Application of GPR55 antagonist CID16020046 in treatment of diabetic nephropathy

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of a GPR55 antagonist CID16020046 in treatment of diabetic nephropathy. In order to clinically select small-molecule targeted drugs for intervention and treatment of diabetic nephropathy, the invention discloses the treatment effect of the GPR55 antagonist CID16020046 on diabetic nephropathy, and the GPR55 antagonist CID16020046 can improve renal functions, relieve histological changes of kidneys, resist renal fibrosis and play a role in protecting renal tubule injuries caused by diabetic nephropathy.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to application of a GPR55 antagonist CID16020046 in the treatment of diabetic nephropathy. Background Art

[0002] Diabetic nephropathy (DN) is one of the most common chronic microvascular complications of diabetes. About 40% of diabetic patients worldwide have DN. DN is the main cause of chronic kidney disease and end-stage renal disease. The typical manifestation of DN is excessive deposition of extracellular matrix in the glomeruli and tubules, causing glomerular sclerosis and renal interstitial fibrosis. In the late stage of the disease, microscopic thickening of the basement membrane, mesangial hyperplasia, diffuse thickening of the capillary basement membrane, and the formation of nodular sclerosis (KW nodules) can be seen. At present, the first-line treatment for diabetic nephropathy is mainly to control blood sugar, blood lipids and blood pressure. Oral hypoglycemic drugs combined with RAAS antagonists such as angiotensin-converting enzyme antagonists (ACEI) or angiotensin receptor blockers (ARB) are often used in clinical treatment of diabetic nephropathy, but these treatments are still insufficient to improve renal function. Therefore, it is necessary to deeply explore and summarize the pathogenic factors that lead to the occurrence and development of diabetic nephropathy, and provide new ideas for the clinical selection of small molecule targeted drugs for intervention and treatment.

[0003] Small molecule drugs are chemically synthesized active substances, usually with a relative molecular mass of less than 1000. The essence of their function is the interaction between drugs and target proteins. Most small molecule drugs can penetrate the cell membrane and target intracellular proteins, and have oral bioavailability. Many of these advantages make small molecule drugs irreplaceable in treatment. CID16020046 is an effective and selective GPR55 antagonist that inhibits the constitutive activity of GPR55. CID16020046 has obvious protective properties against atherosclerosis, such as anti-inflammatory, antioxidant and inhibition of monocyte attachment to endothelial cells. In addition, CID16020046 is also of great significance in the treatment of neurological disorders, inflammation and malignant tumors. However, the GPR55 antagonist CID16020046 has not been used in DN. Summary of the invention

[0004] In view of the above problems, the present invention provides the use of GPR55 antagonist CID16020046 in the treatment of diabetic nephropathy.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides application of a GPR55 antagonist CID16020046 in preparing a drug for treating diabetic nephropathy.

[0007] Furthermore, the GPR55 antagonist CID16020046 can improve renal function, alleviate renal histological changes, reduce renal fibrosis and protect against renal tubular damage.

[0008] The present invention also provides a medicine for treating diabetic nephropathy, which uses the GPR55 antagonist CID16020046 as an active ingredient.

[0009] Furthermore, a pharmaceutically acceptable carrier is also included.

[0010] Furthermore, the drug is in a pharmaceutically acceptable dosage form.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] The present invention uses a series of experiments to prove the therapeutic effect of GPR55 antagonist CID16020046 on DN, provides a new target for the treatment of DN, provides new ideas for scientific research and clinical practice, emphasizes precision medicine, and delays the progression of DN.

[0013] The GPR55 antagonist CID16020046 can resist renal fibrosis and protect renal tubular damage caused by diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The expression level of GPR55 in the kidney tissue of DN mice, where (A) is the immunohistochemical staining of GPR55, scale bar = 50 μm; (B) is the semi-quantitative analysis of GPR55 expression, data are expressed as mean ± standard deviation (n = 10), ***p < 0.001, compared with the NC group.

[0015] Figure 2 In order to investigate whether the GPR55 antagonist CID16020046 could significantly improve the renal function of DN, the mice were divided into three groups (n=10 in each group): control group (NC), diabetic nephropathy group (DN) and diabetic nephropathy administration group (DN+CID), where (A) is the weekly change of fasting blood glucose, B is the renal index (kidney weight / body weight), (CE) is the levels of serum creatinine, urea nitrogen and urine microalbumin / urine creatinine, data are expressed as mean ± SD (n=10), *P<0.05, **p<0.01 and ***p<0.001, compared with the NC group.

[0016] Figure 3 GPR55 antagonist CID16020046 can alleviate the renal histopathological changes in DN, including representative micrographs of (A) HE, (B) PAS, (C) Masson staining, and (D) Sirius red staining, scale bar = 50 μm.

[0017] Figure 4 GPR55 antagonist CID16020046 can reduce renal fibrosis in DN, where (A) is a representative western blot of E-cadherin, Vimentin, α-SMA and N-cadherin protein expression, (B) is a quantitative analysis of E-cadherin, Vimentin, α-SMA and N-cadherin protein expression, data are expressed as mean ± standard deviation (n = 10), *P < 0.05, **p < 0.01 and ***p < 0.001, compared with the NC group.

[0018] Figure 5 GPR55 antagonist CID16020046 has a protective effect on renal tubular injury in DN, where (A) is the immunohistochemical staining of KIM-1 and NGAL, scale = 50 μm, (B) is the semi-quantitative analysis of KIM-1 and NGAL expression, data are expressed as mean ± SD (n = 10), *P < 0.05, **p < 0.01 and ***p < 0.001, compared with the NC group. DETAILED DESCRIPTION

[0019] In order to further illustrate the technical solution of the present invention, the present invention is further described below through embodiments.

[0020] Example 1

[0021] The invention discloses an application of a GPR55 antagonist CID16020046 in the preparation of a drug for treating diabetic nephropathy. The GPR55 antagonist CID16020046 can improve renal function, alleviate histological changes in the kidney, reduce renal fibrosis and has a protective effect on renal tubular damage.

[0022] A drug for treating diabetic nephropathy, comprising a GPR55 antagonist CID16020046 as an active ingredient and a pharmaceutically acceptable carrier. The drug is in a pharmaceutically acceptable dosage form.

[0023] Example 2

[0024] (1) Test methods and steps:

[0025] ① Experimental animals and grouping: 20 SPF 8-week-old male C57BLKS / db (db / db) mice and 10 C57BLKS / db (wt / wt) mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The db / db mice were randomly divided into two groups: DN group (n=10) and DN+GPR55 antagonist CID16020046 (n=10), and wild mice from the same litter were used as normal control group (n=10). Mice in the DN+GPR55 antagonist CID16020046 group were intraperitoneally injected with GPR55 antagonist CID16020046 (20 mg / kg / d), and mice in the normal control group and DN group were given normal saline for 4 consecutive weeks.

[0026] ② Kidney weight / body weight determination and biochemical index detection: Blood was collected from the tail vein of each mouse every week to measure fasting blood sugar. Urine was collected in a metabolic cage 24 hours before the mice were killed. The urine microalbumin and urine creatinine were detected according to the instructions of the kit, and the urine protein excretion rate was calculated; the body weight and bilateral kidney weight of each mouse were weighed to calculate the kidney weight / body weight; serum samples were taken and the Scr and BUN levels were detected according to the instructions of the kit.

[0027] ③ Histological staining: After the mice were killed, the renal tissue was isolated, fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick renal tissue sections. The sections were stained with hematoxylin-eosin (HE), periodic acid-Schiff (PAS), Masson, and Sirius red. Twenty fields of view were randomly selected to observe renal pathological changes and take images of renal tissue.

[0028] ④ Protein extraction and Western blotting: 15 mg of mouse kidney was taken and 200 μL of RIPA lysis buffer was added to extract the total protein of mouse kidney. Equal amounts of protein were separated by SDS-PAGE, wet-transferred to the membrane, blocked with 5% skim milk for 1.5 h, incubated with E-cadherin (1:1000), Vimentin (1:3000), α-SMA (1:5000), N-cadherin (1:4000) and β-actin (1:10000) antibodies, incubated at 4°C overnight, washed with TBST (3 times, 10 min each time), incubated with secondary antibody at room temperature for 1 h, washed with TBST (3 times, 5 min each time), and developed with electrochemiluminescent solution. Finally, the automatic gel imaging system was used to take pictures and gray value analysis was performed with Image J software.

[0029] ⑤ Immunohistochemistry: Kidney tissue sections were subjected to immunohistochemical staining for GPR55, KIM-1, and NGAL. Twenty fields of view were randomly selected, and the average optical density was measured using Image-Pro Plus 6.0 software to detect the relative expression of the protein.

[0030] Statistical methods: GraphPad Prism version 9.0 was used for statistical analysis. Measurement data were expressed as mean ± standard deviation (X ± SD). If normality and variance homogeneity tests were met, the difference between the two groups was evaluated and tested by t test, and the comparison of more than two groups was evaluated and tested by one-way analysis of variance. If not met, unpaired nonparametric t test (Mann-Whitney) was used for evaluation; P < 0.05 was considered statistically significant.

[0031] (2) Results:

[0032] ①GPR55 expression is upregulated in kidney tissue of DN mice

[0033] The expression level of GPR55 in mouse renal tissue sections was detected by immunohistochemistry. The results showed that the staining intensity of GPR55 in the DN group was significantly higher than that in the NC group, and GPR55 was expressed in both glomeruli and renal tubules, but the difference was more significant in renal tubules. Figure 1 ) showed that compared with the NC group, the mean optical density (MOD) of GPR55 in the renal tissue of DN mice was significantly increased, and the difference was statistically significant (P<0.05).

[0034] ②GPR55 antagonist CID16020046 can significantly improve renal function in DN mice

[0035] like Figure 2 As shown in the results, there were no significant differences in physiological parameters such as fasting blood glucose and renal index (kidney weight / body weight) between DN mice and DN mice treated with CID16020046. Compared with the NC group, the levels of Scr, BUN, and urine microalbumin / urine creatinine in the DN group mice were significantly increased (P<0.05). Compared with the DN group, after intervention with the GPR55 antagonist CID16020046, Scr, BUN, and urine protein excretion rate were all reduced, and the differences were statistically significant (P<0.05). These data indicate that the GPR55 antagonist CID16020046 can significantly improve the renal function of DN mice.

[0036] ③ Treatment with GPR55 antagonist CID16020046 can alleviate the histological changes in the kidneys of DN mice

[0037] Depend on Figure 3It can be seen that HE staining showed glomerular capsule occlusion, glomerular sclerosis, tubular damage, and abnormal tubular expansion in the DN model group. The positive reaction in PAS staining was purple-red, indicating that after the administration of the GPR55 antagonist CID16020046, the glycogen deposition in the renal interstitium of DN mice was significantly reduced. In addition, Masson staining and Sirius red staining showed that the GPR55 antagonist could improve the collagen deposition in the renal tissue of DN mice. The above results confirmed that the GPR55 antagonist CID16020046 could alleviate diabetic kidney damage in DN mice.

[0038] ④GPR55 antagonist CID16020046 can reduce renal fibrosis in DN mice

[0039] To explore the effect of GPR55 antagonist CID16020046 on renal fibrosis, the protein expression levels of epithelial-mesenchymal transition markers E-cadherin, Vimentin, α-SMA, and N-cadherin were detected by Western blot. Compared with the NC group, the expression of mesenchymal cell markers (Vimentin, α-SMA, and N-cadherin) in the DN group was significantly upregulated, and the expression of epithelial cell marker (E-cadherin) was significantly reduced, and the difference was statistically significant (P<0.05). After intervention with GPR55 antagonist CID16020046, the higher levels of Vimentin, α-SMA, and N-cadherin and the lower expression level of E-cadherin in DN mice were reversed. Figure 4 These results suggest that intervention with the GPR55 antagonist CID16020046 alleviated renal fibrosis in DN mice.

[0040] ⑤GPR55 antagonist CID16020046 has a protective effect on renal tubular damage in DN

[0041] Depend on Figure 5 It can be seen that compared with the NC group, the expression levels of KIM-1 and NGAL in the DN group were significantly upregulated (P < 0.05). The expression levels of KIM-1 and NGAL were significantly reduced after treatment with the GPR55 antagonist CID16020046 (P < 0.05). The above data show that the increased expression of KIM-1 and NGAL in DN indicates a direct damaging effect on the renal tubules, and this effect was reversed after intervention with the GPR55 antagonist CID16020046.

[0042] In summary, the present invention describes the therapeutic effect of the GPR55 antagonist CID16020046 on DN, which can improve renal function, reduce histological changes in the kidney, resist renal fibrosis, and protect against tubular damage caused by diabetic nephropathy.

[0043] The above shows and describes the main features and advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0044] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Application of GPR55 antagonist CID16020046 in the preparation of drugs for the treatment of diabetic nephropathy.

2. The use according to claim 1, characterized in that: The GPR55 antagonist CID16020046 can improve renal function, alleviate histological changes in the kidney, reduce renal fibrosis and has a protective effect on renal tubular damage.

3. A drug for treating diabetic nephropathy, characterized in that: The active ingredient is the GPR55 antagonist CID16020046.

4. A drug for treating diabetic nephropathy according to claim 3, characterized in that: Also included are pharmaceutically acceptable carriers.

5. A drug for treating diabetic nephropathy according to claim 3, characterized in that: The drug is in a pharmaceutically acceptable dosage form.

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