Application of Rab8a in preparation of medicine for treating or preventing or improving individual hepatic fibrosis

Abstract

The invention discloses application of Rab8a in preparation of drugs for treating or preventing or improving individual hepatic fibrosis, and belongs to the technical field of drugs and disease treatment. The invention provides application of Rab8a in preparation of a medicine for treating or preventing or improving individual hepatic fibrosis. The Rab8a can enhance autophagy by down-regulating the expression level of the I-type TGF-beta receptor on the HSCs membrane, so that the activation of the HSCs is reduced, the hepatic fibrosis is favorably retarded or improved, and the Rab8a has the potential of serving as a target spot for treating the hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine and disease treatment, and in particular to an application of Rab8a in the preparation of a medicine for treating, preventing or improving individual liver fibrosis. Background Art

[0002] Liver fibrosis is a repair response of the liver to chronic damage, characterized by excessive deposition of extracellular matrix (ECM) components, which can ultimately lead to cirrhosis and liver failure. Liver fibrosis is a common pathological process in various chronic liver diseases, such as viral hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease (NAFLD). As research deepens, it is found that liver fibrosis is not a simple linear progression, but a complex process consisting of multiple stages, each with its own unique cellular and molecular characteristics.

[0003] The development of liver fibrosis begins with the activation of hepatic stellate cells (HSCs). In a healthy liver, HSCs remain quiescent and store vitamin A. However, under conditions of chronic injury, they are activated and transformed into myofibroblast-like cells, beginning to synthesize collagen and other ECM components. This transition marks the onset of fibrosis and is accompanied by a series of changes at the molecular and cellular levels. For example, activated HSCs express high levels of α-smooth muscle actin (α-SMA), which is not only a hallmark of HSC activation but also a key factor in promoting ECM deposition. As fibrosis progresses, ECM components accumulate, leading to significant changes in liver architecture. These structural changes not only affect liver function but also further promote HSC activation by altering the mechanical environment, creating a vicious cycle. Studies have shown that increased ECM stiffness can activate the RhoA / ROCK signaling pathway through integrin-mediated signaling, thereby enhancing HSC contractility and ECM synthesis. Furthermore, changes in ECM stiffness can regulate other key transcription factors and signaling pathways, such as the TGF-β / Smad signaling pathway, further driving fibrosis. It's important to note that liver fibrosis is not irreversible. In the early stages, if the primary disease can be effectively controlled and the progression of fibrosis can be inhibited, some patients can achieve fibrosis reversal. However, once cirrhosis progresses to the advanced stage, the likelihood of reversal decreases significantly, and treatment becomes significantly more difficult. Therefore, early diagnosis and intervention are crucial to improving patient outcomes.

[0004] In summary, liver fibrosis is a complex, multistage process involving the interplay of multiple cell types and molecular mechanisms. A deeper understanding of the key steps and regulatory mechanisms in this process will not only reveal the underlying mechanisms of liver fibrosis but also provide a theoretical basis and technical support for the development of more effective treatments. Future work will continue to explore new therapies for liver fibrosis in order to improve clinical outcomes for patients.

[0005] Finding new methods to treat, prevent or improve individual liver fibrosis is a technical problem that needs to be solved by existing technologies. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above technical deficiencies, provide an application of Rab8a in the preparation of a drug for treating, preventing or improving individual liver fibrosis, and solve the technical problem of how to achieve the treatment, prevention or improvement of individual liver fibrosis in the prior art.

[0007] In order to achieve the above technical objectives, the technical solution of the present invention provides an application of Rab8a in the preparation of a drug for treating, preventing or improving individual liver fibrosis.

[0008] In any embodiment, the Rab8a overexpression vector is used in the preparation of a drug for treating, preventing or improving individual liver fibrosis.

[0009] In any embodiment, a Rab8a agonist is used in the preparation of a drug for treating, preventing or improving liver fibrosis in an individual.

[0010] In any embodiment, the individual is a mammal.

[0011] In any embodiment, the individual is a mouse.

[0012] In any embodiment, the individual is a human.

[0013] In any embodiment, the Rab8a overexpression vector is an AAV8-Rab8a overexpression vector.

[0014] In any embodiment, the dosage form of the drug is injection, oral preparation, spray or suppository.

[0015] Compared with the prior art, the beneficial effects of the present invention include: the present invention reveals that Rab8a can enhance autophagy by downregulating the expression level of type I TGF-β receptor on the HSCs membrane, thereby reducing the activation of HSCs, helping to slow down or improve liver fibrosis, indicating that Rab8a has the potential to be a target for the treatment of liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1This is the construction of the liver fibrosis mouse model in Example 1 of the present invention. Figure 1 A: Liver fibrosis mouse model was induced by intraperitoneal injection of carbon tetrachloride (CCL4) and corn oil (Oil); Figure 1 B: Immunoblotting (WB) was used to verify the modeling effect of liver fibrosis model.

[0017] Figure 2 The Rab8a mRNA and protein expression levels in HSCs activated in Example 1 of the present invention were detected. Figure 2 A: qPCR detection of Rab8a mRNA levels in the control group (PBS) and HSCs activation group (TGF-β); Figure 2 B: Western blot was used to detect the protein expression level of Rab8a in the control group (PBS) and HSCs activation group (TGF-β); Figure 2 C: Quantification of Rab8a protein expression levels in different treatment groups.

[0018] Figure 3 In Example 1 of the present invention, a stable transgenic strain with knockdown of Rab8a was constructed and phenotypic experiments were performed. Figure 3 A: Rab8a knockdown effect was detected by WB; Figure 3 B: Real-time polymerase chain reaction (qPCR) was used to detect the knockdown effect of Rab8a; Figure 3 C: Western blot was used to detect the levels of Smad2 / 3 and p-Smad2 / 3, key proteins in the TGF-β signaling pathway, after knockdown of Rab8a. Figure 3 D: Flow cytometry was used to detect the activation of HSCs in different treatment groups.

[0019] Figure 4 This is because knocking down Rab8a in Example 1 of the present invention affects the expression of TGF-β receptor. Figure 4 A: Western blot was used to detect the expression of TGF-β receptor (TβRI / TβRII) after Rab8a knockdown; Figure 4 B: Western blot quantification of TβRI / TβRII expression after knockdown of Rab8a; Figure 4 C: Flow cytometry was used to detect the expression level of TβRI after Rab8a knockdown; Figure 4 D: Flow cytometry quantification of TβRI expression after knockdown of Rab8a; Figure 4 E: Immunofluorescence staining was used to detect the expression of TβRI in different treatment groups; Figure 4 F: Immunofluorescence staining quantification of TβRI expression after knockdown of Rab8a.

[0020] Figure 5 In Example 1 of the present invention, knocking down Rab8a affects the activation of HSCs through autophagy. Figure 5A: Western blot was used to detect Na after Rab8a knockdown + -K + -ATPase expression; Figure 5 B: Western blot was used to detect the expression of autophagy-related proteins LC3 and p62 after Rab8a knockdown; Figure 5 C: Flow cytometry was used to detect the expression of TβRI in different treatment groups; Figure 5 D: Flow cytometry quantification of TβRI expression after knockdown of Rab8a; Figure 5 E: Immunofluorescence staining was used to detect the expression of TβRI and p62 in different treatment groups.

[0021] Figure 6 Example 1 of the present invention verifies at the in vivo level that overexpression of Rab8a inhibits the progression of liver fibrosis. Figure 6 A: A Rab8a-overexpressing liver fibrosis mouse model was constructed using AAV8; Figure 6 B: Immunohistochemistry (IHC) staining was used to detect liver fibrosis in the liver sections of mice in different treatment groups; Figure 6 C: Quantification of liver fibrosis in mice under different treatment groups. DETAILED DESCRIPTION

[0022] The "ranges" disclosed herein are defined in terms of lower and upper limits, where a given range is defined by selecting a lower limit and an upper limit, and the selected lower and upper limits define the boundaries of the particular range. Ranges defined in this manner can be inclusive or exclusive of the end values ​​and can be combined arbitrarily, i.e., any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 to 120 and 80 to 110 are listed for a particular parameter, it is understood that ranges of 60 to 110 and 80 to 120 are also contemplated. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, the following ranges are all contemplated: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In this application, unless otherwise indicated, the numerical range "a to b" is a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, when a parameter is expressed as an integer ≥ 2, this is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0023] Unless otherwise specified, the terms "include" and "comprising" used in this application may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other components not listed may also be included or that only the listed components are included.

[0024] Unless otherwise specified, the term "or" is used in this application to be inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, the condition "A or B" is satisfied if any of the following conditions are met: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0025] Fibrosis is a repair response of the liver to chronic injury, characterized by the excessive deposition of extracellular matrix (ECM) components. This process is primarily driven by activated HSCs, which are activated under conditions of chronic inflammation or injury. These cells synthesize and secrete large amounts of ECM components, promoting the development of liver fibrosis.

[0026] Autophagy is a cellular self-regulatory mechanism by which cells clear damaged organelles and protein aggregates, maintaining a stable internal cellular environment. This process is particularly important in a variety of pathological conditions, including liver fibrosis. Furthermore, autophagy can regulate the function of macrophages and HSCs, alleviating the liver's inflammatory response by promoting the release of anti-inflammatory cytokines and reducing the levels of pro-inflammatory cytokines, thereby reducing the extent of liver fibrosis. Rab8a, a small GTPase, plays an important role in regulating autophagy, endocytosis, and vesicle trafficking. Our study revealed that Rab8a can enhance autophagy by affecting the expression level of type I TGF-β receptor on the membrane of HSCs, thereby regulating the activation state of HSCs and helping to slow liver fibrosis, indicating the potential of Rab8a as a therapeutic target for liver fibrosis.

[0027] This specific embodiment provides a use of Rab8a in the preparation of a drug for treating, preventing or improving individual liver fibrosis.

[0028] This specific embodiment also provides a use of a Rab8a overexpression vector in the preparation of a drug for treating, preventing or improving individual liver fibrosis.

[0029] This specific embodiment also provides a use of a Rab8a agonist in the preparation of a drug for treating, preventing or improving individual liver fibrosis.

[0030] In some embodiments, the individual is a mammal.

[0031] In some embodiments, the individual is a mouse.

[0032] In some embodiments, the individual is a human.

[0033] In some embodiments, the Rab8a overexpression vector is an AAV8-Rab8a overexpression vector.

[0034] In some embodiments, the drug is in the form of an injection, oral solution, spray, or suppository.

[0035] In this study, we investigated the mechanism by which Rab8a affects the progression of liver fibrosis and its potential as a targeted therapy for liver fibrosis. Rab8a downregulates the type I TGF-β receptor on the membrane of HSCs, enhancing autophagy, reducing HSC activation, and improving liver fibrosis. Therefore, targeting Rab8a may be an effective treatment for liver fibrosis. We believe this study is significant because it reveals the mechanism of action of Rab8a in liver fibrosis and proposes a therapeutic strategy based on this mechanism that may delay liver fibrosis, laying the foundation for further research.

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] In the present invention, references to “some embodiments”, “this embodiment”, examples, etc. describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments, and may be combined with each other without conflict.

[0038] If similar descriptions of "first / second" appear in the application documents, the following explanation is added. In the following description, the terms "first\second\third" are merely used to distinguish similar objects and do not represent a specific order of the objects. It can be understood that "first\second\third" can be interchanged with the specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0039] In this embodiment, the term "and / or" is merely a description of the association relationship between associated objects, indicating that three relationships may exist. For example, object A and / or object B may represent three situations: object A exists alone, object A and object B exist at the same time, and object B exists alone.

[0040] Below, the embodiment of the present application is described. The embodiment described below is exemplary and is only used to explain the present application, and is not to be construed as limiting the present application. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used that do not specify the manufacturer are conventional products that can be obtained commercially.

[0041] Example 1

[0042] Establishment of a mouse model of liver fibrosis

[0043] Male C57BL / 6 mice aged 8 to 14 weeks were used to induce a liver fibrosis model by intraperitoneal injection of a mixed solution of 20% CCL4 (Sigma-Aldrich, Catalog No. 289116) and corn oil (Sigma-Aldrich, Catalog No. 23-0230) (dose of 5 μL per gram of body weight). For AAV8-treated liver fibrosis mice, they were first given a two-week pretreatment with CCL4, followed by a tail vein injection of 100 μL of AAV8-Control or AAV8-Rab8a overexpression vector (viral genome concentration of 1.0×10^12 / mL; IGE Biotechnology, Guangzhou, China). After four weeks of continued CCL4 treatment, the mouse livers were harvested and analyzed. All experimental procedures were performed in strict accordance with the guidelines of the Experimental Animal Care and Use Committee of Sun Yat-sen University. Figure 1 As shown, compared with the control group, immunohistochemistry and Sirius Red (PSR) staining of α-SMA and Col1A1 in the livers of mice in the CCL4 group showed increased deposition of α-SMA and Col1A1 in the livers of mice in the CCL4 group, indicating that the mouse liver fibrosis model was successful and HSCs were activated. At the protein level, Rab8a was also decreased with the upregulation of α-SMA and Col1A1.

[0044] Isolation of mouse liver HSCs

[0045] After isolating livers from mice with a hepatic fibrosis model, they were digested by retrograde perfusion with a solution containing pronase (Sigma-Aldrich, Catalog No. 11459643001) and collagenase (Sigma-Aldrich, Catalog No. 11213865001). The cell suspension was centrifuged at 50 x g, and the supernatant containing the non-parenchymal hepatocytes was collected. HSCs were purified from the non-parenchymal hepatocytes by density gradient centrifugation and cultured in DMEM medium supplemented with 10% fetal bovine serum (GIBCO, Catalog No. 10099).

[0046] Induced mouse hepatic stellate cell line JS-1

[0047] JS-1 cells, a mouse hepatic stellate cell line, were cultured in DMEM supplemented with 10% fetal bovine serum (GIBCO, Cat. No. 10099) in a 5% CO2 incubator at 37°C. JS-1 cells were treated with TGF-β (5 μg / mL) for 48 hours and DMOG (2 mM) for 24 hours to investigate how Rab8a regulates TβRI by reducing autophagy.

[0048] Mouse liver section staining

[0049] Paraffin-embedded mouse liver tissue samples were cut into 5 μm thick sections. Subsequently, the sections were stained with hematoxylin and eosin (H&E). In addition, the liver sections were stained with PSR to detect collagen fiber deposition in the tissue. IHC was performed using anti-Col1A1 (CST, 72026S) and anti-α-SMA (CST, 19245S). The above staining was used to evaluate the liver fibrosis in mice. Figure 1 As shown in the Figure 3, it can be observed that the expression level of Rab8a protein increases with the progression of liver fibrosis in mice.

[0050] Detection of JS-1 cell activation status by flow cytometry

[0051] Use 0.25% trypsin-EDTA to digest the logarithmic growth phase JS-1 cells in the culture medium, then terminate the digestion with serum and adjust the cell concentration. After the cells have been washed and centrifuged, add the appropriate volume and concentration of antibodies to the centrifuge tube according to the requirements of the antibody instructions, and incubate at 4°C in the dark for 1 hour. After the incubation is complete, wash the cells again by centrifugation. Then, resuspend the cell pellet and mix it thoroughly, and transfer it to the flow tube. The entire operation process must be protected from light. Finally, flow cytometry is used for detection, and existing software is used for data analysis. Figure 2 As shown, primary HSCs (CP-M041) and the mouse HSCs cell line JS-1 were stimulated with TGF-β (5 μg / mL, 48 hours), and Rab8a levels were measured at the transcriptional and protein levels. The results showed that Rab8a expression decreased with the upregulation of α-SMA and the activation of HSCs.

[0052] Construction of Rab8a knockdown cell lines

[0053] A shRNA plasmid for interfering with Rab8a expression was constructed and transfected into JS-1 cells, creating JS-1-sh-Rab8a 1 and JS-1-sh-Rab8a 2 (1 and 2 represent two different shRNA plasmids interfering with Rab8a expression, both of which are from existing technologies) as well as a negative control cell line. According to the reagent instructions, the shRNA plasmid transfection process was completed using Lipofectamine 3000 and incubated for 24 hours after transfection. Finally, the mRNA and protein levels of the relevant molecules were measured using qPCR and western blot.

[0054] like Figure 3 As shown in Figure 2, we constructed a Rab8a knockdown JS-1 and tested the knockdown efficiency by Western blotting and qPCR. We stimulated HSCs in the control and two knockdown groups with TGF-β (5 μg / mL, 48 hours) and measured intracellular Smad2 / 3 phosphorylation levels. The results showed that HSCs with Rab8a knockdown were more sensitive to TGF-β stimulation. Under the same stimulation conditions, Smad2 / 3 protein phosphorylation levels were higher and α-SMA upregulation was more pronounced. Flow cytometry results showed that Rab8a knockdown cells exhibited a higher proportion of cells in the G2 / M phase upon TGF-β stimulation.

[0055] We examined the levels of type I and type II TGF-β receptors on the membranes of wild-type and Rab8a-knockdown JS-1 cells. Figure 4 The results showed that after TGF-β stimulation, the content of type I TGF-β receptor on the membrane increased, and knocking down Rab8a further increased the level of type I TGF-β receptor on the membrane. We further verified the regulatory effect of Rab8a on type I TGF-β receptor levels on the membrane of HSCs by flow cytometry and immunofluorescence. As expected, knocking down Rab8a increased the level of type I TGF-β receptor on the membrane of HSCs.

[0056] Immunofluorescence

[0057] JS-1 cells were treated in 12-well plates. Cells were fixed with immunostaining fixative, permeabilized with enhanced immunostaining permeabilization buffer, and blocked with immunostaining blocking buffer. Cells were then incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 hour at room temperature. The next day, DAPI was added. Finally, images were captured using an inverted fluorescence microscope.

[0058] After treating Rab8a knockdown cells with the autophagy inducer DMOG, we examined the level of type I TGF-β receptor on the membrane of HSCs. Figure 5As shown in Figure 2, the results indicate that enhanced autophagy can reduce the upregulation of TGF-β receptors on the cell membrane by knocking down Rab8a. This suggests that autophagy may be a downstream pathway of Rab8a-mediated downregulation of type I TGF-β receptors. Furthermore, we examined intracellular autophagy levels and found that knocking down Rab8a decreased intracellular autophagy levels, while DMOG-induced autophagy increased intracellular autophagy levels. We further confirmed changes in type I TGF-β receptors on the membrane of HSCs using flow cytometry and immunofluorescence.

[0059] To explore whether targeting Rab8a can prevent the further development of established fibrosis in vivo, we used HSC-targeted AAV8 to carry overexpressed Rab8a to treat a CCL4-induced mouse fibrosis model. Subsequently, we used mouse AAV8-Rab8aOE (using adenoviral vector AAV8 to construct overexpression of the Rab8a gene) to study the in vivo function of Rab8a in CCL4-induced liver fibrosis. Figure 6 As shown, compared with CCL4 mice, the immunohistochemistry and PSR staining results of α-SMA and Col1A1 in the liver of AAV8 mice showed that the deposition of Col1A1 in the mouse liver was reduced, the expression of α-SMA was reduced, and liver fibrosis was alleviated.

[0060] The specific embodiments of the present invention described above do not limit the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. Application of Rab8a in the preparation of drugs for treating, preventing or improving individual liver fibrosis.

2. Application of Rab8a overexpression vector in the preparation of drugs for treating, preventing or improving individual liver fibrosis.

3. Use of Rab8a agonists in the preparation of drugs for treating, preventing or improving individual liver fibrosis.

4. The use according to any one of claims 1 to 3, characterized in that The individual is a mammal.

5. The use according to any one of claims 1 to 3, characterized in that The individual is a mouse.

6. The use according to any one of claims 1 to 3, characterized in that The individual is a human.

7. The use according to claim 2, characterized in that The Rab8a overexpression vector is an AAV8-Rab8a overexpression vector.

8. The use according to any one of claims 1 to 3, characterized in that The dosage form of the medicine is injection, oral preparation, spray or suppository.

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