Application of targeted intervention FBP2 mRNA in preparation of medicine for preventing and treating diabetic nephropathy

Abstract

The invention relates to application of targeted intervention FBP2mRNA in preparation of a medicine for preventing and treating diabetic nephropathy, and belongs to the technical field of biomedicine. The invention provides an application of FBP2mRNA as a therapeutic target in preparation of a medicine for preventing and treating diabetic nephropathy. The NCBI (National Center of Biotechnology Information) login number of the FBP2mRNA is NM003837.4. The invention also provides an application of the shRNA for specifically interfering with the expression of the FBP2mRNA in the preparation of a medicine for preventing and treating diabetic nephropathy, wherein the nucleotide sequence of the shRNA is shown as SEQ ID NO. 1. It is found for the first time that targeted inhibition of the expression level of FBP2mRNA can effectively reduce the lipid level in mouse glomerular podocytes, reduce the expression level of lipid synthesis related genes and reduce the activity of lipid synthesis protein, and then the effect of treating diabetic nephropathy is achieved, so that FBP2mRNA can be used as a drug treatment target, and the treatment effect of diabetic nephropathy is improved. The application is applied to research, development and preparation of diabetic nephropathy prevention and treatment medicines.

Description

Technical Field

[0001] The invention relates to application of targeted intervention of FBP2 mRNA in preparing drugs for preventing and treating diabetic nephropathy, and belongs to the technical field of biomedicine. Background Art

[0002] Diabetic nephropathy (DKD) is a podocyte disease. Podocytes are glomerular epithelial cells that are an indispensable component of the glomerular filtration unit and the most "fragile" part of the glomerular structure. Podocyte damage occurs in the early stages of diabetic nephropathy, and the fusion and shedding of the foot processes of podocytes will lead to exposure of the basement membrane and the excretion of a large amount of protein, further aggravating proteinuria and accelerating the progression of diabetic nephropathy. Although the existing technology has made considerable progress in the mechanism of podocyte damage, there is no cell-specific and effective treatment method in clinical practice. Therefore, further studying the mechanism of podocyte damage and finding specific and effective treatment strategies are of great theoretical significance and application value.

[0003] In recent years, significant progress has been made in the study of pathophysiology related to podocyte lipid metabolism, which has become a hot topic in this field. Podocyte lipid overload can lead to cellular dysfunction, which is defined as lipotoxicity. This phenomenon is characterized by mitochondrial oxidative stress, actin cytoskeleton remodeling, insulin resistance and inflammatory response, which ultimately leads to podocyte hypertrophy, shedding and death. Importantly, pathological lipid accumulation in podocytes is the result of dysregulation of genes or proteins involved in cellular lipid metabolism, including lipid synthesis, uptake, storage, decomposition and excretion, and is not determined by the amount of circulating lipids. This suggests the importance of podocyte lipid metabolism in diabetic nephropathy. Therefore, in-depth study of podocyte lipid metabolism is of great significance for further clarifying the pathogenesis of diabetic nephropathy. Given that lipid metabolism regulation in podocytes is cell-specific, preventing podocyte lipid metabolism disorders and improving their function are important links in the treatment of diabetic nephropathy.

[0004] Fructose-1,6-bisphosphatase (FBP) is a key enzyme in glucose metabolism, which can catalyze the irreversible hydrolysis of fructose-1,6-bisphosphate into fructose-6-phosphate and inorganic phosphate. There are two highly conserved FBP isozymes in mammals: FBP1 and FBP2, which have 77% homologous sequences. Among them, FBP1 is a key enzyme in gluconeogenesis and is mainly expressed in the liver and kidneys. The expression of FBP2 is not limited to gluconeogenic tissues, but is widely expressed in various tissues and organs.

[0005] RNA interference technology (RNAi) is a gene silencing technology that introduces double-stranded RNA with homologous sequences to the endogenous target gene to induce the degradation of the mRNA of the endogenous target gene, thereby preventing gene expression. At present, this technology is widely used in the fields of signal pathway research, disease model construction, gene therapy, and drug target confirmation. The success of this technology mainly depends on the precise binding of small interfering RNA or lentivirus to the target gene, so choosing an efficient and stable interference mode is important. At the same time, the application of this technology to specifically silence pathogenic genes that are highly expressed in podocytes during diabetic nephropathy is of great significance for exploring new mechanisms of podocyte lipid metabolism imbalance and finding potential therapeutic targets for diabetic nephropathy. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides the use of targeted intervention of FBP2 mRNA in the preparation of drugs for the prevention and treatment of diabetic nephropathy.

[0007] The technical solution of the present invention is as follows:

[0008] Application of FBP2 mRNA as a therapeutic target in the preparation of drugs for the prevention and treatment of diabetic nephropathy.

[0009] Preferably according to the present invention, the NCBI accession number of the FBP2 mRNA is NM_003837.4.

[0010] Application of shRNA that specifically interferes with FBP2 mRNA expression in the preparation of drugs for the prevention and treatment of diabetic nephropathy.

[0011] Preferably according to the present invention, the nucleotide sequence of the shRNA that specifically interferes with the expression of FBP2 mRNA is as shown in SEQ ID NO.1.

[0012] A drug for preventing and treating diabetic nephropathy is a nucleic acid molecule, carbohydrate, lipid, small molecule chemical drug, antibody drug, polypeptide, protein or virus that can specifically interfere with the expression of FBP2 mRNA.

[0013] Preferably according to the present invention, the drug for preventing and treating diabetic nephropathy is shRNA that specifically interferes with the expression of FBP2 mRNA, and its nucleotide sequence is shown in SEQ ID NO.1.

[0014] Beneficial effects:

[0015] The present invention discovers for the first time that targeted inhibition of FBP2 mRNA expression levels can effectively reduce lipid levels in mouse glomerular podocytes, reduce the expression levels of lipid synthesis-related genes, and reduce the activity of lipid synthesis proteins, thereby achieving the effect of treating diabetic nephropathy. Therefore, FBP2 mRNA 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.

[0016] In addition, the present invention designs a shRNA lentivirus that interferes with FBP2 gene expression by knocking down FBP2 mRNA expression levels, successfully reversing lipid accumulation in diabetic nephropathy. Therefore, FBP2 mRNA can be used as a therapeutic target, and FBP2 mRNA interference shRNA can be used to prepare drugs for treating diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results are as follows: Western blotting, reverse transcription-real-time quantitative PCR, tissue immunohistochemistry and tissue immunofluorescence were used to detect the expression changes of FBP2 in mouse glomeruli during diabetic nephropathy (DKD);

[0018] In the figure, A is the result of protein immunoblotting; B is the result of reverse transcription-real-time fluorescence quantitative PCR; C is the result of tissue immunohistochemistry; and D is the result of tissue immunofluorescence.

[0019] Figure 2 The results of western blot and tissue immunohistochemistry were used to detect the expression changes of FBP2 in mouse glomeruli during spontaneous type 2 diabetes.

[0020] In the figure, A is the result of protein immunoblotting; B is the result of tissue immunohistochemistry.

[0021] Figure 3 After podocytes were cultured in vitro and stimulated with high glucose, the expression of FBP2 in podocytes was detected by reverse transcription-real-time fluorescence quantitative PCR and western blot.

[0022] In the figure, A is the protein immunoblotting result of different high glucose concentrations; B is the reverse transcription-real-time fluorescence quantitative PCR result; C is the protein immunoblotting result of different high glucose stimulation time.

[0023] Figure 4 The podocyte-specific FBP2 knockout mice (Cre+ / FBP2 fl / fl ) Construction schematic diagram and genotype identification results;

[0024] In the figure, A is a schematic diagram of specific site knockout; B is the result of genotype detection by agarose gel.

[0025] Figure 5The effects of glycogen staining (PAS staining) and electron microscopy on the podocyte-specific FBP2 deletion in Cre + / FBP2 + / + Diabetic nephropathy mice and Cre + / FBP2 fl / fl Effects of glomerular morphological damage in diabetic nephropathy mice;

[0026] In the figure, A is the results of glycogen staining and TEM electron microscopy; B is the statistics of basement membrane thickness; C is the statistics of foot process width; D is the statistics of foot process number; E is the statistics of glomerular area occupied by glomerular mesangium.

[0027] Figure 6 Is the application of Cre + / FBP2 + / + Diabetic nephropathy mice and Cre + / FBP2 fl / fl In diabetic nephropathy mice, morphological changes and changes in urinary microalbumin were detected by immunofluorescence staining of slit diaphragm proteins;

[0028] In the figure, A is the result of immunofluorescence detection of slit diaphragm protein, and B is the result of urine albumin detection.

[0029] Figure 7 The effect of sh-FBP2 lentivirus, which specifically interferes with FBP2 mRNA expression, on the expression level of FBP2 protein in glomerular podocytes (HPCs).

[0030] Figure 8 The effect of silencing FBP2 mRNA on the mRNA expression level of lipid metabolism-related proteins in glomerular podocytes (HPCs).

[0031] Fig. 9 The results of using Oil Red O and Nile Red staining to show the relationship between FBP2 and lipid deposition in podocytes;

[0032] In the figure, A is the test result of Oil Red O, and B is the test result of Nile Red. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with specific experimental examples, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the embodiments are all common commercially available products unless otherwise specified.

[0034] Human podocytes (HPCs) were a gift from Dr Saleem MA, University of Bristol (UK). They are also available from Yimo Biotech Ltd.

[0035] The FBP2 gene knockout mouse was commissioned to Shanghai Model Organisms Development Co., Ltd. and constructed according to the existing gene knockout method.

[0036] Heterozygous BKS db mice (db / m mice, BKS.Cg-Dock7m + / + Lepr db / J, Stock No. 000642) was purchased from Jackson Laboratory, USA.

[0037] FBP2 fl / fl Mice (C57BL / 6J-FBP2em1(flox)Smoc) and Podocin-Cre mice (129S6.Cg-Tg(NPHS2-cre)295Lbh / BroJ; Stock No: 008523) were purchased from Shanghai Model Organisms Technology Development Co., Ltd.

[0038] Target cell maintenance medium is available from Gibco.

[0039] Example 1: Construction of a streptozotocin (STZ) / high-fat diet (HFD) mouse model

[0040] A streptozotocin (STZ) / high-fat diet (HFD) mouse model was constructed to simulate diabetic nephropathy (DKD) in mice. Then, the expression changes of FBP2 in the glomeruli of mice during diabetic nephropathy (DKD) were detected by western blotting, reverse transcription-real-time quantitative PCR, tissue immunohistochemistry and tissue immunofluorescence.

[0041] The specific implementation process is as follows:

[0042] (1) Construction of STZ / HFD mouse model: 24 healthy male C57BL / 6J mice aged 8 weeks were prepared and fed with a high-fat diet for 4 weeks. Then, a single kidney was removed according to the existing method. One week later, STZ (100 mg / kg) was injected intraperitoneally. The mice were fed with a high-fat diet for 12 weeks to obtain the STZ / HFD mouse model. This mouse model was used to simulate diabetic nephropathy (DKD) in mice, which was recorded as the STZ / HFD group. At the same time, 24 healthy male C57BL / 6J mice aged 8 weeks were fed with a normal diet for 17 weeks as a normal control, which was recorded as the CON group.

[0043] (2) Blood was collected from the eyeballs of mice in the STZ / HFD group and the CON group and then cardiac perfusion was performed. The kidneys of the mice were removed and separated along the largest section. Half of the kidney was fixed with paraformaldehyde. The fixed kidneys were then embedded in paraffin and sliced ​​for immunohistochemistry and immunofluorescence experiments. The results are shown in Table 1. Figure 1 As shown in C and D.

[0044] (3) The renal cortex of the remaining half of the kidney was removed, ground into a homogenate, and then lysed using RIPA lysis buffer (Biyuntian) on ice for 15 minutes.

[0045] (4) The lysed renal cortical tissue was collected, and half of it was used to isolate total RNA from the cells using an RNA rapid extraction kit (ES Science); the integrity of the extracted RNA was tested by agarose gel electrophoresis, and the concentration of the extracted RNA was measured using the TOYOBO Tnano-800; the obtained RNA was used as a template and reverse transcribed to synthesize cDNA products using the Revertra Ace qPCR RT Kit (TOYOBO FSQ-101); Ultra SYBR Mixture (CWBIO) was used for real-time fluorescence quantitative PCR, and the 2-ΔΔCt method was used for analysis. The results are shown in Figure 2. Figure 1 As shown in B.

[0046] (5) The remaining half of the lysed renal cortical tissue was first tested for protein concentration using a BCA protein concentration detection kit (Shangbao Bio, 34001), and then the protein was separated using 10% SDS-PAGE and transferred to a PVDF membrane. Finally, it was labeled with an FBP2 antibody (abcam, ab248391) and observed and counted by chemiluminescence. The results are shown in Figure 2. Figure 1 As shown in A.

[0047] Depend on Figure 1 As shown in A, B, C and D, compared with the CON group mice, the expression levels of FBP2 protein and FBP2 mRNA in the kidneys of the STZ / HFD group mice were significantly increased.

[0048] Example 2: Construction of db / db mouse model

[0049] Spontaneous type 2 diabetes db / db mice were constructed to simulate the occurrence of spontaneous type 2 diabetes in mice. Then, the expression changes of FBP2 in glomeruli during spontaneous type 2 diabetes were detected by western blotting, reverse transcription-real-time quantitative PCR and tissue immunohistochemistry.

[0050] The specific implementation process is as follows:

[0051] (1) Construction of spontaneous type 2 diabetes db / db mice: Heterozygous BKS db mice were self-crossed to obtain a spontaneous type 2 diabetes db / db mouse model, denoted as the db / db group; heterozygous BKS db mice were used as normal controls, denoted as the db / m group; both groups of mice were fed a normal diet for 20 weeks each;

[0052] Among them, the db / db group of mice will show an obese phenotype at 3 to 4 weeks of age, and elevated blood sugar at 4 to 8 weeks of age. Subsequently, they will suffer kidney damage and produce proteinuria as they age.

[0053] (2) Twenty mice from the db / db group and db / m group, aged 20 weeks, were selected. Blood was collected from their eyeballs and then cardiac perfusion was performed. The kidneys of the mice were removed and split along the largest section. Half of the kidneys were fixed with paraformaldehyde. The fixed kidneys were then embedded in paraffin and sliced ​​for immunohistochemical experiments. The results are shown in Table 1. Figure 2 As shown in B.

[0054] (3) The renal cortex of the remaining half of the kidney was removed, ground into a homogenate, and then lysed on ice for 15 minutes using RIPA lysis buffer (Biyuntian). The protein concentration of the lysed renal cortex tissue was first determined using a BCA protein concentration detection kit (Shangbao Bio, 34001), and then the protein was separated using 10% SDS-PAGE and transferred to a PVDF membrane. Finally, it was labeled with FBP2 antibody (abcam, ab248391) and observed and counted by chemiluminescence. The results are shown in the figure. Figure 2 As shown in A.

[0055] Depend on Figure 2 As shown in A and B, the expression level of FBP2 protein in the kidneys of db / db mice was significantly increased compared with db / m mice.

[0056] Example 3: Differences in the expression of FBP2 in podocytes after high glucose stimulation

[0057] Glomerular podocytes (HPCs) cultured in vitro were selected and treated with high glucose. The difference in FBP2 expression between the high glucose group and the normal group was detected.

[0058] The specific implementation process is as follows:

[0059] (1) In vitro cultured glomerular podocytes (HPCs) were selected and inoculated into six-well plates for culture. When the confluence reached 70%, they were divided into three groups. The three groups of cells were incubated with 15 mmol / L and 30 mmol / L glucose solutions (High glucose) and 30 mmol / L mannitol solutions (Mannitol) for 24 h, respectively, to obtain three groups of high-glucose-treated glomerular podocytes, which were respectively recorded as High glucose-15 group, High glucose-30 group and Mannitol group. At the same time, the in vitro cultured glomerular podocytes (HPCs) were used as normal controls, recorded as the control group.

[0060] The above four groups of cells were ground into homogenate and then lysed on ice for 15 minutes using RIPA lysis buffer (Biyuntian). The lysed glomerular podocyte tissues were collected, and half of them were used to isolate total RNA from the cells using an RNA rapid extraction kit (ES Science). The integrity of the extracted RNA was tested by agarose gel electrophoresis, and the concentration of the extracted RNA was measured using Toyobo Tnano-800. The obtained RNA was used as a template and reverse transcribed to synthesize cDNA products using Revertra Ace qPCR RT Kit (TOYOBO FSQ-101). Ultra SYBR Mixture (CWBIO) was used for real-time fluorescence quantitative PCR, and the 2-ΔΔCt method was used for analysis. The results are shown in Figure 2. Figure 3 As shown in B.

[0061] The remaining half of the lysed renal cortical tissue was first tested for protein concentration using a BCA protein concentration detection kit (Shangbao Bio, 34001), and then the protein was separated using 10% SDS-PAGE and transferred to a PVDF membrane. Finally, it was labeled with an FBP2 antibody (abcam, ab248391) and observed and counted by chemiluminescence. The results are shown in Figure 3 As shown in A.

[0062] (2) In vitro cultured glomerular podocytes (HPCs) were selected and inoculated into six-well plates for culture. When the confluence reached 70%, they were divided into three groups. The three groups of cells were co-incubated with 30 mmol / L glucose solution (High glucose) for 12 h, 24 h, and 48 h, respectively, to obtain three groups of high-glucose-treated glomerular podocytes, which were respectively recorded as High glucose-12h group, High glucose-24h group, and High glucose-48h group. At the same time, the in vitro cultured glomerular podocytes (HPCs) were used as normal controls, recorded as the control group.

[0063] The above four groups of cells were ground into homogenate and then lysed on ice for 15 minutes with RIPA lysis buffer (Biyuntian). The lysed glomerular podocyte tissues were collected and the protein concentration was first determined using a BCA protein concentration detection kit (Shangbao Bio, 34001). Then, 10% or 12% SDS-PAGE was used to separate the proteins and the proteins were transferred to PVDF membranes. Finally, the FBP2 antibody (abcam, ab248391) was used to label the antibodies and the bands were observed and counted by chemiluminescence. The results are shown in Figure 3 As shown in C.

[0064] Depend on Figure 3As shown in A, B and C, the expression of FBP2 protein level increased after podocytes were stimulated by high glucose, and the expression increased with the time gradient of high glucose stimulation.

[0065] Example 4. Construction of podocyte-specific knockout FBP2 gene mouse model and podocyte-specific knockout FBP2 diabetic kidney model mouse

[0066] 1. According to Figure 4 A in the article was used to construct a podocyte-specific FBP2 gene knockout mouse model. The specific implementation process is as follows:

[0067] FBP2 fl / fl The mice were crossed with Podocin-Cre mice to obtain the FBP2 fl / + -Podocin-Cre + F1 mice were crossed with FBP2 fl / fl The mice were bred to obtain F2 mice, and the F2 mice were further bred and mated to obtain the genotype FBP2 fl / fl -Podocin-Cre + Target gene mice. After DNA extraction from the mouse tail, the genotype was identified. The results are as follows Figure 4 As shown in B.

[0068] Depend on Figure 4 As shown in B, the podocyte-specific knockout FBP2 gene mouse model was successfully constructed and FBP2 fl / fl / Cre + Mouse.

[0069] 2. Use FBP2 fl / fl / Cre + Mice and FBP2 + / + / Cre + The STZ / HFD diabetic nephropathy mouse model was constructed. The specific construction method and process were the same as in Example 1. Cre + / FBP2 + / + Diabetic nephropathy mice, Cre + / FBP2 fl / fl diabetic nephropathy mice, and the control mice of these two groups of mice; they are respectively denoted as Cre + / FBP2 + / + -control, Cre + / FBP2 fl / fl -control, Cre + / FBP2 fl / fl -STZ / HFD, Cre + / FBP2fl / fl -STZ / HFD.

[0070] Example 5: Detection of the effect of podocyte-specific FBP2 deletion on Cre + / FBP2 + / + Diabetic nephropathy mice and Cre + / FBP2 fl / fl Effects of glomerular morphological damage in diabetic nephropathy mice

[0071] Glycogen staining (PAS staining) and TEM electron microscopy were used to detect the Cre obtained in Example 4. + / FBP2 + / + Diabetic nephropathy mice and Cre + / FBP2 fl / fl Effect of podocyte-specific FBP2 gene deletion on glomerular morphological damage in diabetic nephropathy mice.

[0072] The specific implementation process is as follows:

[0073] 1. PAS staining method to detect the effect of podocyte-specific FBP2 deficiency on glomerular morphological damage

[0074] (1) Cre obtained in Example 4 + / FBP2 fl / fl -STZ / HFD, Cre + / FBP2 fl / fl -STZ / HFD diabetic nephropathy mice and Cre + / FBP2 + / + -control, Cre + / FBP2 fl / fl -Control mice were blooded from their eyeballs and then cardiac perfused. The kidneys were removed and fixed with paraformaldehyde. The fixed kidneys were then embedded in paraffin and sliced ​​to obtain paraffin slices. The paraffin slices were then dewaxed and hydrated: the slices were sequentially placed in xylene I for 20 minutes - xylene II for 20 minutes - anhydrous ethanol I for 10 minutes - anhydrous ethanol II for 10 minutes - 95% alcohol for 5 minutes - 90% alcohol for 5 minutes - 80% alcohol for 5 minutes - 70% alcohol for 5 minutes - washed with distilled water to complete dewaxing and hydration to obtain tissue slices.

[0075] (2) Periodic acid solution treatment: Immerse the tissue sections in periodic acid solution for 10 minutes and rinse with running water for 5 minutes;

[0076] (3) Schiff reagent treatment: Immerse the tissue sections in Schiff reagent for 10 minutes and rinse with running water for 5 minutes;

[0077] (4) Hematoxylin staining: Immerse the tissue sections in hematoxylin stain for 1 minute and rinse with running water for 5 minutes;

[0078] (5) Rapid dehydration: Dehydrate the tissue sections in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes to make them transparent. Take the sections out of xylene and air dry them, then seal them with neutral gum.

[0079] (6) Use a slide scanner (Olympus vs200) to collect and analyze images of tissue sections and observe the results. Figure 5 As shown in A; at the same time, the glomerular area occupied by the glomerular mesangium is calculated, and the result is as follows Figure 5 As shown in E.

[0080] 2. TEM electron microscopy method to detect the effect of podocyte-specific FBP2 deficiency on glomerular morphological damage

[0081] (1) Electron microscopy sample processing: Cre obtained in Example 4 + / FBP2 + / + Diabetic nephropathy mice and Cre + / FBP2 fl / fl Blood was collected from the eyeballs of diabetic nephropathy mice and then cardiac perfusion was performed. The kidneys of the mice were removed and the kidney tissues were fixed with 2.5% glutaraldehyde at 4°C to obtain tissue sections.

[0082] (2) Wash the tissue sections three times with 0.1 mol / L PBS, each time for 15 minutes; then fix them with 1% osmium tetroxide at room temperature for 2 hours;

[0083] (3) Dehydrate tissue sections using a series of 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol and 100% acetone;

[0084] (4) The tissue sections were cut to a thickness of 70 nm, and then stained with 2% saturated dioxyacetic acid and lead citrate. Finally, the tissue sections were observed using Image J software to determine the thickness of the glomerular basement membrane (GBM) and the number of foot processes. The results were as follows: Figure 5 As shown in A, the glomerular basement membrane (GBM) thickness and foot process number are Figure 5 As shown in B and C; at the same time, the number of foot processes was counted, and the results are as follows Figure 5 As shown in D.

[0085] Depend on Figure 5 As shown in Figures A and E, knockout of FBP2 in podocytes reduced glomerular mesangial proliferation and glomerular sclerosis.

[0086] Depend on Figure 5As shown in B, C, and D, podocyte-specific knockout of FBP2 restored the increase in basement membrane thickness and the decrease in the number of foot processes caused by diabetic nephropathy.

[0087] Example 6: Detection of the effect of podocyte-specific FBP2 deletion on Cre + / FBP2 + / + Diabetic nephropathy mice and Cre + / FBP2 fl / fl Effects of slit diaphragm protein specifically expressed in glomerular podocytes of diabetic nephropathy mice

[0088] Select Cre obtained in Example 4 + / FBP2 + / + Diabetic nephropathy mice and Cre + / FBP2 fl / fl In diabetic nephropathy mice, immunofluorescence staining was used to detect slit diaphragm proteins (Nephrin, Podocin, Synapotopodin) specifically expressed in glomerular podocytes to verify podocyte damage and changes in urinary microalbumin.

[0089] The specific implementation process is as follows:

[0090] (1) Cre obtained in Example 4 + / FBP2 fl / fl -STZ / HFD, Cre + / FBP2 fl / fl -STZ / HFD diabetic nephropathy mice and Cre + / FBP2 + / + -control, Cre + / FBP2 fl / fl -Control mice were blooded from their eyeballs and then cardiac perfused. The kidneys were removed and fixed with paraformaldehyde. The fixed kidneys were then embedded in paraffin and sliced ​​to obtain paraffin slices. The paraffin slices were then dewaxed and hydrated: the slices were sequentially placed in xylene I for 20 minutes - xylene II for 20 minutes - anhydrous ethanol I for 10 minutes - anhydrous ethanol II for 10 minutes - 95% alcohol for 5 minutes - 90% alcohol for 5 minutes - 80% alcohol for 5 minutes - 70% alcohol for 5 minutes - washed with distilled water to complete dewaxing and hydration to obtain tissue slices.

[0091] (2) Antigen retrieval was performed on tissue sections using antigen retrieval solution (Biyuntian);

[0092] (3) Add 3% bovine serum albumin (BSA) to the tissue sections and block at room temperature for 90 minutes;

[0093] (4) Gently shake off the blocking solution, add primary antibody to the tissue sections after washing, and then place them flat in a humidified chamber and incubate at 4°C overnight;

[0094] (5) Wash the tissue sections in PBS buffer for 3 times, 5 minutes each time. Then add the corresponding secondary antibody and incubate at room temperature in the dark for 90 minutes;

[0095] (6) Wash the tissue sections in PBS buffer for 3 times, 5 minutes each time. Use a mounting medium containing DAPI for mounting;

[0096] (7) Image acquisition: The fluorescence image of the slit membrane protein was acquired by laser confocal microscopy on the tissue sections. The results are as follows: Figure 6 As shown in A.

[0097] (8) Collecting Cre obtained in Example 4 using a metabolic cage + / FBP2 + / + Diabetic nephropathy mice, Cre + / FBP2 fl / fl The urine of diabetic nephropathy mice and control mice was tested for proteinuria using a life analyzer. The results are as follows Figure 6 As shown in B.

[0098] Depend on Figure 6 As shown in A and B, podocyte-specific knockout of FBP2 effectively alleviated the loss of slit diaphragm protein and the production of proteinuria caused by diabetic kidney.

[0099] Example 7: Effect of sh-FBP2 that specifically interferes with FBP2 mRNA expression on the expression level of FBP2 protein in glomerular podocytes (HPCs)

[0100] According to the nucleotide sequence of FBP2 mRNA, sh-FBP2 lentivirus and sh-NC lentivirus were designed to specifically knock down the expression level of FBP2 mRNA using HIV-1 (human immunodeficiency virus type 1) as the vector. Then, the same dose of sh-FBP2 lentivirus and sh-NC lentivirus were transfected into HPC cells, and the expression level of FBP2 protein was detected by western blotting.

[0101] Among them, the nucleotide sequence of sh-FBP2 lentivirus is: 5'-TGAGGGCTATGCCAAGTATTT-3' (SEQ ID NO.1), and the nucleotide sequence of sh-NC lentivirus is: 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO.2). Both sh-FBP2 lentivirus and sh-NC lentivirus were artificially synthesized by Genema Company according to the sequence information.

[0102] The specific implementation process of using Western blotting to detect the expression level of FBP2 protein is as follows:

[0103] (1) Podocyte plating: Add 0.5×10 5 podocytes and 0.5 ml complete culture medium were incubated at 37°C and 5% CO 2 Stay overnight;

[0104] (2) Virus dilution: Add polybrene (final concentration 5 μg / ml) and 50 μl of sh-FBP2 lentiviral stock solution (50 μl) to 400 μl of target cell maintenance medium to obtain sh-FBP2 lentiviral dilution solution;

[0105] According to the same ratio and method, the sh-NC lentiviral dilution solution was obtained;

[0106] (3) Transfection: Remove the complete culture medium from the plated podocytes obtained in step (1) and add the sh-FBP2 lentiviral dilution solution; after culturing for 12 to 24 h, remove the sh-FBP2 lentiviral dilution solution and add 0.5 ml of complete culture medium and incubate at 37°C and 5% CO 2 Overnight, and selection with puromycin was performed to obtain podocytes infected with sh-FBP2 lentivirus, which was recorded as sh-FPB2 group;

[0107] According to the same ratio and method, podocytes infected with sh-NC lentivirus were obtained and recorded as sh-NC group;

[0108] (4) Western blotting was used to detect the expression level of FBP2 protein in podocytes of the sh-FPB2 group and the podocytes of the sh-NC group. The specific method was as shown in Example 1. Figure 7 As shown in A.

[0109] Depend on Figure 7 It can be seen that sh-FBP2 lentivirus can effectively reduce the expression level of FBP2 protein.

[0110] Example 8: Effect of silencing FBP2 mRNA on mRNA expression levels of lipid metabolism-related proteins in glomerular podocytes (HPCs)

[0111] The same doses of sh-FBP2 lentivirus and sh-NC lentivirus were transfected into HPC cells, respectively. Reverse transcription-real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of lipid metabolism-related proteins in glomerular podocytes (HPCs).

[0112] Reverse transcription-real-time fluorescence quantitative PCR was used to detect the regulation of FBP2 on the mRNA expression level of lipid metabolism-related genes. The specific implementation steps are as follows:

[0113] (1) Obtaining podocytes of the sh-FPB2 group and the podocytes of the sh-NC group according to steps (1) to (3) of Example 7;

[0114] (2) The podocytes of the sh-FPB2 group and the podocytes of the sh-NC group were plated on two plates each. When the cell confluence reached about 70%, one plate was taken from each plate and 30 mmol / L glucose solution (HG) was added for high glucose stimulation treatment. A total of four groups of cells were obtained, which were named CON group, HG group, sh-F group, and sh-F+HG group.

[0115] (3) The above four groups of cells were washed three times with PBS buffer, the supernatant was discarded, and then the cells were lysed with Trizol lysis buffer, and total RNA was isolated from the cells using an RNA rapid extraction kit (ES Science); the integrity of the extracted RNA was tested by agarose gel electrophoresis, and the concentration of the extracted RNA was measured using Toyobo Tnano-800; the obtained RNA was used as a template to perform reverse transcription and synthesize cDNA products using Revertra Ace qPCR RT Kit (TOYOBO FSQ-101); UltraSYBR Mixture (CWBIO) was used to perform real-time fluorescence quantitative PCR on lipid metabolism-related genes; the results are shown in Figure 2. Figure 8 As shown;

[0116] Among them, the lipid metabolism-related genes are cholesterol regulatory element binding protein 2 (SREBP2), low-density lipoprotein receptor (LDLR), intracellular cholesterol transporter 1 (NPC 1), intracellular cholesterol transporter 2 (NPC2), sterol regulatory element binding protein cleavage activating protein (SCAP), HMG-CoA reductase (HMGCR), ATP binding cassette transporter A1 (ABCA1)

[0117] The primer sequences used are as follows:

[0118] Srebp2-F: 5'-CCTGGGAGACATCGACGAGAT-3',

[0119] Srebp2-R: 5'-TGAATGACCGTTGCACTGAAG-3';

[0120] Ldlr-F: 5'-ACGGCGTCTCTTCCTATGACA-3',

[0121] Ldlr-R: 5'-CCCTTGGTATCCGCAACAGA-3';

[0122] Npc1-F: 5'-GTCCAGCGCAGGTGTTTTC-3',

[0123] Npc1-R: 5'-GCCGAACATCACAACAGAGAC-3';

[0124] Npc2-F: 5'-CAAAGGACAGTCTTACAGCGT-3',

[0125] Npc2-R: 5'-GGATAGGGCAGTTAATTCCACTC-3';

[0126] Scap-F: 5'-TATCTCGGGCCTTCTACAACC-3',

[0127] Scap-R: 5'-GGGGCGAGTAATCCTTCACA-3';

[0128] Hmgcr-F: 5'-TGATTGACCTTTCCAGAGCAAG-3',

[0129] Hmgcr-R: 5'-CTAAAATTGCCATTCCACGAGC-3';

[0130] Abca1-F: 5'-TTCCCGCATTATCTGGAAAGC-3',

[0131] Abca1-R: 5'-CAAGGTCCATTCTTGGCTGT-3'.

[0132] From Figure 8, we can see that sh-FBP2 can effectively reduce the mRNA levels of various lipid metabolism-related proteins in HG-induced HPCs, thereby achieving the effect of treating diabetic nephropathy.

[0133] Example 9: Nile red staining shows the relationship between FBP2 mRNA and lipid deposition in podocytes

[0134] Nile red staining was used to investigate the effect of silencing FBP2 mRNA on lipid deposition in podocytes.

[0135] The specific implementation process is as follows:

[0136] (1) Using the podocytes infected with the lentivirus obtained in Example 7, cell plating and climbing were performed. When the cell confluence reached about 70%, one plate was taken from each group and 30 mmol / L glucose solution (HG) was added for high glucose stimulation treatment, which were recorded as sh-NC+control group, sh-NC+HG group, sh-FBP2+control group, and sh-FBP2+HG group respectively;

[0137] The above 4 groups of cells were fixed with paraformaldehyde after climbing the slides, and 4 groups of fixed cells were obtained;

[0138] (2) Isopropanol rinse: Rinse the fixed cells in 60% isopropanol for 25 seconds;

[0139] (3) Nile red staining: After the cells are fixed, stain with Nile red dye for 15 minutes. Wash the stained slides several times with PBS buffer to remove excess dye. Use a mounting medium containing DAPI to seal the slides and observe the Nile red staining using a confocal microscope. The results are as follows: Figure 8 shown.

[0140] Depend on Fig. 9 It can be seen that silencing FBP2 mRNA in podocytes alleviated high glucose-induced lipid deposition in podocytes.

Claims

1. The use of FBP2 mRNA as a therapeutic target in the preparation of a drug for the prevention and treatment of diabetic nephropathy, characterized in that: The NCBI accession number of the FBP2 mRNA is NM_003837.

4.

2. Use of shRNA that specifically interferes with FBP2 mRNA expression in the preparation of a drug for preventing and treating diabetic nephropathy, characterized in that: The nucleotide sequence of the shRNA that specifically interferes with the expression of FBP2 mRNA is shown in SEQ ID NO.

1.

3. A drug for preventing and treating diabetic nephropathy, characterized in that: It is a nucleic acid molecule, carbohydrate, lipid, small molecule chemical drug, antibody drug, peptide, protein or virus that can specifically interfere with the expression of FBP2 mRNA.

4. The drug for preventing and treating diabetic nephropathy according to claim 3, characterized in that: The diabetic nephropathy prevention and treatment drug is a shRNA that specifically interferes with the expression of FBP2 mRNA, and its nucleotide sequence is shown in SEQ ID NO.1.

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