Application of nicotinamide mononucleotide in preparation of medicine for preventing acute kidney injury

Abstract

The invention belongs to the field of biological medicines, and discloses application of nicotinamide mononucleotide in preparation of a medicine for preventing acute kidney injury. Through NMN intervention in the acute stage, the NAD < + > metabolic level and SIRT1 protein expression in the kidney of a mouse can be remarkably improved, inflammatory response is relieved, acute injury of renal tubules is reduced, and therefore acute kidney injury caused by cis-platinum is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of nicotinamide mononucleotide in the preparation of a drug for preventing acute kidney injury. Background Art

[0002] Acute kidney injury (AKI), also known as "acute renal failure", is characterized by a rapid decline in renal function within hours or days. With its high mortality and morbidity, AKI has become a major problem affecting human health worldwide. Clinically, changes in renal function are generally assessed by serum creatinine and urine volume.

[0003] The pathophysiological mechanisms of AKI mainly include apoptosis of renal tubular epithelial cells, cell necrosis and acute inflammatory response. Pathological histological analysis shows that one of the typical pathological features of AKI is acute tubular necrosis, especially the renal tubules in the outer medulla of the kidney with strong metabolic activity and high oxygen demand are extremely susceptible to acute inflammatory response and undergo apoptosis.

[0004] Cisplatin is a widely used anti-tumor drug in clinical practice. It is widely used in the treatment of solid organ cancers such as ovarian cancer, breast cancer, lung cancer, and testicular cancer. However, in the process of clinical use, it is found that the side effects of cisplatin such as nephrotoxicity, ototoxicity, and neurotoxicity greatly limit the clinical use of cisplatin. In particular, nephrotoxicity is the most common and serious, which directly affects the effect and prognosis of chemotherapy. Cisplatin is used in about 10%-20% of cancer patients, of which 30% will suffer from acute kidney injury. Acute kidney injury is one of the serious side effects of cisplatin in clinical use. Finding drugs that can effectively prevent cisplatin-induced acute kidney injury has become an urgent problem to be solved. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a use of nicotinamide mononucleotide in the preparation of a medicament for preventing acute kidney injury.

[0006] In a first aspect of the present invention, there is provided a use of nicotinamide mononucleotide in the preparation of a medicament for preventing acute kidney injury, wherein the acute kidney injury is acute kidney injury caused by cisplatin.

[0007] Furthermore, the prevention is to increase the protein expression level of SIRT1 in the kidney and / or increase the expression of NAD+ in the kidney.

[0008] Furthermore, the acute kidney injury is clinically manifested as acute decline in renal function.

[0009] Furthermore, the acute kidney injury pathologically manifests as renal tubular damage.

[0010] Furthermore, the prevention is to alleviate the acute inflammatory response caused by cisplatin.

[0011] Furthermore, the prevention is to reduce the apoptosis of renal tubular cells caused by cisplatin.

[0012] Furthermore, the prevention is to reduce the oxidative stress of renal tubular cells caused by cisplatin.

[0013] Compared with the prior art, the present invention discloses for the first time that short-term NMN administration can increase the NAD+ content and SIRT1 protein expression in renal tissue, reduce oxidative stress damage and acute inflammatory response of renal tubular cells, thereby improving renal function and preventing acute kidney injury caused by cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The dosing regimen of the cisplatin + NMN group in Example 1 is shown;

[0015] Figure 2 The results of the serum biochemical index detection of four groups of mice obtained in Example 2 are shown in Figure A, which shows the serum urea nitrogen level, and Figure B shows the serum creatinine level;

[0016] Figure 3 The HE staining images of the kidneys of four groups of mice obtained in Example 3 are shown, Figure A is the control group, Figure B is the cisplatin group; Figure C is the cisplatin + NMN1 group, Figure D is the cisplatin + NMN2 group, and Figure E is a statistical graph of the results of renal injury scores;

[0017] Figure 4 The immunohistochemical test results of the kidneys of four groups of mice obtained in Example 4 are shown, wherein Figure A is the acute kidney injury marker KIM-1; Figure B is the neutrophil marker Ly6G; Figure A' is the statistical analysis diagram of KIM-1, and Figure B' is the statistical analysis diagram of Ly6G;

[0018] Figure 5 The graphs of the mRNA expression levels of various genes in kidney tissue detected by real-time quantitative fluorescence PCR (qPCR) in Example 5 are shown, wherein Figure A is a graph of the expression level of the kidney injury marker KIM-1 gene, Figure B is a graph of the expression level of the kidney SIRT1 gene, and Figure C is a graph of the expression level of the inflammatory factor IL-6 gene;

[0019] Figure 6 A statistical graph showing the results of the detection of NAD+ content in kidney tissue in Example 6 is shown;

[0020] Figure 7 The results of the protein expression level detection of SIRT1 in Example 7 are shown in Figure A, Figure A is the immunohistochemical diagram of SIRT1 in the kidneys of four groups of mice; Figure B is the expression of SIRT1 protein, Figure A' is a statistical diagram of immunohistochemical staining of SIRT1 in the kidneys, and Figure B' is the relative expression analysis of SIRT protein;

[0021] Figure 8 The cell experiment detection results of the effect of NMN on cisplatin-induced tubular cell apoptosis and oxidative stress in Example 8 are shown, Figure A is the statistical result of cisplatin-induced LDH release from tubular epithelium; Figure B is the statistical result of the level of LDH released after stimulating HK-2 cells with gradient concentrations of 0, 5, and 10 mmol NMN solution and 100 μmol cisplatin for 24 hours; Figure C shows that NMN increases the expression level of NAD+ in renal tubular cells, Figure D shows that cisplatin causes the release of reactive oxygen species in renal tubular cells, and NMN can significantly reduce the release of reactive oxygen species, indicating that NMN reduces the oxidative stress of renal cells caused by cisplatin, and Figure E is a statistical analysis of the effect of NMN on the oxidative stress of renal tubular cells. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional operations in the art, or according to the conditions recommended by the manufacturer.

[0023] Example 1: Animal model, grouping and drug administration.

[0024] Thirty-two male BL6J mice were randomly divided into four groups, with 8 mice in each group. The experimental animals were divided into control group (Ctrl), cisplatin group (Cis), cisplatin + NMN1 group (NMN1, 500 mg / kg), and cisplatin + NMN2 group (NMN2, 1000 mg / kg).

[0025] The NMN administration method of mice in the cisplatin + NMN1 group (NMN1, 500 mg / kg) and the cisplatin + NMN2 group (NMN2, 1000 mg / kg) was as follows Figure 1 As shown, 24 hours after the mice were intraperitoneally injected with NMN solution, cisplatin was injected at a dose of 23 mg / kg (this time was recorded as day 0), and then the mice were intraperitoneally injected with NMN solution for three consecutive days. 72 hours after the injection of cisplatin, the mouse serum and kidneys were collected and the weight of both kidneys was weighed and stored at -80°C.

[0026] Cisplatin group (Cis): Cisplatin (23 mg / kg) was injected on day 0.

[0027] The control group was injected with an equal volume of saline.

[0028] Example 2: Detection of serum biochemical indicators.

[0029] The serum urea nitrogen (BUN) and creatinine levels of the four groups of mice were tested according to the kit instructions. The results are as follows: Figure 2 As shown. Figure 2It can be seen that compared with the control group, the levels of BUN and creatinine in the kidneys of mice in the cisplatin group (Cis) were significantly increased. Compared with the cisplatin group (Cis), the serum BUN and creatinine levels of mice in the Cis+NMN2 (1000 mg / kg) group were significantly reduced, P<0.05. It can be seen that cisplatin caused a significant increase in the levels of serum urea nitrogen and creatinine in mice, indicating renal damage, while NMN can significantly reduce the levels of urea nitrogen and creatinine.

[0030] Example 3: Pathological observation of kidney tissue.

[0031] The renal tissue was fixed with 10% formaldehyde solution, embedded in paraffin, sliced, stained with hematoxylin-eosin (HE), observed, and scored. Figure 3 As shown in Figure AD. It is easy to see from the figure that the proportion of the cortex and medulla of the renal tissue in the control group is normal, the glomeruli and tubules are arranged normally, the structure is clear, and there is no abnormal changes such as necrosis and degeneration. In the cisplatin group, the renal tubules were obviously dilated and necrotic, the nuclear swelling and fragmentation of the tubular epithelial cells, the tubular lumen was foreign and the interstitial edema, and the tubular interstitium was infiltrated by immune cells, indicating a significant inflammatory response. The NMN treatment groups (cisplatin + NMN1 group and cisplatin + NMN2 group) significantly reduced cisplatin-induced tubular necrosis, tubular dilatation, tubular foreign bodies, loss of cortical brush border and interstitial edema. The statistical results of renal injury are shown in the figure. Figure 3 Figure E showed that NMN significantly reduced cisplatin-induced AKI, P < 0.05.

[0032] Example 4: Immunohistochemical detection of KIM-1 and neutrophils (Ly6G) in kidney tissues of four groups of mice.

[0033] Immunohistochemical staining: The kidney tissue sections of the four groups of mice were incubated in xylene wash solution twice, each time for 15 minutes; then the sections were incubated in 100%, 95%, 80%, and 70% ethanol for 2 minutes respectively for dehydration; then dewaxing, antibody incubation, and finally DAB color development, the color reaction was controlled under the microscope, washed with distilled water, the reaction was terminated, and the sections were sealed. Then ImageJ was used to score the kidneys stained with immunohistochemical staining. The results are as follows: Figure 4 shown.

[0034] Figure 4 Figure A is an immunohistochemical section of KIM-1 protein, a marker of acute kidney injury, and Figure A' is its statistical analysis chart. Figure 4 Figure A in Figure 2 shows that there is a large amount of KIM-1 positive staining in the cisplatin group, and the KIM-1 positive staining in the two NMN treatment groups (cisplatin + NMN1 group and cisplatin + NMN2 group) is significantly reduced. Figure 4Figures A and A' show that the expression of KIM-1 protein in the kidneys of mice in the cisplatin group was significantly higher than that in the control group. Both NMN-treated groups (cisplatin + NMN1 group and cisplatin + NMN2 group) significantly reduced the increase in KIM-1 protein caused by cisplatin, indicating that NMN significantly alleviated renal tubular damage.

[0035] Figure 4 Figure B shows the immunohistochemical staining of the neutrophil marker Ly6G, and Figure B' shows the statistical analysis chart. Figure 4 Figure B shows that there is a large amount of Ly6G positive staining in the tubular interstitium of the cisplatin group, indicating a large number of neutrophil infiltration and severe inflammatory response. Figure 4 As shown in Figures B and B', cisplatin caused a large number of neutrophil infiltration in the kidneys, while the number of neutrophils in the NMN group was significantly reduced, indicating that NMN alleviated the inflammatory response caused by cisplatin. P < 0.05.

[0036] Example 5: Real-time quantitative fluorescence PCR (qPCR) detection of gene mRNA in kidney tissue.

[0037] Mouse kidney RNA was extracted using an RNA extraction kit (Biyuntian) and reverse transcribed into cDNA. qPCR was performed using the SYBR GreenI dye method, with the internal reference gene being 18S. The data were analyzed using the ΔΔct method and plotted using GraphpadPrism software. The results are shown in the figure. Figure 5 shown. Figure 5 Figure A in the middle is a statistical chart of KIM-1 gene expression levels. It can be seen that the cisplatin model group was significantly increased, while the two NMN treatment groups were significantly decreased. Figure 5 Figure B is a statistical chart of the expression level of the SIRT1 gene in the kidney. It can be seen that NMN significantly increased the expression of the SIRT1 gene. Figure 5 Figure C in Figure 2 is a statistical chart of inflammatory factor IL-6 gene expression, which shows that the cisplatin model group was significantly increased, while the two NMN treatment groups were significantly decreased. P<0.05. Figure 5 It can be seen that cisplatin caused high expression of inflammatory factor IL-6, while NMN significantly inhibited the release of inflammatory factors, indicating that NMN alleviated the inflammatory response.

[0038] Example 6: Detection of NAD+ content in kidney tissue

[0039] The NAD detection kit (Biyuntian) was used to detect the NAD+ content in the kidney tissues of four groups of mice. The results are as follows Figure 6 As shown, NMN significantly increased the expression of NAD+ in mouse kidneys, P<0.05, while cisplatin had no significant effect on it.

[0040] Example 7: Immunoblotting was used to detect the expression of IRT1 protein in mouse kidney tissue.

[0041] Mouse kidney tissue was added to Western and IP cell lysate and homogenized, proteins were extracted, separated by SDS-PAGE, transferred to PVDF membrane, blocked at room temperature, added primary antibody (dilution ratio: 1:1000), and incubated at 4°C overnight. After washing, secondary antibody (dilution ratio: 1:1000) was added, incubated on a shaker at room temperature for 1 hour, and then washed and developed with ECL colorimetric solution. The bands were photographed with a gel imager. ImageJ software was used for statistical analysis, and the relative expression of the target protein was calculated by dividing the gray value of the target protein by the gray value of the internal reference protein. The immunohistochemical staining of SIRT1 protein and the statistical graph of the relative protein expression are shown in the figure. Figure 7 As shown in Figure 2, SIRT1 protein in the NMN treatment group increased significantly. P<0.05.

[0042] Example 8: Cell experiment to detect the effect of NMN on apoptosis and oxidative stress of renal tubular cells

[0043] Construction of cisplatin-induced renal tubular cell apoptosis model: HK-2 cells were stimulated with cisplatin at gradient concentrations of 0, 50, 100, and 200 μmol for 24 hours, and apoptosis was detected using a lactate dehydrogenase (LDH) detection kit (Biyuntian). Figure 8 As shown in Figure A, cisplatin can cause HK-2 to release a large amount of LDH, indicating cell apoptosis, and the degree of apoptosis is positively correlated with the concentration of cisplatin. A concentration of 100 μmol cisplatin was selected for subsequent experiments.

[0044] HK-2 cells were stimulated with NMN solutions of gradient concentrations of 0, 5, and 10 mmol and 100 μmol of cisplatin for 24 hours, and the level of LDH released was detected. The results are as follows: Figure 8 As shown in Figure B, it was found that NMN treatment could alleviate the release of LDH, and the inhibitory effect was positively correlated with the NMN concentration, indicating that NMN alleviated renal cell apoptosis.

[0045] The NAD+ kit was used to detect the effect of NMN solution treatment on the NAD+ expression level in HK-2 cells. Figure 8 As shown in Figure C, NMN increases the level of NAD+ in renal tubular cells.

[0046] The three groups of HK-2 cells were treated with a reactive oxygen species (ROS) detection kit (Biyuntian) and photographed under a fluorescence microscope to observe their ROS release. The results are as follows Figure 8 As shown in Figures D and E, cisplatin caused a large amount of ROS release, while NMN significantly inhibited the release of ROS, P<0.05.

Claims

1. Use of nicotinamide mononucleotide in the preparation of a drug for preventing acute kidney injury, wherein the acute kidney injury is acute kidney injury caused by cisplatin.

2. The use according to claim 1, It is characterized in that The prevention is to increase the protein expression level of SIRT1 in the kidney and / or increase the expression of NAD+ in the kidney.

3. The use according to claim 1, It is characterized in that The clinical manifestation of the acute kidney injury is acute renal function decline.

4. The use according to claim 1, It is characterized in that The pathological manifestation of the acute kidney injury is renal tubular damage.

5. The use according to claim 1, It is characterized in that The prevention is to reduce the acute inflammatory response caused by cisplatin.

6. The use according to claim 1, It is characterized in that The prevention is to reduce the apoptosis of renal tubular cells caused by cisplatin.

7. The use according to claim 1, It is characterized in that The prevention is to reduce the oxidative stress of renal tubular cells caused by cisplatin.

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