CN111494407A

CN111494407A - Use of trehalose in the preparation of a medicament for alleviating conditions associated with acute renal injury induced by ischemia reperfusion

Info

Publication number: CN111494407A
Application number: CN202010016606.XA
Authority: CN
Inventors: 杨运文; 黄松明; 张爱华; 贾占军; 刘素雯; 周宁; 高慧萍; 王佩培
Original assignee: Nanjing Childrens Hospital of Nanjing Medical University
Current assignee: Nanjing Childrens Hospital of Nanjing Medical University
Priority date: 2020-01-08
Filing date: 2020-01-08
Publication date: 2020-08-07

Abstract

The invention provides an application of trehalose in preparation of a medicine for relieving renal ischemia-reperfusion injury, and belongs to the field of biological medicines. The application proves that trehalose can relieve acute kidney injury related symptoms induced by ischemia-reperfusion by enhancing autophagy, including acute kidney injury renal function, oxidative stress, renal tubular cell apoptosis, renal inflammatory cell infiltration and other symptoms, thereby providing a theoretical basis for widening the possible application range of trehalose. Since trehalose is currently approved as a safe food ingredient by the U.S. food and drug administration and has been used in food preservation and other works, our findings will most likely provide effective clinical drugs for the prevention and treatment of AKI.

Description

Use of trehalose in the preparation of a medicament for alleviating conditions associated with acute renal injury induced by ischemia reperfusion

Technical Field

The invention relates to a new application of trehalose, in particular to an application of trehalose in preparing a medicament for relieving acute renal injury related symptoms induced by ischemia-reperfusion.

Background

Acute Kidney Injury (AKI) is a common clinical syndrome, mainly manifested by rapid decline of kidney function and accumulation of metabolic waste products, and the increase of serum creatinine (Scr) and decrease of urine volume are diagnostic bases of AKI. The incidence rate of AKI is high and tends to rise year by year, and a cross section study in 2013 shows that the detection rate of AKI of inpatients in China is as high as 2%, 290 million patients with AKI are predicted to be inpatients, and about 70 million patients die of AKI. At present, no specific medicine for treating AK1 exists, and renal replacement therapy is required in severe cases. The risk of developing chronic kidney disease in AKI patients is greatly increased, and some patients can directly progress to end-stage kidney disease, which brings great economic burden to families and society of patients. Many AKIs have complete or partial recovery of renal function and disengagement from dialysis if risk factors are identified, removed or actively intervened in a timely manner, and thus early diagnosis, early intervention, is critical to improving the prognosis of AKI. Its main etiology is ischemia, renal toxic substance caused or primary kidney disease, renal tubular injury and inflammatory cell infiltration are the main pathological features of acute renal injury.

Renal ischemia reperfusion injury is one of the most common causes of AKI, and is widely seen clinically in renal transplantation, segmental nephrectomy, shock, acute renal artery obstruction and other clinical surgical procedures. The pathological mechanism of ischemia-reperfusion injury is complex and changeable, and relates to various injury mechanisms, including oxidative stress, inflammatory reaction, apoptosis reaction and the like. Despite the improvements in current therapies, the mortality rate from ischemia-reperfusion remains high, and the search for new therapies is imminent.

Trehalose (trehalase, Tre), a non-reducing sugar consisting of two glucose molecules with 1, 1-glycosidic linkages, is a safe, reliable natural sugar and has been approved by the U.S. food and drug administration and accepted by european regulatory systems as a safe food ingredient. However, the role of trehalose on ischemia reperfusion-induced AKI and its underlying mechanism have not been clearly elucidated.

Disclosure of Invention

The invention provides an application of trehalose in preparation of a medicine for treating acute kidney injury, and solves the technical problem that no proper medicine is available in the prior art for treating structural and functional damage of a kidney caused by ischemia-reperfusion kidney injury.

The purpose of the invention can be realized by the following technical scheme:

use of trehalose in the manufacture of a medicament for the alleviation of conditions associated with ischemia reperfusion-induced acute kidney injury.

Use of trehalose in the preparation of a medicament for reducing acute kidney injury renal function induced by ischemia reperfusion.

Use of trehalose in the preparation of a medicament for reducing ischemia reperfusion-induced tubular cell apoptosis, oxidative stress, renal inflammatory cell infiltration.

Has the advantages that:

the invention discusses the use of trehalose in medicaments for alleviating acute renal injury related diseases induced by ischemia-reperfusion by using trehalose in an ischemia-reperfusion mouse model and an in vitro cell hypoxia/reoxygenation model. The results show that the treatment of intervention by trehalose in an ischemia-reperfusion mouse model and an in vitro cell hypoxia/reoxygenation model can obviously improve acute kidney injury renal function, renal tubular cell apoptosis, renal inflammatory cell infiltration and oxidative stress reaction. Since trehalose is currently approved as a safe food ingredient by the U.S. food and drug administration and has been used in food preservation and other works, our findings will most likely provide effective clinical drugs for the prevention and treatment of AKI.

Drawings

FIG. 1 trehalose improves kidney injury and kidney function in ischemia reperfusion-induced acute kidney injury and down-regulates NGA L and KIM-l expression levels in kidney tissue;

figure 2 trehalose significantly alleviated renal ischemia reperfusion-induced apoptosis;

figure 3 trehalose significantly alleviated renal ischemia reperfusion-induced oxidative stress;

figure 4 trehalose significantly reduced the inflammatory response induced by renal ischemia reperfusion;

FIG. 5 Effect of different concentrations of trehalose on renal tubular epithelial cell Activity

FIG. 6 trehalose significantly improved cell death and oxidative stress in vitro caused by hypoxia reoxygenation of renal tubular epithelial cells.

Detailed description of the invention

Example 1 materials and methods

1) Materials and reagents

Trehalose was purchased from Sigma, NGA L antibody from Abcam, KIM-1, MPO, and 4-hydroxynenal antibodies from R & D Systems, ATG5 from Baaode, Nanjing, L C3B from Sigma-Aldrich, COX-2 from Cayman, ATPB, I L-6, TNF α, SOD2, β -actin antibodies from proteintech, fluorescent secondary antibody from Invitrogen, CCK8 kit from Keygen Biotech, apoptosis assay kit from Vazyme, MDA, and ROS assay kit from Beyoe, and mitochondrial membrane potential assay kit from Thermiher.

2) Cell culture and processing

Mouse tubular epithelial cells (mPTCs) were cultured in DMEM/F12 medium containing 10% fetal bovine serum, 0.5% penicillin and streptomycin at 37 ℃ in 5% carbon dioxide and 95% air. To investigate the mechanism of action of trehalose in renal tubular epithelial cell injury caused by hypoxia/reoxygenation, when mPTC was grown to 70% density, it was cultured in DMEM/F12 medium without fetal bovine serum, and pretreated with trehalose (dissolved in phosphate buffer) for 1h or the same treatment with sucrose to maintain osmolality. Cells were cultured in a hypoxic incubator (1% O)₂、94％N₂And 5% CO₂) After 24 hours of incubation, the cells are placed under the condition of normal oxygen for reoxygenation for 6 hours, the control cells are incubated under the condition of normal oxygen, and finally the cells are collected for Western Blot detection, TUNE L analysis of apoptosis, autophagy flow detection and other tests.

3) Ischemia reperfusion acute kidney injury model and experimental grouping

Mice were randomly divided into four groups of 6 mice, each group being a placebo group (sham), a trehalose group (sham + Tre), an ischemia-reperfusion model group (IR), and an ischemia-reperfusion + trehalose group (IR + Tre). Trehalose was dissolved in physiological saline and administered by intraperitoneal injection at 2 g/kg. The mice in the control group or the trehalose-treated group were administered with sucrose or trehalose, respectively, by intraperitoneal injection once a day starting from 3 days before the operation. After mice in the ischemia reperfusion surgery group were anesthetized with isoflurane (induction rate of 5%, maintained at 1.5% to 2.5%), abdominals were opened using a midline incision, and bilateral renal pedicle clamping was performed using arteriolar clamps for 35 minutes. During surgery, mice were placed on a heated surgical pad at a stable temperature of 37 ℃ and hydrated with warm saline. After removing the jaws, abdominal suturing was performed after visual confirmation of blood reperfusion of the kidney. Sham group mice received the same procedure but were not clamped. After 24 hours, all groups of mice were euthanized and kidney and blood samples were collected. All animal experiments followed the Chinese regulations for the management and use of experimental animals.

4) Renal function detection

Blood serum components are collected after a mouse blood specimen is centrifuged, and serum creatinine and serum urea nitrogen indexes are detected on a full-automatic biochemical instrument of children hospital in Nanjing.

5) Renal tubular injury score

The degree of tubular injury was observed in the pathological staining of PAS in the kidney and evaluated according to a semi-quantitative injury score with 0 score for normal tubular tissue, 1 score for 30% of tubular injury, 2 scores for 30% -60% of tubular injury and 3 scores for greater than 60%.

6) Histological and immunostaining with fluorescent dye

Kidney tissues are fixed by paraformaldehyde, wrapped by paraffin, and subjected to histology and immunofluorescence staining after tissue sectioning, primary antibody concentrations of NGA L, TNF α, I L-6, L C3B and the like are all 1:100, fluorescent secondary antibody concentration is 1:400, and DAPI staining is carried out according to the marking instruction.

7) Western blotting

The kidney tissue extracts proteins and operates according to literature procedures. Results of Western immunoblotting (Western blot) were subjected to grayscale analysis using ImageJ software.

8) Detection of autophagic flow

RFP-GFP-L C3 plasmid was transiently transfected into mouse tubular epithelial cells, and 24h after treatment cell images were captured by laser scanning confocal microscopy representative cells were randomly selected and photographed and analyzed using ImageJ to quantify the number of spots per cell.

9) Statistical analysis

Data are represented using the mean SD. Multiple comparisons were performed using one-way analysis of variance (ANOVA) and two data comparisons were performed using T-test. P <0.05 is statistically significant.

Example 2 trehalose to improve renal injury and renal function in models of ischemia-reperfusion-induced acute renal injury

In order to evaluate and detect the effect of trehalose in protecting renal ischemia-reperfusion injury, related biochemical indexes of a mouse kidney are detected, after the kidney ischemia-reperfusion molding is carried out for 24 hours, serum muscle and urea nitrogen indexes are obviously increased, the pathological injury of the kidney such as renal tubular dilatation necrosis and the like is more serious, and after the trehalose is treated, the corresponding renal injury and renal function indexes are obviously reduced (fig. 1A, B, D). In addition, the renal tubular injury score also suggested that trehalose treatment could ameliorate cisplatin-induced pathological renal injury (fig. 1C). Therefore, trehalose not only improves kidney function, but also alleviates pathological damage to the kidney. These results indicate that trehalose can protect against renal damage caused by acute renal injury.

In order to further prove that the trehalose has a protective effect on acute renal injury induced by ischemia-reperfusion, specific biological indexes, NGA L and KIM-1, in the early stage of acute renal injury are detected, as shown by fluorescence quantitative PCR and western blot detection in figure 2, KIM-1 and NGA L are highly expressed in the kidney of mice with acute renal injury induced by ischemia-reperfusion, and the expression level is remarkably reduced after trehalose treatment.

Example 3 trehalose enhances autophagy and relieves apoptosis and oxidative stress in a model of ischemia reperfusion-induced acute kidney injury.

The apoptosis induced by renal ischemia reperfusion injury was detected by TUNE L staining As shown in FIG. 3E, the number of TUNE L positive cells in the renal tubules of ischemia-reperfused mice was significantly increased and significantly decreased by trehalose treatment.

Renal ischemia reperfusion injury impairs the antioxidant capacity of the injured kidney and exacerbates oxidative stress. Western blot analysis results showed that trehalose treatment completely prevented ischemia reperfusion-induced down-regulation of mitochondrial respiratory chain-associated proteins, such as mitochondrial SOD (SOD2) and ATPB (fig. 4A-C). In addition, the results of the detection of MDA and 4-hydroxynonenal (4-hydroxynonenal) as biomarkers of oxidative damage (fig. 4D-E) further indicate that trehalose treatment can effectively alleviate oxidative stress in renal ischemia reperfusion injury.

Example 4 trehalose reduced inflammatory injury and reduced neutrophil infiltration in an ischemia reperfusion-induced acute kidney injury model.

Real-time fluorescence quantitative PCR results show that trehalose significantly attenuates the up-regulation of inflammatory factors I L-1 β, I L-6, COX-2, MCP1 and TNF- α caused by ischemia reperfusion injury (fig. 5). western blot results show that the protein levels of COX-2 and I L-6 in the kidneys of mice in the ischemia reperfusion model group are significantly reduced after treatment with trehalose (fig. 5). I L-6 and TNF α show a immunohistochemical staining trend consistent with the above results (fig. 5). furthermore, by western blot analysis, we also examined the protein level of MPO, a marker of neutrophil activation, the results show that the elevated MPO protein level caused by renal ischemia reperfusion injury is significantly down-regulated after trehalose treatment (fig. 5). these results show that trehalose treatment significantly suppresses the inflammatory response caused by renal ischemia reperfusion injury.

Example 5 trehalose treatment can activate autophagy and improve apoptosis and oxidative stress in an in vitro hypoxia/reoxygenation model of renal tubular epithelial cells.

We have shown by cellular activity assays that trehalose concentrations within 50mM are not significantly toxic to mPTC (FIG. 6). We have examined levels of apoptosis of mPTCs by TUNE L staining and have shown that treatment with 40mM trehalose significantly inhibits hypoxia/reoxygenation-induced apoptosis of mPTCs.

In summary, the invention provides an application of trehalose in preparing a medicament for relieving acute renal injury related symptoms induced by ischemia-reperfusion, wherein the medicament is administered by intraperitoneal injection, and can relieve acute renal injury related symptoms induced by ischemia-reperfusion, including acute renal injury, renal function, oxidative stress, renal tubular cell apoptosis, renal inflammatory cell infiltration and other symptoms.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of trehalose in the manufacture of a medicament for the alleviation of conditions associated with ischemia reperfusion-induced acute kidney injury.

2. Use of trehalose according to claim 1 in the manufacture of a medicament for reducing the function of the kidney of an ischemia reperfusion-induced acute kidney injury.

3. Use of trehalose according to claim 1 for the preparation of a medicament for reducing ischemia reperfusion-induced tubular apoptosis, oxidative stress, renal inflammatory cell infiltration.