Carbonic anhydrase 9 as a target for the treatment of diabetic pancreatic beta cell injury and its application

Abstract

The application discloses a new use of carbonic anhydrase 9 (CA9) as a target point for damage of pancreatic beta cells in diabetes, and belongs to the technical field of biological medicine. The application first discloses a key role of CA9 in the pathogenesis of diabetes: in serum and pancreatic tissue of type 2 diabetes patients and model mice, CA9 is significantly increased; cell experiments prove that knocking down CA9 expression can reverse the inhibition of high glucose environment on the viability and insulin secretion function of pancreatic beta cells (RIN-M5F cells). Based on this, the application proposes an application of CA9 as a new diabetes treatment target and diagnostic marker. Specifically, the application includes the following: application of a CA9 inhibitor in preparation of a diabetes treatment drug, a pharmaceutical composition containing the inhibitor, a drug screening method taking CA9 as a target point, and application of detection of CA9 level in diabetes diagnosis. The application provides a brand new strategy and tool for diagnosis and treatment of diabetes.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the novel function of carbonic anhydrase 9 (CA9) in diabetic pancreatic β-cell damage, and reagents, pharmaceutical compositions and methods for the diagnosis, prevention and treatment of diabetes and related complications using CA9 as a target. Background Technology

[0002] Diabetes mellitus is a global metabolic disease characterized by hyperglycemia, one of the core pathological mechanisms of which is the dysfunction and reduction in the number of pancreatic β cells. Numerous factors contribute to β cell damage, including glucotoxicity, lipotoxicity, oxidative stress, and endoplasmic reticulum stress. However, the specific molecular mechanisms by which β cells sense and respond to changes in the microenvironment, particularly acid-base imbalances, under the pathological conditions of diabetes remain poorly understood.

[0003] CA9 is a transmembrane isoenzyme traditionally considered a key regulator of the hypoxic microenvironment of solid tumors. It promotes tumor cell invasion and survival by catalyzing carbon dioxide hydration, acidifying the extracellular environment, and thus facilitating tumor cell invasion. However, the expression and function of CA9 in normal metabolic tissues, particularly in pancreatic β-cells, have never been reported or revealed in the prior art. There is no evidence in the prior art to suggest that CA9 is associated with the pathogenesis of diabetes, especially with direct damage to pancreatic β-cells.

[0004] Therefore, there is an urgent need in this field to elucidate new mechanisms of pancreatic β-cell damage in diabetes and to develop new diagnostic biomarkers and treatment strategies based on this. Summary of the Invention

[0005] This invention reveals for the first time the core role of CA9 in pancreatic β-cell damage in diabetic patients. Studies found that CA9 levels were significantly elevated in the serum of patients with type 2 diabetes mellitus (T2DM); its protein expression was upregulated in the pancreatic tissue of diabetic mice, with a simultaneous increase in serum levels. Further cell experiments showed that under high glucose conditions, the viability of the rat pancreatic β-cell line RIN-M5F was inhibited, and knockdown of CA9 expression significantly reversed the inhibition of cell viability caused by high glucose, while CA9 overexpression exacerbated this inhibitory effect. This discovery completely overturns the traditional understanding that CA9 only plays a role in tumors, providing a theoretical basis for its novel applications in the field of diabetes.

[0006] Based on the above findings, one of the objectives of this invention is to provide a reagent for detecting CA9 expression levels in the preparation of formulations for the diagnosis or prognostic assessment of diabetes and its complications.

[0007] A second objective of this invention is to provide a method for screening potential drugs that protect pancreatic β cells, the method comprising measuring the effect of a candidate substance on CA9 expression or activity in the presence of the candidate substance.

[0008] A third objective of this invention is to provide a pharmaceutical composition for the prevention or treatment of diabetes and its complications, the pharmaceutical composition comprising a CA9 activity inhibitor.

[0009] Preferably, the CA9 inhibitor is selected from small molecule inhibitors, antibodies, antibody-drug conjugates, antisense oligonucleotides, siRNA, shRNA, or the CRISPR-Cas9 gene editing system.

[0010] Preferably, the diabetes includes type 1 diabetes and type 2 diabetes.

[0011] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or other antidiabetic drugs, such as metformin, insulin, GLP-1 receptor agonists, or SGLT2 inhibitors. Beneficial effects of the present invention

[0012] First time revealing: This invention reveals for the first time the core role of CA9 in pancreatic β-cell damage in diabetes, opening up a new direction for research on the pathogenesis of diabetes.

[0013] Novel target: CA9 is a novel therapeutic target for diabetes, and drugs developed targeting it may slow the progression of diabetes by directly protecting β cells.

[0014] Diagnostic value: Detecting CA9 levels in blood or tissues may serve as a novel biomarker for the early diagnosis of diabetes or for assessing the severity of β-cell damage.

[0015] Synergistic therapy: When CA9 inhibitors are used in combination with existing hypoglycemic drugs, they can produce a synergistic effect, directly protecting β cells while lowering blood sugar, thus combining "treating the symptoms" with "treating the root cause". Attached Figure Description

[0016] Figure 1 Comparison of serum CA9 levels between patients with type 2 diabetes mellitus (T2DM) and healthy volunteers (mean ± standard deviation) n =45). ***: P <0.001, compared with healthy volunteers.

[0017] Figure 2 Scatter plot showing the correlation between serum CA9 levels and fasting insulin (FINS), insulin resistance index (HOMA-IR), glycated hemoglobin (HbA1c), and disease duration in patients with type 2 diabetes mellitus (T2DM).

[0018] Figure 3 Changes in body weight (A), fasting blood glucose (FBG) (B), and serum insulin (C) in diabetic model mice (mean ± standard deviation). n =8). "Normal" means "normal control group", "DM" means "diabetic model group", and "STZ 5d" means "streptozotocin 40 mg / kg / d intraperitoneally for 5 consecutive days". *: P <0.05, compared with the normal group.

[0019] Figure 4 Pathological changes in pancreatic tissue of diabetic model mice (HE, 200×). "Normal" indicates "normal control group" and "DM" indicates "diabetic model group".

[0020] Figure 5 Western blot results of CA9 protein expression in pancreatic tissue of diabetic model mice (mean ± standard deviation). n =3). "NC" represents "normal control group" and "DM" represents "diabetes model group". **: P <0.01, compared with NC.

[0021] Figure 6 ELISA results of serum CA9 levels in diabetic model mice (mean ± standard deviation). n =8). "NC" represents "normal control group" and "DM" represents "diabetes model group". **: P <0.01, compared with NC.

[0022] Figure 7 The relative expression of CA9 mRNA in RIN-M5F cells after transfection with CA9 silence (CA9-sh2) and overexpression (CA9-OE). P <0.01, ***: P <0.001, ns: no statistical difference.

[0023] Figure 8 Effects of CA9 silencing and overexpression on the proliferation of RIN-M5F cells damaged by high glucose (HG, glucose 30 mmol / L) (mean ± standard deviation, n =6). ***: P <0.001, ns: no statistical difference.

[0024] Figure 9 Effects of CA9 silencing and overexpression on glucose-stimulated insulin secretion (GSIS) function in RIN-M5F cells under high glucose conditions. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below through specific embodiments, but the scope of protection of this invention is not limited thereto. Example 1: Serum CA9 levels in patients with type 2 diabetes mellitus (T2DM) and its correlation with glucose metabolism indicators

[0026] Forty-five patients with type 2 diabetes mellitus (T2DM) who visited the hospital between May and July 2024 (T2DM group) and 45 healthy individuals who underwent physical examinations during the same period (healthy control group) were selected. Clinical data and glucose metabolism indicators were collected, and serum CA9 levels were measured using ELISA. Statistical analysis was performed using SPSS 27.0. Intergroup comparisons were performed using t-tests or Mann-Whitney U tests, and correlation analysis was performed using Pearson or Spearman correlation analysis.

[0027] The results showed that serum CA9 levels in the T2DM group were significantly higher than those in the healthy control group. P <0.01, see Figure 1 Correlation analysis showed that serum CA9 levels were significantly positively correlated with fasting insulin (FINS), insulin resistance index (HOMA-IR), glycated hemoglobin (HbA1c), and disease duration. P <0.05), but not significantly correlated with indicators such as fasting blood glucose (FBG). P >0.05), see Table 1 for details and Figure 2 .

[0028] Table 1. Correlation between serum CA9 levels and various clinical indicators in patients with type 2 diabetes mellitus (T2DM) n = 45)

[0029] Conclusion: Serum CA9 levels were significantly elevated in patients with type 2 diabetes mellitus (T2DM), and were positively correlated with the severity of insulin resistance, poor long-term glycemic control, and prolonged disease duration. This suggests that CA9 may be involved in the development and progression of diabetes. Example 2: Changes in CA9 expression in serum and pancreatic tissue of diabetic model mice

[0030] A diabetes mellitus (DM) model was established in mice by inducing a high-fat diet combined with multiple administrations of low-dose streptozotocin (STZ, 40 mg / kg / day, ip) for 5 consecutive days. Compared with the normal control group (NC), the DM group mice showed stagnant and significantly reduced body weight gain after model establishment, reaching only 79.8% of the normal control group at the end of the experiment. P <0.05, Figure 3 A); Fasting blood glucose (FBG) consistently higher than 11.1 mmol / L ( P <0.05, Figure 3B). Four weeks after the onset of diabetes, insulin levels in DM model mice decreased significantly ( P <0.05, Figure 3 C). Histopathological examination of the pancreas tissue showed that the pancreas of mice in the DM model group was structurally disordered, with widened interstitial spaces, edema and fatty degeneration of acinar cells, and perivascular erythrocyte exudation and inflammatory cell infiltration; the islets were enlarged, the tissue structure was loose, and some areas showed "cavitation-like" changes. Figure 4 ).

[0031] Western blot (WB) and ELISA assays revealed that CA9 protein expression in pancreatic tissue and serum CA9 levels were significantly increased in the DM group compared to the NC group. P <0.01, Figure 5 , Figure 6 ).

[0032] Conclusion: The levels of CA9 in the pancreatic tissue and serum of diabetic model mice were significantly elevated, which is consistent with the observations in patients with type 2 diabetes mellitus (T2DM). Example 3: Effects of CA9 expression regulation on pancreatic β cells (RIN-M5F) in rats with high glucose injury.

[0033] RIN-M5F cell lines with stable CA9 silence (CA9-sh) and CA9 overexpression (CA9-OE) were constructed using lentiviral transfection technology. qRT-PCR verification confirmed successful transfection. Figure 7 ).

[0034] Effect on cell viability: After culturing in a high glucose (HG, glucose 30 mmol / L) environment for 48 h, cell viability was detected using the CCK-8 assay. The results showed that, compared with the high glucose control group, CA9 silencing significantly improved cell viability. P <0.001), while CA9 overexpression had no significant effect ( P >0.05, Figure 8 ).

[0035] Effects on insulin secretion: After culturing in a high-glucose environment for 48 h, basal and glucose-stimulated insulin secretion (GSIS) was detected by ELISA. The results showed that the high-glucose environment significantly inhibited insulin secretion. P <0.001), knocking down CA9 expression significantly reversed the inhibition of insulin secretion by high glucose ( P <0.001); while overexpression of CA9 further exacerbated insulin secretion defects caused by high glucose ( P <0.001, Figure 9 ).

[0036] Conclusion: Silencing CA9 expression can ameliorate the damage to pancreatic β-cell proliferation and insulin secretion caused by high glucose, while overexpression of CA9 exacerbates this damage. This further confirms that CA9 plays a crucial role in high glucose-induced β-cell injury.

Claims

1. The use of a carbonic anhydrase 9 (CA9) inhibitor in the preparation of a medicament for the prevention or treatment of diabetes, wherein the diabetes is accompanied by pancreatic β-cell damage.

2. The application according to claim 1, characterized in that, The diabetes referred to is either type 1 diabetes or type 2 diabetes.

3. The application according to claim 1, characterized in that, The CA9 inhibitor is selected from small molecule compounds, anti-CA9 antibodies, antibody-drug conjugates, antisense oligonucleotides, siRNA, shRNA, or gene editing tools based on the CRISPR-Cas9 system.

4. The application according to any one of claims 1-3, characterized in that, The drug also contains one or more additional anti-diabetic active ingredients.

5. The application according to claim 4, characterized in that, The additional antidiabetic active ingredient is selected from metformin, insulin, GLP-1 receptor agonists, or SGLT2 inhibitors.

6. A pharmaceutical composition for the prevention or treatment of diabetes, characterized in that, It contains a therapeutically effective amount of a CA9 inhibitor and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, It also contains one or more additional anti-diabetic active ingredients.

8. A method for screening potential drugs to protect pancreatic β cells or treat diabetes, characterized in that, The method includes the following steps: in the presence of a candidate substance, determining its effect on the expression or biological activity of CA9; wherein a candidate substance capable of inhibiting the expression or activity of CA9 is identified as a potential drug.

9. The use of a reagent for detecting CA9 expression levels in the preparation of formulations for diagnosing diabetes or assessing pancreatic β-cell damage.

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