CN112402430A

CN112402430A - Application of alisol B-23-acetate in preventing and treating acute kidney injury

Info

Publication number: CN112402430A
Application number: CN202011449656.3A
Authority: CN
Inventors: 管又飞; 栾志琳
Original assignee: Dalian Medical University
Current assignee: Dalian Medical University
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2021-02-26

Abstract

The invention belongs to the field of biology, and particularly relates to application of an FXR agonist in preparation of a medicine for preventing and/or treating kidney diseases. Specifically, the invention discloses an application of an FXR agonist in preparation of a medicine for preventing and/or treating kidney diseases. The FXR agonist is alisol B-23-acetate. Compared with the prior art, the invention firstly verifies that the alisol B-23-acetic ester can relieve acute kidney injury. The invention provides alisol B-23-acetate as an FXR agonist, which has the possibility of being applied to prevention and treatment of acute kidney injury.

Description

Application of alisol B-23-acetate in preventing and treating acute kidney injury

Technical Field

The invention belongs to the field of biology, and particularly relates to an application of an FXR agonist in preparation of a medicine for preventing and/or treating kidney diseases.

Background

The kidney is an important organ for maintaining the homeostasis of the body, and has the main function of excretion, excretion of metabolic end products and excess substances, and regulation of urine volume and urine components by regulating filtration and reabsorption of water and electrolytes, thereby maintaining the homeostasis of blood pressure and water and salt metabolism of the body. The main structural basis of the kidney for playing a role in excretion, regulating water and salt metabolism and forming urine is glomerulus and renal tubules, blood components are filtered by the glomerulus to form raw urine, and the raw urine is finally formed by reabsorption of each section of renal tubule, and the structural and functional abnormalities of the glomerulus and each section of renal tubule seriously disturb the homeostasis of the body and influence the health of the human body. Acute Kidney Injury (AKI) is a clinical syndrome of acute decline in renal function caused by various causes, manifested by decline in Glomerular Filtration Rate (GFR), azotemia, disturbance in water, electrolyte and acid-base balance, and multiple system complications in severe cases, and is a common critical condition involving multiple clinical families. The mortality rate of critical AKI is as high as 30% to 80%, and about 50% of surviving AKI patients remain with permanent renal function decline. Global disease burden reports suggest that the lethal disability rate of AKI has increased in recent years and that treatment costs are high.

Studies have shown that metabolic nuclear receptor agonists play an important role in alleviating AKI. The metabolic nuclear receptors are a general term for a group of nuclear receptors closely related to sugar, lipid and energy metabolism, and mainly include Farnesoid X Receptors (FXR), Pregnane X Receptors (PXR), peroxisome proliferator-activated receptors (PPARs), Liver X Receptors (LXR), retinoic acid X receptors (RXR), and the like. These nuclear receptors are activated by ligands of endogenous small molecule metabolites such as fatty acids, bile acids, oxidative cholesterol, etc., and play a key regulatory role in the processes of insulin sensitivity, lipogenesis, lipid metabolism, energy balance, blood pressure regulation, inflammation occurrence, cell growth and differentiation, etc., and are receiving attention in the field. A great deal of evidence indicates that metabolic nuclear receptors are central regulatory factors of metabolic diseases, dysfunction of the metabolic nuclear receptors is one of the main causes of metabolic syndrome, and the search for novel therapeutic drugs for major metabolic diseases based on the nuclear receptors can effectively prevent and treat the occurrence and development of the major metabolic diseases.

FXR is a member of the nuclear receptor superfamily and is designated as a farnesoid derivative receptor because it can be activated by a supraphysiological level of farnesoid. Subsequent studies found that physiological levels of Bile acids such as chenodeoxycholic acid (CDCA) are endogenous ligands of FXR, and therefore FXR is also known as Bile Acid Receptor (BAR). A large number of researches prove that the function of FXR is not only limited to regulation of expression of molecules related to synthesis and transportation of bile acid in liver and small intestine, but also widely involved in regulation of important life processes such as glycolipid metabolism, cell proliferation and the like. FXR is also widely expressed in the heart, ovary, thymus, spleen and kidney, in addition to the liver and small intestine.

The rhizoma alismatis root tuber is a traditional Chinese medicine clinical common top-grade medicinal material and is widely applied to the treatment of kidney diseases. The description of the clinical changes of the kidney diseases in modern medicine accords with the efficacy of the rhizoma alismatis in the scope of the indications of the rhizoma alismatis in the traditional Chinese medicine, such as kidney meridian entry, bladder meridian entry, diuresis, dampness excreting and heat discharging. Research has shown that the alisma orientale extract mixture and the triterpenoid compound as the main active component thereof have the function of exciting the nuclear receptor FXR, wherein the triterpenoid compound comprises alisol B-23-acetate.

However, whether alisol B-23-acetate can be used for preventing and treating acute kidney injury diseases has not been reported.

Disclosure of Invention

The invention aims to provide a potential compound which can be used for preventing and treating acute kidney injury diseases.

The research of the applicant in the year discovers that FXR is expressed at high level in epithelial cells of proximal tubules and collecting ducts of kidney, is expressed to different degrees in other tubular segments, glomeruli and mesenchymal cells, is involved in reabsorption of water and salt of kidney, and plays an important role in establishing and maintaining the state of marrow hypertonicity in kidney. Research reports that FXR activation can be obviously improved, FXR gene defect is obviously aggravated, and the occurrence and development of diabetic nephropathy of mice are realized. FXR plays a protective role in alleviating cisplatin and renal ischemia reperfusion-induced acute kidney injury.

Therefore, the physiological function of FXR in the kidney and the effect of FXR in the generation and treatment of kidney diseases are further researched, the efficient low-toxicity natural agonist for targeting and activating the FXR is searched, and the method has important significance for deeply exploring new targets and new drug research and development for treating the kidney diseases.

The specific technical scheme of the invention is as follows:

the invention discloses an application of FXR agonist in preparing a medicine for preventing and/or treating kidney diseases.

Preferably, the FXR agonist is alisol B-23-acetate.

Preferably, the kidney disease is acute kidney injury.

Preferably, the medicine takes the FXR agonist as an active ingredient and also comprises pharmaceutically acceptable auxiliary materials.

The second of the present invention discloses a medicament for preventing and/or treating kidney diseases, which comprises an FXR agonist.

Preferably, the medicament has an FXR agonist as an active ingredient.

Preferably, the dosage form of the medicament comprises: powder, paste, granule, pill, tablet, capsule, granule, soft extract, decoction or injection.

On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.

The invention verifies that the alisol B-23-acetate serving as a nuclear receptor FXR agonist can prevent and treat acute kidney injury, and the specific research scheme is that a wild type C57BL/6 mouse is utilized to prove that the alisol B-23-acetate can relieve the acute kidney injury.

In order to achieve the above purpose, in some preferred embodiments of the present invention, the following specific technical solutions are adopted:

1. renal ischemia-reperfusion-induced acute kidney injury (RIRI) model: mice were fasted for 12h before surgery and had free access to water. Chloral hydrate 3% (100. mu.l/10 g) was anesthetized by intraperitoneal injection. The abdominal cavity was opened, the kidneys were visible, the right kidney was excised, and the left renal artery was rapidly clamped with an arterial clamp. The vessel clamp was loosened after 30min of ischemia to restore blood flow and the renal recovery was observed. And (4) sewing the opening, after the mouse is awake, putting the mouse back into the clean cage, putting the mouse back into the breeding room for breeding, and regularly observing the state and death condition of the mouse and recording the state and death condition of the mouse. The control group was not subjected to ischemic treatment, and the other operations were the same. Reperfusion was performed for 24 hours and then the material was taken.

2. Detection of kidney function index: the method comprises the steps of respectively detecting the urea nitrogen content and the serum/urine creatinine content of mouse serum by using a commercial kit and a high performance liquid chromatography method, detecting the N-acetyl-beta-D-glucosaminidase (NAG) content of mouse urine by using a fluorescent probe, and detecting urine protein by using an SDS-PAGE polyacrylamide gel electrophoresis method.

3. Detection of renal pathological changes: pathological changes in mouse Kidney sections were detected using routine pathological detection techniques such as immunohistochemical staining of Kidney injury marker 1(Kidney injure marker 1, KIM1), hematoxylin-eosin (H & E) staining, glycogen (PAS) staining, and Masson staining.

4. Detection of tissue and apoptotic pathways: detecting the TUNEL staining positive rate of mouse kidney section cells by adopting a terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay (TUNEL) method; detecting the expression level and activity of apoptosis-related proteins (pro-Caspase 3, cleared-Caspase 3, Bcl2, Bax and the like) in the kidney by using a Western Blot method and the like;

5. detection of tissue and cell oxidative stress and endoplasmic reticulum stress levels: detection of oxidative stress-related molecules (malondialdehyde MDA, myeloperoxidase MPO, glutathione mercaptotransferase GSH-ST, and Hydrogen peroxide H) Using different commercial assay kits₂O₂Etc.) content; immunohistochemical staining of paraffin sections of mouse kidneys was performed using 4-hydroxynonenoic acid (4-HNE), 8-OXO-2' -hydroxydeoxyguanosine (8-OXO-dG,8-OXO), and glycoregulatory protein (Grp78) antibodies.

6. Detection of tissue and cellular immune responses: the inflammation-related molecules were quantitatively analyzed using real-time quantitative PCR and ELISA detection methods. Immunohistochemical staining of paraffin sections of mouse kidney was performed using antibodies such as F40/80.

Compared with the prior art, the invention has the following remarkable advantages and effects:

the invention firstly verifies that alisol B-23-acetate can relieve acute kidney injury. The invention provides alisol B-23-acetate as an FXR agonist, which has the possibility of being applied to prevention and treatment of acute kidney injury.

Drawings

FIG. 1 is a graph showing the results of reduction of renal function in RIRI induced by alisol B-23-acetate (AB23) (AIMS-26 significantly improves RIRI induced increase of BUN (A), sCr (B), CCR (C), urinary NAG (D) and urinary protein (E); p <0.001vs Sham, # # p <0.001vs RIRI, n ═ 6);

FIG. 2 is a graph showing the results of alisol B-23-acetate (AB23) in reducing renal structural damage caused by RIRI in the present invention (wherein: A. H & E staining results for paraffin wax section, B. PAS staining results for paraffin wax section, C. KIM1 immunohistochemical staining results for tubular injury marker, D.KIM1 immunohistochemical staining results semi-quantitative statistics, E.ATN fraction statistics, p <0.001vs Sham, # p <0.01vs RIRI, n ═ 6);

FIG. 3 is a graph showing the results of the reduction of apoptosis by RIRI of Alisol B-23-acetate (AB23) in the present invention (wherein: A. the result of TUNEL staining by paraffin section of kidney; B. caspase3 activity assay; C. Western Blot assay for the expression level of apoptosis-related proteins;. p <0.01vs Sham, # p <0.05vs RIRI, n ═ 6);

FIG. 4 is a graph showing the results of the reduction of cellular inflammatory response by RIRI by Alisol B-23-acetate (AB23) in accordance with the present invention (wherein: A. real-time quantitative PCR measures changes in mRNA levels of molecules associated with inflammatory pathways; B. immunohistochemical staining measures the expression of macrophage marker F4/80;. p <0.05,. p <0.01vs. Sham,. p <0.05,. p # p <0.01vs. RIRI, n ═ 6);

FIG. 5 is a schematic diagram showing the results of the method for relieving oxidative stress and endoplasmic reticulum stress caused by RIRI by Alisol B-23-acetate (AB23) (GSH-ST (A), H in kidney tissue detected by conventional biochemical kit)₂O₂(B) And the content of MDA (C); immunohistochemical staining method for detecting the expression of oxidative stress markers 4-HNE (D), 8-OXO (E) and endoplasmic reticulum stress marker Grp78 (F); p<0.05，**p<0.01，***p<0.001vs.Sham，#p<0.05，##p<0.01，###p<0.001vs.RIRI，n＝7-8)。

Detailed Description

The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.

The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.

Example 1

In this example, it was found that alisol B-23-acetate (AB23) can relieve renal function decline caused by acute renal injury (RIRI) induced by renal ischemia-reperfusion.

Serum Creatinine (sCr) and serum Urea Nitrogen (BUN) are common biochemical indicators for clinical examination of renal function, and are often used together with proteinuria to determine whether a patient has acute renal injury. The experimental mice were divided into four groups in this study, namely Sham (Sham) group, Sham + AB23 group, model (RIRI) group and model administration (RIRI + AB23) group.

Two groups of mice (Sham group/Sham + AB23 group) which are subjected to Sham operation are respectively subjected to intraperitoneal injection of DMSO or AB23 before 4 days, and are subjected to right-side kidney resection and left-side kidney dissociation on the fifth day and then are subjected to abdominal cavity suturing after 35 minutes; specifically, the mice were intraperitoneally injected with AB23 at a dose of 60mg/kg, and the concentration of AB23 was about 38.85 mmol/L. Wherein, AB23 is stored at-20 deg.C, and AB23 is weighed out for four days according to the required dosage by a precision balance, and dissolved to the required concentration by DMSO to obtain AB23 solution. The prepared AB23 solution is stored at 4 ℃ to avoid repeated freeze thawing.

In the model group (RIRI group/RIRI + AB23 group), mice were administered DMSO or AB23 intraperitoneal injections respectively 4 days before surgery, and a renal ischemia-reperfusion injury model was constructed on the fifth day. Serum, urine and kidneys were taken 24 hours after surgery, sCr and BUN in serum were measured, and protein in urine was measured.

The results show that the serum urea nitrogen (fig. 1A), serum creatinine (fig. 1B), endogenous blood Creatinine Clearance (CCR) (fig. 1C), urinary N-acetyl- β -D-glucosidase (NAG) (fig. 1D) and urinary protein (fig. 1E) levels were significantly increased in the RIRI group, while the increase in these renal function indices could be significantly improved after AB23 treatment. The above results suggest that AB23 can alleviate the decline in renal function caused by acute renal injury.

Example 2

This example demonstrates that alisol B-23-acetate (AB23) reduces renal structural damage caused by acute renal injury (RIRI) induced by renal ischemia-reperfusion.

The mouse kidney paraffin section is stained with hematoxylin-eosin (H & E) (figure 2A) and glycogen (PAS) (figure 2B), and the result shows that the RIRI group renal tubular epithelial cells are seriously damaged, obvious inflammation and apoptosis are caused, the renal tubular lumen is enlarged, the tube wall is thinned, the phenomena of protein tube type and the like are caused, and AB23 can improve the RIRI to cause the abnormal state of tissue cells. Immunohistochemical staining showed that the expression of Kidney Injury marker Kidney Injury Molecule 1(Kidney Injury Molecule 1, KIM1) was significantly increased, while AB23 could down-regulate the phenomenon of increased KIM1 expression caused by RIRI (fig. 2C-D). Acute tubular necrosis score (ATNscore) also suggested that AB23 could significantly alleviate the renal injury caused by RIRI (fig. 2E). Therefore, AB23 could reduce pathological damage to kidney structures caused by RIRI.

Example 3

This example demonstrates that alisol B-23-acetate (AB23) reduces apoptosis in renal ischemia-reperfusion-induced acute renal injury (RIRI).

TUNEL staining showed a significant increase in the number of apoptotic cells in kidney tissue in RIRI mice, and AB23 decreased RIRI-induced apoptosis (fig. 3A). Fluorescence probe detection showed that the activity of Caspase 3(Caspase 3) containing cysteine in apoptotic performers was significantly increased after RIRI, while AB23 significantly decreased the increase in Caspase3 activity caused by RIRI (FIG. 3B). The Western Blot results showed that RIRI induced alterations in expression of the apoptosis-related molecules clear-Caspase 3, Bax and Bcl2 could be significantly alleviated by AB23 (FIG. 3C). The results indicate that AB23 can reverse the expression of apoptosis related molecules, inhibit programmed cell death and restore the normal physiological state of cells.

Example 4

This example demonstrates that alisol B-23-acetate (AB23) reduces the cellular inflammatory response caused by acute renal injury (RIRI) induced by renal ischemia-reperfusion:

real-time quantitative PCR is utilized to detect the expression levels of related inflammatory molecule mRNAs such as chemotactic factors CCL2, CXCL2, adhesion factor ICAM1, early inflammatory response factors TNF alpha and IFN gamma, interleukins IL2 and IL6, nonspecific inflammatory response factor NOS2 and the like in kidney tissues, and AB23 is found to be capable of obviously inhibiting the expression increase of the molecules in the RIRI group (fig. 4A). Staining of mouse epidermal growth factor-like receptor (EMR1), also known as F4/80, as a macrophage molecular marker by immunohistochemistry, revealed increased expression of F4/80 in the RIRI group, while AB23 reduced the RIRI-induced increase in F4/80 expression (FIG. 4B). The above results suggest that AB23 can reduce the inflammatory response caused by RIRI by alleviating inflammatory infiltration and reducing expression of the relevant inflammatory molecules.

Example 5

This example studies that alisol B-23-acetate (AB23) relieves oxidative stress and endoplasmic reticulum stress caused by acute kidney injury (RIRI) induced by renal ischemia-reperfusion:

by using a biochemical kit to detect glutathione mercaptotransferase (GSH-ST) (figure 5A) promoting the combination of active oxygen and glutathione, active oxygen molecular hydrogen peroxide (H2O2) (figure 5B) and membrane lipid peroxidation product Malondialdehyde (MDA) (figure 5C) induced by active oxygen accumulation in kidney tissue homogenate, RIRI can induce the content increase of GSH-ST, H2O2 and MDA, and AB23 can obviously relieve the increase of the indexes. Meanwhile, the expression of endogenous lipid peroxidation product 4-hydroxynonenal (4-HNE) (figure 5D), oxygen free radical sensitive indicator 8-hydroxydeoxyguanine (8-OXO-dG,8-OXO) (figure 5E) and endoplasmic reticulum stress important marker glucose regulatory protein 78(Grp78) (figure 5F) is detected by immunohistochemistry, and the results show that the expression levels of the molecules in the RIRI group are remarkably increased, and AB23 remarkably improves the expression change of the molecules. The above results suggest that AB23 can reduce the generation of cellular oxidative stress and endoplasmic reticulum stress caused by RIRI.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

Use of an FXR agonist in the preparation of a medicament for the prevention and/or treatment of kidney disease.
2. The use of claim 1, wherein the FXR agonist is alisol B-23-acetate.
3. The use of claim 1, wherein the kidney disease is acute kidney injury.
4. The use according to claim 1, wherein the medicament comprises an FXR agonist as an active ingredient and further comprises a pharmaceutically acceptable excipient.
5. A medicament for the prevention and/or treatment of kidney disease, characterized in that the medicament comprises an FXR agonist.
6. The medicament according to claim 5, characterized in that it comprises as active principle an FXR agonist.
7. The medicament of claim 5, wherein the dosage form of the medicament comprises: powder, paste, granule, pill, tablet, capsule, granule, soft extract, decoction or injection.