Application of the autophagy key regulatory protein Beclin-1 fragment peptide MP1 in the preparation of anti-renal fibrosis drugs

Abstract

This invention discloses the use of a peptide MP1 derived from a fragment of the key autophagy regulatory protein Beclin-1 in the preparation of anti-renal fibrosis drugs. The study found that peptide MP1 exhibits significant anti-renal fibrosis effects in both in vivo and in vitro, with no obvious toxic side effects. Its mechanism of action mainly involves inhibiting the WNT / β-Catenin and TGF-β / Smad signaling pathways, suppressing EMT processes, and thus inhibiting renal fibrosis, without inducing autophagy. Peptide MP1 has significant clinical application value in the preparation of anti-renal fibrosis drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the use of a fragment polypeptide MP1 of the autophagy key regulatory protein Beclin-1, specifically the application of polypeptide MP1 in the prevention or treatment of renal fibrosis. Background Technology

[0002] Fibrosis can affect all organs and has impacted a quarter of the global population. Among all organ fibrosis, renal fibrosis has the second highest incidence rate after liver fibrosis, with an annual prevalence of approximately 1.21%. Renal fibrosis is a common pathway and pathological change leading to end-stage renal disease (ESRD) in all chronic kidney disease (CKD), characterized by the massive accumulation of extracellular matrix (ECM) and loss of nephron function. Extensive ECM deposition damages the renal parenchymal structure, leading to cellular dysfunction, parenchymal scarring, and ultimately, end-stage renal disease and renal failure. Renal fibrosis is difficult to detect, and currently there are no effective treatments to slow or reverse it. Existing treatments are mostly limited to dialysis and kidney transplantation, severely impacting patients' quality of life and imposing a huge economic and social burden. Therefore, there is an urgent need to develop effective therapeutic drugs.

[0003] A growing body of research indicates that epithelial-mesenchymal transition (EMT) is a crucial driver of renal fibrosis. EMT is a transdifferentiation process during which epithelial cells lose their cell polarity, phenotypic characteristics, and intercellular adhesion properties, acquiring some mesenchymal cell characteristics in return. Many growth factors, cytokines, and signaling pathways can induce EMT, among which the WNT / β-Catenin signaling pathway plays a vital role. Normally, the WNT signaling pathway plays a crucial role in embryonic development and tumorigenesis. In the classic WNT / β-Catenin signaling pathway, WNT forms a complex with Frizzled and LRP5 / 6 on the cell surface. Activation of this complex triggers the dissociation of the multifunctional kinase GSK-3β from the regulatory APC / Axin / GSK-3β complex, increasing the stability of β-Catenin. Stable β-Catenin is then transported into the nucleus via Rac1 and other factors, where it binds to the transcription factor LEF / TCF, replacing cofactor repressors and thereby regulating the expression of WNT target genes. Studies have shown that in CKD models, the expression of cytokines related to the WNT signaling pathway, such as β-Catenin, C-Myc, and Cyclin D1, is significantly upregulated. Activation of the WNT pathway can induce renal tubular epithelial cell dedifferentiation, activate the EMT process, and thus promote the fibrosis process.

[0004] Li Wang et al. reported that inhibiting Beclin-1 expression through shBECN1 increased the expression and transcriptional activity of the Lef1 gene, downregulated the expression levels of DKK3 and sfrp, inhibitors of the WNT / β-Catenin signaling pathway, promoted the migration of β-Catenin from the cytoplasm to the nucleus, and thus activated the WNT pathway. Upregulation of Beclin-1, a key regulatory protein in inducing autophagy, continuously activates the autophagy response. Man J Livingston et al. showed that in a mouse model of unilateral ureteral obstruction (UUO), sustained activation of autophagy in the proximal tubules of the kidney promotes the progression of renal interstitial fibrosis. Summary of the Invention

[0005] This invention has discovered through cellular and animal experiments that the peptide MP1, derived from a fragment of the key autophagy regulatory protein Beclin-1, can effectively inhibit renal fibrosis. Based on this, this invention proposes applications of the peptide MP1.

[0006] The polypeptide MP1 is a polypeptide fragment derived from positions 112-123 of the BH3 domain of the Beclin-1 protein, with the amino acid sequence Leu-Ser-Arg-Arg-Met-Lys-Val-Thr-Gly-Asp-Leu-Trp. Currently, only its activity in inducing autophagy in tumor cells has been reported. This invention demonstrates through experiments that the polypeptide MP1 has anti-renal fibrosis activity.

[0007] According to specific embodiments of the present invention, the application of peptide MP1 in the preparation of a drug that inhibits the expression of renal fibrosis-related proteins includes, preferably, the renal fibrosis-related proteins include α-SMA, FN, COL and / or MMP2; preferably, the renal fibrosis-related proteins also include α-SMA, FN, COL and MMP2; preferably, the renal fibrosis-related proteins also include Snail1 and / or Vimentin.

[0008] According to specific embodiments of the present invention, the application of peptide MP1 in the preparation of a drug that inhibits the expression of renal fibrosis-related genes, preferably, the renal fibrosis-related genes inhibited include Acta2, Fn1, COL1A1 and / or Mmp2; preferably, the renal fibrosis-related genes inhibited also include SNAI1 and / or VIM.

[0009] According to specific embodiments of the present invention, the application of peptide MP1 in the preparation of drugs that inhibit the expression of TGF-β / Smad pathway-related genes, preferably, the TGF-β / Smad pathway-related genes include CTNNB1, MYC and / or CCND1.

[0010] According to specific embodiments of the present invention, the application of peptide MP1 in the preparation of a drug that inhibits the expression of TGF-β / Smad pathway-related proteins, preferably, the expression of TGF-β / Smad pathway-related proteins includes phosphorylated Smad protein.

[0011] According to specific embodiments of the present invention, the application of peptide MP1 in the preparation of drugs that inhibit the expression of WNT / β-Catenin pathway-related genes, preferably, the WNT / β-Catenin pathway-related genes include Ctnnb1, Myc, and Ccnd1.

[0012] According to specific embodiments of the present invention, the application of peptide MP1 in the preparation of drugs that inhibit the expression of WNT / β-Catenin pathway-related proteins, preferably, the WNT / β-Catenin pathway-related proteins include β-Catenin, C-Myc and / or Cyclin D1.

[0013] This invention provides the application of the autophagy key regulatory protein Beclin-1 fragment polypeptide MP1 in the preparation of anti-renal fibrosis drugs; preferably, the renal fibrosis includes renal interstitial fibrosis, including renal interstitial fibrosis caused by ureteral obstruction.

[0014] Another object of the present invention is to provide a formulation for treating renal fibrosis, said formulation having a polypeptide MP1 as an active ingredient, said formulation being a drug or a pharmaceutical composition.

[0015] According to a specific embodiment of the present invention, the preparation for treating renal fibrosis is in the following dosage form: tablet, capsule, granule, oral liquid, syrup, drop pill, ointment and patch for skin surface, aerosol, nasal spray, suppository, microsphere preparation, injection dosage form, or lyophilized powder injection.

[0016] The aforementioned formulations may include pharmaceutical excipients, diluents, or carriers, and may be used in any suitable manner for patients requiring treatment. Suitable methods include, but are not limited to, oral, rectal, nasal, topical (including oral and sublingual), subcutaneous, or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and intrathecal) administration.

[0017] The beneficial effects of this invention are:

[0018] This invention demonstrates through experiments that the peptide MP1 has no significant toxic side effects in cell experiments and mouse animal experiments. In both TGF-β1-induced NIH-3T3 and HK2 in vitro fibrosis models, the peptide MP1 of this invention can inhibit the expression of fibrosis markers α-smooth muscle actin (α-SMA), fibronectin, type I collagen (Collagen I), and matrix metalloproteinase 2 (MMP2) at the gene and protein levels.

[0019] In a TGF-β1-induced HK2 cell fibrosis model, the peptide MP1 inhibited the expression of EMT markers Snail1 and Vimentin at both the gene and protein levels, indicating that the peptide MP1 can suppress fibrosis by inhibiting EMT.

[0020] In NIH-3T3 cells, the peptide MP1 had no effect on the expression levels of LC3II / LC3I and P62, indicating that MP1 does not induce autophagy in NIH-3T3 cells.

[0021] In a mouse model of renal fibrosis induced by unilateral ureteral ligation (UUO), the peptide MP1 can reduce serum creatinine (Scr) and serum blood urea nitrogen (BUN) levels, improve renal function, and alleviate renal damage.

[0022] Meanwhile, the peptide MP1 inhibited the expression of fibrosis markers α-SMA, Fibronectin, Collagen I, and MMP2, as well as EMT-related factors Snail1 and Vimentin, at both the protein and gene levels, indicating that MP1 can inhibit renal fibrosis and EMT processes in vivo, and its effect is superior to that of the control drug captopril.

[0023] The peptide MP1 of this invention mainly inhibits EMT and renal fibrosis by inhibiting the WNT / β-Catenin and TGF-β / Smad signaling pathways. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The study demonstrated the inhibitory effect of peptide MP1 on TGF-β1-induced markers of fibrosis in NIH-3T3 cells.

[0026] Figure 2The study showed that the peptide MP1 inhibited TGF-β1-induced fibrosis and EMT markers in HK2 cells.

[0027] Figure 3 This study demonstrates the effects of peptide MP1 on renal function indicators and renal tissue morphology in mice with UUO-induced renal fibrosis.

[0028] Figure 4 The study demonstrated the inhibitory effect of peptide MP1 on renal tissue fibrosis and EMT markers in mice with UUO-induced renal fibrosis.

[0029] Figure 5 The study demonstrated the inhibitory effect of peptide MP1 on the TGF-β1-induced WNT / β-Catenin and TGF-β / Smad pathways in NIH-3T3 cells.

[0030] Figure 6 The study demonstrated the inhibitory effect of peptide MP1 on the TGF-β1-induced WNT / β-Catenin and TGF-β / Smad pathways in HK2 cells.

[0031] Figure 7 The study demonstrated the inhibitory effect of peptide MP1 on the WNT / β-Catenin and TGF-β / Smad pathways in the kidney tissue of mice with UUO-induced renal fibrosis.

[0032] Figure 8 This study demonstrates the effect of peptide MP1 on TGF-β1-induced autophagy in NIH-3T3 cells. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0034] Example 1: Screening for Beclin-1 fragment peptides, a key autophagy regulatory protein with anti-renal fibrosis activity.

[0035] This invention proposes to use interference with the WNT / β-Catenin pathway as a strategy for the treatment of renal fibrosis. First, the Beclin-1 protein fragment was specifically selected. 112-123 The anti-fibrotic activity of the derivative peptides MP1 and MP2 was initially screened.

[0036] Table 1. Amino acid sequences of polypeptide compounds

[0037]

[0038] After constructing an in vitro cell fibrosis model by inducing mouse embryonic fibroblasts (NIH-3T3) with TGF-β1, the above-mentioned polypeptide molecules were administered. After 24 hours of treatment, total cell protein was extracted, and the expression of fibrosis markers α-smooth muscle actin (α-SMA) and fibronectin was detected by Western blotting.

[0039] The results showed that TGF-β1 induction significantly upregulated the expression of fibrosis markers α-SMA and Fibronectin, while intervention with the peptide MP1 significantly inhibited the expression of these markers. Other peptides had no significant effect on the expression of these markers. Therefore, it was preliminarily determined that the peptide MP1 possesses anti-fibrotic activity.

[0040] Example 2: Synthesis of polypeptide MP1

[0041] The peptide MP1 was synthesized using the Fmoc solid-phase synthesis method, purified by reversed-phase HPLC, and characterized by mass spectrometry.

[0042] The sequence of peptide MP1 is: Leu-Ser-Arg-Arg-Met-Lys-Val-Thr-Gly-Asp-Leu-Trp, and its structural formula is as follows:

[0043]

[0044] The specific synthesis steps are as follows:

[0045] (1) Activation of 2-CTC resin: Weigh an appropriate amount (0.5 mmol) of resin into a 20 mL BD syringe, add dichloromethane (DCM), shake at low speed for 30 min to allow the resin to swell fully, and then dry the resin.

[0046] (2) The first amino acid condensation reaction at the C-terminus: Weigh three times the amount of Trp in excess, dissolve it in DCM and add it to the syringe. Shake at low speed for 5 minutes and then add DIEA to start the reaction. Shake for 1 hour, then dry the resin and wash the resin with DMF 3 times.

[0047] (3) Indene test: Take a small amount of DMF-washed resin into a test tube, add indene test reagent (phenol: pyridine: ninhydrin = 1:2:1) and heat in boiling water for 1 minute. Observe the color of the resin. The resin with complete amino acid condensation reaction is colorless, and the resin with deprotection reaction is blue-purple.

[0048] (4) Deprotection reaction: Add deprotection reagent (DMF: redistilled piperidine: DBU = 94:3:3) to the dried resin, shake at low speed for 5 min, repeat 3 times, dry the resin after the reaction, wash with DMF 3 times and then check with indole to determine whether the reaction is complete.

[0049] (5) Condensation reaction of other amino acids: Weigh out 3 times the amount of amino acids, HOBt, and HBTU, dissolve them in DMF, add DIEA and transfer them to a syringe, shake for 1 hour, then dry the resin and clean with DMF.

[0050] (6) Peptide chain elongation: Repeat steps (3), (4), and (5) until the amino acid sequence Leu-Ser-Arg-Arg-Met-Lys-Val-Thr-Gly-Asp-Leu-Trp is fully conjugated.

[0051] (7) Peptide chain cleavage: DCM was added to the dried resin to swell for 3 min, and this was repeated twice; anhydrous methanol was added and shaken at low speed for 3 min; DCM was added again to swell the resin for 3 min; anhydrous methanol was added and shaken at low speed for 3 min, and this was repeated twice. The resin was then dried to make it loose granules. The cleavage agent (TFA:ddH2O:EDT:Tris = 94:2.5:1:2.5) was added to the dried resin, and the mixture was shaken at low speed for 3 h. The cleavage agent was collected in a round-bottom flask, and TFA was added to wash the resin for 5 min, which was repeated twice. The residue was collected in a round-bottom flask containing the cleavage agent.

[0052] (8) Extraction: Use a rotary evaporator to evaporate as much excess solvent as possible from the cleavage agent to concentrate it. Add ice-cold diethyl ether to a round-bottom flask and shake vigorously to precipitate the peptide. Add an appropriate amount of ultrapure water and shake slowly to dissolve the peptide completely 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 standing overnight at -80°C, place it in a freeze dryer to obtain crude peptide.

[0053] (9) HPLC purification: Approximately 50 mg of crude peptide was weighed, dissolved, and filtered through a 0.45 μm filter. A C18 preparative chromatographic column was selected for separation and purification. The mobile phase consisted of 0.1% aqueous phase and acetonitrile solvent. The column was pre-equilibrated with a ratio of 5% acetonitrile and 95% water. After setting the flow gradient, the sample was loaded, and the flow gradient was run. The absorption peak at 220 nm was detected, and the main peak product was collected. The purity was analyzed by HPLC, and the product was characterized by mass spectrometry. The product meeting the purity requirements (>95%) was freeze-dried to obtain peptide MP1.

[0054] Example 3: Anti-renal fibrosis effect of peptide MP1 at the cellular level

[0055] Cells: mouse embryonic fibroblasts (NIH-3T3), human renal tubular epithelial cells (HK2);

[0056] Culture media: DMEM medium containing 10% FBS, DME / F12 medium containing 10% FBS;

[0057] Culture conditions: 37℃, 5% CO2 incubator.

[0058] 1. Cell culture: When the cell confluence reaches 80-90%, discard the upper culture medium, digest with trypsin until the cells are round, add culture medium to stop digestion, repeatedly pipette to detach the cells from the cell wall, transfer to a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, add an appropriate amount of culture medium to resuspend as a single-cell suspension, and add an appropriate amount of cells to a culture dish for cell passage.

[0059] 2. MTT assay for the cytotoxicity of MP1 against NIH-3T3 and HK2 cells:

[0060] 7×10 3 Cell suspensions were prepared and mixed, then added to 96-well plates at 100 μL / well. After culturing for 24 h, peptide MP1 at concentration gradients of 12.5, 25, 50, 100, and 200 μM were added and cultured for another 24 h. MTT was then added and incubated for 4 h. After removing the culture medium, 150 μL of DMSO was added to each well and shaken for 10 min. The absorbance at 490 nm was measured using a microplate reader.

[0061] The experimental results are as follows Figure 1 A and Figure 2 As shown in A, where Figure 1 A represents the survival rate of NIH-3T3 cells. Figure 2 A represents the survival rate of HK2 cells. As shown in the figure, within the concentration range of 0-200 μM, peptide MP1 did not significantly inhibit the survival rate of NIH-3T3 cells, nor did it significantly inhibit the survival rate of NIH-3T3 cells within the concentration range of 0-100 μM.

[0062] 3. Western blot analysis of the effects of MP1 on TGF-β1-induced fibrosis and EMT marker expression in NIH-3T3 and HK2 cells.

[0063] The fibrosis markers detected in this embodiment include α-SMA, Fibronectin (FN), Collagen I (COL), and MMP2, while the EMT markers include Snail1 and Vimentin.

[0064] 3.1 Administration: 3 × 10 5 Cell suspensions were prepared in 2 mL / well of a 6-well plate and mixed thoroughly. After culturing for 24 h, the cells were starved for 10 h using serum-free medium. Two drug concentrations, 50 μM and 100 μM, were used to induce a cell fibrosis model with TGF-β1, while MP1 was added simultaneously. The experimental groups were: control group, TGF-β1 group, TGF-β1 + 50 μM MP1 group, and TGF-β1 + 100 μM MP1 group.

[0065] 3.2 Total protein extraction from cells: After incubating cells with MP1 for 24 h, the supernatant of the culture medium was discarded, the cells were washed 3 times with PBS, dried, and then RIPA lysis buffer containing 1% PMSF was added. The protein lysate was scraped off with a scraper and collected into a 1.5 mL centrifuge tube. The cells were lysed on ice for 30 min, centrifuged at 12000 rpm for 30 min, and the supernatant was collected.

[0066] 3.3 Protein Immunoblotting: Protein concentration was detected using a BCA kit, and the protein loading amount was adjusted to 30 μg. The protein was denatured by heating in a 100℃ metal bath. Proteins of different molecular weights were separated using 10% SDS-PAGE gel electrophoresis: stacking gel at 80V for 30 min, and separating gel at 120V for 1.5 h. After electrophoresis, the target protein was transferred to a PVDF membrane at 250mA for 3 h. After transfer, the membrane was blocked with 5% skim milk powder for 1.5 h, followed by washing with TBST for 10 min × 3 times. The membrane was then incubated with primary antibody overnight at 4℃. The next day, the PVDF membrane was removed, washed with TBST for 10 min × 3 times, incubated with secondary antibody at room temperature for 1 h, washed with TBST for 10 min × 3 times, and then incubated with chemiluminescence immunoassay kit for color development.

[0067] Figure 1 This study demonstrates the effect of peptide MP1 on the expression of TGF-β1-induced fibrosis markers α-SMA, FN, COL, and MMP2 in NIH-3T3 cells. Figure 1 B is a Western blot image of protein immunoblotting. Figure 1 CF is a bar chart representing the band structure. As shown in the figure, compared to the control group, TGF-β1 induction significantly increased the expression levels of fibrosis-related proteins α-SMA, FN, COL, and MMP2. Treatment with the peptide MP1, regardless of high or low concentrations, significantly decreased the expression levels of these proteins. This indicates that the peptide MP1 has a significant inhibitory effect at the protein level on the TGF-β1-induced NIH-3T3 cell renal fibrosis model.

[0068] Figure 2 This study demonstrates the effects of peptide MP1 on the expression of TGF-β1-induced fibrosis markers α-SMA, FN, COL, MMP2, and EMT markers Snail1 and Vimentin (Vim) in HK2 cells. Figure 2 BC is a Western blot image of protein immunoblotting. Figure 2DI is a bar chart representing the band structure. As shown in the figure, compared to the control group, TGF-β1 induction significantly increased the expression levels of fibrosis-related proteins α-SMA, FN, COL, MMP2, and EMT-related proteins Snail1 and Vim. Treatment with peptide MP1 significantly decreased the expression levels of these proteins. This indicates that peptide MP1 has a significant inhibitory effect at the protein level on TGF-β1-induced kidney fibrosis and EMT in HK2 cells.

[0069] 4. qRT-PCR detection of the effect of peptide MP1 on TGF-β1-induced fibrosis marker genes in HK2 and NIH-3T3 cells.

[0070] 4.1 Total RNA extraction from cells: Add 500 μL of RNA lysis buffer to each well of a 6-well plate, repeatedly pipette and transfer to an enzyme-free 1.5 mL EP tube, add 100 μL of chloroform, shake vigorously, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 15 min, aspirate the colorless aqueous phase, add 500 μL of isopropanol, mix well, let stand for 10 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, a white gel-like precipitate will form on the sides and bottom of the tube, add 500 μL of 75% ethanol, gently shake to allow the precipitate to detach from the tube wall, centrifuge at 7500 rpm for 5 min, aspirate the supernatant, air dry the precipitate at room temperature, and dissolve the precipitate in enzyme-free sterile water.

[0071] 4.2 Reverse Transcription: RNA concentration was detected using a micro UV-Vis spectrophotometer. The A260 / A280 ratio of the RNA sample was between 1.8 and 2.2. The reverse transcription system was prepared according to the proportions in the table below. After mixing, the mixture was added to the PCR instrument. The reverse transcription conditions were set as follows: 25℃ for 5 min, 55℃ for 15 min, and 85℃ for 5 min. After reverse transcription was complete, the synthesized cDNA was stored at -20℃.

[0072] Table 2 Reverse Transcription System Configuration Table

[0073]

[0074] 4.3 qPCR: Prepare the reaction system according to Table 3.

[0075] Table 3. Amplification System Configuration Table

[0076]

[0077] Amplification program: Pre-denaturation: 95℃, 5 min. Amplification 40 cycles: 95℃, 10 s; 60℃, 30 s. Melting: 95℃, 1 min; 55℃, 30 s; 95℃, 30 s. Calculate relative expression (22) based on gene Ct values. -ΔΔCt ).

[0078] Figure 1GJ showed the effect of peptide MP1 on the expression of fibrosis-related genes Acta2, Fn1, COL1A1, and Mmp2 in TGF-β1-induced NIH-3T3 cells. As shown in the figure, when the drug dosage was 50 μM and 100 μM, MP1 significantly inhibited the abnormal expression of these genes induced by TGF-β1, indicating that peptide MP1 significantly inhibits TGF-β1-induced renal fibrosis in NIH-3T3 cells at the gene level.

[0079] Figure 2 The study investigated the effects of peptide MP1 on the expression of fibrosis-related genes Acta2, Fn1, COL1A1, and Mmp2, as well as EMT-related genes SNAI1 and VIM, in TGF-β1-induced HK2 cells. The figures show that at doses of 50 μM and 100 μM, MP1 significantly inhibited the abnormal expression of these genes induced by TGF-β1, indicating that peptide MP1 significantly inhibits TGF-β1-induced renal fibrosis in HK2 cells at the gene level.

[0080] Example 4: Anti-renal fibrosis effect of peptide MP1 in mice

[0081] Experimental animals: Male C57BL / 6 mice, 6-8 weeks old, purchased from Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences. The temperature was maintained at 25℃, with alternating lighting for 12 hours, free access to food and water, and bedding changed twice a week.

[0082] 1. Experimental grouping, modeling, and drug administration:

[0083] Experimental groups: Sham group, surgical group (UUO), 0.625 mg / kg MP1 group, 1.25 mg / kg MP1 group, and 20 mg / kg Captopril group, with 10 mice in each group. Establishment of a mouse model of unilateral ureteral obstruction: After anesthesia, the skin on the left side of the mouse's back was incised, the muscle layer was torn open, and the left ureter was freed. Ligations were made at the renal pelvis and the upper third of the ureter. The ureter was then cut between the ligation lines, and the muscle layer and skin were sutured. Drug administration: Drug administration began on the second day after surgery, via subcutaneous injection, once daily for 2 weeks.

[0084] 2. Kidney function index detection: Blood was collected from the orbital cavity of mice after anesthesia and placed in 1.5 mL EP tubes. The tubes were centrifuged at 5000 rpm for 15 min twice. The supernatant was collected, and the serum creatinine (Scr) and serum urea nitrogen (BUN) levels were measured according to the kit instructions.

[0085] The experimental results are as follows Figure 3As shown in AB, peptide MP1 at concentrations of 0.625 mg / kg and 1.25 mg / kg significantly inhibited the abnormal increase in Scr and BUN levels caused by renal fibrosis.

[0086] 3. Morphological appearance and H&E staining results of the left kidney in mice.

[0087] During tissue collection, a portion of the harvested kidney tissue was stored at -80℃, while another portion was fixed in formaldehyde, embedded in paraffin, and used to prepare histopathological sections. A portion of the histopathological sections were subjected to H&E staining. The sections were then dried at 65℃ for 1 hour and dewaxed sequentially: xylene for 20 min, xylene for 20 min, anhydrous ethanol for 2 min, anhydrous ethanol for 2 min, 95% ethanol for 2 min, 90% ethanol for 2 min, and 80% ethanol for 2 min. Staining was performed sequentially: hematoxylin for 7 min, rinsed with tap water, differentiated with 1% hydrochloric acid ethanol for 1 s, rinsed with tap water, eosin staining for 25-30 s, and rinsed with tap water for 5 min. Dehydration and clearing were performed sequentially: 80% ethanol for 7 s, 90% ethanol for 7 s, 95% ethanol for 7 s, anhydrous ethanol for 7 s, anhydrous ethanol for 7 s, xylene for 2 min, and xylene for 2 min.

[0088] The experimental results are as follows Figure 3 As shown in Figure C, the renal tubules in the surgical group underwent dilation, and renal tubular epithelial cells sloughed off. These changes were inhibited after drug administration. These results indicate that peptide MP1 can alleviate kidney damage induced by UUO.

[0089] 4. Effects of MP1 on fibrosis and EMT marker expression at the animal level

[0090] Animal-level detection of tissue fibrosis markers includes Masson staining of kidney pathological sections to detect total collagen deposition, and immunochemical staining of pathological sections to detect the deposition of markers α-SMA, FN, and Vim; Western blotting to detect the expression of markers α-SMA, FN, COL, and MMP2; and qRT-PCR to detect the expression of fibrosis genes Acta2, Fn1, COL1A1, Mmp2, and EMT genes Vim and Snai1.

[0091] 4.1 Masson staining and immunohistochemical staining of pathological sections. The dewaxing and dehydration procedures for pathological sections are the same as those in H&E. Masson staining: Weigert iron hematoxylin staining solution.

[0092] Stain for 5-10 minutes; after aspirating the staining solution, differentiate with acidic ethanol differentiation solution for 5-15 seconds, rinse in tap water, and aspirate excess water; re-blue with Masson's blue solution for 3-5 minutes, rinse with distilled water for 1 minute, and aspirate excess water; stain with Ponceau S and Fuchsia solution for 5-10 minutes, and aspirate excess staining solution; rinse with phosphomolybdic acid solution for 1-2 minutes, and aspirate excess solution; rinse with weak acid working solution for 1 minute, and place in distilled water; shake off excess water from the slide, and dehydrate; air dry the slide, and mount with neutral resin. Immunochemistry: After dewaxing, the slides were boiled in citrate buffer for 2 min to repair the antigen, and then cooled naturally to room temperature. Excess water was aspirated, and endogenous peroxidase inhibitor was added and incubated at room temperature for 10 min. Blocking normal goat serum working solution was added and incubated at room temperature for 15 min. The slides were placed in a humidified chamber, primary antibody was added, and incubated overnight at 4°C. The humidified chamber was removed, and the slides were placed at room temperature for 30 min to allow them to warm up and the primary antibody was recovered. Biotin-labeled goat anti-rabbit IgG was added and incubated at room temperature for 15 min. Horseradish enzyme-labeled streptavidin working solution was added and incubated at room temperature for 15 min. Freshly prepared DAB chromogenic solution was added, and the slides were observed under a microscope until obvious brown deposits appeared. The slides were then placed in distilled water to stop staining. Excess water was removed, hematoxylin was added, and the slides were incubated at room temperature for 3 min. After the incubation, the slides were placed in distilled water to stop staining. The slides were washed with tap water for 2 min, removed, and dehydrated. The slides were air-dried and mounted with neutral resin.

[0093] The experimental results are as follows Figure 3 As shown in DG, compared with the Sham group, the deposition of total collagen fibers, α-SMA, FN, and Vim was significantly increased in the UUO group; when MP1 and Captopril were administered, the deposition of collagen fibers and the above markers was significantly reduced.

[0094] 4.2 Western blot analysis of the expression of tissue fibrosis markers

[0095] Tissue protein extraction: Weigh approximately 15 mg of mouse kidney tissue, add 100 μL of RIPA lysis buffer containing 1% PMSF, add one grinding steel ball (one of each size), grind at 60 Hz for 120 s, remove the grinding steel ball, allow to stand at low temperature to lyse the tissue protein for 30 min, centrifuge at 12000 rpm for 30 min, and repeat twice. Collect the supernatant to obtain the total tissue protein.

[0096] Western blotting of proteins: The procedure is the same as in cell experiments 3.3.

[0097] like Figure 4 As shown, where, Figure 4 AB represents the protein immunoblot bands. Figure 4CH represents the bar chart showing the grayscale statistical intensity of the bands. The figure shows that MP1 significantly downregulates the abnormal expression of UUO-induced renal fibrosis and EMT-related proteins. This indicates that the peptide MP1 possesses in vivo anti-renal fibrosis activity at the protein level.

[0098] 4.3 qRT-PCR was used to detect the expression of fibrosis genes.

[0099] Tissue RNA extraction: Weigh approximately 15 mg of kidney tissue, add 1000 μL of RNA extraction buffer, add one grinding steel ball (one of each size), grind at 60 Hz for 120 seconds, let stand at room temperature for 10 minutes, and then...

[0100] Transfer the lysate to a new enzyme-free EP tube, and proceed as in 2.6. Reverse transcription: Same as cell experiment 4.2 in Example 3. qPCR: Same as cell experiment 4.3 in Example 3.

[0101] The experimental results are as follows Figure 4 As shown in IN, MP1 significantly inhibited fibrosis induced by UUO and abnormal expression of the EMT gene, indicating that the peptide MP1 also inhibits renal fibrosis induced by UUO at the gene level.

[0102] Example 5: Mechanism of action of peptide MP1 against renal fibrosis

[0103] Cells: mouse embryonic fibroblasts (NIH-3T3), human renal tubular epithelial cells (HK2);

[0104] Culture media: DMEM medium containing 10% FBS, DME / F12 medium containing 10% FBS;

[0105] Culture conditions: 37℃, 5% CO2 incubator.

[0106] Kidney tissue from C57BL / 6 mice in the UUO model.

[0107] Studies on the mechanism of action of MP1 include the effects of MP1 on the WNT / β-Catenin and TGF-β / Smad pathways and the effects of MP1 on autophagy activity.

[0108] 1. Effects of MP1 on the WNT / β-Catenin and TGF-β / Smad pathways

[0109] The effects of MP1 on the expression of proteins related to the WNT / β-Catenin and TGF-β / Smad pathways were detected by immunochemical staining of pathological sections, Western blotting, and qRT-PCR. In the extraction of phosphorylation-related proteins for the Western blotting experiment, total cellular protein was extracted after cells were incubated with MP1 for 1 hour; other experimental procedures were the same as those in the cell and animal experiments. The immunochemical staining of pathological sections and the qRT-PCR experiments were performed in the same manner as those in the cell and animal experiments.

[0110] Figure 5 The study investigated the effects of MP1 on the expression of WNT / β-Catenin and TGF-β / Smad pathway-related proteins in NIH-3T3 cells induced by TGF-β1. Figure 5 A is a Western blot image of protein bands. Figure 5 BE is a bar chart representing a strip graph. Figure 5 FH is a bar graph showing the changes in the expression of related genes. As shown in the graph, MP1 significantly inhibited the abnormal expression of WNT / β-Catenin pathway-related proteins β-Catenin, C-Myc, Cyclin D1, and TGF-β / Smad pathway-related protein phosphorylated Smad protein in NIH-3T3 cells induced by TGF-β1. Simultaneously, MP1 significantly inhibited the abnormal expression of WNT / β-Catenin pathway-related genes Ctnnb1, Myc, and Ccnd1. This indicates that the peptide MP1 jointly regulates the differentiation of fibroblasts into myofibroblasts by inhibiting the activity of both the TGF-β / Smad and WNT / β-Catenin pathways.

[0111] Figure 6 The effect of the peptide MP1 on the expression of WNT / β-Catenin and TGF-β / Smad pathway-related protein genes induced by TGF-β1 in HK2 cells. Figure 6 A is a Western blot image of protein bands. Figure 6 BE is a bar chart representing a strip graph. Figure 6 FH is a bar graph showing the changes in the expression of related genes. As shown in the figure, MP1 significantly inhibited the abnormal expression of WNT / β-Catenin pathway-related proteins β-Catenin, C-Myc, Cyclin D1, and TGF-β / Smad pathway-related protein phosphorylation of Smad protein, which are induced by TGF-β1 in HK2 cells. Simultaneously, MP1 significantly inhibited the abnormal expression of WNT / β-Catenin pathway-related genes CTNNB1, MYC, and CCND1. This indicates that MP1 jointly regulates the EMT process by inhibiting the activity of both the TGF-β / Smad and WNT / β-Catenin pathways.

[0112] Figure 7 This study investigated the effect of peptide MP1 on the expression of WNT / β-Catenin and TGF-β / Smad pathway-related protein genes in the kidney tissue of mice with UUO-induced renal fibrosis. Figure 7 A is a Western blot image of protein bands. Figure 7 BF is a bar chart of a bar graph. Figure 7 GJ is a bar chart showing changes in the expression of related genes. Figure 7 KL represents the immunochemical staining pattern. As shown in the figure, compared to the Sham group, the UUO group exhibited significantly increased expression levels of WNT / β-Catenin pathway-related proteins β-Catenin, C-Myc, Cyclin D1, and TGF-β / Smad pathway-related proteins TGF-β1 and phosphorylated Smad protein. Upon administration of MP1, the expression levels of these proteins significantly decreased. The results of qRT-PCR and immunohistochemical experiments were consistent with the Western blot results. This indicates that the peptide MP1 regulates the progression of renal fibrosis in UUO mice by inhibiting the activity of both the TGF-β / Smad and WNT / β-Catenin pathways.

[0113] 2. Effect of peptide MP1 on TGF-β1-induced autophagy in NIH-3T3 cells

[0114] The effect of MP1 on the expression of TGF-β1-induced autophagy-related proteins in NIH-3T3 cells was detected by Western blotting. The Western blotting procedure was performed in the same manner as in cell and animal experiments.

[0115] like Figure 8 As shown, where Figure 8 A is a Western blot image of protein bands. Figure 8 BC represents the bar chart showing the grayscale statistical values ​​of the bands. The figure shows that peptide MP1 has no effect on the expression levels of autophagy-related proteins LC3II / LC3I and the autophagy substrate P62. This indicates that peptide MP1 does not induce autophagy in NIH-3T3 cells.

[0116] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of the autophagy key regulatory protein Beclin-1 fragment polypeptide MP1 in the preparation of anti-renal fibrosis drugs, characterized in that, The amino acid sequence of the polypeptide MP1 is Leu-Ser-Arg-Arg-Met-Lys-Val-Thr-Gly-Asp-Leu-Trp, and the renal fibrosis is renal interstitial fibrosis.

2. The application according to claim 1, characterized in that, The renal interstitial fibrosis mentioned is renal interstitial fibrosis caused by ureteral obstruction.

Images