Application of growth hormone releasing hormone GHRH agonist in preparation of medicine for preventing and treating kidney diseases

Abstract

The invention relates to the technical field of treatment of diabetic nephropathy, in particular to application of a growth hormone releasing hormone GHRH agonist in preparation of a medicine for preventing and treating kidney diseases. The invention provides an application of a growth hormone releasing hormone GHRH agonist in preparation of a medicine for preventing and / or treating kidney diseases, verifies that the growth hormone releasing hormone GHRH agonist GHRH-6A can relieve diabetic nephropathy by relieving oxidative stress and inhibiting ferroptosis for the first time, and has important significance in prevention and treatment of diabetic nephropathy. And a new thought is provided for prevention and treatment of diabetic nephropathy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of diabetic nephropathy treatment, and particularly relates to application of a growth hormone releasing hormone GHRH agonist in preparation of a medicine for preventing and treating kidney diseases. BACKGROUND

[0002] Diabetic nephropathy (DN) is one of the most common complications of diabetes and is the main cause of end-stage renal disease (ESRD). DN is characterized by persistent proteinuria, and its typical morphological changes include glomerular and tubular basement membrane (GBM) thickening, tubular atrophy, renal inflammatory cell infiltration, glomerular sclerosis, and renal interstitial fibrosis. DN is often accompanied by renal vascular dysfunction, renal inflammation, increased oxidative stress, and renal fibrosis. Oxidative stress is believed to play a very important role in the pathogenesis of DN. At present, the treatment of DN in the clinic mainly controls blood glucose and some symptomatic treatment methods to delay the progression of the disease and prolong the survival time of patients, and there is still a lack of effective treatment drugs for DN.

[0003] Growth hormone releasing hormone (GHRH) is a neuroendocrine peptide composed of 44 amino acids, mainly secreted by the hypothalamus, and its receptor (GHRH-R) is widely present in pituitary, lung, eye, placenta and other tissues and organs. The natural GHRH has a very short half-life in the body. The inventors' previous work invented a 29-amino acid GHRH agonist (application number: 202411687455.5). It can promote the release of GH from pituitary cells by binding to GHRH-R, promote angiogenesis, treat cardiovascular diseases, and other effects, but it has not been reported that it can play a role in preventing and treating kidney diseases. Based on this, the present application is proposed. SUMMARY

[0004] The purpose of the present application is to provide the application of a growth hormone releasing hormone GHRH agonist in the preparation of a medicine for preventing and treating kidney diseases 。

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides the application of a growth hormone releasing hormone GHRH agonist shown in formula I in the preparation of a medicine for preventing and / or treating kidney diseases; Formula I.

[0006] The present application also provides the application of a growth hormone releasing hormone GHRH agonist shown in formula I in the preparation of a medicine for preventing and / or treating diabetic nephropathy. Formula I.

[0007] This invention also provides the application of the growth hormone-releasing hormone (GHRH) agonist shown in Formula I in promoting body movement; Formula I.

[0008] The present invention also provides the application of the growth hormone-releasing hormone (GHRH) agonist shown in Formula I in the preparation of a drug that promotes the expression of GHRH receptors in the kidney; Formula I.

[0009] The present invention also provides the application of the growth hormone-releasing hormone (GHRH) agonist shown in Formula I in the preparation of a reagent that reduces the expression level of NADPH oxidase subunits in the kidney; Formula I.

[0010] The present invention also provides the application of the growth hormone releasing hormone GHRH agonist shown in Formula I in promoting the expression of glutathione peroxidase 4 and ferritin heavy chain. Formula I.

[0011] The present invention also provides the use of the growth hormone-releasing hormone (GHRH) agonist of Formula I in the preparation of a medicament for the prevention and / or treatment of organ damage caused by diabetes; said organs include the heart, brain, lungs, blood vessels, or limbs; Formula I.

[0012] The present invention also provides a medicament for the prevention and / or treatment of diabetic nephropathy, comprising a growth hormone-releasing hormone (GHRH) agonist and a pharmaceutically acceptable carrier.

[0013] Preferably, the growth hormone-releasing hormone (GHRH) agonist is as shown in Formula I; Formula I.

[0014] Preferably, the dosage form of the drug includes tablets, capsules, pills, or injections.

[0015] The present invention has the following advantages: This invention is the first to verify that the growth hormone-releasing hormone (GHRH) agonist GHRH-6A, as described in application number 202411687455.5, can alleviate diabetic nephropathy by reducing oxidative stress and inhibiting ferroptosis. This is of great significance for the prevention and treatment of diabetic nephropathy and provides a new approach for its prevention and treatment. Attached Figure Description

[0016] Figure 1 Effects of the agonist GHRH-6A on the structure of the kidneys of DB / DB mice (A: H&E staining of kidney tissue sections of mice in each group; B: PAS staining of kidney tissue sections of mice in each group; C: semi-quantitative analysis of glomerular sclerosis in mice in each group); Figure 2 Effects of the agonist GHRH-6A on biochemical and metabolic indicators of DB / DB mice (A: fasting blood glucose levels of mice in each group; B: cholesterol content of mice in each group; C: triglyceride content of mice in each group; D: albumin content of mice in each group; E: kidney index of mice in each group); Figure 3 Effects of the agonist GHRH-6A on renal fibrosis in DB / DB mice (A: relative expression of transforming growth factor-β1 in the kidneys of mice in each group; B: relative expression of fibronectin in the kidneys of mice in each group); Figure 4 Effects of the agonist GHRH-6A on the exercise capacity of mice; Figure 5 Effects of the agonist GHRH-6A on the expression of growth hormone releasing hormone receptors in the kidneys of DB / DB mice; Figure 6 Effects of the agonist GHRH-6A on the expression level of reactive oxygen species in the kidneys of DB / DB mice; Figure 7 Effects of the agonist GHRH-6A on the level of ferroptosis in the kidneys of DB / DB mice (A: quantitative analysis of iron content in the kidneys of mice in each group; B: ferroptosis-related protein glutathione peroxidase level in the kidneys of mice in each group; C: relative expression content of ferritin heavy chain in the kidneys of mice in each group). DETAILED DESCRIPTION

[0017] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0018] The growth hormone releasing hormone GHRH agonist GHRH-6A in the embodiments of the present application is HD-6A described in the application number 202411687455.5, entitled “A long-chain modified GHRH agonist and its application”; the chemical structural formula is shown as formula I.

[0019] Formula I.

[0020] The growth hormone releasing hormone GHRH agonist GHRH-6A is prepared according to the construction method of HD-6A described in the application number 202411687455.5, entitled “A long-chain modified GHRH agonist and its application”. The amino acid sequence is: NMeTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-Nle-Asp-Arg-Ada-NH2; that is, N-methyl tyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-leucine-glutamine-aspartic acid-isoleucine-norleucine-aspartic acid-arginine-12-aminododecanoic acid.

[0021] The purity of the growth hormone releasing hormone GHRH agonist GHRH-6A is >95%, and the purity is determined by RP-HPLC. MS found value: 597.46 [M+6H] 6+ 716.72 [M+5H] 5+ 895.55 [M+4H] 4+ ; MS theoretical value: 3579.26.

[0022] The DB / DB mice in the embodiments of the present application are type II diabetes model mice, which are purchased from the Animal Model Research Center of Nanjing University. The SPF level 10-week-old wild type WT mice are purchased from the Animal Model Research Center of Nanjing University.

[0023] Example 1

[0024] The SPF level 10-week-old DB / DB mice and WT mice are adaptively fed for 2 weeks before treatment. A part of the DB / DB mice is selected as the treatment group, and the growth hormone releasing hormone GHRH agonist GHRH-6A is subcutaneously injected (dissolved in 10% propylene glycol solution containing 1 ‰ dimethyl sulfoxide) for administration, with a dose of 15 μg per mouse per day, for 8 weeks. The remaining DB / DB mice (model group) and WT mice (control group) are injected with an equal amount of 10% propylene glycol solution containing 1 ‰ dimethyl sulfoxide. During the treatment, the body weight of each mouse is measured every week, and the growth of the mice in each group is observed.

[0025] After the end of the experiment, the mice were sacrificed, the kidneys were removed, and the surrounding excess adipose tissue and outer capsule were removed. The right kidney was embedded, fixed, sectioned to prepare 3 μm thick paraffin sections, the sections were deparaffinized to water, and PAS and H&E staining was performed, and the morphological changes in the kidney tissue of the mice in each group were observed under an optical microscope. The results are shown in Figure 1 . .

[0026] PAS staining was used to evaluate glomerular injury, and deep purple in the glomerulus was considered to be glomerular sclerosis. ImageJ software was used to analyze the percentage of glomerular sclerosis area. The results are shown in Figure 1 . .

[0027] . Figure 1 It can be seen that the GHRH agonist can protect and treat the kidney function of the DB / DB diabetic mice. .

[0028] Example 2 .

[0029] After 8 weeks of treatment, the mice were sacrificed by 1% sodium pentobarbital (100 mg / kg), blood was taken, and after blood coagulation, the supernatant was taken after centrifugation at 1000 x g for 10 min, and the cholesterol (TC), triglyceride (TG), and urine albumin content (ACR) in the serum of the mice in each group were detected, and the kidney index of the mice was calculated, kidney index = (kidney weight / body weight) x 100%. The fasting blood glucose level of the mice in each group was determined. The results are shown in Figure 2 . .

[0030] . Figure 2 It can be seen that GHRH agonist treatment can significantly improve the kidney function of DB / DB mice. .

[0031] Example 3 .

[0032] Western blotting was used for immunoblotting protein detection. The mouse kidney was lysed with tissue cell lysis solution, the protein concentration was determined, and the protein was denatured at 100°C for 10 min. After loading, it was separated by SDS-PAGE and transferred to a PVDF membrane. 5% BSA was blocked at room temperature for 2 h, then 1st antibody [β-actin (1:3 000), TGFβ1 (1:200), FN (1:200)] was added, and incubated at room temperature for 2 h, and incubated overnight at 4°C. After washing the membrane, the corresponding horseradish peroxidase-labeled secondary antibody was added, and incubated at room temperature for 2 h. TBST was used to wash the membrane several times, ECL developing solution was incubated for 1 min, and the excess developing solution on the membrane was removed. The chemical luminescence gel imaging system was used to take a picture, and the gray value analysis was performed by Image J. The relative content of the target protein was represented by the ratio of the target protein to β-actin. The results are shown in Figure 3 . .

[0033] . Figure 3It is known that GHRH agonist can reduce the degree of kidney injury and fibrosis.

[0034] Example 4

[0035] After 8 weeks of treatment, the mice were tested for exercise capacity on a treadmill. The mice were placed on the treadmill and the time they spent on the treadmill was recorded.

[0036] The results are shown in Figure 4

[0037] Figure 4 It is known that GHRH agonist can improve the exercise capacity of mice.

[0038] Example 5

[0039] Western blot was used to detect the expression level of GHRHR protein in the kidney of mice in each group, and the specific steps are as in Example 3. The results are shown in Figure 5

[0040] Figure 5 It is known that the expression of GHRH receptor in the kidney of DB / DB mice is lower than that in WT wild-type mice, and GHRH agonist GHRH-6A can up-regulate the expression of GHRH receptor in kidney tissue.

[0041] Example 6

[0042] The relative fluorescence intensity of reactive oxygen species (ROS) in the kidney of DB / DB mice was obtained by dihydroethidium (DHE) staining, and then the relative fluorescence intensity was quantitatively analyzed to represent the relative amount of ROS. The effect of growth hormone releasing hormone GHRH agonist GHRH-6A on oxidative stress in the kidney of mice was detected. The results are shown in Figure 6

[0043] Figure 6 It is known that GHRH agonist can reduce the amount of ROS in the kidney of DB / DB mice, confirming that GHRH agonist can alleviate the oxidative stress state of the kidney.

[0044] Example 7

[0045] DB / DB mice with congenital leptin receptor gene deletion were used as a model of type 2 diabetes. Long-term hyperglycemia can cause kidney damage. WT mice were used as normal control group (WT group), and 10-week-old male mice were used. DB / DB mice were randomly divided into model group (DB / DB group) and GHRH-6A injection treatment group (DB / DB - ​​​GHRH group), GHRH-6A treatment group was subcutaneously administered GHRH-6A (15 μg per mouse) every other day, the control group and the model group were injected with the same amount of solvent control, and the treatment lasted for 8 weeks. During the treatment, the body weight of the mice was measured every week, and the growth of the mice was observed. After 8 weeks of treatment, all mice were sacrificed. The mouse tissue samples were collected, and urine, blood supernatant and kidney were taken. The wet weight of the bilateral kidneys was weighed, and then the outer capsule of the kidney was removed. The left kidney of the mouse was placed in a-80℃ refrigerator for storage, and was used for protein extraction for Western blot detection and iron content detection. The right kidney was placed in 4% paraformaldehyde for storage, and was made into paraffin sections for pathological detection. The paraffin sections were stained by DHE fluorescence to detect the amount of ROS. The results are shown in Figure 7

[0046] Figure 7 It is shown that the growth hormone releasing hormone GHRH agonist GHRH-6A can reduce the level of ferroptosis in the kidney of DB / DB mice.

[0047] From the above examples, it is verified for the first time that the growth hormone releasing hormone GHRH agonist GHRH-6A can alleviate diabetic nephropathy by reducing oxidative stress and inhibiting ferroptosis, which has important significance for the prevention and treatment of diabetic nephropathy.

[0048] The above is only the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. The use of the growth hormone-releasing hormone (GHRH) agonist shown in Formula I in the preparation of medicaments for the prevention and / or treatment of kidney diseases; Formula I.

2. The use of the growth hormone-releasing hormone (GHRH) agonist shown in Formula I in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy; Formula I.

3. The application of the growth hormone-releasing hormone (GHRH) agonist shown in Formula I in promoting body movement; Formula I.

4. Application of the growth hormone-releasing hormone (GHRH) agonist shown in Formula I in the preparation of a drug that promotes the expression of GHRH receptors in the kidney; Formula I.

5. Application of the growth hormone-releasing hormone (GHRH) agonist shown in Formula I in the preparation of reagents that reduce the expression level of NADPH oxidase subunits in the kidney; Formula I.

6. Application of the growth hormone-releasing hormone (GHRH) agonist shown in Formula I in promoting the expression of glutathione peroxidase 4 and ferritin heavy chain; Formula I.

7. The use of the growth hormone-releasing hormone (GHRH) agonist shown in Formula I in the preparation of a medicament for the prevention and / or treatment of organ damage caused by diabetes; said organs include the heart, brain, lungs, blood vessels, or limbs; Formula I.

8. A drug for the prevention and / or treatment of diabetic nephropathy, characterized in that, This includes growth hormone-releasing hormone (GHRH) agonists and pharmaceutically acceptable carriers.

9. The medicament according to claim 8, characterized in that, The growth hormone-releasing hormone (GHRH) agonist is shown in Formula I. Formula I.

10. The medicament according to claim 8, characterized in that, The dosage forms of the drug include tablets, capsules, pills, or injections.

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