CN115466321A - FOXO3a-DRI peptide fragment, pharmaceutical composition and application thereof - Google Patents
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- CN115466321A CN115466321A CN202211165350.4A CN202211165350A CN115466321A CN 115466321 A CN115466321 A CN 115466321A CN 202211165350 A CN202211165350 A CN 202211165350A CN 115466321 A CN115466321 A CN 115466321A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
Abstract
The invention discloses a FOXO3a-DRI peptide fragment, a pharmaceutical composition and an application thereof, wherein the peptide fragment is obtained by performing full D-type synthesis on an obtained FOXO3a147-179 reverse sequence by taking a147-179 amino acid sequence of FOXO3a as a peptide fragment master matrix; the FOXO3a-DRI peptide segment has the effects of remarkably reducing urine protein, improving podocyte apoptosis and injury, and inhibiting glomerular injury, kidney fibrosis and DKD development, can be used for preparing a medicine for effectively treating or preventing DKD, and effectively promotes the research development of the medicine for treating or preventing diabetic nephropathy.
Description
Technical Field
The invention belongs to the technical field of diabetic nephropathy prevention and treatment medicines, and particularly relates to an FOXO3a-DRI peptide fragment, a pharmaceutical composition and application thereof.
Background
Diabetes Mellitus (DM), a systemic metabolic disease, has now become a global public health problem that seriously compromises human health. Diabetic nephropathy (DKD) is one of the most common and serious microvascular complications of DM, and the improvement in risk in recent 30 years is no better than other complications, and has now become the leading cause of end-stage renal disease (ESRD) patients in developed countries (New Engl J med.2014; 370. However, the current clinically effective treatment of DKD is limited, and despite multifaceted interventions such as blood glucose and blood pressure control, urinary protein reduction, lipid metabolism disorder correction and lifestyle improvement, the risk of death from ESRD combined DM remains high, and the 10-year cumulative mortality rate for patients with albuminuria and impaired glomerular filtration is still as high as 47% (J Am Soc nephrol.2013; 24. Therefore, aiming at the key pathogenic mechanism of DKD, a safe and effective method for treating DKD is found, and the problem that research workers for renal diseases need to solve at present is to reduce the incidence rate of ESRD.
Oxidative stress is a key factor in the initiation, development, and progression of DKD (episxed Redox Sign 2016 25. Podocytes are kidney cells that are highly susceptible to oxidative stress and are susceptible to damage that causes proteinuria. Numerous studies have shown that oxidative stress-induced podocyte injury, and specifically podocyte apoptosis, is a key factor in the development of DKD proteinuria and glomerular sclerosis (Diabetologia 2016. The forkhead protein FOXO3a is used as an oxidative stress response transcription factor, is highly expressed in podocytes of kidney penetrating tissues of DKD patients, and can promote the apoptosis of the podocytes by mediating the transcription of downstream apoptosis-promoting target genes such as Bim, fasL and the like (FASEB J,2020,34, 13300-13316. FOXO3a and P53 are combined with each other to regulate the transcription of pro-apoptotic genes and activate mitochondrial apoptotic pathways, which are potential mechanisms for FOXO3a to regulate apoptosis. Therefore, finding a method for blocking the mutual combination of FOXO3a-P53 has important significance for inhibiting the apoptosis of podocytes and delaying or even preventing the occurrence and development of DKD.
The FH region and CR3 region of FOXO3a can combine with P53 to promote P53 stabilization and activation (JMol biol.2008,384: 590-603), wherein the FH region of FOXO3a is also a DNA binding region, which indicates that the FH region is a key region for FOXO3a to regulate the cellular biological activity, and no research on the specific polypeptide applied to improve kidney tissue damage and inhibit kidney fibrosis and report on delaying or/and reversing DKD is proposed in the areas.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides the FOXO3a-DRI peptide fragment which is used for preparing medicaments and promoting the research progress of medicaments for treating or preventing diabetic nephropathy.
In view of this, the scheme of the invention is as follows:
the invention provides an FOXO3a-DRI peptide segment, the amino acid sequence of which is shown as SEQ ID NO:1 is shown.
In one embodiment of the invention, the FOXO3a-DRI peptide fragment is obtained by performing full D-type synthesis on the obtained FOXO3a147-179 reverse sequence by performing amino acid reverse sequencing on a147-179 amino acid sequence of FOXO3a as a peptide fragment master.
Another objective of the invention is to provide a pharmaceutical composition comprising the FOXO3a-DRI peptide fragment as described above and a pharmaceutically acceptable carrier.
As a preferred aspect of the invention, the FOXO3a-DRI peptide fragment comprises at least one derivative of FOXO3a-DRI, said derivative comprising a shorter peptide fragment comprising the amino acid sequence of FOXO3a-DRI, an FOXO3a-DRI polypeptide after amino acid substitution, and a FOXO3a-DRI and shorter peptide fragments thereof after chemical modification.
The invention also provides application of the FOXO3a-DRI peptide fragment or the pharmaceutical composition in preparing a medicament for reducing proteinuria.
The invention also provides application of the FOXO3a-DRI peptide fragment or the pharmaceutical composition in preparing a medicine for improving podocyte injury.
The invention also provides application of the FOXO3a-DRI peptide fragment or the pharmaceutical composition in preparation of drugs for inhibiting podocyte apoptosis.
Preferably, the FOXO3a-DRI peptide fragment in the medicament for inhibiting podocyte apoptosis has a concentration of 0.5-10 mu M.
The invention also provides application of the FOXO3a-DRI peptide fragment or the pharmaceutical composition in preparing medicines for inhibiting glomerular injury and renal fibrosis.
The invention also aims to provide application of the FOXO3a-DRI peptide fragment or the pharmaceutical composition in preparing a medicament for treating or preventing diabetic nephropathy.
Compared with the prior art, the invention has the advantages that but not limited to:
1. the FOXO3a-DRI peptide provided by the invention has the effects of obviously reducing urine protein, improving podocyte apoptosis and injury, and inhibiting glomerular injury, renal fibrosis and DKD development, can be used for preparing a medicine for effectively treating or preventing DKD, and effectively promotes the research development of the medicine for treating or preventing diabetic nephropathy.
2. The FOXO3a-DRI peptide provided by the invention can be obtained by artificial synthesis after amino acid reverse sequencing is carried out on the FOXO3 a-based 147-179 amino acid sequence serving as a peptide master plate, and the method is simple, convenient and low in cost.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a synthetic route diagram for FOXO3 a-DRI.
FIG. 2 shows the results of the FOXO3a-DRI detection of the improvement of the DKD model mouse proteinuria and the detection of the relevant indexes of the podocyte injury process. Wherein, fig. 2A is urine albumin results; FIG. 2B is a graph showing representative results of immunoblot detection of mouse kidney tissue podocyte markers; FIG. 2C is a statistical plot of Podocin protein; FIG. 2D is a statistical chart of Synaptopodin protein; FIG. 2E is a statistical diagram of WT1 protein; FIG. 2F is the transmission electron microscopy results of kidney tissue, and immunofluorescence staining patterns of Podocin and Synaptopodin of each group of mice.
FIG. 3 shows the results of FOXO3a-DRI in improving DKD model mouse glomerular injury and kidney fibrosis process related index detection. Wherein, FIG. 3A shows the results of the immunofluorescence staining of PAS and Masson staining and mouse kidney fibrosis indexes fibrinectin and Collagen I of each group of mice; FIG. 3B is a graph of a glomerular injury score statistic; FIG. 3C is a statistical graph of the percentage of fibrinectin positive staining; FIG. 3D is a statistical chart of percent positive staining for Collagen I.
FIG. 4 shows the results of FOXO3a-DRI detection of the DKD model mouse kidney apoptosis index process. Wherein, fig. 4A is a representative result of immunoblot detecting mouse renal tissue apoptosis indicators; FIG. 4B is a histogram of P53 protein; FIG. 4C is a histogram of Bax protein; FIG. 4D is a statistical plot of clear caspase3 protein.
FIG. 5 shows the results of FOXO3a-DRI assay for inhibiting the high sugar-induced apoptosis of podocytes. Wherein, fig. 5A is the result of CCK8 detection on the viability of the podocytes of each group; FIG. 5B is a representative result of immunoblot detection of various sets of apoptosis indicators; FIG. 5C is a statistical plot of Bax protein; FIG. 5D is a statistical plot of the cleared caspase3 protein; fig. 5E shows the fluorescent staining results of TUNEL detection of apoptosis in each group of podocytes.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention more clearly apparent, the technical solutions of the present invention are further described in detail below with reference to examples, and it should be understood that the specific embodiments described in the present specification are only for explaining the present invention and are not intended to limit the present invention.
In one embodiment of the invention, a FOXO3a-DRI peptide fragment is provided, the amino acid sequence of which is tlrkdpssseiarldayslingwanssckrp. By single side kidney cut STZ induced DKD mouse model experiment on FOXO3a-DRI peptide fragment. The results show that: compared with a DKD model group, FOXO3a-DRI group urine protein is obviously reduced, podocyte apoptosis and podocyte injury are obviously reduced, expansion of glomerular mesangial matrix, glomerular sclerosis and renal interstitial fibrosis are obviously improved, and apoptotic proteins such as P53, bax and clear caspase3 are obviously reduced, so that the FOXO3a-DRI peptide segment can antagonize an apoptotic pathway, effectively improve podocyte apoptosis and injury and inhibit renal fibrosis.
In one embodiment of the invention, the FOXO3a-DRI peptide fragment is obtained by performing full D-type synthesis on the obtained FOXO3a147-179 reverse sequence by taking the 147-179 amino acid sequence of the FOXO3a as a master peptide fragment, and the synthetic route is shown in FIG. 1.
In another embodiment of the invention, a pharmaceutical composition is provided comprising a FOXO3a-DRI peptide fragment having the amino acid sequence: tlrkdssieartlgaldgwanssrckrp.
As a preferred example, the pharmaceutical composition wherein the FOXO3a-DRI peptide comprises at least one derivative of FOXO3a-DRI comprising a shorter peptide comprising the amino acid sequence of FOXO3a-DRI, an amino acid-substituted FOXO3a-DRI polypeptide, and a chemically modified FOXO3a-DRI and its shorter peptide.
As used herein, "peptide fragment" refers to a polymer of two or more amino acids. Can be modified into analogs, derivatives, functional mimetics, pseudopeptides, and the like of the same, and the like, comprising at least two amino acids. The meaning of the term "peptide fragment" is well known to those skilled in the art. In general, a peptide segment is composed of two or more amino acids linked by an amide bond, which is composed of an amino group of one amino acid and a carboxyl group of an adjacent amino acid. The peptide fragments described herein may comprise naturally occurring amino acids and/or non-naturally occurring amino acids.
The term "derivative" as used herein refers to a variant of a polypeptide. For example, a derivative of a polypeptide may include a variant in which one or more amino acids are substituted relative to the polypeptide. Also included are modifications to the polypeptide, including but not limited to non-naturally occurring amino acids, D-amino acids, amino and/or carboxy-terminal (N-or C-terminal) modified amino acids, particularly modifications to the amino and/or carboxy group at the N-terminus, fatty acid modified peptidomimetics and pseudopeptides, and the like, as are common.
As a preferred example, in the pharmaceutical composition, the pharmaceutically acceptable carrier includes but is not limited to: one or more of diluents, excipients, binders, fillers, disintegrants, wetting agents, absorption enhancers, adsorption carriers, surfactants, or lubricants; the pharmaceutical composition can be prepared into tablets, granules, capsules, oral liquid or injections according to the actual preparation needs of the medicines.
In one embodiment of the invention, on the basis of FOXO3a-DRI peptide segment, urokinase can be obviously reduced, podocyte apoptosis and podocyte injury are obviously relieved, expansion of mesangial matrix, glomerular sclerosis and renal interstitial fibrosis are obviously improved, and apoptotic proteins such as P53, bax and clear caspase3 are obviously reduced, so that the FOXO3a-DRI peptide segment can antagonize an apoptotic pathway, the podocyte apoptosis and injury are effectively improved, and the renal fibrosis is inhibited. I.e. the FOXO3a-DRI peptide fragment or the pharmaceutical composition as described hereinbefore, can be used for the preparation of a medicament for inhibiting or ameliorating the above individual symptoms, and further for the preparation of a medicament for treating or preventing diabetic nephropathy.
The invention is based on the following model theory and pathological basis when researching FOXO3a-DRI peptide segment and performing drug effect verification:
DKD mice induced by Streptozotocin (STZ) after unilateral nephrectomy are classical animal models of DKD and characterized by marked evidence of long-term hyperglycemia with kidney damage, including podocyte apoptosis, inflammatory cell infiltration, tubular cell atrophy, fibronectin (fibrinectin) and Collagen type I (Collagen I) deposition, and glomerular sclerosis. The modeling method is simple and easy to implement, has high success rate, can better simulate the course of disease and the pathological manifestations of the kidney of clinical DKD, and is suitable for the research of DKD.
Pathologically, the ultrastructure of podocytes is usually observed by a transmission electron microscope, and the damage degree of the podocytes is determined by combining the expression conditions of podocyte specific markers, namely podocyclin (Podocin), synaptopodin (Synaptopodin) and Wilms tumor type 1 gene (WT 1). PAS staining was used to observe glomerular hypertrophy and mesangial matrix expansion. Masson staining was used to visualize collagen deposition in tissues. Immunofluorescent staining for fibronectin and collagen type I to visualize more specific renal fibrosis. The immune blotting method (Western blotting) detects apoptosis proteins such as P53, bax, cleared caspase3 and the like, and TUNEL staining detects the breakage of DNA for detecting the apoptosis condition of cells.
Example 1: synthetic method of FOXO3a-DRI peptide fragment
The FOXO3a-DRI peptide fragment synthesis method is shown in figure 1, wherein CR represents a conserved c-terminal transactivation region; FH represents the fork DNA binding region. Selecting 147-179 amino acid sequences as a peptide fragment master FOXO3a according to the interaction area of FOXO3a and P53 147-179 The master plate FOXO3a is prepared 147-179 Amino acids in the reverse order, i.e. position 179T is placed at 147 bits, L at 178 bits is placed at 148 bits, and so on, the FOXO3a is obtained 147-179 Reverse sequence, will FOXO3a 147-179 And (3) carrying out full D-type synthesis on the reverse sequence to obtain a FOXO3a-DRI peptide fragment, wherein the amino acid sequence is tlrkdpssseiarldysingwanrrssckrp and is marked as SEQ ID NO:1. the full D-type synthesis of FOXO3a-DRI peptide fragment was artificially synthesized by Shanghai Jier Biochemical Co., ltd.
Example 2: therapeutic effect of FOXO3a-DRI on DKD model mice
1. Experimental animals: c57BL/6 male mice, 8 weeks old, SPF grade.
Animals were weighed and numbered first, 18 healthy mice weighing 21-23g were selected and randomly divided into 3 groups of 6 mice each. Comprises a control group, a diabetic nephropathy model control group and a medicine group.
2. Experimental modeling, grouping and drug delivery interventions
2.1 DKD modeling method and grouping
2.1.1 unilateral Kidney resection
First, control and unilateral nephrectomy groups were randomized. Mice were anesthetized by intraperitoneal injection at a dose of 0.006ml/g with 1% phenobarbital sodium (in saline), and then fixed on an operation plate in the prone position. Preparing skin on the back of a mouse, disinfecting the skin by an alcohol cotton ball, cutting a 2.0cm incision at the position of a right kidney to expose a muscular layer, shearing a 1cm incision (the incision length is less than the kidney length) at the position of the incision exposure position in parallel with the kidney by a direct scissors, supporting the kidney towards the incision direction, clamping a kidney pedicle at the incision, knotting the kidney pedicle by a suture with the length of about 10cm, completely shearing the kidney by a bending scissors, leaving the knotted kidney pedicle, leaving a 0.1-0.2mm thread end, slightly wiping off blood by a physiological saline cotton ball, observing no dynamic bleeding, sewing the incision, slightly putting the mouse on a metal bath at 37 ℃ for rewarming, putting the mouse back into a clean mouse cage after reviving, and providing sufficient water and feed. The control group was subjected to sham operation.
2.1.2 intervention with STZ given 1 week post nephrectomy
Mice returned to baseline normality 1 week post-surgery and were ready for STZ injection. Mice were starved for 6h in advance. The following solutions A and B are prepared:
solution A: weighing 1.05g of citric acid, and fixing the volume to 50ml of pure water;
and B, liquid B: 1.47g of sodium citrate is weighed to a volume of 50ml of pure water.
Respectively taking 30ml of the solution A and 40ml of the solution B, fully and uniformly mixing, adjusting the pH value to 4.5, and preparing for use. 100mg of STZ is weighed away from light and dissolved in 20ml of AB mixed solution to prepare 5mg/ml, the mixture is fully shaken and uniformly mixed, after the mixture is filtered by a filter with the diameter of 0.22 mu m, the intraperitoneal injection of the mice with the dose of 50mg/kg is started immediately, after all the mice with kidney cuts are beaten, the mice are divided into 2-3 cages, and sufficient water and feed are given. Intraperitoneal injection is carried out for 5 days at the same dosage and method, and AB mixed solution is given as a control group. Random blood glucose measurements were given on day 14, i.e., at the end of week 2 of the disease process, and mice with blood glucose in the range of 300-600mg/dl (i.e., 16-33 mmol/L) were randomly assigned as model and dosing groups.
2.2 FOXO3a-DRI peptide fragment intervention
The FOXO3a-DRI peptide fragment water-soluble powder is dissolved by sterile PBS to prepare a mother solution with the concentration of 5 mg/ml. And (4) raising each component of the experimental group in cages. The control group was observed only. Model groups were given only intraperitoneal injections of PBS, once every other day. The group was administered 2 weeks after STZ intervention by intraperitoneal injection at a dose of 0.5mg/kg every other day for 10 consecutive weeks. Each group of mice was sacrificed 12 weeks after starting the STZ intervention, and the mice urine and kidneys were taken. Urine is used for detecting urine protein and urine creatinine. Kidneys were fixed with 10% neutral buffered formaldehyde, OCT fixed, 2.5% glutaraldehyde fixed and liquid nitrogen frozen tissues, respectively.
3. Observation indicator and measuring method
3.1 the urine adopts a mouse urine albumin ELISA detection kit to detect urine albumin, and a DICT-500 creatinine kit to detect urine creatinine. The urine albumin to urine creatinine ratio was used for comparison between groups to assess the severity of urine protein.
3.2 Formaldehyde-fixed tissues were dehydrated, embedded, sectioned, and then stained with PAS and Masson, respectively. PAS staining was used to observe glomerular injury and Masson staining was used to detect the degree of fibrosis in the kidney tissue.
3.3 After the OCT fixed tissues are sliced by a freezer, immunofluorescent staining is carried out on podocyte markers (Podocin and Synaptopodin) and fibrosis indexes (Fibronectin and Collagen 1).
3.4. Glutaraldehyde fixed tissue is prepared into an electron microscope specimen, and then the transmission electron microscope is adopted to observe the ultrastructure of glomerulus and podocyte.
3.5 extracting protein after freezing the tissue homogenate by liquid nitrogen, and detecting the protein expression level of podocyte markers (Podocin, synaptopodin and WT 1) and apoptosis indexes (P53, bax and cleared caspase 3) by adopting an immunoblotting method.
4. Results of the experiment
4.1 FOXO3a-DRI for improving DKD model mouse proteinuria and podocyte injury
The results of the FOXO3a-DRI on improving the proteinuria and podocyte injury process of the DKD model mouse are shown in figure 2, wherein A in figure 2 is the comparison result of urinary albumin of each group; b is the result of detecting the protein expression of podocyte markers Podocin and Synaptopodin of kidney tissues of each group of mice by immunoblotting, and the numbers 1 and 2 represent 2 different mice in each group; c is a statistical chart of Podocin protein; d is a statistical chart of the Synaptopodin protein; e is a statistical chart of WT1 protein; f is a transmission electron microscope result image of kidney tissues of each group of mice, and the scale is 1 mu m; immunofluorescent staining patterns of Podocin and Synaptopodin with a scale of 25 μm. Wherein Ctrl represents a control group; DKD represents diabetic nephropathy model group; DKD + FOXO3a-DRI represents the group administered. * P<0.01V.S Ctrl, ## P<0.01V.S DKD，n＝5-6。
FIG. 2A shows that FOXO3a-DRI treated group had significantly lower urinary protein than the DKD model group. The immunoblot results in fig. 2B show that FOXO3a-DRI treated groups significantly up-regulated protein expression of podocyte markers (Podocin, synaptopodin and WT 1) compared to the DKD model group, with statistical significance of the differences (fig. 2C-E). The Transmission Electron Microscopy (TEM) results of fig. 2F show that the DKD model group thickened basement membrane, podocyte foot process fused, and the septal disappearance, while the FOXO3a-DRI treated group significantly reversed these pathological changes. Figure 2F shows that the FOXO3a-DRI treated group significantly restored continuous linear expression of podocyte markers Podocin and Synaptopodin in DKD mice.
4.2FOXO3a-DRI improves the glomerular injury and renal fibrosis of DKD model mice
FOXOThe process of 3A-DRI improving the glomerular injury and the renal fibrosis of the DKD model mouse is shown in figure 3, in figure 3A, PAS staining, masson staining, immunofluorescence staining of Fibronectin and Collagen I of each group of mice are sequentially performed from top to bottom, and the scale is 100 micrometers; FIG. 3B is a graph of a glomerular injury score statistic; FIG. 3C is a statistical graph of the percentage of fibrinectin positive staining; FIG. 3D is a statistical chart of percent positive staining for Collagen I. Wherein Ctrl represents the control group; DKD represents diabetic nephropathy model group; DKD + FOXO3a-DRI represents the group administered. * P<0.01V.S Ctrl, ## P<0.01V.S DKD，n＝5-6。
The PAS staining in figure 3A shows that expansion of glomerular mesangial regions and glomerular hypertrophy are significantly improved in FOXO3A-DRI treated group compared to DKD model group, and that the difference in glomerular injury is statistically significant (figure 3B). The Masson staining of figure 3A shows that glomerular collagen deposition was significantly lower in FOXO3A-DRI treated group than in DKD model group. The immunofluorescence staining results of fibrosis indexes fibrinectin and Collagen 1 in fig. 3A show that the degree of glomerular and tubulointerstitial fibrosis in the FOXO3A-DRI treated group was significantly reduced compared to the DKD model group, and the difference was statistically significant (fig. 3C and 3D).
4.3 Index for inhibiting DKD model mouse kidney apoptosis by FOXO3a-DRI
The results of the FOXO3a-DRI process verification for inhibiting the DKD model mouse kidney apoptotic protein are shown in FIG. 4, wherein FIG. 4A shows the results of immunoblot detection of mouse kidney tissues P53, bax, clear caspase3 and other apoptotic proteins, and the numbers 1 and 2 represent 2 different mice in each group; FIG. 4B is a histogram of the P53 protein; FIG. 4C is a histogram of Bax protein; FIG. 4D is a statistical plot of clear caspase3 protein. Wherein Ctrl represents a control group; DKD represents diabetic nephropathy model group; DKD + FOXO3a-DRI represents the group administered. * P<0.01V.S Ctrl, ## P<0.01V.S DKD，n＝5-6。
The immunoblot results in FIG. 4A show that levels of apoptosis indicators such as renal tissue P53, bax and clear caspase3 were significantly reduced and statistically significant in the FOXO3a-DRI treated groups compared to the DKD model group (FIGS. 4B-4D).
Example 3: FOXO3a-DRI inhibits high-sugar-induced podocyte apoptosis
1. Subject: conditionally immortalized mouse podocyte MPC5.
2. Preparation and grouping of cell models
After differentiation of the podocytes at 37 ℃ for 7-14 days, normal glucose NG (5.6 mmol/L glucose), osmolality control (24.4 mmol/L mannitol +5.6mmol/L glucose) and high-glucose HG (30 mmol/L glucose) were respectively intervened, wherein the high-glucose group was intervened with 0, 0.5, 1, 2.5, 5, 10, 20. Mu.M FOXO3a-DRI, and cell viability was measured after 24 hours of total intervention. In other experiments, three concentrations of 0.5, 1 and 2.5 mu M are selected for intervention to detect the apoptosis condition within 24 hours.
3. Observation indicator and measuring method
3.1 measurement of cell viability comparison was made by calculating the cell death ratio after CCK8 measurement.
3.2 foot apoptosis proteins the levels of Bax and clear caspase3 were detected by immunoblotting.
3.3 DNA fragmentation in podocyte apoptosis was detected by TUNEL fluorescent staining.
4. Results of the experiment
The results of FOXO3a-DRI assay for inhibiting the high sugar-induced apoptosis process of podocytes are shown in FIG. 5. Wherein, fig. 5A represents the results of CCK8 assay on the viability of the podocytes from each group; FIG. 5B is the result of comparing the detection of apoptosis proteins such as P53 and cleared caspase3 in each group of cells by immunoblotting; FIG. 5C is a histogram of Bax protein; FIG. 5D is a statistical plot of the cleared caspase3 protein; fig. 5E shows the fluorescent staining results of TUNEL detection of apoptosis in each group of podocytes. NG: normal sugar, containing 5.6mmol/L glucose; man: osmotic pressure control, containing 24.4mmol/L mannitol +5.6mmol/L glucose; HG: high sugar, containing 30mmol/L glucose. * P<0.01V.S Ctrl, # P<0.05V.S DKD， ## P<0.01V.S DKD, n.s. no statistical difference, n = 3-6)
The results of CCK8 in FIG. 5A show that FOXO3a-DRI in the range of 0.5-10 μ M significantly improved the viability of podocytes in high sugar environments, and in particular FOXO3a-DRI in the range of 0.5-2.5 μ M had a concentration-dependent improvement, while FOXO3a-DRI at 20 μ M did not significantly improve, but did not produce cytotoxicity. The immunoblot results in FIG. 5B show that FOXO3a-DRI inhibits the expression of the high-sugar induced podocyte apoptotic protein Bax and clear caspase3 in a concentration-dependent manner, with statistical significance (FIGS. 5C, 5D). The TUNEL fluorescent staining results of fig. 5E show that high sugars significantly induced podocyte apoptosis, whereas FOXO3a-DRI inhibited podocyte apoptosis in a dose-dependent manner.
In conclusion, the FOXO3a-DRI peptide segment can obviously reduce the proteinuria of a DKD mouse, obviously improve podocyte injury, inhibit podocyte apoptosis and obviously inhibit glomerular injury and kidney fibrosis. Therefore, the FOXO3a-DRI peptide fragment can be a new medicine for effectively treating DKD or inhibiting the DKD from developing.
Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Various changes and modifications may be effected therein by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present disclosure, and these changes and modifications are intended to be within the scope of the present disclosure.
Claims (10)
- A foxo3a-DRI peptide, characterized in that its amino acid sequence is as set forth in SEQ ID NO:1 is shown.
- 2. The FOXO3a-DRI peptide fragment of claim 1, wherein the FOXO3a147-179 amino acid sequence is used as a master peptide fragment for amino acid reverse sequencing, and the obtained FOXO3a147-179 reverse sequence is synthesized in full D form.
- 3. A pharmaceutical composition comprising the FOXO3a-DRI peptide fragment of claim 1 and a pharmaceutically acceptable carrier.
- 4. The pharmaceutical composition of claim 3, wherein the FOXO3a-DRI peptide fragment comprises at least one derivative of FOXO3a-DRI, said derivative comprising a shorter peptide fragment comprising the FOXO3a-DRI amino acid sequence, a FOXO3a-DRI polypeptide after amino acid substitution, and a FOXO3a-DRI and shorter peptide fragments thereof after chemical modification.
- 5. Use of the FOXO3a-DRI peptide fragment according to any of claims 1-2 or the pharmaceutical composition according to any of claims 3-4 for the manufacture of a medicament for reducing proteinuria.
- 6. Use of the FOXO3a-DRI peptide fragment of any one of claims 1-2 or the pharmaceutical composition of any one of claims 3-4 for the manufacture of a medicament for ameliorating podocyte injury.
- 7. Use of the FOXO3a-DRI peptide fragment of any one of claims 1-2 or the pharmaceutical composition of any one of claims 3-4 in the manufacture of a medicament for inhibiting apoptosis in a podocyte.
- 8. The use of claim 7, wherein the concentration of the FOXO3a-DRI peptide fragment in the medicament for inhibiting podocyte apoptosis is 0.5-10 μ M.
- 9. Use of the FOXO3a-DRI peptide fragment of any one of claims 1-2 or the pharmaceutical composition of any one of claims 3-4 for the manufacture of a medicament for inhibiting glomerular injury and renal fibrosis.
- 10. Use of the FOXO3a-DRI peptide fragment according to any of the claims 1-2 or the pharmaceutical composition according to any of the claims 3-4 for the preparation of a medicament for the treatment or prevention of diabetic nephropathy.
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