CN116251167A - Application of autophagy key regulatory protein Beclin-1 fragment polypeptide MP1 in preparation of anti-renal fibrosis drugs - Google Patents

Abstract

The invention discloses application of polypeptide MP1 derived from autophagy key regulatory protein Beclin-1 fragment in preparation of medicines for resisting kidney fibrosis. According to the research of the invention, the polypeptide MP1 has obvious effect of resisting kidney fibrosis in vivo and in vitro, has no obvious toxic or side effect, and has an action mechanism of inhibiting an EMT process mainly through inhibiting WNT/beta-Catenin and TGF-beta/Smad signal paths, thereby inhibiting kidney fibrosis and not causing autophagy. The polypeptide MP1 has good clinical application value in preparing medicines for resisting kidney fibrosis.

Description

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

Technical Field

The invention belongs to the technical field of medicines, relates to application of a fragment polypeptide MP1 of autophagy key regulatory protein Beclin-1, and in particular relates to application of the polypeptide MP1 in preventing or treating kidney fibrosis.

Background

Fibrosis can affect all organs, affecting a global quarter of the population. In all organ fibrosis, kidney fibrosis is second only to liver fibrosis, with an annual incidence of about 1.21%. Renal fibrosis is a common pathway and pathological change in all Chronic Kidney Disease (CKD) that progresses to End Stage Renal Disease (ESRD) and is characterized by a massive accumulation of extracellular matrix (ECM) and loss of function of the nephron. The massive deposition of ECM can disrupt the essential structure of the kidney, leading to cellular dysfunction, substantial scarring, and eventually to end-stage renal disease, and to renal failure. The kidney fibrosis is not easy to detect, no treatment method capable of effectively delaying or reversing the fibrosis exists clinically at present, the existing scheme is limited to dialysis and kidney transplantation, the life quality of patients is seriously influenced, huge economic and social burdens are caused, and the development of effective treatment medicines is urgently needed.

More and more studies have shown that epithelial-mesenchymal transition (EMT) is an important driving factor in the progression of renal fibrosis. EMT is a transdifferentiation process during which epithelial cells lose cell polarity, phenotypic characteristics and intercellular adhesion properties, which in turn acquire partial mesenchymal cell properties. Many growth factors, cytokines and signaling pathways can induce the EMT process, with the WNT/β -Catenin signaling pathway playing an important role. In general, the WNT signaling pathway plays an important role in embryonic development and tumorigenesis. In the classical WNT/beta-Catenin signal pathway, WNT, frizzled and LRP5/6 form a complex on the cell surface, and after the complex is activated, the complex triggers the dissociation of the multifunctional kinase GSK-3 beta from the regulatory APC/Axin/GSK-3 beta complex, thereby improving the stability of beta-Catenin. And the stable beta-catenin is transferred into the nucleus through Rac1 and other factors, and is combined with a transcription factor LEF/TCF in the nucleus to replace an auxiliary inhibitor, so that the expression of the WNT target gene is regulated. Studies show that in a CKD model, the expression of cytokines such as beta-Catenin, C-Myc, cyclin D1 and the like related to a WNT signal pathway is obviously up-regulated, and the activation of the WNT pathway can induce the renal tubular epithelial cells to dedifferentiate and activate an EMT process, so that the fibrosis process is promoted.

Li Wang et al report that the expression level of Beclin-1 can be inhibited by shBECN1, so that the expression and transcriptional activity of the Lef1 gene can be improved, the expression levels of WNT/beta-Catenin signal channel inhibitor proteins DKK3 and sfrp can be reduced, the migration of beta-Catenin from cytoplasm to nucleus can be promoted, and the WNT channel can be activated. Upregulation of the expression level of Beclin-1, a key regulatory protein for inducing autophagy, continues to activate the autophagy response. Man J Livingston et al show that continuous activation of autophagy of the proximal tubule of the kidney promotes progression of interstitial fibrosis in a mouse unilateral ureteral obstruction (UFO) model.

Disclosure of Invention

According to the invention, experiments at cell level and animal level show that the polypeptide MP1 derived from the autophagy key regulatory protein Beclin-1 fragment can effectively inhibit kidney fibrosis, and the invention provides application of the polypeptide MP1.

The polypeptide MP1 is a polypeptide fragment derived from the 112-123 position of the BH3 domain of the Beclin-1 protein, the amino acid sequence of the polypeptide MP1 is Leu-Ser-Arg-Arg-Met-Lys-Val-Thr-Gly-Asp-Leu-Trp, only the activity of the polypeptide MP1 in inducing autophagy of tumor cells is reported at present, and the invention proves that the polypeptide MP1 has the anti-renal fibrosis activity through experiments.

According to the application of the polypeptide MP1 in the specific embodiment of the invention, the application of the polypeptide MP1 in preparing medicines for inhibiting the expression of kidney fibrosis related proteins, preferably, the kidney fibrosis related proteins include alpha-SMA, FN, COL and/or MMP2; preferably, the protein associated with inhibiting kidney fibrosis further comprises alpha-SMA, FN, COL, MMP2; preferably, the protein associated with inhibiting kidney fibrosis further comprises Snail1 and/or Vimentin.

According to the application of the polypeptide MP1 in the specific embodiment of the invention, the application of the polypeptide MP1 in preparing medicines for inhibiting the expression of kidney fibrosis related genes, preferably, the genes for inhibiting the kidney fibrosis related genes comprise Acta2, fn1, COL1A1 and/or Mmp2; preferably, inhibiting a gene associated with renal fibrosis further comprises SNAI1 and/or VIM.

According to the application of the polypeptide MP1 in the specific embodiment of the invention, the application of the polypeptide MP1 in preparing medicines for inhibiting the expression of TGF-beta/Smad pathway related genes, preferably, the TGF-beta/Smad pathway related genes comprise CTNNB1, MYC and/or CCND1.

According to the use of the polypeptide MP1 of the specific embodiment of the invention, the use of the polypeptide MP1 for the preparation of a medicament for inhibiting the expression of a TGF-beta/Smad pathway-related protein, preferably the expression of a TGF-beta/Smad pathway-related protein comprises a phosphorylated Smad protein.

According to the application of the polypeptide MP1 in the specific embodiment of the invention, the application of the polypeptide MP1 in preparing a medicament for inhibiting the expression of WNT/beta-Catenin pathway related genes, preferably, the WNT/beta-Catenin pathway related genes comprise Cnnb 1, myc and Ccnd1.

According to the application of the polypeptide MP1 in the specific embodiment of the invention, the application of the polypeptide MP1 in preparing a medicament for inhibiting the expression of WNT/beta-Catenin pathway related proteins, preferably, the WNT/beta-Catenin pathway related proteins comprise beta-Catenin, C-Myc and/or Cyclin D1.

The invention provides an application of autophagy key regulatory protein Beclin-1 fragment polypeptide MP1 in preparing medicines for resisting kidney fibrosis; preferably, the kidney fibrosis includes renal interstitial fibrosis including renal interstitial fibrosis caused by ureteral obstruction.

It is still another object of the present invention to provide a formulation for treating renal fibrosis, the formulation having the polypeptide MP1 as an active ingredient, the formulation being a medicament or a pharmaceutical composition.

The preparation for treating kidney fibrosis according to the specific embodiment of the invention is one of a tablet, a capsule, a granule, an oral liquid, a syrup, a dripping pill, an ointment and a patch for skin surface, an aerosol, a nasal spray, a suppository, a microsphere preparation, an injection preparation or a freeze-dried powder injection.

Pharmaceutical excipients, diluents or carriers may be included in the above formulations and may be used in any suitable manner in a patient in need of treatment. Suitable means include, among others, oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal) administration.

The invention has the beneficial effects that:

experiments prove that the polypeptide MP1 has no obvious toxic or side effect in cell experiments and mouse and animal experiments. The polypeptide MP1 can inhibit the expression of fibrosis markers alpha-smooth actin (alpha-SMA), fibronectin (Fibronectin), type I Collagen (Collagen I) and matrix metalloproteinase 2 (MMP 2) on the gene and protein level in two in vitro fibrosis models of NIH-3T3 and HK2 induced by TGF-beta 1.

In the TGF-beta 1-induced HK2 cell fibrosis model, polypeptide MP1 inhibited the expression of the EMT markers Snail1 and Vimentin at the gene and protein level, indicating that polypeptide MP1 can inhibit fibrosis by inhibiting EMT.

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

Polypeptide MP1 can reduce serum creatinine (Scr) and serum urea nitrogen (BUN) levels in a unilateral ureteral ligation (UUO) -induced kidney fibrosis mouse model, improve kidney function and alleviate kidney injury.

Meanwhile, the polypeptide MP1 can inhibit the expression of fibrosis markers alpha-SMA, fibronectin, collagen I and MMP2 and the expression of EMT related factors Snail1 and Vimentin on the protein and gene level, which shows that MP1 can inhibit kidney fibrosis and EMT process in vivo and has better effect than the control drug captopril.

The polypeptide MP1 of the invention can inhibit the EMT process and the kidney fibrosis process together mainly by inhibiting WNT/beta-Catenin and TGF-beta/Smad signal transduction pathways.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the inhibition of TGF-beta 1 induced NIH-3T3 cell fibrosis markers by polypeptide MP 1;

FIG. 2 shows the inhibition of TGF-beta 1 induced HK2 cell fibrosis by polypeptide MP1 and EMT markers;

FIG. 3 shows the effect of polypeptide MP1 on UUO-induced kidney function index and kidney tissue morphology in mice with kidney fibrosis;

FIG. 4 shows the inhibition of UUO-induced kidney fibrosis in kidney tissue of mice by polypeptide MP1 as an EMT marker;

FIG. 5 shows the inhibition of TGF-beta 1 induced NIH-3T3 cell WNT/beta-Catenin and TGF-beta/Smad pathways by polypeptide MP 1;

FIG. 6 shows the inhibition of TGF-beta 1 induced HK2 cells WNT/beta-Catenin and TGF-beta/Smad pathways by polypeptide MP 1;

FIG. 7 shows the inhibition of UFO-induced kidney fibrosis by polypeptide MP1 on WNT/beta-Catenin and TGF-beta/Smad pathways in kidney tissue of mice;

FIG. 8 shows the effect of polypeptide MP1 on TGF- β1-induced autophagy of NIH-3T3 cells.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.

Example 1 screening of autophagy Critical regulatory protein Beclin-1 fragment Polypeptides having anti-renal fibrosis Activity

The invention aims to take the interference WNT/beta-Catenin pathway as a strategy for treating kidney fibrosis, firstly, a Beclin-1 protein fragment Beclin-1 is selected in a targeted manner ^112-123 And derived polypeptides MP1 and MP2 thereof for preliminary screening of anti-fibrosis activity.

TABLE 1 amino acid sequence of polypeptide compounds

After an in vitro cell fibrosis model is constructed by inducing mouse embryo fibroblasts (NIH-3T 3) through TGF-beta 1, the polypeptide molecules are given, the total cell proteins are extracted after 24 hours of action, and the expression of fibrosis markers alpha smooth actin (alpha-SMA) and Fibronectin (Fibronectin) is detected by western blotting experiments.

The result shows that after TGF-beta 1 induction, the expression of fibrosis markers alpha-SMA, fibronectin is obviously up-regulated, the expression of the markers is obviously inhibited after polypeptide molecule MP1 is interfered, and other polypeptides have no obvious influence on the expression of the markers. Thus, it was preliminarily determined that the polypeptide MP1 had an anti-fibrotic activity.

Example 2 Synthesis of polypeptide MP1

Polypeptide MP1 is synthesized by Fmoc solid phase synthesis, separated and purified by reverse phase HPLC and characterized by mass spectrometry.

The sequence of polypeptide MP1 is: leu-Ser-Arg-Arg-Met-Lys-Val-Thr-Gly-Asp-Leu-Trp, and has the following structural formula:

the specific synthesis steps are as follows:

(1) 2-CTC resin activation: an appropriate amount (0.5 mmol) of the resin was weighed into a 20mL BD syringe, dichloromethane (DCM) was added, the resin was allowed to swell sufficiently by shaking at low speed for 30min, and the resin was drained after completion.

(2) C terminal first amino acid condensation reaction: three times the amount of Trp was weighed, dissolved in DCM, added to a syringe, shaken at low speed for 5min, DIEA was added to start the reaction, and after shaking for 1h, the resin was drained and washed 3 times with DMF.

(3) Indene detection: and (3) selecting a small amount of DMF (dimethyl formamide) washed resin, adding an indene detection reagent (phenol: pyridine: ninhydrin=1:2:1) into a test tube, heating for 1min, observing the color of the resin, and allowing the resin with complete amino acid condensation reaction to be colorless and the resin to be blue-purple after deprotection reaction.

(4) Fmoc deprotection reaction: the resin was dried by adding a deprotecting reagent (DMF: redistilled piperidine: dbu=94:3:3), shaking at low speed for 5min, repeating 3 times, washing 3 times with DMF, and indene inspection to determine whether the reaction was complete.

(5) Condensation reaction of other amino acids: the amino acid, HOBt, HBTU were weighed in 3-fold excess and dissolved in DMF, DIEA was added and transferred to a syringe, and after shaking for 1h, the resin was drained and washed with DMF.

(6) Peptide chain extension: repeating the steps (3), (4) and (5) until the amino acid sequence Leu-Ser-Arg-Arg-Met-Lys-Val-Thr-Gly-Asp-Leu-Trp is joined.

(7) Peptide chain cleavage: adding DCM into the drained resin to swell for 3min, and repeating for 2 times; adding anhydrous methanol, and shaking at low speed for 3min; adding DCM to swell the resin for 3min again; adding anhydrous methanol, shaking at low speed for 3min, repeating for 2 times, and draining to obtain loose granule. The cleavage agent (TFA: ddH) ₂ O: EDT: tris=94: 2.5:1:2.5 Adding into the drained resin, shaking at low speed for 3h, collecting the cutting agent in a round-bottom flask, adding TFA to wash the resin for 5min, repeating for 2 times, and collecting in the round-bottom flask with the cutting agent.

(8) Extraction: the rotary evaporator evaporates the redundant solvent in the cutting agent as much as possible to concentrate the redundant solvent, adds glacial ethyl ether into a round bottom flask, and severely oscillates the mixture, so that the polypeptide is precipitated, adds a proper amount of ultrapure water, slowly oscillates the mixture to enable the polypeptide to be completely dissolved in water, transfers the liquid in the round bottom flask into a separating funnel, oscillates the mixture, stands the mixture, and collects the lower water phase. After standing overnight at-80 ℃, the crude peptide was obtained by placing it in a freeze-dryer.

(9) And (3) HPLC purification: weighing about 50mg of crude peptide, dissolving, and filtering with 0.45 μm filter head; and C18 preparation type chromatographic column is selected for separation and purification, and the mobile phase is water phase containing 0.1% of acetonitrile solvent. The method comprises the steps of using a proportional balance chromatographic column with 5% acetonitrile and 95% water in advance, loading samples after setting a flow gradient, running the flow gradient, detecting an absorption peak at a wavelength of 220nm and collecting a main peak product. HPLC analysis of purity, mass spectrometry characterization. The product meeting the purity requirement (> 95%) is freeze-dried to obtain the polypeptide MP1.

EXAMPLE 3 anti-renal fibrosis at the cellular level of polypeptide MP1

And (3) cells: mouse embryonic fibroblasts (NIH-3T 3), human tubular epithelial cells (HK 2);

culture medium: DMEM medium containing 10% FBS, DME/F12 medium containing 10% FBS;

culture conditions: 37 ℃ and 5% CO ₂ An incubator.

1. Cell culture: when the cell confluence reaches 80-90%, discarding the upper layer culture medium, after pancreatin digestion until the cells are round, adding the culture medium to stop digestion, repeatedly blowing to enable the cells to fall off from the wall, transferring the cells into a centrifuge tube, centrifuging at 800rpm for 5min, discarding the supernatant, adding a proper amount of culture medium to resuspend the cells into single cell suspension, and sucking a proper amount of cells to be added into a culture dish for cell passage.

MTT assay to detect MP1 toxicity to NIH-3T3, HK2 cells:

according to 7X 10 ³ cell/Kong Peizhi cell suspension, adding into 96-well plate after mixing, 100 μl/well, culturing for 24 hr, adding polypeptide MP1 with concentration gradient of 12.5, 25, 50, 100, 200 μM, culturing for 24 hr, adding MTT, incubating for 4 hr, absorbing culture medium, adding 150ul DMSO per well, shaking for 10min, and detecting absorbance at 490nm wavelength by enzyme-labeling instrument.

The experimental results are shown in FIG. 1A and FIG. 2A, wherein FIG. 1A shows NIH-3T3 cell viability and FIG. 2A shows HK2 cell viability. From the graph, in the concentration range of 0-200 mu M, the polypeptide MP1 has no obvious inhibition effect on the survival rate of NIH-3T3 cells, and in the concentration range of 0-100 mu M, the polypeptide MP1 has no obvious inhibition effect on the survival rate of NIH-3T3 cells.

3. Western blot detection of MP1 effect on TGF-beta 1-induced NIH-3T3, HK2 cell fibrosis and EMT marker expression

The fibrosis markers tested in this example included α -SMA, fibronectin (FN), collagen I (COL), MMP2, and the EMT markers included Snail1, vimentin.

3.1 administration treatment: according to 3X 10 ⁵ Cell suspension/Kong PeizhiAfter mixing, adding the mixture into a 6-well plate, culturing for 24 hours, starving cells for 10 hours by using a culture medium without serum, selecting two dosing concentrations of 50 and 100 mu M, inducing a cell fibrosis model by using TGF-beta 1, and simultaneously adding MP1. The experimental group is as follows: control, TGF-. Beta.1, TGF-. Beta.1+50. Mu.M MP1, TGF-. Beta.1+100. Mu.M MP1.

3.2 total cell protein extraction: after incubation of cells with MP1 for 24h, the culture supernatant was discarded, washed 3 times with PBS, blotted dry, and the protein lysate was scraped with a spatula and collected in a 1.5mL centrifuge tube, lysed for 30min on ice, centrifuged at 12000rpm for 30min, and the supernatant was aspirated.

3.3 Western immunoblotting: the BCA kit detects the protein concentration, the protein loading amount is regulated to be 30 mug, the protein is denatured by heating in a metal bath at 100 ℃, the proteins with different molecular weights are separated by using 10% SDS-PAGE gel electrophoresis, the gel is concentrated by 80V for 30min, and the gel is separated by 120V for 1.5h. After electrophoresis, protein transfer printing is carried out, and target proteins are transferred onto the PVDF film, wherein the transfer printing conditions are 250mA and 3h. After the membrane transfer is finished, 5% of skimmed milk powder seals the membrane for 1.5h, TBST is washed for 10min multiplied by 3 times after the membrane transfer is finished, primary antibodies are incubated overnight at 4 ℃, PVDF membranes are taken out the next day, after TBST is washed for 10min multiplied by 3 times, secondary antibodies are incubated for 1h at room temperature, TBST is washed for 10min multiplied by 3 times, and a chemiluminescent kit is added for color development.

FIG. 1 shows the effect of polypeptide MP1 on TGF- β1-induced expression of the NIH-3T3 cell fibrosis marker α -SMA, FN, COL, MMP. Wherein FIG. 1B is a western blotting chart, and FIGS. 1C-F are bar charts of the chart. From the figure, the expression level of fibrosis-related protein alpha-SMA, FN, COL and MMP2 was significantly increased after TGF-beta 1 induction compared with the control group, and the expression level of the protein was significantly decreased after polypeptide MP1 treatment, both at high concentration and at low concentration. The polypeptide MP1 has obvious inhibition effect on a TGF-beta 1 induced NIH-3T3 cell kidney fibrosis model at the protein level.

FIG. 2 shows the effect of polypeptide MP1 on TGF- β1-induced expression of HK2 cell fibrosis marker α -SMA, FN, COL, MMP2 and EMT markers Snail1, vimentin (Vim). Wherein FIGS. 2B-C are Western blot patterns, and FIGS. 2D-I are bar graphs of the patterns. It can be seen from the figure that the expression levels of the fibrosis-related protein alpha-SMA, FN, COL, MMP and the EMT-related proteins Snail1 and Vim are significantly increased after TGF-beta 1 induction compared with the control group, and the expression levels of the proteins are significantly decreased after the polypeptide MP1 treatment. The polypeptide MP1 has obvious inhibition effect on the TGF-beta 1 induced HK2 cell kidney fibrosis and EMT model at the protein level.

Detection of the Effect of polypeptide MP1 on TGF-beta 1-induced HK2, NIH-3T3 cell fibrosis marker Gene by qRT-PCR

4.1 total RNA extraction of cells: adding RNA lysate into a 6-well plate, 500 mu L/well, repeatedly blowing and transferring to an enzyme-free 1.5mL EP tube, adding 100 mu L of chloroform, vigorously shaking, incubating for 5min at room temperature, centrifuging at 12000rpm for 15min, sucking the upper colorless aqueous phase, adding 500 mu L of isopropanol, standing for 10min after mixing, centrifuging at 12000rpm for 10min, discarding the supernatant, forming white colloidal precipitate on the tube side and the tube bottom, adding 500 mu L of 75% ethanol, slightly shaking to enable the precipitate to fall off the tube wall, centrifuging at 7500rpm for 5min, drying the supernatant at room temperature, airing the precipitate at room temperature, and dissolving the precipitate in the enzyme-free sterile water.

4.2 reverse transcription: the concentration of RNA is detected by a micro ultraviolet visible spectrophotometer, and the concentration of RNA sample A260/A280 is between 1.8 and 2.2. Preparing a reverse transcription system according to the proportion in the table, mixing uniformly, adding into a PCR instrument, setting reverse transcription conditions to 25 ℃, 5min,55 ℃, 15min,85 ℃ and 5min, and preserving the synthesized cDNA at-20 ℃ after reverse transcription.

TABLE 2 reverse transcription scheme

4.3qPCR: the reaction system was formulated according to table 3.

TABLE 3 configuration Table of amplification System

Amplification procedure: pre-denaturation: 95 ℃ for 5min. Amplification for 40 cycles: 95 ℃ for 10s;60 DEG C30s. Melting: 95 ℃ for 1min;55 ℃ for 30s;95℃for 30s. Relative expression was calculated from Ct values of the genes (2 ^-ΔΔCt )。

FIGS. 1G-J show the effect of polypeptide MP1 on the expression of fibrosis-associated genes Acta2, fn1, COL1A1, mmp2 in TGF-beta 1-induced NIH-3T3 cells. From the figure, MP1 significantly inhibited TGF-beta 1-induced abnormal expression of the above genes at doses of 50. Mu.M and 100. Mu.M, indicating that polypeptide MP1 significantly inhibited TGF-beta 1-induced kidney fibrosis of NIH-3T3 cells at the gene level.

FIG. 2J-O shows the effect of polypeptide MP1 on expression of fibrosis-associated genes Acta2, fn1, COL1A1, mmp2 and EMT-associated genes SNAI1, VIM in TGF-beta 1-induced HK2 cells. From the figure, MP1 significantly inhibited TGF-beta 1-induced abnormal expression of the above gene at doses of 50. Mu.M and 100. Mu.M, indicating that polypeptide MP1 significantly inhibited TGF-beta 1-induced renal fibrosis of HK2 cells at the gene level.

EXAMPLE 4 anti-renal fibrosis of polypeptide MP1 in mice

Experimental animals: male C57BL/6 mice, 6-8 weeks old, purchased from the national institute of veterinary sciences, china, were kept at 25℃and were alternately illuminated for 12 hours, were fed water ad libitum, and were replaced twice a week.

1. Experimental grouping, modeling and dosing:

experimental grouping: sham, surgical (UUO), 0.625mg/kg MP1, 1.25mg/kg MP1, 20mg/kg Captopril, 10 per group. Building a mouse unilateral ureteral obstruction model: after the mice were anesthetized, the skin of the left back of the mice was cut, the muscle layer was torn, the left ureter was freed, 1/3 of the position on the renal pelvis and ureter was ligated, the ureter was cut between the ligatures, and the muscle layer and skin were sutured. Administration: the administration is started the following day after the operation, the administration mode is subcutaneous injection, and the administration is carried out 1 time a day for 2 weeks.

2. Renal function index detection: after the mice were anesthetized, the mice were subjected to orbital bleeding, collected in a 1.5mL EP tube, centrifuged at 5000rpm for 15min, centrifuged 2 times, and the supernatants were collected, and serum creatinine (Scr) and serum urea nitrogen (BUN) contents were determined according to the kit instructions.

As shown in FIGS. 3A-B, the concentrations of 0.625mg/kg and 1.25mg/kg of the polypeptide MP1 can significantly inhibit the abnormal increase of the levels of Scr and BUN caused by kidney fibrosis.

3. Appearance form of left kidney of mouse and H & E staining result

When the materials are obtained, a part of the kidney tissue is stored at-80 ℃, and the other part is fixed by formaldehyde and embedded by paraffin to prepare the histopathological section. Taking part of pathological sections to carry out H & E staining, drying the pathological sections at 65 ℃ for 1H, and dewaxing: dewaxing is carried out according to the sequence of 20min of dimethylbenzene, 2min of absolute ethyl alcohol, 2min of 95 percent ethyl alcohol, 2min of 90 percent ethyl alcohol and 2min of 80 percent ethyl alcohol. Dyeing: the hematoxylin is sequentially dyed according to the sequence of 7min, tap water washing, 1% ethanol differentiation of hydrochloric acid for 1s, tap water washing, eosin dyeing for 25-30s and tap water washing for 5min. And (3) dehydration and transparency: dehydration was performed sequentially in the order of 80% ethanol 7s, 90% ethanol 7s, 95% ethanol 7s, absolute ethanol 7s, xylene 2min, and xylene 2 min.

As shown in FIG. 3C, the renal tubular expansion of the operative group resulted in the shedding of tubular epithelial cells, and the above changes were inhibited after administration. The above results indicate that polypeptide MP1 can alleviate kidney injury caused by UUFO.

4. Animal level detection of MP1 effects on fibrosis and EMT marker expression

Animal level detection tissue fibrosis markers comprise Masson staining detection of kidney pathological sections, total collagen deposition of tissues, and histoimmunochemistry staining detection of pathological sections, namely alpha-SMA, FN, vim deposition of markers; detecting the expression condition of a marker alpha-SMA, FN, COL, MMP2 by western blotting; qRT-PCR detects the expression of fibrosis genes Acta2, fn1, COL1A1, mmp2 and EMT genes Vim, snai 1.

4.1 Masson staining of pathological sections and histoimmunochemical staining. Dewaxing and dehydration operations of pathological sections are identical to those in H & E. Masson staining: weibert iron hematoxylin staining solution

Dyeing for 5-10min; after the dyeing liquid is sucked up, the dyeing liquid is differentiated for 5 to 15 seconds by using an acidic ethanol differentiation liquid, and is put into tap water for washing, and the excess water is sucked up; returning Masson bluing liquid to blue for 3-5min, washing with distilled water for 1min, and sucking excessive water; dyeing the ponceau dyeing liquid for 5-10min, and sucking the redundant dyeing liquid; washing with phosphomolybdic acid solution for 1-2min, and sucking off excessive solution; washing with weak acid working solution for 1min, and adding into distilled water; spin-drying excessive water on the film, and dehydrating; air-drying the pieces and sealing the pieces with neutral resin. Histoimmunochemistry: placing the dewaxed slice into a citrate buffer solution, boiling for 2min to repair antigen, and naturally cooling to room temperature; absorbing excessive water, adding endogenous peroxidase blocking agent, and incubating for 10min at room temperature; dripping normal goat serum working solution for sealing, and incubating for 15min at room temperature; putting the pieces into a wet box, dripping primary antibody, and incubating overnight at 4 ℃; taking out the wet box, standing at room temperature for 30min, rewarming, and recovering the primary antibody; dripping biotin-marked goat anti-rabbit IgG, and incubating for 15min at room temperature; dripping horseradish enzyme-labeled streptavidin working solution, and incubating for 15min at room temperature; dripping freshly prepared DAB color development liquid, observing under a microscope, and putting into distilled water to terminate dyeing after obvious brown deposition appears; spin-drying excessive water, dripping hematoxylin, incubating at room temperature for 3min, and putting into distilled water to terminate dyeing; washing for 2min, spin-drying excessive water, and dehydrating; air-drying the pieces and sealing the pieces with neutral resin.

The experimental results are shown in fig. 3D-G, and compared with Sham group, the deposition amount of total collagen fibers and alpha-SMA, FN, vim in UUO group is obviously increased; when MP1 and Captopril were administered, the amount of collagen fibers and the above markers deposited was significantly reduced.

4.2 Western blotting detection of expression of tissue fibrosis markers

Tissue protein extraction: about 15mg of kidney tissue of a mouse is weighed, 100 mu L of RIPA lysate containing 1% PMSF is added, grinding steel balls (one each in size) are added, grinding is carried out for 120s at 60Hz, the grinding steel balls are taken out, the kidney tissue is kept at a low temperature for cracking tissue protein for 30min, and centrifugation is carried out for 2 times at 12000 rpm. The supernatant is sucked to be the total tissue protein.

Western blotting: the same procedure was used for cell experiment 3.3.

As shown in fig. 4, fig. 4A-B are western blot banding diagrams, and fig. 4C-H are banding gray scale statistical bar diagrams. From the figure, MP1 significantly down-regulates UUO-induced kidney tissue fibrosis and abnormal expression of EMT-related proteins. The polypeptide MP1 is shown to have in vivo anti-renal fibrosis activity at the protein level.

4.3qRT-PCR detection of the expression of the fibrosis genes.

Tissue RNA extraction: weighing kidney tissue 15mg, adding 1000 μl of RNA extract, adding grinding steel balls (one each, 60Hz grinding for 120s, standing at room temperature for 10min, and collecting the extract

Lysates were transferred to new enzyme-free EP tubes and the subsequent procedure was the same as 2.6. Reverse transcription: cell experiment 4.2 as in example 3. qPCR: cell experiment 4.3 as in example 3.

As shown in the experimental results of FIG. 4I-N, MP1 significantly inhibited UUO-induced fibrosis and abnormal expression of EMT gene, indicating that polypeptide MP1 also inhibited UUO-induced kidney fibrosis at the gene level.

Example 5 mechanism of action of polypeptide MP1 against renal fibrosis

And (3) cells: mouse embryonic fibroblasts (NIH-3T 3), human tubular epithelial cells (HK 2);

culture medium: DMEM medium containing 10% FBS, DME/F12 medium containing 10% FBS;

culture conditions: 37 ℃ and 5% CO ₂ An incubator.

C57BL/6 mouse kidney tissue of UFO model.

MP1 mechanism of action studies included MP1 effects on WNT/beta-Catenin, TGF-beta/Smad pathways and MP1 effects on autophagy activity.

Effect of MP1 on WNT/beta-Catenin, TGF-beta/Smad pathway

The influence of MP1 on the expression of WNT/beta-Catenin, TGF-beta/Smad channel related protein is detected by pathological section histoimmunochemical staining, western blotting and qRT-PCR experiments. In the extraction step of protein related to phosphorylation in western blotting experiments, the total protein of cells is extracted after the cells are incubated with MP1 for 1h, and other experimental steps are correspondingly operated in the cell and animal level experiments; the pathological section tissue immunochemistry staining and qRT-PCR experimental steps are correspondingly operated in the cell and animal level experiment.

FIG. 5 shows the effect of MP1 on TGF-beta 1-induced expression of the genes of the NIH-3T3 cell WNT/beta-Catenin and TGF-beta/Smad pathway related proteins. Wherein FIG. 5A is a western blotting chart, FIGS. 5B-E are bar charts of the chart, and FIGS. 5F-H are bar charts of the variation of the expression of the related genes. From the figure, MP1 significantly inhibits the phosphorylation of Smad proteins by WNT/beta-Catenin pathway-related proteins beta-Catenin, C-Myc, cyclin D1 and TGF-beta/Smad pathway-related proteins that are abnormally expressed in NIH-3T3 cells due to TGF-beta 1 induction. Meanwhile, MP1 obviously inhibits abnormal expression of WNT/beta-Catenin pathway related genes Cnnb 1, myc and Ccnd1. The polypeptide MP1 is shown to regulate the differentiation process of the fibroblast to the myofibroblast by inhibiting the activities of a TGF-beta/Smad channel and a WNT/beta-Catenin channel.

FIG. 6 is a graph showing the effect of MP1 on TGF-beta 1-induced expression of WNT/beta-Catenin and TGF-beta/Smad pathway related proteins in HK2 cells. Wherein FIG. 6A is a western blotting chart, FIGS. 6B-E are bar charts of the chart, and FIGS. 6F-H are bar charts of the variation of the expression of the related genes. From the figure, MP1 significantly inhibits the phosphorylation of Smad proteins by WNT/beta-Catenin pathway-related proteins beta-Catenin, C-Myc, cyclin D1 and TGF-beta/Smad pathway-related proteins that are abnormally expressed in HK2 cells due to TGF-beta 1 induction. Meanwhile, MP1 obviously inhibits abnormal expression of WNT/beta-Catenin pathway related genes CTNNB1, MYC and CCND 1.MP1 was shown to co-regulate the EMT process by inhibiting the activity of the TGF-beta/Smad pathway and the WNT/beta-Catenin pathway.

FIG. 7 shows the effect of polypeptide MP1 on the expression of WNT/beta-Catenin and TGF-beta/Smad pathway-related proteins in UFO-induced kidney fibrosis mouse kidney tissue. Wherein FIG. 7A is a Western blot pattern, FIGS. 7B-F are histogram statistics of pattern, FIGS. 7G-J are histogram statistics of related gene expression changes, and FIGS. 7K-L are histoimmunochemical staining patterns. As can be seen from the figure, the expression levels of WNT/beta-Catenin pathway-associated proteins beta-Catenin, C-Myc, cyclin D1 and TGF-beta/Smad pathway-associated proteins TGF-beta 1, phosphorylated Smad proteins were significantly increased in the UFO group compared to the Sham group, and significantly decreased after MP1 administration. The qRT-PCR experiment and the immunohistochemical experiment result are consistent with the Western blotting result. The polypeptide MP1 is shown to jointly regulate the progress of kidney fibrosis of UFO mice by inhibiting the activities of a TGF-beta/Smad pathway and a WNT/beta-Catenin pathway.

2. Effect of polypeptide MP1 on TGF-beta 1-induced autophagy of NIH-3T3 cells

The effect of MP1 on TGF-beta 1-induced expression of NIH-3T3 cell autophagy-related proteins was examined by western blotting. The western blot experiment was performed in the same manner as in the cell and animal level experiment.

As shown in fig. 8, fig. 8A is a western blot bar chart, and fig. 8B-C are bar charts of bar gradation statistics. From the figure, the polypeptide MP1 had no effect on the expression levels of autophagy-related proteins LC3II/LC3I and autophagy substrate P62. It was shown that polypeptide MP1 did not induce autophagy in NIH-3T3 cells.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (9)

1. Application of autophagy key regulatory protein Beclin-1 fragment polypeptide MP1 in preparing medicines for resisting kidney fibrosis.

2. The use according to claim 1, wherein the amino acid sequence of the polypeptide MP1 is Leu-Ser-Arg-Met-Lys-Val-Thr-Gly-Asp-Leu-Trp.

3. The use of claim 1, wherein the kidney fibrosis comprises renal interstitial fibrosis.

4. The use according to claim 3, characterized in that said renal interstitial fibrosis is that caused by ureteral obstruction.

5. An anti-renal fibrosis preparation, which is characterized in that the preparation takes polypeptide MP1 as an active ingredient and is a medicine or a medicine composition.

6. The anti-renal fibrosis preparation of claim 5, wherein the amino acid sequence of the polypeptide MP1 is Leu-Ser-Arg-Met-Lys-Val-Thr-Gly-Asp-Leu-Trp.

7. The formulation for use in treating renal fibrosis of claim 5, wherein the renal fibrosis is comprises interstitial renal fibrosis.

8. The formulation for use in treating anti-renal fibrosis according to claim 5, wherein the renal interstitial fibrosis is that resulting from ureteral obstruction.

9. The formulation for preventing renal fibrosis according to claim 5 wherein the formulation is one of a tablet, capsule, granule, oral liquid, syrup, drop pill, ointment and patch for skin surface, aerosol, nasal spray, suppository, microsphere formulation, injection or lyophilized powder injection.

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