Active peptide capable of directionally inducing IPSCS cracking, preparation method of active peptide and application of active peptide in preparation for repairing liver injury

Abstract

The invention discloses an active peptide capable of directionally inducing IPSCS cracking, a preparation method of the active peptide and application of the active peptide in a preparation for repairing liver injury. Experiments prove that the active peptide provided by the invention has a remarkable protective effect on a liver injury model induced by cyclophosphamide and shows a definite anti-oxidative stress effect. The result shows that a key path of chemotherapy injury can be intervened from the source by simulating or enhancing an endogenous protection mechanism of the liver. Compared with the existing exogenous synthetic drugs or single-component natural products, the product provided by the invention has the potential advantages of more comprehensive action mechanism and better biogenic property. Meanwhile, the extract is derived from directionally differentiated functional cells, and a brand new thought and material basis are provided for developing a novel chemotherapy liver protective agent which does not affect the chemotherapy curative effect and has good biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of biological agents, and more particularly to an active peptide that can induce cleavage of IPCS, its preparation method, and its application in agents for repairing liver damage. Background Technology

[0002] Chemotherapy is one of the core methods of cancer treatment, but drug-induced liver injury is a common and serious complication in clinical practice. It can lead to chemotherapy interruption, dose reduction, or even acute liver failure, directly affecting the patient's treatment effect and survival prognosis.

[0003] Currently, there are significant shortcomings in clinical management strategies for chemotherapy-related liver injury. First, a passive approach of treating injury after it occurs is generally adopted, lacking effective standardized prevention protocols. While some drugs used to treat liver injury (such as S-adenosylmethionine and bicyclol) ​​have shown preventative potential in studies, they are not yet widely accepted as routine prophylactic medications. Second, existing protective agents or interventions (such as certain antioxidants) often target single pathways of liver injury (such as oxidative stress or inflammation), while the mechanisms of chemotherapy-induced liver injury are complex, involving the interaction of multiple pathways such as oxidative stress, inflammatory response, apoptosis, and cholestasis. Protective strategies based on a single mechanism often have limited or incomplete effects. Furthermore, many natural products that have shown good hepatoprotective effects in preclinical studies (such as curcumin and ellagic acid) are difficult to translate and apply clinically due to their low bioavailability and lack of high-quality human clinical evidence.

[0004] Therefore, there is an urgent need in this field for a new solution that is preferably used for prevention or early intervention, has multi-target or more closely resembles the endogenous protective mechanism of the liver, and has better biocompatibility. Summary of the Invention

[0005] In view of this, one of the objectives of this invention is to provide an active peptide that can induce the lysis of pluripotent stem cells in a directed manner. The amino acid sequence of the main component, a small molecule protein peptide, is: GETGPSGPVGPAGAVGPR. Its active ingredient is more closely similar to the liver's own biological microenvironment and protective repair network.

[0006] Furthermore, the active peptide is derived from the lysate of multifunctional stem cells induced by hepatocyte growth factor (HGF).

[0007] The second objective of this invention is to provide a method for the directed induction of pluripotent stem cells to prepare bioactive peptides. This method uses hepatocyte growth factor (HGF) to induce differentiation in pluripotent stem cells, resulting in bioactive peptides containing small molecule protein peptides. The amino acid sequence of the small molecule protein peptides is: GETGPSGPVGPAGAVGPR.

[0008] Furthermore, the above-mentioned method for preparing bioactive peptides from directed induced pluripotent stem cells includes the following steps: (1) Hepatic induction: Pluripotent stem cells were induced with 50 ng / mL hepatocyte growth factor HGF for 48 hours to obtain hepatic progenitor cells expressing HNF4α and AFP; (2) Targeted induction of active peptides: Collect cells in the logarithmic growth phase after induction in step (1) and sonicate them under ice bath conditions; the sonic power is 300W, and a cycle mode of 3 seconds working and 6 seconds intermittent is adopted, with a total operation time of 5 minutes; (3) Centrifugation: Centrifuge the mixture after ultrasonic disruption in step (2) at 4°C and 8000 rpm for 10 minutes and collect the supernatant; (4) Filtration and clarification: The supernatant obtained in step (3) is filtered through a 0.22 μm filter membrane to remove cell debris and obtain an active peptide containing small molecule protein peptides. The amino acid sequence of the small molecule protein peptide is: GETGPSGPVGPAGAVGPR.

[0009] A third objective of this invention is to provide the application of the above-mentioned active peptides in preparations for repairing liver damage.

[0010] Furthermore, the above application refers to the use of bioactive peptides in liver-damaging preparations targeting cyclophosphamide.

[0011] The fourth objective of this invention is to provide a liver damage repair formulation comprising the above-described active peptide and one or more pharmaceutically acceptable excipients, carriers and / or diluents.

[0012] Compared with existing technologies, the HGF-induced lysis bioactive peptides of pluripotent stem cells and their preparation method provided by this invention have the following significant advantages and beneficial effects: 1. Highly efficient and specific preparation method: This invention employs a phased, specific concentration gradient of hepatocyte growth factor to target induced pluripotent stem cells, clearly defining the induction target as hepatic progenitor cells expressing HNF4α and AFP. This ensures that the subsequently extracted bioactive peptides have a clearly defined hepatic functional profile. Combined with a low-temperature ultrasonic disruption process using specific parameters, it efficiently and gently releases small molecule protein peptides from cells, maximizing their bioactivity and avoiding peptide denaturation or degradation that may occur with harsh chemical extraction methods.

[0013] 2. Clear Product Composition and Identifiable Active Ingredients: The active peptide extract prepared by this invention, identified by liquid chromatography-mass spectrometry, contains small molecule proteins / peptides with a specific amino acid sequence (GETGPSGPVGPAGAVGPR). This ensures that the product of this invention is no longer a crude extract with unknown composition, but a functional product with a clear material basis, providing crucial evidence for quality control, standardized production, and subsequent research on its mechanism of action.

[0014] 3. Significant liver-protective effects: Animal experiments have confirmed that the active peptide extract provided by this invention exhibits a clear liver-protective effect on a cyclophosphamide-induced mouse liver injury model. This effect is manifested in reducing serum transaminase (ALT and AST) levels and improving liver tissue pathological damage.

[0015] 4. Antioxidant Stress Relief: Further mechanistic studies have shown that the liver-protective effect of the active peptide extract of this invention is closely related to its antioxidant stress resistance. Experiments have confirmed that it can effectively reduce oxidative damage in liver tissue, such as reducing malondialdehyde (MDA) content and increasing the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), thereby alleviating the key factors leading to hepatocyte damage at their root.

[0016] 5. Broad Application Prospects: The active peptide extract provided by this invention, due to its clearly defined liver-specific origin, distinct active ingredients, and experimentally verified hepatoprotective and antioxidant effects, and because it originates from directed-differentiated functional cells, possesses better biocompatibility and targeting potential. It has enormous development potential and application value in the fields of preparing hepatoprotective drugs, adjuvant treatment products for liver injury, and antioxidant health foods. Furthermore, its preparation process is stable and controllable, facilitating large-scale production. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0018] Figure 1 The antioxidant capacity test results of the directed-induced active peptide provided in Example 1 of this invention are as follows: (A) T-AOC; (B) DPPH free radical scavenging capacity; (C) HRF scavenging rate. *: P<0.05, **: P<0.01, ***: P<0.001, ***: P<0.0001.

[0019] Figure 2 This is the mass spectrum of PSCP provided in Embodiment 2 of the present invention.

[0020] Figure 3 The effect of PSCP provided in Example 3 of this invention on mouse body weight and kidney coefficient. In the figure, (A) shows the changes in body weight of mice in each group; (B) shows the comparison of mouse body weight on day 18 (****: p<0.0001).

[0021] Figure 4 The effect of PSCP on mouse liver tissue pathology provided in Example 3 of the present invention.

[0022] Figure 5 The effect of PSCP provided in Example 3 of this invention on ALT, AST, SOD, MDA and CAT in mouse serum. In the figure, (A) is ALT, (B) is AST, (C) is SOD, (D) is MDA, and (E) is CAT. **: p<0.01; ***: p<0.001, ****: p<0.0001. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0025] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0026] The main reagents are as follows: DMEM / F12 culture medium GIBCO Invitrogen Corporation Fetal Bovine Serum (FBS) - Zhejiang Tianhang Biotechnology Co., Ltd. DMSO Sigma 10 kD filter membrane Pall 0.22 μm filter membrane Pall Superoxide Dismutase (SOD) Kit Nanjing Jiancheng Bioengineering Co., Ltd. Malondialdehyde (MDA) Reagent Kit Nanjing Jiancheng Bioengineering Co., Ltd. Catalase (CAT) Kit Nanjing Jiancheng Bioengineering Co., Ltd. Alanine aminotransferase (ALT) kit Nanjing Jiancheng Bioengineering Co., Ltd. Aspartate aminotransferase (AST) kit, Nanjing Jiancheng Bioengineering Co., Ltd. Protein (BCA) Reagent Kit, Nanjing Jiancheng Bioengineering Co., Ltd.

[0027] Example 1: Preparation of Active Peptides The induced pluripotent stem cells (iPSCs) used in this invention were purchased from Shanghai Caiyou Industrial Co., Ltd.

[0028] 1.1 Preparation method (1) Hepatic induction: iPSCs were induced for 12, 24 and 48 hours respectively with 10, 20 and 50 ng / mL hepatocyte growth factor (HGF) according to the cell isolation and culture conditions to promote the transformation of mesoendodermal cells into hepatic progenitor cells and obtain hepatic progenitor cells (HPCs) expressing HNF4α and AFP. (2) Targeted induction of active peptides: Collect cells in the logarithmic growth phase after induction in step (1) and sonicate them under ice bath conditions; the sonic power is 300W, and a cycle mode of 3 seconds working and 6 seconds intermittent is adopted, with a total operation time of 5 minutes; (3) Centrifugation: Centrifuge the mixture after ultrasonic disruption in step (2) at 4°C and 8000 rpm for 10 minutes and collect the supernatant; (4) Filtration and clarification: The supernatant obtained in step (3) is filtered through a 0.22 μm filter membrane to remove cell debris and obtain an active peptide containing 9 small molecule protein peptides.

[0029] 1.2 Detection of antioxidant activity of targeted induced active peptides IPSCs cells were induced with different concentrations of HGF (10, 20, and 50 ng / mL) for 12, 24, and 48 hours, respectively. Nine bioactive peptides were obtained after lysis. The antioxidant capacity of these nine bioactive peptides was determined by detecting their total antioxidant capacity (T-AOC), 1,1-diphenyl-2-picrylhydrazine (DPPH) free radical scavenging capacity, and hydroxyl radical (HRF) scavenging rate.

[0030] Total antioxidant capacity (T-AOC), 1,1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging capacity, and hydroxyl radical (HRF) scavenging rate assay kit (Wuhan Elite Biotechnology Co., Ltd.) were used to determine these parameters according to the manufacturer's instructions. T-AOC levels are expressed in mM, DPPH radical scavenging capacity is expressed in mmol VC / L, and HRF scavenging rate is expressed as a percentage (%).

[0031] The results are as follows Figure 1As shown, the active peptide (PSCP) obtained after inducing NK cells with 50 ng / mLNGF for 48 h had the strongest antioxidant capacity, and we used this induction concentration and time in all subsequent experiments.

[0032] 1.3 Key Points of Total Quality Control (QC) (1) iPSCs stage: no mycoplasma, no viral contamination, normal karyotype, and positive rate of pluripotency markers (CD9, CD24) ≥95%; (2) Active peptide stage: purity ≥98%, endotoxin content <0.1 EU / mg, no host protein residue; (3) HLCs stage: viable cell rate ≥90%, teratoma formation risk is 0, no tumorigenicity; (4) Formulation stage: Cell preparations are sterile and pyrogen-free, and the stability of active peptide preparations meets the pharmacopoeia requirements (stored at 4℃ for more than 6 months).

[0033] Three batches of active peptides were prepared, and the test results all met the quality control requirements.

[0034] Example 2: Determination of the amino acid sequence of active peptides by liquid chromatography-mass spectrometry (LC-MS / MS) 0.5 μg of active peptide (PSCP) was dissolved in solution A (0.1% formic acid aqueous solution). The peptide was loaded into a 2 cm pre-packed column (100 μm inner diameter; 3 μm C18-AQ packing material; Dr Maisch) using solution A and separated using a self-made analytical column (ReproSil-Pur C18-AQ packing material; 1.9 μm; Dr Maisch) with an inner diameter of 150 μm and a length of 12 cm.

[0035] The liquid chromatography system was Ultimate 3000 (Thermo Fisher), with a liquid gradient of 600 nl / min for 90 min (mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile solution) (0–8 min, 6–12% B; 8–60 min, 15–32% B; 60–79 min, 32–40% B; 79–80 min, 40–95% B; 80–85 min, 95% B; 85–86 min, 95–6% B; 86–90 min, 6% B).

[0036] The mass spectrometer was an Orbitrap Q Exactive (Thermo Fisher), the mass spectrometry scanning method was positive ion scanning mode, the ion transmission tube temperature was set to 320℃, and the spray voltage was 2.2 kV.

[0037] The mass spectrometer has a first-level full-scan detection range of 300-1,400 m / z, a resolution of 70,000, automatic gain control (AGC) of 5e5, and a maximum injection time of 50 ms.

[0038] Secondary use only selects peptides with a charge of 2-6 for high-energy collision dissociation, with the energy level set at 27%, resolution at 17,500, automatic gain control (AGC) at 2e4, maximum injection time at 80ms, and dynamic exclusion time at 15s.

[0039] The mass spectrometer automatically performed alternating mass spectrometry (MS) processing on the eluted fractions from PSCP, and the results are as follows: Figure 2 As shown, MS / MS analysis of the main components (m / z 781.90) revealed the presence of seven amino acids: glycine (Gly, G), glutamic acid (Glu, E), threonine (Thr, T), proline (Pro, P), serine (Ser, S), valine (Val, V), and alanine (Ala, A). The amino acid sequence was: GETGPSGPVGPAGAVGPR, with glycine accounting for approximately 56%.

[0040] Example 3: Study on the protective effect of active peptides on the liver 3.1 Establishment of a cyclophosphamide-induced mouse liver injury model Male ICR mice (18-22 g) were randomly divided into 4 groups: Control: Intraperitoneal injection of an equal volume of normal saline. Cyclophosphamide administration group (Model): mice were intraperitoneally injected with cyclophosphamide at a dose of 200 mg / kg body weight, administered once on days 14, 15, and 16.

[0041] PSCP (100 mg / kg): PSCP was administered intraperitoneally at a dose of 100 mg / kg of mouse body weight once daily for 18 days; cyclophosphamide was administered intraperitoneally at a dose of 200 mg / kg of mouse body weight on days 14, 15, and 16.

[0042] PSCP (200 mg / kg): PSCP 200 mg / kg mouse body weight was administered intraperitoneally once a day for 18 days; cyclophosphamide 200 mg / kg mouse body weight was administered intraperitoneally on days 14, 15 and 16.

[0043] On day 17, the mice were placed in metabolic cages for rearing. On day 18, urine and 0.5-1 mL of blood were collected from the orbital rim of the mice. The samples were centrifuged at 3000 r / min for 5 min at 4 ℃, and the supernatant was extracted and stored at -20 ℃ for later analysis. The mice were then euthanized by spinal dislocation, and the liver tissue was removed, weighed, and prepared for use.

[0044] 3.2 Changes in the general condition of mice Before the experiment, all mice in all groups were in good spirits, with normal water and food intake and smooth, soft fur. Compared with the Control group, the cyclophosphamide group showed decreased water and food intake, lethargy, disheveled fur and hair loss, and decreased urine output. Compared with the cyclophosphamide group, mice in both PSCP dosage groups had smoother, softer fur, no significant difference in water and food intake, normal spirits, and normal urine output.

[0045] 3.3 Changes in mouse body weight The mice were weighed, and the results were as follows: Figure 3 As shown. Figure 3 (A) shows the weight changes of the four groups of mice, and (B) shows the weight of the mice before sacrifice on day 18. Compared with the control group, the weight of the other three groups of mice was significantly reduced (p<0.001). Compared with the model cyclophosphamide group, there was no significant difference in the weight of mice in the two PSCP dosage groups.

[0046] 3.4 Histopathological and morphological observation HE staining: Drying and dewaxing: Place the slides in a drying machine at 60℃ for 2 hours. Then, place the slides in xylene I, II, and III for 10 minutes each, anhydrous ethanol I, II, and III for 5 minutes each, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol, and wash with distilled water.

[0047] Hematoxylin staining of cell nuclei: Immerse the sections in hematoxylin for 3-8 min, then wash with tap water to restore blue color.

[0048] Eosin staining of cytoplasm: Immerse the sections in eosin staining solution for 1-3 min.

[0049] Dehydration and clearing: Quickly pass the sections through 85% alcohol, 95% alcohol for 2 seconds, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 10 minutes, and xylene II for 10 minutes to clear them.

[0050] Mounting: Add an appropriate amount of neutral resin to the slide and mount it.

[0051] The pathological characteristics of kidney tissue were observed using HE staining, and the results were as follows: Figure 4 As shown in the figure. Compared with the control group, the model cyclophosphamide group showed more hepatocellular necrosis, with black granules resembling intrahepatic hemorrhage. The PSCP (100 mg / kg) group showed a small amount of inflammatory cell infiltration in the central vein and portal area of ​​the liver lobules. The PSCP (200 mg / kg) group showed basically no abnormalities in the liver tissue. This indicates that PSCP has a protective effect against cyclophosphamide-induced liver injury.

[0052] 3.5 Detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) Mouse serum samples were collected, and changes in ALT, AST, SOD, MDA, and CAT levels in the mouse serum were detected according to the kit instructions. Results are as follows: Figure 5 As shown.

[0053] Figure 5 Figures (A) and (B) show that, compared with the control group, the serum ALT and AST levels in the model cyclophosphamide group were significantly increased (p<0.01, p<0.001). Compared with the model cyclophosphamide group, the serum ALT levels in the PSCP (100 mg / kg) and PSCP (200 mg / kg) groups were significantly decreased (p<0.0001), and the serum AST levels in the PSCP (200 mg / kg) group were significantly decreased (p<0.001). These results indicate that PSCP can significantly improve liver damage.

[0054] Figure 5 (C) shows that, compared with the control group, the serum SOD activity in the model cyclophosphamide group was significantly decreased (p<0.001). Compared with the model cyclophosphamide group, the SOD activity in the PSCP (200 mg / kg) group was significantly increased (p<0.01).

[0055] Figure 5 The results (D) show that, compared with the control group, the serum MDA activity in the model cyclophosphamide group was significantly increased (p<0.01). Compared with the model cyclophosphamide group, the serum MDA activity in the PSCP (200 mg / kg) group was significantly decreased (p<0.01).

[0056] Figure 5 The middle (E) panel showed that there was no significant difference in serum CAT activity between the control group and the model cyclophosphamide group. Compared with the model cyclophosphamide group, serum CAT activity was significantly increased in the PSCP (100 mg / kg) and PSCP (200 mg / kg) groups (p<0.05, p<0.001).

[0057] This demonstrates that PSCP has an antioxidant effect on mice.

[0058] In summary, the active peptides provided by this invention have been experimentally verified to have a significant protective effect against cyclophosphamide-induced liver injury models and exhibit a clear anti-oxidative stress effect. This suggests that it may intervene in key pathways of chemotherapy-induced damage at the source by mimicking or enhancing the liver's endogenous protective mechanisms. Compared with existing exogenous synthetic drugs or single-component natural products, the product of this invention has the potential advantages of a more comprehensive mechanism of action and better biocompatibility. Furthermore, the extract is derived from directed-differentiated functional cells, providing a novel approach and material basis for developing a new chemotherapy-protective agent with good biocompatibility that does not affect the efficacy of chemotherapy.

[0059] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. An active peptide for targeted induction of lysis of pluripotent stem cells, characterized in that, The amino acid sequence of the main component, a small molecule protein peptide, is: GETGPSGPVGPAGAVGPR.

2. The method for preparing bioactive peptides from directed-induced pluripotent stem cells as described in claim 1, characterized in that, The active peptides are derived from the lysate of multifunctional stem cells induced by hepatocyte growth factor (HGF).

3. A method for preparing bioactive peptides from directionally induced pluripotent stem cells, characterized in that, Hepatocyte growth factor (HGF) was used to induce differentiation of pluripotent stem cells to obtain bioactive peptides containing small molecule protein peptides. The amino acid sequence of the small molecule protein peptides is: GETGPSGPVGPAGAVGPR.

4. The method for preparing bioactive peptides from directed-induced pluripotent stem cells as described in claim 3, characterized in that, Includes the following steps: (1) Hepatic induction: Pluripotent stem cells were induced with 50 ng / mL hepatocyte growth factor HGF for 48 hours to obtain hepatic progenitor cells expressing HNF4α and AFP; (2) Targeted induction of active peptides: Collect cells in the logarithmic growth phase after induction in step (1) and sonicate them under ice bath conditions; the sonic power is 300W, and a cycle mode of 3 seconds working and 6 seconds intermittent is adopted, with a total operation time of 5 minutes; (3) Centrifugation: Centrifuge the mixture after ultrasonic disruption in step (2) at 4°C and 8000 rpm for 10 minutes and collect the supernatant; (4) Filtration and clarification: The supernatant obtained in step (3) is filtered through a 0.22 μm filter membrane to remove cell debris and obtain an active peptide containing small molecule protein peptides. The amino acid sequence of the small molecule protein peptide is: GETGPSGPVGPAGAVGPR.

5. The application of the active peptide according to claim 1 in a liver damage repair preparation.

6. The use of the active peptide according to claim 1 in a liver-repairing formulation targeting cyclophosphamide.

7. A liver damage repair agent, characterized in that, It comprises the active peptide as described in claim 1 or 2 and one or more pharmaceutically acceptable excipients, carriers and / or diluents.

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