. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] ADAMTS4 is a member of the disintegrin and metallo-protrinase with thrombospondin motifs (ADAMTS) family of type I platelet-binding protein motifs, belonging to the same Zn group as matrix metalloproteinases and disintegrin-protrinases. 2+ This study investigated the secondary structure of ADAMTS4, a family of metalloproteinases. Analysis revealed that ADAMTS4 contains a thrombospondin-1 (TSP-1) domain at its terminal. TSP-1 activates transforming growth factor-beta (TGF-β), one of the most important cytokines responsible for liver fibrosis. Hepatocytes (HSCs) and damaged hepatocytes can autocrine and paracrine TGF-β, leading to a positive feedback increase and exacerbating liver fibrosis. TGF-β1 is a key factor influencing the development of liver fibrosis within the TGF-β family. Studies have shown that TGF-β1 promotes ECM synthesis, inhibits its degradation, suppresses hepatocyte regeneration, and promotes apoptosis, resulting in an imbalance between hepatic parenchymal and interstitial components. This imbalance leads to liver fibrosis through multiple pathways. The TSP-1 domain in ADAMTS4 contains an amino acid sequence that activates TGF-β1. Therefore, we hypothesize that ADAMTS4 may influence liver fibrosis by activating TGF-β1 expression, thereby stimulating HSCs to synthesize and secrete large amounts of ECM. We will conduct experiments to verify this hypothesis.

[0038] 1. Establishment of a mouse model of liver fibrosis

[0039] A mouse model of liver fibrosis was established by intraperitoneal injection of carbon tetrachloride (a 5% CCl4 solution prepared with corn oil), at a dose of 5 ml / kg, twice a week for 4 weeks. Figure 1 A). The control group received an intraperitoneal injection of the same amount of corn oil as the model group. Four weeks later, mice were euthanized by cervical dislocation, and their livers were removed. Immunohistochemical staining of relevant fibrosis markers showed that the CCl4 intraperitoneal injection group exhibited significantly higher levels of Masson's staining, Sirius red staining (PSR), and α-SMA compared to the control group. Figure 1 B), the results of Masson staining showed that the collagen volume fraction in the CCl4 intraperitoneal injection group was approximately 14%, while that in the corn oil injection group was approximately 5%. Figure 1 C). Sirius red staining results showed that the Sirius red area in the CCl4 intraperitoneal injection group was 17% higher than that in the corn oil injection group. Figure 1 C). α-SMA staining results showed that the CCl4 intraperitoneal injection group had approximately 20% higher levels of [something] than the corn oil injection group. Figure 1 C). Experimental results demonstrate the successful establishment of a mouse model of liver fibrosis.

[0040] 2. Expression of ADAMTS4 in liver fibrosis samples

[0041] like Figure 2 As shown, the protein expression of ADAMTS4 in the established mouse liver fibrosis model was detected by Western blotting (see [reference]). Figure 2 A) The results showed that the protein content of ADAMTS4 in the WT+CCl4 group was higher than that in the WT group, and Image analysis showed a statistically significant difference in the gray values ​​of the protein bands (see A). Figure 2 B). The expression of ADAMTS4 mRNA in this fibrosis model was detected using the RT-CR method. The results also showed that the mRNA content of ADAMTS4 in the WT+CCl4 group was higher than that in the WT group (see...). Figure 2 C), consistent with the WB results. Next, we performed enzyme-linked immunosorbent assay (ELISA) on the serum of 42 patients with clinical liver fibrosis (stage S1 cirrhosis), using healthy individuals as a control group. We found elevated ADAMTS4 protein levels in 25% of the serum samples from patients with cirrhosis (see [link to study]. Figure 2D). Due to the low positive rate of serum enzyme-linked immunosorbent assay (ELISA), we used an exosome extraction kit (Life Technology, USA) to isolate and collect exosomes from patient serum to detect ADAMTS4. A total of 76 clinical patient serum samples were tested, including 30 normal control samples, 30 samples from stage S1 cirrhosis, and 16 samples from hepatocellular carcinoma. The detection was performed using the patented liposome complex nanoparticle (TCLN) biochip technology developed by Ohio State University (OSU). The ADAMTS4 molecular probe (MB) was individually encapsulated in a cationic liposome nanoparticle solution. These cationic liposome nanoparticles coated on the biochip attracted negatively charged exosomes under an electrostatic field, forming larger nanoparticles. The expression of ADAMTS4 was observed under a total internal reflection fluorescence (TIRF) microscope (see [link to TIRF microscopy]). Figure 2 E), the results showed that in patients with cirrhosis, the positive rate of ADAMTS4 increased by 63% compared with serum enzyme-linked immunosorbent assay (see E). Figure 2 F). Surprisingly, ADAMTS4 was also highly expressed in liver cancer patients, with a positive rate of 86% (see F). Figure 2 F), which suggests that it also plays an important role in the process of liver cancer development.

[0042] 3. Effects of ADAMTS4 on carbon tetrachloride-induced liver fibrosis

[0043] 3.1 The absence of ADAMTS4 did not affect the changes in liver indices in mice.

[0044] The liver index of mice in the CCl4 injection group was significantly higher than that of mice in the Oil injection group (P < 0.05). There was no statistically significant difference in liver index between ADAMTS4-deficient mice in the CCl4 injection group and wild-type mice in the CCl4 injection group. (See Table 1)

[0045] Table 1 Comparison of liver index among different groups of mice

[0046]

[0047]

[0048] *Compared with the Oil-injected mouse group, P<0.05

[0049] 3.2 ADAMTS4 deficiency can reduce liver damage and fibrosis.

[0050] Serum biochemical indicators of mice in each group are detailed in Table 2. After 4 weeks of CCl4 induction, ADAMTS4- / - and ADAMTS4+ / + mice showed significantly higher levels of ALT, AST, HA, PIIIP, IVC, and LN compared to the corn oil injection group (all P < 0.05). The CCl4 group in ADAMTS4- / - mice significantly reduced ALT levels, while HA and PIIIP levels were significantly lower than those in the ADAMTS4+ / + CCl4 group (all P < 0.05). AST levels showed a similar trend, but did not reach statistical significance. IVC and LN levels in this strain of mice were within the normal range. Further comparisons of the groups are detailed in Table 2. Figure 3 (AF), suggesting that the absence of ADAMTS4 may reduce the occurrence of early liver damage and fibrosis.

[0051] Table 2. Serological markers in each group of mice to detect liver damage and fibrosis.

[0052]

[0053] *Compared with the WT+Oil group, P<0.05

[0054] Compared with the WT+CCl4 group, P<0.05

[0055] 3.3 ADAMTS4 deficiency reduces the degree of hepatocellular damage and liver fibrosis.

[0056] HE staining results showed that the hepatocytes of mice in the Oil injection group were intact and regularly round or oval; the hepatocytes of mice in the CCl4 injection group showed severe fatty degeneration, irregular shape, punctate necrosis, severe inflammatory infiltration, incomplete lobular structure, and blue fibrous cords in the portal area and periphery of the lobules; the CCl4 group of ADAMTS4-deficient mice showed significantly reduced inflammation, milder liver damage, and better recovery of lobular structure compared to the wild-type mice in the CCl4 injection group (see...). Figure 4 ).

[0057] Masson staining results showed that in the Oil-injected group, the hepatocytes of mice were neatly arranged and the liver lobules were intact; in the CCl4-injected group, the liver tissue of mice was damaged, with fatty degeneration of cells, localized necrosis and collagen deposition in spots and focal areas, uneven arrangement of hepatic cords, and formation of pseudolobules; compared with the wild-type mice CCl4-injected group, the degree of liver tissue damage in the ADAMTS4-deficient mice CCl4-injected group was improved, with a significant reduction in fat vacuoles and fibrosis, suggesting a reduction in ECM accumulation in the mouse liver. (See...) Figure 5 A), Image J quantified its collagen volume fraction, showing that the CCl4-injected group of ADAMTS4-deficient mice had approximately 10% less collagen volume compared to the CCl4-injected group of wild-type mice. Figure 5B). Sirius red staining further showed that there was no significant difference between the Oil-injected mice and wild-type and ADAMTS4-deficient mice (see [link]). Figure 5 C), Image J's quantification of collagen area also showed no statistically significant difference (see C). Figure 5 D). However, in a mouse model of liver fibrosis induced by CCl4 injection for 4 weeks, the absence of ADAMTS4 resulted in a significant reduction in collagen area (see D). Figure 5 C), its collagen area decreased by approximately 14% ( Figure 5 D), which is consistent with the Masson staining results. This indicates that the absence of ADAMTS4 can reduce the degree of hepatocellular damage and liver fibrosis.

[0058] 4. Molecular mechanisms by which ADAMTS4 affects liver fibrosis

[0059] 4.1 The absence of ADAMTS4 reduces fibrillation and the expression of matrix metalloproteinases.

[0060] The expression levels of matrix metalloproteinases MMP2, MMP9, MMP14 and matrix metalloproteinase inhibitors TIMP1, TIMP2, and TIMP3 in the livers of mice in each group were detected. Figure 6 It can be seen that after 4 weeks of CCl4 injection, there was no significant difference in ADAMTS4- / - mRNA levels between WT mice and MMP2, MMP9, and TIMP2 (see [link to relevant documentation]). Figure 6 AB, Figure 6 E), while ADAMTS4- / - mice showed significantly reduced MMP14 mRNA expression in the liver compared to WT mice ( Figure 6 C) indicates that ECM degradation is less in the liver of ADAMTS4- / - mice, while the increase in TIMP1 and TIMP3 in the liver of WT mice suggests less extracellular matrix (ECM) degradation and more fibrosis (see C). Figure 6 (D, F). It is evident that the absence of ADAMTS4 reduces the formation of liver fibrosis and the expression of matrix metalloproteinases.

[0061] 4.2. ADAMTS4 deficiency inhibits hepatic stellate cell (HSC) activation.

[0062] Immunohistochemical staining of liver tissues from mice in each group revealed increased α-SMA expression in the CCl4-injected group, which exhibited a specific brownish-yellow color. This difference was statistically significant compared to the oil-injected group (P < 0.05) (see [link to relevant documentation]). Figure 7In ADAMTS4-deficient mice, the expression of α-SMA in the liver tissue of mice injected with CCl4 was decreased, and the number of brown-yellow granules was reduced. Compared with wild-type mice injected with CCl4, the difference was statistically significant (P < 0.05) (see...). Figure 7 This suggests that the activity of hepatic stellate cells was inhibited, and liver fibrosis was reduced.

[0063] 4.3 The absence of ADAMTS4 affects collagenase synthesis by influencing the TGF-β1 pathway.

[0064] We first examined the expression levels of ADAMTS4 protein in the liver tissues of mice in each group. The ADAMTS4-deficient group showed a significantly lower ADAMTS4 protein level compared to the wild-type mouse group (P < 0.05), indicating that ADAMTS4 was indeed knocked out in the liver tissue of transgenic mice. The α-SMA protein expression level in the liver tissue of wild-type mice injected with CCl4 was significantly higher than that in the oil-injected group (P < 0.05), indicating the successful establishment of the CCl4-induced liver fibrosis model. However, the α-SMA and TGF-β1 protein expression levels in the liver tissue of ADAMTS4-deficient mice injected with CCl4 were significantly lower than those in the wild-type mouse group injected with CCl4 (P < 0.05), suggesting that ADAMTS4 affects mouse liver fibrosis through TGF-β1. However, the Collagen I protein expression level in the liver tissue of ADAMTS4-deficient mice injected with CCl4 was higher than that in the wild-type mouse group injected with CCl4 (P < 0.05), suggesting that ADAMTS4 affects mouse liver fibrosis by influencing collagenase I synthesis. (See details) Figure 8 A, B)

[0065] Furthermore, because Sirius red is a strong acid dye, it can react with collagen fibers to enhance birefringence and improve resolution, causing the collagen fibers to exhibit a significant birefringence phenomenon. Under polarized light, they display different colors, roughly distinguishing different fiber types (Type I collagen fibers: tightly packed, exhibiting strong birefringence, appearing yellow; Type III collagen fibers: exhibiting weak birefringence, appearing as fine green fibers). Therefore, when we observed collagen fibers using a polarized light microscope, we found that the collagen fibers in the CCl4-injected group of wild-type mice were tightly packed, appearing yellow, and also showing fine green fibers; while the collagen fibers in the CCl4-injected group of ADAMTS4-deficient mice were red, with almost no visible fine green fibers (see details). Figure 9 The results suggest that ADAMTS4 deficiency affects the synthesis of type III collagen fibers. Therefore, ADAMTS4 deficiency can alleviate liver fibrosis in mice.

[0066] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. The application of a metalloproteinase ADAMTS4 as a target in the preparation of a drug for treating early liver fibrosis, characterized in that, The treatment of early liver fibrosis can be achieved by knocking out or inhibiting ADAMTS4 expression.

2. The application according to claim 1, characterized in that, The drug treats early liver fibrosis through one or more of the following pathways: (1) Reduces hepatocellular damage; (2) Reduce the formation of liver fibrosis; (3) Reduce the expression of matrix metalloproteinases; (4) Inhibits the activity of hepatic stellate cells; (5) Promotes the synthesis of collagenase.

3. The application according to claim 2, characterized in that, The reduction in liver fibrosis is achieved by decreasing the expression of TIMP-1 and TIMP-3.

4. The application according to claim 2, characterized in that, The matrix metalloproteinases include MMP14.

5. The application according to claim 2, characterized in that, The collagenase includes collagenase I.

6. The application according to claim 1, characterized in that, The drug is in a pharmaceutically acceptable dosage form.

7. The application according to claim 6, characterized in that, The dosage form of the drug is tablets, capsules, granules, injections, or sprays.

8. The application according to claim 1, characterized in that, The medication is to be taken orally or in a non-gastrointestinal form.

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