阅读说明：本技术 铁死亡抑制剂Liproxstatin-1在制备减轻肾纤维化药物中的应用 (Application of iron death inhibitor Liproxstatin-1 in preparation of medicine for relieving renal fibrosis ) 是由 张波 周艳 陈湘 何垚 陈志� 李冰晟 甘宇 于 2021-09-13 设计创作，主要内容包括：本发明公开了一种铁死亡抑制剂Liproxstatin-1在制备治疗肾间质纤维化药物中的应用,属于生物医药技术领域。本发明系统阐明了小鼠单侧输尿管结扎所致的肾纤维模型中铁沉积的发生及铁死亡相关蛋白的表达；同时采用铁死亡抑制剂Liproxstatin-1腹腔给药,证明了Liproxstatin-1可以抑制肾小管上皮细胞铁死亡,从而抑制促纤维化因子TGF-β1、CTGF和PDGF的分泌及肌成纤维细胞的活化,最终减轻小鼠肾纤维化程度,为治疗肾纤维化提供潜在药物和新思路。(The invention discloses an application of an iron death inhibitor Liproxstatin-1 in preparing a medicament for treating renal interstitial fibrosis, belonging to the technical field of biological medicines. The system of the invention clarifies the generation of iron deposition and the expression of iron death related protein in a renal fiber model caused by unilateral ureteral ligation of a mouse; meanwhile, an iron death inhibitor, namely Liproxstatin-1, is administered in the abdominal cavity, so that the fact that Liproxstatin-1 can inhibit the iron death of renal tubular epithelial cells is proved, the secretion of fibrosis promoting factors TGF-beta 1, CTGF and PDGF and the activation of myofibroblasts are inhibited, the renal fibrosis degree of a mouse is finally relieved, and a potential medicament and a new idea are provided for treating renal fibrosis.)

1. Application of iron death inhibitor Liproxstatin-1 in preparing medicine for relieving renal fibrosis.

2. Use of the inhibitor of iron death, Liproxstatin-1, according to claim 1, in the manufacture of a medicament for reducing renal fibrosis, wherein the use is in the inhibition of renal tubular epithelial cell iron death.

3. The use of the inhibitor of iron death, Liproxstatin-1, according to claim 1, in the preparation of a medicament for reducing renal fibrosis, wherein said use is the inhibition of the secretion of the pro-fibrotic factors TGF- β 1, CTGF and PDGF.

4. The use of the inhibitor of iron death, Liproxstatin-1, according to claim 1, in the manufacture of a medicament for reducing renal fibrosis, wherein the use is the inhibition of myofibroblast activation.

5. The use of the inhibitor of iron death, Liproxstatin-1, according to claim 1, in the manufacture of a medicament for reducing renal fibrosis, wherein said use prevents impairment of renal function and protects the kidney.

6. The use of the iron death inhibitor Liproxstatin-1 in the preparation of a medicament for reducing renal fibrosis according to claim 1, wherein the medicament for reducing renal fibrosis is in the form of injection.

7. The use of the iron death inhibitor, Liproxstatin-1, in the manufacture of a medicament for reducing renal fibrosis according to claim 1, wherein the medicament for reducing renal fibrosis is administered by intraperitoneal injection.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of an iron death inhibitor Liproxstatin-1 in preparation of a medicine for relieving renal fibrosis.

Background

Renal fibrosis is a common pathway and major pathological process for the progression of various Chronic Kidney Diseases (CKD) to the end stage, characterized by normal renal structural destruction, fibroblast proliferation, excessive deposition of extracellular matrix. Renal fibrosis can be caused by a complex set of etiologies, such as obstruction, inflammation, immunity, metabolism, or other systemic diseases. There is currently no effective way to slow the progressive impairment of renal function in chronic kidney disease or to completely delay the progression of renal fibrosis, and treatment options for patients with end-stage renal disease are limited to dialysis and renal transplantation. Although research on the pathogenesis of renal fibrosis has been advanced in recent years, the use of various anti-renal fibrosis drugs has not been effective in improving poor prognosis, and there are very few drugs that can be clinically used for inhibiting renal fibrosis. Therefore, the intensive research on the cellular and molecular mechanism of renal fibrosis generation and the research on a novel anti-renal fibrosis treatment way are active fields of the current renal fibrosis prevention and treatment research.

Iron death (ferroptosis) was a novel form of programmed cell death first discovered by Dixon in 2012 and is mainly characterized by the accumulation of a large amount of lipid peroxides accompanied by iron ion dependence during cell death. The iron death is obviously different from the cell death modes such as apoptosis, necrosis and autophagy in the aspects of cell morphology, genetics, biochemistry and the like. Intracellular iron overload, Glutathione (GSH) depletion, and lipid peroxidation are three key factors that cause iron death. When the antioxidant capacity of the cells is reduced and lipid reactive oxygen species accumulate, oxidative cell death, i.e., iron death, can be caused. Although the morphological, biological and mechanistic pathways of iron death are partially understood, the research on the whole signaling pathway molecules related to iron death is still in the early stage, and a plurality of brute-force fields which are not developed yet exist. In recent years, iron death has been found to play a very important role in the pathophysiological processes of cancer, organ ischemia-reperfusion injury, neurodegenerative diseases and the like. Studies have shown that iron death is an important death mode of renal tubular epithelial cells in acute renal injury, but whether iron death occurs in the process of chronic renal fibrosis and the role of iron death of renal tubular epithelial cells in chronic renal fibrosis are not reported. The effect and the mechanism of the renal tubular epithelial cell iron death in the occurrence of the renal fibrosis are revealed, and a new way and a new therapeutic target point are provided for the research of the prevention and the treatment of the renal fibrosis. Liproxstatin-1(Lip-1) is a potent inhibitor of iron death and blocks lipid peroxidation, thereby inhibiting hypoxia injury associated with iron death. In recent years, Liproxstatin-1 has received attention from researchers because it exhibits a variety of pharmacological activities. The existing research shows that: liproxstatin-1 can protect mouse myocardium from ischemia/reperfusion injury by lowering voltage-dependent anion selective channel 1 levels and restoring GPX4 levels; liproxstatin-1 attenuates morphine tolerance by inhibiting spinal cord cell iron death; liproxstatin-1 can inhibit RSL 3-induced human renal proximal tubular epithelial cell death and GPX4 deletion-induced acute renal failure. Therefore, Liproxstatin-1 is a potent iron death inhibitor, but the role and mechanism of iron death in renal fibrosis are not clear so far, and there is no report on the treatment of renal fibrosis by Liproxstatin-1.

Disclosure of Invention

In order to solve the technical problems, the invention provides an application of an iron death inhibitor Liproxstatin-1 in preparing a medicament for relieving renal fibrosis, and aims to provide a new way and a treatment target for research on prevention and treatment of renal fibrosis, and the following technical scheme is adopted: application of iron death inhibitor Liproxstatin-1 in preparing medicine for relieving renal fibrosis.

Preferably, the use is for inhibiting iron death in renal tubular epithelial cells.

Preferably, the use is for inhibiting the secretion of the profibrotic factors TGF-beta 1, CTGF and PDGF.

Preferably, the use is for inhibiting activation of myofibroblasts.

Preferably, the use is capable of preventing impairment of kidney function and protecting the kidney.

Preferably, the dosage form of the drug for relieving renal fibrosis is injection.

Preferably, the application mode of the drug for relieving renal fibrosis is intraperitoneal injection administration.

The invention has the beneficial effects that:

according to the invention, a large number of researches show that the renal tubular epithelial cell iron death is an important factor causing renal fibrosis, and the iron death inhibitor Liproxstatin-1 can better inhibit the renal tubular epithelial cell iron death, and further inhibit the secretion of the fibrosis promoting factors TGF-beta 1, CTGF and PDGF by inhibiting the renal tubular epithelial cell iron death, and can inhibit the activation of myofibroblasts, so that the action and the mechanism of the renal tubular epithelial cell iron death in the occurrence of renal fibrosis are specifically disclosed, and a new way and a specific treatment target point are provided for the research on the prevention and treatment of renal fibrosis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed 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 creative efforts.

FIG. 1 shows the results of experiments in which Liproxstatin-1 reduced UUO-induced iron death of tubular epithelial cells in renal tissue.

FIG. 2 shows the results of experiments in which Liproxstatin-1 reduces morphological changes and renal function impairment in the UUO model.

FIG. 3 shows the results of experiments in which Liproxstatin-1 reduced the deposition of renal collagen in the UUO model.

FIG. 4 shows the results of experiments in which Liproxstatin-1 reduced the release of profibrokine and the proliferation and activation of fibroblasts.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; all reagents used in the examples are commercially available unless otherwise specified.

The molecular biology experiments referred to in the following examples of the present invention include:

(1) analysis of renal function

Blood was taken from the ocular arteries and serum creatinine (Scr) and urea nitrogen (BUN) levels (Beckman AU5800, usa) were determined colorimetrically.

(2) Prussian blue staining

Kidney tissue was dissected, a portion of the tissue specimens were fixed in 4% paraformaldehyde solution and paraffin tissue sections were embedded. The tissue was incubated in prussian blue solution for 1 hour and then washed with Phosphate Buffered Saline (PBS). A second incubation was performed in 0.5% benzidine tetrahydrochloride. After rinsing, the observation was performed under a light microscope.

(3) E-cadherin co-staining with TUNEL

For detection of TUNEL positive cells, assays were performed using the TUNEL apoptosis detection kit according to the instructions provided by the manufacturer. To determine whether cell death occurred primarily in renal tubular epithelial cells, E-cadherin (epithelial cell marker) co-staining with TUNEL was detected in renal tissue using immunofluorescence staining. (4) Determination of the Kidney GSSG/GSH ratio

The ratio of oxidized glutathione (GSSG) to total glutathione (GSH, reduced glutathione + GSSG) was determined by GSH and GSSG assay kits according to the manufacturer's instructions. The absorbance at 412nm was measured with a microplate reader.

(5) Immunohistochemistry, immunofluorescence and histopathology

Expression of GPX4, collagen I, TGF-. beta.1, CTGF and PDGF, E-cadherin and α -SMA were examined using immunohistochemistry, immunofluorescence and histopathology, etc., and collagen deposition was reflected using Masson staining, Sirius red staining.

(6) Western blot analysis

Renal tissue, HK2 cells and human renal fibroblasts were lysed in RIPA lysis buffer. Total protein concentration was determined using BCA kit. Detecting the protein expression condition through the steps of electrophoresis, membrane conversion, sealing, antibody incubation, development and the like.

(7) Determination of superoxide dismutase (SOD) and Malondialdehyde (MDA) levels

SOD (A001-1) and MDA (A003-1) kits are purchased from the institute of bioengineering, Jiancheng, Nanjing, China. HK2 cells were collected according to the manufacturer's instructions and tested for SOD (hydroxylamine method) and MDA (thiobarbituric acid method) levels.

(8) Analysis of iron content

10mg of kidney tissue slices are taken from each group, washed with cold PBS and the samples to be tested are mixed evenly. Relative iron concentrations in kidney tissue and HK2 cell lysates were assessed using an iron detection kit according to the instructions.

Example 1

Animal treatment

Male C57BL/6 mice were 8 weeks old. All experimental animal procedures were approved by the ethical committee of the scientific research center of the university of south-central university (chansha, china). Mice were anesthetized with sodium pentobarbital (80 mg/kg; i.p.) and all efforts were made to minimize pain in the animals during and after surgery.

Animal model

A Unilateral Ureteral Obstruction (UUO) model is constructed. The lower skin, fascia and muscles in the back of the mouse were cut open, exposing the left kidney, gently separated along the infrarenal level with a glass needle, exposing the left ureter, ligated with 2 4-0 silk threads, and the ureter was disconnected between the 2 ligation threads to prevent retrograde urinary tract infection. Gentamicin is absorbed completely after the wound is fully flushed, internal muscles and fascia are continuously sutured by 4-0 silk threads, and the skin is discontinuously sutured by 3-0 silk threads. The sham operation group and the Liproxstatin-1 group only isolated the left ureter but not ligated, and the rest of the operation was identical to the model creation group. The 24C 57BL/6 mice were randomly divided into four groups: (1) a sham operation group; (2) liproxstatin-1 group; (3) a UUO group; (4) UUO + Liproxstatin-1 group. Mice in the groups of Lipoxstatin-1 and UUO + Lipoxstatin-1 were intraperitoneally injected with 200. mu.L of Lipoxstatin-1 (10mg/kg/d in DMSO, then diluted with 0.9% NaCl) for 14 consecutive days after surgery. Mice in the sham operation group and the UUO group were injected with an equal volume of 0.9% NaCl solution intraperitoneally. After the last treatment, all animals were anesthetized with sodium pentobarbital and sacrificed. Blood was collected from the ophthalmic artery by retroorbital bleeding. One part of the left kidney was fixed in 4% paraformaldehyde solution, the remaining part was snap frozen in liquid nitrogen and stored at-80 ℃ for further analysis.

The above molecular biology experiments were performed on four groups of mice, and the results are shown in FIGS. 1 to 4.

As shown in fig. 1, the iron concentration in kidney tissues of UUO mice (fig. 1A, fig. 1B), GSSG/GSH ratio (fig. 1C), the number of TUNEL positive cells (fig. 1D), the average level of oxidative stress (fig. 1E, fig. 1F) were significantly increased, and GPX4 expression was significantly down-regulated (fig. 1G) compared to the sham group. Compared with the UUO group, the indexes are obviously improved after the treatment of the Liproxstatin-1. These data indicate that iron death occurs in tubular epithelial cells during UUO-induced renal fibrosis, and that Liproxstatin-1 is able to inhibit the occurrence of iron death.

As shown in fig. 2A-2C, the kidney weight/body weight ratio (kidney weight index), BUN and Scr levels were significantly higher in the UUO group than in the sham group, and the injury to renal function was reduced by treatment with Liproxstatin-1. HE staining found that the glomerulus and tubular structure of the Liproxstatin-1 group were normal, indicating that Liproxstatin-1 treatment (10mg/kg/d) did not cause damage to the renal structure. UUO groups have obviously widened space, inflammatory cells and perivascular exudate infiltrate, and glomerular capillaries expand. In the UUO + Liproxstatin-1 group, the UUO-induced morphological changes were significantly improved (FIG. 2D). These results indicate that Liproxstatin-1 can prevent UUO-induced morphological changes and renal function impairment in vivo.

Collagen deposition is an important feature of renal fibrosis, and as shown in fig. 3A, no significant collagen deposition was observed in the sham group or the Liproxstatin-1 group, whereas the UUO group was very significant; the deposition of collagen in kidney tissue was reduced in Liproxstatin-1 treated mice compared to the UUO group. Furthermore, immunohistochemical staining and western blot analysis showed a significant increase in type I collagen expression in kidney tissue of the UUO group, while type I collagen expression was significantly decreased in the UUO + Liproxstatin-1 group (fig. 3A, fig. 3B). These data indicate that Liproxstatin-1 reduces UUO-induced interstitial collagen deposition in the kidney.

As shown in FIG. 4A, the expression of the fibrosis promoting factors TGF-beta 1, CTGF and PDGF in the kidney tissues of the rats in the UUO group is obviously increased compared with that in the sham operation group; compared with the UUO group, the Liproxstatin-1 treatment significantly reduces the expression of TGF-beta 1, CTGF and PDGF. Immunofluorescence staining and western blot analysis suggest that the expression of a main molecular marker alpha-SMA of myofibroblasts is obviously increased in the UUO group. Liproxstatin-1 treatment reduced α -SMA expression compared to the UUO group (fig. 4B, fig. 4C). These results indicate that Liproxstatin-1 is able to reduce UUO-induced secretion of profibrotic factor and myofibroblast activation in kidney tissue.

The above experimental results show that: in the process of renal fibrosis, renal tubular epithelial cells are subjected to iron death, and an iron death inhibitor Liproxstatin-1 can inhibit the occurrence of iron death. The Liproxstatin-1 inhibits the death of renal tubular epithelial cells, thereby preventing the morphological change and renal function damage caused by UUO in vivo, weakening the deposition of renal interstitial collagen, inhibiting the secretion of the fibrosis promoting factor in renal tissues and the activation of myofibroblasts, and achieving the effect of relieving renal fibrosis.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.