阅读说明：本技术 Bix-01294在制备治疗急性肾损伤相关病症药物中的应用 (Application of BIX-01294 in preparation of medicine for treating acute kidney injury related diseases ) 是由 于晓文 张爱华 贾占军 黄松明 张玥 李书敏 曹诗涵 于 2021-09-18 设计创作，主要内容包括：本发明属于BIX-01294的新用途技术领域,公开了BIX-01294在制备治疗急性肾损伤相关病症药物中的应用,本发明提出了BIX-01294可以减轻顺铂诱导的急性肾损伤相关病症,包括急性肾小管损伤、肾功能、肾小管细胞凋亡、炎症因子的表达及分泌等病症,主要是通过抑制肾小管细胞的凋亡来减轻急性肾损伤。(The invention belongs to the technical field of new application of BIX-01294, and discloses application of BIX-01294 in preparation of a medicament for treating acute renal injury related diseases.)

Application of BIX-01294 in preparing a medicament for treating acute kidney injury-related diseases.

2. The use according to claim 1, wherein BIX-01294 is used in the manufacture of a medicament for alleviating a cisplatin-induced acute kidney injury-related disorder.

3. The use of claim 2, wherein BIX-01294 is used in the manufacture of a medicament for reducing acute kidney injury renal function induced by cisplatin.

4. The use of claim 2, wherein BIX-01294 is used in the preparation of a medicament for reducing cisplatin-induced tubular apoptosis.

5. The use of claim 2, wherein BIX-01294 is used in the manufacture of a medicament for reducing cisplatin-induced kidney inflammatory factor expression and secretion.

6. The use of claim 2, wherein BIX-01294 is used in the manufacture of a medicament for improving cisplatin-induced mitochondrial function in renal tubules.

Technical Field

The invention relates to the technical field of new application of BIX-01294, in particular to application of BIX-01294 in preparing a medicament for treating acute kidney injury related diseases.

Background

Acute Kidney Injury (AKI) refers to a clinical syndrome in which sudden renal function decline, with or without oliguria or anuresis, is a short-term sudden decline or loss of renal function caused by pathological changes in the glomerulus, tubule, renal interstitium, or blood vessels, and is manifested by sudden failure to maintain normal water-electrolyte balance, accumulation of in vivo metabolites, and symptoms such as azotemia, water and electrolyte disorders, metabolic acidosis, and the like. AKI is a clinical syndrome which can be secondary to various diseases, has the characteristics of acute onset, fast progression and high fatality rate, and is one of the important causes of chronic kidney diseases. It is a group of serious clinical syndrome, has high mortality, and can cause irreversible change of renal function due to delayed diagnosis or improper treatment, so that patients can enter into maintenance dialysis, thereby bringing great negative influence to social economy.

The prevalence community for AKI is 1%, 7.1% at hospital. The morbidity rate is 10% -80% of the annual hospital acquired AKI fatality rate, the fatality rate of the combined multi-organ failure is more than 50%, and the fatality rate requiring renal replacement therapy is 80%, so the AKI has high morbidity rate and mortality rate. Its main causes are ischemia, Renal toxic substance-induced or primary Renal disease, Renal Tubular Epithelial Cell (RTEC) injury is the main pathological basis of AKI. Under disease conditions, actively functioning RTECs are more susceptible to Acute Tubular Necrosis (ATN) caused by injury due to nephrotoxins and the like, resulting in AKI and renal failure.

Cisplatin (CP) is one of the most commonly used and most effective chemotherapeutic drugs in clinical treatment of solid tumors (including head, neck, ovary, etc.). Clinical investigations have shown that its clinical use is greatly limited due to its dose-dependent nephrotoxic effects. Cisplatin-induced AKI occurs at 25% to 35% and its major mechanisms of causing injury include induction of apoptosis by oxidative stress of renal tubular epithelial cells and the ability to trigger an inflammatory response that is involved in kidney injury. How to effectively prevent and alleviate AKI caused by cisplatin and better play the cisplatin anti-tumor effect becomes a problem to be solved urgently at present.

The loss of tubular cells, including tubular apoptosis and necrosis, in patients and animal models is considered to be a major cell involved in the development of AKI. In addition, the shed renal tubular cells and proteins form a tubular shape in the lumen to block the urine flow, and the damaged tubule may further promote renal interstitial fibrosis, interstitial inflammation and capillary vessel loss. How to prevent tubular cell loss is therefore critical to the treatment of AKI.

After the cisplatin enters the cell, the cisplatin can activate pro-apoptotic proteins such as Bax in a Bcl-2 family, so that a mitochondrial permeability transition pore is promoted to be opened, and pro-apoptotic factors such as cytochrome C and apoptosis inducing factors are released from mitochondria to cytoplasm to generate caspase cascade reaction, thereby finally causing apoptosis.

BIX-01294 is a specific inhibitor of G9 alpha Histone Methyltransferase (G9a Histone Methyltransferase). G9 α (also known as KMT1C or EHMT2) is the second histone methyltransferase reported. It belongs to one of the members of the Suv39H protein family containing the SET domain, is an important euchromatin HMT, and is mainly responsible for methylation of K9 and K27 sites in euchromatin domain histone H3. G9 alpha is mainly highly expressed in muscle, heart, liver and other tissues. G9 α regulates gene transcription by two different mechanisms: in one aspect, G9 α can promote methylation of histone or DNA in the promoter region of a gene, thereby inhibiting gene transcription; on the other hand, G9 α can act as a scaffold protein to recruit transcriptional activators and thereby activate gene transcription. In recent years, with the progress of research, it has been found that G9 α can regulate autophagy and cell differentiation, and plays an important role in various biological processes such as tumorigenesis, embryonic development, cognitive and adaptive behavior, and adipogenesis. The study shows that the administration of the G9 alpha inhibitor BIX-01294 can enhance the sensitivity of glioma cells to temozolomide and promote apoptosis; BIX-01294 has also been reported to ameliorate hyperalgesia caused by nerve injury; in both lung and kidney, it was found that BIX-01294 administered to inhibit G9 α improves pulmonary and renal interstitial fibrosis; it has also been found that BIX-01294 blocks the growth cycle of Plasmodium and treats malaria. However, BIX-01294 has not been reported to improve AKI caused by cisplatin.

Disclosure of Invention

The invention aims to provide a new application of BIX-01294 and an application of the BIX-01294 in preparing a medicament for treating acute kidney injury related diseases.

The invention discovers the application of BIX-01294 in medicaments for relieving the AKI related diseases induced by the cisplatin through two aspects of animal experiments and in vitro cell experiments.

The invention provides an application of BIX-01294 in preparing a medicament for treating acute kidney injury related diseases.

Further, the invention provides an application of BIX-01294 in preparing a medicament for alleviating cisplatin-induced Acute Kidney Injury (AKI) -related disorders.

Further, the invention provides an application of BIX-01294 in preparing a medicine for relieving cisplatin-induced AKI renal function.

Further, the invention provides an application of BIX-01294 in preparing a medicament for alleviating cisplatin-induced tubular cell apoptosis.

Further, the invention provides an application of BIX-01294 in preparing a medicine for reducing the expression and secretion of a cisplatin-induced kidney inflammatory factor.

Further, the invention provides an application of BIX-01294 in preparing a medicament for improving cisplatin-induced renal small-tube cell mitochondrial function.

The invention discloses the following technical effects:

the invention provides a new application of BIX-01294, and provides a new application of BIX-01294 in relieving cisplatin-induced acute renal injury related symptoms including acute tubular injury, renal function, tubular cell apoptosis, inflammatory factor expression and secretion and the like, wherein the symptoms are mainly used for relieving acute renal injury by inhibiting tubular cell apoptosis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 is a graph of the results of renal function and glycogen staining (PAS) after cisplatin modeling and treatment with BIX-01294, wherein A is the PAS staining after cisplatin modeling and treatment with BIX-01294 and B is the blood biochemical results after cisplatin modeling and treatment with BIX-01294;

FIG. 2 is a QPCR method and an ELISA method for studying the effect of BIX-01294 on the expression and secretion of cisplatin-induced renal inflammatory molecules;

FIG. 3 shows the QPCR method and Western blot to study the effect of BIX-01294 on cisplatin-induced tubular apoptosis and kidney injury-related molecules;

FIG. 4 shows the effect of BIX-01294 on cisplatin-induced tubular apoptosis, active oxygen and mitochondrial DNA copy number in vitro measured by flow cytometry and QPCR method, wherein A, B shows the apoptosis of cells measured by flow cytometry, C shows the level of mitochondrial active oxygen measured by flow cytometry, and D shows the change of mitochondrial DNA copy number in cells measured by QPCR method.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The alcohol fraction of the present invention refers to volume fraction.

The Western blot, the real-time fluorescent quantitative PCR, the PAS staining and the flow cytometry method have the following specific operation steps:

real-time fluorescence quantitative pcr (qpcr):

total RNA of kidney tissues is extracted, and the concentration and purity of the RNA solution are determined by spectrophotometry. Mu.g of gRNA was reverse-transcribed into cDNA using a reverse transcription kit (Takara, DaLian), and the change of different genes was examined according to the following reaction system.

a. Reaction system

PCR thermal cycling parameters

55℃-95℃,snap every 0.5℃,repeat 81 circles.

PAS dyeing:

the tissue was fixed with 4% paraformaldehyde for 48h, sectioned with paraffin, deparaffinized to water, rinsed with distilled water, and rinsed 3 times with 70% alcohol. Soaking in periodic acid alcoholic solution for 10min (the temperature of the solution is 17-20 deg.C), washing with 70% alcohol, adding into reducing solution for 1min (the temperature of the solution is 17-20 deg.C), washing with 70% alcohol, adding into colorless salt base fuchsin solution for 1-1.5h, and placing into 37 deg.C incubator in winter when the room temperature is low. Washing with flowing water for 10min, re-staining cell nucleus with Mayer \ s hematoxylin re-staining solution for 3-5min, differentiating with 1% hydrochloric acid alcohol, washing with flowing water, dehydrating, and sealing.

Annexin V/PI apoptosis double staining experiment:

inoculating tubular epithelial cells (RTEC) of logarithmic growth phase into 6-well plate, respectively administering cisplatin and BIX-01294, allowing the drugs to act for 24h, digesting the cells with EDTA-free pancreatin, centrifuging at 1000rpm for 5min, collecting the cells, and discarding the culture medium. The cells were washed twice with pre-cooled PBS solution, and 400. mu.L of 1 XBinding Buffer suspension cells were added thereto at a cell density of about 1X 10⁶cells/mL. And adding 5 mu Lannexin V and 5 mu L PI into each group of cell suspension, mixing the mixture evenly, and incubating the mixture for 15min at room temperature in a dark condition. Detecting by a flow cytometer within 1 h.

Mitochondrial membrane potential (JC-1) assay:

the decrease in mitochondrial membrane potential is a marker event in the early stages of apoptosis. The reduction of cell membrane potential can be easily detected by the conversion of JC-1 from red fluorescence to green fluorescence, and the conversion of JC-1 from red fluorescence to green fluorescence can also be used as a detection index of early apoptosis. Selecting cells in logarithmic phase, inoculating into 6-well plate, administering cisplatin and BIX-01294, allowing the drug to act for 24h, digesting the cells with EDTA-free pancreatin, centrifuging at 1000rpm for 5min, collecting the cells, and discarding the culture medium. The cells were washed twice with pre-cooled PBS solution, 1mL JC-1 staining working solution was added, and mixed well. The cells were incubated at 37 ℃ for 20min in a cell incubator. During the incubation, an appropriate amount of JC-1 staining buffer (1X) was prepared at a rate of 4mL of ultrapure water per 1mL of JC-1 staining buffer (5X) and placed in an ice bath. After incubation at 37 ℃ was complete, the supernatant was aspirated and washed 2 times with JC-1 staining buffer (1X). 2mL of cell culture medium was added and detected by flow cytometry over 1 h.

Reactive Oxygen Species (ROS) detection:

selecting cells in logarithmic phase, inoculating into 6-well plate, administering cisplatin and BIX-01294, allowing the drug to act for 24h, digesting the cells with EDTA-free pancreatin, centrifuging at 1000rpm for 5min, collecting the cells, and discarding the culture medium. The cells were washed once with pre-cooled PBS solution, 500. mu.L mitoSOX staining solution (1X) was added and mixed well. The cells were incubated at 37 ℃ for 20min in a cell incubator. After incubation at 37 ℃, the supernatant was removed by centrifugation, the cells were washed twice with PBS solution, and 500 μ L of cell culture medium was added and detected by flow cytometry within 1 hour.

The present invention will be described in detail below with reference to specific embodiments.

Example 1 Effect of BIX-01294 on cisplatin-induced acute Kidney injury Kidney function

Male C57BL/6 mice weighing 18-22 g were divided into 3 groups, i.e., a control group, a cisplatin model group, and a BIX-01294 treatment group (n-8).

Control group: injecting the same volume of medium into abdominal cavity 1 time a day for 4 days;

cisplatin model group: intraperitoneal injection, 20mg/kg, single administration;

BIX-01294 treatment group: BIX-01294 was administered 1 day in advance (i.p., 1.5. mu.g/. mu.L), followed by a single dose of cisplatin (i.p., 20mg/kg), BIX-01294 was treated for another 3 days, and blood was taken 72 hours after cisplatin injection, leaving kidney tissue.

FIG. 1A shows the result of PAS staining after modeling with cisplatin and treatment with BIX-01294. According to PAS staining results, the cis-platinum renal tubular structure is damaged, the protein tube type is formed, and BIX-01294 can obviously improve the kidney injury induced by cisplatin.

Blood samples were centrifuged (20min, 3000r/min) and serum Creatinine and Urea nitrogen were measured using Creatinine Kit (Creatinine Assay Kit (cat: K625-100, biolars)) and Urea nitrogen Kit (QuantiChrrom Urea Assay Kit (cat: DIUR-500, Hayward, Calif)). FIG. 1B shows the biochemical results of blood after cisplatin modeling and treatment with BIX-01294. from the results, it can be seen that the success of cisplatin modeling and the significant increase of serum creatinine and urea nitrogen suggest that the kidney is damaged. After BIX-01294 treatment, the renal function can be obviously improved, and the levels of creatinine and urea nitrogen are reduced compared with a model group, wherein p is less than 0.05. .

Example 2 Effect of BIX-01294 on cisplatin-induced expression and secretion of Kidney inflammatory molecules

The influence of BIX-01294 on the expression and secretion of cisplatin-induced renal inflammatory molecules was studied by QPCR and ELISA.

As shown in FIG. 2, the effect of BIX-01294 on the expression and secretion of cisplatin-induced renal inflammatory molecule was investigated by QPCR. In the cisplatin-induced AKI model, the expression levels of inflammatory factors MCP-1, IL-18 and ICAM-1 in the cisplatin model group are obviously increased compared with the control group, and p is less than 0.05. The BIX-01294 treatment group can obviously reduce the expression levels of MCP-1, IL-18 and ICAM-1, and p is less than 0.0001.

The result shows that BIX-01294 can obviously reduce the expression levels of MCP-1, IL-18 and ICAM-1 in the AKI model induced by cisplatin.

Example 3 Effect of BIX-01294 on cisplatin-induced tubular apoptosis and Kidney injury-associated molecules

The QPCR method is used for detecting the expression levels of the molecules related to the renal apoptosis and the renal tubular injury and the G9 alpha to determine the improvement condition of BIX-01294 on the AKI induced by the cisplatin, and the result is shown in figure 3, wherein the expression of the G9 alpha and the apoptosis related molecule Bax of the cisplatin model group is obviously increased; the expression of the injury related molecules KIM-1 and NGAL is obviously increased. And BIX-01294 treated group was able to significantly reduce the expression levels of G9 α, Bax, KIM-1 and NGAL.

The result shows that BIX-01294 can obviously reduce the expression level of cisplatin-induced renal apoptosis and loss-related molecules.

Example 4 Effect of BIX-01294 on cisplatin-induced tubular apoptosis, reactive oxygen species and mitochondrial DNA copy number in vitro

In vitro application of RTEC cells (mouse renal tubular epithelial cell line), selecting cells in logarithmic growth phase, inoculating the cells in a 6-well plate, respectively administering cisplatin (5. mu.g/mL) and BIX-01294 (2. mu.M), and detecting the apoptosis incidence rate of the tubular cells by using a flow cytometer after the drug action is 24 h. As shown in FIGS. 4A-B, the rate of apoptosis was significantly increased in the cisplatin model group, and about 38% of the rate of apoptosis was observed, while the rate of apoptosis was significantly decreased in the BIX-01294-treated group compared to the cisplatin model group, and about 20% of the rate of apoptosis was observed. As shown in FIG. 4C, the reactive oxygen species level in the cisplatin model cells was significantly increased compared to the control group, with P < 0.001. Compared with the cisplatin model group, the BIX-01294 treatment group can obviously reduce the intracellular active oxygen level, and P is less than 0.001. As shown in FIG. 4D, the copy number of mitochondrial DNA of the cells in the cisplatin model group was significantly reduced compared to the control group, with P < 0.0001. Compared with the cisplatin model group, the BIX-01294 treatment group can obviously increase the copy number of the cell mitochondrial DNA, and P is less than 0.05.

The result shows that the cisplatin stimulates the RTEC cells for 24h, can obviously induce the occurrence of apoptosis, and can cause the increase of the intracellular ROS level and the reduction of the mitochondrial DNA copy number. After being treated by BIX-01294, the composition can obviously reduce the occurrence of cisplatin-induced apoptosis, reduce the intracellular ROS level and increase the mitochondrial DNA copy number, and indicates that BIX-01294 is possible to improve cisplatin-induced mitochondrial function damage.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.