Use of polypeptide fk2 in the preparation of drugs for resisting renal fibrosis

Abstract

The application discloses a use of a natural polypeptide FK2 in preparation of a drug for resisting kidney fibrosis. The application researches and finds that the polypeptide FK2 shows a significant effect of resisting kidney fibrosis in both in-vivo and in-vitro tests, has no obvious toxic side effect, and the mechanism of action is mainly that the polypeptide FK2 inhibits an IKKbeta / NF-kappaB signal path, relieves inflammation reaction and an EMT process caused by injury, and then inhibits fibrosis, so that the polypeptide FK2 has a good clinical application value in preparation of the drug for resisting kidney fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the use of a polypeptide FK2, specifically the application of polypeptide FK2 in the prevention or treatment of renal fibrosis. Background Technology

[0002] Studies have found that fibrosis can affect all organs and is associated with 45% of disease-related deaths. Renal fibrosis is the final pathological feature of chronic kidney disease (CKD), characterized by massive accumulation and cross-linking of the extracellular matrix (ECM), glomerular sclerosis, tubular atrophy, and interstitial fibrosis, ultimately leading to kidney failure. Renal fibrosis is difficult to detect, and there are currently no effective treatments. Existing treatment options are mostly limited to dialysis and kidney transplantation, severely impacting patients' quality of life and imposing a huge burden on society and families.

[0003] There are many causes of CKD, including diabetic nephropathy, hypertension, glomerulonephritis, and acute kidney injury. Early treatment of fibrosis mainly targets different pathological factors. For example, the main strategy for renal fibrosis caused by diabetic nephropathy is to control blood sugar levels; for renal fibrosis caused by hypertensive nephropathy, the main focus is on blood pressure regulation; antioxidants, as one of the treatment strategies, can regulate oxidative stress and its involvement in the AKI and AKI-CKD conversion process.

[0004] Inflammation is also a key pathological feature of CKD. When the kidneys suffer persistent or excessive damage, the infiltration of immune cells such as monocytes, neutrophils, and macrophages into the damaged site, along with persistent inflammatory responses, can induce and exacerbate fibrosis. Inflammatory responses accompany diabetic nephropathy, oxidative stress, hypertension, and fibrosis; therefore, regulating inflammatory responses can influence the development and progression of renal fibrosis. The NF-κB signaling pathway is the most important inflammatory signaling pathway. In animal models of unilateral ureteral obstruction (UUO) and ischemia-reperfusion (I / R) kidney injury, NF-κB is activated. In damaged epithelial cells, the NF-κB signaling pathway is activated, directly leading to fibroblast activation. By regulating targeted inflammatory factors, connective molecules, and pro-inflammatory kinases, it directly promotes fibrosis in the liver, brain, and kidneys. In addition, the NF-κB signaling pathway also regulates the epithelial-mesenchymal transition (EMT) process. By inhibiting the ubiquitination and degradation of the EMT inducer Snail1, it promotes the expression of mesenchymal factors, thereby promoting EMT in epithelial cells that have undergone inflammatory responses.

[0005] Furthermore, in the later stages of CKD, the kidneys secrete and synthesize large amounts of pro-fibrotic growth factors, such as TGF-β, which promotes ECM synthesis. Therefore, targeting TGF-β and its signaling pathway can effectively inhibit fibrosis. However, TGF-β also plays an important role in the body's normal physiological regulation, and inhibiting TGF-β may trigger severe autoimmune diseases.

[0006] Therefore, the progression from kidney injury to CKD and the development of renal fibrosis is usually caused by a combination of factors, and treatment strategies targeting only specific factors are not very effective. Although significant progress has been made in research on CKD and renal fibrosis in recent years, the number of approved treatments for renal fibrosis remains very limited. Summary of the Invention

[0007] This invention has discovered through cellular and animal experiments that peptide FK2 can effectively inhibit renal fibrosis. Based on this, this invention proposes applications of peptide FK2.

[0008] This invention relates to the application of peptide FK2, according to specific embodiments of the present invention, in the preparation of anti-renal fibrosis drugs. The amino acid sequence of peptide FK2 is Thr-Met-Lys-Leu-Leu-Leu-Val-Thr-Leu. Peptide FK2 is a product obtained from corn cob via trypsin hydrolysis and possesses anti-inflammatory activity. Studies have shown that its anti-inflammatory activity is mainly achieved through the IKKβ / NF-κB signaling pathway. Simultaneously, FK2 can inhibit LPS-induced NF-κB signaling pathway activation in the kidney. This invention experimentally demonstrates that peptide FK2 possesses anti-renal fibrosis activity.

[0009] According to the application of FK2 in a specific embodiment of the present invention, the renal fibrosis includes renal interstitial fibrosis, including renal interstitial fibrosis caused by ureteral obstruction.

[0010] Another object of the present invention is to provide an agent for treating renal fibrosis, the agent having a polypeptide FK2 as an active ingredient, the agent being a drug or a drug composition.

[0011] 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.

[0012] 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.

[0013] The beneficial effects of this invention are:

[0014] This invention demonstrates through experiments that peptide FK2 has no significant toxic side effects in mouse animal experiments. In both TGF-β-induced HK2 and NIH-3T3 in vitro models, peptide FK2 of this invention inhibits the expression of fibrosis markers fibronectin, type I collagen, α-smooth muscle actin (α-SMA), and matrix metalloproteinase 2 (MMP2) at the gene and protein levels. In the TGF-β-induced HK2 cell fibrosis model, it inhibits the expression of the EMT marker Vimentin. In LPS-induced RAW264.7 cells, it inhibits the expression of inflammatory factors and genes such as IL1β, IL6, TNF-α, and MCP-1. In a unilateral ureteral ligation (UUO)-induced renal fibrosis mouse model, peptide FK2 inhibits the expression of fibrosis markers fibronectin, Collagen I, α-SMA, MMP2, and Vimentin at the gene and protein levels, thus inhibiting... Ccl2, IL1b, IL6, Tnf The expression of inflammatory factor genes is inhibited. Mechanism of action studies show that the peptide FK2 of this invention mainly alleviates the inflammatory response and EMT process caused by kidney injury and inhibits kidney fibrosis by inhibiting IKKβ / NF-κB signaling transduction. Attached Figure Description

[0015] 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.

[0016] Figure 1 This shows the anti-fibrotic activity of peptide compounds screened using the NIH-3T3 cell fibrosis model.

[0017] Figure 2 This study demonstrates the anti-renal fibrosis activity of PAY, FK2, and P13L in a TGF-β1-induced mTEC cell fibrosis model.

[0018] Figure 3 A schematic diagram illustrating the toxicity of FK2 to NIH-3T3 and HK2 cells;

[0019] Figure 4 Schematic diagram of the inhibitory effect of FK2 on TGF-β-induced HK2 cell fibrosis model

[0020] Figure 5 A schematic diagram illustrating the inhibitory effect of FK2 on a TGF-β-induced NIH-3T3 cell fibrosis model;

[0021] Figure 6 This is a schematic diagram illustrating the effect of FK2 on fibrosis marker genes in an HK2 cell fibrosis model.

[0022] Figure 7 A schematic diagram illustrating the effect of FK2 on fibrosis genes in the NIH-3T3 cell fibrosis model.

[0023] Figure 8 This is a schematic diagram illustrating the effect of FK2 on renal function indicators in mice with UUO-induced renal fibrosis.

[0024] Figure 9 This is a schematic diagram illustrating the effect of FK2 on the morphology of kidney tissue in mice with UUO-induced renal fibrosis.

[0025] Figure 10 This is a schematic diagram illustrating the effect of FK2 on the deposition of UUO-induced renal tissue fibrosis markers.

[0026] Figure 11 This is a schematic diagram illustrating the expression of FK2-induced renal fibrosis marker proteins by UUO.

[0027] Figure 12 This is a schematic diagram illustrating the expression of marker genes for renal fibrosis induced by FK2 and UUO.

[0028] Figure 13 This is a schematic diagram illustrating the effect of FK2 on the EMT process;

[0029] Figure 14 A schematic diagram illustrating the effect of FK2 on the inflammatory response in renal fibrosis;

[0030] Figure 15 This is a schematic diagram illustrating the binding of FK2 and IKKβ.

[0031] Figure 16 This is a schematic diagram illustrating the effect of FK2 on the NF-κB signaling pathway. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0033] Example 1: Screening for polypeptide compounds with anti-renal fibrosis activity

[0034] This invention proposes to use interference with the NF-κB signaling pathway as a strategy for the treatment of renal fibrosis. First, among many peptide compounds that have a regulatory effect on the NF-κB signaling pathway, peptide compounds such as M3, GPETAFLR, MLIF, SQ9, IL15, AIP6, PAY, FK2, and P13L were specifically selected.

[0035] Table 1. Amino acid sequences of polypeptide compounds

[0036]

[0037] First, the antifibrotic activity of peptide compounds such as M3, GPETAFLR, MLIF, SQ9, IL15, AIP6, PAY, FK2, and P13L, which intervene in the NF-κB signaling pathway, was preliminarily screened. An in vitro cell fibrosis model was constructed by inducing mouse embryonic fibroblasts (NIH-3T3) with TGF-β. The above-mentioned peptide molecules were administered at a dose of 50 μM. After 24 h of treatment, the total protein of the cells was extracted, and the expression of fibrosis markers fibronectin and α-smooth muscle actin (α-SMA) was detected by Western blotting.

[0038] The results are as follows Figure 1 As shown, after TGF-β induction, the expression of the markers Fibronectin and α-SMA was significantly upregulated. After peptide intervention, peptides FK2, PAY, and P13L significantly inhibited the expression of the above markers. Therefore, it was preliminarily determined that peptides FK2, PAY, and P13L have anti-fibrotic activity.

[0039] Based on this, the cell line was changed to mouse renal tubular epithelial cells (mTEC) to further screen for the anti-renal fibrosis activity of peptides FK2, PAY, and P13L. The experimental procedures were the same as those used in the NIH-3T3 cell screening process.

[0040] The results are as follows Figure 2 As shown, after changing the cell line, peptides PAY and P13L no longer inhibited the expression of Fibronectin, while only peptide FK2 consistently showed stable anti-renal fibrosis activity.

[0041] Example 2: Synthesis of polypeptide FK2

[0042] The peptide FK2 was synthesized using the Fmoc solid-phase synthesis method, purified by reversed-phase HPLC, and characterized by mass spectrometry.

[0043] The sequence of polypeptide FK2 is as follows: Thr-Met-Lys-Leu-Leu-Leu-Val-Thr-Leu. The specific synthesis steps are as follows:

[0044] (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.

[0045] (2) The first amino acid condensation reaction at the C-terminus: Weigh three times the amount of Leu in excess, dissolve it in DCM and add it to the syringe. Shake at low speed for 5 min and then add DIEA to start the reaction. Shake for 1 h and then dry the resin. Wash the resin with DMF 3 times.

[0046] (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 min. 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.

[0047] (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 end, wash with DMF 3 times and then check with indole to determine whether the reaction is complete.

[0048] (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 h, then dry the resin and clean with DMF.

[0049] (6) Peptide chain elongation: Repeat steps (3), (4), and (5) until the amino acid sequence Thr-Met-Lys-Leu-Leu-Leu-Val-Thr-Leu is fully conjugated.

[0050] (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.

[0051] (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℃, place it in a freeze dryer to obtain crude peptide.

[0052] (9) HPLC purification: Weigh approximately 50 mg of crude peptide, dissolve it, and filter it using a 0.45 μm filter. Use a C18 preparative column for separation and purification. The mobile phase consists 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. Purity was analyzed by HPLC, and the product was characterized by mass spectrometry. Products meeting the purity requirements (>95%) were freeze-dried to obtain pure peptide FK2.

[0053] Example 3: Anti-renal fibrosis effect of peptide FK2 at the cellular level

[0054] Cells: mouse embryonic fibroblasts (NIH-3T3), human renal tubular epithelial cells (HK2);

[0055] Culture media: DMEM medium containing 10% FBS, DME / F12 medium containing 10% FBS;

[0056] Culture conditions: 37℃, 5% CO2 incubator.

[0057] 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, and transfer to a centrifuge tube. Centrifuge at 800 rpm for 5 min, discard the supernatant, add an appropriate amount of culture medium to resuspend the cells into a single-cell suspension, and add an appropriate amount of cells to a culture dish for cell passage.

[0058] 2. CCK8 assay for the cytotoxicity of FK2 against NIH-3T3 and HK2 cells:

[0059] 7×10 3 Cell suspensions were prepared and mixed, then added to 96-well plates at 100 μL / well. After culturing for 24 h, FK2 at concentration gradients of 12.5, 25, 50, 100, and 200 μM were added and cultured for another 24 h. CCK8 was then added and incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader.

[0060] Experimental results are as follows Figure 3As shown in the figure, A represents the survival rate of NIH-3T3 cells, and B represents the survival rate of HK2 cells. The figure indicates that within the concentration range of 0-200 μM, FK2 did not significantly inhibit the survival rate of either NIH-3T3 or HK2 cells.

[0061] 3. Western blot analysis of the effect of FK2 on the expression of TGF-β-induced fibrosis markers in HK2 and NIH-3T3 cells.

[0062] The fibrosis markers detected in this experiment included Fibronectin, Collagen I, α-SMA, and MMP2.

[0063] 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, 25 μM and 50 μM, were used to induce a cell fibrosis model with TGF-β, while simultaneously adding FK2. The experimental groups were: control group, TGF-β group, TGF-β + 25 μM FK2 group, and TGF-β + 50 μM FK2 group.

[0064] 3.2 Total protein extraction from cells: After incubating cells with FK2 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.

[0065] 3.3 Western Blotting: Protein concentration was detected using a BCA kit. 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 by 10% SDS-PAGE gel electrophoresis. The stacking gel was run at 80 V for 30 min, and the separating gel at 120 V for 1.5 h. After electrophoresis, the target protein was transferred to a PVDF membrane at 250 mA for 3 h. After transfer, the membrane was blocked with 5% skim milk powder for 1.5 h, washed with TBST for 10 min × 3 times, and 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 chemiluminescence was added for color development.

[0066] Experimental results Figure 4The effect of FK2 on the expression of fibrosis markers Fibronectin, Collagen I, MMP2, and α-SMA in TGF-β-induced HK2 cells was investigated. Figure 4 Image A shows the protein immunoblot bands. Figure 4 The BE (bar graph) is a statistical representation of the band structure. As shown in the graph, when the drug dose reaches 25 μM, FK2 reduces the expression of fibrosis markers, with a significant inhibitory effect on the expression of Fibronectin protein; when the drug dose increases to 50 μM, FK2 significantly inhibits all of the above indicators. This indicates that FK2 has a significant inhibitory effect at the protein level on the TGF-β-induced HK2 cell renal fibrosis model.

[0067] Experimental results Figure 5 The study investigated the effects of FK2 on the expression of fibrosis markers Fibronectin, Collagen I, MMP2, and α-SMA in TGF-β-induced NIH-3T3 cells. Figure 5 A is a Western blot image of protein bands. Figure 5 The BE (bar graph) is a statistical representation of the band structure. The graph shows that when the drug dose reached 25 μM, FK2 reduced the expression of fibrosis markers; when the drug dose increased to 50 μM, FK2 significantly inhibited all of these markers. This indicates that FK2 has a significant inhibitory effect at the protein level on the TGF-β-induced NIH-3T3 cell renal fibrosis model.

[0068] 4. qRT-PCR detection of the effect of FK2 on TGF-β-induced fibrosis marker genes in HK2 and NIH-3T3 cells.

[0069] 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 forms on the sides and bottom of the tube, add 500 μL of 75% ethanol, gently shake to detach the precipitate 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.

[0070] 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℃.

[0071] Table 2 Reverse Transcription System Configuration Table

[0072]

[0073] x = 1000 ng / (RNA concentration)

[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 (2T) based on gene Ct values. -ΔΔCt ).

[0078] Experimental results Figure 6 FK2-induced fibrosis-related genes in HK2 cells induced by TGF-β. Fn1 , COL1A1, Mmp2 Acta2 The effect of FK2 on the expression of these genes was investigated. As shown in the figure, at a dose of 25 μM, FK2 significantly inhibited the abnormal expression of these genes induced by TGF-β, and the inhibitory effect was enhanced when the dose was increased to 50 μM. This indicates that FK2 significantly inhibits TGF-β-induced kidney fibrosis in HK2 cells at the gene level.

[0079] Experimental results Figure 7 FK2-induced fibrosis-related genes in TGF-β-induced NIH-3T3 cells Fn1, COL1A1, Mmp2, Acta2 The effect of FK2 on the expression of these genes was investigated. As shown in the figure, at a dose of 25 μM, FK2 significantly inhibited the abnormal expression of these genes induced by TGF-β, and the inhibitory effect was enhanced when the dose was increased to 50 μM. This indicates that FK2 significantly inhibits TGF-β-induced renal fibrosis in NIH-3T3 cells at the gene level.

[0080] Example 4: Anti-renal fibrosis effect of peptide FK2 in mice

[0081] Experimental animals: Male C57BL / 6 mice, 6-8 weeks old, purchased from Lanzhou Veterinary Research Institute, 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 operation group, surgical group (UUO), 0.5 mg / kg FK2 group, and 20 mg / kg Captopril group, with 15 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. Detection of renal function indicators: Blood was collected from the orbital cavity of mice after anesthesia and collected in 1.5 mL EP tubes. The tubes were centrifuged at 5000 rpm for 15 min, and the centrifugation was repeated twice. The supernatant was collected and the serum creatinine (Scr) and serum urea nitrogen (BUN) levels were measured according to the kit instructions.

[0085] Experimental results are as follows Figure 8 As shown, FK2 significantly inhibited the abnormal increase in BUN and Scr levels caused by renal fibrosis. Among them, FK2 was more effective than Captopril in inhibiting Scr.

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

[0087] Left kidney tissue was harvested from mice and photographed to observe its morphology. 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. Some histopathological sections were subjected to H&E staining. The sections were then dried at 65℃ for 1 h 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, xylene for 2 min.

[0088] Experimental results are as follows Figure 9 As shown. Figure 9 A shows the effect of FK2 on the appearance of the kidneys. As can be seen from the figure, after UUO surgery, the kidneys atrophied and turned significantly yellow. After administration of 0.5 mg / kg of FK2, the kidneys became redder in color compared to the model group, and the appearance of the kidneys improved. Figure 9 B shows the H&E staining results of kidney tissue sections. As can be seen from the figure, in the model group, the renal tubules dilated and the renal tubular epithelial cells sloughed off. After drug administration, these changes were inhibited. These results indicate that FK2 can alleviate kidney damage induced by UUO.

[0089] 4. Detection of the effect of FK2 on the expression of fibrosis markers 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 Fibronectin and α-SMA; Western blotting to detect the expression of markers Fibronectin, Collagen I, MMP2, and α-SMA; and qRT-PCR to detect fibrosis genes. Fn1, COL1A1, Mmp2, Acta2 The expression of .

[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 min; after aspirating the staining solution, differentiate with acidic ethanol differentiation solution for 5-15 s, rinse in tap water, and aspirate excess water; re-blue with Masson's blue solution for 3-5 min, rinse with distilled water for 1 min, and aspirate excess water; stain with Ponceau S and Fuchsia solution for 5-10 min, and aspirate excess staining solution; rinse with phosphomolybdic acid solution for 1-2 min, and aspirate excess solution; rinse with weak acid working solution for 1 min, and place in distilled water; shake off excess water from the slide, 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 allowed to cool naturally to room temperature. Excess water was blotted out, 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, and primary antibody was added and incubated overnight at 4°C. The humidified chamber was removed and placed at room temperature for 30 min to allow for rewarming, 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 observed under a microscope until obvious brown deposition appeared. The slides were then placed in distilled water to stop staining. Excess water was removed, and hematoxylin was added and incubated at room temperature for 3 min. After incubation, the slides were placed in distilled water to stop staining. The slides were washed with tap water for 2 min, blotted out excess water, and dehydrated. The slides were air-dried and mounted with neutral resin.

[0093] Experimental results are as follows Figure 10 As shown, total collagen fibers, Fibronectin, and α-SMA were mainly deposited in the renal tubular interstitial region. Compared with the Sham group, the deposition of total collagen fibers, Fibronectin, and α-SMA was significantly increased in the UUO group. When FK2 and Captopril were administered, the deposition of collagen fibers and the above markers was significantly reduced, and FK2 was slightly more effective than Captopril.

[0094] 4.2 Detection of tissue fibrosis markers by Western blotting

[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] Experimental results are as follows Figure 11 As shown. Among them Figure 11A is a Western blot image of protein bands. Figure 11 BE is a bar chart representing the grayscale statistical values ​​of the bands. The figure shows that FK2 downregulates the abnormal expression of renal fibrosis-related proteins induced by UUO. Specifically, FK2 significantly outperforms the positive control drug Captopril in inhibiting Collagen I, MMP2, and α-SMA. This indicates that FK2 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 of each size, grind at 60 Hz for 120 s, let stand at room temperature for 10 min, 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] Experimental results are as follows Figure 12 As shown, FK2 significantly inhibited the abnormal expression of fibrosis genes induced by UUO, among which, for COL1A1, Mmp2 The inhibitory effect of FK2 was superior to that of Captopril, indicating that FK2 also inhibits renal fibrosis induced by UUO at the gene level.

[0102] Example 5: Mechanism of action of peptide FK2 against renal fibrosis

[0103] Cells: human renal tubular epithelial cells (HK2), mouse mononuclear macrophages (RAW264.7).

[0104] Culture media: DME / F12 medium containing 10% FBS, DMEM 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 FK2 include its effects on EMT and inflammatory responses, as well as its regulation of the NF-κB signaling pathway.

[0108] 1. The effect of FK2 on the EMT process

[0109] The effect of FK2 on the EMT process was detected by immunochemical staining of pathological sections, Western blotting, and qRT-PCR. The proteins detected were EMT markers E-cadherin, vimentin, and TGF-β, and the marker genes included... Snail1, vim, Tgfb1 The experimental procedures are the same as those in cell and animal experiments.

[0110] Experimental results are as follows Figure 13 As shown, where Figure 13 AC represents the staining of kidney tissue sections containing EMT markers Vimentin and TGF-β, along with a statistical diagram of the positive area. As shown in the figure, FK2 significantly inhibited the deposition of Vimentin and TGF-β in kidney tissue. Figure 13 Figure DG shows the banding and statistical graphs of EMT markers E-Cadherin, TGF-β, and Vimentin protein expression in kidney tissue. Figure HJ shows the banding and statistical graphs of EMT markers E-Cadherin and Vimentin protein expression in HK2 cells. As can be seen from the figures, FK2 significantly inhibited the expression of mesenchymal cell markers at both the tissue and cellular levels, but FK2 had no significant effect on the expression of the epithelial cell marker E-Cadherin. Figure KP shows the effect of FK2 on EMT marker genes in kidney tissue and HK2 cells. Snail1, vim, Tgfb1 As shown in the figure, FK2 significantly inhibits the expression of EMT-related genes at the gene level. This indicates that FK2 can inhibit the continuous progression of EMT during renal fibrosis both in vivo and in vitro, but it cannot restore the epithelial phenotype of cells that have already undergone phenotypic transformation.

[0111] 2. The effect of FK2 on the inflammatory response

[0112] The effect of FK2 on the inflammatory response was detected using immunochemical staining of pathological sections, ELISA, and qRT-PCR. Proteins detected included inflammatory factors such as CD68, IL-1β, TNF-α, and IL-6, and marker genes included... Ccl2, IL1b, IL6 Tnf The procedures for tissue immunochemical staining and qRT-PCR are the same as those for cell and animal experiments.

[0113] The ELISA assay was used to detect the expression of inflammatory factors in mouse serum, and the procedure was performed according to the kit instructions (UE Biosciences, M6152, M6149). For the effect of FK2 on in vitro inflammatory responses, the LPS (1 μg / mL)-induced RAW264.7 cell inflammation model was selected. Before detecting the effect of FK2 on the inflammatory response, the toxic effect of FK2 on normal RAW264.7 cells was first detected by the CCK8 assay, with the experimental procedure being the same as in the cell experiments. The experimental groups for the inflammatory response detection were: control group, LPS group, LPS + 25 μM FK2 group, and LPS + 50 μM FK2 group.

[0114] Experimental results are as follows Figure 14 As shown. Among them Figure 14 AD is a tissue immunochemical staining detection method.

[0115] The images show the deposition of inflammatory markers such as CD68, IL-1β, and TNF-α in kidney tissue sections using FK2, along with a statistical diagram of positive areas. The figures indicate that FK2 administration reduced the expression of inflammatory factors in kidney tissue. Figure EH shows the qPCR detection of these markers in kidney tissue. Ccl2, IL1b, IL6, Tnf The expression of inflammatory factor genes is shown in the figure. As can be seen from the figure, FK2 can significantly downregulate the level of inflammatory factor genes in tissues. Figure 14 IJ shows the effect of FK2 on the expression of inflammatory factors IL6 and TNF-α in serum using ELISA. As can be seen from the figure, the above-mentioned inflammatory factors were abnormally increased in UUO tissue. After FK2 administration, the expression of inflammatory factors was significantly downregulated. Figure 14 K represents the toxicity test results of FK2 on RAW264.7 cells. As shown in the figure, FK2 has no toxic side effects on the cells within the concentration range of 0-50 μM. Figure 14 LP was used to detect inflammatory factor genes in a LPS-induced RAW264.7 cell inflammation model using qPCR. Ccl2, IL1b, IL6, Tnf and fibrosis-related genes tgfb1 As shown in the figure, the above-mentioned genes were significantly upregulated in the model group. FK2 reduced the abnormal expression of the above-mentioned genes, indicating that FK2 inhibited the inflammatory response that occurs in the process of renal fibrosis at both in vivo and in vitro levels.

[0116] 3. Regulatory role of FK2 in the NF-κB signaling pathway

[0117] The binding of FK2 to IKKβ was detected by immunofluorescence assay; the effect of FK2 on the expression of proteins related to the NF-κB signaling pathway was detected by Western blotting.

[0118] 3.1 Immunofluorescence of cells: When the confluence of RAW264.7 cells reached 80-90%, trypsin-digested cells were seeded into laser confocal dishes at a seeding density of 8 × 10⁶ cells / day. 4 / well. After culturing for 24 h, the complete culture medium was discarded, and the cells were starved for 10 h using serum-free culture medium. FITC-bound FK2 was added under dark conditions. The experimental groups were: blank control group and FITC-FK2 group. After 24 h of treatment, the culture medium was discarded, the cells were washed three times with PBS, and then fixed with 4% formaldehyde for 15 min; permeabilized with 0.5% Triton for 5 min; blocked with 5% BSA for 1 h; incubated with IKKβ (1:200) antibody at room temperature for 1 h; incubated with fluorescent secondary antibody at room temperature for 1 h; incubated with DAPI (10 μg / mL) for 5 min; washed three times with PBS, and then PBS was added to the dish. Fluorescence was observed under a laser scanning ultra-high resolution microscope. All the above operations were performed under dark conditions.

[0119] Experimental results Figure 15 To detect the binding of FK2 and IKKβ by cellular immunofluorescence, the figure shows that, compared with the control group, the fluorescence of FITC-labeled FK2 partially overlapped, indicating that FK2 can partially bind to the protein IKKβ.

[0120] The procedures for protein immunoblotting are the same as those for cellular and animal-level experiments.

[0121] Experimental results are as follows Figure 16 As shown in Figures AD, which illustrate the effect of FK2 on the NF-κB signaling pathway in UUO tissues, FK2 significantly inhibits the expression of the upstream protein IKKβ in the NF-κB signaling pathway and suppresses the phosphorylation of downstream proteins IκBα and P65. Similar results were obtained at the cellular level (Figure EH). This indicates that FK2 can affect the transduction of the NF-κB signaling pathway by inhibiting the expression of the upstream protein IKKβ, thereby inhibiting the phosphorylation of downstream proteins IκBα and P65.

[0122] 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. sequence list <110> Institute of Materia Medica, Chinese Academy of Medical Sciences Lanzhou University <120> Application of peptide FK2 in the preparation of anti-renal fibrosis drugs <141> 2022-05-30 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <400> 1 Thr Met Lys Leu Leu Leu Val Thr Leu 1 5

Claims

1. The application of polypeptide FK2 in the preparation of anti-renal fibrosis drugs, characterized in that, The amino acid sequence of the polypeptide FK2 is Thr-Met-Lys-Leu-Leu-Leu-Val-Thr-Leu.

2. The application according to claim 1, characterized in that, The renal fibrosis includes renal interstitial fibrosis.

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

4. The application according to claim 1, characterized in that, The drug is in the form of an injection.

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