Human recombinant GDF15 protein and application thereof in preparation of medicine for preventing and treating sepsis acute kidney injury

Abstract

The invention relates to a human recombinant GDF15 protein and application thereof in preparation of drugs for preventing and treating sepsis acute kidney injury, and belongs to the technical field of biological medicines. In order to solve the problem that an effective prevention and treatment scheme for improving SA-AKI by inhibiting oxygen death is lacked in existing clinical and research, the invention provides a human recombinant GDF15 protein of which the coding gene nucleotide sequence is shown as SEQ ID No: 1. Animal experiment results show that by intraperitoneal injection of the human recombinant GDF15 protein, kidney inflammation of sepsis mice can be relieved, and expression of PGAM5 can be down-regulated. Cell experiment results show that when the human recombinant GDF15 protein is given, LPS-induced USF2 increase can be inhibited. The human recombinant GDF15 protein provided by the invention can improve SA-AKI by inhibiting oxygen death and inflammation, provides a new idea and an intervention target for prevention and treatment of SA-AKI, and shows a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to human recombinant GDF15 protein and application thereof in preparing a drug for preventing and treating sepsis-induced acute kidney injury. Background Art

[0002] Sepsis is a fatal organ dysfunction caused by the body's dysregulated host response to infection. It has many complications and a high mortality rate, which seriously endangers human health. The kidney is the organ most susceptible to sepsis. About 50% of sepsis patients develop sepsis-associated acute kidney injury (SA-AKI), which is an important cause of death from sepsis.

[0003] In previous studies on the development mechanism of SA-AKI, it was found that apoptosis inducing factor 1 (AIFM1) is a secretory marker of oxygen death, which is significantly increased in the serum of SA-AKI patients. In the SA-AKI animal model, it was found that renal tubular epithelial cells have oxygen death, accompanied by inflammation and oxidative stress. It is speculated that renal tubular oxygen death is involved in the occurrence and development of SA-AKI.

[0004] Phosphoglycerate mutase 5 (PGAM5) is a key molecule involved in oxygen death and is significantly increased in SA-AKI renal tubular epithelial cells; the JASPER database predicts that PGAM5 is regulated by upstream transcription factor 2 (USF2). Knocking out USF2 inhibits PGAM5 expression, increases AIFM1 phosphorylation levels, and improves oxygen death indicators. However, existing clinical and research studies lack effective prevention and treatment plans that can improve SA-AKI by inhibiting oxygen death. Summary of the invention

[0005] In order to solve the problem that existing clinical and research lacks an effective prevention and treatment plan for improving SA-AKI by inhibiting oxygen death, the present invention provides a human recombinant GDF15 protein and its use in the preparation of a drug for preventing and treating sepsis-induced acute kidney injury.

[0006] The technical solution of the present invention:

[0007] A human recombinant GDF15 protein, the nucleotide sequence of the gene encoding the human recombinant GDF15 protein is shown in SEQ ID No: 1.

[0008] A use of the human recombinant GDF15 protein provided by the present invention in preparing a drug for preventing and treating acute kidney injury caused by sepsis.

[0009] Furthermore, the drug for preventing and treating acute kidney injury caused by sepsis contains human recombinant GDF15 protein as the only active ingredient or one of the active ingredients.

[0010] Furthermore, the content of human recombinant GDF15 protein in the drug for preventing and treating acute kidney injury caused by sepsis is 0.1wt% to 99wt%.

[0011] Furthermore, the drug for preventing and treating acute kidney injury caused by sepsis also includes pharmaceutically acceptable excipients and / or carriers.

[0012] Furthermore, the drug for preventing and treating acute kidney injury caused by sepsis is in the form of an intraperitoneal injection.

[0013] Furthermore, the drug for preventing and treating acute kidney injury in sepsis can downregulate the expression of PGAM5 in the kidneys of SA-AKI mice and inhibit oxygen death of renal tubular epithelial cells.

[0014] Furthermore, the drug for preventing and treating acute kidney injury caused by sepsis can reduce the expression of USF2 in HK2 cells induced by LPS.

[0015] Furthermore, the drug for preventing and treating sepsis-induced acute kidney injury can reduce the inflammatory response in the kidneys of SA-AKI mice.

[0016] Furthermore, the drug for preventing and treating sepsis-induced acute kidney injury can reduce oxidative stress in the kidneys of SA-AKI mice.

[0017] Beneficial effects of the present invention:

[0018] The present invention synthesizes human recombinant GDF15 protein, which can reduce inflammatory response and oxygen death of macrophages and endothelial cells, and has important anti-oxygen death and anti-inflammatory activity. Animal experimental results show that intraperitoneal injection of human recombinant GDF15 protein can reduce renal inflammation and oxidative stress in septic mice, and downregulate the expression of PGAM5. Cell experimental results show that administration of human recombinant GDF15 protein can inhibit LPS-induced increase in USF2. Based on this, it can be seen that the human recombinant GDF15 protein provided by the present invention can improve SA-AKI by inhibiting oxygen death and inflammation, providing new ideas and intervention targets for the prevention and treatment of SA-AKI, and showing good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of human recombinant GDF15 overexpression plasmid;

[0020] Figure 2The protein electrophoresis staining photos of bacterial protein supernatant and precipitate at different IPTG induction times when preparing human recombinant GDF15 protein, in which M is the protein molecular weight standard, 1 is bacterial protein before induction, 2 is bacterial protein after induction, 3 and 4 are 0.1mmol / L IPTG, bacterial protein supernatant and precipitate after 9h induction at 30℃, 5 and 6 are 0.1mmol / L IPTG, bacterial protein supernatant and precipitate after 12h induction at 30℃, 7 and 8 are 0.1mmol / L IPTG, bacterial protein supernatant and precipitate after 18h induction at 30℃;

[0021] Figure 3 This is a protein electrophoresis staining photo of the purified human recombinant GDF15 protein. In the figure, M is the protein molecular weight standard, 1 is the bacterial protein before induction, 2 is the bacterial protein after induction, 3 is the purified sample effluent, and 4 is the protein after elution with 50mM imidazole;

[0022] Figure 4 This is a comparison of the mRNA expression of inflammatory factor TNF-α in the kidneys of different mice after intraperitoneal injection of human recombinant GDF15 protein in Example 2;

[0023] Figure 5 This is a comparison of the mRNA expression of NOS2, a molecule related to oxidative stress, in the kidneys of different mice after intraperitoneal injection of human recombinant GDF15 protein in Example 2;

[0024] Figure 6 This is a comparison of the mRNA expression of PTGS2, a molecule related to oxidative stress, in the kidneys of different mice after intraperitoneal injection of human recombinant GDF15 protein in Example 2;

[0025] Figure 7 This is a comparison of PGAM5 mRNA expression in the kidneys of different mice after intraperitoneal injection of human recombinant GDF15 protein in Example 2;

[0026] Figure 8 This is a comparison diagram of the effect of human recombinant GDF15 protein on LPS-induced HK-2 cell USF2 mRNA expression in Example 3;

[0027] Fig. 9 This is a comparison of the effect of human recombinant GDF15 protein on the mRNA expression of inflammation-related factors in LPS-induced RAW264.7 cells in Example 3;

[0028] Fig.10 This is a comparison chart of the effect of human recombinant GDF15 protein in Example 3 on the mRNA expression of inflammation-related factors in LPS-induced HUVEC cells. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0030] Example 1

[0031] This example provides a method for preparing and purifying human recombinant GDF15 protein.

[0032] Growth differentiation factor 15 (GDF15) is a member of the transforming growth factor-β superfamily. Previous studies have shown that increased serum GDF15 levels are associated with sepsis and multiple organ dysfunction in critically ill patients. This example refers to the Gdf15 gene sequence provided by Genbank, and commissioned Wuhan Vinocell Biotechnology Co., Ltd. to optimize, synthesize and design codons, and construct a GDF15 overexpression plasmid with a HIS tag by prokaryotic expression. The schematic diagram of the plasmid is shown in Figure 1 .

[0033] The specific steps of preparing human recombinant GDF15 protein using GDF15 overexpression plasmid in this example are as follows:

[0034] (1) The GDF15 overexpression plasmid was transformed into DH5α competent cells for amplification of the recombinant plasmid, and the amplified recombinant plasmid DNA was collected and transformed into BL21 competent cells. An appropriate amount of sterile LB medium was added, mixed and placed in a 37°C shaker for shaking culture to restore the cells to normal growth state and express the antibiotic resistance gene encoded by the plasmid. The cultured bacterial liquid was spread on LB solid medium containing the corresponding antibiotics, and the grown single colony was picked and inoculated into 5 mL LB medium. The colony was cultured at 37°C overnight, and then transferred to LB medium at a volume ratio of 1:100. The culture was continued at 37°C for 2 to 8 hours until the bacteria reached the logarithmic growth phase, that is, the OD600 value was between 0.6 and 0.8. The sample was kept before induction or as a control without IPTG induction.

[0035] (2) Add IPTG (isopropylthiogalactoside) to the bacterial solution to a final concentration of 0.1 mM, continue shaking culture at 30°C, 250 rpm for 9 h for induction culture, keep a sample for induction, centrifuge at 4°C, 4000 rpm for 10 min, keep the precipitate, add an appropriate amount of 1× PBS to resuspend, add PMSF (phenylmethylsulfonyl fluoride) to dilute it to 0.1 mM, pre-cool on ice for 10 min; place on ice, and ultrasonically disrupt the bacterial solution to make it clear from turbid for 5-10 min (10-20 s / time).

[0036] Add 10% TritonX-100 to the bacterial solution and dilute it to 0.5% TritonX-100. Oscillate at 4°C for 15 min, centrifuge again at 4°C, 12000 rpm for 10 min, take the supernatant and precipitate, keep the supernatant and precipitate samples after induction, resuspend the precipitate with an equal volume of 1×PBS, detect by protein electrophoresis, stain with Coomassie Brilliant Blue, and decolorize.

[0037] The protein expression was verified by 10% SDS-PAGE. Figure 2 As shown in the figure, after induction, there is a clear band around 37 kDa, which is basically the same size as the target protein, indicating that the human recombinant GDF15 protein is successfully expressed. The target protein is also located in the supernatant, indicating that the obtained human recombinant GDF15 protein is soluble.

[0038] The specific steps for purifying the prepared human recombinant GDF15 protein in this example are as follows:

[0039] The supernatant containing human recombinant GDF15 protein was purified by nickel affinity chromatography and dialyzed, and distilled water was injected into the pump line or syringe. Remove the stopper and inject distilled water dropwise to prevent air from entering the system; wash (about 3-5mL of distilled water) the ethanol in the column; balance the column with binding buffer (not less than 5mL); transfer the unclarified lysate to the column under the action of a syringe or pump; rinse with binding buffer until the absorption curve stabilizes at the baseline; then elute with 50, 100, 200, 300mM imidazole, with a loading speed of 1min / mL. After purification, the GDF15 protein was dialyzed to remove most of the imidazole residues so as not to interfere with subsequent experiments.

[0040] The results of 10% SDS-PAGE showed that Figure 3 As shown, after purification, a single band was obtained near 37 kDa with fewer miscellaneous bands. The protein concentration of the human recombinant GDF15 protein sample obtained after dialysis was measured using the BCA method, and the concentration of the purified human recombinant GDF15 protein was determined to be 0.7 mg / mL.

[0041] Example 2

[0042] In this example, animal experiments were conducted to investigate the effects of human recombinant GDF15 protein on renal inflammation, oxidative stress and PGAM5 expression in SA-AKI mice.

[0043] 1. Methods for constructing SA-AKI mouse model

[0044] In this example, the SA-AKI mouse model was established by direct administration of endotoxin.

[0045] (1) Experimental animals: Select healthy, age-appropriate SPF-grade male mice weighing between 20 and 30 g. Ensure that the experimental animals have no kidney disease or other diseases that may affect the experimental results before the experiment.

[0046] (2) Experimental reagents: Prepare LPS (lipopolysaccharide) as a source of endotoxin, and prepare physiological saline for diluting LPS.

[0047] (3) Animal anesthesia: Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40 mg / kg).

[0048] (4) LPS administration: LPS (12 mg / kg) was intraperitoneally injected into the anesthetized mice. The needle was inserted into the abdomen, lateral to the midline. The injection speed should be slow to avoid excessive stimulation to the mice. A control group injected with saline was also set up.

[0049] (5) Detection: After LPS injection, the mental state, activity, diet and drinking water of mice were closely observed. Blood samples of mice were collected at 6h, 12h and 24h after LPS injection to detect renal function indicators such as serum creatinine and urea nitrogen. The renal function indicators such as serum creatinine and urea nitrogen of mice in the model group were significantly higher than those in the control group, indicating that the model was successfully constructed.

[0050] 2. Grouping and dosing method:

[0051] This embodiment sets three groups:

[0052] (1) Negative control group-Saline group: intraperitoneal injection of an equal volume of normal saline;

[0053] (2) Model control group-LPS group: intraperitoneal injection of an equal volume of normal saline;

[0054] (3) Treatment group - LPS + rhGDF15 group: The human recombinant GDF15 protein purified in Example 1 (10 nmol / kg) was intraperitoneally injected every day for three days in advance, followed by intraperitoneal injection of LPS (12 mg / kg).

[0055] 3. Investigate the effects of human recombinant GDF15 protein on renal inflammation, oxidative stress and PGAM5 expression in SA-AKI mice.

[0056] The three groups of mice were killed, and the kidney tissues were quickly collected and frozen in liquid nitrogen. The kidney tissues were placed in a mortar, an appropriate amount of TRIzol reagent was added, and the grinding rod was used for homogenization. RNA was extracted according to the TRIzol instructions to obtain RNA precipitation. According to the instructions of the reverse transcription reagent, the RNA was reverse transcribed into cDNA, and the relative expression levels of renal inflammation-related factor TNF-α, renal oxidative stress-related factors NOS2 and PTGS2, and PGAM5 mRNA were calculated by fluorescence quantitative PCR. The results are shown in Figure 2. Figure 4 , Figure 5 , Figure 6 and Figure 7 shown.

[0057] Figure 4 , Figure 5 , Figure 6 and Figure 7 The results showed that compared with the Saline group, intraperitoneal injection of LPS caused an increase in the expression of inflammatory-related factors, oxidative stress-related factors and PGAM5 mRNA in SA-AKI mice, while human recombinant GDF15 protein could significantly reduce renal inflammation and oxidative stress in septic mice and downregulate the expression of PGAM5.

[0058] Example 3

[0059] In this example, cell experiments were performed to investigate the effect of human recombinant GDF15 protein on the expression of USF2 in HK-2 cells (human renal tubular epithelial cells) induced by LPS, and on the expression of inflammatory factors in RAW267.4 and HUVEC cells induced by LPS.

[0060] In this example, the method of LPS inducing HK-2, RAW264.7, and HUVEC cells:

[0061] On a sterile operating table, HK-2, RAW264.7, and HUVEC cells were inoculated in DMEM culture medium at 37°C and 5% CO. 2 LPS solution with a concentration of 10ug / mL was prepared with LPS powder, HK-2, RAW264.7, and HUVEC cells were seeded in 6-well plates, and the cell density was adjusted to 50%-70%. LPS solution was added to the cell culture medium to a final concentration of 100ng / mL, and the LPS induction time was 24h.

[0062] Grouping and administration method in this embodiment:

[0063] (1) Control group: HK-2, RAW264.7, and HUVEC cells were treated with equal amounts of saline;

[0064] (2) LPS group: LPS-induced HK-2, RAW264.7, and HUVEC cells were treated with an equal amount of saline;

[0065] (3) LPS+rhGDF15 group: LPS-induced HK-2, RAW264.7, and HUVEC cells were treated with the human recombinant GDF15 protein (10 ng / mL) purified in Example 1 for 24 h.

[0066] Investigate the effect of human recombinant GDF15 protein on LPS-induced USF2 mRNA expression in HK-2 cells:

[0067] The cells in each group were collected, total RNA was extracted, and the RNA was reverse transcribed into cDNA using a reverse transcription kit. The cDNA of each group of cells was used as a template for qRT-PCR amplification. According to the results of qRT-PCR, the relative expression levels of USF2 and inflammatory factor mRNA in each group of cells were calculated. The results are shown in Figure 8 , Fig. 9 , Fig.10 shown.

[0068] Figure 8 The results showed that compared with the Control group, the relative expression of USF2 mRNA in HK-2 cells induced by LPS was significantly increased, while human recombinant GDF15 protein could significantly reduce the increase of USF2 mRNA expression in HK-2 cells induced by LPS.

[0069] Fig. 9 and Fig.10 The results showed that compared with the Control group, the relative expression levels of inflammatory factors IL-6, IL-1β, CXCL2, NOS2, GPX4, and PTGS2 mRNA in RAW264.7 cells induced by LPS were significantly increased, and the relative expression levels of inflammatory factors IL-6, IL-1β, TNF-α, ICAM-1, VCAM-1, and PTGS2 mRNA in HUVEC cells were significantly increased, while human recombinant GDF15 protein could significantly reduce the increase of mRNA expression of inflammatory-related factors in RAW264.7 and HUVEC cells induced by LPS.

[0070] Animal experiments and cell experiments have confirmed that the human recombinant GDF15 protein provided can improve SA-AKI by inhibiting oxygen death and inflammation, providing new ideas and intervention targets for the prevention and treatment of SA-AKI, and showing good clinical application prospects.

Claims

1. A human recombinant GDF15 protein, characterized in that: The nucleotide sequence of the human recombinant GDF15 protein encoding gene is shown in SEQ ID No:

1.

2. Use of the human recombinant GDF15 protein as claimed in claim 1 in the preparation of a drug for preventing and treating acute kidney injury caused by sepsis.

3. The use of the human recombinant GDF15 protein according to claim 2 in the preparation of a drug for preventing and treating acute kidney injury caused by sepsis, characterized in that: The drug for preventing and treating acute kidney injury caused by sepsis uses human recombinant GDF15 protein as the only active ingredient or one of the active ingredients.

4. Use of the human recombinant GDF15 protein according to claim 2 or 3 in the preparation of a drug for preventing and treating acute kidney injury caused by sepsis, characterized in that: The content of human recombinant GDF15 protein in the drug for preventing and treating acute kidney injury caused by sepsis is 0.1 wt% to 99 wt%.

5. The use of the human recombinant GDF15 protein according to claim 4 in the preparation of a drug for preventing and treating acute kidney injury caused by sepsis, characterized in that: The drug for preventing and treating acute kidney injury caused by sepsis also includes pharmaceutically acceptable adjuvants and / or carriers.

6. The use of the human recombinant GDF15 protein according to claim 5 in the preparation of a drug for preventing and treating acute kidney injury caused by sepsis, characterized in that: The medicine for preventing and treating acute kidney injury caused by sepsis is in the form of an intraperitoneal injection.

7. The use of the human recombinant GDF15 protein according to claim 6 in the preparation of a drug for preventing and treating acute kidney injury caused by sepsis, characterized in that: The drug for preventing and treating acute kidney injury caused by sepsis can downregulate the expression of PGAM5 in the kidneys of SA-AKI mice and inhibit oxygen death of renal tubular epithelial cells.

8. The use of the human recombinant GDF15 protein according to claim 7 in the preparation of a drug for preventing and treating acute kidney injury caused by sepsis, characterized in that: The drug for preventing and treating acute kidney injury caused by sepsis can reduce the expression of USF2 in HK2 cells induced by LPS.

9. The use of the human recombinant GDF15 protein according to claim 8 in the preparation of a drug for preventing and treating acute kidney injury caused by sepsis, characterized in that: The drug for preventing and treating sepsis-induced acute kidney injury can reduce the inflammatory response of the kidneys of SA-AKI mice.

10. Use of the human recombinant GDF15 protein according to claim 9 in the preparation of a drug for preventing and treating acute kidney injury caused by sepsis, characterized in that: The drug for preventing and treating sepsis-induced acute kidney injury can reduce the oxidative stress in the kidneys of SA-AKI mice.

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