Use of benzoyl oxypaeoniflorin in preparation of a drug for preventing or treating FABP4 high expression related kidney diseases

Abstract

The application discloses application of benzoyl oxypaeoniflorin in preparation of a medicine for preventing or treating FABP4 high-expression related kidney diseases. It is found for the first time that the benzoyl oxypaeoniflorin can effectively prevent or treat FABP4 high-expression related kidney diseases, especially renal interstitial fibrosis, by inhibiting a FABP4 target.

Description

Technical Field

[0001] This invention relates to the use of benzoyloxypaeoniflorin (BOPF) in the preparation of drugs for the prevention or treatment of kidney diseases associated with high expression of fatty acid-binding protein 4 (FABP4); specifically, it relates to the use of benzoyloxypaeoniflorin in the preparation of FABP4 inhibitors, drugs that inhibit FABP4-Fascin1 interaction, TGF-β / Smad signaling pathway inhibitors, drugs that inhibit pEMT (partial epithelial-mesenchymal transition) in renal tubular epithelial cells, and drugs for the prevention or treatment of renal interstitial fibrosis. Background Technology

[0002] Chronic kidney disease (CKD) has become a major global public health challenge. Once CKD progresses to end-stage renal disease, patients require renal replacement therapy. In 2023, approximately 4.59 million patients worldwide received renal replacement therapy; however, resources are extremely unevenly distributed, and most patients cannot receive timely treatment. Renal interstitial fibrosis is a common pathological pathway in the progression of CKD to end-stage renal disease, and its severity directly determines the rate of decline in kidney function.

[0003] Therefore, elucidating the pathogenesis of renal interstitial fibrosis and finding effective intervention targets and active drugs are of great significance for delaying the progression of CKD and reducing the global disease burden.

[0004] Fatty acid-binding protein 4 (FABP4) is an important member of the fatty acid-binding protein family. Initially thought to be a lipid chaperone specifically expressed by adipocytes and macrophages, it plays a central role in fatty acid uptake, transport, and metabolic regulation. Recent studies have gradually revealed that FABP4 also plays a crucial role in renal pathological states. Multiple studies have confirmed that FABP4 expression is significantly upregulated in mouse models of renal interstitial fibrosis induced by unilateral ureteral occlusion (UUO) and in renal tubular epithelial cells stimulated by lipopolysaccharide (LPS) or transforming growth factor-beta (TGF-β). Further intervention with gene silencing technology or selective small molecule inhibitors (such as BMS309403) can effectively reduce inflammatory responses, improve lipid metabolism disorders, and delay fibrosis progression, suggesting that FABP4 may participate in the pathological process of renal interstitial fibrosis by regulating inflammation and lipid metabolism. Subsequent studies have further confirmed that FABP4 is highly expressed in various kidney disease models, including toxin- or ischemia-reperfusion injury (IRI)-induced kidney injury, diabetic nephropathy, and hyperuricemic nephropathy, and its expression level is closely related to the degree of renal tubular damage, lipid deposition, and fibrotic lesions.

[0005] Clinical studies have also provided important evidence for the pathological significance of FABP4 in kidney diseases. Furuhashi et al. found that serum FABP4 levels in patients with end-stage renal disease were approximately 20 times higher than those with normal renal function, and high FABP4 levels were independently associated with the risk of cardiovascular event-related death. In diabetic nephropathy, serum FABP4 levels were closely related to the rate of renal function decline; even in the early stages when albuminuria was normal, elevated FABP4 indicated an increased risk of renal function decline. More direct histological evidence comes from a recent study by Huang et al., who, through analysis of renal biopsy tissues from 70 patients with diabetic nephropathy, found that high expression of renal tubular FABP4 was significantly positively correlated with the level of urinary N-acetyl-β-D-glucosaminidase (UNAG), a marker of renal tubular injury, and the risk of progression to end-stage renal disease. A recent longitudinal cohort study published by Tanaka et al. has for the first time confirmed that urinary FABP4 levels can independently predict the future decline in renal function and the risk of all-cause mortality in diabetic patients, further establishing the clinical value of FABP4 as a renal prognostic biomarker.

[0006] Although the pathological role of FABP4 in kidney diseases has received increasing attention, the molecular mechanism by which it promotes renal interstitial fibrosis still needs to be elucidated in depth.

[0007] At the same time, although drug development targeting FABP4 has attracted widespread attention, there is still a lack of an active drug that targets FABP4 and can effectively alleviate kidney diseases related to high expression of FABP4, such as renal interstitial fibrosis. Summary of the Invention

[0008] To overcome the technical deficiency of existing drugs targeting FABP4 that effectively alleviate FABP4-high expression-related kidney diseases such as renal interstitial fibrosis, this invention provides the application of benzoyl paeoniflorin oxide (BOPF) in the preparation of drugs for the prevention or treatment of FABP4-high expression-related kidney diseases. This invention is the first to discover that benzoyl paeoniflorin oxide can effectively prevent or treat FABP4-high expression-related kidney diseases, especially renal interstitial fibrosis, by inhibiting the FABP4 target.

[0009] To achieve the above objectives, the present invention adopts the following technical solution.

[0010] This invention provides the application of benzoyl paeoniflorin in the preparation of drugs for the prevention or treatment of FABP4-related kidney diseases.

[0011] In this invention, the FABP4 high expression-related kidney disease refers to a kidney pathological state closely related to abnormally high FABP4 expression, such as one or more of acute kidney injury, chronic kidney disease, and renal cell carcinoma; the chronic kidney disease may include one or more of focal segmental glomerulosclerosis, chronic nephritis, diabetic nephropathy, hyperuricemic nephropathy, lupus nephritis, polycystic kidney disease, and renal fibrosis; the renal fibrosis is, for example, renal interstitial fibrosis.

[0012] In some implementations, the FABP4 overexpression-associated kidney disease is renal interstitial fibrosis, and the application is achieved by inhibiting pEMT in renal tubular epithelial cells.

[0013] The renal tubular epithelial cell pEMT refers to a pathological state in which renal tubular epithelial cells undergo incomplete phenotypic transformation during the progression of chronic kidney disease (CKD) after being stimulated by inflammation, injury, or other factors, transitioning from typical epithelial cell characteristics to mesenchymal cell characteristics.

[0014] In some preferred embodiments, the inhibition of pEMT in renal tubular epithelial cells is achieved by inhibiting the TGF-β / Smad signaling pathway.

[0015] In some preferred embodiments, the suppression of the TGF-β / Smad signaling pathway is achieved by blocking the FABP4-Fascin1 interaction.

[0016] In some preferred embodiments, the inhibition of pEMT in renal tubular epithelial cells manifests as the downregulation of one or more proteins among Collagen I, α-SMA, N-cadherin, Vimentin, Fibronectin, Snail, Slug, and Twis.

[0017] In some preferred embodiments, the inhibition of pEMT in renal tubular epithelial cells manifests as the restoration of E-cadherin expression.

[0018] In some preferred embodiments, inhibition of the TGF-β / Smad signaling pathway manifests as a decrease in the phosphorylation levels of one or more proteins among ALK5, Smad2, Smad3, and Smad4.

[0019] This invention also provides the application of benzoyl paeoniflorin in the preparation of FABP4 inhibitors.

[0020] The present invention also provides its use in the preparation of a drug that inhibits the interaction between FABP4 and Fascin1.

[0021] This invention also provides an application in the preparation of TGF-β / Smad signaling pathway inhibitors.

[0022] In some implementations, the suppression of the TGF-β / Smad signaling pathway is achieved by blocking the FABP4-Fascin1 interaction.

[0023] In some implementations, inhibition of the TGF-β / Smad signaling pathway manifests as a decrease in the phosphorylation levels of one or more proteins among ALK5, Smad2, Smad3, and Smad4.

[0024] The present invention also provides the application of benzoyl paeoniflorin in the preparation of a drug that inhibits pEMT in renal tubular epithelial cells.

[0025] In some implementations, the inhibition of pEMT in renal tubular epithelial cells is achieved by inhibiting the TGF-β / Smad signaling pathway.

[0026] In some embodiments, the inhibition of pEMT in renal tubular epithelial cells manifests as the downregulation of one or more proteins among Collagen I, α-SMA, N-cadherin, Vimentin, Fibronectin, Snail, Slug, and Twis.

[0027] In some implementations, the inhibition of pEMT in renal tubular epithelial cells manifests as the restoration of E-cadherin expression.

[0028] In some preferred embodiments, the suppression of the TGF-β / Smad signaling pathway is achieved by blocking the FABP4-Fascin1 interaction.

[0029] In some preferred embodiments, inhibition of the TGF-β / Smad signaling pathway manifests as a decrease in the phosphorylation levels of one or more proteins among ALK5, Smad2, Smad3, and Smad4.

[0030] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0031] The reagents and raw materials used in this invention are all commercially available.

[0032] The positive and progressive effects of this invention are as follows: This invention unexpectedly discovered that benzoyl paeoniflorin can be used as a high-affinity small molecule inhibitor of FABP4 for the prevention or treatment of kidney diseases associated with high expression of FABP4.

[0033] Specifically, benzoyl paeoniflorin can inhibit the TGF-β / Smad signaling pathway by blocking the interaction between FABP4 and Fascin1, thereby inhibiting pEMT in renal tubular epithelial cells and exerting an anti-renal interstitial fibrosis effect. Attached Figure Description

[0034] Figure 1 This study presents the dynamic expression changes of FABP4 and its role as a biomarker of fibrosis in UUO and IRI mouse models. (AC) In the UUO model, FABP4 mRNA and protein expression in kidney tissue increased in a time-dependent manner, showing a significant increase by day 7 post-surgery (n=3). (DF) In the IRI model, FABP4 expression peaked on day 7 post-surgery and declined by day 14 (n=3). (GH) On day 7 post-surgery, the expression of Collagen I, α-SMA, N-cadherin, and Vimentin in the kidney tissue of both UUO and IRI model mice was significantly increased, while E-cadherin expression was significantly decreased, indicating successful induction of fibrosis. Simultaneously, FABP4 was significantly increased in both models (n=6). P <0.05, P <0.01, P <0.001, P<0.0001.

[0035] Figure 2 Results related to the expression and localization of FABP4 in renal tubular epithelial cells of UUO and IRI mice. (A) Immunohistochemical staining showed that FABP4 protein expression was significantly increased in renal tubular epithelial cells of UUO and IRI mice, mainly located in the cytoplasm; (B) Immunofluorescence double labeling showed that FABP4 significantly co-localized with the renal tubular epithelial cell marker LTL, and the fluorescence intensity of FABP4 was significantly enhanced in the UUO and IRI groups.

[0036] Figure 3 The results related to the upregulation of FABP4 expression in HK2 cells under TGF-β1 and H / R stimulation were analyzed. (AC) Western blotting and immunofluorescence were used to detect FABP4 protein expression in HK2 cells; n=3. P <0.05, P <0.01, P <0.001, P <0.0001.

[0037] Figure 4 Results related to FABP4 gene knockdown alleviating TGF-β1 and I / R-induced pEMT process in HK2 cells. (A) Immunofluorescence assay to verify the transfection efficiency of siRNA in HK2 cells; (BC) qPCR and WB to detect the FABP4 knockdown efficiency of siFABP4-1, siFABP4-2, and siFABP4-3; (DF) qPCR and WB results showed that under TGF-β1 stimulation, FABP4 knockdown significantly reversed the upregulation of mRNA and protein expression of fibrosis markers Collagen I, α-SMA, N-cadherin, and Vimentin, and restored E-cadherin mRNA and protein expression; (GH) WB showed that under H / R stimulation, FABP4 gene knockdown also significantly alleviated the pEMT process in HK2 cells; n=3, P <0.05, P <0.01, P <0.001, P <0.0001.

[0038] Figure 5Results related to the construction of renal tubular epithelial cell-specific FABP4 conditional knockout mice. (A) Schematic diagram of FABP4CKO mouse construction; (B) PCR identification of the genotypes of FABP4f / f mice and FABP4 CKO (Cdh16-Cre+ FABP4f / f) mice; (CE) qPCR and WB verification of FABP knockout effect; n=6, P <0.05, P <0.01, P <0.001, P <0.0001.

[0039] Figure 6 To investigate the effects of FABP4 knockout specifically on renal tubular epithelial cells in alleviating UUO-induced renal interstitial fibrosis. (A) Representative morphological and histopathological images of the kidneys of mice in each group 7 days after UUO surgery; (BC) Quantitative analysis of renal tubular injury scores and renal interstitial fibrosis area, n=3; (D) Transcriptome sequencing (RNA-seq) heatmap showing the expression profile of fibrosis-related genes in the renal cortex of mice in each group, with 79 differentially expressed genes including TGF-β signaling pathway members, ECM components, EMT-related factors, inflammation and chemokines; (EF) qPCR validation of mRNA expression levels of key fibrosis and inflammation-related genes, n=6; (GH) Western blot analysis of EMT markers and fibrosis-related protein expression in renal tissue, n=6. P <0.05, P <0.01, P <0.001, P <0.0001.

[0040] Figure 7 To investigate the effects of FABP4 knockout specifically on renal tubular epithelial cells on renal fibrosis induced by renal interstitial renal fibrosis. (A) Representative morphological and histopathological images of the kidneys of mice in each group 7 days after IRI surgery; (BC) Quantitative analysis of renal tubular injury scores and renal interstitial fibrosis area, n=3; (DE) Western blot analysis of EMT markers and fibrosis-related protein expression in renal tissue, n=6; P <0.05, P <0.01, P <0.001, P <0.0001.

[0041] Figure 8 To illustrate the results of AAV9-mediated local FABP4 knockdown in the kidneys in alleviating IRI-induced renal interstitial fibrosis. (A) Schematic diagram and experimental timeline of the AAV9-mediated local FABP4 knockdown strategy in the kidneys; (B) Immunofluorescence assay to verify the efficiency of local AAV9 viral infiltration in the kidneys; (CD) qPRC and WB assays to verify the FABP4 protein knockdown efficiency, n=3; (EF) WB assay to detect the effect of FABP4 KD on IRI-induced EMT and fibrosis marker expression, n=6; P <0.05, P <0.01, P <0.001, P <0.0001.

[0042] Figure 9a Results of IP-MS screening and identification of FABP4 interacting proteins 1. (A) Coomassie brilliant blue staining showing IP product protein bands; (B) Schematic diagram of IP-MS experimental procedure; (C) Visualization of IP-specific interacting proteins.

[0043] Figure 9b Results related to IP-MS screening and identification of FABP4 interacting proteins 2. (A) RNA-seq GO enrichment showed significant enrichment of cytoskeleton remodeling and ECM remodeling; (B) Secondary mass spectra of representative peptides of FSCN1.

[0044] Figure 10 Results related to the verification of the interaction between FABP4 and Fascin1 and the localization of their domains. (A) Molecular docking shows the binding mode of Fascin1 and FABP4; (BC) CoIP verifies the interaction between FABP4 and Fascin1 in HK2 cells; (D) Immunofluorescence double labeling verifies the colocalization of FABP4 and Fascin1 in HK2 cells; (E) Immunofluorescence double labeling verifies the colocalization of FABP4 and Fascin1 in the kidney tissues of UUO and IRI mice; (FH) Co-IP verifies that the 268-378 amino acid region of Fascin1 is the key domain for binding FABP4.

[0045] Figure 11a Results related to FABP4 enhancing the TGF-β / SMAD signaling pathway by stabilizing Fascin1. (AB) CHX tracing assay to detect the effect of FABP4 on Fascin1 protein stability; (C) RNA-seq GSEA enrichment analysis; n=3, P<0.05, P <0.01, P <0.001, P <0.0001.

[0046] Figure 11b Results related to FABP4 enhancing the TGF-β / SMAD signaling pathway by stabilizing Fascin1: (AB) CoIP verification of the interaction between Fascin1 and ALK5 in HK2 cells stimulated by TGF-β1; (CF) Functional rescue experiment, Western blotting verification of Fascin1-mediated regulation of TGF-β / SMAD signaling by FABP4; n=3, P <0.05, P <0.01, P <0.001, P <0.0001.

[0047] Figure 12 Results related to the interaction between benzoyl paeoniflorin oxide (BOPF) and the FABP4 protein. (A) Chemical structure of benzoyl paeoniflorin oxide (BOPF); (B) Cartoon diagram of FABP4 binding to BOPF; (C) Surface diagram of FABP4 binding to BOPF; (D) 2D diagram of FABP4 binding to HYN2101; (E) 3D diagram of FABP4 binding to BOPF; (F) SPR sensor image showing a strong binding affinity between BOPF and FABP4 (KD = 3.87 × 10⁻⁶). -7 M)

[0048] Figure 13 The results of the safety evaluation of BOPF on HK2 cells and its protective effect against TGF-β1-induced cell damage were presented. (A) The effect of different concentrations of BOPF on HK2 cell viability after 24 h of treatment with CCK8 assay was detected, n=3; (B) Morphological changes of HK2 cells after TGF-β1 stimulation and BOPF intervention were observed by optical microscopy; (C) The effect of BOPF on the decrease in HK2 cell viability induced by TGF-β1 was detected by CCK8 assay, n=3. P <0.05, P <0.01, P <0.001, P <0.0001.

[0049] Figure 14 To investigate the effects of BOPF on the interaction between FABP4 and Fascin1 and its regulation of the TGF-β / Smad signaling pathway and pEMT process. (A) Co-IP detection of the effect of BOPF on the interaction between FABP4 and Fascin1 in TGF-β1-induced HK2 cells; (B) Immunofluorescence double labeling detection of the effect of BOPF on the co-localization of FABP4 and Fascin1 in TGF-β1-induced HK2 cells; (CD) Western blotting detection of the effect of BOPF on the expression of key proteins in the TGF-β / Smad signaling pathway and relative quantitative analysis, n=3; (EF) Western blotting detection of the effect of BOPF on the expression of pEMT marker proteins and relative quantitative analysis, n=3. P <0.05, P <0.01, P <0.001, P <0.0001.

[0050] Figure 15 Results regarding the protective effect of BOPF against renal interstitial fibrosis in UUO mice. (A) Experimental flowchart; (BC) Relative quantitative analysis of renal tubular injury scores and collagen area in each group, n=3; (D) Gross morphological and pathological observation of the kidneys of mice in each group; (E) Western blot analysis of the effect of BOPF on the expression of renal fibrosis marker proteins (Colagen I, α-SMA, N-cadherin, Vimentin, and E-cadherin) in each group of mice; (F) Western blot analysis of the effect of BOPF on the expression of key proteins in the TGF-β / Smad signaling pathway; (GH) Western blot analysis of relative protein quantification, n=3. P <0.05, P <0.01, P <0.001, P <0.0001.

[0051] Figure 16 Representative H&E staining images of heart, liver, lung, and kidney tissues after administration of BOPF 20 mg / kg (in vivo safety evaluation).

[0052] Figure 17Results related to the protective effect of FABP4-mediated BOPF against renal interstitial fibrosis in UUO mice. (A) Experimental flowchart; (B) Renal tubular injury scores of mice in each group, n=3; (C) Histological staining (HE, PAS, and Masson) to observe renal interstitial collagen deposition and renal tubular injury in each group; (D) Relative quantitative analysis of renal interstitial collagen area, n=3; (E) Immunohistochemical detection of renal Collagen I, α-SMA, and E-cadherin expression in each group; (FH) Relative quantitative analysis of immunohistochemistry, n=3; P <0.05, P <0.01, P <0.001, P <0.0001. Detailed Implementation

[0053] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0054] In this invention, the relevant terms are explained as follows: "FABP4 high expression-related kidney disease" refers to a kidney pathological state closely related to abnormally high FABP4 expression, such as one or more of acute kidney injury, chronic kidney disease, and renal cell carcinoma; the chronic kidney disease may include one or more of focal segmental glomerulosclerosis, chronic nephritis, diabetic nephropathy, hyperuricemic nephropathy, lupus nephritis, polycystic kidney disease, and renal fibrosis; the renal fibrosis may be, for example, renal interstitial fibrosis.

[0055] "Partial Epithelial-Mesenchymal Transition (pEMT)" refers to a pathological state in which renal tubular epithelial cells undergo an incomplete phenotypic transformation during the progression of chronic kidney disease (CKD) after being stimulated by inflammation, injury, or other factors, transitioning from typical epithelial cell characteristics to mesenchymal cell characteristics.

[0056] Example

[0057] The present invention verified the link between FABP4 and anti-renal interstitial fibrosis, as well as the effect and related mechanism of benzoyl paeoniflorin in effectively alleviating anti-renal interstitial fibrosis by inhibiting FABP4, through the following steps.

[0058] 1. Preparation of experimental drugs for animals

[0059] (1) Benzoyl paeoniflorin (BOPF) dosing regimen

[0060] ① Dosage parameters

[0061] The dosage was 20 mg / kg, the solvent system was a DMSO (dimethyl sulfoxide)-corn oil mixture (DMSO final concentration ≤2%), and the route of administration was intraperitoneal injection. For example, for a 20 g mouse, the single dose was 0.4 mg.

[0062] ② Preparation of stock solution

[0063] Based on the solubility data provided by MedChemExpress (MCE), prepare a 100 mg / mL DMSO stock solution: Weigh 5 mg of BOPF, dissolve it in 50 μL of DMSO, aliquot after complete dissolution, and store at -20℃ protected from light.

[0064] ③ Preparation of working solution

[0065] Prepare 0.2 mg / 100 μL working solution (final DMSO concentration 2%): Take 20 μL of stock solution (containing 2 mg of drug), add 980 μL of corn oil, vortex to mix, and prepare 1 mL working solution (drug concentration 0.4 mg / 200 μL). Prepare and use immediately.

[0066] ④ Drug administration procedure

[0067] Each mouse was injected intraperitoneally with 200 μL containing 0.4 mg of the drug (i.e., 20 mg / kg).

[0068] (2) Dosing regimen of FABP4 inhibitor BMS-309403

[0069] ① Dosing parameters

[0070] The dosage was 40 mg / kg, the solvent system was a DMSO-corn oil mixture (DMSO final concentration ≤2%), and the route of administration was by gavage. For example, for a 20 g mouse, the single dose was 0.8 mg.

[0071] ② Preparation of stock solution

[0072] Based on the solubility data provided by MCE, prepare a 200 mg / mL DMSO stock solution: Weigh 35 mg of BMS-3094035 mg, dissolve it in 25 μL of DMSO, aliquot it after complete dissolution, and store it at -20℃ protected from light.

[0073] ③ Preparation of working solution

[0074] Prepare 0.4 mg / 100 μL working solution (final DMSO concentration 2%): Take 20 μL of stock solution (containing 4 mg of drug), add 980 μL of corn oil, vortex to mix, and prepare 1 mL working solution (drug concentration 0.8 mg / 200 μL). Prepare and use immediately.

[0075] ④ Drug administration procedure

[0076] Each mouse was administered 200 μL of the drug via gavage, containing 0.8 mg of the drug (i.e., 40 mg / kg).

[0077] 2. Experimental Methods

[0078] 2.1 SPR Experiment

[0079] 2.1.1 Protein Coupling

[0080] The target protein was covalently immobilized on the CM5 chip surface using an amino-coupled method. The protein was diluted to 50 μg / mL (10 mM sodium acetate, pH 4.0) and injected at a flow rate of 10 μL / min for 900 sec, resulting in a target conjugation volume of approximately 15,000 RU. The conjugation procedure was executed automatically by the system.

[0081] 2.1.2 Small molecule sample preparation

[0082] The small molecule stock solution (20 mM, 100% DMSO) was diluted 20-fold with 1.05× PBS-P+ to 1 mM (containing 5% DMSO), and then serially diluted 3-fold with running buffer containing 5% DMSO for a total of 10 concentration gradients (0.05–1000 μM), with a zero concentration control and repeat concentration points set up.

[0083] 2.1.3 Preparation of Solvent Calibration Curve

[0084] Prepare 4.5% and 5.8% DMSO stock solutions, and mix them by volume to prepare eight solvent calibration standards (DMSO final concentration 4.5%–5.8%) to eliminate systematic errors caused by differences in DMSO refractive index.

[0085] 2.1.4 Multi-cycle dynamics detection

[0086] The detection parameters were set as follows: binding time 60 sec, dissociation time 120 sec, flow rate 30 μL / min, and detection temperature 25°C. A dual-channel detection mode (flow cell 2-1) was used, with the running buffer serving as a start-up control. Samples of each concentration were injected sequentially, and extra washes with 50% DMSO were performed between cycles to remove tubing residue. Baseline stability was monitored throughout the experiment to ensure the binding signal was concentration-dependent.

[0087] 2.1.5 Data Analysis

[0088] Data processing was performed using Biacore T200 Evaluation Software. At least five continuous concentration data points were selected, and the dissociation equilibrium constant KD was calculated by fitting the steady-state affinity model with isotherms.

[0089] 2.2 Establishment, grouping, and material collection of experimental animal models

[0090] 2.2.1 Establishment of animal models

[0091] Establishment of a unilateral ureteral obstruction model (UUO model): Patients fasted for 12 hours preoperatively. Anesthesia was administered via intraperitoneal injection of pentobarbital 50 mg / kg. After satisfactory anesthesia, the patient was prepared, placed in the right lateral decubitus position, and disinfected with povidone-iodine. A surgical incision of approximately 1 cm was made about one finger-width below the left rib. The skin, subcutaneous tissue, and muscles were longitudinally incised layer by layer until the peritoneum was opened. The left kidney was located and squeezed out of the incision, fully exposing the left kidney. The ureter was freed, and double ligation was performed near the renal hilum and at the lower 1 / 3 level of the ureter using 6-0 sutures. The ligation force should be moderate, just enough to block urine flow. The left kidney and ureter were then repositioned. After the procedure, the incision was sutured layer by layer and disinfected with povidone-iodine. In the sham surgery group (Sham), only the left ureter was dissected without ligation. The entire experiment was conducted under the influence of an electric blanket and a heat lamp.

[0092] Establishment of a renal ischemia-reperfusion injury model (IRI model): Patients fasted for 12 hours preoperatively. Anesthesia was administered via intraperitoneal injection of pentobarbital 50 mg / kg. After satisfactory anesthesia, the patient was prepared, placed in the right lateral decubitus position, and disinfected with povidone-iodine. A surgical incision of approximately 1 cm was made about one finger-width below the left rib. The skin, subcutaneous tissue, and muscles were longitudinally incised layer by layer until the peritoneum was opened. The left kidney was located and squeezed out of the incision, fully exposing it. The renal artery was freed at the renal hilum and clamped with a vascular clamp. A timer was used for approximately 42 minutes, observing the kidney's condition; the kidney would darken after clamping. A small piece of sterile gauze was used to cover the incision to reduce fluid evaporation and heat loss. After the clamping time was completed, the vascular clamp was carefully removed. The kidney should then slowly return to a pinkish-red color. The kidney was then repositioned. 500 μL of heated sterile saline (37 ℃) was injected into the peritoneal cavity to prevent dehydration. After the procedure, the incision was sutured layer by layer and disinfected with povidone-iodine. The sham surgery group (Sham) only dissected the left renal artery without clamping it. The entire experiment was conducted under the influence of an electric blanket and a heat lamp.

[0093] 2.2.2 Experimental Grouping

[0094] (1) UUO model (C57BL / 6J mice): divided into Sham group, BOPF group, UUO group, BMS309403 group, UUO+BOPF 5mg / kg group, UUO+BOPF 10mg / kg group, and UUO+BOPF 20mg / kg group.

[0095] (2) UUO model (FABP4 CKO mouse): divided into Sham-Ctrl group, UUO-Ctrl group, UUO+FABP4 CKO group, and UUO+FABP4 CKO+BOPF group.

[0096] 2.2.3 Obtaining experimental animal samples

[0097] Mice were euthanized by cervical dislocation after thorough anesthesia. They were fixed in a supine position, and the skin of the chest and abdomen was disinfected with povidone-iodine. The thoracic and abdominal cavities were incised layer by layer to fully expose the heart and both kidneys. Cardiac perfusion was performed via left ventricular puncture: perfusion was performed using physiological saline until both kidneys changed from dark red to pale white, indicating adequate perfusion. The left kidney was then quickly separated and removed, rinsed in pre-cooled physiological saline, and the renal capsule was carefully dissected. Blood vessels and connective tissue at the renal hilum were removed. The left kidney was transversely cut along the coronal plane. The portion near the renal hilum was placed in 4% paraformaldehyde fixative and fixed at 4°C for 24 h. After routine dehydration, it was embedded in paraffin for histopathological examination. The distal two-thirds of the kidney was rapidly flash-frozen in liquid nitrogen and then stored at -80°C for subsequent molecular biological assays such as RNA sequencing, real-time quantitative PCR, and Western blot.

[0098] 2.3 HE staining

[0099] (1) Dewaxing paraffin sections to water: Place the sections in xylene I for 20 min - xylene II for 20 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 75% ethanol for 5 min, and rinse with tap water.

[0100] (2) Hematoxylin staining: Stain the sections in hematoxylin solution for 3-5 min, rinse with tap water, differentiate with differentiation solution, rinse with tap water, blue with blue solution, and rinse with tap water.

[0101] (3) Eosin staining: The sections were dehydrated in 85% and 95% graded ethanol for 5 min each, and then stained in eosin staining solution for 5 min.

[0102] (4) Dehydration and mounting: The sections were sequentially placed in anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min and then cleared and mounted with neutral resin.

[0103] (5) Result interpretation: The cell nucleus is blue and the cytoplasm is red.

[0104] 2.4 PAS staining

[0105] (1) Dewaxing paraffin sections to water: Place the sections in xylene I for 20 min - xylene II for 20 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 75% ethanol for 5 min, and rinse with tap water.

[0106] (2) Periodic acid staining: Immerse the sections in periodic acid staining solution for 15 min, and wash twice with distilled water.

[0107] (3) Chev staining: Immerse the sections in Chev staining solution for 30 min, protect from light, and rinse with tap water for 5 min.

[0108] (4) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3-5 min, rinse with tap water, differentiate with differentiation solution, rinse with tap water, blue with blue solution, and rinse with tap water.

[0109] (5) Dehydration and mounting: The sections were sequentially placed in anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min, and then cleared and mounted with neutral resin.

[0110] (6) Result interpretation: Glycogen and cell wall are purple-red, and cell nucleus is light blue.

[0111] 2.5 Masson staining

[0112] (1) Dewaxing of paraffin sections: The sections were placed in xylene I for 20 min - xylene II for 20 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 75% ethanol for 5 min, and then rinsed with tap water.

[0113] (2) Potassium dichromate staining: Soak the slices in potassium dichromate overnight and rinse with tap water.

[0114] (3) Iron hematoxylin staining: Iron hematoxylin A solution and B solution are mixed in equal proportion to form iron hematoxylin staining solution. The slices are immersed in iron hematoxylin for 3 min, rinsed with tap water, differentiated with differentiation solution, rinsed with tap water, blued with blue solution, and rinsed with tap water.

[0115] (4) Ponceau S Acid Fuchsia Staining: Immerse the slices in Ponceau S Acid Fuchsia for 5-10 min, then rinse with tap water.

[0116] (5) Phosphomolybdic acid staining: Immerse in phosphomolybdic acid aqueous solution for 1-3 min.

[0117] (6) Aniline blue staining: After phosphomolybdic acid, do not wash with water, directly immerse in aniline blue staining solution for 3-6 min.

[0118] (7) Differentiation: The sections were differentiated with 1% glacial acetic acid and dehydrated with anhydrous ethanol.

[0119] (8) Clearing and mounting: Immerse the sections in anhydrous ethanol for 5 min, xylene for 5 min to clear, and then mount with neutral resin.

[0120] (9) Collagen fibers are blue; muscle fibers, cellulose and red blood cells are red.

[0121] 2.6 Immunohistochemistry (IHC)

[0122] (1) Dewaxing of paraffin sections: The sections were placed in xylene I for 15 min - xylene II for 15 min - xylene III for 15 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 85% ethanol for 5 min - ethanol for 5 min - and then rinsed with distilled water.

[0123] (2) Antigen retrieval: Perform antigen retrieval on tissue sections according to standard procedures in this field. During this process, excessive evaporation of the buffer solution should be prevented, and the slides should not be dried. After natural cooling, place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 minutes each time.

[0124] (3) Blocking endogenous peroxidase: Place the slide in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 20 min. Then place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time.

[0125] (4) Serum blocking: Add 3% BSA evenly to the histochemistry zone and block at room temperature for 30 min. (Use rabbit serum to block if the primary antibody is of goat origin, and BSA to block if it is of other origin).

[0126] (5) Add primary antibody: Gently shake off the blocking solution, drop the corresponding primary antibody prepared in PBS at a certain ratio onto the slide, place the slide flat in a humidified chamber and incubate overnight at 4°C, adding a small amount of water to the humidified chamber to prevent antibody evaporation.

[0127] (6) Add secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add the secondary antibody (HRP-labeled) of the corresponding species to the primary antibody to cover the tissue and incubate at room temperature for 50 min.

[0128] (7) DAB staining: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. The positive result is brownish-yellow. Rinse the slide with tap water to stop the staining.

[0129] (8) Counterstaining cell nuclei: Counterstain with hematoxylin for about 3 minutes, rinse with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, use hematoxylin blue solution to blue, and rinse with tap water.

[0130] (9) Dehydration and mounting: Place the sections in 75% ethanol for 5 min - 85% ethanol for 5 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - xylene I for 5 min to dehydrate and become transparent. Remove the sections from the xylene and let them dry slightly before mounting with neutral resin.

[0131] Results interpretation: Hematoxylin staining of cell nuclei was blue, and DAB positive expression was brownish-yellow.

[0132] 2.7 Double labeling of tissue immunofluorescence

[0133] (1) Dewaxing of paraffin sections: The sections were placed in xylene I for 15 min - xylene II for 15 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 85% ethanol for 5 min - 75% ethanol for 5 min - and then rinsed with distilled water.

[0134] (2) Antigen retrieval: Tissue slides were placed in a retrieval box filled with sodium citrate antigen retrieval buffer (pH 6.0) and subjected to antigen retrieval in a microwave oven according to standard procedures in the field. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be dried out. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time.

[0135] (3) Serum blocking: After the section is slightly dried, draw a circle around the tissue with a histochemical pen, add BSA (if the primary antibody is from goat, use 10% donkey serum for blocking; if the primary antibody is from other sources, use 3% BSA for blocking), and block for 30 min.

[0136] (4) Add the first primary antibody: Remove the blocking solution, add the prepared primary antibody, and incubate overnight at 4°C in a humidified box.

[0137] (5) Add the corresponding secondary antibody: Place the slide in PBS (pH 7.4) and wash it 3 times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add the corresponding HRP-labeled secondary antibody and incubate at room temperature for 50 min.

[0138] (6) Add the corresponding TSA dye: After incubation, place the slide in PBS (pH 7.4) and wash it 3 times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add TSA to the circle and incubate at room temperature in the dark for 10 min. After incubation, place the slide in PBS and wash it 3 times on a decolorizing shaker for 5 min each time.

[0139] (7) Antigen retrieval: Tissue slides were placed in a retrieval box filled with sodium citrate antigen retrieval buffer (pH 6.0) and subjected to antigen retrieval in a microwave oven according to standard procedures in the art. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be dried out. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time.

[0140] (8) Serum blocking: After the section is slightly dried, draw a circle around the tissue with a histochemical pen, add BSA (if the primary antibody is from goat, use 10% donkey serum for blocking; if the primary antibody is from other sources, use 3% BSA for blocking), and block for 30 min.

[0141] (9) Add the second primary antibody: Remove the blocking solution, add the prepared primary antibody, and incubate it overnight at 4 °C in a humidified box.

[0142] (10) Add the corresponding secondary antibody: Place the slide in PBS (pH 7.4) and wash it 3 times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add the corresponding HRP-labeled secondary antibody and incubate at room temperature for 50 min.

[0143] (11) Counterstaining cell nuclei with DAPI: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time. After slightly drying the slide, add DAPI staining solution to the circle and incubate at room temperature in the dark for 10 min.

[0144] (12) Mounting: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, mount it with anti-fluorescence quenching mounting medium.

[0145] (13) Microscopic examination and photography: The slides were observed and images were collected under a fluorescence microscope.

[0146] (14) The cell nuclei stained with DAPI are blue under ultraviolet excitation, and positive expression is indicated by red light (TYR-570) or green light (488) labeled with the corresponding fluorescein.

[0147] 2.8 Human renal cortical proximal tubular epithelial cells (HK2) and in vitro model establishment and grouping

[0148] 2.8.1 HK2 cell origin

[0149] The HK2 cells used in this experiment were purchased from Wuhan Pronosai Life Science Technology Co., Ltd. (Catalog No. CL-0109).

[0150] 2.8.2 Cell resuscitation

[0151] First, remove the cryovials containing the cells to be thawed, wrap them with EP gloves, and rapidly rewarm them in a 37°C water bath until thawed. Then, transfer the thawed cell suspension from the cryovials to a 15 ml centrifuge tube, add 1 ml of complete culture medium, and centrifuge at 800 rpm for 5 min. Simultaneously, add 3 ml of complete culture medium to another small dish and preheat it in a 37°C incubator. After centrifugation, resuspend the cells in 1 ml of complete culture medium, and evenly seed them into small dishes containing complete culture medium. Incubate at 37°C in a 5% CO2 incubator.

[0152] 2.8.3 Cell medium exchange

[0153] Observe the culture medium status and HK2 cell growth daily. When the culture medium color changes from red to pale yellow, change the medium every 1-2 days. Discard the original culture medium and slowly add 1×PBS pre-warmed to 37°C along the side wall of the culture dish, gently washing the cells twice to remove residual serum and metabolic waste. Then add fresh complete culture medium and continue culturing in a 37°C, 5% CO2 incubator. HK2 cells grow with a typical "paving stone" morphology, a characteristic feature of renal tubular epithelial cells.

[0154] 2.8.4 Cell passage

[0155] When HK2 cells reach 80%-90% confluence, discard the original culture medium and gently wash twice with 1×PBS pre-warmed to 37°C. Add approximately 1 mL of trypsin per 10 cm culture dish to evenly cover the cell monolayer. Incubate at 37°C for 1-2 min. Observe under an inverted microscope until the cells shrink and become rounded, and the gaps widen. Immediately add twice the volume of fresh complete culture medium to stop digestion. Gently pipette to collect the cells and transfer them to centrifuge tubes. Centrifuge at 1000 rpm for 3 min. Discard the supernatant, resuspend the cells in fresh complete culture medium, aliquot into new culture dishes at a 1:2 ratio, add culture medium to make up the difference, gently shake to mix, and continue culturing in a 37°C, 5% CO2 incubator. Avoid moving the cells for 24 h after subculturing to facilitate cell adhesion.

[0156] 2.8.5 Cell cryopreservation

[0157] Using a 10 cm culture dish as a reference, approximately 3 mL of universal serum-based cryopreservation medium is needed per dish of cells. Discard the original culture medium and gently wash the cells 2-3 times with 1 mL of 1×PBS pre-warmed to 37°C. Add trypsin to thoroughly rinse the cell monolayer, incubate at 37°C for 1-2 min, and add twice the volume of complete culture medium to stop digestion once the cells become rounded. Gently and repeatedly pipette to completely detach the cells and collect them into 15 mL centrifuge tubes. Centrifuge at 1000 rpm for 2 min. Discard the supernatant, add 3 mL of universal serum-based cryopreservation medium, gently mix by pipetting, and aliquot 1 mL into cryovials. Place the cryovials in a controlled-temperature chamber and incubate at -80°C overnight. Transfer to liquid nitrogen for storage the next day.

[0158] 2.8.6 siRNA cell transfection

[0159] (1) Cell preparation: Seed HK2 cells in 6-well plates and culture them until the cell confluence reaches 50%-60% before transfection. At this time, the cells are in the logarithmic growth phase, which is conducive to the uptake of transfection reagents.

[0160] (2) siRNA preparation: To obtain the best gene silencing efficiency, the final concentration of FABP4 siRNA was 50 nM. The lyophilized FABP4 siRNA (designed and synthesized by Nanjing Kris Biotechnology Co., Ltd., sequence shown in Table 1) powder was dissolved in nuclease-free ddH2O to prepare a 100 μM stock solution (according to the instructions: add 25 μL of ddH2O per tube). During transfection, 1 μL of the stock solution was added to 2 mL of antibiotic-free complete culture medium, with a final concentration of 50 nM.

[0161] (3) Preparation of transfection complex: First, take 1 μL of FABP4 siRNA (100 μM) and dilute it in 200 μL of jetPRIME buffer. Gently pipette to mix, vortex the jetPRIME reagent for 5 seconds, and then briefly centrifuge. Then add 4 μL of jetPRIME reagent to the siRNA-buffer mixture, vortex for 1 second, briefly centrifuge, and finally incubate at room temperature for 10-15 min to form the transfection complex.

[0162] (4) Transfection procedure: Add the transfection complex dropwise and evenly to the culture medium containing 10% FBS but without antibiotics. Gently shake the culture dish to distribute it evenly and incubate it in a 37°C, 5% CO2 incubator. At the same time, a negative control group (transfected with NC-siRNA) and a blank control group (only transfection reagent added) were set up.

[0163] (5) Post-transfection treatment: 24 h after transfection, the medium was replaced with complete medium (containing 10% FBS and 1% double antibody), and cultured for another 24-48 h. Cells were then collected for subsequent detection.

[0164] Table 1

[0165] 2.8.7 DNA cell transfection

[0166] (1) Cell preparation: HK2 cells were seeded in 6-well plates and transfected when the cell confluence reached 70%-80% and the cells were in the logarithmic growth phase.

[0167] (2) Plasmid DNA preparation: The concentration of FABP4 overexpression plasmid DNA is 600 ng / μL, and 2 μg of plasmid DNA is transfected into each well; the required volume is calculated as: 2 μg ÷ 600 ng / μL = 3.33 μL. Take 3.3 μL of FABP4 overexpression plasmid DNA and dilute it in 200 μL of jetPRIME buffer, and gently pipette to mix.

[0168] (3) Preparation of transfection complex: First, vortex the jetPRIME reagent containing plasmid DNA for 5 seconds and then briefly centrifuge; then add 8 μL jetPRIME reagent to the DNA-buffer mixture, vortex for 1 second and then briefly centrifuge; finally, incubate at room temperature for 10-15 min to form DNA-transfection reagent complex.

[0169] (4) Transfection procedure: Add the transfection complex dropwise to the culture medium containing 10% FBS but without antibiotics, gently shake the culture dish to distribute it evenly, and incubate it in a 37℃, 5% CO2 incubator; at the same time, set up an empty vector control group (transfected empty plasmid Vector) and a blank control group (only transfection reagent added).

[0170] (5) Post-transfection treatment: 24 h after transfection, the medium was replaced with complete medium (containing 10% FBS and 1% double antibody), and cultured for another 24-48 h. Cells were then collected for subsequent detection.

[0171] 2.8.8 Co-transfection of DNA and siRNA into cells

[0172] (1) Cell preparation: HK2 cells were seeded in 6-well plates and co-transfected when the cells reached 70%-80% confluence and were in the logarithmic growth phase.

[0173] (2) Preparation of plasmid DN and siRNA: ① Overexpression of FSCN1 plasmid DNA: concentration 426 ng / μL, 2 μg per well. Calculated volume required: 2 μg ÷ 426 ng / μL = 4.69 μL (approximately 4.7 μL).

[0174] ② FSCN1 siRNA (sequence shown in Table 2): Dissolve the lyophilized powder in nuclease-free ddH2O to prepare a 100 μM stock solution (according to the instructions: add 25 μL ddH2O per tube). For transfection, add 1 μL of the stock solution to 2 mL of culture medium, resulting in a final concentration of 50 nM.

[0175] ③ Prepare FABP4 siRNA and FABP4 overexpression plasmid DNA according to standard procedures in this field.

[0176] (3) Transfection complex preparation: First, dilute 2 μg of overexpressing FSCN1 plasmid and 50 nM FSCN1 siRNA together in 200 μL jetPRIME buffer and gently pipette to mix. Vortex the jetPRIME reagent for 5 seconds and centrifuge briefly. Then add 8 μL of jetPRIME reagent, vortex for 1 second, and centrifuge briefly. Finally, incubate at room temperature for 10-15 min to form the transfection complex.

[0177] (4) Transfection procedure: Add the transfection complex dropwise and evenly to the culture medium containing 10% FBS but without antibiotics. Gently shake the culture dish to distribute it evenly and incubate at 37°C in a 5% CO2 incubator. Empty vector control group: Transfected with Vector or (and) NC-siRNA. Blank control group: Transfected with transfection reagent only.

[0178] (5) Post-transfection treatment: 24 h after transfection, the medium was replaced with complete medium (containing 10% FBS and 1% double antibody), and cultured for another 24-48 h. Cells were then collected for subsequent detection.

[0179] Table 2

[0180] 2.8.9 Establishment of in vitro experimental models

[0181] Establishment of a TGF-β1-stimulated HK2 cell model (TGF-β1 model): HK2 cells were seeded at an appropriate density in 6-well plates, with 2 mL of complete culture medium per well, and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 70%-80%, the original culture medium was discarded, and the cells were gently washed twice with pre-warmed 1×PBS. The medium was then replaced with 2 mL of serum-free DMEM / F12 medium, and the cells were starved for 6 h to synchronize the cell cycle and enhance the cell responsiveness to stimulation. Subsequently, 2 μL of TGF-β1 (10 μg / mL stock solution) was added to bring the final concentration to 10 ng / mL, and the cells were cultured for another 24 h. A control group (containing an equal volume of sterile PBS) was also established. After induction, morphological changes in the cells were observed under an inverted microscope: the control group HK2 cells showed a typical "paving stone" epithelial morphology, while the TGF-β1-induced cells showed obvious spindle-shaped or fusiform changes.

[0182] Establishment of the hypoxia / reoxygenation (H / R) stimulation model of HK2 cells: HK2 cells were seeded at an appropriate density in 6-well plates, with 2 mL of complete culture medium per well. When the cell confluence reached 50%-60%, the medium was replaced with DMEM / F12 medium containing 0.5% FBS, and the cells were starved and synchronized for 12 h. After synchronization, the cells were gently washed twice with pre-warmed 1×PBS and randomly divided into two groups: the normal control group was replaced with complete culture medium (10% FBS) and cultured in a normoxic incubator (37℃, 5% CO2, 95% air); the H / R model group was replaced with hypoxic culture medium (0.5% FBS) and cultured in a tri-gas incubator (37℃, 1% O2, 5% CO2, 94% N2) for 9 h, followed by replacement with complete culture medium (10% FBS) and reoxygenation under normoxic conditions for 3 h. The hypoxia / reoxygenation cycle was repeated 3 times. After the last reoxygenation, cells were collected for subsequent testing.

[0183] 2.9 CCK8 Experiment

[0184] (1) HK2 cells were loaded with 2×10 3 Cells were seeded at a density of 100 μL / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 12 h. When the cells adhered and reached 70%-80% confluence, they were divided into groups: control group: containing only complete culture medium; model group: with TGF-β1 added (final concentration 10 ng / mL); drug intervention group: pretreated with small molecule drug for 6 h, followed by TGF-β1 added and cultured for another 24 h.

[0185] (2) Discard the original culture medium and add 100 μL of fresh culture medium containing 10% CCK8 solution to each well. At the same time, set up blank control wells (containing complete culture medium, corresponding concentration of drug and CCK8 solution, but without cells) to subtract background absorbance.

[0186] (3) Place the 96-well plate in a 37℃, 5% CO2 incubator for 1 h (the incubation time was determined in the preliminary experiment to be within the linear range of 0.5-4 h), and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value of each well at 450 nm.

[0187] 2.10 Protein Extraction from Cells and Tissues

[0188] Cell protein extraction: Discard the culture medium, wash cells twice with 1×PBS, and add 130 μL of RIPA lysis buffer (containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor, i.e., RIPA : Cocktail 100× : Phosphatase inhibitor 100× = 100 : 1 : 1) to each well. Lyse on ice for 10 min. Scrape cells off ice with a cell scraper and collect them into 1.5 mL pre-chilled EP tubes. Sonicate the cells (20% power, 3 sec, 10 sec interval, 3 times). Centrifuge at 12000 rpm for 20 min at 4℃. Carefully transfer the supernatant to a new pre-chilled EP tube. Dilute 2 μL of the supernatant with 18 μL of RIPA lysis buffer for BCA protein quantification. Aliquot the remaining supernatant and store temporarily at -20℃ or -80℃.

[0189] Tissue protein extraction: Weigh 30-35 mg of kidney tissue and place it in a 1.5 mL pre-chilled EP tube. Add 500 μL of RIPA lysis buffer (containing 1× protease inhibitor cocktail and 1× phosphatase inhibitor, i.e., RIPA : Cocktail 100× : Phosphatase inhibitor 100× = 100 : 1 : 1). Add 1-2 stainless steel grinding beads to each tube (the number of grinding beads should be consistent across tubes to ensure balancing). Pre-chill at -20℃ for 5 min, then place in a tissue homogenizer and homogenize at 60 Hz for 1 min. Repeat this cycle 3 times at 30 sec intervals until the tissue is homogenized. Lyse on ice for 10 min, centrifuge at 12000 rpm for 20 min at 4℃, and carefully transfer the supernatant to a new pre-chilled EP tube. Dilute 2 μL of the supernatant with 18 μL of RIPA lysis buffer for BCA protein quantification. Aliquot the remaining supernatant and store temporarily at -20℃ or -80℃.

[0190] 2.11 BCA protein quantification

[0191] (1) Preparation of standard products: ① Protein standard stock solution (25 mg / mL): Add 1.2 mL of protein standard preparation solution to a 30 mg BSA standard tube, dissolve thoroughly, and prepare a 25 mg / mL protein standard stock solution. After aliquoting, store at -20°C for long-term storage, avoiding repeated freeze-thaw cycles.

[0192] ② Protein standard working solution (0.5 mg / mL): Take 20 μL of 25 mg / mL protein standard stock solution, add 980 μL of diluent (the same lysis buffer used for the sample to be tested, such as RIPA lysis buffer), mix well, and prepare a 0.5 mg / mL standard working solution. Prepare and use immediately, or aliquot and store at -20℃.

[0193] (2) Preparation of BCA working solution: Calculate the required volume of working solution based on the number of samples. Prepare the solution according to the BCA reagent A:B solution = 50:1 (volume ratio), and use immediately after preparation. For example: 6 test samples + 8 standards + 1 duplicate well = 15 wells. Each well requires 200 μL of working solution, for a total of 15 × 200 = 3000 μL. Therefore, 3000 μL of A solution and 60 μL of B solution are required.

[0194] (3) Preparation of standard curve: Add 0.5 mg / mL standard working solution to the wells of a 96-well plate at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL, and bring the total volume to 20 μL with RIPA lysis buffer. The corresponding final concentrations are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, with 3 replicates for each concentration.

[0195] (4) Sample determination: Add 2 μL of the protein to be tested to the sample well and make up to 20 μL with RIPA lysis buffer.

[0196] (5) BCA reaction: Add 200 μL of BCA working solution to each well and incubate at 37℃ for 30 min.

[0197] (6) Absorbance measurement: The absorbance value at 562 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. A standard curve was plotted with the standard concentration as the x-axis and the absorbance value as the y-axis. The regression equation was calculated, and the protein concentration of the sample was calculated accordingly.

[0198] 2.12 Western blot

[0199] Preparation of the lower gel layer (taking a 1.0 mm thick gel as an example): Take 2.7 mL each of equal volumes of lower gel solution (2×) and lower gel buffer (2×), mix well, and then add 60 μL of modified coagulant. Gently mix to avoid generating air bubbles. Pour the mixed solution into the gel-forming glass plate, with the liquid level about 1 cm below the upper edge of the short glass plate.

[0200] Preparation of the top layer: Take equal volumes of top layer gel solution (2×) and colored top layer gel buffer (2×), 0.75 mL each (shake well before use), mix thoroughly, then add 15 μL of modified coagulant and mix gently. No need to wait for the bottom layer gel to solidify; directly and gently pour the mixture into the gel-forming glass plate, immediately inserting the comb teeth of the appropriate thickness. Note that the top layer gel should be poured gently to avoid flushing into the bottom layer gel.

[0201] Gel polymerization: Allow the gel to solidify at room temperature for approximately 15 minutes, then remove the comb teeth before electrophoresis. The prepared gel exhibits gradient-like separation capabilities, with a separation range of 10-250 kDa.

[0202] Protein electrophoresis: Install the prepared gel into the electrophoresis tank, add 1×PG610 electrophoresis buffer, and check for leakage. Remove the comb teeth. After denaturing the protein samples by boiling at 100℃ for 10 min, load 20-40 μg of total protein per well. Electrophoresis conditions: constant voltage 150 V, approximately 35 min. Terminate electrophoresis when the bromophenol blue indicator migrates to the bottom of the gel.

[0203] Transfer: Use 1× wet transfer buffer. Adjust transfer conditions according to the molecular weight of the target protein: constant current 400mA, 50 min.

[0204] (6) Blocking and antibody incubation: Block the PVDF membrane with rapid blocking buffer at room temperature for 20 min after transfer. Add diluted primary antibody (at the concentration recommended in the antibody manufacturer's instructions) and incubate overnight at 4°C on a shaker. Wash three times with TBST for 10 min each time. Add HRP-labeled secondary antibody (at the concentration recommended in the antibody manufacturer's instructions) and incubate at room temperature for 1 h. Wash three times with TBST for 10 min each time.

[0205] (7) Development and Imaging: Prepare the luminescent solution according to the ECL chemiluminescence kit instructions, evenly cover the PVDF membrane, and incubate in the dark for 1-2 min. Expose in a chemiluminescence imaging system and acquire images. Perform quantitative analysis of gray values ​​using ImageJ or corresponding software, and use glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control to calculate the relative expression level of the target protein.

[0206] (8) Antibody information is shown in Table 3 below.

[0207] Table 3

[0208] 2.13 Co-immunoprecipitation (COIP)

[0209] (1) Prepare cell lysate and prepare 500 μL Lysis buffer + 5 μL protease inhibitor Cocktail.

[0210] (2) Aspirate the culture medium and wash the cells once with ice-cold 1xPBS.

[0211] (3) Remove the PBS and add 0.5 ml of pre-chilled Lysis buffer to each large dish (10 cm), and incubate on ice for at least 5 minutes.

[0212] (4) Scrape the cells off the large dish, transfer the extract to a new 1.5 ml enzyme-free EP tube, and place it on ice.

[0213] (5) Perform ultrasonic fracturing on ice 3 times, each time for 5 seconds.

[0214] (6) Centrifuge at 4℃ and 14,000 rpm for 10 minutes, and transfer the supernatant to a new enzyme-free EP tube. The supernatant is the cell lysate. If necessary, the lysate can be stored at -80℃. Note: After the cell lysate is prepared, Western blot (WB) can be performed to determine the presence of the target protein in the cell lysate.

[0215] (7) Pretreatment of cell lysate: First, add 5 μL of Protein A and 5 μL of Protein G to 500 μL of cell lysate (containing 200-1000 μg of total protein). Then, incubate at 4°C by rotation for 30-60 minutes. Finally, centrifuge at 12,000 rpm for 1 minute at 4°C, and retain the supernatant for later use.

[0216] (8) Antigen-antibody binding: Set up an input group (one large dish), an IP group (one large dish), and an IgG group (one large dish).

[0217] (9) After aspirating 50 μL from the Input group, mix the remaining protein into a tube and then divide it equally into 500 μL each for the IgG and IP groups.

[0218] (10) Add 3 μg of target protein antibody to IP group and 3 μg of IgG antibody to IgG group, and gently mix overnight at 4°C.

[0219] (11) Immune complex precipitation: After incubating the antibody overnight, add 10 μL each of Protein A and Protein G, and gently mix overnight at 4°C. If the beads evaporate, add 1 part anhydrous ethanol and 4 parts DEPC water to prepare 20% ethanol.

[0220] (12) Centrifuge at 12,000 rpm for 1 minute and retain the precipitate.

[0221] (13) Wash the precipitate with 0.5 mL of 1xWash buffer, shake slowly on a shaker at 4°C for 20 minutes, centrifuge at 12000 rpm for 1 minute, and retain the precipitate.

[0222] (14) Repeat step 12 for a total of 3 washes. Note: When washing and removing the supernatant, be careful not to aspirate the packing material. Except for the last time when the lower layer of liquid is completely aspirated, do not completely aspirate at other times. If sending for mass spectrometry sequencing at this time, place the two tubes of IP and IgG protein in a -80 degree freezer.

[0223] (15) WB detection: Resuspend the precipitate in 20-40 μL of 1xSDS loading buffer, vortex, and then centrifuge for 30 seconds to allow the beads and liquid on the tube wall to reach the bottom of the tube. That is: 40 μL of 1xSDS loading buffer is added to the IgG and IP groups, and 1 / 4 of 5xSDS loading buffer is added to the Input group.

[0224] (16) Boil at 100℃ for 10 minutes for the Input group and for 15 minutes for the IP and IgG groups.

[0225] (17) Load the sample and run the gel.

[0226] 2.14 Immunoprecipitation combined with mass spectrometry (IP-MS) analysis

[0227] 2.14.1 Immunoprecipitation

[0228] Same as step 2.13.

[0229] 2.14.2 Coomassie blue staining

[0230] (1) Place the gel in a suitable container, add 50 ml of deionized water, and microwave on high for 3 min.

[0231] (2) Discard the deionized water, add 50 ml of deionized water, and wash on a shaker for 5 min.

[0232] (3) Discard the deionized water, add about 20 ml of Coomassie brilliant blue staining solution, and stain for 10-30 min.

[0233] (4) Discard the Coomassie brilliant blue staining solution, add about 100 ml of deionized water, and shake on a shaker to decolorize.

[0234] 2.14.3 MS detection

[0235] The mass spectrometry identification was commissioned to Shanghai Baiqu Biotechnology Co., Ltd.

[0236] 2.15 RNA extraction from cells and tissues

[0237] 2.15.1 Sample pyrolysis

[0238] For cells: First, aspirate the culture medium and wash once with an appropriate amount of PBS. Then add 50 μL of Lysis Buffer, pipette and vortex 10 times, transfer to an enzyme-free EP tube, and vortex for 10 seconds to fully lyse the cells.

[0239] For the tissue: First, 5 mg of kidney tissue was excised into a 1.5 ml EP tube, 300 μL of Lysis Buffer was added, and the tissue was pre-cooled at -20°C for 5 min. Then, it was placed in a tissue homogenizer and homogenized at 60 Hz for 1 min, with a 30-second interval, for 3 cycles until the tissue was homogenized. Next, it was centrifuged at 12,000 rpm for 2 min, and the supernatant was transferred to a new enzyme-free 1.5 ml EP tube.

[0240] 2.15.2 Column loading / RNA binding

[0241] (1) Add an equal volume of anhydrous ethanol to the lysed cells or tissue and mix thoroughly (precipitation may occur, which is normal; just continue the operation). You can invert the centrifuge tube several times or use a pipette to blow and blow vigorously 10 times to disperse any possible precipitate, and then add the liquid to the centrifuge column.

[0242] (2) Centrifuge at 12,000 rpm for 1 min.

[0243] 2.16 Reverse transcription

[0244] (1) Genomic DNA removal

[0245] Prepare the mixtures shown in Table 4 below in RNase-free centrifuge tubes: Table 4

[0246] Mix gently by pipetting. Incubate at 42°C for 2 minutes.

[0247] (2) Preparation of reverse transcription reaction system

[0248] In step 1, add 5 × HiScript Ⅲ qRT SuperMix as shown in Table 5 directly to the reaction tube.

[0249] Table 5

[0250] Gently mix by pipetting.

[0251] (3) Perform reverse transcription reaction

[0252] The reaction was carried out under the conditions shown in Table 6.

[0253] Table 6

[0254] The product can be used immediately for qPCR reactions, or stored for a short period at -20℃ or for a long period at -80℃.

[0255] 2.17 Real-time quantitative polymerase chain reaction (RT-qPCR)

[0256] (1) Prepare the mixture shown in Table 7 below in the qPCR tube.

[0257] Table 7

[0258] (2) Perform qPCR reaction under the conditions shown in Table 8.

[0259] Table 8

[0260] (3) The primer list is shown in Table 9 below.

[0261] Table 9

[0262] 2.18 Kidney tissue transcriptome sequencing (RNA-seq)

[0263] (1) Total RNA extraction and quality control: Total RNA was extracted from tissues using the TRIzol method (Invitrogen, CA, USA). RNA purity (A260 / A280 ratio) was detected using a NanoDrop spectrophotometer (Thermo Scientific, DE, USA), and RNA integrity (RIN value) was detected using an Agilent 5400 Fragment Analyzer system (Agilent Technologies, CA, USA). RNA samples with a total amount ≥1 μg and meeting quality standards were selected for subsequent library construction.

[0264] (2) Library construction and sequencing: The library was constructed using the NEB Next Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, MA, USA). PolyA-tailed mRNA was enriched with Oligo(dT) magnetic beads and randomly fragmented to 300 bp fragments using divalent cation exchange. cDNA was synthesized by reverse transcription, and after end repair, A-tailing, and adapter ligation, target fragments were screened using AMPure XP beads. The final library was obtained by PCR amplification and purification. After the insert size was qualified by Qubit 2.0 quantification and Agilent 5400 detection, PE150 paired-end sequencing was performed by Shanghai Bio-Tech Co., Ltd. on the Illumina NovaSeq X Plus platform.

[0265] (3) Data quality control: FastP (v0.23.4) was used to filter the raw data, removing adapter sequences, low-quality reads (Q<20) and reads containing N bases > 5, to obtain clean data for subsequent analysis.

[0266] (4) Sequence alignment and quantification: Clean reads were aligned to the reference genome using HISAT2 (v2.2.1), and transcripts were reconstructed and gene expression levels were calculated using StringTie2 (v2.1.4). Expression was normalized using FPKM and TPM.

[0267] (5) Differential expression analysis: DESeq2 (v1.38.3) was used for samples with biological replicates, and edgeR (v3.40.2) was used for samples without replicates. Differential gene screening criteria: |log2FoldChange| > 1 and padj < 0.05 (DESeq2) or padj < 0.005 (edgeR).

[0268] (6) Functional enrichment analysis: ClusterProfiler (v4.6.2) was used for GO functional enrichment (hypergeometric test, Padj<0.05) and KEGG pathway enrichment analysis (Padj<0.05). Gene set enrichment analysis was performed using the GSEA method.

[0269] 3. Experimental Results

[0270] 3.1 FABP4 expression is upregulated in renal interstitial fibrosis

[0271] (1) Animal model validation: qPCR and Western blot confirmed that FABP4 expression was significantly increased in the kidney tissues of UUO and IRI mice. Figure 1Immunohistochemistry and immunofluorescence localization showed that FABP4 was mainly expressed in renal tubular epithelial cells. Figure 2 ).

[0272] (2) Cell model validation: Western blot and immunofluorescence confirmed that FABP4 expression was significantly increased in HK2 cells stimulated by TGF-β1 and H / R, and was located in the cytoplasm and nucleus. Figure 3 ).

[0273] 3.2 FABP4 promotes the progression of renal interstitial fibrosis

[0274] (1) In vitro functional experiments: siRNA knockdown of FABP4 significantly reversed TGF-β1 and H / R-induced partial EMT (pEMT) in HK2 cells, as evidenced by downregulation of Collagen I, α-SMA, N-cadherin, and Vimentin expression, and restoration of E-cadherin expression. Figure 4 ).

[0275] (2) In vivo gene knockout experiment: FABP4 CKO significantly reduced renal interstitial collagen deposition, renal tubular damage, and pEMT progression in UUO and IRI mice. RNA-seq analysis showed that FABP4 knockout inhibited the expression of 79 fibrosis-related genes, including TGF-β family members, extracellular matrix (ECM) components, EMT-related molecules, and inflammatory factors. Figure 5-7 ).

[0276] (3) In vivo gene knockdown experiment: FABP4 KD also significantly reduced IRI-induced renal tubular epithelial cell pEMT process and renal interstitial fibrosis. Figure 8 ).

[0277] 3.3 FABP4 promotes renal interstitial fibrosis by enhancing the TGF-β / Smad signaling pathway through Fascin1.

[0278] (1) Screening of interacting proteins: IP-MS combined with RNA-seq enrichment analysis identified Fascin1 as a key interacting protein of FABP4. Figure 9a and Figure 9b ).

[0279] (2) Interacting protein verification: CoIP and immunofluorescence co-localization confirmed that the interaction between FABP4 and Fascin1 is enhanced under TGF-β1 stimulation. The truncated mutant experiment showed that the 268-378 amino acid region of Fascin1 is the key domain mediating its binding with FABP4. Figure 10 ).

[0280] (3) Regulation of Fascin1 protein stability by FABP4: The CHX tracking experiment showed that FABP4 knockdown significantly promoted Fascin1 protein degradation, while FABP4 overexpression stabilized Fascin1 protein. Figure 11a (AB).

[0281] (4) Regulation of the TGF-β / Smad signaling pathway by the FABP4-Fascin1 axis: Western blot and CoIP results showed that FABP4 promotes the binding of Fascin1 to ALK5 by stabilizing Fascin1, thereby enhancing the TGF-β / Smad signaling pathway, as evidenced by increased phosphorylation levels of ALK5 and Smad2 / 3. Figure 11a C, Figure 11b (AF).

[0282] (5) Functional rescue experiment: TGF-β1 induces phosphorylation of ALK5 and Smad2 / 3, FABP4 knockdown inhibits this phosphorylation, and Fascin1 overexpression can reverse this inhibitory effect; conversely, FABP4 overexpression enhances phosphorylation, and Fascin1 knockdown blocks this enhancing effect. This confirms the regulatory role of Fascin1 in the TGF-β / Smad signaling pathway mediated by FABP4. Figure 11b (CF).

[0283] 3.4 BOPF exerts its anti-renal interstitial fibrosis effect by targeting FABP4.

[0284] 3.4.1 Safety and morphological effects of BOPF on HK2 cells

[0285] like Figure 12 A represents the chemical structure of BOPF; Figure 12 The BD model demonstrates the molecular docking binding mode of BOPF and FABP4 protein, with a binding energy of -12.378 kcal / mol. To further verify the binding ability of BOPF and FABP4, this invention uses surface plasmon resonance (SPR) technology for detection. The results show that BOPF and FABP4 have a strong binding affinity, with a dissociation equilibrium constant (Kd) of 3.87 × 10⁻⁶. -7 M ( Figure 12 E).

[0286] Furthermore, this invention used the CCK8 assay to evaluate the toxic effects of BOPF on HK2 cells. The results showed that BOPF had no significant toxicity to HK2 cells within the concentration range of 0.625-160 μM, indicating a broad safety margin; the cell viability was optimal in the 10-40 μM concentration range. Figure 13Therefore, subsequent cell experiments used two doses: 10 μM (low concentration) and 40 μM (high concentration). Optical microscopy showed that HK2 cells underwent significant morphological changes after TGF-β1 stimulation, exhibiting a spindle-shaped or fusiform appearance; after intervention with 10 μM and 40 μM BOPF, the cell morphology was significantly improved (A). Figure 13 The CCK8 assay further confirmed that BOPF at both 10 μM and 40 μM concentrations significantly improved the TGF-β1-induced decrease in HK2 cell viability. Figure 13 (C).

[0287] 3.4.2 BOPF inhibits the binding of FABP4 to Fascin1 and regulates the TGF-β / Smad signaling pathway

[0288] To verify whether BOPF regulates the TGF-β / Smad signaling pathway by inhibiting the interaction between FABP4 and Fascin1, this invention first used a CoIP experiment to detect their binding. The results showed that TGF-β1 stimulation significantly enhanced the interaction between FABP4 and Fascin1 in HK2 cells, while BOPF intervention significantly inhibited their binding. Figure 14 The A). Immunofluorescence double labeling assay further confirmed that BOPF significantly inhibited the TGF-β1-induced co-localization expression of FABP4 and Fascin1 (A). Figure 14 B).

[0289] Western blot results showed that BOPF significantly inhibited TGF-β1-induced activation of the TGF-β / Smad signaling pathway, as evidenced by a significant decrease in the phosphorylation levels of ALK5, Smad2, and Smad3. Figure 14 Meanwhile, BOPF significantly inhibited the pEMT process in renal tubular epithelial cells, as evidenced by a significant downregulation of the expression of Colagen I, α-SMA, N-cadherin, and Vimentin, and a significant recovery of E-cadherin expression. Figure 14 EF).

[0290] 3.4.3 Protective effect of BOPF on renal interstitial fibrosis in UUO mice

[0291] To further verify the anti-fibrotic effect of BOPF in vivo, this invention established a UUO mouse model ( Figure 15 The results showed that BOPF at doses of 5, 10, and 20 mg / kg could alleviate kidney enlargement in UUO mice to varying degrees, and significantly reduce renal interstitial collagen deposition and renal tubular damage. Figure 15Western blot (WB) results showed that BOPF improved renal fibrosis markers in UUO mice in a dose-dependent manner: 20 mg / kg was superior to 10 mg / kg in reducing the expression of Colagen I, α-SMA, N-cadherin, and Vimentin, and in restoring E-cadherin levels; 10 mg / kg was superior to 5 mg / kg; and 20 mg / kg BOPF was as effective as the positive control drug BMS309403 (40 mg / kg). Figure 15 The E and G). Therefore, 20 mg / kg was selected as the BOPF intervention dose for subsequent mechanistic studies. Further Western blot analysis showed that both BOPF and BMS309403 significantly inhibited the activation of the TGF-β / Smad signaling pathway in the kidneys of UUO mice, as evidenced by a significant decrease in the phosphorylation levels of ALK5, Smad2, and Smad3 (E and G). Figure 15 (F and H).

[0292] Furthermore, compared with the Sham group, mice in the 20 mg / kg BOPF monotherapy group were in good general condition, active, had normal appetite, and steadily gained weight, with no obvious toxic reactions. HE staining of major organs showed that the heart, liver, lungs, and kidneys had clear tissue structures, normal cell morphology, and no obvious pathological damage. These results suggest that BOPF has good in vivo safety within the effective dose range. Figure 16 ).

[0293] 3.4.4 Verification of the mechanism of FABP4-mediated renal protective effect of BOPF

[0294] To clarify the mediating role of FABP4 in the anti-fibrotic effect of BOPF, this invention uses the FABP4 CKO mouse model for verification. Figure 17 A). Histological staining (HE, PAS, and Masson) results showed that in FABP4 CKO mice, 20 mg / kg BOPF failed to further alleviate UUO-induced renal tubular injury and renal interstitial fibrosis. Figure 17 Immunohistochemical results consistently showed that BOPF could not further inhibit the expression of fibrosis markers such as Colagen I and α-SMA, nor could it further restore E-cadherin expression. Figure 17 (EH). The above results confirm that FABP4 is an essential target for BOPF to exert its nephroprotective effect.

[0295] in conclusion

[0296] FABP4 can stabilize Fascin1 protein, promote the binding of Fascin1 to ALK5, thereby enhancing the TGF-β / Smad signaling pathway and driving renal tubular epithelial cell pEMT and renal interstitial fibrosis.

[0297] BOPF is a high-affinity small molecule inhibitor of FABP4. It can exert an anti-renal interstitial fibrosis effect by blocking the interaction between FABP4 and Fascin1 and inhibiting the TGF-β / Smad signaling pathway. Its efficacy depends on the FABP4 target.

Claims

1. Application of benzoyl paeoniflorin in the preparation of drugs for the prevention or treatment of FABP4-related kidney diseases.

2. The application as described in claim 1, characterized in that, The FABP4 high expression-related kidney diseases are selected from one or more of acute kidney injury, chronic kidney disease, and renal cell carcinoma. The chronic kidney disease may be selected from one or more of focal stage glomerulosclerosis, chronic nephritis, diabetic nephropathy, hyperuricemic nephropathy, lupus nephritis, polycystic kidney disease, and renal fibrosis; the renal fibrosis may be, for example, renal interstitial fibrosis.

3. The application as described in claim 1, characterized in that, The kidney disease associated with high FABP4 expression is renal interstitial fibrosis, and the application is achieved by inhibiting pEMT in renal tubular epithelial cells. Preferably, the inhibition of pEMT in renal tubular epithelial cells is achieved by inhibiting the TGF-β / Smad signaling pathway; More preferably, the inhibition of the TGF-β / Smad signaling pathway is achieved by blocking the FABP4-Fascin1 interaction.

4. The application as described in claim 3, characterized in that, The inhibition of pEMT in renal tubular epithelial cells is manifested by the downregulation of one or more of the following proteins: Collagen I, α-SMA, N-cadherin, Vimentin, Fibronectin, Snail, Slug, and Twis. And / or, the inhibition of pEMT in the renal tubular epithelial cells is manifested as the restoration of E-cadherin expression; And / or, inhibition of the TGF-β / Smad signaling pathway manifests as a decrease in the phosphorylation levels of one or more proteins among ALK5, Smad2, Smad3, and Smad4.

5. Application of benzoyl paeoniflorin in the preparation of FABP4 inhibitors.

6. Application of benzoyl paeoniflorin in the preparation of drugs that inhibit the interaction between FABP4 and Fascin1.

7. Application of benzoyl paeoniflorin in the preparation of TGF-β / Smad signaling pathway inhibitors.

8. The application as described in claim 7, characterized in that, The inhibition of the TGF-β / Smad signaling pathway is achieved by blocking the interaction between FABP4 and Fascin1; And / or, inhibition of the TGF-β / Smad signaling pathway manifests as a decrease in the phosphorylation levels of one or more proteins among ALK5, Smad2, Smad3, and Smad4.

9. Application of benzoyl paeoniflorin in the preparation of drugs that inhibit pEMT in renal tubular epithelial cells.

10. The application as described in claim 9, characterized in that, The inhibition of pEMT in renal tubular epithelial cells is achieved by inhibiting the TGF-β / Smad signaling pathway; And / or, the inhibition of pEMT in renal tubular epithelial cells is manifested by the downregulation of one or more proteins among Collagen I, α-SMA, N-cadherin, Vimentin, Fibronectin, Snail, Slug, and Twis; And / or, the inhibition of pEMT in the renal tubular epithelial cells is manifested as the restoration of E-cadherin expression; Preferably, the inhibition of the TGF-β / Smad signaling pathway is achieved by blocking the FABP4-Fascin1 interaction; And / or, inhibition of the TGF-β / Smad signaling pathway manifests as a decrease in the phosphorylation levels of one or more proteins among ALK5, Smad2, Smad3, and Smad4.

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