Use of a kdelr3 inhibitor in the manufacture of a medicament for preventing and / or treating fibrotic disease in an individual

Abstract

This invention belongs to the field of biomedical technology and discloses the application of KDELR3 inhibitors in the preparation of drugs for the prevention and / or treatment of individual fibrotic diseases. In a wild-type mouse model of pulmonary fibrosis, compared with the control group receiving intratracheal injection of Scr siRNA, intratracheal injection of KDELR3 siRNA significantly reduced the degree of fibrosis in the mouse lung tissue. KDELR3 inhibition significantly reduced collagen deposition and improved lung tissue structural damage. Further mechanistic studies showed that KDELR3 inhibitors can inhibit the differentiation of fibroblasts into myofibroblasts, thereby reducing the production of fibrosis-related extracellular matrix. These results indicate that KDELR3 is a key molecule regulating the occurrence and development of fibrosis, and drugs that can inhibit KDELR3 expression or activity can be used to prevent and treat various fibrotic diseases, including pulmonary fibrosis, laying the foundation for the development of novel drugs that are effective and safe in treating fibrotic diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of KDELR3 inhibitors in the preparation of drugs for the prevention and / or treatment of individual fibrotic diseases. Background Technology

[0002] KDELR3, short for Lys-Asp-Glu-Leu endoplasmic reticulum protein retention receptor 3, is a membrane receptor protein located between the endoplasmic reticulum (ER) and the Golgi apparatus. It plays a crucial role in intracellular protein transport and quality control. As a member of the KDEL receptor family, KDELR3 primarily recognizes secreted proteins carrying KDEL or HDEL sequences (such as GRP78 and GRP94). When these proteins are transported into the Golgi apparatus, KDELR3 recognizes their signal sequences and re-enters the ER. In addition, KDELR3 can bind to various cellular signaling molecules, including G proteins, Src family kinases, protein kinase A (PKA), and mitogen-activated white blood cell kinases (MAPKs), and participates in cell membrane transport, signal transduction, and stress responses by regulating these signaling cascades. Studies have shown that KDLER3 is involved in tumor immune escape in breast cancer, and KDLER3 is highly expressed in scleral fibroblasts and participates in regulating the production of extracellular matrix in scleral cells in high myopia.

[0003] Fibrosis is defined as the excessive proliferation of fibroblasts within an organ, accompanied by the accumulation of large amounts of extracellular matrix, destroying tissue structure. In the late stages of various diseases, such as viral hepatitis, chronic nephritis, and scleroderma, organ fibrosis occurs, ultimately leading to organ failure. Common fibrotic diseases include pulmonary fibrosis, renal fibrosis, hepatic fibrosis, and skin fibrosis. The fibroblast-myofibroblast transition (FMT) is a key step in the occurrence and development of IPF. During the IPF process, pro-fibrotic signals induce fibroblasts to transform into myofibroblasts, which exhibit strong ECM synthesis and contractile abilities, promoting excessive ECM deposition and alveolar structural destruction, ultimately leading to decreased lung tissue compliance and impaired gas exchange. Reducing fibroblast differentiation can effectively treat pulmonary fibrosis models induced by various factors. Although current research indicates that fibroblast differentiation plays an important role in the process of pulmonary fibrosis, there are currently no drugs that can effectively block this behavior, thereby preventing or reversing the progression of pulmonary fibrosis.

[0004] Currently, the main treatments for fibrosis include drug therapy and organ transplantation. Organ transplantation, as the only last resort for fibrosis patients, faces limitations in its application and promotion due to difficulties in donor sourcing, high surgical risks, and high costs. Existing drugs cannot reverse the progression of fibrosis, and their clinical efficacy and safety are insufficient to meet treatment needs. Therefore, there is an urgent need to conduct in-depth research into the mechanisms of fibrosis, identify new therapeutic targets, and fully integrate innovative pharmaceutical development trends to develop novel drugs that can effectively treat fibrosis with high safety. Summary of the Invention

[0005] This invention discloses the application of KDELR3 inhibitors in the preparation of drugs for the prevention and / or treatment of individual fibrotic diseases, laying the foundation for the development of novel drugs that can effectively treat fibrotic diseases and have high safety.

[0006] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides the use of KDELR3 inhibitors in the preparation of medicaments for the prevention and / or treatment of individual fibrotic diseases.

[0007] In the above technical solutions, the KDELR3 inhibitor includes at least one of the following: 1) Substances that affect the expression level and / or function of the KDELR3 gene; 2) Substances that reduce or inactivate KDELR3 activity.

[0008] 3) Substances that promote the degradation of KDELR3.

[0009] In the above technical solutions, the KDELR3 inhibitor includes at least one of the following: 1) At least one of siRNA, shRNA, or miRNA that can inhibit KDELR3 expression; 2) Vectors containing the above-mentioned siRNA, shRNA, and / or miRNA; 3) Host cells containing the above-mentioned vectors.

[0010] In the above technical solution, the upstream primer sequence of the KDELR3 gene is shown in SEQ ID No. 1, which is 5'-GGTGAACTACAGTTACACGCCG-3', and the downstream primer sequence is shown in SEQ ID No. 2, which is 5'-AGTGGTGATGGTCTCAGCCTCT-3'.

[0011] In the above technical solutions, the fibrotic diseases include pulmonary fibrosis, renal fibrosis, myocardial fibrosis, liver fibrosis, and skin fibrosis.

[0012] In the above technical solution, the fibrotic disease is pulmonary fibrosis.

[0013] In the above technical solution, the fibrotic disease is idiopathic pulmonary fibrosis.

[0014] In the above technical solutions, the individual is a mammal.

[0015] In a second aspect, the present invention provides a pharmaceutical composition comprising a KDELR3 inhibitor and a pharmaceutically acceptable carrier.

[0016] In the above technical solutions, the pharmaceutical composition is an injectable form, capsule, tablet, nasal spray, or aerosol.

[0017] The beneficial effects of this invention are as follows: This invention explores how KDELR3 inhibitors can slow down the fibrosis process by inhibiting the differentiation of fibroblasts, providing a new KDELR3-targeted treatment strategy for fibrotic diseases, and further laying the foundation for the development of novel drugs that can effectively treat fibrotic diseases and have high safety. Attached Figure Description

[0018] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the accompanying drawings are merely schematic illustrations, used to help illustrate the technical solutions and preferred embodiments of the present invention, and do not constitute a limitation on the scope of protection of the technical solutions of the present invention. Within the scope defined by the claims of the present invention, any equivalent transformations or modifications based on the principles of the present invention should be considered to fall within the protection scope of the present invention.

[0019] Figure 1 The images show the H&E, Masson, and Sirius red staining results of lung tissues from mice that underwent BLM-induced fibrosis and were injected with KDELR3 siRNA and Scr siRNA via airway.

[0020] Figure 2 Ashcroft scores of lung tissue from mice induced by BLM with airway injection of KDELR3 siRNA and mice induced by airway injection of Scr siRNA were given, where **p<0.01; ***p<0.05; **p<0.05; **p<0.01; ***p<0.05; **p<0.01.

[0021] Figure 3Western blot results for fibronectin, type I collagen (Col1a1), α-SMA, and KDELR3 in mice that underwent BLM-induced fibrosis and were injected with KDELR3 siRNA and Scr siRNA via airway. In the data, **p<0.01; ***p<0.05; **p<0.05; **p<0.01; ***p<0.05; **p<0.01.

[0022] Figure 4 The results of RT-PCR for fibronectin, type I collagen (Col1a1), α-SMA, and KDELR3 in mice that underwent BLM-induced fibrosis via airway injection of KDELR3 siRNA and Scr siRNA. In the figures, **p<0.01; ***p<0.05; **p<0.05; **p<0.01; ***p<0.05; **p<0.01.

[0023] Figure 5 The results show the quantitative values ​​of hydroxyproline in mice injected with KDELR3 siRNA and Scr siRNA via the airway after BLM-induced fibrosis. In the figures, **p<0.01; ***p<0.05; **p<0.05; **p<0.01; ***p<0.05; **p<0.01.

[0024] Figure 6 The differentiation of human lung fibroblasts into myofibroblasts after KDELR3 knockdown was achieved by transfecting them with KDELR3 siRNA. The values ​​are as follows: **p<0.01; ***p<0.05; **p<0.05; **p<0.01; ***p<0.05; **p<0.01. Detailed Implementation

[0025] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0026] Currently, the main treatments for fibrosis include drug therapy and organ transplantation. Organ transplantation, as the only last resort for fibrosis patients, faces limitations in its application and promotion due to difficulties in donor sourcing, high surgical risks, and high costs. Existing drugs cannot reverse the progression of fibrosis, and their clinical efficacy and safety are insufficient to meet treatment needs. Therefore, there is an urgent need to conduct in-depth research into the mechanisms of fibrosis, identify new therapeutic targets, and fully integrate innovative pharmaceutical development trends to develop novel drugs that can effectively treat fibrosis with high safety.

[0027] Definition and use of terms Fibrotic Diseases: In this invention, fibrotic diseases include, but are not limited to, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, hepatic fibrosis, and skin fibrosis. Hepatic fibrosis, in particular, refers to a pathological state characterized by abnormal proliferation of connective tissue in the liver tissue accompanied by extensive extracellular matrix deposition under the influence of various pathogenic factors. This process can be induced by a variety of factors, including viral infection, chronic inflammatory response, oxidative stress, and long-term alcohol consumption. Its histological characteristics are mainly manifested by increased and deposited fibrous tissue in the portal areas and hepatic lobules, but without the formation of obvious fibrous septa. If the condition continues to progress, it can further develop into cirrhosis, characterized by the formation of pseudolobules and the appearance of fibrous septa between the central venous area and the portal area, ultimately leading to the destruction of the normal liver structure. In my country, the main cause of chronic liver disease is viral hepatitis, and viral hepatitis-related liver fibrosis is closely related to intrahepatic inflammatory response, cell damage, and persistent viral replication. It is noteworthy that this pathological process still has a certain degree of reversibility in its early stages; therefore, through combined antiviral therapy and immune function modulation interventions, the progression of liver fibrosis can be delayed or reversed to some extent.

[0028] The main pathological features of pulmonary fibrosis include mesenchymal cell proliferation, extracellular matrix hyperplasia and deposition, and lung parenchymal remodeling. Current treatments for pulmonary fibrosis primarily involve anti-inflammatory, antioxidant, anti-fibroblast proliferation and collagen deposition therapies, as well as lung transplantation.

[0029] Renal fibrosis refers to the abnormal accumulation of extracellular matrix and connective tissue within the kidneys, leading to structural damage and a gradual decline in kidney function. Most chronic kidney diseases are accompanied by varying degrees of fibrosis in their end-stage progression, eventually resulting in renal failure. This process is typically closely related to persistent inflammatory responses, renal tubular epithelial cell damage and apoptosis, and dysregulation of various signaling molecules associated with fibrosis formation. Therefore, the occurrence and progression of renal fibrosis can be delayed or prevented through various interventions, such as inhibiting inflammatory responses, reducing cell apoptosis, and regulating pro-fibrotic factors.

[0030] Myocardial fibrosis is primarily characterized by abnormally active fibroblasts accompanied by excessive deposition of extracellular matrix in myocardial tissue, thereby disrupting the normal structure of the myocardium. This pathological change is commonly seen in various cardiac conditions, including hypertension-related cardiac injury, ischemic cardiomyopathy, dilated cardiomyopathy, viral myocarditis, and diabetic cardiomyopathy. Myocardial fibrosis causes myocardial tissue to become stiff and lose elasticity, thus interfering with the heart's diastolic and systolic functions, and is an important factor affecting the prognosis and functional recovery of the cardiovascular system.

[0031] Skin fibrosis is typically characterized by the formation of scar tissue. This scar tissue is a dense fibrous connective tissue that gradually remodels and matures from granulation tissue during the repair process. After trauma or other injury, fibroblasts are activated and proliferate rapidly, gathering in the injured area and secreting extracellular matrix components, thereby promoting tissue repair and ultimately forming a scar structure.

[0032] KDELR3 inhibitor: In this invention, KDELR3 inhibitor refers to: 1) substances that affect the expression level or / function of the KDELR3 gene, which may include siRNA, shRNA and miRNA that can inhibit KDELR3 expression, vectors containing the above-mentioned siRNA, shRNA and / or miRNA and host cells containing the above-mentioned vectors, but this invention is not limited thereto. 2) Substances that reduce or inactivate KDELR3 activity; 3) Substances that promote the degradation of KDELR3, such as KDELR3 antibodies that cause degradation.

[0033] Individual: In this invention, the term "individual" refers to a mammal, including but not limited to rats, mice, non-human primates, humans, dogs, cats, horses, cattle, sheep, pigs, and goats. Humans or mice are preferred.

[0034] Prevention and treatment: The term "prevention" in this invention refers to preventing or reducing the occurrence of fibrosis after use in the presence of possible fibrotic factors. The term "treatment" in this invention refers to reducing the degree of fibrosis, curing fibrosis to normalize it, or slowing the progression of fibrosis.

[0035] This invention discloses the use of KDELR3 inhibitors in the preparation of medicaments for the prevention and / or treatment of individual fibrotic diseases. Fibrotic diseases include pulmonary fibrosis, renal fibrosis, myocardial fibrosis, liver fibrosis, and skin fibrosis. The individuals are mammals.

[0036] KDELR3 inhibitors include at least one of the following: 1) Substances that affect the expression level and / or function of the KDELR3 gene; for example, the KDELR3 transcription factor HIF-1α inhibitor Otipra can reduce the expression of KDELR3 mRNA and protein.

[0037] 2) Substances that reduce or inactivate KDELR3 activity.

[0038] 3) Substances that promote the degradation of KDELR3.

[0039] Specifically, KDELR3 inhibitors include at least one of the following: 1) At least one of siRNA, shRNA, or miRNA that can inhibit KDELR3 expression; 2) Vectors containing the above-mentioned siRNA, shRNA, and / or miRNA; 3) Host cells containing the above-mentioned vectors.

[0040] The upstream primer sequence of the KDELR3 gene is shown in SEQ ID No. 1, which is 5'-GGTGAACTACAGTTACACGCCG-3', and the downstream primer sequence is shown in SEQ ID No. 2, which is 5'-AGTGGTGATGGTCTCAGCCTCT-3'.

[0041] This invention also discloses a pharmaceutical composition comprising a KDELR3 inhibitor and a pharmaceutically acceptable carrier. The pharmaceutical composition may be in the form of an injectable form, capsule, tablet, nasal spray, or aerosol, and may be administered to test subjects in an individually acceptable manner, such as by injection, oral administration, nasal spray, or other methods.

[0042] This invention demonstrates through the following examples that inhibiting KDELR3 expression in fibroblasts can significantly reduce the differentiation of fibroblasts into myofibroblasts. The differentiation of fibroblasts into myofibroblasts plays a crucial role in the pathogenesis of fibrotic diseases, including liver fibrosis, pulmonary fibrosis, renal fibrosis, and skin fibrosis.

[0043] To better explain the present invention, the following detailed description is provided in conjunction with specific embodiments.

[0044] Example 1: Effect of airway injection of KDELR3 siRNA on the degree of pulmonary fibrosis Laboratory animals and materials: 1. Laboratory animals: Source: Wild-type mice (WT, C57BL / 6) bred at the Tongji Medical College Animal Room.

[0045] Childbearing age: 6-8 weeks.

[0046] 2. Experimental methods: Wild-type mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (70 mg / kg), followed by intratracheal injection of bleomycin (BLM) at a final concentration of 1.5 mg / kg. BLM was purchased from MCE and dissolved in physiological saline before use. Mice that received the same volume of Scr siRNA (SEQ ID No. 3, 5'-TTCTCCGAACGTGTCACGTdTdT-3') via intratracheal injection served as controls. On days 14 and 17 after BLM administration, KDELR3 siRNA (SEQ ID No. 4, 5'-GCACCAUCUUGGGAUAAAC-3') and Scr siRNA were injected intratracheally again, respectively. Mice were sacrificed 21 days later, and the degree of pulmonary fibrosis in each mouse was analyzed.

[0047] The severity of interstitial fibrosis in each contiguous region was independently assessed using the Ashcroft scoring system in a blinded manner.

[0048] 3. Experimental Results: The results of H&E, Msson, and Sirius red staining of lung tissue from wild-type mice are as follows: Figure 1 As shown, combined with Figure 1 It can be seen that, after BLM induction, compared with mice injected with Scr siRNA via airway, mice injected with KDELR3 siRNA via airway showed significantly reduced lung injury and fibrosis.

[0049] Ashcroft's score results are as follows: Figure 2 As shown, by Figure 2 It can be seen that the Ashcroft scores of mice injected with KDELR3 siRNA via the airway were lower than those of mice injected with Scr siRNA via the airway, indicating that the degree of pulmonary fibrosis was greatly reduced.

[0050] In conclusion, airway injection of KDELR3 siRNA is beneficial for the prevention or treatment of pulmonary fibrosis in mice.

[0051] Example 2 Effects of airway injection of KDELR3 siRNA on the protein and mRNA levels of fibronectin, type I collagen, αSMA, and KDLER3; To further evaluate the degree of fibrosis in the lungs of mice after BLM injection, this invention detected the protein and mRNA levels of fibronectin, type I collagen, αSMA, and KDLER3 in the lung tissue of each mouse using Western blot and RT-PCR.

[0052] Lung tissue from mice was collected after the experiment in Example 1. Proteins were extracted from the tissue using RIPA lysis buffer, and the expression levels of target proteins, such as fibronectin, type I collagen, αSMA, and KDELR3, were detected by Western blot. The results are as follows: Figure 3 As shown. For the specific method of Western blot, refer to Hu Y et al., 2022, Nature Communications, 13:114.

[0053] Simultaneously, quantitative real-time RT-PCR was performed using SYBR Premix Ex Taq (TaKaRa), with β-actin as an internal control to normalize the relative expression of each target gene. The results are as follows: Figure 4 As shown in the table below, the primers corresponding to each target gene are listed in Table 1. Specific testing methods for gene expression are described in Chen et al., 2015, International Journal of Clinical and Experimental Pathology 8:6700-6707.

[0054] Table 1. List of primers corresponding to the target gene Combination Figure 3 and Figure 4 It can be seen that, compared with mice injected with ScrsiRNA via airway, mice injected with KDELR3siRNA via airway showed reduced transcription levels and protein expression levels of fibronectin, type I collagen, and α-SMA, which further confirms that pulmonary fibrosis in mice injected with KDELR3siRNA via airway was significantly reduced.

[0055] Example 3: Effect of airway injection of KDELR3 siRNA on hydroxyproline levels; 1. Experimental materials: Hydroxyproline Detection Kit: Nanjing Jiancheng Biotechnology Co., Ltd.

[0056] 2. Experimental methods: The expression level of hydroxyproline in the lung tissue of mice in each group in Example 1 was determined using a hydroxyproline detection kit.

[0057] The measurement results are as follows Figure 5 As shown, combined with Figure 5 As can be seen, consistent with the expression results of Example 2 above, compared with mice injected with KDELR3 siRNA via airway, mice injected with Scr siRNA via airway after BLM induction showed more severe fibrosis and significantly upregulated hydroxyproline levels in lung tissue.

[0058] Example 6: Effect of KDELR3 knockdown on fibroblast differentiation Fibroblast differentiation into myofibroblasts is crucial for the development of fibrosis. Therefore, we investigated the effect of KDELR3 on TGFβ-induced fibroblast differentiation.

[0059] like Figure 6 As shown, the western blot results indicated that, compared with cells transfected with Scr siRNA, human lung fibroblasts transfected with KDELR3 siRNA after stimulation with 10 ng / mLTGF-β showed a significant decrease in marker proteins for differentiation into myofibroblasts.

[0060] In summary, the data above indicate that inhibiting KDELR3 can suppress fibroblast differentiation, thereby slowing the progression of pulmonary fibrosis. Furthermore, fibroblast differentiation plays an important role in renal fibrosis, myocardial fibrosis, liver fibrosis, and skin fibrosis; therefore, inhibiting KDELR3 could slow the onset of these fibrotic conditions, providing a new therapeutic target.

[0061] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Application of KDELR3 inhibitors in the preparation of drugs for the prevention and / or treatment of individual fibrotic diseases.

2. The application according to claim 1, characterized in that: The KDELR3 inhibitor includes at least one of the following: 1) Substances that affect the expression level and / or function of the KDELR3 gene; 2) Substances that reduce or inactivate KDELR3 activity; 3) Substances that promote the degradation of KDELR3.

3. The application according to claim 2, characterized in that: The KDELR3 inhibitor includes at least one of the following: 1) At least one of siRNA, shRNA, or miRNA that can inhibit KDELR3 expression; 2) Vectors containing the above-mentioned siRNA, shRNA, and / or miRNA; 3) Host cells containing the above-mentioned vectors.

4. The application according to claim 1, characterized in that: The upstream primer sequence of the KDELR3 gene is shown in SEQ ID No. 1, which is 5'-GGTGAACTACAGTTACACGCCG-3', and the downstream primer sequence is shown in SEQ ID No. 2, which is 5'-AGTGGTGATGGTCTCAGCCTCT-3'.

5. The application according to claim 1, characterized in that: The fibrotic diseases mentioned include pulmonary fibrosis, renal fibrosis, myocardial fibrosis, liver fibrosis, and skin fibrosis.

6. The application according to claim 1, characterized in that: The fibrotic disease mentioned is pulmonary fibrosis.

7. The application according to claim 1, characterized in that: The fibrotic disease mentioned is idiopathic pulmonary fibrosis.

8. The application according to claim 1, characterized in that: The individual in question is a mammal.

9. A pharmaceutical composition, characterized in that: This includes KDELR3 inhibitors and pharmaceutically acceptable carriers.

10. The pharmaceutical composition according to claim 9, characterized in that: The pharmaceutical composition is available in injectable, capsule, tablet, nasal spray, or aerosol form.

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