CN116115606A - Application of Xanthoxol in preparing medicament for treating cisplatin-induced acute kidney injury - Google Patents

Application of Xanthoxol in preparing medicament for treating cisplatin-induced acute kidney injury Download PDF

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CN116115606A
CN116115606A CN202310051172.0A CN202310051172A CN116115606A CN 116115606 A CN116115606 A CN 116115606A CN 202310051172 A CN202310051172 A CN 202310051172A CN 116115606 A CN116115606 A CN 116115606A
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cisplatin
xanthoxol
kidney injury
acute kidney
injury
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王龙
黄进宇
闪波
郑洁
莫绪明
杨运文
刘益海
梁平
高贝贝
王帅
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Hangzhou First Peoples Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A61K31/37—Coumarins, e.g. psoralen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract

The invention relates to application of Xanthotoxol in preparing a medicament for treating cisplatin-induced acute kidney injury. The invention provides application of Xanthoxol as a natural small molecular compound in medicines for relieving symptoms related to acute kidney injury induced by cisplatin by using Xanthoxol on a cisplatin-induced acute kidney injury mouse model and an in vitro cell model. The research result shows that the structure and function of acute kidney injury kidney can be obviously improved by utilizing Xanthoxol to perform intervention treatment on a cisplatin mouse model and in-vitro cells, and the death of tubular cell iron can be obviously improved. The scheme of the invention provides effective clinical medicines for preventing and treating acute kidney injury AKI.

Description

Application of Xanthoxol in preparing medicament for treating cisplatin-induced acute kidney injury
Technical Field
The invention relates to the field of pharmaceutical application, in particular to application of Xanthoxol in preparing a medicament for treating cisplatin-induced acute kidney injury.
Background
Cisplatin was widely used in clinic for treating various malignant tumors including lung cancer, bladder cancer, cervical cancer, head and neck malignant tumors and the like from the seventies of the last century, is one of the most effective and common chemotherapeutics for treating solid tumors, but because cisplatin has a killing effect on tumor cells and can cause a series of serious adverse reactions on normal tissues and organs, the clinical application of cisplatin is greatly limited. Adverse reactions of cisplatin mainly include a series of systemic organ injury reactions such as nephrotoxicity, which is the most common toxic and side effect, and about 1/3 of patients have renal dysfunction to cause acute renal failure after being treated by cisplatin, and the dose-related nephrotoxicity greatly limits clinical application of cisplatin. The mechanism of normal cell damage by cisplatin has long been unknown, and serial studies have found that DNA damage, inflammatory mediators, iron death, oxidative stress, autophagy, etc. caused by cisplatin may be responsible for normal tubular epithelial cell damage by cisplatin (Zheng Dong et al Cisplatin nephrin gateway: new insights and therapeutic implementations. Nat Rev nephorol. 2022).
In view of the effective anti-tumor effect of the platinum drugs and derivatives thereof, the platinum drugs are still the most widely applied clinical anti-tumor chemotherapeutics at present, however, the clinical use of the drugs is greatly limited by toxic and side effects such as kidney injury caused by the platinum drugs, and the effective drugs for treating cisplatin-induced acute kidney injury are lacking in clinic at present, so that the development of new effective drugs for treating cisplatin-induced acute kidney injury related drugs has important clinical significance.
Xanthoxol is also called 8-hydroxy psoralen, and has chemical formula of C 11 H 6 O 4 The structural formula is shown in the following figure:
Figure BDA0004058043430000011
the natural small molecular compound has a molecular weight of 202.16, is a linear furocoumarin with biological activity, exists in various plants, is mainly extracted from fructus cnidii fruits, has strong pharmacological activities such as anti-inflammatory, antioxidant, 5-HT (5-hydroxytryptamine) antagonism and neuroprotection (Zhongnv Ma et al, metabolism and Metabolic Inhibition of Xanthotoxol in Human Liver Microsons. Evence-Based Complementary and Alternative medicine.2016), and is mainly studied at present, but has no literature report on the related effects of the compound in treating central nerve injury related to cerebral hemorrhage.
Disclosure of Invention
The invention provides application of a natural small molecular compound Xanthoxol in preparing a medicament for treating cisplatin-induced acute kidney injury, and discovers that the Xanthoxol improves the cisplatin-induced acute kidney injury by up-regulating GPX4 protein expression for the first time.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
use of Xanthoxol in the manufacture of a medicament for treating cisplatin-induced acute kidney injury.
Xanthoxol ameliorates acute kidney injury by up-regulating protein levels of anti-iron death-related proteins.
The anti-iron death related protein is GPX4.
The improvement of acute kidney injury is the alleviation of injury or death of tubular epithelial cells.
Xanthotoxol down regulates NGAL protein and KIM-l protein levels in acute kidney injury kidney tissue.
GPX4 is a protein for resisting lipid peroxidation and can inhibit cell iron death, and the research of the invention discovers that Xanthoxol inhibits cell iron death of tubular epithelial cells by up-regulating GPX4 expression, so that AKI (acute kidney injury acute kidney injury) is improved, and the levels of NGAL and KIM1 protein serving as markers of acute kidney injury are also down-regulated.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides application of Xanthoxol as a natural small molecular compound in medicines for relieving symptoms related to acute kidney injury induced by cisplatin by using Xanthoxol on a mouse model and an in vitro cell model of acute kidney injury induced by cisplatin.
The research result shows that the structure and function of acute kidney injury kidney can be obviously improved by utilizing Xanthoxol to perform intervention treatment on a cisplatin mouse model and in-vitro cells, and the death of tubular cell iron can be obviously improved.
The scheme of the invention provides effective clinical medicines for preventing and treating acute kidney injury AKI.
Drawings
FIG. 1 shows that Xanthoxol significantly improves kidney injury and kidney function in cisplatin-induced acute kidney injury; in fig. 1, a is serum creatinine in each group of mice 72 hours after cisplatin injection; b is serum urea nitrogen of each group of mice 72 hours after cisplatin injection; c is glycogen PAS pathology staining of kidney tissue of each group of mice 72 hours after cisplatin injection; d is a pathologically stained tubular injury score based on panel C.
FIG. 2 shows that Xanthotoxol down-regulates NGAL and KIM-l protein levels in kidney tissue in a cisplatin-induced acute kidney injury model; in FIG. 2, A is the level of KIM-1 and NGAL protein, which are markers of acute kidney injury in kidney tissue of each group of mice, detected by western blot 72 hours after cisplatin injection; b is KIM-1 protein band gray scale quantitative statistics based on western blot detection; c is NGAL protein band gray scale quantitative statistics based on western blot detection.
FIG. 3 shows that Xanthoxol treatment significantly upregulates protein levels of GPX 4; in fig. 3, a is the level of GPX4 protein in the kidney tissue of each group of mice detected by western blot, and B is the quantitative statistics of the grayscale of the GPX4 protein band based on the western blot detection.
FIG. 4 shows that Xanthoxol treatment significantly reduced cisplatin-induced injury and death of tubular epithelial cells in an in vitro tubular epithelial cell model; in FIG. 4, A is the activity level of cells after 24 hours of treatment of HK with CCK8 at various concentrations of Xanthotoxol; b is the detection of changes in GPX4 mRNA levels in HK2 cells treated with Xanthotoxol (20. Mu.M) and 24 hours after cisplatin (10. Mu.g/ml) using fluorescent quantitative PCR (QRT-PCR); c is the detection of the level of LDH in the supernatant of HK2 cells after Xanthoxol (20. Mu.M) treatment and 24 hours after cisplatin (10. Mu.g/ml) treatment by ELISA; d is the activity level of HK2 cells detected with CCK8 after Xanthoxol (20. Mu.M) treatment and 24 hours after cisplatin (10. Mu.g/ml) treatment.
Detailed Description
The above-described matters of the present invention will be further described in detail by way of examples, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples, and all techniques realized based on the above-described matters of the present invention are within the scope of the present invention.
The experimental methods used in the examples below are conventional methods, and the reagents, methods and apparatus used are conventional in the art, unless otherwise indicated.
Example 1 materials and methods
1) Materials and reagents
Xanthoxol was purchased from MCE, NGAL was purchased from Abcam, KIM-1 was purchased from R & DSsystems, and GPX4 and beta-actin were purchased from proteontech; cell activity detection kit CCK8 is purchased from Nanjakyl corporation.
2) Cell culture and treatment
Human tubular epithelial cells (HK 2) were cultured in DMEM/F12 medium containing 10% fetal bovine serum, 0.5% penicillin and streptomycin at 37℃under 5% carbon dioxide and 95% air. To investigate the effect of Xanthoxol on cisplatin-induced tubular epithelial cell injury, culture was changed to DMEM/F12 medium without fetal bovine serum when HK2 was grown to 70% density and either pretreatment with Xanthoxol (stock solution in DMSO) for 1h or equivalent treatment with DMSO was added. After incubating the cells in an incubator for 24 hours, the cells were finally collected for fluorescent quantitative PCR (QRT-PCR) detection and for assays such as CCK8 analysis of fine activity.
3) Cisplatin-induced acute kidney injury mouse model and experimental group
The mice were randomly divided into four groups of 6 mice each, which were a blank control group (veccle group), a Xanthotol control group (Xanthotol group), a cisplatin injection model group (veccle+CDDP group) and a cispatin+Xanthotol group (Xanthotol+CDDP group), respectively.
Xanthoxol was first dissolved in DMSO, then a DMSO solution of Xanthoxol was dissolved in 40% PEG400 to give Xanthoxol injection.
a) The Xanthoxol control group and the cispratin+Xanthoxol group mice were given Xanthoxol injection preparations by intraperitoneal injection at a dose of 10mg/kg daily;
b) Mice from the placebo group were injected with cisplatin (cisplatin injection here refers to cisplatin injection model group and cisplatin +
The solvent (40%) was administered by intraperitoneal injection once daily 24 hours before cisplatin injection in Xanthoxol group
PEG400)；
c) Mice of the cispatatin + Xanthoxol group were given separately by intraperitoneal injection once daily, starting 24 hours prior to cisplatin injection
Xanthotoxol；
d) Cisplatin injection model group mice were injected 20mg/kg of cisplatin at one time via the abdominal cavity, and mice were sacrificed and drawn material 72h after cisplatin injection.
Mice in the blank control group and the Xanthoxol control group received the same dose of saline injection;
e) After 72 hours, all groups of mice were euthanized and kidney and blood samples were collected. All animal experiments obey the regulations of animal management and use of Chinese experiments.
4) Renal function testing
And centrifuging a mouse blood sample, collecting serum components, and detecting serum creatinine and serum urea nitrogen indexes by using a corresponding ELISA kit.
5) Tubular injury scoring
The degree of tubular injury is observed in pathological staining of kidney PAS, and tubular injury assessment is carried out according to semi-quantitative injury scoring standard, wherein the normal tubular tissue is 0 score, the tubular injury degree is 1 score of 30%, the tubular injury degree is 2 score of 30% -60%, and the tubular injury degree is 3 score of more than 60%.
6) Histopathological staining
Kidney tissue was fixed with paraformaldehyde, paraffin-wrapped, and histologically stained after tissue sections.
7) Cell activity assay
Human tubular epithelial cells were cultured, pretreated with Xanthoxol (20M) for 1h, then cisplatin was added for 24h, and the activity level of the cells was analyzed by CCK8, and the cell experiment was repeated three times and counted.
8) Statistical analysis
Data were represented using mean value SD. The comparison between the groups was performed using one-way analysis of variance (ANOVA) and the comparison between the two groups was performed using T-test. P <0.05 is statistically significant.
Example 2Xanthotoxol improves kidney injury and kidney function in cisplatin-induced acute kidney injury models.
In order to detect and evaluate the effect of Xanthoxol in protecting cisplatin-induced acute kidney injury, the invention detects kidney-related biochemical indexes in mouse serum, serum muscle and urea nitrogen indexes are obviously increased after the injection of Cisplatin (CDDP) for 72 hours, renal pathological injuries such as tubular dilation necrosis and the like are also more serious, and corresponding kidney injury and kidney function indexes are obviously improved after 10mg/kg Xanthoxol treatment (figures 1A-1C). In addition, a pathological staining based tubular injury score also suggests that Xanthoxol treatment can ameliorate cisplatin-induced renal pathological injury (FIG. 1D). Therefore, xanthoxol can not only improve kidney function, but also alleviate kidney pathological damage. These results indicate that Xanthoxol can protect cisplatin-induced acute kidney injury and that it does not itself show significant toxic side effects.
To further demonstrate the role of Xanthoxol in protecting cisplatin-induced acute kidney injury, the present invention examined early-stage specific biological indicators of acute kidney injury, NGAL and KIM-1. As shown in the western blot detection results of FIG. 2, KIM-1 and NGAL are highly expressed in the kidneys of cisplatin-induced acute kidney injury mice, and protein levels thereof are significantly reduced after Xanthoxol treatment, which further demonstrates that Xanthoxol can ameliorate cisplatin-induced acute kidney injury.
Example 3 Xanthotol ameliorates cisplatin-induced acute kidney injury by up-regulating GPX4 protein expression.
Further, the present invention explored the possible mechanism by which Xanthoxol improved cisplatin-induced acute kidney injury, and the experimental results of the present invention showed that GPX4 protein levels were significantly up-regulated in mouse kidney tissues after Xanthoxol treatment as determined (FIG. 3).
Since a series of studies indicate that up-regulating GPX4 expression can improve cisplatin-induced iron death in tubular epithelial cells and thus improve cisplatin-induced acute kidney injury, the results of the present invention for the first time found that Xanthoxol can up-regulate the expression level of GPX4 in mouse kidney tissue.
Thus, the above results suggest that Xanthoxol may improve cisplatin-induced acute kidney injury by up-regulating GPX4 protein expression.
Example 4 in the cisplatin model of in vitro tubular epithelial cells, xanthoxol treatment improved cisplatin-induced cell death.
First, in vitro cultured tubular epithelial cells, possible cytotoxicity of Xanthoxol was detected using CCK8, as shown in FIG. 4A, xanthoxol concentration less than 200. Mu.M did not cause toxic side effects on tubular cells; further, pretreatment with in vitro cultured tubular epithelial cells for 1h followed by addition of 10 μg/ml cisplatin for 24h and harvesting cells, on the one hand by fluorescent quantitative PCR detection, found that Xanthoxol treatment significantly up-regulated GPX4 gene expression (FIG. 4B); on the other hand, cell activity was measured by CCK8 and measuring Lactate Dehydrogenase (LDH) levels in cell supernatants, and the results showed that Xanthoxol was able to significantly improve cisplatin-induced cell damage and death (fig. 4c &4 d).
In summary, the invention provides an application of Xanthoxol in preparing a medicament for alleviating symptoms related to cisplatin-induced acute kidney injury, wherein the medicament is administered by intraperitoneal injection, the dosage of mice is 10mg/kg, and the acute kidney injury is improved by up-regulating the expression of anti-iron death-related GPX4 protein.
The present invention is not limited to the preferred embodiments, and any simple modification, equivalent replacement, and improvement made to the above embodiments by those skilled in the art without departing from the technical scope of the present invention, will fall within the scope of the present invention.

Claims (5)

  1. Use of Xanthotoxol in the manufacture of a medicament for the treatment of cisplatin-induced acute kidney injury.
  2. 2. Use of Xanthoxol according to claim 1 for the preparation of a medicament for the treatment of cisplatin-induced acute kidney injury, characterized by: xanthoxol ameliorates acute kidney injury by up-regulating protein levels of anti-iron death-related proteins.
  3. 3. Use of Xanthoxol according to claim 2 for the preparation of a medicament for the treatment of cisplatin-induced acute kidney injury characterized by: the anti-iron death related protein is GPX4.
  4. 4. Use of Xanthoxol according to claim 2 for the preparation of a medicament for the treatment of cisplatin-induced acute kidney injury characterized by: the improvement of acute kidney injury is the alleviation of injury or death of tubular epithelial cells.
  5. 5. Use of Xanthoxol according to claim 1 for the preparation of a medicament for the treatment of cisplatin-induced acute kidney injury, characterized by: xanthotoxol down regulates NGAL protein and KIM-l protein levels in acute kidney injury kidney tissue.
CN202310051172.0A 2023-02-02 2023-02-02 Application of Xanthoxol in preparing medicament for treating cisplatin-induced acute kidney injury Pending CN116115606A (en)

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