Application of dapagliflozin in preparation of medicine for preventing and / or treating obesity-related salt-sensitive hypertension

Abstract

The invention relates to the field of medicines, and particularly discloses application of dapagliflozin to preparation of a medicine for preventing and / or treating obesity-related salt-sensitive hypertension. According to the application, the new application of dapagliflozin in prevention and treatment of a specific and high-risk disease subtype of obesity-related salt-sensitive hypertension is defined for the first time. Experiments prove that dapagliflozin can improve the sodium treatment function of the kidney and promote urine sodium excretion by inhibiting abnormal continuous coupling of a Na / K-ATPase / c-Src signal axis in the kidney. The discovery reveals a new kidney protection mechanism of dapagliflozin independent of the hypoglycemic effect, and provides a new drug use and a treatment scheme for clinical prevention and treatment of obesity-related renal sodium metabolism disorders and salt-sensitive hypertension.

Description

Technical Field

[0001] This application relates to the pharmaceutical field, and more specifically, it relates to the use of dapagliflozin in the preparation of a medicament for the prevention and / or treatment of obesity-related salt-sensitive hypertension. Background Technology

[0002] Dapagliflozin, an inhibitor of sodium-glucose cotransporter 2, has been widely used clinically to treat type 2 diabetes and, due to its proven cardiovascular and renal protective effects, has been expanded to treat heart failure and chronic kidney disease. These effects are mainly attributed to its osmotic diuretic, metabolic-improving, and hemodynamic-enhancing effects.

[0003] In the prior art, patent document CN112156090A discloses the "use of dapagliflozin in the preparation of a drug for improving skeletal muscle mitochondrial function." This document states that dapagliflozin can promote mitochondrial biosynthesis, improve oxidative phosphorylation, and reduce inflammation by activating the AMP-dependent protein kinase (AMPK) signaling pathway in skeletal muscle, upregulating the expression of factors such as peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), ultimately for the prevention or treatment of obesity and related metabolic syndromes. This prior art focuses entirely on improving the function of skeletal muscle, an energy metabolism organ, aiming to address systemic energy metabolism disorders.

[0004] However, obesity-induced metabolic disorders also profoundly affect blood pressure regulation, with salt-sensitive hypertension being an important and challenging clinical subtype. Salt-sensitive hypertension is a type of hypertension in which blood pressure levels are significantly dependent on dietary sodium intake. Its prevalence is significantly increased in obese individuals, and its cardiovascular event risk and treatment difficulty are higher than those of non-salt-sensitive hypertension, constituting a significant clinical burden.

[0005] The kidneys' sodium processing function is a central regulator for maintaining sodium homeostasis and long-term blood pressure stability. Under physiological conditions, the kidneys respond to changes in salt intake by precisely regulating the reabsorption and excretion of sodium ions in the renal tubules. One of the key executors of this function is the sodium / potassium ATPase (Na / K-ATPase) on the basolateral side of the renal tubular epithelial cells. Recent studies have further revealed that Na / K-ATPase is not only an ion transport pump but also a crucial scaffold for signal transduction complexes. Its binding and activation with c-Src kinase forms the Na / K-ATPase / c-Src signaling axis, regulating downstream cellular metabolism, oxidative stress, and inflammatory states, thereby indirectly but profoundly affecting sodium transport processes.

[0006] In obesity, this sophisticated regulatory system is disrupted. Abnormal lipid metabolism and insulin resistance lead to excessive production of reactive oxygen species (ROS), triggering oxidative modification of the Na / K-ATPase α1 subunit. This modification disrupts the normal function of the Na / K-ATPase / c-Src signaling axis, resulting in signal transduction dysregulation. The consequences are twofold: firstly, it directly or indirectly exacerbates renal tubular sodium reabsorption; secondly, it activates downstream inflammatory pathways (such as STAT3), creating a vicious cycle of oxidative stress and inflammation. Therefore, obesity, by specifically disrupting the renal Na / K-ATPase / c-Src signaling axis, leads to impaired sodium processing function, which constitutes a key molecular pathological basis for the occurrence and development of salt-sensitive hypertension.

[0007] Existing treatments, including traditional diuretics, while promoting sodium excretion, primarily focus on inhibiting other sodium transporters (such as the Na-Cl cotransporter NCC). This constitutes a "general" intervention targeting the downstream excretion pathway, failing to precisely regulate the upstream core pathological link of "Na / K-ATPase / c-Src signaling axis dysregulation-oxidative stress-inflammation" specifically disrupted by obesity. Therefore, current technology still lacks a drug application and technical solution capable of precisely regulating the imbalance in renal sodium metabolism under obesity-related pathological conditions. Summary of the Invention

[0008] To address the aforementioned technical problems, this application provides the use of dapagliflozin in the preparation of a medicament for the prevention and / or treatment of obesity-related salt-sensitive hypertension.

[0009] The technical solution adopted in this application is as follows: On the one hand, this application provides the use of dapagliflozin in the preparation of a medicament for the prevention and / or treatment of obesity-related salt-sensitive hypertension, wherein obesity-related salt-sensitive hypertension refers to a subtype of hypertension driven by impaired renal sodium metabolism regulation in an obese state and in which blood pressure levels are highly sensitive to dietary sodium intake.

[0010] Furthermore, the above-mentioned uses are achieved by directly regulating the sodium homeostasis regulatory unit in the proximal convoluted tubule epithelial cells of the kidney, which includes at least the key molecular platform of the Na / K-ATPase α1 subunit and its mediated signal transduction function.

[0011] Furthermore, the above-mentioned uses restore the renal sodium metabolism homeostasis and improve salt-sensitive hypertension by inhibiting the abnormal and persistent coupling between the Na / K-ATPase α1 subunit and c-Src kinase in the obese state, thereby restoring the Na / K-ATPase / Src signaling axis from a pathologically persistent activation state to a physiologically regulated state.

[0012] Furthermore, the above uses also include selectively alleviating renal oxidative stress in obesity-related pathological states, wherein the alleviation of oxidative stress is manifested by at least one of reducing protein carbonylation levels, reducing malondialdehyde content, and / or increasing superoxide dismutase activity.

[0013] Furthermore, the above uses also include suppressing renal inflammatory responses, which are manifested by reducing the expression level of IL-17.

[0014] Furthermore, the above-mentioned prevention and / or treatment of obesity-related salt-sensitive hypertension includes improving glomerular filtration function and / or promoting urinary sodium excretion.

[0015] Secondly, this application provides a pharmaceutical composition for the prevention and / or treatment of obesity-related salt-sensitive hypertension, the composition comprising a therapeutically effective amount of dapagliflozin and a pharmaceutically acceptable carrier.

[0016] Thirdly, this application provides the use of dapagliflozin in the preparation of a medicament for restoring the regulatory balance of the renal Na / K-ATPase / c-Src signaling axis in obese individuals.

[0017] Furthermore, the aforementioned reconstruction includes correcting the abnormal signal amplification mediated by Na / K-ATPase α1 and inhibiting the sustained activation of the ERK / STAT3 inflammatory cascade.

[0018] Furthermore, the above-mentioned drugs are used to prevent or treat renal sodium metabolism disorders or salt-sensitive hypertension.

[0019] In summary, this application has the following beneficial effects: This application, for the first time, clearly defines the novel use of dapagliflozin in the prevention and treatment of a specific, high-risk subtype of obesity-related salt-sensitive hypertension. It expands the clinical application of dapagliflozin from known conditions such as diabetes, heart failure, and chronic kidney disease to a completely new indication with a well-defined mechanism. This provides an effective drug option based on a novel mechanism for the clinical prevention and treatment of refractory salt-sensitive hypertension closely related to obesity, demonstrating significant clinical application value.

[0020] This invention proposes and validates a novel strategy for regulating renal sodium metabolism, focusing on obesity-associated salt-sensitive hypertension, a specific and clinically highly heterogeneous disease subtype.

[0021] This invention discovers that, under the pathological state of obesity, the Na / K-ATPase α1 subunit in renal tubular epithelial cells no longer functions solely as an ion transport pump, but rather as a metabolic-inflammatory signaling integration center. Abnormal and persistent coupling occurs between the subunit and c-Src kinase, resulting in a pathologically and persistently activated state of the Na / K-ATPase / c-Src signaling axis. This disrupts renal sodium metabolism homeostasis and amplifies oxidative stress and inflammatory responses, ultimately promoting the occurrence and development of salt-sensitive hypertension.

[0022] This invention demonstrates that dapagliflozin can restore the Na / K-ATPase / c-Src signaling axis from a pathologically activated state to a physiologically regulated state by breaking the aforementioned abnormal and persistent coupling relationship, thereby achieving the reconstruction of the renal sodium metabolism regulatory system. This mechanism of action differs from the existing strategies of inhibiting a single sodium transporter or downstream sodium excretion pathway; instead, it is a "signal reset" regulatory approach targeting key regulatory units of sodium metabolism.

[0023] Furthermore, the signal reset effect described in this invention is independent of the hypoglycemic effect of dapagliflozin and has significant pathophysiological selectivity. In non-pathophysiological states, it does not significantly interfere with the basic regulatory function of the Na / K-ATPase / c-Src signal axis, demonstrating good safety characteristics. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the mechanism of action of dapagliflozin in improving obesity-related renal sodium metabolism disorders in this invention; Figure 2 This demonstrates the construction of a diet-induced obese mouse model and the experimental design for dapagliflozin intervention: Figure 2 A: Experimental timeline diagram, including time points for animal grouping, dietary intervention, and drug treatment. Experimental group descriptions: LF+HS group: 16 weeks of normal diet + 1 week of high-salt diet (control group); HF+HS group: 16 weeks of high-fat diet + 1 week of high-salt diet (obesity model group); HF+HS+Dapa group: 16 weeks of high-fat diet + 1 week of high-salt diet + concurrent dapagliflozin treatment for 1 week (dapagliflozin treatment group). Figure 2 B: Body weight change curves of mice in different treatment groups; Figure 2 C: Comparison of mouse body weights in each group at the experimental endpoint; Figure 2 D: Appearance and kidney morphology of mice in the control group and the obese group, with perirenal fat; Figure 3 The effects of a high-salt diet and dapagliflozin intervention on renal function and sodium metabolism in normal and obese mice were shown, specifically including: Figure 3 A: Urinary sodium intake ratio in mice of different treatment groups + output-to-Na + intake ratio); Figure 3 B: Urine output-to-water intake ratio in mice under different treatment groups. Figure 3 C: Urinary sodium excretion rate (UNa) in mice under different treatment groups + V); Figure 3 D: Plasma sodium concentration in mice in different treatment groups. Figure 3 E: Creatinine clearance (Ccr) in mice under different treatment groups. Figure 4 The effects of dapagliflozin on the expression and localization of Na / K-ATPase α1 protein in the renal cortex were demonstrated: Figure 4 A: Representative bands of renal cortical Na / K-ATPase α1 protein in Western blot; Figure 4 B: Quantitative analysis results of Na / K-ATPase α1 protein expression; Figure 4 C: Representative image of Na / K-ATPase α1 immunohistochemical staining in kidney tissue; Figure 4 D: Quantitative analysis of the positive area of ​​immunohistochemical staining; Figure 5 This is a representative map and quantitative analysis of the expression of p-Src, p-ERK1 / 2 and p-STAT3 proteins in kidney tissue as detected by Western Blot. Figure 6 The effects of dapagliflozin on renal oxidative stress indicators and plasma oxidative damage markers were shown: Figure 6 A: Representative bands of renal cortical tissue protein carbonylation in Western blot (using Ponceau S stained membrane as a sample loading reference). Figure 6 B: Quantitative analysis of protein carbonylation levels; Figure 6 C: Results of plasma malondialdehyde (MDA) content determination; Figure 6 D: Results of plasma superoxide dismutase (SOD) activity assay; Figure 7 This demonstrates the regulatory effect of dapagliflozin on renal and systemic inflammatory states: Figure 7 A: Representative bands of renal cortical IL-6 protein in Western blot; Figure 7 B: Quantitative analysis of IL-6 protein expression in the renal cortex; Figure 7 C: Results of plasma IL-6 concentration measurement; Figure 7 D: Representative images of IL-6 and IL-17 immunohistochemical staining in kidney tissue; Figure 7 E: Quantitative analysis of the area positive by IL-6 immunohistochemical staining; Figure 7 F: Quantitative analysis of the area positive by IL-17 immunohistochemical staining; Figure 8 Dapagliflozin demonstrated its ameliorative effect on IL-6-induced oxidative stress in renal tubular epithelial cells. Figure 8 A: Representative bands of LLC-PK1 and HK-2 cell protein carbonylation in Western blot; Figure 8 B: Quantitative analysis of protein carbonylation levels in LLC-PK1 cells; Figure 8 C: Quantitative analysis of protein carbonylation levels in HK-2 cells; Figure 8 D: Results of MDA content determination in LLC-PK1 cells; Figure 8 E: Results of MDA content determination in HK-2 cells; Figure 8 F: Results of SOD activity assay in LLC-PK1 cells; Figure 8 G: Results of SOD activity assay in HK-2 cells; Figure 9 This study demonstrates the inhibitory effect of dapagliflozin on the IL-6-induced Na / K-ATPase / c-Src / ERK1 / 2 / STAT3 signaling pathway and its mechanistic model. Figure 9 A: Representative Western blot bands of p-Src, p-ERK1 / 2 and p-STAT3 proteins in LLC-PK1 and HK-2 cells; Figure 9B: Quantitative analysis of phosphorylation levels of p-Src, p-ERK1 / 2 and p-STAT3 in LLC-PK1 cells; Figure 9 Quantitative analysis of phosphorylation levels of p-Src, p-ERK1 / 2 and p-STAT3 in C:HK-2 cells. Detailed Implementation

[0025] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0026] The term "obesity-related salt-sensitive hypertension" refers to a subtype of salt-sensitive hypertension that occurs in obese individuals, with abnormal renal sodium metabolism regulation as the core pathophysiological mechanism, and whose blood pressure levels are highly sensitive to changes in dietary sodium intake.

[0027] This subtype of hypertension belongs to a specific type of salt-sensitive hypertension. Currently, there is no unified international nomenclature or classification for "obesity-related salt-sensitive hypertension." This application, based on the existing disease concept framework of salt-sensitive hypertension and considering obesity as a clear risk factor and the resulting pathophysiological changes, primarily affecting the kidneys, provides a mechanistic-bound definition of this type of hypertension.

[0028] The renal pathophysiological changes mentioned include at least: (1) enhanced sodium reabsorption function in the proximal tubules of the kidneys in obese individuals; (2) changes in the renal tubular epithelial cells. Subunit-mediated signal transduction function undergoes pathophysiological abnormalities, manifested as: (3) Abnormal and persistent coupling with c-Src kinase; (4) The above abnormal coupling further triggers the pathophysiological continuous activation of downstream signaling pathways (including but not limited to ERK, STAT3), accompanied by increased oxidative stress and amplified inflammatory response; (5) thereby disrupting renal sodium metabolism homeostasis, making blood pressure highly sensitive to dietary sodium intake, forming a typical salt sensitivity phenotype.

[0029] Therefore, the "obesity-related salt-sensitive hypertension" described in this application is not a simple renaming of the existing disease concept, but a functional disease subtype definition of salt-sensitive hypertension based on a clear understanding of the pathophysiological background, key target organs, and core molecular mechanisms.

[0030] This patent is the first to propose a clear, repeatable, and interventionable new drug use for obesity-related salt-sensitive hypertension subtype with well-defined pathophysiological mechanisms.

[0031] The technical solution of this invention is as follows: This invention provides the use of dapagliflozin in the preparation of a medicament for the prevention and / or treatment of obesity-related salt-sensitive hypertension.

[0032] Specifically, dapagliflozin exerts its therapeutic effect through the following mechanisms, such as Figure 1 As shown: It inhibits the overactivation of the Na / K-ATPase / c-Src signaling pathway in the kidney.

[0033] It reduces renal oxidative stress, including lowering protein carbonylation levels, reducing malondialdehyde content, and increasing superoxide dismutase activity.

[0034] It inhibits the inflammatory response in the kidneys and reduces the expression of inflammatory factors such as IL-17.

[0035] Based on the above mechanism, dapagliflozin can be formulated into a corresponding drug for improving obesity-related renal sodium metabolism disorders and preventing salt-sensitive hypertension.

[0036] Furthermore, the present invention has demonstrated the novel use of dapagliflozin through the following experiments: Animal model validation: In a mouse model of obesity-related renal sodium metabolism induced by a high-fat, high-salt diet, dapagliflozin treatment significantly improved renal sodium metabolism disorders and renal function indicators.

[0037] Specifically, this manifests as follows: 1) Promote sodium and water excretion: Dapagliflozin intervention significantly increased 24-hour urinary sodium excretion rate and urine volume, effectively reversing sodium and water retention in obese mice.

[0038] 2) Improves glomerular filtration function: Dapagliflozin significantly increases creatinine clearance (Ccr) in obese mice and restores damaged renal filtration function.

[0039] 3) Restoring sodium metabolism balance: Dapagliflozin synergistically alleviates obesity-related renal sodium metabolism disorders by promoting urinary sodium excretion and improving glomerular filtration.

[0040] 2. Mechanism Study: Dapagliflozin effectively inhibits the abnormal activation of the Na / K-ATPase / c-Src signaling pathway in the kidney; dapagliflozin significantly reduces renal oxidative stress, including decreasing protein carbonylation levels, reducing malondialdehyde (MDA) content, and increasing superoxide dismutase (SOD) activity. Dapagliflozin significantly inhibits renal inflammatory responses and reduces the expression of inflammatory factors such as IL-17.

[0041] 3. In vitro experimental verification: In IL-6 stimulated renal tubular epithelial cells (LLC-PK1 and HK-2), dapagliflozin pretreatment was able to reverse IL-6-induced oxidative stress and inhibit the activation of the Na / K-ATPase / c-Src / ERK / STAT3 signaling pathway. Its effect was comparable to that of the Src kinase specific inhibitor PP2, demonstrating that its mechanism of action depends on the regulation of the Na / K-ATPase / c-Src signaling axis.

[0042] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0043] Example I. Experimental Methods: 1. Animal model construction and experimental design; SPF-grade 4-week-old male C57BL / 6J mice were selected, and an obesity model was established by inducing an obesity model with a high-fat diet. Figure 2 As shown in A, the specific steps are as follows: (1) Eighteen mice were randomly divided into three groups: normal control group (n=6), obesity model group (n=6) and dapagliflozin intervention group (n=6).

[0044] (2) The control group was fed a normal diet with 10% kcal, while the obese group and the dapagliflozin intervention group were fed a high-fat diet with 60% kcal for 16 weeks. At this time, the weight of the obese mice was significantly different from that of the normal mice.

[0045] (3) After the modeling was completed, the control group (LF+HS group, LH) was adjusted to a high-salt diet (10% kcal, 8% NaCl), while the obese group (HF+HS group, HH) and the dapagliflozin intervention group (HF+HS+Dapagliflozin group, HHD) were on a high-fat, high-salt diet (60% kcal, 8% NaCl). At the same time, the HHD group was given 0.5 mg / kg dapagliflozin by gavage daily. The high-salt diet and dapagliflozin intervention period were both 1 week.

[0046] 2. Sample collection and processing; After the experiment, the mice were anesthetized and the following samples were collected: (1) Blood samples: Blood was collected by cardiac puncture, anticoagulated with heparin, and separated into plasma by centrifugation for biochemical index detection.

[0047] (2) Tissue samples: Kidney tissue was taken, part of which was stored at -80℃ for protein extraction, and part of which was fixed with 4% paraformaldehyde for pathological analysis.

[0048] 3. Histopathological analysis; (1) Paraffin section preparation: After fixation, the kidney tissue was dehydrated, cleared, impregnated with paraffin and then embedded. The section thickness was 5 μm.

[0049] (2) Staining analysis: H&E staining, Masson trichrome staining and immunohistochemical staining were performed respectively (to detect the levels of IL-6, IL-17 and Na / K-ATPase α1 in the kidney).

[0050] (3) Image acquisition: Images were scanned and acquired using a whole-section scanner, and the staining intensity was quantitatively analyzed using ImageJ software.

[0051] 4. Biochemical indicator testing; (1) Electrolyte detection: Na in plasma and urine was determined by flame atomic absorption spectrometry. + K + concentration.

[0052] (2) Oxidative stress indicators: The MDA content and SOD activity in plasma were detected using a malondialdehyde (MDA) assay kit (TBA method) and a total superoxide dismutase (T-SOD) test kit (hydroxylamine method).

[0053] (3) Inflammation and renal function indicators: The plasma IL-6 level was detected using an IL-6 ELISA kit. Creatinine and urea nitrogen levels were measured using a urea nitrogen test kit (urease method) and a creatinine assay kit (sarcosine oxidase method).

[0054] 5. Cellular experimental verification; (1) Cell culture: Human renal tubular epithelial cells HK-2 and porcine renal tubular epithelial cells LLC-PK1 were cultured in DMEM complete medium (containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin).

[0055] (2) Experimental groups: control group, IL-6 stimulation group, dapagliflozin pretreatment group, Src kinase inhibitor PP2 pretreatment group, dapagliflozin alone treatment group and PP2 alone treatment group were set up.

[0056] (3) Indicator detection: The regulatory effect of dapagliflozin on the Na / K-ATPase / c-Src signaling pathway in renal tubular epithelial cells was determined by Western blotting, and the MDA and SOD contents in the cell lysate were measured using a kit.

[0057] 6. Western blot analysis of proteins; (1) Protein sample preparation: The renal cortical tissue or the fully grown renal tubular epithelial cells were added to pre-cooled lysis buffer and homogenized on ice. The mixture was centrifuged at 14,000 rpm for 10 minutes at 4°C, and the supernatant was collected as the total protein sample.

[0058] (2) Protein concentration determination: The protein concentration was determined using the Bradford method. 10 μL of protein sample was mixed with 200 μL of QuickStart Bradford 1× working solution and incubated at room temperature for 15 minutes. The absorbance value at 595 nm was measured using an ELISA reader, and the protein concentration was calculated based on the standard curve.

[0059] (3) Western blot detection: Electrophoresis: Take 30 μg of total protein sample and separate it by 10% SDS-PAGE gel electrophoresis.

[0060] Transfer: The protein was transferred to a PVDF membrane by wet transfer at 4°C for 120 minutes.

[0061] Blocking and Incubation: After transfer, block with TBST solution containing 5% skim milk (or 5% BSA) at room temperature for 1 hour. Then add primary antibody (Na / K-ATPase α1, p-Src, p-ERK1 / 2, p-STAT3, IL-6, etc.) diluted with TBST solution containing 5% skim milk (or 5% BSA) and incubate overnight at 4°C. After 3 washes with TBST, add the corresponding HRP-labeled secondary antibody and incubate at room temperature for 1 hour.

[0062] Development and detection: Using an ECL chemiluminescence kit, the mixture of A and B solutions in equal volume ratio is added to the membrane. After reacting for 1 minute, the membrane is pressed with X-ray film in a dark room. The exposure time is controlled according to the signal intensity (usually 30 seconds to 10 minutes). After development and fixing, protein bands are obtained.

[0063] (4) Quantitative analysis of signals: The X-ray film was scanned into a digital image and imported into Image J software (Version 1.53t). β-actin was used as an internal reference. The gray value ratio of the target protein band to the corresponding internal reference band was calculated for semi-quantitative analysis.

[0064] 7. Data analysis; Comparisons between two groups were performed using a two-tailed unpaired Student's t-test, and comparisons among multiple groups were performed using one-way ANOVA supplemented by Tukey's post-hoc test. Data are expressed as mean ± standard error, as indicated in the figure captions. Significance levels are indicated as follows: This indicates no significant difference.

[0065] II. Experimental Results: 1. DaPa's effect in improving obesity-induced renal sodium metabolism disorders; A successful obese mouse model was established. At 20 weeks of age, the body weight of the obese group (HF) was significantly increased by 51.6% compared with the normal control group (LF). Figure 2 B and Figure 2 As shown in Figure C, the control group mice had a symmetrical body shape and neat, glossy fur, while the HF group generally exhibited abdominal distension, especially with significant fat accumulation around the kidneys, a condition known as fatty kidney. Figure 2 As shown in D. After high-salt dietary intervention, obese mice showed significant renal sodium metabolism disorder, manifested as decreased urinary sodium excretion ( Figure 3 A, Figure 3 C) Decreased urine output ( Figure 3 B) and sodium and water retention, accompanied by a decrease in creatinine clearance (Ccr) ( Figure 3 E) indicates impaired glomerular filtration function.

[0066] One week after dapagliflozin intervention, the 24-hour urinary sodium excretion rate and urine volume in the treatment group (HHD) were significantly higher than those in the obesity model group (HH). Figure 3 A- Figure 3 C), plasma sodium concentration synchronously returns to normal ( Figure 3 (D) The renal sodium load was effectively relieved. Furthermore, Dapa treatment significantly improved creatinine clearance in obese mice ( Figure 3 E), indicating that it can improve glomerular filtration function.

[0067] The above results confirm that dapagliflozin can effectively alleviate obesity-related renal sodium metabolism disorders by promoting urinary sodium excretion and improving renal function, providing experimental evidence for its clinical application.

[0068] The regulatory effects of dapagliflozin on the expression and signaling pathway of Na / K-ATPase α1 subunit; This invention confirms that, in addition to the known SGLT2 inhibition mechanism, dapagliflozin exerts a renal protective effect by regulating the expression of the Na / K-ATPase α1 subunit and the Na / K-ATPase signal transduction function.

[0069] In the obesity model, the expression of Na / K-ATPase α1 subunit in kidney tissue was significantly reduced ( Figure 4 A, Figure 4 B), Immunostaining showed decreased signal in the basement membrane region and abnormal intracytoplasmic aggregation (B). Figure 4 C, Figure 4 (D) indicates impaired ion transport function. After intervention with dapagliflozin, the membrane localization of the α1 subunit was improved ( Figure 4 C, Figure 4 D), expression levels significantly recovered ( Figure 4 A, Figure 4 (B) This suggests that the medication can repair the sodium pump function.

[0070] Signaling pathway studies showed that phosphorylation levels of Src, ERK1 / 2, and STAT3 were significantly increased in the kidneys of obese mice, such as... Figure 5 As shown, this indicates that the Na / K-ATPase / Src signaling axis is in a state of continuous activation (in pathological conditions, this signaling pathway is continuously overactivated, suggesting signal transduction disorders or desensitization). Dapagliflozin treatment effectively inhibits the abnormal phosphorylation of the above signaling molecules, such as... Figure 5 As shown, the normal regulatory function of the signal pathway has been restored.

[0071] The above results indicate that dapagliflozin improves renal sodium metabolism by upregulating the expression of the Na / K-ATPase α1 subunit and inhibiting the abnormal and persistent coupling of the Na / K-ATPase / c-Src / ERK / STAT3 signaling pathway, which provides a new mechanistic basis for its application in the treatment of obesity-related renal sodium metabolism disorders.

[0072] Experimental validation of dapagliflozin's improvement of obesity-related oxidative stress and inflammatory response; This invention demonstrates, through a systematic evaluation of renal oxidative stress and inflammatory markers, that dapagliflozin can effectively improve the pathological microenvironment associated with obesity.

[0073] 3.1 Effect on improving oxidative stress; The experimental results showed that, compared with the normal control group (LH), the level of protein carbonylation in the kidney tissue of the obese model group (HH) mice was significantly increased by approximately 1.48 times. Figure 6 A, Figure 6 B), the malondialdehyde (MDA) content increased by approximately 1.41 times ( Figure 6 C), superoxide dismutase (SOD) activity decreased by approximately 10.8% (C). Figure 6 (D) indicates that renal oxidative stress levels are significantly increased in obese individuals. After dapagliflozin intervention, the renal protein carbonylation level in the treatment group (HHD) was reduced by approximately 19.1% compared to the model group. Figure 6 A, Figure 6 B), MDA content decreased by approximately 37.5% ( Figure 6 C), SOD activity recovered by 11.2% ( Figure 6 D).

[0074] 3.2 Regulatory role in inflammatory response; Regarding inflammatory markers, the expression level of IL-6 protein in the kidney tissue of the obesity model group was significantly increased by approximately 2.08 times. Figure 7 A, Figure 7 B), plasma IL-6 levels increased approximately 2.25 times ( Figure 7 C). Immunohistochemical analysis showed that IL-6 in the kidneys of HH group mice exhibited a deep brownish-yellow cytoplasmic staining (C). Figure 7D), the area of ​​IL-17 positive expression is expanded, especially in areas of interstitial inflammation. Figure 7 D). Following dapagliflozin treatment, renal IL-6 protein expression decreased by approximately 34.2% ( Figure 7 A, Figure 7 B), plasma IL-6 levels decreased by approximately 31.4% ( Figure 7 C), the IL-6 positive area decreased by approximately 20.9% ( Figure 7 E), the IL-17 positive area decreased by approximately 24.5% ( Figure 7 F).

[0075] The above results indicate that dapagliflozin significantly improves the renal oxidative stress-inflammatory microenvironment by simultaneously reducing protein oxidative damage, inhibiting lipid peroxidation, enhancing antioxidant enzyme activity, and effectively inhibiting the overexpression of inflammatory factors such as IL-6 and IL-17. This provides important experimental evidence for its application in the treatment of obesity-related renal sodium metabolism disorders.

[0076] Dapagliflozin improves IL-6-induced oxidative stress in renal tubular epithelial cells; This invention further validates the ameliorative effect of dapagliflozin on oxidative stress at the cellular level by establishing an in vitro inflammatory injury model.

[0077] 4.1 Cell model establishment and experimental design; An in vitro inflammatory injury model was constructed using porcine renal tubular epithelial cells (LLC-PK1) and human renal tubular epithelial cells (HK-2) with IL-6 (10 ng / mL) as an inflammatory stimulant to simulate the obesity-related microinflammatory environment. The experiment included a control group, an IL-6 stimulation group, an IL-6 + dapagliflozin pretreatment group (0.5 μM, 24 h), and a dapagliflozin pretreatment group alone (0.5 μM, 24 h).

[0078] 4.2 Improvement effect on protein oxidative damage; DNPH carbonyl derivatization analysis showed that IL-6 stimulation significantly increased the carbonylation level of proteins in LLC-PK1 cells by approximately 1.68-fold, while dapagliflozin pretreatment reduced it by approximately 21.8%. Figure 8 A, Figure 8 B); IL-6 stimulation significantly increased the carbonylation level of proteins in HK-2 cells by approximately 1.67-fold, while dapagliflozin pretreatment reduced it by approximately 20.5% ( Figure 8 A, Figure 8 (C) indicates that the drug can effectively reduce protein damage mediated by oxidative stress.

[0079] 4.3 Recovery of antioxidant system function; Biochemical tests showed that IL-6 stimulation increased MDA content in LLC-PK1 cells by approximately 1.73 times. Figure 8 D), SOD activity decreased by approximately 18.9% ( Figure 8 F), dapagliflozin pretreatment reduced MDA content by approximately 28.1% ( Figure 8 D), SOD activity recovered to 92.7% of normal levels ( Figure 8 F); IL-6 stimulation increased MDA content in HK-2 cells by approximately 1.19 times (F). Figure 8 E), SOD activity decreased by approximately 19.1% ( Figure 8 G), dapagliflozin pretreatment reduced MDA content by approximately 27.3% ( Figure 8 E), SOD activity recovered to 93.1% of normal levels ( Figure 8 G).

[0080] 4.4 Analysis of drug action characteristics; It is worth noting that dapagliflozin alone had no significant effect on basal SOD activity and MDA levels, indicating that its antioxidant protective effect is clearly pathologically dependent. This characteristic helps to reduce potential non-targeted side effects in clinical applications.

[0081] The above cell experiments show that dapagliflozin can effectively alleviate IL-6-induced protein oxidative damage and lipid peroxidation, enhance cellular antioxidant defense capabilities, and its protective effect is pathologically selective, providing cellular experimental evidence for its application in the treatment of obesity-related renal sodium metabolism disorders.

[0082] 5. Dapagliflozin regulates the Na / K-ATPase / Src / ERK / STAT3 signaling cascade; This invention reveals the regulatory role of dapagliflozin in the Na / K-ATPase / c-Src signaling pathway through cell experiments, elucidating its molecular mechanism for improving renal sodium metabolism disorders.

[0083] 5.1 Verification of signal pathway interactions; After IL-6 stimulation of LLC-PK1 cells, the Na / K-ATPase / c-Src signaling pathway was significantly activated, as evidenced by an approximately 1.58-fold increase in Src phosphorylation, an approximately 2.18-fold increase in ERK1 / 2 phosphorylation, and an approximately 2.78-fold increase in STAT3 phosphorylation. Figure 9 A, Figure 9 B). After intervention with the Src kinase-specific inhibitor PP2 (10 μM, pretreatment for 2 h), not only was Src autophosphorylation inhibited by approximately 35.1%, but IL-6-induced ERK1 / 2 and STAT3 phosphorylation were also inhibited by approximately 40.6% and 40.7%, respectively. Figure 9 A, Figure 9 B); After IL-6 stimulation of HK-2 cells, Src phosphorylation level increased by approximately 1.67-fold, ERK1 / 2 phosphorylation level increased by approximately 2.53-fold, and STAT3 phosphorylation level increased by approximately 1.91-fold. Figure 9 A, Figure 9 C). After intervention with the Src kinase-specific inhibitor PP2, Src autophosphorylation was inhibited by approximately 47.6%, and IL-6-induced ERK1 / 2 and STAT3 phosphorylation were also inhibited by approximately 20.3% and 25.1%, respectively. Figure 9 A, Figure 9 C); confirms that this signal transduction depends on Src kinase.

[0084] 5.2 The signal modulation effect of dapagliflozin; Dapagliflozin intervention significantly inhibited IL-6-induced phosphorylation of Src, ERK1 / 2, and STAT3, with similar effects observed in both LLC-PK1 and HK-2 cell lines. Specifically, dapagliflozin reduced IL-6-induced Src phosphorylation by approximately 19.0%, ERK1 / 2 phosphorylation by approximately 28.0%, and STAT3 phosphorylation by approximately 28.3% in LLC-PK1 cells. Figure 9 A, Figure 9 B); reduced IL-6-induced Src phosphorylation by approximately 38.4%, ERK1 / 2 phosphorylation by approximately 21.8%, and STAT3 phosphorylation by approximately 25.3% in HK-2 cells. Figure 9 A, Figure 9 C).

[0085] 5.3 Analysis of the characteristics of the mechanism of action; Notably, dapagliflozin's inhibitory effect on the activation of the aforementioned signaling molecules is similar to that of PP2 treatment, suggesting that its mechanism of action involves regulating downstream signaling cascades by intervening in the activation state of Src kinase. This finding indicates that Na / K-ATPase is not only an ion transporter but also a key molecular platform integrating metabolic and inflammatory signaling.

[0086] The above results demonstrate that dapagliflozin disrupts the positive feedback amplification loop of inflammatory signals by inhibiting the overactivation of the Na / K-ATPase / c-Src / ERK / STAT3 signaling cascade, providing an important molecular mechanism basis for its application in the treatment of obesity-related renal sodium metabolism disorders.

[0087] In summary, this invention, through a series of in vitro and in vivo experiments, demonstrates for the first time that dapagliflozin, in addition to the traditional SGLT2 inhibition mechanism, reveals a novel mechanism by which it improves obesity-related renal sodium metabolism disorders by regulating the Na / K-ATPase / c-Src signaling axis. Specifically, this invention confirms that dapagliflozin exerts its therapeutic effects synergistically through the following multiple pathways: (1) upregulating the expression of the renal Na / K-ATPase α1 subunit and improving its membrane localization; (2) inhibiting the abnormal activation of the Na / K-ATPase / c-Src / ERK / STAT3 signaling cascade; (3) alleviating protein carbonylation and lipid peroxidation damage and enhancing antioxidant enzyme activity; and (4) reducing the expression of inflammatory factors such as IL-17. These effects collectively promote the recovery of renal sodium metabolism function.

[0088] Compared with existing technologies, this invention innovatively proposes the "signal pathway reset" theory, redefining Na / K-ATPase as a metabolic-inflammatory signaling integration platform, and demonstrating that dapagliflozin can act as a "signal pathway regulator" to restore it from a pathologically activated state to physiological homeostasis. Experiments have shown that this effect is pathology-dependent, does not affect basic signaling pathways under normal conditions, and has good safety characteristics.

[0089] This invention provides a new pharmaceutical use for dapagliflozin, offering sufficient experimental evidence for its application in the prevention and treatment of obesity-related renal sodium metabolism disorders and salt-sensitive hypertension, and has significant clinical application value and development prospects.

[0090] Finally, it should be noted that the above-described embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The use of dapagliflozin in the preparation of a medicament for the prevention and / or treatment of obesity-related salt-sensitive hypertension, characterized in that, Obesity-related sensitive hypertension refers to a subtype of hypertension driven by impaired renal sodium metabolism regulation in obese individuals, where blood pressure levels are highly sensitive to dietary sodium intake.

2. The use of dapagliflozin according to claim 1 in the preparation of a medicament for the prevention and / or treatment of obesity-related salt-sensitive hypertension, characterized in that, The intended use is achieved by directly regulating key molecular nodes involved in sodium homeostasis regulation in the proximal convoluted tubule epithelial cells of the kidney, wherein the key molecular nodes include at least the Na / K-ATPase α1 subunit and its mediated signal transduction function.

3. The use of dapagliflozin according to claim 2 in the preparation of a medicament for the prevention and / or treatment of obesity-related salt-sensitive hypertension, characterized in that, The application aims to restore renal sodium metabolism homeostasis and improve salt-sensitive hypertension by inhibiting the abnormal and persistent coupling between the Na / K-ATPase α1 subunit and c-Src kinase in obese individuals, thereby restoring the Na / K-ATPase / Src signaling axis from a pathologically activated state to a physiologically regulated state.

4. The use of dapagliflozin according to claim 1 in the preparation of a medicament for the prevention and / or treatment of obesity-related salt-sensitive hypertension, characterized in that, The uses also include selectively alleviating renal oxidative stress in obesity-related pathological states, wherein the reduction of oxidative stress is manifested by at least one of reducing protein carbonylation levels, reducing malondialdehyde content, and / or increasing superoxide dismutase activity.

5. The use of dapagliflozin according to any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of obesity-related salt-sensitive hypertension, characterized in that, The use also includes suppressing renal inflammatory responses, which is manifested by reducing the expression level of IL-17.

6. The use of dapagliflozin according to any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of obesity-related salt-sensitive hypertension, characterized in that, The prevention and / or treatment of obesity-related salt-sensitive hypertension includes improving glomerular filtration function and / or promoting urinary sodium excretion.

7. A pharmaceutical composition for the prevention and / or treatment of obesity-related salt-sensitive hypertension, characterized in that, The composition comprises a therapeutically effective amount of dapagliflozin and a pharmaceutically acceptable carrier.

8. Use of dapagliflozin in the preparation of a medicament for restoring the regulatory balance of the renal Na / K-ATPase / c-Src signaling axis in obese individuals.

9. The use of dapagliflozin according to claim 8 in the preparation of a medicament for restoring the renal Na / K-ATPase / c-Src signaling axis regulatory balance in obese individuals, characterized in that... The reconstruction includes correcting the aberrant signal amplification mediated by Na / K-ATPase α1 and inhibiting the sustained activation of the ERK / STAT3 inflammatory cascade.

10. The use of dapagliflozin according to claim 8 in the preparation of a medicament for restoring the renal Na / K-ATPase / c-Src signaling axis regulatory balance in obese individuals, characterized in that... The drug is used to prevent or treat renal sodium metabolism disorders or salt-sensitive hypertension.

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