Preparation method and application of rhein-polypeptide and solution thereof

Abstract

The present invention belongs to the field of medical technology, and discloses a preparation method and application of a rhein-polypeptide and its solution. The molecular formula is Rh-FFYGRGD, Rh represents rhein, and FFYGRGD represents polypeptide. Preparation method: (1), using a conventional solid phase polypeptide synthesis method to synthesize rhein-polypeptide, i.e., Rh-FFYGRGD; (2), adding Rh-FFYGRGD to 1#PBS buffer, adding Na2CO3 solution to adjust the pH of the system until the pH is the same as that of 1#PBS buffer, then adding 2#PBS buffer to constant volume, at which time the concentration of Rh-FFYGRGD in the system is 1~6mg / mL, heating to 70~100℃, after Rh-FFYGRGD is completely dissolved, then naturally cooling to room temperature to obtain a rhein-polypeptide solution; wherein, the Na2CO3 solution is prepared by dissolving Na2CO3 in 3#PBS buffer; the pH of 1#PBS buffer, 2#PBS buffer and 3#PBS buffer are the same and are 7.0~8.0. The rhein-polypeptide of the present invention can inhibit cell proliferation and migration ability, maintain epithelial cell morphology, and affect cell cycle progression in the TGF-β2-induced EMT model, and has application potential in proliferative vitreoretinopathy.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a preparation method and application of rhein-polypeptide and a solution thereof. Background Art

[0002] Proliferative vitreoretinopathy (PVR) is an excessive wound healing response after ocular trauma and rhegmatogenous retinal detachment surgery, leading to recurrent retinal detachment or severe vision loss. Retinal pigment epithelium (RPE) is the main cell type involved in PVR. The epithelial-mesenchymal transition (EMT) process of RPE is an important pathological mechanism of retinal fibrosis, which is characterized by cell migration, proliferation and formation of proliferative membranes, leading to obvious retinal lesions. Transforming growth factor (TGF-β2) is often used to induce the EMT model of RPE cells in vitro.

[0003] Rhein (abbreviated as Rh) is the main active ingredient of the traditional Chinese medicine rhubarb. It is a 1,8-dihydroxyanthraquinone derivative with anti-inflammatory, anti-oxidative stress, anti-oxidation and anti-fibrosis effects. It is widely used in the treatment of intractable diseases such as diabetes, kidney disease, arthritis, cancer, etc., and can cause apoptosis and cell cycle arrest of various cancer cells. However, the poor solubility and low biocompatibility of rhein monomers limit its clinical application.

[0004] In recent years, peptide self-assembled nanomaterials have unique drug effects, simple synthesis, good biocompatibility, low immunogenicity and toxicity, and rapid drug response, and have shown great application prospects in drug delivery, immune adjuvants, tissue engineering, etc. If a rhein-peptide can be developed, it is expected to play an important role in proliferative vitreoretinopathy. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the object of the present invention is to provide a preparation method and application of rhein-polypeptide and its solution.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A rhein-polypeptide, the molecular formula of which is Rh-FFYGRGD, wherein Rh represents rhein, and FFYGRGD represents a polypeptide (the amino acid sequence is shown in SEQ ID No. 1); the structural formula is as follows: .

[0007] A method for preparing a rhein-polypeptide solution, comprising the following steps: (1) Synthesize rhein-peptide, i.e., Rh-FFYGRGD, by conventional solid phase peptide synthesis method, where Rh represents rhein and FFYGRGD represents peptide; (2) Add Rh-FFYGRGD to 1# PBS buffer and add Na 2 CO 3 The solution adjusts the pH of the system until it is the same as the pH of 1# PBS buffer, and then adds 2# PBS buffer to make up the volume. At this time, the concentration of Rh-FFYGRGD in the system is 1~6 mg / mL, and is heated to 70~100°C. After Rh-FFYGRGD is completely dissolved, it is naturally cooled to room temperature to obtain a rhein-polypeptide solution; wherein the Na 2 CO 3 The solution is composed of Na 2 CO 3 It is prepared by dissolving in 3# PBS buffer; the pH of 1# PBS buffer, 2# PBS buffer and 3# PBS buffer are the same and are 7.0~8.0.

[0008] Preferably, in step (2), the concentrations of 1#PBS buffer, 2#PBS buffer and 3#PBS buffer are the same and are 0.01-0.03M.

[0009] Preferably, in step (2), Rh-FFYGRGD is added to 1#PBS buffer under ultrasonic conditions.

[0010] Preferably, in step (2), Na 2 CO 3 The concentration of the solution is 1~2M.

[0011] A use of the rhein-polypeptide in preparing a drug for inhibiting epithelial-mesenchymal transition induced by TGF-β2.

[0012] Preferably, the rhein-polypeptide is used in the preparation of a drug for treating proliferative vitreoretinopathy.

[0013] Beneficial effects: The rhein-polypeptide of the present invention can inhibit cell proliferation and migration ability, maintain epithelial cell morphology, and affect cell cycle progression in the TGF-β2-induced EMT model. It has application potential in proliferative vitreoretinopathy and can promote the clinical application of hydrophobic natural products in retinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : High-resolution mass spectrum of Rh-FFYGRGD.

[0015] Figure 2 : Effects of rhein and rhein-peptide drugs on cell proliferation.

[0016] Figure 3 :Effects of rhein and rhein-peptide drugs on TGF-β2-induced RPE cell morphology.

[0017] Figure 4 : Effects of rhein and rhein-peptide drugs on cell migration.

[0018] Figure 5 : Effects of rhein and rhein-peptide drugs on cell cycle progression, A: Cell cycle measured by flow cytometry, B: Bar graph of the proportion of cells in G0 / G1, S and G2 / M phases. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the described embodiments, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0020] The sources of the reagents involved in the following examples and performance characterizations are as follows: Human retinal pigment epithelial cells (ARPE-19) were purchased from ATCC, USA; Rhein was purchased from Shanghai Sangong Biotechnology Co., Ltd.; Fmoc-protected amino acids were purchased from Shanghai Jier Biochemical Co., Ltd.; 2-Cl-Trt resin, purchased from Tianjin Nankai University Solid Phase Synthesis Company, activity 1.2 mmol / mL; DMEM medium was purchased from Beijing Solebow Technology Co., Ltd.; Trypsin was purchased from Beijing Solebow Technology Co., Ltd.; Fetal bovine serum was purchased from Wuhan Sanying Biotechnology Co., Ltd.; Cell proliferation detection kit CCK8 reagent was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; Cell cycle detection kit PI / RNase reagent was purchased from BD Pharmingen, USA; TGF-β2 was purchased from MedChemexpress Biotechnology Company, USA.

[0021] Example 1 - Synthesis of Rhein-Peptide The standard SPPS solid phase peptide synthesis method was used to synthesize rhein-peptide, and the molecular formula was Rh-FFYGRGD, where Rh represents rhein and FFYGRGD represents polypeptide; the structural formula is as follows: .

[0022] The specific steps are as follows: (1) Weigh 1 mmol of 2-Cl-Trt resin into a solid phase synthesis tube, then add 10 mL of anhydrous dichloromethane (hereinafter referred to as DCM), and shake on a shaker for 20 min to allow the 2-Cl-Trt resin to fully swell; (2) Use an ear bulb to remove DCM from the solid phase synthesis tube; (3) Dissolve 1.5 eq (1.5 mmol) of the first Fmoc-protected amino acid, i.e., Fmoc-Asp(tBu)-OH, in 15 mL of anhydrous DCM, add 3 eq (3 mmol) of DIEPA, transfer to the above solid phase synthesis tube after full dissolution, and react at room temperature for 1 h; (4) Use an ear bulb to remove the reaction solution in the solid phase synthesis tube, wash with anhydrous DCM 5 times, 1 min each time, add 20 mL of blocking solution with a volume ratio of anhydrous DCM: methanol: DIEPA = 17:2:1, and block at room temperature for 20 min; (5) Use an ear bulb to remove the blocking solution in the solid phase synthesis tube, first wash with anhydrous DCM 5 times (each time DCM amount 10 mL, each time 1 min), then wash with N, N-dimethylformamide (hereinafter referred to as DMF) 5 times (each time DMF amount 10 mL, each time 1 min), add 15 mL of DMF solution containing 20% ​​by volume piperidine, cut the Fmoc protecting group for 35 minutes, continue to wash the cut Fmoc with DMF (each time DMF amount 15 mL, each time 1 min, a total of 5 washes), and proceed to the next step of reaction; (6) Dissolve the second Fmoc-protected amino acid, i.e., Fmoc-Gly-OH 2mmol, HBTU 2mmol, and DIEPA 4mmol, in 10 ml DMF. After fully dissolved, add the mixture into the solid phase synthesis tube and react for 2 h. (7) Remove the reaction solution from step (6), wash with DMF 5 times (10 mL of DMF each time, 1 min each time), then add 15 mL of DMF solution containing 20% ​​by volume of piperidine, cleave the Fmoc protecting group for 35 min, and then wash the cleaved Fmoc with DMF (15 mL of DMF each time, 1 min each time, a total of 5 washes); (8) sequentially replacing the second Fmoc-protected amino acid Fmoc-Gly-OH with the third Fmoc-protected amino acid Fmoc-Arg(pbf)-OH, the fourth Fmoc-protected amino acid Fmoc-Gly-OH, the fifth Fmoc-protected amino acid Fmoc-Tyr(tBu)-OH, the sixth Fmoc-protected amino acid Fmoc-Phe-OH, the seventh Fmoc-protected amino acid Fmoc-Phe-OH and rhein molecules in equal amounts, and repeating the process of steps (6) to (7); (9) After the reaction of rhein was completed, DMF and DCM were used for washing 5 times, each time with a dosage of 10 mL and each time for 1 min. After washing, the mixture was heated to 95% TFA, 2.5% TIS, 2.5% H 2 10 mL of the solution composed of 2-Cl-Trt and 2-Cl-Trt was added into the solid phase synthesis tube for reaction for 30 min. The product was cut from the 2-Cl-Trt resin and dried by rotary evaporator in vacuum. After the solvent was removed, the product was precipitated with icy ether to obtain a crude product with a yield of about 85%. The crude product was then separated and purified by HPLC (the mobile phase was water: methanol = 80:20 by volume) and lyophilized to obtain pure Rh-FFYGRGD.

[0023] The high-resolution mass spectrum of Rh-FFYGRGD is shown in Figure 1 shown.

[0024] Example 2 - Preparation of rhein-polypeptide solution 5 mg of Rh-FFYGRGD prepared in Example 1 was added to 800 μL PBS buffer (pH = 7.4, concentration 0.01 M), ultrasonically treated for 15 min, and 1 M Na 2 CO 3 The solution was adjusted to pH = 7.4, and then PBS buffer (pH = 7.4, concentration 0.01M) was added to make the volume 1mL. At this time, the concentration of Rh-FFYGRGD in the system was 5mg / mL. The solution was heated to 70°C, and after Rh-FFYGRGD was completely dissolved, it was naturally cooled to room temperature to obtain a rhein-polypeptide solution; wherein the Na 2 CO 3 The solution is composed of Na 2 CO 3 It was prepared by dissolving in PBS buffer (pH=7.4, concentration 0.01M).

[0025] Product performance characterization 1. Effects of rhein and rhein-peptide on cell proliferation (1.1) Preparation of ARPE-19 cell suspension and experimental reagents Preparation of ARPE-19 cell suspension: Incubate in a 10 cm diameter culture dish at 37°C and 5% CO 2 ARPE-19 cells were cultured in DMEM medium (containing 10% fetal bovine serum) until 90% confluence, the old medium was discarded, and the cells were washed twice with 4 mL PBS buffer (pH = 7.4, concentration 0.01 M). 2 mL of trypsin was added to the culture dish and incubated in a 37°C incubator (5% CO 2 ) for 2 minutes for cell digestion. At this time, 80% of the cells became round and shrunk. Then, 2 mL of DMEM medium (containing 10 v% fetal bovine serum) was added to the culture dish to terminate the digestion. The cells were gently blown with a sterile pipette and transferred to a 15 mL centrifuge tube. After centrifugation at 1000 rpm for 5 minutes, a cell pellet was obtained. 4 mL of DMEM medium (containing 10 v% fetal bovine serum) was added and the cell pellet was gently blown to prepare an ARPE-19 cell suspension. 10 µL of the ARPE-19 cell suspension was aspirated into a hemacytometer and counted under a microscope.

[0026] Preparation of rhein solutions and rhein-peptide solutions with different concentration gradients: The rhein stock solution and the rhein-peptide stock solution were diluted into solutions with concentration gradients of 2, 10, 50 and 100 µM using DMEM culture medium (containing 10 v% fetal bovine serum), respectively. The rhein stock solution was prepared by dissolving rhein in PBS buffer (pH=7.4, concentration 0.01 M) (its concentration was 5 mg / mL), and the rhein-peptide stock solution was the rhein-peptide solution with a concentration of 5 mg / mL prepared in Example 2; the rhein solution or rhein-peptide solution with a concentration of 0 µM was replaced by DMEM culture medium (containing 10 v% fetal bovine serum).

[0027] (1.2) Set up the experimental group, blank group and control group: Experimental group: ARPE-19 cell suspension was inoculated into 96-well plates, 100 μL per well (the number of cells was about 5×10 3 ) at 37°C incubator (5% CO 2 ) for 16 h, discard the old medium in the 96-well plate, add 100 µL of rhein solution or rhein-peptide solution with different concentration gradients (2, 10, 50, 100 μM) to each well, place in the incubator for 48 h, discard the old medium in the 96-well plate, wash the cells twice with 150 µL PBS buffer (pH = 7.4, concentration 0.01 M), add 100 µL DMEM medium and 10 µL CCK8 reagent to each well, and incubate at 37 °C in a cell culture incubator (5% CO 2 ) and continued incubation for 2 h, and the absorbance at 450 nm was measured by an ELISA reader; Blank group: 100 μL DMEM medium and 10 μL CCK8 reagent were directly added to each well of the 96-well plate and incubated at 37°C in a cell culture incubator (5% CO 2 ) for 2 h, and the absorbance at 450 nm was measured by an ELISA reader; Control group: ARPE-19 cell suspension was inoculated into 96-well plates, 100 μL per well (cell number was about 5×10 3 ) at 37°C incubator (5% CO 2 ) for 16 h, discard the old medium in the 96-well plate, add 100 µL DMEM medium (containing 10v% fetal bovine serum) to each well, place in the incubator for 48 h, discard the old medium in the 96-well plate, wash the cells twice with 150 µL PBS buffer (pH=7.4, concentration 0.01M), add 100 µL DMEM medium and 10 µL CCK8 reagent to each well, and place in a 37°C cell culture incubator (5% CO 2 ) and continued incubation for 2 h, and the absorbance at 450 nm was measured by an ELISA reader; Calculation formula: .

[0028] Figure 2 The effects of rhein and rhein-peptide drugs on cell proliferation, Figure 2 The 0µM concentration of rhein solution or rhein-peptide solution was used as the control group. Figure 2 It can be seen that after treating ARPE-19 cells with 0, 2, 10, 50, and 100 μM rhein solution for 48 hours, it was found that the five different concentrations of rhein solution had no significant effect on cell proliferation; and after treating ARPE-19 cells with rhein-peptide solution for 48 hours, it was found that 10 μM rhein-peptide solution reduced the viability of RPE cells to 0.83. In addition, increasing the drug dose had a stronger inhibitory effect on cell proliferation, and 50 and 100 μM rhein-peptide solutions reduced the viability of RPE cells to 0.57 and 0.55, respectively. The above shows that rhein-peptide has a stronger inhibitory effect on cell proliferation, and 10 μM rhein-peptide solution can be used as the best dose in RPE cells.

[0029] 2. Effects of rhein and rhein-peptide on TGF-β2-induced RPE cell morphology The preparation of ARPE-19 cell suspension, rhein solution and rhein-peptide solution are the same as those in item (1.1).

[0030] The ARPE-19 cell suspension was inoculated into a 6-well plate, 2 mL per well (the number of cells was about 8 × 10 4 ) at 37°C incubator (5% CO 2) for 16 h, discard the old culture medium in the 6-well plate, add 2 mL of rhein solution or rhein-peptide solution with different concentration gradients (0, 10 µM) to each well, treat in the incubator for 4 h, then add 2 µL of 10 µg / mL TGF-β2 aqueous solution to each well of the 6-well plate to make the final concentration of 10 ng / mL, continue to culture for 48 h, observe the morphological changes of the cells under an inverted microscope, and take pictures.

[0031] Figure 3 The effects of rhein and rhein-peptide drugs on TGF-β2-induced RPE cell morphology. Figure 3 It can be seen that after ARPE-19 cells were treated with TGF-β2 for 48 hours, the cells changed from oval or elliptical to fibrous or spindle-shaped, that is, the cells changed from epithelial cell morphology to mesenchymal cell morphology ( Figure 3 Left figure); 10μM rhein solution had no significant effect on TGF-β2-induced cell morphological changes ( Figure 3 Middle figure); however, 10 μM rhein-peptide solution made the TGF-β2-treated RPE cells still appear oval or elliptical ( Figure 3 The above indicates that rhein-peptide can maintain the epithelial cell morphology of RPE and prevent the change to mesenchymal cell morphology induced by TGF-β2.

[0032] 3. Scratch test to detect the effect of rhein and rhein-peptide on cell migration The preparation of ARPE-19 cell suspension, rhein solution and rhein-peptide solution are the same as those in item (1.1).

[0033] The ARPE-19 cell suspension was inoculated into a 6-well plate, with 2 mL per well (the number of cells was approximately 2 × 10 5 ) at 37°C incubator (5% CO 2 ) until the monolayer cells were 90% confluent, scratched with a 1 mL sterile pipette tip, the old culture medium in the 6-well plate was discarded, and the plates were washed once with 1 mL PBS buffer (pH = 7.4, concentration 0.01 M), 2 mL of rhein solution or rhein-peptide solution with different concentration gradients (0, 10 µM) was added to each well, and the plates were treated in an incubator for 4 h, and then 2 µL of 10 µg / mL TGF-β2 aqueous solution was added to each well of the 6-well plate to make the final concentration of 10 ng / mL. After further culturing for 48 h, the cell migration distance was observed under an inverted microscope and pictures were taken.

[0034] Figure 4 The effect of rhein and rhein-peptide drugs on cell migration. Figure 4It can be seen that when the same scratch was made with a sterile pipette tip, ARPE-19 cells were treated with TGF-β2 for 48 h, and the cells migrated and fused. Figure 4 The distance of wound healing induced by TGF-β2 was shorter than that of the control group ( Figure 4 Left column), while 10 μM rhein-peptide solution ( Figure 4 Right column) The distance of wound healing induced by TGF-β2 was shorter than that of the control group ( Figure 4 Left column) and rhein-treated group ( Figure 4 The above indicated that both rhein and rhein-peptide could inhibit TGF-β2-induced cell migration, and rhein-peptide had a stronger inhibitory effect on cell migration.

[0035] 4. Flow cytometry to detect the effects of rhein and rhein-peptide on cell cycle The preparation of ARPE-19 cell suspension, rhein solution and rhein-peptide solution are the same as those in item (1.1).

[0036] The ARPE-19 cell suspension was inoculated into a 6-well plate, with 2 mL per well (the number of cells was approximately 2 × 10 5 ) at 37°C incubator (5% CO 2 ) for 16 h, discard the old culture medium in the 6-well plate, add 2 mL of rhein solution or rhein-peptide solution with different concentration gradients (0, 10 µM) to each well, treat in the incubator for 4 h, then add 2 µL of 10 µg / mL TGF-β2 aqueous solution to each well of the 6-well plate to make the final concentration of 10 ng / mL, continue to culture for 24 h, discard the old culture medium, wash the cells once with 1 mL PBS buffer (pH = 7.4, concentration 0.01 M), add 500 μL of trypsin to each well and digest at 37 ° C for 2 min, then add 500 μL of DMEM medium (containing 10v% fetal bovine serum) to each well to terminate digestion, gently blow the cells with a sterile pipette, transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 5 min to obtain the cell pellet, add 1 mL PBS buffer (pH = 7.4, concentration 0.01 M) to wash the cells twice, and then add pre-cooled 1 mL The cells were fixed with 75v% ethanol at -20℃ for 12h, and the cell pellets were collected by centrifugation at 1000rpm for 5min. After washing once with 1mL PBS buffer (pH=7.4, concentration 0.01M), 500 µLPI / RNase reagent was added to the cell pellets, and the cells were incubated at room temperature in the dark for 15min. The cell suspension was transferred to a flow tube, and the cell cycle was detected by flow cytometry, and the graphs were drawn using Modfit 5 software.

[0037] Figure 5The effects of rhein and rhein-peptide drugs on cell cycle progression, A: cell cycle measured by flow cytometry, B: bar graph of the percentage of cells in G0 / G1, S and G2 / M phases. Figure 5 It can be seen that: the rhein-peptide treatment group ( Figure 5 A right panel) significantly increased the number of cells in the S phase and G2 / M phase, while the rhein-treated group ( Figure 5 A middle panel) and control group ( Figure 5 A left figure) had no significant difference in the cell cycle; through statistical analysis, it was found that the proportion of cells in the control group, rhein treatment group and rhein-peptide treatment group in the G0 / G1 phase was 75.12%, 75.85% and 53.6%, respectively, the proportion of cells in the S phase was 8.49%, 8.33% and 12.98%, respectively, and the proportion of cells in the G2 / M phase was 16.39%, 15.82% and 33.42%, respectively ( Figure 5 B). The above indicated that rhein-peptide affected the cell cycle process of RPE and could arrest the cell cycle in S phase and G2 / M phase, while 10 μM rhein monomer had no significant effect on the cell cycle process.

Claims

1. A rhein-polypeptide, characterized in that: The molecular formula is Rh-FFYGRGD, Rh represents rhein, and FFYGRGD represents polypeptide; the structural formula is as follows: 。 2. A method for preparing a rhein-polypeptide solution, characterized in that: Here are the steps: (1) Rhein-FFYGRGD was synthesized by conventional solid phase peptide synthesis method, where Rh represents rhein and FFYGRGD represents peptide; (2) Add Rh-FFYGRGD to 1#PBS buffer, add Na2CO3 solution to adjust the pH of the system until it is the same as the pH of 1#PBS buffer, then add 2#PBS buffer to make up the volume, at which time the concentration of Rh-FFYGRGD in the system is 1~6 mg / mL, heat to 70~100°C, wait for Rh-FFYGRGD to be completely dissolved, and then naturally cool to room temperature to obtain a rhein-polypeptide solution; wherein the Na2CO3 solution is prepared by dissolving Na2CO3 in 3#PBS buffer; the pH of 1#PBS buffer, 2#PBS buffer and 3#PBS buffer is the same and is 7.0~8.

0.

3. The method for preparing the rhein-polypeptide solution according to claim 1, characterized in that: In step (2), the concentrations of 1#PBS buffer, 2#PBS buffer and 3#PBS buffer are the same and are 0.01~0.03M.

4. The method for preparing the rhein-polypeptide solution according to claim 1, characterized in that: In step (2), Rh-FFYGRGD is added to 1# PBS buffer under ultrasonic conditions.

5. The method for preparing the rhein-polypeptide solution according to claim 1, characterized in that: In step (2), the concentration of the Na2CO3 solution is 1~2M.

6. Use of the rhein-polypeptide according to claim 1 in the preparation of a drug for inhibiting epithelial-mesenchymal transition induced by TGF-β2.

7. The use according to claim 7, characterized in that: Application of rhein-peptide in the preparation of drugs for the treatment of proliferative vitreoretinopathy.

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