Polypeptide compound as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide compound as well as a preparation method and application thereof. The amino acid sequences of the polypeptide compound are as shown in SEQ ID NO.1-9. The polypeptide GN and the analogue GN11 thereof show a remarkable renal fibrosis resisting effect at in-vivo and in-vitro levels. The analogue disclosed by the invention is low in dosage, free from obvious toxic and side effects, low in production cost and relatively high in generalizability, and can be used for preventing or treating chronic kidney diseases and related inflammation and fibrosis diseases induced by the chronic kidney diseases.

Description

Technical Field

[0001] The invention relates to a polypeptide compound and a preparation method and application thereof, belonging to the field of polypeptide synthesis and application. Background Art

[0002] Chronic kidney disease (CKD) is a disease characterized by progressive decline in kidney function. Diabetic nephropathy, hypertension, glomerulonephritis, acute kidney injury, etc. can all lead to the occurrence of CKD. As the disease continues to progress, irreversible loss of nephrons occurs, the kidney's regenerative ability weakens, the kidney microenvironment is damaged, metabolism becomes abnormal, oxidative stress and inflammatory responses occur, ultimately leading to end-stage renal disease and / or premature death.

[0003] Renal fibrosis is a pathological feature of CKD at the end stage, manifested as massive accumulation and cross-linking of the extracellular matrix (ECM), glomerular sclerosis, tubular atrophy, renal interstitial fibrosis, etc., and ultimately leads to renal failure. Renal fibrosis is difficult to detect, and there is no effective treatment drug in clinical practice. Existing treatment options are mostly limited to dialysis and kidney transplantation, which seriously affects the quality of life of patients and brings a huge burden to society and families.

[0004] In existing studies, the treatment of CKD and renal fibrosis has mainly focused on targeting molecules and signal regulation pathways related to the ECM production process. A large number of preclinical and clinical trial results have also shown that new therapeutic strategies such as regulating inflammatory responses, inhibiting profibrotic growth factors, and targeting epigenetic changes can also alleviate the development of fibrosis. In addition, some treatments that were originally thought to mainly regulate molecular pathways directly related to fibrosis can also regulate other processes, such as inflammatory responses, making it difficult to determine which factors and pathways are dominant in the treatment of CKD and renal fibrosis.

[0005] Inhibition of the renin-angiotensin system (RAS) is currently the main method for treating CKD. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) can slow the progression of CKD in proteinuric nephropathy, but there is currently no method to prevent the progression of acute kidney injury to CKD, and no clinical trials have confirmed whether blocking RAS can reduce renal fibrosis. Endothelin 1 receptor antagonists atrasentan and sodium-glucose transporter 2 canagliflozin can inhibit the development of CKD caused by diabetic nephropathy. Both canagliflozin and atrasentan have pleiotropic effects. Studies have shown that canagliflozin has anti-fibrotic effects in animal diabetic nephropathy models, adenine-induced CKD, and human liver disease. Atrasentan has the effect of reducing renal fibrosis in experimental diabetic nephropathy models, but it is unclear whether sodium-glucose transporter 2 inhibitors or endothelin 1 receptor antagonists can reduce human renal fibrosis.

[0006] The process from kidney damage to CKD and renal fibrosis is usually caused by the combined effects of multiple factors, and treatment strategies targeting only specific factors are not ideal. In particular, the failure of clinical trials of TGF-β1 blockade shows that strategies targeting a single pathogenic process may have a negative impact on certain physiological processes occurring at the same time, further emphasizing the need for alternative approaches in the treatment of CKD and renal fibrosis. Although a lot of progress has been made in the research on CKD and renal fibrosis in recent years, there are still only a handful of approved therapeutic drugs for renal fibrosis.

[0007] Inflammation and fibrosis are important pathological features of CKD. When the kidneys are damaged continuously or excessively, the body's inflammatory signaling pathways are activated, and immune cells such as monocytes, neutrophils, and macrophages gather at the damaged site. These cells promote the secretion of inflammatory factors by activating transcription factors, thereby aggravating the fibrosis process.

[0008] Sustained chronic inflammatory response can also damage telomerase function and shorten telomeres, which is one of the reasons for accelerated aging. Inflammatory aging is a subclinical symptom caused by the continuous secretion of proinflammatory factors. Senescent cells promote the aging of adjacent cells and organs through blood circulation or microvessels. Cellular senescence is an important feature of kidney disease. Compared with normal cells, the physiological function and proliferation ability of senescent cells gradually decline, and cells are out of the cell cycle. Senescent cells have abnormal metabolism and can secrete a large number of inflammatory factors and chemokines, which further induce inflammatory response, form a vicious cycle, and aggravate the pathological changes of the body. In chronic kidney diseases such as acute kidney injury, glomerulonephritis, diabetic nephropathy, nephrotic syndrome, and polycystic kidney disease, there is the occurrence of cellular senescence, and the expression levels of aging marker proteins such as P16, P21, P53, SASP, and β-galactosidase expression increase. In addition, renal tubular cells are easily damaged in aging and pathological conditions. Studies have found that the senescence of renal tubular cells and renal interstitial cells in mice with unilateral ureteral obstruction (UUO) increases, and inhibiting the senescence of renal tubular cells can significantly inhibit the development of renal fibrosis. Therefore, regulating cell senescence may provide a new treatment strategy for kidney disease. Summary of the invention

[0009] Purpose of the invention: The technical problem to be solved by the present invention is to provide a polypeptide compound and a preparation method and application thereof, wherein the polypeptide compound can be used to prevent and / or treat renal fibrosis and other organ fibrosis.

[0010] Technical solution: To solve the above technical problems, the present invention provides a polypeptide compound, which contains a parent peptide represented by the following amino acid sequence:

[0011] NH2-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Arg-Leu-Arg-Phe-Ile-Pro-Lys-CO NH2

[0012] Where Xaa1 = Glu or absent;

[0013] Xaa2 = Ala or absent;

[0014] Xaa3 = Pro or not present;

[0015] Xaa4 = Ala or absent;

[0016] Xaa5 = Leu or absent;

[0017] Xaa6 = Leu or absent;

[0018] Xaa7 = Thr or absent;

[0019] Xaa8 = Ser or not present.

[0020] In the compounds protected by the present invention, if Xaan (n≥2) does not exist, then the amino acids before it do not exist.

[0021] Among them, a polypeptide compound or a pharmaceutically acceptable salt thereof, the amino acid sequence of the polypeptide compound is shown in SEQ ID NO.1-9. The polypeptide compound is a novel polypeptide compound that can improve organ fibrosis and fibrotic symptoms associated with organ diseases. The present invention has proved through a large number of experimental studies that the polypeptide compound has no adverse reactions and can be used to treat or improve organ fibrosis and fibrotic symptoms associated with organ diseases, especially suitable for treating or improving renal fibrosis and fibrotic symptoms associated with organ diseases.

[0022] Among them, the novel anti-aging polypeptides and their analogs provided by the present invention are evaluated for pharmacodynamics through in vitro cell model activity screening and in vivo animal model activity detection.

[0023] The present invention also provides a method for preparing the polypeptide compound, comprising the following steps:

[0024] (1) Add dichloromethane to MBHA resin, drain after the resin is fully swollen, add deprotection reagent, shake at low speed, drain, and wash; add a mixture of amino acid, HOBt and HBTU and N,N-diisopropylethylamine, shake, drain, and wash;

[0025] (2) coupling the amino acids one by one to the MBHA resin according to step (1);

[0026] (3) DCM is added to the drained resin to swell it, and anhydrous methanol is added to shake it at a low speed; drain, cut, wash, concentrate, add icy ether to precipitate the polypeptide, add water to dissolve all the polypeptide in water, filter, purify, and obtain pure peptide.

[0027] The present invention also provides use of the polypeptide compound or a pharmaceutically acceptable salt thereof in preparing a drug for preventing and / or treating chronic kidney disease.

[0028] The present invention also provides use of the polypeptide compound or a pharmaceutically acceptable salt thereof in preparing a drug for preventing and / or treating renal fibrosis.

[0029] Wherein, the renal fibrosis includes renal fibrosis diseases caused by obstructive nephropathy, diabetic nephropathy, hypertensive nephropathy, nephritis, renal tumors, adverse drug reactions or infection with pathogenic microorganisms.

[0030] Wherein, the polypeptide compound or a pharmaceutically acceptable salt thereof inhibits the expression of fibrosis markers.

[0031] Wherein, the polypeptide compound or a pharmaceutically acceptable salt thereof can alleviate and / or inhibit the abnormal expression of fibrosis genes.

[0032] Wherein, the concentration of the polypeptide compound is 25-50 μM.

[0033] The present invention also provides a preparation for treating organ fibrosis, which contains the polypeptide compound or a pharmaceutically acceptable salt thereof.

[0034] The dosage forms of the preparation include tablets, capsules, granules, oral liquids, syrups, pills, ointments and patches for skin surfaces, aerosols, nasal sprays, suppositories, microsphere preparations, injection liquids or freeze-dried powder injections.

[0035] The above-mentioned preparations may include pharmaceutical excipients, diluents or carriers, and may be used for patients in need of treatment in any suitable manner, including but not limited to oral, rectal, nasal, topical (including oral and sublingual), subcutaneous or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal) administration.

[0036] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0037] 1. The present invention synthesized a series of GN polypeptide analogs and studied the efficacy of GN and its analogs, including in vitro activity and cytotoxicity, in vivo activity evaluation, etc. The results showed that compared with the parent peptide GN, the analog GN11 had better anti-fibrotic activity, which proved that GN polypeptide analogs can be used to prepare drugs for preventing or treating renal fibrosis and organ fibrosis.

[0038] 2. The present invention screens the shortest active fragment of the parent peptide GN by peptide chain reduction. The screened analog GN11 has improved activity compared to the parent peptide, and has a high synthesis yield, is easy to scale up production, and greatly reduces production costs.

[0039] 3. Compared with small molecule compounds, analogs are non-toxic and have a large safety window; compared with large molecule proteins, the preparation cost is more economical and the scalability is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The anti-renal fibrosis activity of GN was detected in the TGF-β1-induced NIH-3T3 cell fibrosis model: A: expression level of fibrosis marker proteins; B: relative expression of fibrosis marker proteins;

[0041] Figure 2 The anti-renal fibrosis activity of GN was detected in the TGF-β1-induced HK2 cell fibrosis model: A: expression level of fibrosis marker proteins; B: relative expression of fibrosis marker proteins;

[0042] Figure 3 Schematic diagram of the effect of GN on renal function indices in mice with UUO-induced renal fibrosis: A: Scr content; B: BUN content;

[0043] Figure 4 Schematic diagram of the effect of GN on H&E staining of kidney tissue sections induced by UUO;

[0044] Figure 5 Schematic diagram of GN on the expression of UUO-induced renal tissue fibrosis marker proteins: A: expression level of renal tissue fibrosis-related proteins; B: relative expression of renal tissue fibrosis-related proteins;

[0045] Figure 6 This is a schematic diagram of the expression of fibrosis marker genes in renal tissue induced by UUO by GN;

[0046] Figure 7 The cytotoxicity of GN and its analogs to NIH-3T3 cells was detected;

[0047] Figure 8 The anti-renal fibrosis activity of GN analogs was detected in the TGF-β1-induced NIH-3T3 cell fibrosis model: A: expression level of marker protein; B: relative expression of marker Fibronectin; C: relative expression of marker α-SMA;

[0048] Fig. 9 The toxicity of GN and GN11 to NRK-52E cells was detected;

[0049] Fig.10 Schematic diagram of the effect of GN and GN11 on the expression of fibrosis marker proteins in the NRK-52E cell fibrosis model: A: expression level of marker protein; B: relative expression of marker Collagen I; C: relative expression of marker Fibronectin;

[0050] Fig.11 Schematic diagram of the effect of GN11 on fibrosis genes Col1a1, Fn1, Mmp2, and Vim in the NRK-52E cell fibrosis model;

[0051] Fig.12 Schematic diagram of the effect of GN11 on renal function indicators Scr (A) and BUN (B) in mice with UUO-induced renal fibrosis;

[0052] Fig.13 Schematic diagram of the effect of GN11 on H&E staining of kidney tissue sections induced by UUO;

[0053] Fig.14 Schematic diagram of GN11's effect on the expression of UUO-induced renal injury-related genes Clu (A) and Havcr (B) in kidney tissue;

[0054] Fig.15 Schematic diagram of GN11's effect on the expression of fibrosis marker proteins in renal tissue induced by UUO: A: expression level of fibrosis marker proteins; B: relative expression of fibrosis marker proteins;

[0055] Fig.16 This is a schematic diagram of the effect of GN11 on the expression of fibrosis marker genes in UUO-induced kidney tissue;

[0056] Fig.17 is the mass spectrum of GN;

[0057] Fig.18 is the mass spectrum of GN15;

[0058] Fig.19 is the mass spectrum of GN14;

[0059] Fig. 20 is the mass spectrum of GN13;

[0060] Fig.21 is the mass spectrum of GN12;

[0061] Fig. 22 is the mass spectrum of GN11;

[0062] Fig.23 is the mass spectrum of GN10;

[0063] Fig.24is the mass spectrum of GN9;

[0064] Fig.25 This is the mass spectrum of GN8. DETAILED DESCRIPTION

[0065] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0066] Example 1 Synthesis of polypeptide compounds

[0067] For the convenience of explanation, this example takes the synthesis of the mother peptide GN as an example. The polypeptide compound is synthesized by the Fmoc solid phase synthesis method, separated and purified by reverse phase HPLC, and characterized by mass spectrometry. The specific synthesis steps are as follows:

[0068] (1) Activation of MBHA resin: 1.040 mg (0.5 mmol) of resin (Xi'an Lanxiao Technology New Materials Co., Ltd., 1.LXSS09-1-1201) was weighed into a 20 mL BD syringe, dichloromethane (DCM) was added, and the mixture was shaken at a low speed for 30 min to allow the resin to fully swell. After the reaction was completed, the resin was drained.

[0069] (2) Deprotection of Fmoc: Add 5 mL of deprotection reagent (N,N-dimethylformamide: redistilled piperidine: 1,8-diazabicyclo[5.4.0]undec-7-ene = 94:3:3, v / v / v) to the dried resin, shake at low speed for 5 min, repeat 3 times, drain the resin, add 5 mL of N,N-dimethylformamide (DMF) to wash 3 times, and then perform indole test to determine whether the reaction is complete. Indole test: Pick a small amount of DMF-washed resin into a test tube, add indole test reagent (phenol: pyridine: liquid ninhydrin = 1:2:1, v / v / v) and heat in boiling water for 1 min, observe the color of the resin, and the resin will turn blue-purple after the deprotection reaction is complete.

[0070] (3) Amino acid condensation reaction: Weigh 3 times the amount of amino acid, 1-hydroxybenzotriazole (HOBt), and HBTU, dissolve them in 7 mL of DMF, add 0.5 mL of N,N-diisopropylethylamine (DIEA), and transfer to a syringe. After shaking for 1 h, drain the resin and wash with DMF.

[0071] (4) Indole test: Take a small amount of DMF-washed resin and put it into a test tube. Add indole test reagent (phenol: pyridine: liquid ninhydrin = 1:2:1, v / v / v) and heat in boiling water for 1 min. Observe the color of the resin. The resin with complete amino acid condensation reaction is colorless.

[0072] (5) Peptide chain extension: Repeat steps (2), (3), and (4) until the amino acid sequence is fully joined.

[0073] (6) Peptide chain cleavage: Add DCM to the drained resin to swell for 3 min, repeat twice; add anhydrous methanol and shake at low speed for 3 min; add DCM to swell the resin for 3 min again; add anhydrous methanol and shake at low speed for 3 min, repeat twice, and then drain the resin to make it loose granules. Add 10 mL of cutting agent (trifluoroacetic acid: ddH2O: triisopropylsilane Tris = 95:2.5:2.5) to the drained resin, shake at low speed for 3 h, collect the cutting agent in a round-bottom flask, add trifluoroacetic acid (TFA) to wash the resin for 5 min, repeat twice, and collect in a round-bottom flask containing the cutting agent.

[0074] (7) Extraction: Use a rotary evaporator to evaporate the excess solvent in the cutting agent to concentrate it, add 40 mL of ice ether to the round-bottom flask and shake vigorously to precipitate the polypeptide, add an appropriate amount of ultrapure water, shake slowly to dissolve all the polypeptide in the water, transfer the liquid in the round-bottom flask to a separatory funnel, shake, let stand, and collect the lower aqueous phase. After placing it at -80°C overnight, put it into a freeze dryer to obtain a crude peptide.

[0075] (8) HPLC purification: Weigh 50 mg of crude peptide, dissolve it, and filter it with a 0.45 μm filter head; select a C18 preparative chromatographic column for separation and purification, and the mobile phase is a 0.1% ultrapure water and acetonitrile solvent. Use 20% acetonitrile and 50% water to balance the chromatographic column in advance, set the flow gradient (elution gradient: 0-40min acetonitrile ratio from 20% to 50%, flow rate: 10 mL / min), load the sample, run the flow gradient, detect the absorption peak at a wavelength of 220 nm, and collect the main peak product. HPLC analysis purity, mass spectrometry characterization ( Figures 17 to 25 The product that meets the purity requirement (>95%) is freeze-dried to obtain pure peptide GN and its analogs.

[0076] The polypeptide compounds 1-9 synthesized based on the above steps are shown in Table 1 below:

[0077] Table 1 Amino acid sequence of polypeptide compound

[0078]

[0079] Example 2 In vitro screening of the activity of polypeptide compound GN in improving and treating fibrosis

[0080] Cells: mouse embryonic fibroblasts (NIH-3T3), human renal tubular epithelial cells (HK2);

[0081] Culture medium: NIH-3T3 cells were cultured in high-glucose DMEM medium containing 10% FBS, and HK2 cells were cultured in DME / F12 medium containing 10% FBS;

[0082] Culture conditions: 37°C, 5% CO2 incubator.

[0083] 1. Cell culture: When the cell confluence reaches 80-90%, discard the upper medium, trypsinize until the cells are round, add high-glucose DMEM medium (NIH-3T3) containing 10% FBS or DME / F12 medium (HK2) containing 10% FBS to terminate digestion, repeatedly blow to make the cells fall off the wall, transfer to a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, add high-glucose DMEM medium (NIH-3T3) containing 10% FBS or DME / F12 medium (HK2) containing 10% FBS to resuspend into a single cell suspension, and aspirate an appropriate amount of cells and add them to a culture dish for cell passaging.

[0084] 2. Western blotting to detect the effect of GN on the expression of fibrosis markers induced by TGF-β1 in NIH-3T3 and HK2 cells

[0085] The fibrosis markers detected in this experiment include α-smooth actin (α-SMA), fibronectin, type I collagen (Collagen I) and matrix metalloproteinase 2 (MMP2).

[0086] 2.1 Dosage treatment: 3×10 5 The cell suspension was prepared at 10 μg / mL, mixed and added to a 6-well plate at 2 mL / well. After culturing for 24 h, the cells were starved for 10 h with serum-free high-glucose DMEM (NIH-3T3) or DME / F12 medium (HK2). The parent peptide GN was selected at two dosing concentrations of 25 and 50 μM, and TGF-β1 was used to induce a cell fibrosis model. The experimental groups were: Control group, TGF-β1 group, 25 μM GN group (TGF-β1+25 μM GN), and 50 μM GN group (TGF-β1+50 μM GN).

[0087] 2.2 Total cell protein extraction: After incubating the cells with GN for 24 h, discard the culture medium supernatant, wash the cells with PBS three times, absorb dryness, add RIPA lysis buffer containing 1% PMSF, scrape the protein lysate with a scraper and collect it in a 1.5 mL centrifuge tube, lyse on ice for 30 min, centrifuge at 12000 rpm for 30 min, and aspirate the supernatant.

[0088] 2.3 Protein immunoblotting: BCA kit was used to detect protein concentration, and the protein loading amount was adjusted to 20 μg. The protein was heated in a metal bath at 100°C to denature the protein. Proteins of different molecular weights were separated using 10% SDS-PAGE gel electrophoresis. The concentrated gel was run at 80V for 30 minutes, and the separation gel was run at 120V for 1.5 hours. After the electrophoresis, protein transfer was performed, and the target protein was transferred to a PVDF membrane and then analyzed using GenScript eBlot. TM The membrane was transferred using a wet transfer instrument for 17 minutes. After the transfer, the membrane was blocked with 5% skim milk powder for 1.5 hours, washed with TBST for 10 minutes × 3 times, and incubated with primary antibody at 4°C overnight. The PVDF membrane was removed the next day, washed with TBST for 10 minutes × 3 times, and incubated with secondary antibody (HRP-labeled goat anti-rabbit IgG, Beyotime, A0208) at room temperature for 1 hour, washed with TBST for 10 minutes × 3 times, and a chemiluminescence kit was added for color development.

[0089] First, NIH-3T3 cells and HK2 cells were induced by TGF-β1 (5 ng / mL) to construct in vitro cell fibrosis models, and GN was administered at doses of 25 μM and 50 μM. Total cell protein was extracted after 24 h of action, and the expression of Fibronectin, Collagen I, α-SMA, and MMP2 was detected by Western blotting. Figure 1-2 As shown in the data, after NIH-3T3 cells and HK2 cells were induced by TGF-β1, the expression of markers α-SMA, Fibronectin, Collagen I, and MMP2 was significantly upregulated. When a dose of 50 μM GN was given, the expression of the above markers was significantly inhibited. In NIH-3T3 cells, when the dose was reduced to 25 μM, GN lost its inhibitory effect on the expression of indicators such as Fibronectin, MMP2, and α-SMA, indicating that GN has anti-fibrotic activity in vitro.

[0090] Example 3 In vivo activity detection of polypeptide GN in improving and treating renal fibrosis

[0091] Experimental animals: Male C57BL / 6 mice, 6-8 weeks old, were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The breeding temperature was maintained at 25°C, with 12h alternating lighting, free access to food and water, and the bedding was changed twice a week.

[0092] 1. Experimental grouping, modeling and drug administration:

[0093] Experimental groups: sham group (Sham), operation group (UUO, unilateral ureteral ligation), low-dose group (UUO+0.5mg / kg GN), high-dose group (UUO+5.0mg / kg GN), 8 mice in each group. Construction of mouse unilateral ureteral obstruction model: After the mouse was anesthetized, the skin on the left back of the mouse was cut, the muscle layer was torn, the left ureter was freed, and the ureter was ligated at the renal pelvis and the upper 1 / 3 of the ureter, the ureter was cut between the ligatures, and the muscle layer and skin were sutured. Drug administration: Drug administration began on the second day after surgery, and the administration method was subcutaneous injection once a day for 2 weeks.

[0094] 2. Detection of renal function indexes: After the mice were anesthetized, blood was collected from their eye sockets and collected in 1.5 mL EP tubes. The samples were centrifuged at 5000 rpm for 15 min, twice, and the supernatant was collected. The serum Scr and serum BUN contents were measured according to the instructions for the creatinine (Scr) assay kit (C011-2-1) and the instructions for the urea nitrogen (BUN) test kit (C013-2-1). The experimental results are shown in Figure 2. Figure 3 As shown, GN significantly inhibited the abnormal increase of BUN and Scr levels caused by renal fibrosis.

[0095] 3. Mouse H&E staining results

[0096] The left kidney tissue of the mouse was removed during sampling. Part of the removed kidney tissue was stored at -80°C, and part was fixed with formaldehyde, embedded in paraffin, and histopathological sections were made. Some pathological sections were taken for H&E staining, and the pathological sections were dried at 65°C for 1h. Dewaxing: xylene 20min, xylene 20min, anhydrous ethanol 2min, anhydrous ethanol 2min, 95% ethanol 2min, 90% ethanol 2min, 80% ethanol 2min. Staining: Staining was carried out in the order of hematoxylin 7min, tap water washing, 1% hydrochloric acid ethanol differentiation 1s, tap water washing, eosin staining 25-30s, and tap water washing 5min. Dehydration and transparency: Dehydration was carried out in the order of 80% ethanol 7s, 90% ethanol 7s, 95% ethanol 7s, anhydrous ethanol 7s, anhydrous ethanol 7s, xylene 2min, and xylene 2min.

[0097] The experimental results are as follows Figure 4 As shown in the figure, the renal tubules in the model group were dilated and the renal tubular epithelial cells were shed. After administration, the above changes were inhibited. The above results show that GN can alleviate the renal damage caused by UUO.

[0098] 4. Detection of the effect of GN on the expression of fibrosis markers at the animal level

[0099] Western blotting was used to detect the expression of markers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin; qRT-PCR was used to detect the expression of fibrosis genes Fn1, Col1a1, and Acta2.

[0100] 4.1 Western blotting to detect the expression of tissue fibrosis markers

[0101] Tissue protein extraction: Weigh 15 mg of kidney tissue from each group of mice, add 100 μL of RIPA lysis buffer containing 1% PMSF, add grinding steel balls (one large and one small), grind at 60 Hz for 120 s, remove the grinding steel balls, let stand at low temperature for 30 min to lyse tissue protein, centrifuge at 12000 rpm for 30 min, centrifuge twice. The supernatant is the total tissue protein.

[0102] Western blotting: The operation is the same as that of cell experiment 2.3.

[0103] The experimental results are as follows Figure 5 As shown in the figure, GN downregulated the abnormal expression of renal tissue fibrosis-related proteins Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin caused by UUO, indicating that GN has in vivo anti-renal fibrosis activity at the protein level.

[0104] 4.2qRT-PCR detection of fibrosis gene expression.

[0105] Tissue RNA extraction: Weigh 15 mg of kidney tissue from each group of mice, add 1000 μL RNA extraction solution, add grinding steel beads (one large and one small), grind at 60 Hz for 120 s, let stand at room temperature for 10 min, transfer the lysate to a new enzyme-free EP tube, add 200 μL chloroform, shake vigorously, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 15 min, draw the upper colorless aqueous phase, add 500 μL isopropanol, mix well and let stand for 10 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, a white colloidal precipitate is formed on the side and bottom of the tube, add 1000 μL 75% ethanol, shake gently to make the precipitate fall off the tube wall, centrifuge at 7500 rpm for 5 min, draw the supernatant, dry the precipitate at room temperature, and dissolve the precipitate in enzyme-free sterile water.

[0106] Reverse transcription: The RNA concentration was detected by a micro-UV-visible spectrophotometer. Samples with RNA A260 / A280 values ​​between 1.8 and 2.2 met the requirements of subsequent experimental operations. The reverse transcription system was prepared according to the proportions in Table 2, mixed and added to the PCR instrument, and the reverse transcription conditions were set to 37°C, 2 min, 55°C, 15 min, and 85°C, 5 min. After the reverse transcription was completed, the synthesized cDNA was stored at -20°C.

[0107] Table 2 Reverse transcription system configuration table

[0108]

[0109] x = 1000 ng / (RNA concentration)

[0110] qPCR: Prepare the reaction system according to Table 3.

[0111] Table 3 Amplification system configuration table

[0112]

[0113]

[0114] The primer sequences are shown in Table 4:

[0115] Table 4

[0116]

[0117] Amplification program: Pre-denaturation: 95°C, 5 min. Amplification 40 cycles: 95°C, 10 s; 60°C, 30 s. Melting: 95°C, 1 min; 55°C, 30 s; 95°C, 30 s. Relative expression was calculated based on the Ct value of the gene (2 -ΔΔCt ).

[0118] The experimental results are as follows Figure 6 As shown, GN significantly inhibited the abnormal expression of fibrosis genes Col1a1, Fn1, and Acta2 caused by UUO, indicating that GN also inhibits UUO-induced renal fibrosis at the gene level.

[0119] Example 4 MTT method to detect the cytotoxicity of polypeptide compounds in vitro

[0120] Cells: mouse embryonic fibroblasts (NIH-3T3), rat renal tubular epithelial cells (NRK-52E);

[0121] Culture medium: NIH-3T3 cells were cultured in high-glucose DMEM medium containing 10% FBS, and NRK-52E cells were cultured in high-glucose DMEM medium containing 5% FBS;

[0122] Culture conditions: 37°C, 5% CO2 incubator.

[0123] Press 7×10 3Cell suspension was prepared at 100 μL / well and added to a 96-well plate after mixing. After culturing for 24 h, GN and its analogs were added at a concentration gradient of 12.5, 25, 50, 100, and 200 μM. After culturing for 24 h, MTT (5 mg / mL, Solebold, 715F0528) was added and incubated for 3 h. The absorbance at a wavelength of 570 nm was detected by a microplate reader.

[0124] The experimental results are as follows Figure 7 and Fig. 9 As shown by Figure 7 It can be seen that in NIH-3T3 cells, within the concentration of 25 μM, the analogs have no toxic side effects on cells. Fig. 9 It can be seen that within the concentration range of 0-50 μM, GN and GN11 have no toxic side effects on NRK-52E cells.

[0125] Example 5 In vitro screening of polypeptide compounds for improving and treating fibrosis

[0126] The cells and culture conditions used in this example are the same as those in Example 4.

[0127] 1. Western blotting to detect the effects of GN and its analogs on the expression of fibrosis markers in NIH-3T3 and NRK-52E cells induced by TGF-β1

[0128] 2. Dosage treatment: 3×10 5 / mL prepared cell suspension, mixed and added to 6-well plates, 2mL / well, cultured for 24h, and then starved cells for 10h with serum-free medium. The dosage of polypeptide analogs in the anti-fibrotic activity screening experiment of GN analogs was 25μM. The experimental groups were: Control group, TGF-β1 group, TGF-β1+GN group, TGF-β1+GN15 group, TGF-β1+GN14 group, TGF-β1+GN13 group, TGF-β1+GN12 group, TGF-β1+GN11 group, TGF-β1+GN10 group, TGF-β1+GN9 group, TGF-β1+GN8 group, and the rest of the operations were the same as in Example 2.

[0129] The shortest active fragment of the parent peptide GN was screened by using the TGF-β1-induced NIH-3T3 cell fibrosis model, and the GN polypeptide analogue was administered at a dose of 25 μM. After 24 hours of action, the total cell protein was extracted, and the expression of α-SMA and Fibronectin was detected by western blotting. Figure 8 As shown, after TGF-β1 induction, the expression of markers α-SMA and Fibronectin was significantly upregulated. After peptide molecule intervention, peptide GN11 significantly inhibited the expression of the above markers.

[0130] On this basis, the cell line was changed to NRK-52E cells to further screen the anti-renal fibrosis activity of peptides GN and GN11. The experimental steps were the same as those in the NIH-3T3 cell screening process. Fig.10 It can be seen that at a dosage of 25 μM, GN had no inhibitory effect on the overexpressed fibrosis markers Collagen I and Fibronectin, while at the same dose, GN11 was able to significantly reduce the expression of the above markers, thereby determining that the polypeptide GN11 has better in vitro anti-fibrosis activity than the parent peptide GN.

[0131] 3. qRT-PCR detection of the effect of GN11 on TGF-β1-induced fibrosis marker genes in NRK-52E cells

[0132] 3.1 Extraction of total RNA from cells: RNA lysis solution was added to a 6-well plate, 500 μL / well, and the mixture was repeatedly pipetted and transferred to an enzyme-free 1.5 mL EP tube. 100 μL of chloroform was added, and the mixture was shaken vigorously. The mixture was incubated at room temperature for 5 min, and centrifuged at 12000 rpm for 15 min. The upper colorless aqueous phase was aspirated, 250 μL of isopropanol was added, and the mixture was allowed to stand for 10 min after mixing. The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. A white colloidal precipitate was formed on the side and bottom of the tube. 500 μL of 75% ethanol was added, and the precipitate was gently shaken to fall off the tube wall. The mixture was centrifuged at 7500 rpm for 5 min, the supernatant was aspirated, the precipitate was dried at room temperature, and the precipitate was dissolved in enzyme-free sterile water. The subsequent operation was the same as that in 4.2 of Example 3.

[0133] Experimental Results Fig.11 The figure shows the effect of GN11 on the expression of fibrosis-related genes Col1a1, Fn1, Mmp2, Vim, etc. in NRK-52E cells induced by TGF-β1. As shown in the figure, when the dosage was 25 μM, GN11 significantly inhibited the abnormal expression of the above genes induced by TGF-β1, indicating that GN11 significantly inhibited TGF-β1-induced renal fibrosis in NRK-52E cells at the gene level.

[0134] Example 6 In vivo activity detection of polypeptide GN11 in improving and treating renal fibrosis

[0135] The experimental animals and operations were the same as those in Example 3.

[0136] We first evaluated the effect of GN11 on UUO-induced renal injury. Figure 12-13 As shown. Fig.10 It can be seen that GN11 significantly reduced the abnormal overexpression of renal function indicators Scr and BUN induced by UUO; Fig.11It can be seen that after UUO induction, the renal tubules of mice atrophied and the renal tubular epithelial cells fell off. After administration, the above changes were inhibited. The detection results of renal injury-related marker genes Clu and Havcr are as follows Fig.14 As shown in the figure, it can be seen that GN11 significantly reduced the abnormal expression of the above genes after UUO induction. The above results show that GN11 can alleviate kidney damage caused by UUO.

[0137] On this basis, the in vivo anti-renal fibrosis activity of GN11 was evaluated, and the expression of fibrosis markers was detected at both the protein and gene levels. Fig.15 The results showed that after UUO induction, the expression of fibrosis markers Fibronectin, Collagen I, MMP2, α-SMA, and Vimentin were significantly upregulated. After GN11 intervention, the overexpression of the above markers was significantly inhibited, and a low dose of GN11 (0.5 mg / kg) could inhibit the expression of all the above markers, indicating that the in vivo anti-renal fibrosis activity of GN11 is better than that of the parent peptide GN.

[0138] The results of the detection of fibrosis marker genes Col1a1, Fn1, and Acta2 expression are as follows Fig.16 As shown in the figure, GN11 significantly downregulated the overexpression of the above genes, indicating that GN11 also inhibits UUO-induced renal fibrosis at the gene level.

Claims

1. A polypeptide compound or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide compound contains the parent peptide represented by the following amino acid sequence: NH2-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Arg-Leu-Arg-Phe-Ile-Pro-Lys-CONH2; Where Xaa1 = Glu or absent; Xaa2 = Ala or absent; Xaa3 = Pro or not present; Xaa4 = Ala or absent; Xaa5 = Leu or absent; Xaa6 = Leu or absent; Xaa7 = Thr or absent; Xaa8 = Ser or absent; If Xaan (n≥2) does not exist, then the amino acids before it do not exist.

2. A polypeptide compound or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequences of the polypeptide compounds are shown in SEQ ID NOs. 1-9.

3. A method for preparing the polypeptide compound according to claim 1 or 2, characterized in that: The following steps are involved: (1) Add dichloromethane to MBHA resin, drain after the resin is fully swollen, add deprotection reagent, shake at low speed, drain, and wash; add a mixture of amino acid, HOBt and HBTU and N,N-diisopropylethylamine, shake, drain, and wash; (2) coupling the amino acids one by one to the MBHA resin according to step (1); (3) DCM is added to the drained resin to swell it, and anhydrous methanol is added to shake it at a low speed; drain, cut, wash, concentrate, add icy ether to precipitate the polypeptide, add water to dissolve all the polypeptide in water, filter, purify, and obtain pure peptide.

4. Use of the polypeptide compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating chronic kidney disease.

5. Use of the polypeptide compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating renal fibrosis.

6. The use according to claim 5, characterized in that: The renal fibrosis includes renal fibrosis diseases caused by obstructive nephropathy, diabetic nephropathy, hypertensive nephropathy, nephritis, renal tumors, adverse drug reactions or infection by pathogenic microorganisms.

7. The use according to claim 5, characterized in that: Alleviate and / or inhibit the abnormal expression of fibrosis genes.

8. A preparation for treating organ fibrosis, characterized in that: It contains the polypeptide compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

9. The preparation according to claim 8, characterized in that The dosage forms of the preparation include tablets, capsules, granules, oral liquids, syrups, pills, ointments and patches for skin surfaces, aerosols, nasal sprays, suppositories, microsphere preparations, injection liquids or freeze-dried powder injections.

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