Application of USP53 gene silencing drugs in renal ischemia-reperfusion injury

Abstract

The present invention belongs to the field of biological gene engineering technology, and relates to an application of silencing USP53 gene in a drug for renal ischemia-reperfusion injury. The present invention proposes the application of a substance for silencing USP53 expression in human renal tubular epithelial cells in a drug for alleviating the inflammatory response, apoptosis and ferroptosis of renal tubular cells induced by renal ischemia-reperfusion. The above-mentioned substance for silencing USP53 expression in human renal tubular epithelial cells includes a small interfering RNA with a sequence of GUGCGGUACAUUUCUACAATT. The present invention discloses for the first time that silencing USP53 expression in renal tubular epithelial cells can improve renal ischemia-reperfusion injury, and provides a new drug design target for the treatment of renal ischemia-reperfusion injury, which has important theoretical guidance and clinical transformation application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological genetic engineering, and relates to an application of a USP53 gene silencing drug in a drug for treating renal ischemia-reperfusion injury, and specifically relates to an application of a substance for silencing USP53 expression in human renal tubular epithelial cells in a drug for alleviating the inflammatory response, cell apoptosis and ferroptosis of renal tubular cells induced by renal ischemia-reperfusion. Background Art

[0002] Renal ischemia-reperfusion injury refers to the pathological and physiological process in which the organ is aggravated by the restoration of blood flow after a period of ischemia and hypoxia. Clinically, renal ischemia-reperfusion often occurs in hemorrhagic shock, renal transplantation, and cardiovascular surgery. Since there is still a lack of effective drugs to prevent and treat renal ischemia-reperfusion, it has a high morbidity and mortality rate. The pathological mechanism of renal ischemia-reperfusion injury is complex, including inflammatory response, endoplasmic reticulum stress, mitochondrial dysfunction, and cell apoptosis. However, more and more studies have shown that ferroptosis plays a key role in its occurrence and development, and deubiquitinating enzymes can affect the ferroptosis process by regulating the ubiquitination level of key molecules of ferroptosis.

[0003] USP53 is a newly discovered member of the deubiquitinase family, and its gene encoding is located on chromosome 4 in the human genome. Currently, there are few related studies, and they are mainly focused on cholestasis, cancer, and hearing loss, and there are no reports on its role in renal ischemia-reperfusion injury. Summary of the invention

[0004] Aiming at the problems existing in the traditional treatment of ischemia-reperfusion injury, the present invention proposes the application of a novel substance for silencing USP53 gene in a drug for treating renal ischemia-reperfusion injury.

[0005] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0006] The present invention proposes the use of a substance for silencing USP53 expression in human renal tubular epithelial cells in a drug for alleviating renal ischemia-reperfusion injury. The USP53 gene sequence is shown in SEQ ID NO: 1.

[0007] USP53 ubiquitin specific peptidase 53 gene sequence can be queried by the following methods:

[0008] Gene ID: 54532, updated on 3-Apr-2024

[0009] Source link:

[0010] https: / / www.ncbi.nlm.nih.gov / nuccore / NC_060928.1?from=122517672&to=1226006 20&report=genbank.

[0011] Furthermore, the present invention proposes the use of a substance that silences the expression of USP53 in human renal tubular epithelial cells in a drug for alleviating the inflammatory response, apoptosis and ferroptosis of renal tubular cells induced by renal ischemia-reperfusion.

[0012] The above-mentioned vector for silencing USP53 expression in human renal tubular epithelial cells comprises a small interfering RNA vector, wherein the small interfering RNA vector comprises a small interfering RNA with a sequence of GUGCGGUACAUUUCUACAATT.

[0013] The present invention also proposes the use of small interfering RNA that silences USP53 expression in human renal tubular epithelial cells in the preparation of a drug for alleviating inflammatory response, apoptosis and ferroptosis of renal tubular cells induced by renal ischemia-reperfusion. The small interfering RNA sequence is GUGCGGUACAUUUCUACAATT and is named USP53-homo-1605.

[0014] The present invention also proposes the use of a substance for detecting the expression level of USP53 in human renal tubular epithelial cells in a diagnostic reagent or kit for renal ischemia-reperfusion injury.

[0015] Specifically, the present invention proposes the use of a substance for detecting the expression level of USP53 in human renal tubular epithelial cells in a diagnostic reagent or kit for inflammatory response, apoptosis and ferroptosis of renal tubular cells induced by renal ischemia-reperfusion.

[0016] This patent was funded by the Shandong Natural Science Foundation General Project (ZR2023MH137), the National Natural Science Foundation Youth Project (81900659), and the Shandong University Second Hospital Cross-Cultivation (General) Fund (2023JX19).

[0017] Compared with the prior art, the advantages and positive effects of the present invention are:

[0018] The present invention uses small interfering RNA to specifically silence UPS53 in human renal tubular epithelial cells, and proposes that silencing USP53 expression can significantly alleviate cell damage induced by ischemia-reperfusion, thereby improving the inflammatory response, apoptosis and ferroptosis of renal tubular cells. USP53 renal tubular specific knockout mice were constructed by loxP-Cre technology, and a renal ischemia-reperfusion mouse model was established. In vivo experiments verified that silencing USP53 expression in renal tubular epithelial cells can significantly improve renal damage caused by renal ischemia-reperfusion; the present invention firstly reveals that silencing USP53 expression in renal tubular epithelial cells can improve renal ischemia-reperfusion injury, providing a new drug design target for the treatment of renal ischemia-reperfusion injury, and has important theoretical guidance and clinical transformation application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of immunoblotting and statistics are shown, where Ctr: control group; Reoxygenation after hypoxia (90 min) 6, 12, 24h: represent the sample groups collected at 6, 12, and 24h after reoxygenation and resugaration. * indicates P<0.05 compared with the control group (n=6).

[0020] Figure 2 The protein expression of USP53 in human renal tubular epithelial cells was detected by immunoblotting after silencing the gene expression of USP53 using USP53 small interfering RNA. NC: siRNA-NC control group; siRNA-USP53: transfection of USP53 small interfering RNA group. * indicates P<0.05 compared with the siRNA-NC control group (n=6).

[0021] Figure 3 Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of proinflammatory factors in human renal tubular epithelial cells: interleukin-1β (IL-1β), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) after silencing USP53 gene expression in human renal tubular epithelial cells using USP53 small interfering RNA. NC: siRNA-NC control group; siRNA-USP53: transfection of USP53 small interfering RNA group; Ctr: control group; Reoxygenation after hypoxia (90 min) 12h, 24h: represents the sample groups collected at 12h and 24h after OGD. * indicates P<0.05 compared with NC+Ctr; # indicates P<0.05 compared with NC+OGD group at the same time point (n=6).

[0022] Figure 4 After silencing the USP53 gene expression in human renal tubular epithelial cells using USP53 small interfering RNA, an oxygen-glucose deprivation model was constructed to detect cell apoptosis after 24 hours. NC: siRNA-NC control group; siRNA-USP53: transfection of USP53 small interfering RNA group; Ctr: control group; Reoxygenation after hypoxia (90 min): represents the sample group collected at 24 hours after OGD. * indicates P<0.05 compared with NC+Ctr; # indicates P<0.05 compared with NC+OGD group (n=6).

[0023] Figure 5 After silencing the USP53 gene expression in human renal tubular epithelial cells using USP53 small interfering RNA using a special kit, an oxygen-glucose deprivation model was constructed to detect ferroptosis-related indicators: intracellular iron ions and glutathione (GSH) levels. NC: siRNA-NC control group; siRNA-USP53: transfection of USP53 small interfering RNA group; Ctr: control group; Reoxygenation after hypoxia (90 min): represents the sample group collected at 24h after OGD. * indicates P<0.05 compared with NC+Ctr; # indicates P<0.05 compared with NC+OGD group.

[0024] Figure 6 Immunoblotting was used to detect the expression level of USP53 protein in the kidney tissue of mice at different time points after renal ischemia-reperfusion. Sham: sham-operated mice group; Time after I / R 24, 48: mice were killed 24h and 48h after renal ischemia-reperfusion. * indicates P<0.05 compared with the Sham group (n=6).

[0025] Figure 7 Immunohistochemistry was used to detect the expression level of USP53 protein in the kidney tissue of mice at different time points after renal ischemia-reperfusion. Sham: sham-operated mice group; Time after I / R 24, 48: mice were killed 24h and 48h after renal ischemia-reperfusion; IgG: IgG antibody was used as negative control in the primary antibody; USP53: USP53 antibody was used as the primary antibody.

[0026] Figure 8To construct USP53 renal tubular epithelial cell-specific knockout mice by using the loxP-Cre technology, and to detect the gene silencing efficiency by immunofluorescence. a. Cre-LoxP recombination system was used to construct renal tubular cell USP53-specific knockout mice. The renal tubular cell USP53-specific knockout mice Cdh16-Cre / USP53fl / fl are indicated as Cre+ / -USP53fl / fl; Cre- / -USP53fl / fl are control mice. b. Immunofluorescence double labeling detection of Cre+ / -USP53fl / fl mice, in which LTL marks proximal tubular cells, Clabindin marks distal tubular cells, and DBA marks collecting duct cells (showing red fluorescence); USP53 shows green fluorescence; and the cell nucleus shows blue fluorescence (marked by DAPI staining).

[0027] Fig. 9 High performance liquid chromatography was used to detect the biochemical indicators of kidney injury in each group of mice: serum creatinine and urea nitrogen levels. Sham: sham-operated mice group; I / R 24h, 48h: mice killed at 24h and 48h after renal ischemia and reperfusion; WT: wild-type mice; Cre+ / -USP53fl / fl: renal tubular cell USP53-specific knockout mice. * indicates compared with the WT Sham group, # indicates compared with the WT model group at the same time point, P<0.05 (n=6).

[0028] Fig.10 HE staining was used to detect the morphological damage of the kidneys in each group of mice and the scores were calculated. Sham: sham-operated mice group; I / R 24h, 48h: mice killed at 24h and 48h after renal ischemia and reperfusion; WT: wild-type mice; Cre+ / -USP53fl / fl: mice with specific knockout of USP53 in renal tubular cells. * indicates compared with the WT Sham group, # indicates compared with the WT model group at the same time point, P<0.05 (n=6). DETAILED DESCRIPTION

[0029] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0030] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0031] Example 1

[0032] In the present embodiment, unless otherwise specified, the percentages of substances are percentages by volume, and the experimental steps not otherwise specified are all routine biological experimental operations.

[0033] 1. Experimental Procedure

[0034] 1.1 Establishment of mouse renal ischemia-reperfusion model and tissue sampling

[0035] The specific steps are as follows.

[0036] (1) Construction of mouse renal ischemia-reperfusion model

[0037] Twelve-week-old wild-type (WT) C57BL / 6J mice were selected and anesthetized with sodium pentobarbital (30 mg / kg body weight). The limbs of the mice were fixed with nylon thread, and the abdomen was disinfected with iodine and cut open along the midline of the mouse abdomen. The bilateral renal pedicles of the mice were clamped with vascular clamps and observed for 5 minutes. When the kidneys turned black due to ischemia, the timing was started for 30 minutes, and then the vascular clamps were removed to restore blood supply.

[0038] (2) Kidney tissue sampling

[0039] After 24h and 48h of ischemia-reperfusion, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (30mg / kg body weight), the limbs of the mice were fixed, the abdominal cavity was opened along the midline of the abdomen, and pre-cooled saline was used for perfusion. After the blood was completely drained, the kidneys of the mice were taken out and cut longitudinally. Half of the kidneys were fixed in 4% paraformaldehyde, and the other half was stored in a -80℃ refrigerator for later use.

[0040] (3) Preparation of mouse serum

[0041] The blood of the mice was taken out, left to stand at room temperature for 3 hours, and then placed in a low-temperature centrifuge and centrifuged at 3000 rpm at 4°C for 15 minutes. The supernatant was aspirated and stored in a -80°C refrigerator for the detection of serum creatinine and urea nitrogen in mice.

[0042] 1.2 Small interfering RNA-USP53 transfection

[0043] Small interfering RNA-USP53 was commissioned to Shanghai Jima Pharmaceutical Technology Co., Ltd. to construct, and the sequence is GUGCGGUACAUUUCUACAATT. There is no strict requirement for the source of the small interfering RNA-USP53 described in the present invention, and a conventional method for constructing small interfering RNA is used.

[0044] HK-2 cells were cultured in a culture dish, and cell transfection was performed when the cell density reached 40%-50%. The specific steps are as follows: Take two sterile 1.5mL centrifuge tubes, add 500μL serum-free RPMI-1640 to each centrifuge tube, add 2μg small interfering RNA and 4μL Lipo2000 respectively, incubate at room temperature for 5min, mix together, and incubate at room temperature for 20min. Pour out the culture medium in the culture dish, wash three times with PBS, add the prepared transfection reagent, place in a 37℃ constant temperature incubator and culture for 6h, then replace with complete culture medium and continue to culture for 24h before applying exogenous stimulation.

[0045] 1.3 Glucose and oxygen deprivation experiment in human renal tubular epithelial cells (HK-2)

[0046] Oxygen glucose deprivation (OGD) was used to simulate renal ischemia-reperfusion injury in vivo. HK-2 cells in good growth condition were inoculated into a 3 ml diameter dish, with 5×10 cells in each dish. 5 After the cells are tightly attached to the wall, the culture medium is discarded, and the cells are washed twice with sterile PBS buffer. After discarding the PBS, 3 ml of sterile Eagle's buffer is added, and the culture dish is placed in an anoxic chamber, nitrogen is introduced, and the oxygen concentration in the chamber is detected by an instrument. When the oxygen concentration drops to 0.1%, the anoxic chamber is sealed and placed in a 37°C incubator. The timing is started for 90 minutes. After the time is up, the culture dish is taken out and the cell state is observed under a microscope. At this time, the cells should appear needle-like. In a sterile operating table, Eagle's buffer is discarded, and the cells are washed twice with sterile PBS. 3 ml of RPMI-1640 culture medium containing 10% FBS is added. For the control group, only the culture medium is replaced. The cells are cultured at 37°C containing 5% CO2 for 6h, 12h, and 24h, and then lysed with RIPA lysis buffer (strong) or Trizol lysis buffer to collect the cells and store them in a -80°C refrigerator for subsequent detection.

[0047] 1.4 Immunoblotting

[0048] (1) Total protein extraction from tissues

[0049] Cut a small piece of kidney tissue on ice, about 20 mg, and place it in a 1.5 mL centrifuge tube. Use 150 μL of RIPA lysis buffer (strong) containing 1% PMSF to lyse the tissue. Grind the kidney tissue on ice with a tissue grinder. After standing for 15 minutes, place it in a 4°C centrifuge and centrifuge at 12,000 rpm for 15 minutes. Pipette about 120 μL of supernatant and transfer it to a 1.5 mL centrifuge tube.

[0050] (2) Total cell protein extraction

[0051] Aspirate excess PBS in the culture dish, add 70 μL of RIPA lysis buffer (strong) containing 1% PMSF, gently scrape the cells from the bottom of the culture dish with a cell scraper, operate on ice, transfer the scraped cell lysate to a clean 1.5 mL centrifuge tube, lyse on ice for about 15 minutes, put the centrifuge tube into a 4°C centrifuge, high-speed centrifuge at 12000 rpm for 10 minutes, and transfer 50 μL of protein supernatant to a 1.5 mL centrifuge tube.

[0052] (3) Preparation of protein samples

[0053] The protein concentration was detected by BCA method.

[0054] 1) Prepare a standard protein curve. Use a standard protein with a concentration of 1 mg / mL. Use a 96-well plate and add 0, 4, 8, 12, 16, and 20 μL of standard protein to each well. Use deionized water to make up to 20 μL so that the concentrations of each point on the standard curve are 0.2, 0.4, 0.6, 0.8, and 1 mg / mL.

[0055] 2) Prepare BCA mixed solution: According to the instructions, mix solution A and solution B at a volume ratio of 50:1, and add 200 μL of BCA mixed solution to each sample.

[0056] 3) Add 2 μL of protein sample and 18 μL of deionized water to each well and mix gently.

[0057] 4) Add 200 μL of BCA mixture to each well, mix thoroughly, and place in a 37°C incubator incubated for 30 minutes in the dark.

[0058] 5) Use an ELISA reader to measure the absorbance of the protein sample at a wavelength of 562 nm.

[0059] 6) Determine the protein concentration-absorbance standard curve and calculate the standard curve equation, substitute the measured protein sample absorbance, calculate the protein sample concentration, adjust the sample concentration to be consistent, add deionized water and 5× loading. Put the EP tube in a 100℃ metal bath and heat for 10 minutes, take it out and cool it, then put it in a -80℃ refrigerator for storage.

[0060] (4) Preparation of SDS-polyacrylamide gel

[0061] The preparation of polyacrylamide gel is divided into two steps. First, prepare the separation gel. The concentration of the separation gel is mainly 8% and 10%. The thickness of the gel preparation plate used is 1.5mm. After the gel preparation is completed, mix it thoroughly and use a pipette to slowly add it between the fixed glass plates to a height of about 4cm. Add 1mL of isopropanol above the liquid surface to keep the liquid surface level. After 30 minutes, the separation gel solidifies, discard the upper layer of isopropanol, and pour the prepared upper layer of stacking gel between the plates. When the stacking gel is completely filled, insert a comb and let it stand at room temperature for about 30 minutes.

[0062] (5) Electrophoresis

[0063] Insert the prepared gel into the vertical electrophoresis tank, check the tightness between the two sides of the gel, pour the electrophoresis solution into the electrophoresis tank, fill the tank with the electrophoresis solution, start loading the sample, cover the electrophoresis tank cover, insert the wire into the electrophoresis power socket, check whether the electrode is correct, set the starting voltage to 90V, turn on the power to start electrophoresis, and after the sample re-layer gel enters the separation gel, adjust the voltage to 120V to continue electrophoresis, wait for the bromophenol blue to completely run out of the separation gel, turn off the power to stop electrophoresis and prepare for membrane transfer.

[0064] (6) Transfer

[0065] The wet transfer method was used for transfer. A 5cm×8cm PVDF membrane was placed in methanol and soaked for about 5 minutes. After the PVDF membrane was fully activated, the gel was removed from the electrophoresis tank, the gel was removed from the glass plate, and the excess gel was removed. The sponge, filter paper, and PVDF membrane for transfer were placed in the transfer solution, and the sponge and filter paper were fully soaked. The sponge, filter paper, separation gel, and PVDF membrane were placed on the negative electrode in sequence, and the filter paper and sponge were covered after the excess bubbles were discharged. The transfer clamp was closed and placed in the electrophoresis tank. The ice pack required for cooling was placed in the electrophoresis tank. The transfer solution was filled into the transfer tank, and the transfer tank cover was covered. The transfer tank was placed in a foam box containing ice water, and the power cord was plugged into the power jack. Check whether the electrode was inserted correctly, turn on the power, and transfer the membrane at a constant current of 300mA for 1h.

[0066] (7) Closed

[0067] After the transfer is completed, turn off the power and remove the transfer clip, use tweezers to take out the PVDF membrane, place it with the side close to the gel facing down in the pre-prepared blocking solution containing 5% skimmed milk powder, and block it on a horizontal shaker for 1 hour.

[0068] (8) Incubation with primary antibody

[0069] Remove the blocked PVDF membrane from the blocking solution, wash it three times with TBST, 5 minutes each time, place the PVDF membrane on a glass plate, cut the PVDF membrane into the required width according to the molecular weight of the target protein and the marker on the PVDF membrane, absorb excess moisture with toilet paper, put the strip into the antibody box, pour the antibody into the antibody box, place it on a shaker, and cut it overnight.

[0070] (9) Membrane washing

[0071] Recover the primary antibody, take out the strips with tweezers and wash them three times in TBST, each wash for 10 min.

[0072] (10) Incubation with secondary antibody

[0073] Use TBST to prepare 5% skim milk powder, dilute the secondary antibody at a ratio of 1:5000, put the band into a 50mL centrifuge tube containing the secondary antibody, place it on a horizontal shaker, and incubate at room temperature for 2 hours.

[0074] (11) Membrane washing

[0075] The secondary antibody was discarded and the membrane was washed 3 times with TBST, each time for 10 min.

[0076] (12) Development

[0077] Prepare ECL luminescent liquid in a ratio of 1:1 between liquid A and liquid B, place the strip on a disc, remove excess liquid on the strip with toilet paper, drop ECL luminescent liquid on the strip, place it in a developer, adjust the exposure time, and take a photo to save the image.

[0078] (13) Analysis

[0079] The grayscale analysis software imageJ was used to analyze the grayscale of the saved bands, and the grayscale value ratio of the target protein band to the internal reference protein band was the relative expression level of the target protein in different groups.

[0080] 1.5 Immunohistochemical staining

[0081] The tissue sections were placed in a 65°C oven for 2 h, and while still hot, the sections were dewaxed and immersed in water according to the following procedure.

[0082] (1) Dewax and hydrate according to the following steps: xylene I (20 min) → xylene II (20 min) → anhydrous ethanol (10 min) → 95% ethanol (5 min) → 90% ethanol (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min) → deionized water (10 min).

[0083] (2) Place the tissue section in an antigen retrieval box, pour in citric acid-sodium citrate (pH 6.0) antigen retrieval solution, place in a microwave oven, heat on high heat for 3 minutes, then immediately switch to low heat and heat for 30 minutes. Remove from the antigen retrieval box and cool at room temperature.

[0084] (3) Discard the antigen retrieval solution in the antigen retrieval box and add PBS buffer to wash three times, each time for 5 minutes.

[0085] (4) Wipe off the excess PBS on the slide, add 3% H202-methanol solution (the volume ratio of H202 to methanol is 3:97) on the slice specimen, block for 15 minutes, and add once in the middle.

[0086] (5) Wash with PBS three times, 5 min each time.

[0087] (6) Add 5% BSA (prepared in PBS) for blocking and incubate at 37°C for 1 h.

[0088] (7) Prepare the primary antibody at a mass ratio of 1:100 (antibody: 2% PBS). Aspirate the excess BSA and drip the primary antibody onto the tissue sections, about 20 μL per tissue. Store in a humidified box and incubate overnight at 4°C. Homologous IgG is used as a negative control.

[0089] (8) Place the wet box at room temperature for 30 minutes and wash with PBS three times, 5 minutes each time.

[0090] (9) Wipe dry the PBS around the tissue sections and add the corresponding secondary antibody. Place the wet box at 37°C and incubate for 60 min.

[0091] (10) Wash the sections with PBS three times, 5 min each time.

[0092] (11) Prepare DAB dye solution according to the instructions, store in a dark place, and use within 30 min. Shake off excess water on the slices, add DAB dye solution to the tissue slices, start timing, and observe under a microscope. When brown particles appear on the tissue sample, stop timing and terminate the reaction with tap water. Then, stain each group according to the recorded time.

[0093] (12) Wipe off excess water around the tissue sections, add hematoxylin for staining for 3 minutes, immerse the sections in tap water to terminate the reaction, differentiate with 1% hydrochloric acid-alcohol (the volume ratio of hydrochloric acid to alcohol is 1:99), immerse the sections in tap water to terminate the differentiation, and ammonia water will turn blue.

[0094] (13) Dehydrate and make transparent according to the following steps: 70% ethanol (5 min) → 80% ethanol (5 min) → 90% ethanol (5 min) → 95% ethanol (5 min) → anhydrous ethanol (10 min) → xylene (20 min).

[0095] (14) Sealing: Take out the tissue slices, wipe off the excess xylene, add a small amount of neutral gum to the tissue slices and seal with a coverslip, then place in a fume hood to dry.

[0096] (15) Take photos under an optical microscope and save the images.

[0097] 1.6 Immunofluorescence staining

[0098] (1) The immunofluorescence staining procedure is basically the same as that of immunohistochemistry staining before overnight incubation with the primary antibody, but 3% H202-methanol solution is not required to block endogenous peroxidase. When incubating with the primary antibody, USP53 and markers of different parts of the renal tubules or immune cell markers are co-prepared in the same EP tube. 30 μL is added to each tissue sample and placed in a wet box for incubation overnight at 4 °C.

[0099] (2) Remove the wet box and place it at room temperature. Rewarm for 30 minutes. Wash with PBS three times, 5 minutes each time. Shake off the PBS. Add a drop of green fluorescent secondary antibody (prepared with 2% BSA) to each tissue section and incubate at room temperature for 2 hours in the dark.

[0100] (3) Wash tissue sections with PBS 5 times, 5 min each time.

[0101] (4) Wipe off excess PBS, add the corresponding red fluorescent secondary antibody (prepared with 2% BSA) to the tissue section, and incubate in a wet box at room temperature in the dark for 2 h.

[0102] (5) Wash tissue sections with PBS 5 times, 5 minutes each time. Wipe off excess PBS and add DAPI (1:3000 dilution, prepared in PBS) to stain the nucleus. Add about 30 μL to each tissue specimen and incubate at room temperature in the dark for 5 minutes.

[0103] (6) Wash tissue sections with PBS five times, 5 min each time.

[0104] (7) Wipe off excess PBS around the tissue section, add a small amount of anti-fluorescence quencher mounting medium to the sample and use a coverslip to seal the sample. Be careful not to create bubbles and fix the coverslip with nail polish.

[0105] 1.7 Extraction of total RNA from cells and Real-time RT-PCR detection

[0106] (1) Extraction of total RNA

[0107] ① In this step, all experimental instruments and consumables are soaked in DEPC water overnight and sterilized by high pressure steam. After washing the cell samples with saline, discard the saline, add 1 mL of Trizol lysis buffer, scrape the cells with an RNase-free cell scraper, and transfer them to a 1.5 mL RNase-free EP tube.

[0108] ② Add 200 μL of chloroform to each EP tube, vortex vigorously for 15 seconds to mix, and let stand at room temperature for 15 minutes.

[0109] ③ Centrifuge at 12000rpm, 4ºC for 15min, carefully transfer 400μL of supernatant to another RNase-free 1.5mL EP tube, and do not transfer the middle layer.

[0110] ④ Add 400 μL of isopropanol to each tube, gently invert upside down to mix, and let stand at room temperature for 20 minutes.

[0111] ⑤ Centrifuge at 12000rpm, 4ºC for 15min, discard the isopropanol in the tube without touching the precipitate at the bottom of the tube.

[0112] ⑥ Add 1 mL of 75% ethanol to each tube to wash the precipitate.

[0113] ⑦ Centrifuge at 12000rpm, 4ºC for 3min, discard the isopropanol, repeat this step, and place the EP tube opening in a fume hood to dry.

[0114] ⑧ Add an appropriate amount of DEPC water to each tube (about 40 μL for tissues and about 20 μL for cells), gently tap the EP tube, and use a small spectrophotometer to measure the RNA concentration after the RNA is dissolved.

[0115] ⑨ Use a small spectrophotometer to measure the RNA concentration, clean the detection port with a small amount of DEPC water, return to zero, use an RNase-free pipette tip to draw 1 μL of sample and add it to the detection port, detect RNA purity by OD260 / 280 ratio, and select samples with OD260 / 280 in the range of 1.8-2.0 for reverse transcription.

[0116] (2) RNA reverse transcription

[0117] 1 μg of mRNA sample was taken, and the reaction system was as follows: 4 μL of 5× Buffer, 1 μL of olig-DT, 1 μL of 6-mers, 1 μg of RNA, and DEPC water was added to 20 μL. Amplification program: 15 min at 37°C, 5 s at 85°C, and terminated at 4°C.

[0118] (3) Real-time quantitative PCR

[0119] After instant centrifugation of the primers, add sterile deionized water according to the primer instructions, gently tap the tube wall, and centrifuge clockwise after the primers are fully dissolved. Pipette 10 μL of each upstream and downstream primers, add 80 μL of deionized water, and dilute to a final concentration of 10 μM. The reaction system is as follows.

[0120] The volume of cDNA was 1 μL, the volume of Primer (upstream and downstream mixture, 10 μM) was 1 μL, the volume of 2×SYBR Premix Ex TaqTM was 10 μL, and the volume of ddH2O was 8 μL.

[0121] Real-time RT-PCR amplification procedure is as follows.

[0122] Pre-denaturation: 95℃ for 3min; denaturation: 95℃ for 10s; annealing: 59℃ for 30s; extension: 72℃ for 30s; final extension: 72℃ for 10min; melting curve: 65-95℃ for 15min.

[0123] The skeleton protein gene β-Actin was selected as the internal reference to calculate the expression of the target gene. -△△Ct目的 -2 -△△Ct内参 The relative expression level of the target gene was calculated.

[0124] 1.8 HE staining to detect renal morphological damage and score

[0125] (1) HE staining

[0126] The preparation method of paraffin sections of mouse kidney tissue and the dewaxing and hydration process refer to IHC. The dewaxed and hydrated sections are gently shaken dry, and hematoxylin dye is added to the tissue sections for staining for 3 minutes. The staining is terminated with tap water. The sections are immersed in a 1% alcohol solution of hydrochloric acid for about 5 seconds, and the reaction is terminated with tap water. The sections are then immersed in 1% ammonia water for about 10 seconds, and the reaction is terminated with tap water. The slides are gently shaken dry, and eosin dye is added for staining for 1 minute. The reaction is terminated with tap water. The slides are placed in 95% alcohol for about 30 seconds. The specific time is adjusted according to the degree of decolorization. They are soaked in 100% ethanol for 5 minutes, soaked in xylene for 15 minutes, and then sealed with neutral gum and observed under a microscope.

[0127] (2) Morphological damage score

[0128] Under an optical microscope, ten high-power fields of the renal cortex and outer medulla were randomly selected. The degree of morphological damage was scored and statistically analyzed according to the degree of tubular damage, such as tubular cell nuclear shedding, tubular expansion, brush border shedding, and vacuolar degeneration. The scoring criteria were as follows:

[0129] 0-10% is scored as 1;

[0130] 11–25% scored 2;

[0131] 26–45% scored 3;

[0132] 46–75% scored 4;

[0133] >75% score is 5.

[0134] 1.9 Flow cytometry detection of cell apoptosis

[0135] The flow cytometry was used to detect the effect of gene silencing of USP53 expression in HK-2 cells on hypoxia-induced cell apoptosis. The specific steps are as follows.

[0136] (1) Collect cells: After cell stimulation, collect the culture medium supernatant into a 15 mL centrifuge tube, wash three times with 2 mL of PBS and collect in the same centrifuge tube. Add 0.5 mL of trypsin (without EDTA) to each well for digestion. After the cells are completely rounded under the microscope, gently blow and collect the cell suspension in the same centrifuge tube and centrifuge at 1300 rpm 4ºC for 7 minutes, and remove the supernatant. Add 5 mL of pre-cooled PBS to wash the cells twice, centrifuge at 1300 rpm 4ºC for 7 minutes, and discard the supernatant.

[0137] (2) Add 400 μL of binding solution to the centrifuge tube, shake the centrifuge tube to resuspend the cells, and transfer the cell suspension to the flow cytometry tube. According to the instructions of the kit, add 5 μL of FITC dye solution, shake gently to mix, and incubate at 4°C for 15 min in the dark.

[0138] (3) Add 10 μL of PI dye solution, gently shake to mix, and incubate at 4°C for 5 min in the dark.

[0139] (4) Detection of cell apoptosis by flow cytometry.

[0140] 1.10 Determination of intracellular iron content

[0141] Follow the instructions of the iron ion colorimetric detection kit.

[0142] (1) Wash the cells to be tested three times with PBS buffer, add 200 μL / well lysis buffer, and place on a shaker for 2 hours.

[0143] (2) Dilution of the standard: Use the diluent provided in the kit to dilute the 3 mM stock solution of the standard to the following: 300 uM, 150 uM, 75 uM, 37.5 uM, 18.75 uM, 9.38 uM, 4.69 uM. Pay attention to the multiple dilution and set up a 0 concentration control reaction tube.

[0144] (3) Mix the buffer solution and 4.5% potassium permanganate solution in a ratio of 1:1, which is called the mixed solution.

[0145] (4) Prepare the assay system according to Table 1. Take several 1.5 mL EP tubes, set up one blank control tube, seven standard tubes and several sample tubes. If the iron content in the sample is too high, dilute it appropriately with lysis buffer before assaying.

[0146] Table 1 Iron ion content determination system

[0147]

[0148] (5) Mix well and incubate at 60°C for 1 h.

[0149] (6) Cool to room temperature and centrifuge the droplets on the tube cap and tube wall into the bottom of the tube.

[0150] (7) Add 30 μL of iron ion detector, mix well, and incubate at room temperature for 30 min.

[0151] (8) Take 200 μL and add it to a 96-well culture plate. Use a multi-function microplate reader to measure the absorbance at 550 nm.

[0152] (9) Draw a standard curve and calculate the iron ion content.

[0153] 1.11 Determination of glutathione content

[0154] Follow the instructions of the GSH and GSSG detection kits.

[0155] (1) Preparation of working solution: prepare GSSG stock solution, DTNB stock solution, protein removal reagent M solution (prepared immediately before use), NADPH stock solution (40 mg / mL), 5-fold diluted glutathione reductase, total glutathione detection working solution, 0.5 mg / mL NADPH, diluted GSH scavenging auxiliary solution (prepared immediately before use), and GSH scavenging reagent working solution (prepared immediately before use).

[0156] (2) Preparation of standard: Use protein removal reagent M solution to dilute 10mM GSSG stock solution into 15μM, 10μM, 5μM, 2μM, 1μM, and 0.5μM GSSG solutions to prepare a standard curve; add diluted GSH scavenging auxiliary solution at a ratio of 20μL of diluted GSH scavenging auxiliary solution to 100μL of standard, and immediately vortex to mix; then add GSH scavenging reagent working solution at a ratio of 4μL GSH scavenging reagent working solution per 100μL, immediately vortex to mix, and react at 25℃ for 60min.

[0157] (3) Preparation of total GSH content samples and standards: Wash the cells once with PBS, collect the cells by centrifugation, and aspirate the supernatant. Add 3 times the volume of the cell pellet to the protein removal reagent M solution and vortex thoroughly. Then use liquid nitrogen and a 37°C water bath to quickly freeze and thaw the sample twice, then place it at 4°C or in an ice bath for 5 minutes, and centrifuge it at 4°C at 10,000g for 10 minutes. Take the supernatant for the determination of total GSH. The treated samples are temporarily stored in a 4°C refrigerator and should not be stored in a -80°C refrigerator for more than 10 days. Before the measurement, the sample needs to be diluted with protein removal reagent M solution, and the dilution multiple can be up to 20 times.

[0158] (4) Preparation of samples for GSSG content to be measured: Take part of the above-prepared samples for total GSH content to be measured, add 20 μL of diluted GSH removal auxiliary solution per 100 μL of sample, add diluted GSH removal auxiliary solution, and immediately vortex mix. Then add 4 μL of GSH removal reagent working solution per 100 μL of sample, add GSH removal working solution, immediately vortex mix, and react at 25°C for 60 minutes. The above reaction can remove up to 50 μM GSH. If the GSH content in the sample is too high, it needs to be appropriately diluted before removing GSH.

[0159] (5) Determination of samples and standards.

[0160] ① Take a 96-well culture plate, add samples or standards in order according to Table 2, and mix well; add 150 μL of total GSH detection working solution, mix well, and incubate at room temperature for 5 minutes.

[0161] Table 2 Total GSH detection system

[0162]

[0163] ②Add 50 μl of 0.5 mg / mL NADPH solution and mix well.

[0164] ③After 25 minutes of reaction, use a multifunctional microplate reader to measure the absorbance at 412nm. If the absorbance of the sample is relatively low, the incubation time can be extended to 30-60 minutes. The absorbance of the standard and sample will increase linearly with time within a certain range.

[0165] (6) Calculation of total glutathione content in samples.

[0166] The total GSH and GSSG contents of the sample were calculated according to the standard curve, and then the GSH content was calculated based on the total GSH content and GSSG content: GSH=Total Glutathione-GSSG×2.

[0167] 1.12 Construction of USP53 renal tubular epithelial cell-specific knockout mice

[0168] The present invention constructs USP53 renal tubular cell-specific knockout mice, and the construction strategy and identification method are as follows:

[0169] (1) Establishment of tubular cell-specific knockout mice: Tubular cell Cdh16-Cre mice and USP53flox / flox mice were purchased from Shanghai Model Organisms Technology Co., Ltd. USP53flox / flox mice were mated with tubular cell Cdh16-Cre mice to obtain F1 generation mice. The Cdh16-Cre+ / USP53flox / + genotype in the F1 generation mice was mated with USP53flox / flox mice to obtain the second generation offspring (F2). The target genotype mice of Cdh16-Cre+ / USP53flox / flox were finally obtained through breeding of F2 mice.

[0170] (2) Identification of renal tubular cell-specific gene knockout mouse model: The kidney tissue of Cdh16-Cre+ / USP53flox / flox mice was embedded in OCT and frozen sectioned. The renal tubular markers were labeled with immunofluorescence double labeling technology to co-localize with USP53, and the expression changes of USP53 in renal tubular cells were observed under laser confocal microscopy. Immunofluorescence co-localization analysis can accurately identify whether the genetically engineered mice were successfully constructed.

[0171] 2. Experimental results

[0172] (1) After constructing an oxygen-glucose deprivation model for human renal tubular epithelial cells according to step 1.3 of this example, the expression level of USP53 protein in the cells was detected at 6, 12, and 24 hours, respectively. Then, immunoblotting was performed according to step 1.4. The results showed that in the oxygen-glucose deprivation model, the protein expression of USP53 in human renal tubular epithelial cells was significantly increased, and showed a certain time dependence with the extension of the time of reoxygenation and resugaration, such as Figure 1 shown.

[0173] (2) After immunoblotting in step 1.4, the protein expression of USP53 in human renal tubular epithelial cells was significantly decreased after silencing the USP53 gene expression in human renal tubular epithelial cells using USP53 small interfering RNA in step 1.2. Figure 2 shown.

[0174] (3) After silencing the USP53 gene expression in human renal tubular epithelial cells using USP53 small interfering RNA in step 1.2, an oxygen-glucose deprivation model was constructed in step 1.3. The results of Real-time RT-PCR detection in step 1.7 showed that pro-inflammatory factors in cells at 12 and 24 h time points showed that silencing the USP53 gene expression could alleviate the inflammatory response induced by ischemia-reperfusion, such as Figure 3 shown.

[0175] (4) After silencing the expression of USP53 gene in human renal tubular epithelial cells using USP53 small interfering RNA in step 1.2, an oxygen-glucose deprivation model was constructed through step 1.3, and then the level of cell apoptosis was detected by flow cytometry according to the content recorded in step 1.9. The results showed that silencing the expression of USP53 gene could reduce the cell apoptosis induced by ischemia-reperfusion, such as Figure 4 shown.

[0176] (5) After silencing the USP53 gene expression in human renal tubular epithelial cells using USP53 small interfering RNA in step 1.2, an oxygen-glucose deprivation model was constructed in step 1.3. The results of the iron ion detection kit in step 1.10 and the GSH and GSSG detection kits in step 1.11 showed that silencing the USP53 gene expression could reverse the increase in intracellular iron ions caused by ischemia-reperfusion, such as Figure 5 As shown in a; GSH decreases, such as Figure 5 As shown in b, silencing USP53 gene expression can improve cell ferroptosis.

[0177] (6) The immunoblotting results in step 1.4 showed that the expression level of USP53 protein in the renal tissue of the renal ischemia-reperfusion model mice in step 1.1 was significantly increased, and showed a certain time dependence with the extension of reperfusion time, such as Figure 6 shown.

[0178] (7) The immunohistochemical results in step 1.5 showed that the expression level of USP53 protein in ischemia-reperfusion renal tissue was significantly increased and concentrated in the renal tubules, such as Figure 7 shown.

[0179] (8) In step 1.12, USP53 renal tubular epithelial cell-specific knockout mice were constructed using loxP-Cre technology, and the gene silencing efficiency was detected by immunofluorescence in step 1.6. The results showed that USP53 renal tubular-specific knockout mice were successfully constructed. Figure 8 shown.

[0180] (9) The mouse serum was collected by step (3) in Example 1.1, and the levels of serum creatinine and urea nitrogen, which are related indicators of kidney damage in each group of mice, were detected by high performance liquid chromatography. The results showed that silencing USP53 in renal tubular epithelial cells could improve kidney damage caused by ischemia-reperfusion. Fig. 9 shown.

[0181] (10) The results of HE staining and renal morphological injury scoring in step 1.8 showed that silencing USP53 in renal tubular epithelial cells could improve the brush border shedding, tubular expansion, tubular protein formation, and vacuolar degeneration of renal tubular epithelial cells caused by ischemia-perfusion, thereby alleviating tubular cell damage. Fig.10 shown.

[0182] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Use of a substance for silencing USP53 expression in human renal tubular epithelial cells in the preparation of a drug for alleviating renal ischemia-reperfusion injury, wherein the USP53 gene sequence is as shown in SEQ ID NO: 1, and the substance for silencing USP53 expression in human renal tubular epithelial cells is a small interfering RNA vector, and the small interfering RNA vector comprises the sequence as shown in SEQ ID NO:

2.

2. Use of a substance for silencing USP53 expression in human renal tubular epithelial cells in the preparation of a drug for alleviating inflammatory response, cell apoptosis and ferroptosis of renal tubular cells induced by renal ischemia-reperfusion, the USP53 gene sequence is shown in SEQ ID NO: 1, and the substance for silencing USP53 expression in human renal tubular epithelial cells is a small interfering RNA vector, and the small interfering RNA vector comprises the sequence shown in SEQ ID NO:

2.

3. Use of a small interfering RNA that silences USP53 expression in human renal tubular epithelial cells in the preparation of a drug for alleviating inflammatory response, apoptosis and ferroptosis of renal tubular cells induced by renal ischemia-reperfusion, wherein the sequence of the small interfering RNA is shown in SEQ ID NO:

2.

4. Use of substances for detecting the high or low expression levels of USP53 in human renal tubular epithelial cells in the preparation of diagnostic reagents or kits for renal ischemia-reperfusion injury.

5. Use of substances for detecting the high or low expression levels of USP53 in human renal tubular epithelial cells in the preparation of diagnostic reagents or kits for inflammatory response, apoptosis and ferroptosis of renal tubular cells induced by renal ischemia-reperfusion.

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