阅读说明：本技术 藤黄酸作为制备预防或治疗肾脏缺血-再灌注损伤药物的用途 (Application of gambogic acid in preparation of medicine for preventing or treating renal ischemia-reperfusion injury ) 是由 陈刚 李亚坤 韩珍旖 于 2021-11-18 设计创作，主要内容包括：本发明公开了一种藤黄酸作为制备预防或治疗肾脏缺血-再灌注损伤药物的用途。本发明通过对传统中药藤黄的提取物藤黄酸进行动物试验和细胞实验,发现一定剂量的藤黄酸能够显著减轻小鼠肾脏缺血-再灌注损伤,这种保护作用主要是通过增强线粒体自噬来实现,这为预防或治疗肾脏IRI提供了重要的技术支持。临床上可以采用藤黄酸冻干粉针配制注射液,通过静脉注射给药,给药时间是在获取移植器官前24h之内对供体进行至少一次静脉推注,静脉推注的有效剂量每次为4～40mg/m～(2)。本发明已证实藤黄酸在肾脏保护和修复中的新用途,这也为医学科研人员在临床上探索治疗心脏、肝脏、肺等其他器官的缺血-再灌注损伤提供了新的选择方案或思路。(The invention discloses an application of gambogic acid in preparing a medicament for preventing or treating renal ischemia-reperfusion injury. Animal experiments and cell experiments are carried out on gambogic acid which is an extract of traditional Chinese medicine gamboge, and the gambogic acid with a certain dose is found to be capable of remarkably relieving mouse renal ischemia-reperfusion injury, and the protection effect is mainly realized by enhancing mitochondrion autophagy, so that important technical support is provided for preventing or treating renal IRI. Clinically, the gambogic acid freeze-dried powder injection can be prepared into injection for intravenous injection administration, and the administration time isPerforming intravenous injection on the donor at least once within 24 hours before the transplanted organ is obtained, wherein the effective dose of the intravenous injection is 4-40 mg/m each time 2 . The invention proves the new application of the gambogic acid in kidney protection and repair, and provides a new selection scheme or thought for medical researchers to clinically explore and treat ischemia-reperfusion injury of other organs such as heart, liver, lung and the like.)

1. Application of gambogic acid in preparing medicine for preventing or treating renal ischemia-reperfusion injury is provided.

2. Use of gambogic acid according to claim 1 for the preparation of a medicament for the prevention or treatment of renal ischemia-reperfusion injury, wherein: the medicament is any acceptable medicament containing gambogic acid active ingredients in pharmacy.

3. Use of gambogic acid according to claim 2 for the preparation of a medicament for the prevention or treatment of renal ischemia-reperfusion injury, wherein: the preparation is tablet, capsule, dripping pill, lyophilized powder for injection or injection containing gambogic acid active ingredient.

4. Use of gambogic acid according to claim 3 for the preparation of a medicament for the prevention or treatment of renal ischemia-reperfusion injury, wherein: the medicament is garcinolic acid freeze-dried powder injection.

5. Use of gambogic acid according to claim 4 for the preparation of a medicament for the prevention or treatment of renal ischemia-reperfusion injury, wherein: the gambogic acid freeze-dried powder injection is clinically administered by a venous route, and the administration time is to carry out intravenous injection on a donor within 24 hours before a transplanted organ is obtained.

6. Use of gambogic acid according to claim 5 for the preparation of a medicament for the prevention or treatment of renal ischemia-reperfusion injury, wherein: the administration time is that the donor is subjected to first intravenous injection 24-12 hours before the transplanted organ is obtained, and the donor is subjected to second intravenous injection 2-1 hour before the transplanted organ is obtained.

7. Use of gambogic acid according to claim 6 for the preparation of a medicament for the prevention or treatment of renal ischemia-reperfusion injury, wherein: the administration time is that the donor is subjected to a first intravenous bolus injection 12 hours before the transplanted organ is obtained, and the donor is subjected to a second intravenous bolus injection 1 hour before the transplanted organ is obtained.

8. Use of gambogic acid according to claim 5 or 6 or 7 for the preparation of a medicament for the prevention or treatment of renal ischemia-reperfusion injury, wherein: the effective dose of the intravenous injection is 4-40 mg/m each time²In the unit of mg/m²Represents the amount of drug in milligrams per square meter of body surface area of the donor.

9. Use of gambogic acid according to claim 8 for the preparation of a medicament for the prevention or treatment of renal ischemia-reperfusion injury, wherein: the effective dose of the intravenous injection is 8-16 mg/m each time²。

10. Use of gambogic acid according to claim 9 for the preparation of a medicament for the prevention or treatment of renal ischemia-reperfusion injury, wherein: the effective dose of the intravenous injection is 12mg/m each time²。

Technical Field

The invention belongs to the technical field of medicines, mainly relates to a new pharmaceutical application of gambogic acid, and particularly relates to an application of gambogic acid in preparing a medicine for preventing or treating renal ischemia-reperfusion injury.

Background

Ischemia-reperfusion injury (IRI) is one of the most common causes of acute kidney injury, and is commonly seen in patients with renal transplantation, cardiac surgery, trauma, and shock. For example, kidney transplantation is currently the most effective means of treating end-stage renal disease. However, the process of kidney transplantation includes the removal of a kidney from a donor, the preservation of the kidney in vitro, and the revascularization and blood reperfusion for the kidney implantation into a recipient, and thus warm ischemic injury in the donor before the kidney harvest, cold ischemic injury during cold preservation, and reperfusion injury after restoration of blood flow after transplantation into the recipient inevitably occur. Such ischemia-reperfusion injury often results in delayed recovery of transplanted renal function, increased incidence of acute and chronic rejection, and thereby significantly affects the near and long term prognosis of the transplant. Therefore, renal IRI has been one of the research hotspots in the field of organ transplantation.

Since 1 month and 1 day 2015, organ donation of the citizen after the death has become a main transplant organ source in China, and the number of donations of dead organs is rapidly increased. According to the relevant records, 10793 cases of kidney transplantation operations are commonly performed in 2017 in China, and the number 2 lies in the world. The number of organ transplants in our country is rapidly increasing, but the problem of donor shortage is still very serious. Taking kidney dialysis patients as an example, the number of newly-increased dialysis patients in China is over 5 ten thousand every year, and the number of kidney transplantation cases in the year is just ten thousand. To alleviate this severe situation, marginal donors are incorporated into donor pools. However, such donors often experience repeated hypotension, hypoxemia, and cardiopulmonary resuscitation, resulting in more severe renal IRI following renal transplantation, which in turn significantly affects the near-term and long-term effects of the transplantation. Therefore, research and development of a medicament which has feasibility in clinical application and can effectively prevent and treat the IRI of the kidney of a patient have very important significance for transplantation medicine.

Currently, medical researchers have achieved great performance in basic research on renal IRI, and many of them show good effects in animal and cell experiments, but fail to meet the needs of solubility, safety and effectiveness in the subsequent clinical transformation process. Therefore, effective means for protecting the renal IRI that can be used in clinical kidney transplantation are still extremely lacking.

In order to solve the above problems, in addition to the intensive research on chemical drugs, researchers have attempted to find a Chinese medicinal composition that can protect the IRI of the kidney. The active ingredients in traditional Chinese medicine and natural plants have wide pharmacological activity, wherein gambogic acid is the main active ingredient of the traditional Chinese medicine gamboge. Experiments show that the gambogic acid has wide pharmacological action and obvious antitumor action, and the gambogic acid for injection is used for clinical antitumor research in China and shows good human body tolerance and safety. For example, a gambogic acid subject group at a medical university in China cooperates with a pharmaceutical enterprise to study the role of gambogic acid in tumor resistance, and gambogic acid lyophilized powder for injection is developed, and the gambogic acid lyophilized powder for injection shows small toxic and side effects, good human tolerance and good safety in clinical trials at stages I, II and III, but due to the fact that the gambogic acid lyophilized powder for injection has no obvious clinical advantage in treating malignant tumors, the subsequent study of the new drug can only be stopped by clinical excellent evaluation, which is very surprising.

Recent experiments of medical researchers also show that gambogic acid can play a good role in protecting experimental lung and hepatic fibrosis, and researches prove that gambogic acid has a certain anti-inflammatory effect, but whether gambogic acid can play a role in protecting kidney IRI is not reported. Renal IRI is very common in clinic, but at present, there is still a lack of drugs that can effectively treat renal IRI in clinic. Therefore, the drug which has the protective effect on the renal IRI is urgently expected to be provided for clinical use in the field.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, provides the application of gambogic acid in preparing a medicament for preventing or treating renal ischemia-reperfusion injury, and develops a new medicament for renal transplantation operation, which can intervene renal IRI in time.

In order to realize the purpose, the invention discloses application of a traditional Chinese medicine gamboge extract in preparing a new medicine, namely application of gambogic acid in preparing a medicine for preventing or treating renal ischemia-reperfusion injury.

Further, the medicament is any pharmaceutically acceptable medicament containing gambogic acid as an active ingredient.

Furthermore, the medicament is a tablet, a capsule, a dripping pill, a freeze-dried powder injection or an injection containing the gambogic acid active ingredient.

Still further, the medicament is garcinolic acid freeze-dried powder injection.

Preferably, the gambogic acid freeze-dried powder injection is clinically administered by intravenous route, and the administration time is to carry out intravenous bolus injection on a donor within 24 hours before the transplanted organ is obtained.

Furthermore, the administration time is that the donor is subjected to first intravenous bolus injection 24-12 hours before the transplanted organ is obtained, and the donor is subjected to second intravenous bolus injection 2-1 hours before the transplanted organ is obtained.

Further, the administration time is a first bolus of the donor 12h before the transplant organ is harvested and a second bolus of the donor 1h before the transplant organ is harvested.

Preferably, the effective dose of the intravenous bolus injection is 4-40 mg/m each time²In the unit of mg/m²Represents the amount of drug in milligrams per square meter of body surface area of the donor. In the medical field, the body surface area of the human body (donor) is commonly calculated as follows:

when the body weight is less than or equal to 30kg, the body surface area (m)²) 0.035 × body weight (kg) + 0.1;

body surface area (m) when body weight > 30kg²) 1.05+ [ body weight (kg) -30 ] × 0.02.

The effective dose of gambogic acid for donor can be determined within the above range according to various factors, for example, adjustment according to the body weight, age, physical condition and the like of the donor.

Further, the effective dose of the intravenous injection is 8-16 mg/m each time²。

Further, the effective dosage of the intravenous bolus is 12mg/m each time²。

The invention researches the effect of gambogic acid on the renal IRI and discusses the possible mechanism of the effect by constructing a mouse renal IRI model and adopting the gambogic acid to carry out intraperitoneal injection administration on the mouse renal IRI model. Experimental data show that after a kidney IRI mouse is pretreated by the gambogic acid, the blood biochemical detection and the staining results of the kidney tissues HE, TUNEL and MPO show that compared with a kidney IRI mouse control group, the levels of serum creatinine and urea nitrogen are obviously reduced, and the damage, apoptosis and neutrophil infiltration conditions of the kidney tissues are obviously reduced. Results of transmission electron microscopy, immunofluorescent staining and Western blot (Western blot) analysis show that phenomena of mitochondrial damage, active oxygen accumulation and autophagy and inhibition of mitochondrial autophagy are all significantly reduced in IRI mice after gambogic acid pretreatment compared to the IRI mouse control group. In addition, pretreatment of mice with a mitochondrial autophagy agonist also significantly improved ischemia-reperfusion-induced renal dysfunction, renal tissue injury, apoptosis, and neutrophil infiltration. However, when the mitophagy inhibitor was used in combination with gambogic acid, the protective effect of gambogic acid pretreatment on renal IRI mice was significantly impaired. These experimental results demonstrate that, in classical renal IRI mice, gambogic acid can effectively improve renal IRI by enhancing mitophagy, and thus gambogic acid can be used to prepare a medicament for preventing or treating renal IRI.

The invention has the beneficial effects that: animal experiments and cell experiments are carried out on gambogic acid which is an extract of traditional Chinese medicine gambogic acid, the effect of the gambogic acid in the kidney IRI is explored, a certain dose of gambogic acid is found to obviously relieve the mouse kidney ischemia-reperfusion injury, the protection effect is mainly realized by enhancing mitochondrion autophagy, important technical support is provided for clinically preventing or treating related diseases of the kidney IRI, and clinically, the gambogic acid lyophilized powder can be used for preparing injection and the injection is applied through a vein administration way. According to reasonable deduction of experimental results, the gambogic acid is also suitable for application in preparing medicaments for preventing or treating ischemia-reperfusion injury of organs such as heart, liver, lung and the like.

Drawings

FIG. 1, comprising FIGS. 1A-1G, shows the results of analysis of serum and renal tissue, hematology and HE, TUNEL and MPO staining after 8 week-old C57 mice were pretreated with solvent or Gambogic Acid (GA), and then subjected to sham or left renal ischemia-reperfusion surgery, respectively, and 24h reperfusion. Wherein: FIG. 1A is a bar graph comparing Serum Creatinine (Serum Creatinine) and urea nitrogen (BUN) levels for each group; FIG. 1B is a graph comparing HE staining results of various groups of kidney tissues; FIG. 1C is a bar graph comparing the lesion scores of various groups of renal tissue specimens; FIG. 1D is a graph comparing TUNEL staining of kidney tissues in various groups, DAPI staining of nuclei; FIG. 1E is a bar graph comparing the number of apoptotic cells per high power (400-fold) of microscopic field for each group of renal tissue specimens; FIG. 1F is a graph comparing MPO staining results for various groups of kidney tissues; FIG. 1G is a bar graph comparing the number of MPO positive cells per high power field of view for each group of kidney tissue specimens. In each figure, Sham group is solvent pretreatment + Sham group; RIRI group is solvent pretreatment + renal ischemia reperfusion injury group; the GA + RIRI group is a gambogic acid pretreatment and kidney ischemia reperfusion injury group.

Fig. 2, comprising fig. 2A-2D, shows the results of analysis of 8-week-old C57 mice pretreated with solvent or Gambogic Acid (GA), followed by sham or left renal ischemia-reperfusion surgery, collection of renal tissue after 24h reperfusion, transmission electron microscopy, and DHE immunofluorescence staining, respectively. Wherein: FIG. 2A is a comparative transmission electron microscope result of kidney tissues of each group; FIG. 2B is a histogram comparing the mitochondrial damage status of kidney tissue specimens from each group; FIG. 2C is a graph comparing DHE immunofluorescent staining results for various groups of kidney tissues; FIG. 2D is a bar graph comparing DHE mean fluorescence intensity for each group of kidney tissue specimens. In each figure, Sham group is solvent pretreatment + Sham group; RIRI group is solvent pretreatment + renal ischemia reperfusion injury group; the GA + RIRI group is a gambogic acid pretreatment and kidney ischemia reperfusion injury group.

Fig. 3, which includes fig. 3A-3I, shows the results of analysis of 8-week-old C57 mice pretreated with solvent or Gambogic Acid (GA), followed by sham or left renal ischemia-reperfusion surgery, collection of renal tissue after 24h reperfusion, Western blot, transmission electron microscopy, and immunofluorescence staining, respectively. Wherein: FIG. 3A is a Western blot comparison of LAMP1, LC3-II, p62 and β -actin in various groups of kidney tissues; FIG. 3B is a histogram of the expression levels of LAMP1, LC3-II and p62 in various groups of kidney tissues; FIG. 3C is a comparative graph of TEM results of kidney tissues of each group; FIG. 3D is a Western blot comparison of PINK1, Parkin and β -actin in various groups of kidney tissues; FIG. 3E is a bar graph comparing the expression levels of PINK1 and Parkin in various groups of kidney tissues; FIG. 3F is a graph comparing immunofluorescence results for LC3 and Tom20 for various groups of kidney tissue; FIG. 3G is a graph comparing immunofluorescence results for Parkin and Tom20 for various groups of kidney tissue; FIG. 3H is a bar graph of the number of yellow spots (representing LC3 co-localization with Tom 20) per renal tubule and the percentage of mitochondria containing yellow spots (representing Parkin and Tom20 co-localization) relative to all mitochondria in each group of renal tissues; FIG. 3I is a comparative transmission electron microscope result of kidney tissues of each group. In each figure, Sham group is solvent pretreatment + Sham group; RIRI group is solvent pretreatment + renal ischemia reperfusion injury group; the GA + RIRI group is a gambogic acid pretreatment and kidney ischemia reperfusion injury group.

Fig. 4, which includes fig. 4A-4I, shows the results of an 8 week old C57 mouse treated with either solvent, pre-treatment with the mitochondrial autophagy agonist rapamycin (Rapa) or Gambogic Acid (GA), or with the mitochondrial autophagy inhibitor 3-MA 1h before each Gambogic Acid (GA) administration or with the mitochondrial autophagy inhibitor Mdivi-1 30min before administration, followed by sham or left renal ischemia-reperfusion surgery, respectively, collection of serum and renal tissue after 24h reperfusion, hematology and staining of HE, TUNEL, MPO and DHE. Wherein: FIG. 4A is a bar graph comparing serum creatinine and urea nitrogen levels for each group; FIG. 4B is a graph comparing HE staining results for various groups of kidney tissues; FIG. 4C is a bar graph comparing the lesion scores of various groups of renal tissue specimens; FIG. 4D is a graph comparing TUNEL staining of kidney tissues in various groups; FIG. 4E is a histogram of apoptotic cell numbers per hyperscope field for each group of renal tissue specimens; FIG. 4F is a comparison of MPO staining results for various groups of kidney tissues; FIG. 4G is a bar graph comparing the number of MPO positive cells per high power field of view for various groups of kidney tissue specimens; FIG. 4H is a graph comparing DHE immunofluorescent staining results for various groups of kidney tissues; FIG. 4I is a bar graph comparing DHE mean fluorescence intensity for each group of kidney tissue specimens. In each figure, Sham group is solvent pretreatment + Sham group; RIRI group is solvent pretreatment + renal ischemia reperfusion injury group; the Rapa + RIRI group is a rapamycin pretreatment + renal ischemia reperfusion injury group; the GA + RIRI group is a gambogic acid pretreatment and kidney ischemia reperfusion injury group; the 3-MA + GA + RIRI group is 3-MA and gambogic acid pretreatment + kidney ischemia reperfusion injury group; the Mdivi-1+ GA + RIRI group is Mdivi-1 and the gambogic acid pretreatment + renal ischemia reperfusion injury group.

Fig. 5 includes fig. 5A to 5B. Wherein: FIG. 5A shows the passage of human renal tubular epithelial cell line HK-2 cells through Gambogic Acid (GA) and cobalt chloride (CoCl)₂) Detecting a contrast map of apoptosis by flow cytometry after treatment; FIG. 5B is a bar graph comparing the apoptosis rates of the various groups.

Fig. 6 includes fig. 6A to 6D. Wherein: FIG. 6A shows the interaction of human renal tubular epithelial cell line HK-2 cells with 3-methyladenine (3-MA), rapamycin (Rapa), Gambogic Acid (GA), and cobalt chloride (CoCl)₂) Detecting a contrast map of apoptosis by flow cytometry after treatment; FIG. 6B is a histogram comparing the apoptosis rates of the various groups; FIG. 6C is the passage of human renal tubular epithelial cell line HK-2 cells through Gambogic Acid (GA), cobalt chloride (CoCl)₂) Or bavero mycin a1(Baf-a1) after treatment flow cytometry to detect apoptosis; FIG. 6D is a histogram comparing the apoptosis rates of the various groups.

Fig. 7 includes fig. 7A to 7B. Wherein: FIG. 7A shows the transfection of human renal tubular epithelial cell line HK-2 cells with NC siRNA or Parkin siRNA followed by Gambogic Acid (GA) and cobalt chloride (CoCl)₂) Detecting a contrast map of apoptosis by flow cytometry after treatment; FIG. 7B is a bar graph comparing the apoptosis rates of the various groups.

Detailed Description

The present invention will be described in further detail below with reference to the drawings, examples and experimental examples. Of course, the scope of protection of the invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit of the invention. The present invention has been described generally and/or specifically with respect to materials used in experiments and test methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is described herein in as detail as possible. The following examples further illustrate the invention without limiting it. Any equivalent changes in form only, but not in material, made in accordance with the present inventive concept should be considered as within the scope of the present invention.

Example 1

The injection prepared from the gambogic acid freeze-dried powder injection can be used for administering the drug to the donor by intravenous injection before a kidney transplantation operation, the drug administration time is that the donor is subjected to the intravenous injection for the first time 24-20 hours before the transplanted organ is obtained, and the donor is subjected to the intravenous injection for the second time 2-1 hours before the transplanted organ is obtained. The effective dose of the medicine is 4-40 mg/m for each time²Can be adjusted according to the body weight, age, and physical condition of the donor, wherein the unit is mg/m²Represents the amount of drug in milligrams per square meter of body surface area of the donor. For example, the effective dose of each intravenous bolus is selected to be 14-16 mg/m²The composition is used for preventing donor from suffering from renal ischemia-reperfusion injury, or repairing the renal ischemia-reperfusion injury.

Example 2

The injection prepared from the gambogic acid freeze-dried powder injection is used for administering the drug to the donor through intravenous injection before a kidney transplantation operation, the drug administration time is that the first intravenous injection is carried out on the donor 14-12 hours before the transplanted organ is obtained, and the second intravenous injection is carried out on the donor 2-1 hours before the transplanted organ is obtained. The effective dose of each intravenous bolus injection is selected to be 8-10 mg/m²The composition is used for preventing donor from suffering from renal ischemia-reperfusion injury, or repairing the renal ischemia-reperfusion injury.

Example 3

The injection prepared from the gambogic acid lyophilized powder injection is administered to a donor by intravenous injection before a kidney transplantation operation, wherein the administration time is that the donor is subjected to the intravenous injection for the first time 12h before a transplanted organ is obtained, and the donor is subjected to the intravenous injection for the second time 1h before the transplanted organ is obtainedPulse bolus injection. The effective dose of each intravenous injection is 11-13 mg/m²The composition is used for preventing donor from suffering from renal ischemia-reperfusion injury, or repairing the renal ischemia-reperfusion injury.

In order to confirm the excellent efficacy of the gambogic acid of the present invention in the application of drugs for preventing or treating renal ischemia-reperfusion injury, the results obtained in various pharmacological basic studies are further described below. In the experiment, commercially available gambogic acid powder is dissolved to prepare the gambogic acid injection. For convenience of description, abbreviations for concepts, meanings or names in each item of pharmacology are as follows:

GA: gambogic acid;

and (3) a Serum: serum;

creating: creatinine;

BUN: urea nitrogen;

HE: hematoxylin-eosin staining method;

TUNEL: in situ terminal transferase labeling;

DAPI: 4', 6-diamidino-2-phenylindole dihydrochloride;

HPF: high power mirror field of view;

MPO: myeloperoxidase;

DHE: (ii) a dihydroethidine;

LAMP-1: lysosomal associated membrane glycoprotein 1A;

LC-3: microtubule-associated protein 1-light chain 3;

LC-3I: microtubule-associated protein 1-light chain 3-I;

LC-3 II: microtubule-associated protein 1-light chain 3-II;

beta-actin: beta actin;

PINK 1: PTEN-induced kinase 1;

parkin: parkin RBR E3 ubiquitin protein ligase;

tom 20: mitochondrial outer membrane translocase 20;

merge: fusing pictures;

rapa: rapamycin;

3-MA: 3-methyladenine;

mdivi-1: mitochondrial fission inhibitor 1;

Baf-A1: baverromycin a 1;

NC siRNA: negative control siRNA.

Annexin V: annexin V;

PI: propidium iodide.

Experimental example 1

Effect of Gambogic acid on Kidney ischemia-reperfusion injury model mice

The following experiments prove that the gambogic acid can be used as a medicament for preventing or treating the renal IRI through experimental study on the influence of the gambogic acid on the renal injury degree of mice with the renal IRI.

The experimental procedure was as follows:

1. laboratory animal

SPF male C57BL/6J mice (6 weeks old) were purchased from Beijing Huafukang Biotech GmbH, and were housed in SPF animal houses at the laboratory animal center of the affiliated Hospital of the college of Hospital, Huazhong university of science and technology, and were kept on free diet for a 12h day/night period. All animal manipulations involved in this experiment were in accordance with the relevant provisions of the ethical committee on laboratory animals of the university of science and technology in china. After entering the barrier environment, 6-week-old C57 mice were acclimated for 2 weeks, and experimental treatment was started at 8-week-old mice.

Experimental grouping 1: 22 mice were randomly assigned to the solvent pretreatment + Sham (Sham) group (n ═ 6), the solvent pretreatment + Renal Ischemia Reperfusion Injury (RIRI) group (n ═ 8), and the gambogic acid pretreatment + renal ischemia reperfusion injury (GA + RIRI) group (n ═ 8).

Experimental grouping 2: 46 mice were randomly assigned to the solvent pretreatment + Sham (Sham) group (n ═ 6), the solvent pretreatment + Renal Ischemia Reperfusion Injury (RIRI) group (n ═ 8), the mitochondrial autophagy agonist rapamycin pretreatment + renal ischemia reperfusion injury (Rapa + RIRI) group (n ═ 8), the gambogic acid pretreatment + renal ischemia reperfusion injury (GA + RIRI) group (n ═ 8), the mitochondrial autophagy inhibitor 3-MA + renal ischemia reperfusion injury after gambogic acid pretreatment (3-MA + GA + RIRI) group (n ═ 8), and the mitochondrial autophagy inhibitor Mdivi-1+ gambogic acid pretreatment renal ischemia reperfusion injury (Mdivi-1+ RIRI) group (n ═ 8).

2. Kidney ischemia-reperfusion injury model

Each group of experimental mice was anesthetized with 1% sodium pentobarbital by intraperitoneal injection (80mg/kg), the abdomen was fixed upward on a constant temperature blanket at 32 ℃, and molding was started after the mice had stabilized respiratory heartbeat. The abdominal cavity is opened by taking the median incision of the abdomen, the right kidney is exposed, and the right kidney is excised after the renal pedicle and ureter are ligated with 6-0 silk thread. The left kidney was then exposed, the left renal pedicle was closed with a atraumatic vascular clamp after dissociation, and the mouse was immediately placed in a 32 ℃ infant incubator. The vascular clamp was removed after 34min, the color change of the kidney was observed to confirm good perfusion of the kidney, and then the intestinal tract was restored and the abdominal cavity was closed by double-layer continuous suturing. Sham group mice did not clamp the left renal pedicle and the rest of the procedure was the same as in the RIRI group. The whole operation process follows the principle of aseptic operation, the surgical instruments are sterilized by high-pressure steam, the mice are continuously put into the infant incubator after the operation, and the mice are put back to the mouse cage after the mice are awakened and are eaten normally.

3. Pharmaceutical intervention and outcome management

The intervention time of gambogic acid (intraperitoneal injection, 2mg/kg) and rapamycin (intraperitoneal injection, 2mg/kg) is 36h, 12h and 1h before ischemia, and the injection is carried out once respectively; the mitophagy inhibitor 3-MA is injected 1h before each injection of gambogic acid (i.e. intraperitoneal injection, 30 mg/kg); the mitophagy inhibitor Mdivi-1 is injected 30min before each gambogic acid injection (intraperitoneal injection, 25 mg/kg); sham and RIRI mice were injected intraperitoneally with an equal volume of solvent each time.

After 24h of reperfusion, collecting serum of each group of mice, killing the mice, taking the left kidney, removing the kidney capsule, longitudinally cutting the kidney into two halves, fixing one half in 4% neutral formaldehyde solution, and then respectively performing immunofluorescence staining on HE, TUNEL, MPO, LC3+ Tom20 and Parkin + Tom20 on each group of samples; putting the other half of the sample in an environment at-80 ℃, and performing DHE fluorescent staining and Western blot at the later stage (detecting LAMP1, LC3-II, p62, PINK1 and Parkin); or a little kidney tissue (about 1 mm. times.1 mm) is fixed in an electron microscope fixing solution and then detected by a transmission electron microscope. The above detection results are shown in fig. 1 to 4.

Fig. 1 (fig. 1A-1G) shows the effect of gambogic acid on ischemia-reperfusion-induced renal dysfunction, renal tissue injury, and inflammatory cell infiltration. Wherein: fig. 1A shows that ischemia-reperfusion resulted in significantly elevated serum creatinine (Cr) and urea nitrogen (BUN) levels in mice, while Cr and BUN levels were significantly reduced after gambogic acid pretreatment, indicating that the renal function of the mice was significantly improved. Fig. 1B and 1C show that gambogic acid pretreatment significantly reduced ischemia-reperfusion-induced kidney tissue injury in mice. Fig. 1D and 1E show that gambogic acid pretreatment significantly improved ischemia-reperfusion-induced apoptosis in mouse kidney tissue. Fig. 1F and 1G show that gambogic acid pretreatment significantly reduced the ischemia-reperfusion-induced infiltration of MPO-positive cells (i.e., neutrophils) in mouse kidney tissue. In the figure, # represents the <0.001vs Sham group and # represents the <0.001vs RIRI group.

Fig. 2 (fig. 2A-2D) shows the effect of gambogic acid on ischemia-reperfusion-induced mitochondrial damage and reactive oxygen species accumulation in renal tissue. Wherein: fig. 2A and 2B are transmission electron microscopy results showing that gambogic acid pretreatment significantly reduced ischemia-reperfusion-induced mitochondrial injury in mouse kidney tissue. Fig. 2C and 2D are DHE immunofluorescent staining results showing that mean fluorescence intensity of DHE in mouse kidney tissue was significantly reduced after gambogic acid pretreatment, indicating a reduction in active oxygen content. In the figure, # represents the <0.001vs Sham group and # represents the <0.001vs RIRI group.

The experimental data of fig. 1 and 2 show that the gambogic acid pretreatment can significantly improve the ischemia-reperfusion-induced renal tissue injury, so that gambogic acid can be used for preparing a medicament for preventing or treating renal ischemia-reperfusion injury.

Fig. 3 (fig. 3A to 3I) shows the effect of gambogic acid on autophagy and mitochondrial autophagy in renal tissue. Wherein: fig. 3A and 3B are Western blot results showing that the gambogic acid pretreatment of RIRI group significantly increased the expression level of LAMP1 and LC3-II and decreased the expression level of p 62. Fig. 3C is a transmission electron microscopy result showing that ischemia-reperfusion results in a significant reduction in the number of autophagosomes/autophagosomes (indicated by black arrows) in renal tissue, while the number of structures described above increased after gambogic acid pretreatment. The experimental results of fig. 3A to 3C illustrate that: ischemia-reperfusion results in significant inhibition of autophagy in renal tissue, which is mitigated by the gambogic acid pretreatment. Fig. 3D and 3E are Western blot results showing that the expression levels of PINK1 and Parkin in renal tissues of the gambogic acid pretreated group were significantly increased compared to the RIRI group. Fig. 3F to 3H show that ischemia-reperfusion results in significant inhibition of mitophagy, particularly Parkin-dependent mitophagy, in renal tissue, which is significantly reduced after gambogic acid pretreatment. Fig. 3I is a transmission electron microscopy result showing that ischemia-reperfusion results in a significant reduction in the number of mitophagosomes/lysosomes (indicated by black arrows) within renal tissue, while the number of structures described above increased after gambogic acid pretreatment. The results of fig. 3D to 3I illustrate: ischemia-reperfusion results in significant inhibition of mitochondrial autophagy in renal tissue, which could be mitigated by gambogic acid pretreatment. In the figure, <0.01, <0.001vs Sham group, # 0.01, # 0.001vs RIRI group.

Fig. 4 (fig. 4A-4I) shows the effect of inhibition of mitophagy on the anti-renal ischemia-reperfusion injury effect of gambogic acid. Wherein: FIG. 4A shows that serum creatinine (Cr) and urea nitrogen (BUN) levels were significantly reduced in the rapamycin pretreated group, which is an agonist for mitophagy, compared to the RIRI group, while Cr and BUN levels were significantly increased in the two groups after the use of the mitophagy inhibitor 3-MA or Mdivi-1, compared to the GA + RIRI group. The HE staining results of fig. 4B and 4C show that enhanced mitophagy improves ischemia-reperfusion-induced kidney tissue injury, while inhibition of mitophagy attenuates the protective effect of gambogic acid pretreatment on ischemia-reperfusion-induced kidney tissue injury. TUNEL staining results in fig. 4D and 4E show that enhanced mitophagy can improve ischemia-reperfusion-induced apoptosis in renal tissue, while inhibition of mitophagy attenuates the protective effect of gambogic acid pretreatment on ischemia-reperfusion-induced apoptosis in renal tissue. The MPO staining results of fig. 4F and 4G show that enhanced mitophagy reduces ischemia-reperfusion-induced neutrophil infiltration in renal tissue, while inhibition of mitophagy attenuates the inhibitory effect of gambogic acid pretreatment on ischemia-reperfusion-induced neutrophil infiltration in renal tissue. The DHE immunofluorescent staining results of fig. 4H and 4I show that enhanced mitophagy reduces ischemia-reperfusion-induced reactive oxygen species accumulation in renal tissue, while inhibition of mitophagy attenuates the inhibitory effect of gambogic acid pretreatment on ischemia-reperfusion-induced reactive oxygen species accumulation in renal tissue. In the figure, <0.05, <0.01, < 0.001.

The experimental data of fig. 3 and 4 show that the gambogic acid pretreatment can significantly reduce the inhibition effect of ischemia-reperfusion on mitophagy in kidney tissues, and the inhibition of mitophagy can significantly weaken the protective effect of the gambogic acid pretreatment on renal ischemia-reperfusion injury.

Experimental example 2

Effect of gambogic acid on hypoxic injury of renal tubular epithelial cells

Human renal tubular epithelial cell line HK-2 cells were cultured in 1640 medium containing 10% fetal bovine serum in 5% CO₂In a cell culture box at 37 ℃, the cells grow adherently. Using cobalt chloride (CoCl)₂) And (5) processing the cells for 24h to establish an in-vitro cell hypoxia injury model.

Cellular experiments one used the following grouping:

control group: no intervention is carried out;

CoCl₂group (2): imparting CoCl₂Stimulating for 24h (400 mu M);

GA+CoCl₂group (2): cells were pretreated with GA (100nM) for 4h, followed by CoCl₂(400. mu.M) for 24 h.

After the cells are treated as above, the cells are collected and subjected to flow cytometry to detect the apoptosis. The detection results are shown in fig. 5 (fig. 5A to 5B). FIGS. 5A and 5B show that gambogic acid pretreatment can significantly mitigate CoCl₂Mimicking hypoxia-induced apoptosis.

The cell experiment two was grouped as follows:

control group: no intervention is carried out;

CoCl₂group (2): imparting CoCl₂Stimulating for 24h (400 mu M);

Rapa+CoCl₂group (2): cells were pretreated with Rapa (100nM) for 1h, then CoCl₂Stimulating for 24h (400 mu M);

GA+CoCl₂group (2): cells were pretreated with GA (100nM) for 4h, followed by CoCl₂Stimulating for 24h (400 mu M);

3-MA+GA+CoCl₂group (2): cells were pretreated with 3-MA (10mM) for 1h, with GA (100nM) for 4h, and with CoCl₂Stimulating for 24h (400 mu M);

GA+CoCl₂group + Baf-A1: cells were pretreated with GA (100nM) for 4h, followed by CoCl₂Stimulation (400. mu.M) for 24h in CoCl₂Baf-A1(100nM) was added 4h before the end of the treatment;

NC siRNA+GA+CoCl₂group (2): after NC siRNA cell transfection for 24h, cells were pretreated with GA (100nM) for 4h, followed by CoCl₂Stimulating for 24h (400 mu M);

Parkin siRNA+GA+CoCl₂group (2): after 24h transfection of Parkin siRNA cells, cells were pretreated with GA (100nM) for 4h, followed by CoCl₂(400. mu.M) for 24 h.

The unit of the above-mentioned amount is a molar concentration by volume (i.e., a concentration of the substance), which can be abbreviated as M, and represents mol/L. nM (nmol/L), μ M (μmol/L), mM (mmol/L), M (mol/L) are common. 1000 nM-1 μ M; 1000 μ M to 1 mM; 1000mM ═ 1M.

After the cells are treated as above, the cells are collected and subjected to flow cytometry to detect the apoptosis. The detection results are shown in fig. 6 (fig. 6A to 6D) and fig. 7 (fig. 7A to 7B). FIGS. 6A-6D show that pretreatment with rapamycin (Rapa), a mitochondrial autophagy agonist, significantly reduced CoCl₂Simulating hypoxia-induced apoptosis; the combined use of the mitophagy inhibitor 3-MA or Baf-A1 significantly attenuated the protective effect of gambogic acid on hypoxic injury. Fig. 7A-7B show that inhibition of Parkin-dependent mitochondrial autophagy significantly attenuated the protective effect of gambogic acid on hypoxic injury of cells.

The experimental data in conjunction with fig. 5-7 demonstrate that gambogic acid can significantly reduce hypoxia-induced renal tubular epithelial cell injury by enhancing mitochondrial autophagy.

In conclusion, by means of the technical scheme of the invention and by combining the animal and cell experimental data, the gambogic acid can obviously relieve the renal ischemia-reperfusion injury, the protection effect is mainly realized by enhancing the mitochondrion autophagy, so that the new application of the gambogic acid in the aspect of preventing or treating the renal ischemia-reperfusion injury medicine can be determined, and the new application provides a new selection scheme for medical researchers in clinical research on the renal ischemia-reperfusion injury.

The above description is only exemplary of the present invention and should not be taken as limiting the invention, as any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention should be included in the protection scope of the present invention.