Use of PON1 in the manufacture of a medicament for the diagnosis, treatment and / or prevention of hepatic osteodystrophy

Abstract

The application belongs to the technical field of biological medicine, and discloses the use of PON1 in the preparation of drugs for diagnosing, treating and / or preventing hepatic osteodystrophy. In view of the clinical problem that HOD lacks specific diagnostic markers and effective intervention means in the early stage, the application finds and proves that the PON1 level in the liver and blood circulation decreases significantly in the early stage of HOD, and the degree of decrease is related to bone loss. Therefore, PON1 can be used as a sensitive biomarker for diagnosing HOD, and is used for the development of related kits. More importantly, by exogenous supplement of PON1 protein or specific up-regulation of PON1 expression in the liver by using gene therapy means, the liver damage can be directly improved, and osteogenesis can be effectively promoted and osteoclastogenesis can be inhibited, so that the bone mineral density and bone microstructure of the HOD model animal can be significantly recovered. The application first discloses that PON1 acts as a liver-derived factor to regulate bone metabolism through the liver-bone axis, and provides a new strategy and drug development basis for the early diagnosis and targeted treatment of HOD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of PON1 in the preparation of drugs for the diagnosis, treatment and / or prevention of hepatic osteodystrophy. Background Technology

[0002] Hepatic osteodystrophy (HOD) is a systemic metabolic disease caused by chronic liver damage, leading to osteoporosis and bone malnutrition. It is characterized by a general decrease in bone mineral density and deterioration of bone structure in patients with chronic liver injury, potentially resulting in fractures and a significant decline in quality of life. The incidence of HOD in patients with chronic liver injury is as high as 20%–50%, affecting approximately 200 million people worldwide. Currently, HOD is often misdiagnosed as osteoporosis in clinical practice, and treatment often focuses solely on vitamin D and calcium lactate supplementation, neglecting the crucial role of the liver. This leads to chronic, intractable disease and a waste of significant medical resources.

[0003] Limitations and shortcomings of existing technologies: The pathogenesis of HOD is not fully understood, and there is a lack of specific biomarkers for early diagnosis and effective early intervention methods. Current research focuses mainly on the indirect effects of liver injury on bone metabolism, while exploration of the direct mechanisms of action of hepatogenic factors in bone metabolism regulation is limited, failing to meet the clinical needs for early diagnosis and precision treatment of HOD. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide the use of PON1 in the preparation of medicaments for the diagnosis, treatment, and / or prevention of hepatic osteodystrophy, thereby providing a more effective method for the diagnosis, prevention, and treatment of hepatic osteodystrophy.

[0005] To achieve the objectives of this invention, the invention includes the following technical solutions:

[0006] The use of PON1 as a biomarker in the preparation of kits for diagnosing hepatic osteodystrophy. (PON1: paraoxonase 1; NCBI Gene ID: 18979.)

[0007] Furthermore, in the above-mentioned uses, hepatic osteodystrophy is a bone loss-related disease induced by chronic liver injury.

[0008] Furthermore, in the above-described uses, the kit contains a detection reagent for detecting the expression level of PON1 in a biological sample obtained from the individual to be tested.

[0009] Furthermore, in the above-mentioned uses, the expression level of PON1 is the PON1 protein level or the PON1 mRNA level; and / or, the detection reagent includes an antibody against PON1 protein or a nucleic acid probe that specifically binds to PON1 mRNA.

[0010] The present invention also discloses the use of PON1, its agonists or substances capable of upregulating PON1 expression in the preparation of medicaments for the prevention and / or treatment of hepatic osteodystrophy.

[0011] Furthermore, in the above-described uses, the substance capable of upregulating PON1 expression is selected from the PON1 protein, a nucleic acid molecule encoding the PON1 protein, an expression vector containing the nucleic acid molecule, or a compound capable of promoting the transcription or translation of the endogenous PON1 gene.

[0012] Furthermore, in the above-described uses, the nucleic acid molecule comprises a nucleotide sequence encoding the PON1 protein as defined by NCBI Gene ID: 18979; and / or, the PON1 protein comprises an amino acid sequence as defined by NCBI Gene ID: 18979.

[0013] The present invention also discloses a pharmaceutical composition for the prevention and / or treatment of hepatic osteodystrophy, comprising a therapeutically effective amount of PON1 protein, its agonist or substance capable of upregulating PON1 expression, and a pharmaceutically acceptable carrier.

[0014] Furthermore, the above-mentioned pharmaceutical composition is formulated into a dosage form for injection administration.

[0015] The present invention also discloses a method for screening candidate drugs for the prevention and / or treatment of hepatic osteodystrophy, the method comprising the following steps:

[0016] (a) Under test conditions, the candidate material was brought into contact with a system capable of expressing PON1;

[0017] (b) Detect the effect of the candidate substance on the expression level or activity of PON1 in the system;

[0018] (c) Select candidate substances that can upregulate the expression level or activity of PON1 as the candidate drug.

[0019] Furthermore, in the above method, the system capable of expressing PON1 is a cell expressing PON1, cell lysate, or a reporter gene system containing PON1 gene transcriptional regulatory elements.

[0020] Compared with the prior art, the present invention has the following outstanding advantages:

[0021] 1. Significant therapeutic effect: By supplementing recombinant PON1 protein exogenously or regulating the expression of PON1 in the liver, it can directly act on key links of bone metabolism, effectively promote osteoblast activity, inhibit osteoclast function, and has a significant effect on improving osteoporosis. It can significantly increase bone density and bone quality in patients with hepatic osteopathy and reduce the risk of fracture.

[0022] 2. High specificity: Intervention targeting PON1 is highly specific, avoiding potential drug side effects and adverse reactions in traditional treatments, thus improving the safety and tolerability of the treatment.

[0023] 3. High Clinical Application Value: This invention provides a completely new approach and method for the treatment and prevention of hepatic osteodystrophy, and is expected to fill a gap in current clinical practice, possessing significant clinical application value and market potential. It can not only be used to treat patients already diagnosed with hepatic osteodystrophy, but also as a preventative medication for high-risk groups, delaying or preventing the onset and progression of the disease.

[0024] 4. Clear mechanism of action: Based on in-depth research on the pathogenesis of hepatic osteopathy, the molecular mechanism by which PON1 regulates bone metabolism through the liver-bone axis has been clarified, providing a solid theoretical basis for drug development and application. Attached Figure Description

[0025] Figure 1 PON1 expression shows a significant decrease in the early stages of HOD: a. Expression of PON1 mRNA in the liver; b. Expression of PON1 protein in the liver; d. PON1 concentration in mouse serum; e. and g. Immunohistochemical staining of PON1 in bone tissue; f. and h. Immunohistochemical staining of PON1 in liver tissue; i. PON1 concentration in serum of healthy individuals and patients with hepatic osteodystrophy.

[0026] Figure 2 PON1 is specifically secreted by the liver: a. Differentially expressed proteins in the liver and serum overlap with the secretory protein library; b. PON1 expression in various tissues and organs was retrieved from the Genecard database; c. PON1 mRNA expression in various tissues and organs of mice;

[0027] Figure 3 Validation of PON1 overexpression mediated by adenovirus vector in animal models: a. PON1 overexpression adeno-associated virus vector sequence; b. Schematic diagram of animal model establishment; c. PON1 mRNA expression in bone tissue; de. Immunohistochemistry and grayscale statistical analysis of PON1 in liver tissue; f. PON1 protein expression in liver tissue.

[0028] Figure 4Recombinant protein PON1 promotes osteoblast activity and inhibits osteoclast proliferation: a. POB cells were treated with different concentration gradients of rPON1 for 1 week, followed by ALPL staining; bf. osteoblast-related markers were detected after 48 hours of treatment with different concentrations of rPON1; gh. EdU staining was performed after 24 hours of treatment with rPON1 on activated MC3T3 cells; i. CCK8 assay was performed after 24 hours of treatment with rPON1 on activated MC3T3 cells; j. Immunofluorescence was performed after treatment with rPON1 on primary osteoblasts; k. CCK8 assay was performed after 24 hours of treatment with rPON1 on activated Raw264.7 cells.

[0029] Figure 5 Adenovirus vector-mediated PON1 overexpression can improve liver injury in HOD model mice: ab. ALT and AST levels in the serum of mice in each group; cd. Immunohistochemical results and grayscale analysis of α-SMA in liver tissue of mice in each group; e. HE staining results of liver tissue; f. Sirius red staining results of liver tissue.

[0030] Figure 6 Adenovirus vector-mediated PON1 overexpression can improve bone loss in HOD model mice: ab. Representative microCT images of the distal femur of mice in each group; c. Bone area fraction statistics of mice in each group; d. Bone mineral density statistics of mice in each group; e. Trabecular bone number statistics of mice in each group; f. Trabecular bone thickness statistics of mice in each group; g. Trabecular bone separation statistics of mice in each group; h. Bone volume fraction statistics of mice in each group.

[0031] Figure 7 ROC curve of serum PON1 level for diagnosing NASH-HOD based on a small sample clinical study. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1:

[0034] 1. PON1 expression decreased significantly in the early stage of HOD.

[0035] A CCl4-induced HOD mouse model (C57BL / 6 mice) was used to establish liver fibrosis and bone metabolic damage models at different time points (weeks 0, 1, 3, and 6). Liver, serum, tibia, femur, and fibula samples were collected from mice at each time point. RNA was extracted from liver tissue samples from normal and HOD mice using an RNA extraction kit. Changes in PON1 mRNA were detected using a reverse transcription kit and a quantitative real-time PCR kit (with β-actin gene as an internal control). A portion of liver tissue from normal and HOD mice was used to extract proteins using RIPA lysis buffer (containing protease inhibitors and PMSF). A portion of the liver tissue was embedded in paraffin to form paraffin blocks, which were then sectioned and stained with hematoxylin and eosin using a hematoxylin and eosin staining kit for HE staining and immunohistochemical analysis. After adjusting the protein concentrations extracted from each sample to the same level, SDS-PAGE gel electrophoresis was performed for separation (electrophoresis conditions: 80V, constant voltage for 20 minutes, 120V, constant voltage for 60 minutes) and transfer to a membrane (transfer conditions: using Genscript Biotech eBlot™ L1). The protein was transferred onto a PVDF membrane using a rapid wet transfer apparatus (transfer time 15 minutes). The PVDF membrane was then blocked (blocking conditions: TBST solution containing 5% skim milk powder, incubated at room temperature for 2 hours). The PVDF membrane was then incubated at 4°C with primary antibody PON1 (Abcam, catalog number: ab181861, antibody dilution 1:1000) for 16 hours. The PVDF membrane was then washed three times with TBST buffer (80 rpm, 10 minutes each time) on a horizontal shaker at room temperature. After washing, the membrane was incubated with secondary antibody (Abcam, catalog number: ab205718, antibody dilution 1:10000) at room temperature for 2 hours. The washing process was repeated. Finally, the membrane was developed using a chromogenic reagent. The developed images were analyzed by grayscale analysis and statistical analysis. The results are shown below. Figure 1 In the middle. For example Figure 1 As shown in Figure ac, with the progression of chronic liver injury, the expression of both PON1 mRNA and protein in the liver significantly decreased in the early stage of liver injury (1 week of CCl4 treatment). The expression level of PON1 in mouse serum was quantitatively analyzed using an ELISA kit, and the results are as follows: Figure 1 As shown in d, serum PON1 levels also decreased in the early stages of liver injury. Figure 1 f and h show the immunohistochemical results of PON1 in liver tissue, indicating a decrease in intrahepatic PON1 protein levels in the early stages of liver injury; simultaneously Figure 1Further immunohistochemistry of PON1 in bone tissue, as described in e and g, revealed a significant decrease in PON1 protein levels in bone during the early stages of liver injury. In addition, we collected serum samples from healthy individuals and patients with hepatic osteodystrophy, and quantitatively detected the expression levels of PON1 in the serum of both groups using an ELISA kit (human ELISA PON1 Kit, proteintech, KEOO543). Figure 1 i showed a significant decrease in serum PON1 expression levels in patients with hepatic osteodystrophy. Therefore, we hypothesize that PON1 may be an early risk factor for hepatic osteodystrophy; once intrahepatic PON1 expression decreases, intrabone PON1 levels decrease, and bone loss begins.

[0036] Table 1: Primer Table

[0037]

[0038] Example 2

[0039] Screening experiments have shown that PON1 is specifically secreted by the liver and is a liver-derived factor.

[0040] To screen for potential key regulatory factors, we further performed overlap analysis on differentially expressed proteins from liver and serum with secreted protein libraries, ultimately identifying eight potential proteins. Among these eight candidate proteins, we focused particularly on liver-specific secreted proteins, as well as factors that play important roles in lipid metabolism and immune inflammation, and show significant changes before bone density decline. Ultimately, we screened for paraoxonase 1 (PON1); the Genecard database showed that PON1 is mainly distributed in the liver; our mRNA analysis of isolated mouse tissues and organs revealed that PON1 mRNA expression is primarily in the liver. Therefore, our preliminary experiments demonstrate that PON1 is specifically secreted by the liver and is a liver-derived factor.

[0041] Example 3

[0042] We successfully constructed a mouse model that specifically overexpresses PON1 in the liver. First, we constructed a PON1-overexpressing adeno-associated virus vector, the vector map of which is shown below. Figure 3 As shown in figure a, the vector number is ZT014946, the cloning site is BstBI-BamHI, and a target sequence of 1761 bp is inserted between them. The target sequence is disclosed below. AAV-TBG-pon1-3×HA-T2A-EGFP was successfully constructed. The vector was injected via tail vein at a dose of 4.0 × 10⁻⁶. 12PON1 was specifically overexpressed in the liver of 6-week-old mice (C57BL / 6 mice) at a dose of μg / kg. An early-onset HOD mouse model was constructed at 12 weeks (CCl4 induced for 3 weeks). The gene and protein levels of PON1 were then verified by quantitative real-time PCR, Western blot and immunohistochemistry, respectively. Figure 3 b is a schematic diagram of animal model creation. Figure 3 c indicates that the PON1 overexpression lentiviral vector can upregulate the PON1 gene level in bone tissue. Figure 3 Immunohistochemistry and Western blot analysis of PON1 in liver tissue by df also showed that PON1 overexpression lentiviral vectors could significantly upregulate the protein level of PON1 in liver tissue.

[0043]

[0044] Example 4

[0045] PON1 can promote osteoblast proliferation and activity, and inhibit osteoclast proliferation.

[0046] To further clarify the regulatory role of PON1 on bone mass, we euthanized newborn mice (within 48 hours of birth) under ether anesthesia, and dissected the skullcap, placing it in a cell culture dish containing PBS. The periosteum, blood vessels, and other connective tissues were removed. The cells were washed three times with PBS, and the skull was cut into bone fragments smaller than 1 mm. The fragments were centrifuged at 1000 rpm for 5 min, digested three times with type II collagenase, and then centrifuged again. The centrifuged bone fragments and cells were placed in α-MEM medium containing 10% fetal bovine serum and incubated at 37°C. The primary cells extracted at this stage were P0 generation; P1 generation cells were used for experiments. We treated primary osteoblasts with recombinant human PON1 protein (abcam, AB53376, hereinafter referred to as rPON1) at concentrations of 0, 25, 50, 100, and 200 ng / mL for one week, followed by ALPL staining. We then treated primary osteoblasts with gradient concentrations of rPON1, and after 48 hours, we detected the expression levels of osteogenic-related markers. The results are as follows: Figure 4 As shown in Figure 1, the mRNA levels of osteoblast differentiation and mineralization markers Runx2, Sp7, Ocn, Col-1α, and Alpl were significantly increased, demonstrating that PON1 can promote osteoblast proliferation and enhance osteoblast differentiation and mineralization capabilities in vitro. To observe the further effects of PON1 on osteoblasts, activated MC3T3 cell lines were treated with rPON1 at concentrations of 0, 25, 50, 100, and 200 ng / mL for 24 hours. Figure 4 Immunofluorescence staining results showed that PON1 could enter osteoblasts with prolonged treatment time. When primary osteoblasts and primary osteoclasts were treated with gradient concentrations of exogenous rPON1, Edu staining and CCK8 assays showed that… Figure 4 The results from gk showed that PON1 significantly promoted osteoblast proliferation while inhibiting osteoclast proliferation in vitro. This suggests that during the course of HOD, the absence or reduction of intrahepatic PON1 may inhibit osteoblast activity through blood circulation, leading to bone loss.

[0047] Example 5:

[0048] Adenovirus vector-mediated PON1 overexpression can improve liver injury in HOD model mice.

[0049] To observe the alleviating effect of liver-specific overexpression of PON1 on early HOD liver injury, we collected serum, liver, and bone tissue from the AAV8-overexpressing PON1 mouse model in Example 3. The results of blood biochemical index detection in mice showed that serum ALT and AST levels in the PON1-overexpressing group were significantly lower than those in the CCl4-positive control group (…). Figure 5 Serum ALT and AST levels (ab). Subsequently, immunohistochemical staining of α-SMA, a molecular marker of fibrosis, in liver tissue sections was performed. The results showed that the degree of liver fibrosis in the PON1 overexpression group was significantly lower than that in the CCl4-positive control group (ab). Figure 5 cd). HE staining and Sirius red staining results also showed that the degree of liver fibrosis in the CCl4-positive control group mice was more severe than that in the PON1 overexpression group. Figure 5 (ef). This demonstrates that adenovirus vector-mediated PON1 overexpression can effectively improve liver damage in HOD model mice.

[0050] Example 6

[0051] Adenovirus vector-mediated PON1 overexpression can improve bone loss in HOD model mice.

[0052] To observe the alleviating effect of liver-specific overexpression of PON1 on early HOD bone loss, we collected serum, liver, and bone tissue from the AAV8 liver-specific PON1 overexpression mouse model in Example 3. MicroCT results showed that the bone mass, bone volume fraction, trabecular bone number, and trabecular bone thickness of the positive control mice induced by CCl4 were significantly decreased, while the trabecular bone separation was significantly increased. In contrast, the bone mass, bone mass per tissue volume, and trabecular bone number of the mice injected with AAV8-Pon1 were significantly increased. Figure 6 ah).

[0053] Example 7 Preliminary assessment of the diagnostic value of serum PON1 levels in hepatic osteodystrophy associated with non-alcoholic steatohepatitis.

[0054] To preliminarily validate the potential of serum PON1 as a diagnostic biomarker for hepatic osteodystrophy (HOD), we conducted a small clinical study on non-alcoholic steatohepatitis-associated HOD (NASH-HOD).

[0055] 1. Sample and Methods Peripheral blood serum samples were collected from 17 clinically diagnosed NASH-HOD patients and 24 age- and sex-matched healthy volunteers as normal controls, for a total of 41 samples. All sample collection and processing followed relevant ethical guidelines. The concentration of PON1 protein in all serum samples was detected using the ELISA method described in Example 1.

[0056] 2. Statistical Analysis Serum PON1 concentration data were correlated with the clinical diagnostic gold standard (whether NASH-HOD or not), receiver operating characteristic (ROC) curves were plotted, the area under the curve (AUC) was calculated to assess diagnostic accuracy, and the optimal diagnostic cutoff value and its corresponding diagnostic efficacy index were determined based on the maximum value of the Youden index.

[0057] 3. Results ROC curve as follows Figure 7 As shown in the figure. Analysis results showed that the AUC value for serum PON1 levels in distinguishing NASH-HOD patients from healthy controls was 0.632 (95% CI: 0.449–0.815). The optimal diagnostic cutoff value was a serum PON1 concentration ≤ 381.73 ng / mL. At this cutoff value, the diagnostic sensitivity was 47.1%, the specificity was 87.5%, and the accuracy was 70.7%.

[0058] 4. Conclusion This preliminary study, based on a small sample size, demonstrates that serum PON1 concentration is significantly reduced in NASH-HOD patients, exhibiting a certain discriminatory ability as a diagnostic biomarker, particularly with high specificity. This result is the first clinical study to validate the feasibility of using decreased serum PON1 concentration as an auxiliary diagnostic indicator for HOD, providing preliminary empirical evidence for the development of related diagnostic kits. Understandably, the sample size in this study is limited; further improvements in diagnostic efficacy are expected through expanding the sample size, optimizing population stratification, or combining with other biomarkers for multi-indicator analysis.

[0059] Summary of Examples: This invention systematically verifies the role and application potential of PON1 in hepatic osteopathy through a series of examples: 1. Mechanism discovery and diagnostic value verification (Examples 1 and 2): In CCl4-induced HOD mouse models and serum of clinical patients, it was confirmed that the mRNA and protein levels of PON1 were significantly reduced in the early stage of the disease, and that it was specifically secreted by the liver, suggesting that it can be used as a plasma biomarker for early diagnosis.

[0060] 2. Construction and validation of gene therapy strategy (Example 3): An adeno-associated virus (AAV) vector (AAV8-TBG-Pon1) that specifically overexpresses PON1 in the liver was successfully constructed, and it was confirmed that it could effectively upregulate PON1 expression in the liver and bone tissue of HOD model mice.

[0061] 3. In vitro functional verification (Example 4): Cells were treated with recombinant PON1 protein, demonstrating that PON1 can promote osteoblast activity, differentiation and proliferation in a dose-dependent manner, while inhibiting osteoclast proliferation, thus clarifying its direct regulatory role in bone metabolism.

[0062] 4. In vivo therapeutic effect verification (Examples 5 and 6): In HOD model mice, liver-specific overexpression of PON1 can not only significantly improve liver injury indicators (reduce ALT and AST, and alleviate fibrosis), but also directly reverse bone loss, significantly increase bone mineral density, trabecular bone number and thickness, and improve bone microstructure.

[0063] In summary, the examples presented a complete chain of evidence, from disease association, tissue specificity, molecular mechanisms, in vitro function to in vivo efficacy, fully demonstrating the feasibility and effectiveness of targeting PON1 for the diagnosis and treatment of hepatic osteopathy. The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. Use of PON1 as a biomarker in the preparation of kits for the diagnosis of hepatic osteodystrophy.

2. The use according to claim 1, characterized in that, The kit contains detection reagents for detecting the expression level of PON1 in biological samples obtained from the individual to be tested.

3. The use according to claim 2, characterized in that, The expression level of PON1 is either the PON1 protein level or the PON1 mRNA level; and / or, the detection reagent includes an antibody against the PON1 protein or a nucleic acid probe that specifically binds to PON1 mRNA.

4. Use of PON1, its agonists, or substances capable of upregulating PON1 expression in the preparation of medicaments for the prevention and / or treatment of hepatic osteodystrophy.

5. The use according to claim 4, characterized in that, The substance capable of upregulating PON1 expression is selected from the PON1 protein, a nucleic acid molecule encoding the PON1 protein, an expression vector containing the nucleic acid molecule, or a compound capable of promoting the transcription or translation of the endogenous PON1 gene.

6. The use according to claim 5, characterized in that, The nucleic acid molecule contains a nucleotide sequence encoding the PON1 protein as defined by NCBI Gene ID: 18979; and / or, the PON1 protein contains an amino acid sequence as defined by NCBI Gene ID: 18979.

7. A pharmaceutical composition for the prevention and / or treatment of hepatic osteodystrophy, characterized in that, It contains a therapeutically effective amount of PON1 protein, its agonist or a substance that can upregulate PON1 expression, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is formulated into a dosage form for injection.

9. A method for screening candidate drugs for the prevention and / or treatment of hepatic osteodystrophy, characterized in that, The method includes the following steps: (a) Under test conditions, the candidate material was brought into contact with a system capable of expressing PON1; (b) Detect the effect of the candidate substance on the expression level or activity of PON1 in the system; (c) Select candidate substances that can upregulate the expression level or activity of PON1 as the candidate drug.

10. The method according to claim 9, characterized in that, The system capable of expressing PON1 is a cell expressing PON1, cell lysate, or a reporter gene system containing PON1 gene transcriptional regulatory elements.

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