Application of CYP4A11 in preparation of medicine for preventing and treating diabetic nephropathy

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of CYP4A11 in preparation of a medicine for preventing and treating diabetic nephropathy. The invention provides a biomarker CYP4A11 gene for auxiliary diagnosis of diabetic nephropathy or a coding product of the CYP4A11 gene for the first time. The expression of CYP4A11 in renal tubules of a DKD patient is up-regulated, the expression of CYP4A14 in renal tubules of a db / db mouse is up-regulated, and the expression of CYP4A11 mRNA in HK-2 cells which are subjected to in-vitro high-glucose culture is up-regulated; therefore, CYP4A11 or a coding product thereof can be used as an auxiliary diagnosis index of diabetic nephropathy for auxiliary diagnosis of diabetic nephropathy. The invention also finds that the level of ferroptosis in the diabetic nephropathy can be successfully reversed by specifically interfering the expression of CYP4A11 mRNA through the sh-CYP4A11 lentivirus, so that the CYP4A11 gene or an encoding product of the CYP4A11 gene can be used as a drug treatment target to be applied to prevention, alleviation or / and treatment of the diabetic nephropathy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the application of CYP4A11 in the preparation of drugs for the prevention and treatment of diabetic nephropathy. Background Art

[0002] Diabetic nephropathy (DKD) has long been considered a glomerular-related disease. However, the "glomerulocentric" theory fails to explain the higher prevalence of severe tubular lesions compared to typical glomerular structural changes in patients with type 2 diabetes and microalbuminuria. Furthermore, as many as 20.5% to 63.0% of diabetic patients develop progressive renal insufficiency without developing microalbuminuria. Consequently, a significant number of researchers believe that tubular lesions may precede microvascular lesions and play a crucial role in the development and progression of DKD. Under high glucose stimulation, excessive reabsorption by renal tubular epithelial cells leads to abnormal tubular-glomerular feedback, hypertrophy and hypoxia of proximal tubular epithelial cells, mitochondrial dysfunction leading to oxidative stress, and the release of chemotactic and inflammatory factors by the tubules, which promote renal interstitial inflammation and fibrosis. Despite considerable progress in understanding the mechanisms that trigger tubular cell injury, no cell-specific and effective treatments are currently available. Therefore, further investigation of the mechanisms of tubular cell injury in DKD and the search for specific and effective therapeutic strategies are of great theoretical and practical significance.

[0003] Ferroptosis is an iron-dependent, lipid peroxidation-driven, non-apoptotic form of cell death characterized by iron overload and lipid peroxide accumulation. Its pathological features include mitochondrial shrinkage, increased membrane density, and decreased mitochondrial cristae. In recent years, the role of ferroptosis in renal tubular epithelial cell damage in DKD has become a research hotspot. In DKD, renal tubular epithelial cell metabolism is elevated, and the production of reactive oxygen species by the mitochondrial oxidative respiratory chain increases significantly. Due to high energy demands and dependence on aerobic metabolism, renal tubules are highly sensitive to oxidative stress and lipid peroxidation, and are susceptible to damage from metabolic disorders and hypoxia. Therefore, further elucidating the pathogenesis of DKD is crucial. Given the key role of ferroptosis in renal tubular injury in DKD, interventions targeting ferroptosis in renal tubular epithelial cells to reduce cell damage are crucial for the treatment of DKD.

[0004] Cytochrome P450 family 4 subfamily A member 11 (CYP4A11) is an important member of the cytochrome P450 family located on the endoplasmic reticulum (the mouse homologous gene is named Cyp4a14). It is abundant in the human liver and kidney. It is a major medium-chain fatty acid ω hydroxylase and is regulated by peroxisome proliferator-activated receptor α (PPARα). It plays an important role in maintaining cellular fatty acid balance and membrane integrity.

[0005] RNA interference (RNAi), also known as a gene silencing technique, involves introducing double-stranded RNA with homologous sequences to an endogenous target gene, inducing mRNA degradation and thereby preventing gene expression. Currently, this technique is widely used in signaling pathway research, disease model development, gene therapy, and drug target identification. The success of this technique depends primarily on the precise binding of small interfering RNA or lentivirus to the target gene, making the selection of an efficient and stable interference pattern crucial. Furthermore, the application of this technique to specifically silence pathogenic genes that are highly expressed in renal tubular epithelial cells during DKD is of great significance for exploring new mechanisms of ferroptosis in renal tubular epithelial cells and identifying potential therapeutic targets for DKD. Summary of the Invention

[0006] In view of the problems and deficiencies in the prior art, the present invention provides the use of CYP4A11 in the preparation of a drug for the prevention and treatment of diabetic nephropathy.

[0007] Based on the above purpose, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides the use of the CYP4A11 gene or the encoded product of the CYP4A11 gene as a drug target for screening drugs for preventing, alleviating and / or treating diabetic nephropathy.

[0009] According to the above application, preferably, the encoding product of the CYP4A11 gene includes CYP4A11 mRNA and CYP4A11 protein.

[0010] The second aspect of the present invention provides the use of a substance that specifically interferes with CYP4A11 expression in the preparation of a medicament for preventing, alleviating or / and treating diabetic nephropathy.

[0011] According to the above application, preferably, the substance that specifically interferes with CYP4A11 expression is one or more of a nucleic acid molecule, a small molecule compound, an antibody, a protein, a gene editing vector, a lentivirus or an adeno-associated virus.

[0012] According to the above application, more preferably, the lentivirus is sh-CYP4A11 lentivirus, and the nucleotide sequence of the sh-CYP4A11 lentivirus is: 5'-CAGAAATGGGTGGAGACATTC-3'.

[0013] A third aspect of the present invention provides a drug for preventing, alleviating and / or treating diabetic nephropathy, wherein the drug contains a substance that specifically interferes with the expression of CYP4A11.

[0014] According to the above-mentioned drug, preferably, the substance that specifically interferes with CYP4A11 expression is one or more of a nucleic acid molecule, a small molecule compound, an antibody, a protein, a gene editing vector, a lentivirus or an adeno-associated virus.

[0015] According to the above-mentioned drug, more preferably, the lentivirus is sh-CYP4A11 lentivirus.

[0016] According to the above medicine, preferably, the medicine further contains a pharmaceutically acceptable carrier or excipient.

[0017] A fourth aspect of the present invention provides use of a detection reagent for the CYP4A11 gene or a product encoded by the CYP4A11 gene in the preparation of a product for auxiliary diagnosis of diabetic nephropathy.

[0018] According to the above application, preferably, the product detects the expression level of the CYP4A11 gene or the product encoded by the CYP4A11 gene in a sample by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern blot, Western blot, chip, high-throughput sequencing platform, immunohistochemistry, tissue immunofluorescence or enzyme-linked immunosorbent assay.

[0019] According to the above application, preferably, the detection sample of the product is a cell or tissue; and the product is a chip, a preparation or a kit.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention discovered for the first time that targeted inhibition of the expression level of the CYP4A11 gene or the product encoded by the CYP4A11 gene can effectively reduce the level of ferroptosis in mouse renal tubular epithelial cells, reduce the gene expression level of oxidative stress-related pathways, and reduce the production of lipid peroxides, thereby achieving the effect of treating diabetic nephropathy. Therefore, the CYP4A11 gene or the product encoded by the CYP4A11 gene can be used as a drug treatment target and applied to the research and development and preparation of drugs for the prevention and treatment of diabetic nephropathy.

[0022] (2) The present invention designed a shRNA lentivirus that interferes with CYP4A11 gene expression by knocking down CYP4A11 mRNA expression levels, successfully reversing the level of ferroptosis in diabetic nephropathy. Therefore, CYP4A11 mRNA can be used as a therapeutic target, and shRNA interfering with CYP4A11 mRNA can be used to prepare drugs for preventing, alleviating or / and treating diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Figure 4: Changes in CYP4A11 expression in renal tubules of patients with diabetic nephropathy (DKD). Figure A shows the results of HE and PAS pathological staining of renal tissues in the control and DKD groups (scale bar = 50 μm, n = 3). Figure B shows the changes in CYP4A11 expression in renal tubules of the two groups detected by immunohistochemistry and semiquantitative analysis (scale bar = 50 μm, n = 3).

[0024] Figure 2 Figure 1 shows the expression changes of Cyp4a14 (mouse homolog of CYP4A11) in the renal tissues of spontaneous type 2 diabetic mice (db / db mice). Figure A shows the results of HE and PAS pathological staining of renal tissues of db / m and db / db mice, scale bar = 50 μm; Figure B shows the expression levels of Kim-1 and Cyp4a14 in the renal tissues of the two groups detected by immunohistochemistry and semi-quantitative analysis, scale bar = 50 μm, n = 6; Figure C shows the expression levels of Kim-1 and Cyp4a14 in the renal tissues of the two groups detected by protein immunoblotting and semi-quantitative analysis, n = 6; Figure D shows the expression level of Cyp4a14 in the renal tissues of the two groups detected by reverse transcription-real-time fluorescence quantitative PCR, n = 6.

[0025] Figure 3 Figure 1 shows the expression changes of CYP4A11 in immortalized human renal proximal tubular epithelial cells (HK-2) cultured in high glucose in vitro. Figure A shows the expression differences of Kim-1 and CYP4A11 in HK-2 cells under normal glucose and high glucose conditions detected by western blotting and semi-quantitative analysis (n=3); Figure B shows the expression levels of CYP4A11 mRNA in the two cell groups detected by reverse transcription-real-time fluorescence quantitative PCR (n=3); Figure C shows the subcellular localization and expression level changes of CYP4A11 in the two cell groups detected by cell immunofluorescence (scale bar=20μm).

[0026] Figure 4 The Cyp4a14 (mouse homologous gene of CYP4A11) gene was specifically knocked out in renal tubular epithelial cells (Cre + / Cyp4a14 fl / fl ) Construction diagram and genotype identification results; in the figure, A is a schematic diagram of specific site knockout; B is the genotype result of agarose gel detection;

[0027] Figure 5 Figure 2 shows the expression level of Cyp4a14 in DKD mice with Cyp4a14-deficient renal tubular epithelial cells. Figure A shows the results of HE and PAS pathological staining of mice in each group, scale bar = 20 μm; B shows the changes in blood creatinine levels of mice in each group, n = 6; C shows the changes in uACR levels of mice in each group, n = 6; D shows the differences in Cyp4a14 expression in renal tissues of mice in each group detected by western blot.

[0028] Figure 6 Figure 1 shows the changes in ferroptosis levels in DKD mice with specific Cyp4a14 deficiency in renal tubular epithelial cells. Figure A shows the mitochondrial morphology of renal tubular epithelial cells in the renal tissue of each group of mice observed by transmission electron microscopy (Scale bar = 1 μm); B shows the expression changes of ferroptosis-related molecules GPX4, TFR1, and SLC7A11 in each group of mice detected by Western blotting and semi-quantitative analysis (n = 6); C shows the changes in GSH content in the renal tissue of each group of mice detected by biochemical kit (n = 6); D shows the Fe content in the renal tissue of each group of mice detected by biochemical kit (n = 6). 2+ Content change, n = 6;

[0029] Figure 7 Figure 1: Effects of sh-CYP4A11 lentivirus, which specifically interferes with CYP4A11 mRNA expression, on CYP4A11 expression in immortalized human renal proximal tubular epithelial cells (HK-2). Figure (A) Western blotting and semiquantitative analysis of Kim-1 and CYP4A11 expression in each cell group (n = 3); Figure (B) RT-PCR analysis of CYP4A11 mRNA expression in each cell group (n = 3).

[0030] Figure 8 The effect of silencing CYP4A11 mRNA on the ferroptosis level in HK-2 cells. Figure A shows the mitochondrial morphology of each group of cells observed by radio microscope, scale bar = 10 μm; B shows the expression changes of ferroptosis-related molecules GPX4, TFR1 and SLC7A11 in each group of cells detected by protein immunoblotting and semi-quantitative analysis, n = 3; C shows the expression of Fe in each group of cells detected by biochemical kit 2+ D is the change of GSH content in each group of cells detected by biochemical kit, n=3. DETAILED DESCRIPTION

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Experimental methods in the following examples, where specific conditions are not specified, were performed using conventional techniques in the art or in accordance with the conditions recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available. Furthermore, the experimental results presented in the examples are averages of multiple experimental results.

[0033] BKS-db homozygous mutant mice (db / db mice, C57BLKS / J-Lepr em2Cd479 / Gpt, Strain NO.T002407) and littermate control BKS-db / m mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0034] The Cyp4a14 conditional gene knockout mouse was constructed by entrusting Saiye (Suzhou) Biotechnology Co., Ltd. according to the existing gene knockout method.

[0035] Cyp4a14-flox mice (C57BL / 6JCya-Cyp4a14 em1flox / Cya, S-CKO-01979) and Cdh16-MerCreMer mice (C57BL / 6JCya-Cdh16 em1(IRES-MerCreMer) / Cya, C001432) are available at Cyyce (Suzhou) Biotechnology Co., Ltd.

[0036] Immortalized human proximal tubular epithelial cell line (HK-2) was purchased from Shanghai Kuisai Biotechnology Co., Ltd.

[0037] Both the sh-CYP4A11 lentivirus and the sh-NC lentivirus were synthesized by GeneCare Biotechnology based on sequence information. The nucleotide sequence of the sh-CYP4A11 lentivirus is: 5'-CAGAAATGGGTGGAGACATTC-3' (SEQ ID NO. 1), and the nucleotide sequence of the sh-NC lentivirus is: 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO. 2).

[0038] Example 1: Expression of CYP4A11 in renal tubules of DKD patients

[0039] Immunohistochemical staining was used to detect the expression of CYP4A11 in the renal tubules of DKD patients.

[0040] 1. Source of test materials

[0041] Diabetic nephropathy (DKD) renal tissue (clinical sample-related experimental research was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Ethics Approval No.: 2023-KY-0501-004). Diabetic nephropathy (DKD) renal tissue was obtained from paraffin sections of the Renal Pathology Department of our hospital. The inclusion criteria were adult patients with diabetic nephropathy aged ≥18 years with a clear clinical diagnosis and confirmed by renal puncture biopsy); normal control renal tissue (control group) was obtained from the biological sample bank of our hospital and the National Human Genetic Resources Sharing Service Platform (Platform No.: 2005DKA21300), which was normal renal tissue adjacent to the cancer after renal tumor surgery. The clinical data of the enrolled patients were obtained from the hospital archived medical records.

[0042] 2. Test methods

[0043] (1) HE staining

[0044] 1) Three paraffin sections each of renal tissue from patients diagnosed clinically and pathologically with DKD and adjacent normal renal tissue were selected;

[0045] 2) Paraffin sections were placed in a 60°C oven for 30 minutes. Dewaxed in three separate vats of xylene at room temperature for 15 minutes each. The xylene was then eluted in 100%, 100%, 95%, 85%, and 75% alcohol, followed by 5 minutes per vat of distilled water. Finally, the sections were washed three times in distilled water.

[0046] 3) Tissue sections were stained with hematoxylin, eosin and sealed with neutral resin.

[0047] (2) PAS staining

[0048] 1) Three paraffin sections each of renal tissue from patients diagnosed clinically and pathologically with DKD and adjacent normal renal tissue were selected;

[0049] 2) Paraffin sections were placed in a 60°C oven for 30 minutes. Dewaxed in three separate vats of xylene at room temperature for 15 minutes each. The xylene was then eluted in 100%, 100%, 95%, 85%, and 75% alcohol, followed by 5 minutes per vat of distilled water. Finally, the sections were washed three times in distilled water.

[0050] 3) Tissue sections were subjected to periodic acid reaction, Schiff reaction, hematoxylin nuclear staining and neutral resin sealing.

[0051] (3) Immunohistochemical staining

[0052] 1) Three paraffin sections each of renal tissue from patients diagnosed clinically and pathologically with DKD and adjacent normal renal tissue were selected;

[0053] 2) Paraffin sections were placed in a 60°C oven for 30 minutes. Sections were then dewaxed at room temperature and then washed three times in xylene for 15 minutes each. The xylene was then washed in 100%, 100%, 95%, 85%, and 75% alcohol, followed by 5 minutes of immersion each time in distilled water. Finally, the sections were washed three times in distilled water.

[0054] 3) Sectioning: Tissue sections were heated in TRIS-EDTA antigen retrieval solution, blocked with 2% goat serum for 30 minutes, and finally incubated overnight with CYP4A11 antibody (11688-1-AP, Mitaka). The next day, histochemical staining was completed by incubation with secondary antibody, DAB color development, hematoxylin nuclear staining, and neutral resin mounting.

[0055] 3. Test results

[0056] HE and PAS pathological staining results are as follows Figure 1 As shown in A, Figure 1 A shows that compared with the control group, the glomerular basement membrane of the DKD group was thickened, the mesangial matrix increased, and the renal tubules atrophied. Figure 1 As shown in B, Figure 1 B shows that CYP4A11 is mainly expressed in the renal tubules. Compared with the control group, the expression of CYP4A11 in DKD patients was significantly increased, and the difference was statistically significant (P<0.05). Figure 1 It can be seen that compared with the control group, the expression level of CYP4A11 protein in the renal tubules of the DKD group was significantly increased.

[0057] Example 2: Mouse Modeling and Changes in CYP4A14 Expression in Mouse Renal Tubules

[0058] Western blot and real-time quantitative PCR were used to detect the expression of CYP4A14 (mouse homologous gene name of CYP4A11) and renal tubular injury molecule Kim-1 in the renal tubules of diabetic mouse models.

[0059] 1. Animal husbandry and grouping

[0060] The experiment involved two groups: db / db and db / m mice. The db / db group consisted of 6-week-old male mice with spontaneous type 2 db / db diabetes, and the db / m group consisted of 6-week-old db / m control male mice from their littermates. Both db / db diabetic male mice and db / m control males were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. After 2 weeks of adaptive feeding, both groups of mice were fed a normal diet for 12 weeks. At 20 weeks of age, all mice were sacrificed and samples were collected.

[0061] 2. Test methods

[0062] (1) HE staining, PAS staining and immunohistochemical staining

[0063] After collecting blood from the apex of the heart, the mice in the db / db group and the db / m group were perfused. The mouse kidneys were removed, the renal capsule and medulla were removed, and the kidneys were separated along the largest section. Half of the kidney tissue was fixed with paraformaldehyde. The fixed kidneys were then embedded in paraffin and sectioned for HE staining, PAS staining, and immunohistochemical staining. The specific procedures for HE staining, PAS staining, and immunohistochemical staining were the same as in Example 1.

[0064] (2) Western blot

[0065] After collecting blood from the apex of the heart, the mice in the db / db and db / m groups underwent cardiac perfusion. The kidneys were removed, and the renal capsule and medulla were removed. Then, part of the renal cortex of the mouse kidney tissue was obtained and homogenized with RIPA lysis buffer in a pre-cooled low-temperature tissue grinder to extract total tissue protein. The protein concentration of the extracted tissue protein was first determined using a BCA protein concentration assay kit. The proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane. Finally, they were labeled with Kim-1 antibody (PA5-79345, Thermo Fisher Scientific) and Cyp4a14 antibody (11688-1-AP, Mitaka Corporation) and observed by chemiluminescence, and the grayscale analysis of the bands was performed.

[0066] (3) Real-time quantitative PCR

[0067] 1) After collecting blood from the apex of the heart and performing cardiac perfusion, the kidneys were removed, and the renal capsule and medulla were removed. RNA was then extracted from the renal cortex using RNA extraction reagent.

[0068] 2) The integrity of the extracted RNA was tested by agarose gel electrophoresis and the NanoDrop TM One spectrophotometer to measure the concentration of extracted RNA;

[0069] 3) Using the obtained RNA as a template, reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific) to synthesize cDNA products;

[0070] The primer sequences are as follows:

[0071] Cyp4a14-F: 5'-GCACCAGATTCTTCTCACCATAGC-3' (SEQ ID NO.3),

[0072] Cyp4a14-R: 5'-TCAAAGCGGAGCAGGGTCAG-3' (SEQ ID NO. 4);

[0073] 4) Apply PowerUp TM SYBR TM Green premix was used for real-time fluorescence quantitative PCR and 2 -ΔΔCt Method for analysis.

[0074] 3. Test results

[0075] The results of HE and PAS pathological staining are as follows Figure 2 As shown in A, compared with db / m control mice, db / db mice with spontaneous type 2 diabetes showed increased mesangial cells, proliferation of mesangial matrix, and enlarged glomerular volume in the kidneys.

[0076] The results of immunohistochemical staining were as follows Figure 2 As shown in B, compared with db / m control mice, the expressions of Kim-1 and CYP4A14 in the renal tubules of db / db mice were significantly increased, and the differences were statistically significant (P<0.0001).

[0077] Western blot test results Figure 2 As shown in C, Figure 2 C showed that compared with db / m control mice, the expression of Kim-1 in db / db group mice was increased, and the difference was statistically significant (P<0.01). The expression level of CYP4A14 protein was significantly increased, and the difference was statistically significant (P<0.0001).

[0078] Real-time quantitative PCR test results Figure 2 As shown in D, Figure 2 D shows that compared with db / m control mice, the expression level of Cyp4a14 mRNA in the kidneys of db / db group mice was significantly increased, and the difference was statistically significant (P<0.0001).

[0079] Example 3: Differences in CYP4A11 expression in renal tubular epithelial cells after high glucose stimulation

[0080] Immortalized human renal tubular epithelial cells (HK-2) were cultured in vitro and treated with high glucose. The expression differences of Kim-1 and CYP4A11 between the high glucose group and the normal group were detected.

[0081] 1. Test method

[0082] (1) Western blot

[0083] HK-2 cells in the exponential growth phase and in good growth condition were randomly divided into two groups, normal glucose group (NG) and high glucose group (HG), and cultured with the following different culture media for 48 hours: 1) NG: normal glucose medium with 5.6 mM glucose; 2) HG: high glucose medium with 30 mM glucose; RIPA lysis buffer was added to the cells in the NG and HG groups and lysed on ice for 15 minutes, and the lysed cell proteins were collected; the protein concentration was first determined using a BCA protein concentration detection kit; then the proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane, and finally labeled with Kim-1 antibody (PA5-79345, Thermo Fisher Scientific) and CYP4A11 antibody (11688-1-AP, Mitaka) and observed by chemiluminescence, and the grayscale analysis of the bands was performed.

[0084] (2) Real-time quantitative PCR

[0085] HK-2 cells in the exponential growth phase and in good growth condition were randomly divided into two groups, normal glucose group (NG) and high glucose group (HG), and cultured with the following different culture media for 48 hours: 1) NG: normal glucose medium with 5.6 mM glucose; 2) HG: high glucose medium with 30 mM glucose. RNA extraction reagent was added to the cells in the NG and HG groups to extract cellular RNA; the integrity of the extracted RNA was tested by agarose gel electrophoresis and NanoDrop TM The extracted RNA concentration was measured using a spectrophotometer. The obtained RNA was used as a template and reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit. The primer sequences are as follows:

[0086] CYP4A11-F: 5'-GAATGACACCATCTACAGCCTGAC-3' (SEQ ID NO.5),

[0087] CYP4A11-R: 5'-TGCCTCTTCCTCTTGATCTTCTCC-3' (SEQ ID NO. 6);

[0088] Apply PowerUp TM SYBR TM Green premix was used for real-time fluorescence quantitative PCR and 2 -ΔΔCt Method for analysis.

[0089] (3) Cell immunofluorescence detection

[0090] Sterile cell slides with a diameter of 1.5 mm were plated in the corresponding wells of a 24-well plate in advance. A normal glucose group (NG) and a high glucose group (HG) were set up. HK-2 cell suspensions (20-25% cell density) in the exponential growth phase and in good growth condition were added to the corresponding wells of the NG and HG groups for culture. When the cells in the well plate grew to a density of about 50%, the culture medium of the NG and HG groups was replaced with a normal glucose medium containing 5.6 mM glucose and a high glucose medium containing 30 mM glucose, respectively. After 48 hours of culture, the cells were fixed with paraformaldehyde, permeabilized with 0.3% Triton X-100 solution, blocked with 3% BSA solution, and then incubated overnight with CYP4A11 antibody (11688-1-AP, Mitaka). The cells were incubated with secondary antibodies and stained with DAPI the next day, and finally imaged using a laser confocal microscope.

[0091] 2. Test results

[0092] Western blot test results Figure 3 As shown in A, Figure 3 A showed that compared with the normal group (NG), the expression of CYP4A11 in HK-2 cells in the high glucose group (HG) was significantly increased, and the difference was statistically significant (P<0.001). HK-2 cells were obviously damaged, and the expression level of Kim-1 was increased, and the difference was statistically significant (P<0.01).

[0093] Real-time quantitative PCR test results Figure 3 As shown in B, Figure 3 B showed that compared with the normal group (NG), the mRNA expression level of CYP4A11 in HK-2 cells in the high glucose group (HG) was increased, and the difference was statistically significant (P<0.0001).

[0094] The results of cell immunofluorescence assay were as follows Figure 3 As shown in C, Figure 3 C showed that compared with the normal group (NG), the expression of CYP4A11 in HK-2 cells in the high glucose group (HG) was significantly increased, and the difference was statistically significant (P<0.01).

[0095] Example 4: Construction of renal tubular epithelial cell-specific Cyp4a14 gene knockout mice (Cre+ / Cyp4a14fl / fl)

[0096] Construction of renal tubular epithelial cell-specific Cyp4a14 gene knockout mice (Cre + / Cyp4a14 fl / fl ) and genotype verification was performed.

[0097] The specific construction and verification process is as follows:

[0098] (1) The Cre-Loxp recombinase system was used to achieve specific knockout of the Cyp4a14 gene in mouse renal tubular epithelial cells. Two Loxp sites in the same direction were inserted upstream of exon 2 and downstream of exon 3 of the Cyp4a14 gene to obtain Cyp4a14-flox mice (C57BL / 6JCya-Cyp4a14 em1flox / Cya, S-CKO-01979, Saiye Biotechnology Co., Ltd.), the construction diagram is as shown in Figure 4 As shown in A. First, the Cyp4a14 obtained by mating flox / flox Cyp4a14 was obtained by mating mice with renal tubular epithelial cell-specific Cre mice (Cdh16-MerCreMer, C001432, Saiye Biotechnology Co., Ltd.) flox / flox (Cre+) mice were then induced with tamoxifen to obtain conditional Cyp4a14 gene knockout mice in renal tubular epithelial cells when they reached adulthood.

[0099] (2) DNA was extracted from the tail of mice about 10 days old using the ethanol precipitation method, and the genomic product was obtained by PCR amplification. The genotype was identified by 1.5% agarose gel electrophoresis. The results were as follows: Figure 4 As shown in B.

[0100] Depend on Figure 4 B shows that the renal tubular epithelial cell-specific knockout mouse model of Cyp4a14 gene was successfully constructed, and the Cyp4a14 flox / flox (Cre+) mice.

[0101] Example 5: Detecting the effects of specific knockout of Cyp4a14 in renal tubular epithelial cells on renal damage and Cyp4a14 expression levels in DKD mice.

[0102] DKD mice with specific knockout of the Cyp4a14 gene in renal tubular epithelial cells were constructed. The effects of specific knockout of Cyp4a14 in renal tubular epithelial cells on renal injury in DKD were examined by PAS staining and serum creatinine and urine albumin / creatinine ratio (uACR) in mice. Western blotting was used to detect the expression of Cyp4a14 protein in renal tissue.

[0103] 1. Animal husbandry and grouping

[0104] The 6-week-old male wild-type Cre obtained in Example 4 was selected + -Cyp4a14 + / + and Cyp4a14-specific knockout mice Cre + -Cyp4a14fl / fl , the mice were divided into two groups: Control-Cre + -Cyp4a14 + / + group, STZ / HFD-Cre + -Cyp4a14 + / + Group, Control-Cre + -Cyp4a14 fl / fl group and STZ / HFD-Cre + -Cyp4a14 fl / fl There were 6 mice in each group. Among them, STZ / HFD-Cre + -Cyp4a14 + / + group and STZ / HFD-Cre + -Cyp4a14 fl / fl The mice in the control group were fed a high-fat diet (HFD) for 4 weeks at 6 weeks of age, and were intraperitoneally injected with streptozotocin (STZ) at a dose of 55 mg / kg for 5 consecutive days at 10 weeks of age; + -Cyp4a14 + / + Group and Control-Cre + -Cyp4a14 fl / fl The STZ / HFD group was given a standard diet and an equal volume of citrate buffer. Fasting blood glucose was measured every three days after injection. When the blood glucose level of the STZ / HFD group mice remained stable at ≥11.1 mmol / L, the STZ / HFD mouse model was successfully established and designated as DKD mice.

[0105] 2. Test methods

[0106] (1) PAS staining

[0107] Kidney tissues of mice in each group were taken for PAS staining to detect kidney pathological damage in mice in each group; wherein, the specific experimental method of PAS staining was the same as that in Example 1.

[0108] (2) Serum creatinine and urine albumin / creatinine ratio (uACR) testing

[0109] The serum of mice in each group was collected to detect the blood creatinine content in the serum; the urine of mice in each group was collected to detect the urine albumin and creatinine content in the urine, and the uACR value was calculated.

[0110] (3) Western blot

[0111] Western blotting was used to detect the expression level of Cyp4a14 protein in each group of mice. The detection method was the same as the Western blotting method in Example 2.

[0112] 3. Test results

[0113] The PAS staining results of mice in each group are shown in Figure 2. Figure 5 As shown in A, Figure 5 A shows that knockout of Cyp4a14 in renal tubular epithelial cells reduces glomerular mesangial proliferation and glomerular sclerosis.

[0114] The results of serum creatinine and urine albumin / creatinine ratio (uACR) of mice in each group were as follows: Figure 5 B, 5C, by Figure 5 As shown in Figures B and 5C, specific knockout of Cyp4a14 in renal tubular epithelial cells can reduce serum creatinine in DKD mice, and the difference is statistically significant (P<0.01), and at the same time reduce the production of proteinuria uACR, and the difference is statistically significant (P<0.001).

[0115] Western blot results of mice in each group are shown in Figure 2. Figure 5 As shown in D, Figure 5 D shows that after Cyp4a14 was specifically knocked out in renal tubular epithelial cells, the protein expression of Cyp4a14 in the mouse kidney tissue could not be detected by western blotting.

[0116] These results indicate that tubular epithelial cell-specific knockout of Cyp4a14 can alleviate renal damage in DKD mice.

[0117] Example 6: Detecting the effect of specific knockout of Cyp4a14 in renal tubular epithelial cells on ferroptosis in DKD mice

[0118] Select the Control-Cre obtained in Example 5 + -Cyp4a14 + / + group, STZ / HFD-Cre + -Cyp4a14 + / + Group, Control-Cre + -Cyp4a14 fl / fl group and STZ / HFD-Cre + -Cyp4a14 fl / fl Transmission electron microscopy, western blotting and biochemical kits were used to detect ferroptosis in the renal tissue of mice in each group.

[0119] 1. Test method

[0120] (1) Transmission electron microscopy

[0121] Six mice in each group aged 20 weeks were selected. Blood was collected from the apex of the heart and then perfused. The kidneys were removed and the renal capsule and medulla were removed. The kidneys were separated along the largest section and 1 mm 3Kidney tissue was fixed with 2.5% glutaraldehyde at 4°C to obtain tissue sections. After rinsing with 1× PBS buffer, the sections were fixed with 1% osmium hydroxide for 90 minutes. The sections were then dehydrated using a series of 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol and 100% acetone. The tissues were then embedded in 812 epoxy resin and placed on an ultrathin microtome for 60 nm ultrathin sectioning. Finally, the sections were stained with uranyl acetate and lead citrate solution, washed three times with double-distilled water, and placed in a dry environment at room temperature overnight to prepare sections for electron microscopy. The tissues were photographed under a transmission electron microscope, and experimental images were observed and collected.

[0122] (2) Western blot detection

[0123] One quarter of renal cortical tissue was collected from each group of mice, and the expression levels of ferroptosis-related molecules GPX4, SLC7A11, and TFR1 in each group of mice were detected by Western blotting. The detection method was the same as the Western blotting method in Example 2.

[0124] (3) Biochemical kit to detect the content of antioxidant GSH in tissues

[0125] 100 mg of renal cortical tissue was collected from each group of mice. The renal tissue was fully ground using a tissue grinder according to the instructions of the kit (Solarbo). The tissue was centrifuged at 8000 g for 10 minutes at 4°C, and the supernatant was collected and tested at 4°C. Blank tubes, standard tubes, and sample tubes were prepared separately. The mixture was mixed and allowed to stand for 2 minutes, and then the absorbance at 412 nm was measured. The absorbance minus the blank well was used as the horizontal axis, and the concentration was used as the vertical axis. A standard curve was drawn to calculate the content of the antioxidant GSH in each sample.

[0126] (4) Biochemical kit to detect Fe in tissues 2+ content

[0127] 20 mg of renal cortical tissue from each group of mice was collected and homogenized in 5 volumes of Iron Assay buffer according to the instructions of the kit (Sigma-Aldrich). The samples were centrifuged at 16,000 g for 10 minutes at 4°C, and the final volume was adjusted to 100 μL. 50 μL of sample was added to a 96-well plate according to the concentration, and Iron Assay Buffer was added to make the volume per well reach 100 μL. Finally, 5 μL of Iron Assay Buffer was added to each well. After incubation at room temperature in the dark for 30 minutes, 100 μL of Iron Probe was added to each well. The plates were then placed on a horizontal shaker and incubated at room temperature in the dark for 60 minutes. The absorbance at 593 nm was measured.

[0128] 2. Test results

[0129] The results of transmission electron microscopy of mice in each group are shown in Figure 2. Figure 6 As shown in A, Figure 6 A shows that mice in the DKD state showed significant ferroptosis-related pathological damage, with smaller mitochondrial volume and a reduced number of mitochondrial cristae. However, after specific knockout of the Cyp4a14 gene in renal tubular epithelial cells, ferroptosis damage in the mouse renal tubules was significantly reduced and the number of mitochondrial cristae increased.

[0130] Western blot results of mice in each group are shown in Figure 2. Figure 6 As shown in B, Figure 6 B shows that the protein levels of ferroptosis-inhibiting proteins GPX4 and SLC7A11 were reduced in DKD mice, and the reduced GPX4 and SLC7A11 could be reversed after specific knockout of the Cyp4a14 gene in renal tubular epithelial cells, with the difference being statistically significant (P<0.05). TFR1 was increased in the renal tissue of DKD mice, and specific knockout of the Cyp4a14 gene in renal tubular epithelial cells could also reverse the expression level of TFR1, with the difference being statistically significant (P<0.01).

[0131] The results of the antioxidant GSH content in the tissues of mice in each group are as follows Figure 6 As shown in C, Figure 6 C shows that the antioxidant GSH level is reduced in DKD mice, and specific knockout of the Cyp4a14 gene in renal tubular epithelial cells can reverse the reduced antioxidant GSH level, and the difference is statistically significant (P<0.001).

[0132] Fe in tissues of mice in each group 2+ The content results are as follows Figure 6 As shown in D, Figure 6 As shown in Figure 4, Fe 2+ The level of Fe 2+ The difference was statistically significant (P<0.001).

[0133] In summary, obvious ferroptosis changes appeared in the renal tissue of DKD mice, and specific knockout of the Cyp4a14 gene in renal tubular epithelial cells could alleviate ferroptosis damage in the renal tissue of DKD mice.

[0134] Example 7: Effect of sh-CYP4A11 that specifically interferes with CYP4A11 mRNA expression on CYP4A11 protein expression in HK-2 renal tubular epithelial cells

[0135] Based on the nucleotide sequence of CYP4A11 mRNA, we designed a sh-CYP4A11 lentivirus (a viral vector containing HIV-1 (human immunodeficiency virus type 1)) to specifically knock down CYP4A11 mRNA expression, and a control sh-NC lentivirus. HK-2 cells were then transfected with the same dose of sh-CYP4A11 and sh-NC lentivirus, respectively, and CYP4A11 protein expression was assessed by western blotting.

[0136] Among them, the nucleotide sequence of the sh-CYP4A11 lentivirus is: 5'-CAGAAATGGGTGGAGACATTC' (SEQ ID NO.1), and the nucleotide sequence of the sh-NC lentivirus is: 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO.2). Both the sh-CYP4A11 lentivirus and the sh-NC lentivirus were artificially synthesized by GeneCare Company according to the sequence information.

[0137] 1. Test method:

[0138] (1) Western blot

[0139] 1) Prepare complete culture medium with a density of 4×10 4 1 mL of cell suspension was inoculated into 6-well plates and cultured at 37°C for 24 hours until the cells were confluent to about 30%. Based on the cell MOI = 10 and the virus titer of 1 × 10 8 TU / mL, add 20 μL of sh-CYP4A11 lentivirus or control virus to each well, continue culturing for 16 hours, then replace with complete medium. Observe the infection efficiency or perform cell passage operation after about 72 hours of infection;

[0140] 2) The lentivirus-transfected cells were digested and transferred to new T25 culture flasks. When the density reached approximately 70% after 48 hours, an appropriate concentration of puromycin was added. The puromycin-containing medium was replaced every 3 days until all cells in the uninfected control group were killed by puromycin and no dead cells appeared in the virus-infected group. The puromycin concentration was then reduced to a maintenance concentration (1 / 2 of the original concentration). The infected cells were screened and amplified to obtain a stable sh-CYP4A11 lentivirus-transfected strain, designated as the sh-CYP4A11 group. HK-2 cells infected with the sh-NC lentivirus were obtained using the same ratio and method, designated as the sh-NC group.

[0141] 3) The sh-NC group cells with a growth density of approximately 40% were randomly divided into a normal glucose group (NG) and a high glucose group (HG). The cells were cultured with the corresponding normal glucose and high glucose media described in Example 3 for 48 hours, respectively, to obtain the NG-shNC group and the HG-shNC group. The sh-CYP4A11 group cells with a growth density of approximately 40% were cultured with high glucose media for 48 hours to obtain the HG-shCYP4A11 group.

[0142] 4) Western blotting and semi-quantitative analysis were used to detect the expression levels of Kim-1 and CYP4A11 proteins in the three groups of cells. The detection method was the same as the Western blotting method in Example 3.

[0143] (2) Real-time quantitative PCR

[0144] 1) Prepare complete culture medium with a density of 4×10 4 1 mL of cell suspension was inoculated into 6-well plates and cultured at 37°C for 24 hours until the cells were confluent to about 30%. Based on the cell MOI = 10 and the virus titer of 1 × 10 8 TU / mL, add 20 μL of sh-CYP4A11 lentivirus or control virus to each well, continue culturing for 16 hours, then replace with complete medium. Observe the infection efficiency or perform cell passage operation after about 72 hours of infection;

[0145] 2) The lentivirus-transfected cells were digested and transferred to new T25 culture flasks. When the density reached approximately 70% after 48 hours, an appropriate concentration of puromycin was added. The puromycin-containing medium was replaced every 3 days until all cells in the uninfected control group were killed by puromycin and no dead cells appeared in the virus-infected group. The puromycin concentration was then reduced to a maintenance concentration (1 / 2 of the original concentration). The infected cells were screened and amplified to obtain a stable sh-CYP4A11 lentivirus-transfected strain, designated as the sh-CYP4A11 group. HK-2 cells infected with the sh-NC lentivirus were obtained using the same ratio and method, designated as the sh-NC group.

[0146] 3) The sh-NC group cells with a growth density of approximately 40% were randomly divided into a normal glucose group (NG) and a high glucose group (HG). The cells were cultured with the corresponding normal glucose and high glucose media described in Example 3 for 48 hours, respectively, to obtain the NG-shNC group and the HG-shNC group. The sh-CYP4A11 group cells with a growth density of approximately 40% were cultured with high glucose media for 48 hours to obtain the HG-shCYP4A11 group.

[0147] 4) Reverse transcription-real-time quantitative PCR was used to detect the expression levels of CYP4A11 mRNA in the three groups of cells. The specific method was as described in Example 3, and the primer sequences were the same as those in Example 3.

[0148] 2. Test results

[0149] Western blot test results Figure 7 As shown in A, Figure 7 A shows that the protein levels of Kim-1 and CYP4A11 increased in HK-2 cells cultured in high glucose (HG-shNC group), and sh-CYP4A11 could reduce the elevated protein levels of Kim-1 and CYP4A11 under high glucose conditions (HG-shCYP4A11 group), and the differences were statistically significant (P<0.001, P<0.01,).

[0150] Real-time quantitative PCR test results Figure 7 As shown in B, Figure 7 B shows that sh-CYP4A11 can reduce the elevated CYP4A11 mRNA level under high glucose conditions, and the difference is statistically significant (P<0.01).

[0151] In summary, sh-CYP4A11 lentivirus can effectively reduce the expression levels of CYP4A11 protein and mRNA.

[0152] Example 8: Effect of sh-CYP4A11 that specifically interferes with CYP4A11 mRNA expression on ferroptosis in renal tubular epithelial cells HK-2

[0153] Three groups of cells, NG-shNC, HG-shNC and HG-shCYP4A11 obtained in Example 7, were selected and the ferroptosis of each group of cells was detected using transmission electron microscopy, Western blotting and biochemical kits.

[0154] 1. Test method:

[0155] (1) Transmission electron microscopy

[0156] The three groups of cells, NG-shNC, HG-shNC, and HG-shCYP4A11, obtained in Example 7 were used. When the cell density of each group reached 80-90%, the cells were trypsinized and washed three times with PBS buffer, then fixed in 2.5% glutaraldehyde for more than 2 hours. The subsequent steps were the same as the transmission electron microscopy detection method in Example 6.

[0157] (2) Western blot detection

[0158] When the cell density of each group reached 80-90%, the total cell protein was collected, and the expression levels of ferroptosis-related molecules GPX4, SLC7A11, and TFR1 in each group of mice were detected by Western blotting. The detection method was the same as that in Example 2.

[0159] (3) Biochemical kit to detect Fe in tissues 2+ , antioxidant GSH content

[0160] The three groups of cells, NG-shNC, HG-shNC and HG-shCYP4A11 obtained in Example 7, were used to collect cells when the cell density reached about 90%, and the Fe 2+ The detection method of GSH content is the same as that in Example 6.

[0161] 2. Test results

[0162] Transmission electron microscopy results are as follows Figure 8 As shown in A, Figure 8 A shows that HK-2 cultured in high glucose showed obvious ferroptosis morphological damage, mitochondrial atrophy, increased membrane density, and reduced or even disappeared mitochondrial cristae. After specific interference with CYP4A11 mRNA expression, ferroptosis damage in renal tubular epithelial cells was significantly reduced and the number of mitochondrial cristae increased.

[0163] Western blotting results Figure 8 As shown in B, Figure 8 B shows that the protein levels of ferroptosis inhibitory proteins GPX4 and SLC7A11 in renal tubular epithelial cells induced by high glucose were reduced, and knockdown of the Cyp4a14 gene could reverse the reduced GPX4 and SLC7A11, and the difference was statistically significant (P<0.001, P<0.01). TFR1 increased under high glucose conditions, and specific interference with CYP4A11 mRNA expression could also reverse the expression level of TFR1, and the difference was statistically significant (P<0.05).

[0164] Fe 2+ , Antioxidant GSH content test results are as follows Figure 8 C, 8D. Figure 8 C and 8D show that high glucose induces Fe 2+ levels increased, the antioxidant GSH level decreased, and the application of sh-CYP4A11 could inhibit Fe 2+ The levels of serum GSH and creatinine increased and decreased, and the differences were statistically significant (P<0.001, P<0.05).

[0165] In summary, significant ferroptosis changes occurred in HK-2 cells cultured in high glucose, and the application of sh-CYP4A11 could alleviate the ferroptosis damage of renal tubular epithelial cells induced by high glucose.

Claims

1. Use of the CYP4A11 gene or its encoded product as a drug target for screening drugs for preventing, alleviating and / or treating diabetic nephropathy.

2. Use of substances that specifically interfere with CYP4A11 expression in the preparation of drugs for preventing, alleviating or / and treating diabetic nephropathy.

3. The use according to claim 2, characterized in that The substance that specifically interferes with CYP4A11 expression is one or more of a nucleic acid molecule, a small molecule compound, an antibody, a protein, a gene editing vector, a lentivirus or an adeno-associated virus.

4. The use according to claim 3, characterized in that The lentivirus is sh-CYP4A11 lentivirus, and the nucleotide sequence of the sh-CYP4A11 lentivirus is: 5'-CAGAAATGGGTGGAGACATTC-3'.

5. A drug, characterized in that The medicine is used for preventing, alleviating or / and treating diabetic nephropathy, and contains a substance that specifically interferes with the expression of CYP4A11.

6. The drug according to claim 4, characterized in that The substance that specifically interferes with CYP4A11 expression is one or more of a nucleic acid molecule, a small molecule compound, an antibody, a protein, a gene editing vector, a lentivirus or an adeno-associated virus.

7. The drug according to claim 6, characterized in that The substance that specifically interferes with CYP4A11 expression is sh-CYP4A11 lentivirus.

8. The drug according to claim 5, characterized in that The medicine also contains pharmaceutically acceptable carriers or excipients.

9. Use of a detection reagent for the CYP4A11 gene or its encoded product in the preparation of a product for auxiliary diagnosis of diabetic nephropathy.

10. The use according to claim 9, characterized in that The product is used to detect the expression level of the CYP4A11 gene or the product encoded by the CYP4A11 gene in a sample through RT-PCR, real-time quantitative PCR, in situ hybridization, Northern blot, Western blot, chip, high-throughput sequencing platform, immunohistochemistry, tissue immunofluorescence or enzyme-linked immunosorbent assay.

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