CN114668843A

CN114668843A - Nano self-assembled glycopeptide BIVA-PK and application thereof in renal fibrosis caused by ischemia-reperfusion injury

Info

Publication number: CN114668843A
Application number: CN202210056384.3A
Authority: CN
Inventors: 欧阳清; 李莉莉; 陈香美; 桑田
Original assignee: First Medical Center of PLA General Hospital
Current assignee: First Medical Center of PLA General Hospital
Priority date: 2022-01-18
Filing date: 2022-01-18
Publication date: 2022-06-28
Anticipated expiration: 2042-01-18
Also published as: CN114668843B

Abstract

The invention relates to the technical field of polypeptide medicines, in particular to nano self-assembled glycopeptide BIVA-PK and application thereof in renal fibrosis caused by ischemia-reperfusion injury. The invention constructs the nanometer self-assembly glycopeptide BIVA-PK with fibrosis microenvironment specificity trigger response by utilizing a nanometer self-assembly technology, and the glycopeptide can accurately target and induce apoptosis of M2 type macrophages, block the conversion of the macrophage to myofibroblasts, reduce the generation of cell factors for promoting fibrosis, and can more specifically and efficiently inhibit the acute kidney injury from progressing to kidney fibrosis. The glycopeptide has double targeting mechanisms, can prevent nonspecific histiocyte damage, has high hydrophilicity, has high enrichment efficiency in kidney, and improves the action effect and safety. BIVA-PK has an obvious blocking effect on renal fibrosis caused by ischemia-reperfusion injury, and can be used for treating AKI-CKD to delay the progress of uremia.

Description

Nano self-assembled glycopeptide BIVA-PK and application thereof in renal fibrosis caused by ischemia-reperfusion injury

Technical Field

The invention relates to the technical field of polypeptide medicines, in particular to a nano self-assembled glycopeptide BIVA-PK and application thereof in renal fibrosis caused by ischemia-reperfusion injury.

Background

Renal fibrosis (Renal fibrosis) is the outcome of pathological repair of Acute Kidney Injury (AKI), and Ischemia Reperfusion Injury (IRI) is one of the causes of Renal fibrosis. Chronic Kidney Disease (CKD) develops when the inflammatory response persists after IR injury and tissue damage is not completely repaired, with the pathology that kidney Fibrosis, i.e. massive deposition of extracellular matrix (ECM) in the renal interstitium, loss of nephrons and surrounding vascular beds, and eventually the loss of renal function progresses to uremia (Mutsaers HA, olin p. editorial: organic fibers: Triggers, Pathways, and Cellular pathology. front med (lausanne) 2016,3: 55.). Clinical studies have shown that long-term follow-up for 1-3 years, 30-50% of patients with AKI develop fibrosis, and more than 10% of patients receive long-term Renal Replacement Therapy (RRT) (See EJ, Jayasinghe K, Glassford N, Bailey M, Johnson DW, polkinghorn KR, Toussaint ND, Bellomo r.long-term risk of additive outer instruments aid kit in j. a systematic review and method-analysis of heart students con details of excess. kit int.2019, 95(1): 160. lined to 172.) End-stage Renal disease (End, stage recovery) and death will eventually occur. Therefore, blocking the AKI strike CKD and post-renal fibrosis treatment induced by ischemia reperfusion injury is an important public health concern.

At present, the treatment of AKI after progressing to fibrosis still faces a great challenge, and there is no specific and effective mechanism-directed therapeutic drug clinically other than supporting symptomatic treatment (US National Library of science. clinical trials. gov [ online ], https:// clinical trials. gov/ct2/show/NCT 01665391; US National Library of science. clinical trials. gov [ online ], https:// clinical trials. gov/ct2/show/NCT 01113801.). Therefore, how to find new therapeutic drugs aiming at the progress and the regulation mechanism of the fibrosis is an important challenge for the treatment of the kidney fibrosis.

Macrophages (macrophages) are a heterogeneous population of natural immune cells with pathogensMultiple functions including body clearance, injury repair, and inflammation regulation (Wynn TA, Chawla A, Pollard JW. macrophage biology in depth, hoseostasis and disease. Nature.2013,496(7446): 445-55.). Macrophages are highly plastic and play different roles at different stages in the development of AKI-CKD (fig. 1, Distler JHW,

AH,Ramanujam M,Whitfield ML,

lafyatis R.Shared and discontint mechanisms of fibrosis. nat Rev Rheumatol.2019, (15) (12): 705-. In the early stages of acute kidney Injury, M1-type macrophages are involved in an inflammatory response characterized by TNF- α, IL-12 secretion, causing acute tissue Injury (Chiba T, Skypnyk NI, Skvanca LB, Penchev R, Zhang KX, Rochon ER, Fall JL, Paueksakon P, Yang H, Alford CE, Roman BL, Zhang MZ, Harris R, Hukride NA, de Caestecker MP, Retinoic Acid signalling codes macro-10 and Dependent Injury and Repair aft AKI. JAm Soc Nephrol.2016,27(2): 495-508.). Macrophages of type M2a subsequently promote tubular repair by inhibiting inflammatory responses early in the repair of injury (Zhang MZ, Wang X, Wang Y, Niu A, Wang S, Zou C, Harris RC. IL-4/IL-13-mediated polarization of renal macrogenes/dense cells to an M2 apoptosis for recovery from access kit in Kidney Int.2017,91(2):375 (386)). However, chronic inflammation triggers a fibrotic process when tissue repair is poor and inflammatory responses persist, and M2-type macrophages become the central regulator and direct participant in the fibrotic process by producing cytokines and growth factors that promote fibrosis, including TGF-. beta.PDGF-B, IGF-1, Galactin-3, etc. (Distler JHW,

Ramanujam M,Whitfield ML,

Lafyatis R.Shared and distinct mechanisms of fibrNat Rev Rheumatotol.2019, 15(12) 705-730; broekema M, Harmsen MC, van Luyn MJ, Koerts JA, Petersen AH, van Kootecn TG, van Goor H, Navis G, Popa ER. bone marrow-derived myofibrasts distribution to the new internal fibroblast fertilization publication and product procollagen I after chemia/repair in rats.J. Am Soc Newrol.2007, 18(1):165-75.), or myofibroblast transformation (MMT) occurs, greatly promoting the formation of local fibrotic microenvironment (Huen SC, Cantley. macrogenes LG. neutral infusion and repair. Annu. physiology.7, 201469. 449.).

In Vivo Self-Assembly Polypeptide nanotechnology (In Vivo Self-Assembly Nano-Polypeptide) is a nanotechnology (Zhang D, Qi GB, ZHao YX, Qiao SL, Yang C, Wang H. In Single Structure of nanofibres from Purpurin18-Peptide junctions and the Assembly Induced elongation efficiency In Tumor sites. adv. Mater.2015,27(40):6125-30.) that Self-assembles to form a nanostructure In Situ In target cells by a dual targeting mechanism of molecular active recognition and specific endonuclease cutting. The in vivo self-assembly polypeptide nano molecule consists of three parts: (1) the binding peptide recognizes and binds to a target molecule on the surface of a cell and is internalized into a lysosomal pathway along with the target molecule; (2) carrying out specific molecular shearing by using the specific endonuclease in the lysosome, and then carrying out self-assembly and retaining in the cell; (3) release of effector molecules carried by polypeptide nanomolecules to achieve effector functions (Zhang D, Qi GB, Zhao YX, Qiao SL, Yang C, Wang H. in Situ Format of Nanofibers from pure 18-Peptide Conjugates and the Assembly Induced elongation effects in Tumoter sites. adv. mater.2015,27(40): 6125-30.).

The in vivo self-assembly polypeptide nanotechnology improves the specificity of recognition not only through a 'dual targeting' mechanism (recognition and shearing); sensitive and specific micro-environment enrichment and retention are also achieved by self-assembly of the sheared nanostructure, changing the distribution and metabolism of the nanomolecules between the micro-environment site and the normal tissue (FIG. 2, Ye D, Shuhendler AJ, Cui L, Tong L, Tee SS, Tikhomirov G, Felser DW, Rao J. Bioorthogonal cycling-mediated in situ self-assembly of small-interaction probes for imaging of the activity of the enzyme in vivo. Nat. Chem.2014,6(6) (519-26; Shi J, Du X, Hugang Y, Zohou J, Yuan D, Wu D, Zhang Y, Habuak R, Epto IR 137, Xu B. ligand-receptor interactions J. acquisition of microorganism J. 1. 3. sample J. 3. interaction-interaction probes. 11. 3. of the same, 3. of the same, one of the same kind of microorganism, one. Based on the in vivo self-assembly polypeptide nanotechnology, the development of the medicine for treating the kidney fibrosis has important significance for solving the problems in the kidney fibrosis treatment.

Disclosure of Invention

The invention aims to provide a nano self-assembled glycopeptide. Another objective of the invention is to provide the application of the nano self-assembled glycopeptide.

Firstly, in the research process of the occurrence and development mechanism of the kidney fibrosis, the invention discovers that the conversion of M2 type macrophages to myofibroblasts is blocked by targeted induction of the apoptosis of the macrophages in the kidney fibrosis microenvironment, and the generation of the profibrotic cytokines is reduced, so that the acute kidney injury can be more specifically and efficiently inhibited from progressing to the kidney fibrosis.

Based on the above findings, the present invention provides the use of macrophage M2 as a therapeutic target for renal fibrosis caused by ischemia-reperfusion injury.

The invention also provides application of the inhibitor of M2 type macrophages in preparing a medicament for preventing or treating renal fibrosis caused by ischemia-reperfusion injury.

The inhibitors of M2-type macrophages described above preferably target CD 206.

Further preferably, the inhibitor of M2-type macrophages is a pro-apoptotic agent of M2-type macrophages.

The pro-apoptotic agent of the M2-type macrophage may be a pro-apoptotic polypeptide that targets M2-type macrophages.

On the basis of finding that M2 type macrophages play an important promoting role in the process of advancing IRI-AKI to fibrosis, the difficulty of specifically regulating the function and quantity of M2 type macrophages in the renal fibrosis microenvironment remains. Based on the nanometer supermolecule characteristics of polypeptide, the polypeptide is used as a drug delivery structure Is beneficial to realize highly specific and safe targeted drug delivery. In addition, the invention discovers that the kidney CD206 is discovered by constructing a mouse ischemia reperfusion kidney injury fibrosis model⁺Macrophage numbers exhibit a time-dependent increase, thus mannose receptors (MR, CD206) were selected as targets for targeting pro-apoptotic molecules by macrophages of the M2 type during fibrosis in ischemia-reperfusion renal injury; meanwhile, the invention also finds that the expression level of Cathepsin B is in the same trend with the number of M2 macrophages, so that Cathepsin B is selected as an enzyme cutting site to induce the responsive self-assembly of the targeted apoptosis-promoting glycopeptide. Based on the discovery, the invention develops a novel efficient nano self-assembled glycopeptide, which targets M2 type macrophages in a renal fibrosis microenvironment, and regulates and controls a local microenvironment by carrying effector molecules, thereby playing a role in inhibiting the progress of fibrosis.

The present invention provides a polypeptide having the following amino acid sequence and modifications: (manose) -Pra-GFRG-LVFF-GKKWWKKWDipK, wherein (manose) -Pra is mannose modification and Dip is diphenylalanine.

In the above-described polypeptide, mannose modification is achieved by coupling (coupling via Click reaction) an alkynyl group in the side chain of Pra amino acid with azide-modified mannose. The glycosylation modification can provide the polypeptide with the characteristic of targeting M2 macrophage, and is beneficial to improving the hydrophilicity of the nano self-assembled glycopeptide, so that the glycopeptide has the characteristics of small molecular weight and high hydrophilicity, the enrichment efficiency of the glycopeptide in the kidney is obviously improved, and the organ targeting is improved.

The amino acid sequence of the polypeptide is shown in SEQ ID NO.1, wherein Xaa represents diphenylalanine (Dip). Ligand (mannose) of the polypeptide is combined with CD206 specifically expressed on the surface of M2-type macrophage in a targeting way, and then enters a lysosome through a Clathrin way, a GFRG-LVFF sequence can be recognized and sheared by Cathepsin B in the lysosome, so that a derivative sequence LVFFGKKWWKKWDipK of the apoptosis-promoting peptide is formed, the derivative sequence can be self-assembled, the retention time of the derivative sequence in a kidney fibrosis microenvironment is prolonged, secondary targeting for the kidney is provided, the apoptosis-promoting activity of KKGWWKKWDipK is effectively improved, the apoptosis-promoting polypeptide GKKWWKKWDipK has no apoptosis-promoting activity outside cells, and a cytochrome C is released by damaging a mitochondrial membrane after entering cytoplasm to start a Caspase-mediated apoptosis way. The polypeptide can be used as apoptosis promoting glycopeptide of M2 type macrophage in kidney fibrosis microenvironment, and has effects of effectively inducing macrophage apoptosis and controlling fibrosis progression. The specific structure and working principle of the polypeptide are shown in figure 3.

Specifically, the chemical structural formula of the polypeptide provided by the invention is shown as the formula (I):

based on the functions of the polypeptide, the invention provides the application of the polypeptide in preparing in vivo self-assembly polypeptide medicaments.

The invention also provides application of the polypeptide in preparing a medicament for targeted induction of apoptosis of M2 type macrophages.

The invention also provides application of the polypeptide in preparing a medicament for preventing or treating kidney fibrosis.

Preferably, the above-described renal fibrosis is renal fibrosis resulting from the progression of acute renal injury to chronic renal disease.

Further preferably, the renal fibrosis is renal fibrosis caused by ischemia reperfusion injury. The polypeptide provided by the invention is particularly suitable for relieving or improving renal fibrosis caused by ischemia-reperfusion injury.

The present invention provides a medicament comprising a polypeptide as described above.

The medicine can also comprise auxiliary materials allowed in the pharmaceutical field or other active ingredients used for treating kidney fibrosis besides the polypeptide.

The invention has the beneficial effects that:

1. the invention utilizes the nano self-assembly technology to construct the nano self-assembly glycopeptide BIVA-PK of the fibrosis microenvironment specificity trigger response, the glycopeptide can accurately target and induce apoptosis of M2 type macrophages, block the conversion of the macrophage to myofibroblasts, reduce the generation of fibrosis promoting cytokines, and can more specifically and efficiently inhibit the acute kidney injury from progressing to kidney fibrosis.

2. The nano self-assembly glycopeptide BIVA-PK constructed by the invention has a double targeting mechanism, can prevent nonspecific histiocyte damage, and meanwhile, the glycopeptide has higher hydrophilicity and higher enrichment efficiency in the kidney, thereby improving the action effect and the safety.

3. The nano self-assembled glycopeptide BIVA-PK constructed by the invention has an obvious blocking effect on kidney fibrosis caused by ischemia reperfusion injury, can be used for treating AKI-CKD, delays the progress of uremia and provides a new treatment medicament for blocking the progress of AKI-CKD.

Drawings

FIG. 1 is a schematic diagram showing the functions of different macrophage subsets in kidney injury, repair and fibrosis in the background art of the present invention.

FIG. 2 is a schematic diagram of the in vivo dual targeting and function regulating mechanism of the nano self-assembly polypeptide technology in the background art of the present invention.

FIG. 3 is a schematic diagram of the operation of targeting induction of apoptosis of M2 macrophages by the nano self-assembled glycopeptide of the present invention.

FIG. 4 is a photograph showing CD206 after renal ischemia-reperfusion injury in example 1 of the present invention⁺The results of measuring the number of macrophages and the expression level of Cathepsin B, wherein the left graph is CD206⁺A macrophage number change assay result, wherein F4/80 represents EMR1, is a mouse macrophage surface marker, CD206 represents MMR, is a macrophage surface mannose receptor, is an M2 macrophage surface marker, and Merge represents F4/80 and CD206 coexpression; the right panel shows the results of detecting the expression level of Cathepsin B, wherein sham surgery represents the sham control group, and 3 days, 7 days, 14 days and 28 days represent days 3, 7, 14 and 28 after model construction, respectively.

FIG. 5 shows the BIVA-PK sequence of the self-assembled nano-glycopeptide and the morphology of the assembled nano-glycopeptide in example 2 of the present invention, with a 2 μm scale.

FIG. 6 is a graph showing the effect of BIVA-PK, a self-assembly glycopeptide, on the survival rate and body weight of a renal fibrosis model induced by ischemia-reperfusion injury in example 3 of the present invention.

FIG. 7 is a diagram of the slowing of renal function loss caused by ischemia reperfusion injury by the nano self-assembled glycopeptide BIVA-PK in example 3 of the present invention.

FIG. 8 is a graph showing that the nano self-assembled glycopeptide BIVA-PK of example 3 of the present invention can effectively reduce the fibrotic area of the kidney after ischemia reperfusion injury.

FIG. 9 is a graph of the reduction of kidney M2 macrophage infiltration after ischemia reperfusion injury by the nano self-assembled glycopeptide BIVA-PK of example 4 of the present invention.

FIG. 10 is a graph of the effect of the nano self-assembled glycopeptide BIVA-PK of the present invention in targeting ischemia reperfusion injury of the kidney in example 5.

FIG. 11 is the BIVA-PK nano-self-assembly glycopeptide of example 6 of the present invention effectively targeting CD206⁺Macrophages are provided.

FIG. 12 is a graph showing the specific induction of apoptosis of CD206+ macrophages by the nano self-assembled glycopeptide BIVA-PK in example 6 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 construction and analysis of mouse ischemia-reperfusion Kidney injury fibrosis model

A Unilateral Ischemia Reperfusion Injury model with a C57/BL6 background is constructed and a Contralateral Nephrectomy (UAIRIx) model is performed, wherein the specific method comprises the following steps: the experimental animal is an 8-week-old C57 male mouse, and the weight of the experimental animal is 20-22 g. Intraperitoneal injection of 1% pentobarbital to anesthetize mice, expose the left kidney, separate the renal pedicle, clamp the renal artery with artery, change the color of the kidney to black purple, perform resection of the right kidney at the same time, remove the clamp after 40min, and take the patient after the perfusion of the kidney is recovered.

Immunofluorescent staining and Western blotting detection are carried out on the constructed mouse model, and the result is shown in figure 4, wherein the immunofluorescent staining result shows that the kidney CD206 of the model⁺Macrophage numbers showed time-dependent increases, suggesting that CD206 as a macrophage target of type M2 for ischemia-reperfusion renal injury fibersIndicative of chemolysis; western blotting results show that the expression level of Cathepsin B in the model is in the same trend with the number of M2 type macrophages, and suggest that Cathepsin B can be used as an enzyme cutting site to induce the responsive self-assembly of the targeted apoptosis-promoting glycopeptide.

Example 2 design and Synthesis of Nano self-assembled glycopeptide BIVA-PK

Through screening and optimization, the amino acid sequence (SEQ ID NO.1) and modification of the nano self-assembly glycopeptide BIVA-PK are finally determined as follows: (manose) -Pra-GFRG-LVFF-GKKWWWDipK, wherein the (manose) -Pra is mannose modification, the GFRG-LVFF is a cathepsin B shearing sequence (SEQ ID NO.2), the GKKWWKKWDipK is a pro-apoptotic sequence (SEQ ID NO.3), and the structural formula of the self-assembled glycopeptide BIVA-PK is shown as the formula (I). And simultaneously synthesizing a control polypeptide PK, wherein the control polypeptide only contains a pro-apoptotic sequence GKKWWKKWDipK and no mannose modification and a Cathepsin B shearing sequence.

Synthesizing a polypeptide sequence of a main body on Wang resin by adopting a solid-phase polypeptide synthesis method, coupling mannose and synthesizing glycopeptide. The obtained crude product of the self-assembly glycopeptide molecule is purified and separated by preparative chromatography, the synthesized product is identified by mass spectrum, and the morphology of the nano structure and the stacking lamellar structure of the synthesized product are characterized (figure 5).

Example 3 effectiveness of Nano self-assembling glycopeptide BIVA-PK to treat AKI-CKD Kidney fibrosis

Construction of Unilateral Ischemia Reperfusion Injury and Contralateral Nephrectomy (Unilateral Ischemia-repetition infusion and comparative Nephrectomy, uIRIx) model: the experimental animal is an 8-week-old C57 male mouse, and the weight of the experimental animal is 20-22 g. Intraperitoneal injection of 1% pentobarbital to anesthetize mice, expose the left kidney, separate the renal pedicle, clamp the renal artery with artery, change the color of the kidney to black purple, perform resection of the right kidney at the same time, remove the clamp after 40min, and take the patient after the perfusion of the kidney is recovered. Starting on 3 days after reperfusion, tail vein injecting nanometer self-assembly glycopeptide BIVA-PK with the dose of 10mg/kg and the injection frequency of 1 time/2 days for 3 times, and taking the nanometer self-assembly glycopeptide BIVA-PK as a BIVA-PK treatment group; the control group was injected with saline at the tail vein at the corresponding time.

The general condition of the mice was evaluated on day 28 after reperfusion in each group, and the results showed that the survival rate of the BIVA-PK treated mice was significantly higher than that of the control group, and the body weight of the BIVA-PK treated mice was higher than that of the control group (saline group) (fig. 6).

The peripheral blood of the mice of each group is taken 28 days after reperfusion, and the serum creatinine and urea nitrogen levels are measured to evaluate the renal function, and the results show that the nano self-assembled glycopeptide BIVA-PK can significantly reduce the serum creatinine and urea nitrogen levels after the reperfusion of the mice renal ischemia (figure 7), and has a protective effect on the renal function.

The kidney of each group is taken 28 days after reperfusion, the kidney tissue is dehydrated, fixed, embedded in paraffin and sliced, Masson staining detects kidney collagen deposition, namely a fibrotic focus, ImageJ calculates the area of the renal fibrotic focus, and the result shows that the area of the renal fibrosis is remarkably reduced after the nano self-assembled glycopeptide BIVA-PK is treated (figure 8), and the nano self-assembled glycopeptide BIVA-PK is proved to be capable of effectively reducing the degree of the renal fibrosis in the chronic stage after ischemia reperfusion.

Example 4 remodeling of AKI-CKD Kidney fibrosis microenvironment by Nano self-assembling glycopeptide BIVA-PK

Macrophages are the major immune cell type in renal fibrotic foci and play an important role in promoting renal fibrosis. This example for the nano self-assembled glycopeptide BIVA-PK to Kidney CD206 ⁺The influence of macrophage number is detected, and the specific method and the result are as follows:

collecting mouse kidney 14 days after reperfusion, preparing single cell suspension, performing flow antibody staining, and detecting CD11b by flow cytometry⁺F4/80⁺CD206⁺Macrophage number and proportion, the results show that: BIVA-PK treatment group renal CD206⁺The macrophage number is obviously less than that of the normal saline group (control group), and the BIVA-PK can effectively reduce the renal CD206 after the ischemia-reperfusion injury⁺Macrophage infiltration number (FIG. 9, P)<0.05)。

Example 5 targeting of the Nano self-assembled glycopeptide BIVA-PK to the Kidney

Marking BIVA-PK by fluorescein, clamping left renal artery of a mouse to establish ischemia reperfusion injury, not treating the right side, injecting fluorescence-marked BIVA-PK into tail vein at 7 days, and displaying the results: the fluorescence labeled BIVA-PK was significantly enriched in the kidney on the next day after injection, especially in the damaged kidney on the left side, but was weakly distributed in the heart, liver, spleen and other internal organs, demonstrating the organ selectivity of BIVA-PK in vivo (FIG. 10).

Example 6 targeting of the Nano self-assembled glycopeptide BIVA-PK to macrophages

Based on the observed changes of the number and phenotype of the kidney macrophages of the AKI-CKD after the BIVA-PK is administrated in the in vivo experiment, the influence of the BIVA-PK on the phenotype and the function of the macrophages and the molecular mechanism are further verified in vitro.

Macrophage cell line RAW264.7 is cultured in vitro, cytokine IL-4 is utilized to induce CD206 expression, meanwhile, the renal tubular epithelial cell line TCMK-1 is cultured, ALEXA FLUOR 647 marked BIVA-PK (10 mu M) is added into culture supernatant of the two cells, the cells are incubated for 2 hours at 37 ℃, and fluorescence signals in macrophages are detected by high content imaging. The result shows that the macrophage has obvious fluorescence signal enrichment, but no obvious fluorescence signal is detected in the renal tubular epithelial cell, and the BIVA-PK is proved to selectively target and enter the CD206⁺Within macrophages, no effect on tubular epithelial cells (FIG. 11, P)<0.05)。

Culturing macrophage cell line RAW264.7 in vitro, inducing CD206 expression by using cytokine IL-4, and mixing BIVA-PK, PK and CD206 with different concentrations⁺Macrophage incubation, incubation at 37 ℃ for 6 hours, CCK8 to determine cell activity, and drug dose-response relationship was calculated. The results show that BIVA-PK has significant pro-apoptotic effect on M2-type macrophages at lower concentrations (IC50 ═ 8.37 μ M), with the pro-apoptotic effect increasing with increasing concentration, whereas PK is less pro-apoptotic at equivalent doses (figure 12). Separating primary mesangial cells of mice, culturing a tubular epithelial cell line TCMK-1, and mixing the two kinds of intrinsic cells of kidney and CD206 ⁺Respectively inoculating macrophages to a 96-well plate (10000 per well), respectively co-incubating with BIVA-PK with different concentrations, incubating at 37 ℃ for 6 hours, measuring the cell activity by CCK8, and calculating the drug dose effect relationship; the results show that BIVA-PK has obvious apoptosis promoting effect on M2 type macrophages, and BIVA-PK is inherent to kidneyCells such as mesangial cells and tubular epithelial cells had little effect (FIG. 12, P)<0.05)。

In summary, poor repair after acute kidney injury is manifested by replacement of normal tissue architecture of the kidney by a large number of myofibroblasts and extracellular matrix, eventually progressing to renal fibrosis. M2-type macrophages are important regulators of the fibrotic microenvironment, greatly promoting the progress of fibrosis by producing various growth factors and transdifferentiating into myofibroblasts. The nano self-assembly polypeptide developed by the invention can specifically interfere M2 type macrophages in a fibrosis microenvironment, block the promotion effect of the macrophage on the fibrosis microenvironment and achieve the purpose of inhibiting the progress of fibrosis. The invention provides a new solution for treating renal fibrosis caused by chronic inflammation by accurately regulating and controlling a fibrosis microenvironment. The nano self-assembled glycopeptide BIVA-PK provided by the invention can be applied to the treatment of renal fibrosis caused by ischemia-reperfusion injury, and provides a new choice for a treatment medicament for the renal fibrosis. When the nanometer self-assembly glycopeptide BIVA-PK is applied to disease prevention and treatment related to the invention, the nanometer self-assembly glycopeptide BIVA-PK has the characteristics of receptor-dependent cell targeting and self-assembly retention after shearing, has strong cell and organ selectivity, exact effect and definite mechanism, simultaneously avoids the defects of difficult storage of protein medicinal preparations, high cost and poor curative effect lasting effect of small molecule medicaments, and is expected to improve the current situation that kidney fibrosis lacks effective intervention means.

Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Sequence listing

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Claims

Use of an inhibitor of macrophage M2 in the manufacture of a medicament for preventing or treating renal fibrosis caused by ischemia reperfusion injury.
2. The use according to claim 1, wherein the inhibitor of macrophage M2 targets CD 206.
3. The use according to claim 1 or 2, wherein the inhibitor of macrophages of type M2 is a pro-apoptotic agent of macrophages of type M2.
4. A polypeptide having the following amino acid sequence and modifications: (manose) -Pra-GFRG-LVFF-GKKWWWDipK, wherein (manose) -Pra is mannose modification, and Dip is diphenyl alanine.
5. The polypeptide of claim 4, wherein the polypeptide has a chemical structure according to formula (I):
6. use of the polypeptide of claim 4 or 5 for the preparation of a medicament for self-assembling a polypeptide in vivo.
7. Use of a polypeptide according to claim 4 or 5 for the preparation of a medicament for the targeted induction of apoptosis of M2-type macrophages.
8. Use of a polypeptide according to claim 4 or 5 in the manufacture of a medicament for the prevention or treatment of renal fibrosis.
9. The use of claim 8, wherein the renal fibrosis is that resulting from the progression of an acute renal injury to a chronic renal disease;

preferably, the renal fibrosis is renal fibrosis caused by ischemia reperfusion injury.
10. A medicament, characterized in that it comprises a polypeptide according to claim 4 or 5.