Application of multi-target polypeptide in preparation of medicine for treating organ fibrosis

Abstract

The invention relates to application of a multi-target polypeptide in preparation of a medicine for treating organ fibrosis, in particular to application of a polypeptide mini-klotho and a gene therapy vector and method thereof in preparation of medicines for treating fibrosis of kidney, liver, lung and other organs. The invention provides the application of mini-klotho in a model for treating kidney, liver and lung fibrosis, and the mini-klotho has a remarkable curative effect and does not have obvious toxic or side effects. Therefore, the mini-klotho disclosed by the invention can be prepared into a medicinal preparation for treating the organ fibrosis.

Description

Technical Field

[0001] The present invention relates to the use of a multi-target polypeptide in preparing a drug for treating organ fibrosis, and specifically relates to the use of a mini-klotho polypeptide and a gene therapy vector thereof in preparing a drug for treating fibrosis of organs such as kidneys, livers, and lungs. Background Art

[0002] Organ fibrosis is characterized by excessive deposition of extracellular matrix (ECM) due to dysregulated tissue damage and repair responses (Signal Transduct Target Ther. 2022; 7(1):206), which is mainly driven by myofibroblasts. Fibrosis can occur in multiple important organs, including the kidney, liver, lung, heart, etc. Fibrosis-related diseases such as chronic kidney disease, cirrhosis, and idiopathic pulmonary fibrosis have caused a huge disease burden worldwide. It has been reported that deaths caused by fibrosis-related diseases in developed countries have reached 45% of all human deaths (Nature 2020; 587(7835):555-566). However, there is no effective treatment for fibrosis-related diseases so far. Therefore, organ fibrosis is increasingly recognized as a major human health challenge.

[0003] The pathological mechanism of organ fibrosis is complex and has not yet been fully elucidated. However, the TGF-β, FGF2, and Wnt / β-catenin pathways are recognized as the three key pathways that mediate the development of fibrosis. TGF-β1 is one of the most effective profibrotic factors, which mainly regulates fibrosis through classical and non-classical signaling pathways. TGF-β1 can promote the activation of fibroblasts to myofibroblasts, the expression of α-smooth muscle actin (α-SMA), induce cell apoptosis and epithelial-mesenchymal transition, and ECM deposition. Wnt / β-catenin signal transduction can also promote organ fibrosis. In fibroblasts, Wnt ligands induce their activation and overexpression of ECM. FGF2 is also a key fibroblast factor that mainly promotes fibroblast proliferation and activation and induces epithelial-mesenchymal transition. In addition, existing literature has confirmed that TGF-β interacts with the Wnt / β-catenin pathway, and TGF-β can also induce FGF2 expression and activate the FGF2 pathway. It can be seen that the TGF-β, FGF2, and Wnt / β-catenin pathways may have a mutually promoting effect. Therefore, finding countermeasures that can simultaneously block the three signaling pathways of TGF-β, FGF2, and Wnt / β-catenin has important significance and clinical application value for inhibiting the occurrence and development of organ fibrosis.

[0004] The present invention relates to the discovery of a unique multi-target polypeptide named mini-klotho. It is reported in the literature that Klotho has the function of binding to TGF-β receptor, FGF receptor, Wnt ligand, blocking TGF-β / Smad signal transduction (J.Biol.Chem., 2011, 10:8655-8665), Wnt / β-catenin signal transduction (J.Am Soc.Nephrol.2013, 24:771-785), and FGF2 / FGFR1 (J.Pathol.2014, 234:560-572) signal transduction, thereby inhibiting organ fibrosis and delaying the progression of fibrosis-related diseases. However, the full-length anti-aging protein Klotho is a transmembrane protein with a large molecular weight, difficult and expensive to synthesize, and can cause side effects such as hypocalcemia and hypophosphatemia, making it difficult to apply clinically.

[0005] The present invention discovers that mini-klotho, as a novel and effective small molecule polypeptide, can inhibit damage and fibrosis of important organs (such as kidney, liver, and lung), and delay or / and reverse the course of fibrosis-related organ diseases.

[0006] The present invention also relates to the discovery of a unique mini-klotho gene therapy vector. Adeno-associated virus (AAV) is one of the most actively studied gene therapy vectors. As a vector, it has the advantages of high safety, low immunogenicity, wide host range, stable expression and physical properties. However, after intravenous injection, the commonly used gene therapy vectors AAV2 or AAV9 are mainly sequestered in the liver, which may cause liver toxicity, and high doses of AAV can also lead to complement activation. The new serotype Myo4A-AAV with muscle tropism has more efficient and selective muscle transduction, and liver sequestration is significantly reduced. Muscles are weak division sites, and injection of the virus can make them a long-term and stable secretion site. Therefore, using Myo4A-AAV as a vector for intramuscular injection treatment is a unique way to administer mini-klotho because of its simple injection method and small dosage.

[0007] Unilateral ureteral ligation (UUO) is a classic animal model of CKD, which is characterized by the ability to quickly and completely display different pathological stages and characteristics of interstitial fibrosis in obstructed kidneys, including inflammatory cell infiltration, proliferation and apoptosis of tubular cells, activation of myofibroblasts, and deposition of fibronectin and type I collagen. The ischemia-reperfusion (IRI) model is a classic model of progression from acute kidney injury (AKI) to chronic kidney disease (CKD). Ten days after unilateral renal ischemia-reperfusion (IRI) surgery, combined with contralateral resection (UIRI) can clearly display the pathological process of chronic renal fibrosis. The modeling method is simple and easy, with a high success rate, and has the advantages of small surgical incision, short operation time and few complications. The established model is suitable for the study of the progression of acute renal injury to chronic renal fibrosis.

[0008] Carbon tetrachloride (CCL4) is one of the main drugs for inducing liver cirrhosis. The main characteristics of this model are oxidative stress, severe perilobular inflammation and septal fibrosis, and necrosis of zone III and the center of the lobule. The modeling method is simple and easy, with a high success rate, and is similar to the pathophysiology of human cirrhosis. The bleomycin (BLM) model is the most widely used model for inducing idiopathic pulmonary fibrosis (IPF). This model has the histological characteristics of pulmonary fibrosis, such as the loss of bronchial endothelial buds, collagen walls, and alveolar spaces, which are similar to those of IPF patients. In addition, this model has the advantages of simple and easy modeling methods and high reproducibility.

[0009] In pathology, Masson staining is usually used to observe the extracellular collagen deposition in tissues. For more specific tissue fibrosis, immunostaining and Western blot methods (Western blot) of myofibroblast markers α-smooth muscle actin (α-SMA), fibronectin and type I collagen are often used to qualitatively and quantitatively observe and evaluate. Summary of the invention

[0010] The purpose of the present invention is to provide a use of a polypeptide (mini-klotho) in the preparation of a drug for treating diseases related to organ fibrosis. According to a further feature of the use described in the present invention, the prepared drug comprises: a therapeutically effective polypeptide (mini-klotho), and a treatment method using a muscle-tropic Myo4A-AAV novel adeno-associated virus (AAV) serotype as a vector for intramuscular injection.

[0011] The mini-klotho of the present invention can simultaneously inhibit three key signal pathways of organ fibrosis: TGF-β, FGF2, and Wnt / β-catenin. Mini-Klotho polypeptide has not been previously reported, and its amino acid sequence structure is homologous to a sequence of the anti-aging protein (Klotho) molecule. The purpose of the present invention is also to provide the use of derivatives of the polypeptide mini-klotho in the preparation of drugs for treating diseases related to organ fibrosis.

[0012] According to further features of the use described in the present invention, the derivatives of mini-klotho include: shorter peptides containing the mini-klotho amino acid sequence, mini-klotho-related polypeptides after amino acid substitution, and chemically modified mini-klotho and its shorter peptides.

[0013] According to the use described in the present invention, the prepared drug carrier and method, that is, the Myo4A-AAV new adeno-associated virus (AAV) serotype is used as a carrier for intramuscular injection.

[0014] The experiment of the present invention proves that mini-klotho has no obvious toxic side effects in the mouse animal experiment. The inventor used the renal fibrosis model of unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion injury (UIRI), the liver fibrosis model induced by carbon tetrachloride (CCL4), and the pulmonary fibrosis model induced by bleomycin (BLM) to study the efficacy of mini-klotho in preventing and treating fibrosis of multiple organs. The results show that in the renal fibrosis model, compared with the UUO or UIRI model group, the renal interstitial collagen deposition in the mini-klotho treatment group was significantly reduced, and α-smooth muscle actin and fibronectin were significantly reduced, indicating that mini-klotho can effectively inhibit renal interstitial fibrosis caused by UUO and UIRI. In the liver and lung fibrosis models, compared with the CCL4 and BLM model groups, the liver and lung tissue collagen deposition in the mini-klotho treatment group was significantly reduced, and fibronectin was significantly reduced, indicating that mini-klotho has a therapeutic effect on liver and lung fibrosis.

[0015] In summary, mini-klotho has a significant inhibitory effect on fibrosis of multiple important organs (such as kidney, liver, and lung) without obvious toxic side effects, and can therefore be used to prepare drugs that effectively inhibit organ fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1:Mini-klotho inhibits the activation of rat kidney fibroblasts (NRK-49F) in vitro. Rat fibroblasts (NRK-49F) were transfected with mini-klotho lentivirus and a stable transfected cell was established. TGF-β, FGF2, and Wnt3a were treated respectively. Among them, A: mini-klotho antagonizes TGF-β-induced downstream pathway signals and mediated fibrosis-related proteins. B: mini-klotho antagonizes FGF2-induced downstream pathway signals and mediated fibrosis-related proteins. C: mini-klotho antagonizes Wnt3a-induced downstream pathway signals and mediated fibrosis-related proteins.

[0017] Figure 2 :Schematic diagram of the adeno-associated virus structure of Myo4A-AAV as a vector to overexpress mini-klotho.

[0018] Figure 3 :Green fluorescent protein (GFP) fluorescence staining images of various organs of mice. The UUO mouse model was injected with mini-klotho AAV virus overexpressing green fluorescent protein (GFP) in situ in the right quadriceps muscle, and the fluorescence intensity of left and right muscles, left and right kidneys, heart, liver, spleen, lungs and other organs was detected.

[0019] Figure 4 : Masson staining of kidneys of mice in each group of UUO model. In this figure, from left to right are sham operation group; sham operation + mini-klotho group; UUO group; UUO + mini-klotho group.

[0020] Figure 5 : Immunostaining images of renal fibrosis indicators (fibronectin and α-SMA) in each group of mice in the UUO model. In this figure, from left to right are sham operation group; sham operation + mini-klotho group; UUO group; UUO + mini-klotho group.

[0021] Figure 6 : Masson staining of kidneys of mice in each group of UIRI model. In this figure, from left to right are sham operation group; sham operation + mini-klotho group; UIRI group; UIRI + mini-klotho group.

[0022] Figure 7 :Immunostaining images of renal fibrosis indicators (α-SMA and vimentin) in each group of mice in the UIRI model. In this figure, from left to right are sham operation group; sham operation + mini-klotho group; UIRI group; UIRI + mini-klotho group.

[0023] Figure 8 :The expression levels of liver fibrosis-related proteins in each group of mice were detected by immunoblotting of CCL4 model. In this figure, from left to right are normal saline group; normal saline + mini-klotho group; CCL4 model group; CCL4 model + mini-klotho group.

[0024] Fig. 9 : Masson staining of liver of mice in each group of CCL4 model. In this figure, from left to right are normal saline group; normal saline + mini-klotho group; CCL4 model group; CCL4 model + mini-klotho group.

[0025] Fig.10 :BLM model immunoblotting method was used to detect the expression levels of pulmonary fibrosis-related proteins in each group of mice. In this figure, from left to right are sham operation group; sham operation + mini-klotho group; BLM group; BLM + mini-klotho group.

[0026] Fig.11 : Masson staining of the lungs of mice in each group of the BLM model. In this figure, from left to right are the sham operation group; sham operation + mini-klotho group; BLM group; BLM + mini-klotho group. DETAILED DESCRIPTION

[0027] The present invention will be further described below by way of embodiments with reference to the accompanying drawings.

[0028] Construction of polypeptide mini-klotho: 159 amino acids of the KL1 domain of the full-length Klotho were cut to obtain the polypeptide mini-klotho sequence. The amino acid sequence information of the polypeptide mini-klotho is as follows: FQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADH (SEQ ID NO. 1).

[0029] For the in vitro cell experiments, a Flag-tagged lentivirus overexpressing mini-klotho was synthesized by Beijing Qingke Biotechnology Co., Ltd.; for the in vivo animal experiments, a GFP-tagged adeno-associated virus overexpressing mini-klotho was synthesized by Guangzhou Saiye Baimu Biotechnology Co., Ltd.

[0030] Example 1: Inhibitory effect of peptide mini-klotho on activation of rat kidney fibroblasts (NRK-49F) in vitro 1. Experimental materials: Cells: Rat kidney fibroblasts (NRK-49F).

[0031] Culture medium: DMEM / F12 (HAM) (1:1) culture medium containing 10% FBS.

[0032] Culture conditions: 37℃ incubator with 5% CO2.

[0033] Construction of small molecule peptide mini-klotho: 159 amino acids of the KL1 domain of full-length Klotho were cut and handed over to Beijing Qingke Biotechnology Co., Ltd. to synthesize a lentivirus with a Flag tag to overexpress mini-klotho.

[0034] 2. Experimental treatment: (1) Cultured rat kidney fibroblasts (NRK-49F) were cultured at 1.5×10 6 The cells were seeded in 6-well cell culture plates and cultured for 1 day. The cells were transfected with lentivirus at an optimal infection rate of 20. The culture medium was changed after 24 hours. The stable transfected cells were selected with puromycin. The lentiviral vector overexpressing mini-klotho contained a puromycin resistance gene. In the previous experiments, all untreated cells were killed within 3-4 days to obtain the optimal screening concentration of 7.5 μg / ml. The rat fibroblasts (NRK-49F) that were successfully transfected were selected with a 7.5 μg / ml dose of puromycin until there was no cell death, and a stable transfected cell line overexpressing mini-klotho was established.

[0035] (2) Stable cell lines and untreated rat kidney fibroblasts (NRK-49F) were cultured at 1.5×10 6 The cells were seeded in 6-well cell culture plates, and the basal medium was replaced the next day to starve the cells for 24 hours. TGF-β (2 ng / ml), FGF2 (10 ng / ml), and Wnt3a (100 ng / ml) were added for stimulation, and proteins were collected 24 hours later for Western blot experiments.

[0036] 3. Experimental results: (1) Experimental results are as follows Figure 1 As shown in A, mini-klotho can inhibit the expression of downstream molecules of the TGF-β pathway, as well as the mediated expression of fibronectin and α-smooth muscle actin (α-SMA). At the same time, the expression of the Flag tag was detected to confirm the successful overexpression of mini-klotho.

[0037] (2) If Figure 1 As shown in B, mini-klotho can inhibit the expression of downstream molecules of the FGF2 pathway and the expression of fibrosis-related proteins caused by it.

[0038] (3) If Figure 1 As shown in Figure C, mini-klotho can inhibit the expression of downstream molecules of the Wnt3a pathway and the expression of fibrosis-related genes. Therefore, as a novel peptide, mini-klotho can antagonize the activity of TGF-β, FGF2 and Wnt3a, block key pro-fibrosis signaling pathways in the body, and thus inhibit the expression of fibrosis-related proteins.

[0039] Example 2: Myo4A-AAV-mini-klotho-GFP is specifically expressed in the muscles of UUO mice after intramuscular injection 1. Experimental Animals C57 mice, male, weighing 20-22 g, SPF grade.

[0040] The animals were weighed and numbered first, and 24 healthy mice weighing 20-22 g were selected and randomly divided into 4 groups, 6 mice in each group, including sham operation group, sham operation injection mini-klotho group, model group and model injection mini-klotho group.

[0041] 2. Experimental Grouping (1) Sham operation group / sham operation group injected with mini-klotho: After the mice were anesthetized with 1% sodium pentobarbital at room temperature at 1 ml / kg body weight, an incision was made at 2-3 cm along the midline of the abdomen. After local disinfection, the skin, subcutaneous tissue, muscular layer and peritoneum were cut layer by layer. After the left ureter was found, it was sutured layer by layer. After local disinfection, the mouse was marked and placed in the corresponding cage.

[0042] (2) Model group / model injection mini-klotho group: anesthesia and disinfection were performed as above. The skin, subcutaneous tissue, muscular layer and peritoneum were cut layer by layer. After finding the left ureter, the ureter was ligated at two locations, the lower kidney level and the upper 1 / 3 of the ureter, and then sutured layer by layer. After local disinfection, the markings were verified and the mice were placed in the corresponding cages.

[0043] 3. Experimental Procedure The adeno-associated virus overexpressing mini-klotho was diluted to a titer of 10 with sterile PBS solution. 12gc / ml, ready for use. Two weeks before surgery, 50 μl of adeno-associated virus overexpressing mini-klotho was injected into the right quadriceps of mice in the sham operation and mini-klotho injection groups and the model injection groups. 50 μl of empty virus was injected into the sham operation and model groups. The experimental groups were caged. UUO surgery was performed 2 weeks later, and the mice in each group were killed after 7 days of feeding. The left and right muscles, left and right kidneys, heart, liver, spleen, lungs and other organs were taken, fixed with 10% neutral buffered formaldehyde and frozen with liquid nitrogen. After dehydration, embedding, slicing and preparation of formaldehyde-fixed tissues, the GFP fluorescence intensity of the left and right muscles, left and right kidneys, heart, liver, spleen, lungs and other organs was detected.

[0044] 4. Experimental Results (1) Figure 2 This is a schematic diagram of the Myo4A-AAV-mini-klotho-GFP virus structure.

[0045] (2) Mini-klotho-GFP is specifically expressed in the right muscles of mice: like Figure 3 As shown in the figure, mini-klotho-GFP is mainly expressed in muscle, and also in heart, liver, and kidney. However, there is no fluorescence in the organs of mice injected with empty virus. Scale bar, 100μm.

[0046] Example 3: Inhibitory effect of peptide mini-klotho on renal fibrosis in the UUO model of mice 1. Experimental Animals C57 mice, male, weighing 20-22 g, SPF grade.

[0047] The animals were weighed and numbered first, and 24 healthy mice weighing 20-22 g were selected and randomly divided into 4 groups, 6 mice in each group, including sham operation group, sham operation injection mini-klotho group, model group and model injection mini-klotho group.

[0048] 2. Experimental Grouping (1) Sham operation group / sham operation group injected with mini-klotho: After the mice were anesthetized with 1% sodium pentobarbital at room temperature at 1 ml / kg body weight, an incision was made at 2-3 cm along the midline of the abdomen. After local disinfection, the skin, subcutaneous tissue, muscular layer and peritoneum were cut layer by layer. After the left ureter was found, it was sutured layer by layer. After local disinfection, the mouse was marked and placed in the corresponding cage.

[0049] (2) Model group / model injection mini-klotho group: anesthesia and disinfection were performed as above. The skin, subcutaneous tissue, muscular layer and peritoneum were cut layer by layer. After finding the left ureter, the ureter was ligated at two locations, the lower kidney level and the upper 1 / 3 of the ureter, and then sutured layer by layer. After local disinfection, the markings were verified and the mice were placed in the corresponding cages.

[0050] 3. Experimental Procedure The adeno-associated virus overexpressing mini-klotho was diluted to a titer of 10 with sterile PBS solution. 12 gc / ml, prepared and used immediately. Two weeks before surgery, 50 μl of adeno-associated virus overexpressing mini-klotho was injected in situ at multiple points in the right quadriceps of mice in the sham-operated mini-klotho-injected group and the model-operated mini-klotho-injected group. 50 μl of empty virus was injected into the sham-operated and model-operated mice. The experimental groups were housed in cages. UUO surgery was performed 2 weeks later, and the mice in each group were killed after 7 days of feeding. The left kidneys were removed and fixed with 10% neutral buffered formaldehyde and frozen in liquid nitrogen. After dehydration, embedding, sectioning, and preparation, the formaldehyde-fixed tissues were subjected to Masson staining and immunostaining of fibronectin and smooth muscle actin α.

[0051] 4. Experimental Results (1) Masson staining showed that mini-klotho reduced renal interstitial collagen deposition in UUO mice: The experimental results are as follows Figure 4 As shown, the renal interstitial collagen deposition in the mini-klotho injection group was significantly lower than that in the model group. Scale bar, 50μm.

[0052] (2) Mini-klotho reduces renal interstitial fibrosis in UUO mice: The experimental results are as follows Figure 5 As shown, compared with the model group, the levels of renal interstitial fibronectin and smooth muscle actin α (α-SMA) in the model group injected with mini-klotho were significantly reduced. Scale bar, 50μm.

[0053] Example 4: Inhibitory effect of peptide mini-klotho on renal fibrosis in mouse UIRI model 1. Experimental Animals C57 mice, male, weighing 20-22 g, SPF grade.

[0054] The animals were weighed and numbered first, and 24 healthy mice weighing 20-22 g were selected and randomly divided into 4 groups, 6 mice in each group, including sham operation group, sham operation injection mini-klotho group, model group and model injection mini-klotho group.

[0055] 2. Experimental groups (1) Sham operation group / sham operation group injected with mini-klotho: After the mice were anesthetized with 1% sodium pentobarbital at room temperature at 1 ml / kg body weight, an incision was made at 2-3 cm along the midline of the abdomen. After local disinfection, the skin, subcutaneous tissue, muscular layer and peritoneum were cut layer by layer. After the left renal pedicle was found, the renal pedicle was sutured layer by layer. After local disinfection, the mouse was marked and placed in the corresponding cage.

[0056] (2) Model group / model injection mini-klotho group: anesthesia and disinfection were performed as above. The skin, subcutaneous tissue, muscular layer and peritoneum were cut layer by layer. After finding the left renal pedicle, the left renal pedicle was quickly blocked with a non-destructive micro artery clamp. The kidney turned from bright red to purple-black, indicating successful clamping. The mice were placed on a 37.5-degree metal heating plate and timed for 35 minutes. After the operation, the layers were sutured. After local disinfection, the marks were verified and the mice were placed in the corresponding cages.

[0057] 3. Experimental Procedure The adeno-associated virus overexpressing mini-klotho was diluted to a titer of 10 with sterile PBS solution. 12 gc / ml, ready for use. Two weeks before surgery, 50 μl of adeno-associated virus overexpressing mini-klotho was injected into the right quadriceps of mice in the sham-operated mini-klotho group and the model-operated mini-klotho group at multiple points in situ. Mice in the sham-operated and model-operated mini-klotho groups were injected with 50 μl of empty virus. Two weeks later, UIRI surgery was performed, and mice in each group were killed on the 10th day of feeding. Blood samples were collected, and the left kidney was taken. The tissues were fixed with 10% neutral buffered formaldehyde and frozen in liquid nitrogen. After dehydration, embedding, sectioning, and preparation, the formaldehyde-fixed tissues were subjected to Masson staining and immunostaining of smooth muscle actin α and vimentin.

[0058] 4. Experimental Results (1) Masson staining showed that mini-klotho reduced renal interstitial collagen deposition in UIRI mice: The experimental results are as follows Figure 6 As shown, the renal interstitial collagen deposition in the mini-klotho injection group was significantly lower than that in the model control group. Scale bar, 50 μm.

[0059] (2) Mini-klotho reduces renal interstitial fibrosis in UIRI mice: The experimental results are as follows Figure 7 As shown, compared with the model group, the levels of smooth muscle actin α and vimentin in the renal tissue of mice injected with mini-klotho were significantly decreased (scale bar, 50 μm).

[0060] Example 5: Inhibitory effect of mini-klotho on liver fibrosis in the mouse carbon tetrachloride (CCL4) model 1. Experimental Animals C57 mice, male, weighing 20-22 g, SPF grade.

[0061] The animals were weighed and numbered first, and 24 healthy mice weighing 20-22 g were selected and randomly divided into 4 groups, with 6 mice in each group, including a saline group, a saline-injected mini-klotho group, a CCL4 model group, and a CCL4 model-injected mini-klotho group.

[0062] 2. Experimental groups (1) Normal saline group / normal saline injection mini-klotho group: Each mouse was intraperitoneally injected with sterile normal saline at a dose of 50 μl / 20 g every three days for 8 weeks. After injection, the marking was verified and the mouse was placed in the corresponding cage.

[0063] (2) CCL4 model group and CCL4 model injected with mini-klotho group: CCL4 solution was diluted with olive oil at a volume ratio of 1:3, and each mouse was intraperitoneally injected with CCL4 solution at a dose of 50 μl / 20 g every three days for 8 weeks. The mouse was marked and placed in the corresponding cage.

[0064] 3. Experimental Procedure The adeno-associated virus overexpressing mini-klotho was diluted to a titer of 10 with sterile PBS solution. 12 gc / ml, prepared and used immediately. Two weeks before CCL4 injection, 50 μl of adeno-associated virus overexpressing mini-klotho was injected into the right quadriceps of mice in the saline-injected mini-klotho group and the CCL4 model-injected mini-klotho group at multiple points in situ. Mice in the saline group and the CCL4 model group were injected with 50 μl of empty virus. After 2 weeks, CCL4 and saline were injected intraperitoneally every 3 days. At the 8th week, mice in each group were killed, blood samples were collected, and livers were taken. The tissues were fixed with 10% neutral buffered formaldehyde and frozen in liquid nitrogen. After dehydration, embedding, slicing, and preparation, the formaldehyde-fixed tissues were subjected to Masson staining and fibronectin immunostaining. After the frozen tissues were homogenized, proteins were extracted, and the expression levels of fibrosis-related proteins were detected by Western Blot.

[0065] 4. Experimental Results (1) Mini-klotho reduces the expression level of liver fibrosis-related proteins in CCL4 mice: The experimental results are as follows Figure 8As shown in the figure, the expressions of liver fibrosis-related proteins such as fibronectin, tenascin, collagen I, vimentin, and smooth muscle protein α were significantly decreased in the CCL4 model injected mini-klotho group mice.

[0066] (2) Masson staining to detect the degree of liver tissue fibrosis: The experimental results are as follows Fig. 9 As shown, the collagen deposition in the liver of mice injected with mini-klotho in the CCL4 model was significantly lower than that in the CCL4 model group. Scale bar, 50 μm.

[0067] Example 6: Inhibitory effect of peptide mini-klotho on pulmonary fibrosis in the mouse bleomycin (BLM) model 1. Experimental Animals C57 mice, male, weighing 20-22 g, SPF grade.

[0068] The animals were weighed and numbered first, and 24 healthy mice weighing 20-22 g were selected and randomly divided into 4 groups, with 6 mice in each group, including sham operation group, sham operation injection mini-klotho group, BLM model group and BLM model injection mini-klotho group.

[0069] 2. Experimental groups (1) Sham operation group / sham operation injection mini-klotho group: After the mice were anesthetized with 1% sodium pentobarbital at room temperature at 1 ml / kg body weight, a 1 cm incision was made on the neck; after local disinfection, the skin, subcutaneous layer, and muscle layer were cut layer by layer. The connective tissue in front of the trachea was removed to expose the trachea. The front end of the 1 ml insulin syringe was pointed toward the heart end and inserted straight into the trachea through the middle of the two tracheal cartilage rings. 50 μl of normal saline was injected into the trachea. During this period, pay attention to whether the mouse chokes on liquid in the nasal cavity. After the injection, the mouse was placed in an upright position with its head up, and the surgical board was knocked hard on the table for 30 seconds. The mouse was removed from the surgical board, and its upper limbs were grasped and rotated for 2 minutes to allow the injected BLM to be evenly distributed in the lungs. The neck incision was then sutured layer by layer. After local disinfection, the markings were verified and the mice were placed in the corresponding cages.

[0070] (2) BLM model group and BLM model injected with mini-klotho group: anesthesia and disinfection were performed as above. After local disinfection, the skin, subcutaneous tissue, and muscular layer were cut layer by layer. The connective tissue in front of the trachea was removed to expose the trachea. The front end of the 1ml insulin syringe was pointed toward the heart end and inserted straight into the trachea through the middle of the two tracheal cartilage rings. 50μl of normal saline was injected into the trachea. During this period, pay attention to whether there is any liquid choking in the nasal cavity of the mouse. After the injection, the mouse was placed upright with its head up, and the surgical board was knocked hard on the table for 30 seconds. The mouse was removed from the surgical board, and its upper limbs were grasped and rotated for 2 minutes so that the injected BLM could be evenly distributed in the lungs. The neck incision was then sutured layer by layer. After local disinfection, the markings were verified and placed in the corresponding mouse cage.

[0071] 3. Experimental Procedure The adeno-associated virus overexpressing mini-klotho was diluted to a titer of 10 with sterile PBS solution. 12 gc / ml, prepared and used immediately. Bleomycin solution was diluted with sterile saline at a concentration of 5 mg / kg at a ratio of 1:50. Virus injection was performed immediately after tracheal injection of bleomycin solution and saline. 50 μl of adeno-associated virus overexpressing mini-klotho was injected in situ at multiple points in the right quadriceps of mice in the sham operation injection of mini-klotho group and the model injection of mini-klotho group. 50 μl of empty virus was injected into the sham operation group and the model group. Mice in each group were killed 4 weeks later, and the lungs were taken out, fixed with 10% neutral buffered formaldehyde and frozen in liquid nitrogen. After dehydration, embedding, sectioning and preparation, the formaldehyde-fixed tissues were subjected to Masson staining and fibronectin immunostaining. Proteins were extracted after homogenization of frozen tissues, and the expression levels of fibrosis-related proteins were detected by Western Blot.

[0072] 4. Experimental Results (1) Mini-klotho reduces the expression level of pulmonary fibrosis-related proteins in BLM mice: The experimental results are as follows Fig.10 As shown in the figure, the expressions of pulmonary fibrosis-related proteins such as fibronectin, tenascin, vimentin, and smooth muscle protein α in mice injected with mini-klotho were significantly decreased.

[0073] (2) Masson staining to detect the degree of lung tissue fibrosis: The experimental results are as follows Fig.11 As shown, the lung collagen deposition in the mini-klotho injection group was significantly lower than that in the model group. Scale bar, 50μm.

[0074] In summary, mini-klotho can significantly reduce the collagen deposition in kidney, liver, and lung tissues of UUO, UIRI, CCL4, and BLM mice, and significantly reduce the expression levels of fibronectin, type I collagen, and α-SMA in kidney, liver, and lung tissues of UUO, UIRI, CCL4, and BLM mice. Therefore, mini-klotho can become a new drug that effectively inhibits organ fibrosis.

Claims

1. Use of the polypeptide mini-klotho in the preparation of a drug for treating diseases related to organ fibrosis, wherein the amino acid sequence of the polypeptide mini-klotho is as follows: FQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADH (SEQ ID NO. 1).

2. The use according to claim 1, characterized in that The organ fibrosis specifically includes kidney fibrosis, liver fibrosis or lung fibrosis.

3. The use according to claim 1, characterized in that The medicine comprises: a therapeutically effective dose of polypeptide mini-klotho and a muscle-tropic Myo4A-AAV novel adeno-associated virus over-expressing the polypeptide mini-klotho.

4. The use according to claim 1, characterized in that The drug is administered by intramuscular injection.

5. Use of a derivative of the polypeptide mini-klotho in the preparation of a drug for treating fibrosis of the kidney, liver, lung and other organs, wherein the amino acid sequence of the polypeptide mini-klotho is as follows: FQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADH (SEQ ID NO. 1).

6. The use according to claim 5, characterized in that The derivatives of the polypeptide mini-klotho include: shorter peptide segments containing the mini-klotho amino acid sequence, mini-klotho related polypeptides after amino acid substitution, chemically modified mini-klotho and shorter peptide segments thereof.

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