Application of PGK1 protein in preparation of medicine for treating diabetic nephropathy

Abstract

The invention discloses application of PGK1 protein in preparation of a medicine for treating diabetic nephropathy. It is found that PGK1 protein knock-down is beneficial for improving the kidney function of a diabetic kidney model mouse, and PGK1 protein overexpression can cause the kidney function of the diabetic kidney model mouse to be poorer. The discovery shows that the PGK1 protein can be used as a target for screening and preparing the medicine for treating the diabetic nephropathy, and the PGK1 protein inhibitor has the prospect of being developed into the medicine for treating the diabetic nephropathy.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of PGK1 protein in preparing medicine for treating diabetic nephropathy. Background Art

[0002] Diabetic nephropathy (DN) seriously endangers human life and health. It is not only a common complication of diabetes, but also one of the most important kidney diseases leading to end-stage renal disease (ESRD). It has become the leading cause of ESRD worldwide. According to the International Diabetes Federation, the number of diabetic patients will increase to 700 million by 2045, and the prevalence of diabetic nephropathy, as a common complication of diabetes, is also increasing year by year. [Reference: Research Progress on the Main Pathogenesis of Diabetic Nephropathy, Life Sciences, 2023]

[0003] Phosphoglycerate kinase 1 (PGK1) is the first enzyme in glycolysis to produce adenosine triphosphate (ATP). It can catalyze the transfer of the phosphate group of 1,3-diphosphoglycerate to adenosine diphosphate (ADP) to generate 3-phosphoglycerate and ATP. In addition to participating in the glycolysis process, PGK1 can also participate in a variety of biological activities as a protein kinase regulator, including cell growth, division, differentiation, death, DNA repair, etc. In recent years, PGK1 overexpression is believed to be related to a variety of cancers such as breast cancer, prostate cancer, glioma, liver cancer, lung cancer and gastric cancer, and has become an important target in the field of cancer research. Studies have found that PGK1 can affect the function of cancer-related transcription factors, thereby affecting tumor growth, proliferation, metastasis, angiogenesis and drug resistance. In addition, the step of PGK1 catalyzing glycolysis can produce ATP, which provides energy and substances for the growth and proliferation of tumor cells, especially under hypoxic conditions, which is also considered to be an important reason for PGK1 to participate in tumor growth. However, the mechanism of PGK1's involvement in cancer development is very complex and has not yet been clearly studied. [Reference: Zhou Jing, Study on the therapeutic effect and molecular mechanism of alfuzosin on type 2 diabetic mice, Master's thesis of Lanzhou University, 2023]

[0004] In recent years, some studies have shown that activating PGK1 can activate the glycolysis process and have some beneficial effects on the body. Studies have shown that activating PGK1 can stimulate glycolysis and increase ATP levels, thereby slowing down nerve damage and enhancing dopamine function, slowing down Parkinson's neurodegeneration. In addition, studies have shown that terazosin can treat sepsis, colitis and gastric ulcers by activating PGK1. Zhou Jing also found that alfuzosin can activate PGK1 to enhance aerobic glycolysis, consume more glucose, and lower blood sugar. [Reference: Zhou Jing, Study on the therapeutic effect and molecular mechanism of alfuzosin on type 2 diabetic mice, Master's thesis of Lanzhou University, 2023]

[0005] There is currently no literature report on the treatment of diabetic nephropathy with PGK1 inhibitors. Summary of the invention

[0006] The purpose of the present invention is to provide the use of PGK1 protein in preparing a drug for treating diabetic nephropathy.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] Application of PGK1 protein as a target in the preparation of drugs for treating diabetic nephropathy.

[0009] Application of PGK1 protein as a target in screening and discovering drugs for treating diabetic nephropathy.

[0010] Application of PGK1 protein inhibitors in the preparation of drugs for treating diabetic nephropathy.

[0011] Preferably, the drug uses a PGK1 protein inhibitor as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0012] More preferably, the auxiliary material is a solid, liquid or semi-solid auxiliary material.

[0013] More preferably, the dosage forms include tablets, capsules, and injections.

[0014] Beneficial effects:

[0015] The present invention found that knocking down PGK1 protein helps improve the renal function of diabetic kidney model mice, and overexpression of PGK1 protein leads to worse renal function of diabetic kidney model mice. This finding shows that PGK1 protein can be used as a target for screening and preparing drugs for treating diabetic nephropathy, and PGK1 protein inhibitors have the prospect of being developed into drugs for treating diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The expression levels of PGK1 protein in the kidneys of each group of mice in the knockdown experiment, including: (A) Western blot graph; (B) Western blot statistical graph (n=4).

[0017] Figure 2 The expression levels of PGK1 protein in the kidneys of each group of mice in the overexpression experiment, including: (A) Western blot; (B) Western blot statistics (n=4).

[0018] Figure 3 The serum creatinine (A), urea nitrogen (B), and cystatin C (C) levels of each group of mice in the knockdown experiment (n=6).

[0019] Figure 4 Serum creatinine (A), urea nitrogen (B), and cystatin C (C) levels in each group of mice in the overexpression experiment (n=6).

[0020] Figure 5 The following are the expansion degrees of the glomerular mesangial area in each group of mice in the knockdown experiment, including: (A) representative H&E staining pathological images; (B) glomerular mesangial area expansion score (n=6).

[0021] Figure 6 The expansion degree of glomerular mesangial area in each group of mice in the overexpression experiment, including: (A) representative H&E staining pathological image; (B) glomerular mesangial area expansion score (n=6).

[0022] Figure 7 The degree of renal fibrosis in each group of mice in the knockdown experiment, including: (A) representative Sirius image; (B) fibrosis score (n=6).

[0023] Figure 8 The degree of renal fibrosis in each group of mice in the overexpression experiment, including: (A) representative Sirius image; (B) fibrosis score (n=6).

[0024] Fig. 9 The ROS levels in the kidneys of each group of mice in the knockdown experiment, including: (A) representative DHE staining images; (B) DHE staining scores (n=6).

[0025] Fig.10 The ROS levels in the kidneys of each group of mice in the overexpression experiment, including: (A) representative DHE staining images; (B) DHE staining scores (n=6).

[0026] Fig.11 The infiltration degree of renal macrophages in each group of mice in the knockdown experiment, including: (A) representative F4 / 80 staining image; (B) F4 / 80 staining score (n=6).

[0027] Fig.12 The macrophage infiltration degree of each group of mice kidney in the overexpression experiment, including: (A) representative F4 / 80 staining image; (B) F4 / 80 staining score (n=6). DETAILED DESCRIPTION

[0028] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0029] 1. Experimental Materials

[0030] C57BLKS / J db / db and C57BLKS / J db / m mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd.

[0031] AAV9-Ksp-shPGK1 and its control AAV9-Ksp-shCon, AAV9-Ksp-PGK1 overexpression plasmid and its control AAV9-Ksp-control plasmid were purchased from Paigen Biopharmaceuticals.

[0032] 2. Experimental Methods

[0033] 1. Selection of model animals

[0034] The db / db mouse is derived from the autosomal recessive inheritance of the C57BLKS / J inbred strain. Obvious obesity and increased fasting blood sugar, increased water intake and urine output can be seen 6 weeks after birth, which is most obvious at 10-12 weeks, and complications such as diabetic nephropathy can occur. The 10-12 week C57BLKS / J db / db mouse is commonly used as a diabetic nephropathy model in the field, and the C57BLKS / Jdb / m mouse is used as a negative control model of diabetic nephropathy.

[0035] 2. Animal grouping and PGK1 protein knockdown experiment

[0036] 11-week-old C57BLKS / J db / db and db / m mice were pre-adapted for one week and anesthetized with 0.3% sodium pentobarbital. The kidneys were opened to find the renal pelvis, and 50 μl of AAV9-Ksp-shPGK1 (5'-GTCCAAACTAGGAGATGTCTA-3') or AAV9-Ksp-shCon (5'-TTCTCCGAACGTGTCACGT-3') were injected into the renal pelvis to achieve specific knockdown of PGK1 in renal tubular epithelial cells. After the injection, the mice were sutured and continued to be fed for 6 weeks. Serum and renal tissue were taken for serological and renal tissue biochemical tests. The grouping and treatment are shown in the following table.

[0037]

[0038] 3. Animal grouping and PGK1 protein overexpression experiment

[0039] After one week of pre-adaptation, 11-week-old C57BLKS / J db / db and db / m mice were anesthetized with 0.3% sodium pentobarbital, the kidneys were opened to find the renal pelvis, and 50 μl of AAV9-Ksp-PGK1 overexpression plasmid or AAV9-Ksp-control plasmid was injected into the renal pelvis to achieve specific overexpression of PGK1 in renal tubular epithelial cells. After the injection, suture was performed, and after 6 weeks of continued feeding, serum and kidney tissue were taken for serological and renal tissue biochemical tests. The grouping and treatment are shown in the following table.

[0040]

[0041] 4. Determination of PGK1 protein expression level

[0042] Western blot was used to detect the protein expression of PGK1 (ab199438, Abcam) in mouse kidney tissue. An appropriate amount of mouse kidney tissue was cut, added to RIPA lysis buffer and ground, centrifuged and collected, and the total protein was quantified to the same level after the protein content was determined. 5× protein loading buffer was added to the protein sample, boiled and denatured for 10 minutes, cooled to room temperature and loaded for SDS-PAGE gel electrophoresis. After wet transfer, the membrane was blocked, and primary and secondary antibodies were added for incubation. Finally, β-actin (ab6276, Abcam) was used as an internal reference to determine the relative content of the protein, and the gray value of the band was analyzed using Image J software.

[0043] 5. Determination of serum urea nitrogen (BUN) and creatinine (CRE) levels

[0044] Blood was collected from the heart, and the whole blood was allowed to stand at room temperature for 30 minutes, and centrifuged at 3000r / min for 15 minutes to obtain mouse serum samples. The levels of BUN and Cre in mouse serum were determined using a urea nitrogen kit (C013-2-1, Nanjing Jiancheng) and a creatinine kit (C011-2-1, Nanjing Jiancheng), respectively.

[0045] 6. Determination of serum cystatin C level

[0046] The level of mouse serum Cystatin C was measured using an ELISA kit (EK0679, Boster Biotech).

[0047] 7. Renal histopathology

[0048] 7.1. HE staining and Sirius red staining

[0049] Mice were killed by cervical dislocation, and half of the unilateral kidney was fixed in formalin solution at 4°C for 24 h. After paraffin embedding, the renal tissues of each group were sliced ​​to a thickness of 4 μm. Subsequently, hematoxylin-eosin (HE) and Sirius red staining were performed, and the pathological changes of renal structure and renal interstitial fibrosis were observed under an optical microscope and histological analysis was performed; glomerular matrix hyperplasia, mesangial expansion, glomerular wall adhesion, inflammatory cell infiltration, and glomerular basement membrane thickening were observed, and the deposition of collagen fibers in the renal interstitial region was observed under an optical microscope, and collagen fibers (dyed red) were quantitatively analyzed using Image J software.

[0050] 7.2. DHE probe staining

[0051] Kidney paraffin sections were dewaxed and hydrated, and then stained with the active oxygen probe DHE (810253P, Sigma, 5 μM) at 37°C in the dark for 1 hour, sealed with an anti-fluorescence quencher, and photographed under a fluorescence microscope. Image Pro Plus software was used to set up a macro program for positive expression, and the expression of DHE in different groups was analyzed under the same conditions.

[0052] 7.3 F4 / 80 immunofluorescence staining

[0053] Kidney paraffin sections were dewaxed, hydrated, and then heat-fixed with high-pressure antigens. After blocking, the sections were incubated with F4 / 80 antibody (ab300421, Abcam) at a dilution of 1:200 at 4°C overnight, washed, and then incubated with fluorescent secondary antibodies (Goat Anti-Rabbit IgG H&L (Alexa Fluor 594)(ab150080)) was incubated at a concentration of 1:1000 at 37°C for 1 h and covered with DAPI (10236276001, Sigma, 5 μM). Five fields of view were randomly selected from each slice under a microscope and recorded. The macro program for positive expression was set using ImagePro Plus software. The expression levels of F4 / 80 in different groups were analyzed under the same conditions.

[0054] 8. Data Processing

[0055] The experimental results were expressed as (Mean±SD), and Student's t was used for comparison between two groups, and one-way ANOVA was used for comparison between multiple groups. P<0.05 was considered statistically significant.

[0056] 3. Experimental Results

[0057] 1. PGK1 protein knockdown results

[0058] The results are as follows Figure 1As shown. Compared with the control mouse-Con shRNA group (A1), the PGK1 protein expression level of mice in the diabetic kidney mouse-Con shRNA group (A3) was significantly upregulated (P<0.05), indicating that the PGK1 protein expression was upregulated in the diabetic kidney model. Compared with the diabetic kidney mouse-Con shRNA group (A3), the PGK1 protein expression level of mice in the diabetic kidney mouse-PGK1 shRNA group (A4) was significantly downregulated (P<0.05), indicating that PGK1 shRNA achieved the knockdown of PGK1 protein and achieved the expected purpose.

[0059] 2. PGK1 protein overexpression results

[0060] The results are as follows Figure 2 As shown. Compared with the control mouse-PGK1 control group (B1), the PGK1 protein expression level of mice in the diabetic kidney mouse-PGK1 control group (B3) was significantly upregulated (P<0.05), indicating that the PGK1 protein expression was upregulated in the diabetic kidney model. Compared with the diabetic kidney mouse-PGK1 control group (B3), the PGK1 protein expression level of mice in the diabetic kidney mouse-PGK1 overexpression group (B4) was significantly upregulated (P<0.05), indicating that the AAV9-Ksp-PGK1 overexpression plasmid achieved the overexpression of PGK1 protein and achieved the expected purpose.

[0061] 3. Serum BUN, Cre, and Cystatin C levels

[0062] The results of serum index level determination in knockdown test are as follows Figure 3 As shown. Compared with the control mouse-Con shRNA group (A1), the serum BUN, Cre, and Cystatin C levels of mice in the diabetic kidney mouse-Con shRNA group (A3) were significantly upregulated (P<0.05), indicating that the serum BUN, Cre, and Cystatin C levels were upregulated in the diabetic kidney model. Compared with the diabetic kidney mouse-Con shRNA group (A3), the serum BUN, Cre, and Cystatin C levels of mice in the diabetic kidney mouse-PGK1 shRNA group (A4) were significantly downregulated (P<0.05), indicating that PGK1 protein knockdown can effectively reduce the serum BUN, Cre, and Cystatin C levels of diabetic kidney mice.

[0063] The results of serum index level determination in overexpression test are as follows Figure 4As shown. Compared with the control mouse-PGK1 control group (B1), the serum BUN, Cre, and Cystatin C levels of mice in the diabetic kidney mouse-PGK1 control group (B3) were significantly upregulated (P<0.05), indicating that the serum BUN, Cre, and Cystatin C levels were upregulated in the diabetic kidney model. Compared with the diabetic kidney mouse-PGK1 control group (B3), the serum BUN, Cre, and Cystatin C levels of mice in the diabetic kidney mouse-PGK1 overexpression group (B4) were further significantly upregulated (P<0.05), indicating that overexpression of PGK1 protein will lead to further increase in the serum BUN, Cre, and Cystatin C levels of diabetic kidney mice.

[0064] Under normal circumstances, the concentrations of BUN, Cre, and Cystatin C in the blood are very stable. By detecting the levels of BUN, Cre, and Cystatin C in the blood, the renal tubular filtration function can be accurately evaluated. Figure 3 , Figure 4 The results showed that knocking down PGK1 protein helped improve the renal function of diabetic kidney model mice, while overexpression of PGK1 protein led to worse renal function in diabetic kidney model mice.

[0065] 4. Renal histopathological test results

[0066] The results of the measurement of the expansion degree of the mouse glomerular mesangial area in the knockdown experiment are as follows Figure 5 As shown. Compared with the control mouse-Con shRNA group (A1), the diabetic mouse-Con shRNA group (A3) had significantly increased expansion of the glomerular mesangial area (P<0.05), indicating that the expansion of the glomerular mesangial area in the diabetic mouse model was significantly increased. Compared with the diabetic mouse-Con shRNA group (A3), the diabetic mouse-PGK1 shRNA group (A4) had significantly decreased expansion of the glomerular mesangial area (P<0.05), indicating that PGK1 protein knockdown can effectively alleviate the expansion of the glomerular mesangial area in diabetic mice.

[0067] The results of the determination of the expansion degree of the mouse glomerular mesangial area in the overexpression experiment are as follows Figure 6 As shown. Compared with the control mouse-PGK1 control group (B1), the diabetic kidney mouse-PGK1 control group (B3) mice had significantly increased glomerular mesangial expansion (P<0.05), indicating that the diabetic kidney model mice had significantly increased glomerular mesangial expansion. Compared with the diabetic kidney mouse-PGK1 control group (B3), the diabetic kidney mouse-PGK1 overexpression group (B4) mice had further increased glomerular mesangial expansion (P<0.05), indicating that PGK1 protein overexpression would lead to further increased glomerular mesangial expansion in diabetic kidney mice.

[0068] The results of the knockdown experiment on the degree of renal fibrosis in mice were as follows Figure 7 As shown. Compared with the control mouse-Con shRNA group (A1), the renal fibrosis degree of mice in the diabetic kidney mouse-Con shRNA group (A3) was significantly increased (P<0.05), indicating that the renal fibrosis degree of mice in the diabetic kidney model was significantly increased. Compared with the diabetic kidney mouse-Con shRNA group (A3), the renal fibrosis degree of mice in the diabetic kidney mouse-PGK1 shRNA group (A4) was significantly reduced (P<0.05), indicating that PGK1 protein knockdown can effectively alleviate the renal fibrosis degree of diabetic kidney mice.

[0069] The results of the determination of the degree of renal fibrosis in mice in the overexpression experiment are as follows Figure 8 As shown. Compared with the control mouse-PGK1 control group (B1), the degree of renal fibrosis in the diabetic kidney mouse-PGK1 control group (B3) mice was significantly enhanced (P<0.05), indicating that the degree of renal fibrosis in mice in the diabetic kidney model was significantly enhanced. Compared with the diabetic kidney mouse-PGK1 control group (B3), the degree of renal fibrosis in the diabetic kidney mouse-PGK1 overexpression group (B4) mice was further enhanced (P<0.05), indicating that the overexpression of PGK1 protein will lead to further enhancement of renal fibrosis in diabetic kidney mice.

[0070] The results of the knockdown experiment on the level of ROS in the mouse kidney are as follows Fig. 9 As shown. Compared with the control mouse-Con shRNA group (A1), the ROS level in the kidneys of mice in the diabetic kidney mouse-Con shRNA group (A3) was significantly increased (P<0.05), indicating that the ROS level in the kidneys of mice in the diabetic kidney model was significantly increased. Compared with the diabetic kidney mouse-Con shRNA group (A3), the ROS level in the kidneys of mice in the diabetic kidney mouse-PGK1 shRNA group (A4) was significantly reduced (P<0.05), indicating that PGK1 protein knockdown can effectively reduce the ROS level in the kidneys of diabetic kidney mice.

[0071] The results of the determination of ROS levels in mouse kidneys in the overexpression experiment are as follows Fig.10 As shown. Compared with the control mouse-PGK1 control group (B1), the renal ROS level of mice in the diabetic kidney mouse-PGK1 control group (B3) was significantly increased (P<0.05), indicating that the renal ROS level of mice in the diabetic kidney model was significantly increased. Compared with the diabetic kidney mouse-PGK1 control group (B3), the renal ROS level of mice in the diabetic kidney mouse-PGK1 overexpression group (B4) was further increased (P<0.05), indicating that the overexpression of PGK1 protein will lead to a further increase in the renal ROS level of diabetic kidney mice.

[0072] The results of the determination of macrophage infiltration in the mouse kidney in the knockdown experiment are as follows Fig.11As shown. Compared with the control mouse-Con shRNA group (A1), the infiltration degree of macrophages in the kidneys of mice in the diabetic kidney mouse-Con shRNA group (A3) was significantly enhanced (P<0.05), indicating that the infiltration degree of macrophages in the kidneys of mice in the diabetic kidney model was significantly enhanced. Compared with the diabetic kidney mouse-Con shRNA group (A3), the infiltration degree of macrophages in the kidneys of mice in the diabetic kidney mouse-PGK1 shRNA group (A4) was significantly reduced (P<0.05), indicating that PGK1 protein knockdown can effectively alleviate the infiltration degree of macrophages in the kidneys of diabetic kidney mice.

[0073] The results of determination of macrophage infiltration in mouse kidneys in the overexpression experiment are as follows Fig.12 As shown. Compared with the control mouse-PGK1 control group (B1), the infiltration degree of macrophages in the kidneys of the diabetic kidney mouse-PGK1 control group (B3) mice was significantly enhanced (P<0.05), indicating that the infiltration degree of macrophages in the kidneys of mice in the diabetic kidney model was significantly enhanced. Compared with the diabetic kidney mouse-PGK1 control group (B3), the infiltration degree of macrophages in the kidneys of the diabetic kidney mouse-PGK1 overexpression group (B4) mice was further enhanced (P<0.05), indicating that the overexpression of PGK1 protein will lead to a further enhancement of the infiltration degree of macrophages in the kidneys of diabetic kidney mice.

[0074] The degree of mesangial expansion, renal fibrosis, renal ROS level and renal macrophage infiltration are important indicators for evaluating renal function. Figure 5 to Figure 12 The results showed that knocking down PGK1 protein helped improve the renal function of diabetic kidney model mice, while overexpression of PGK1 protein led to worse renal function in diabetic kidney model mice.

[0075] In summary, knocking down PGK1 protein helps improve the renal function of diabetic kidney model mice, while overexpression of PGK1 protein leads to worse renal function in diabetic kidney model mice. This finding suggests that PGK1 protein can be used as a target for screening and preparing drugs for treating diabetic nephropathy, and PGK1 protein inhibitors have the prospect of being developed into drugs for treating diabetic nephropathy.

[0076] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. Application of PGK1 protein as a target in the preparation of drugs for the treatment of diabetic nephropathy.

2. Application of PGK1 protein as a target in screening and discovering drugs for the treatment of diabetic nephropathy.

3. Application of PGK1 protein inhibitors in the preparation of drugs for the treatment of diabetic nephropathy.

4. The use according to claim 3, wherein the drug has a PGK1 protein inhibitor as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

5. The use according to claim 4, wherein the auxiliary material is a solid, liquid or semi-solid auxiliary material.

6. The use according to claim 4, wherein the dosage form comprises tablets, capsules, and injections.

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