Application of emodin in medicine for treating acute kidney injury

Abstract

The invention belongs to the technical field of medicine, and particularly relates to application of emodin in medicine for treating acute kidney injury, the medicine for treating acute kidney injury is a preparation for inhibiting kidney iron sagging, and inhibition of kidney iron sagging specifically refers to activation of SIRT3, reduction of PINK1 acetylation level, up-regulation of glutathione peroxidase GPX4 and SLC7A11 expression level and reduction of transferrin TF expression level. The ROS level of lipid active oxygen is inhibited, so that the effect of treating the acute kidney injury caused by iron poisoning is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to application of rheumatoid arthritis in medicines for treating acute kidney injury. Background Art

[0002] Acute kidney injury (AKI) occurs in about half of critically ill patients and is characterized by a rapid decline in renal excretion function, accumulation of nitrogen metabolites, decreased urine output, metabolic acidosis, elevated potassium and phosphorus concentrations, and a clinical mortality rate of up to 18%. Pathologically, AKI is characterized by the detachment of tubular epithelial cells from the basement membrane into the tubular lumen, the loss of the brush border of the proximal tubular segment, and the formation of a tubular phenotype derived from detached cells, tubular fragments, and proteins, which undoubtedly increases the mortality rate and the probability of transformation to chronic kidney disease in patients. Programmed tubular epithelial cell death (such as ferroptosis) is a key event in the pathophysiological process of AKI injury. Recent studies have shown that inhibiting ferroptosis is a key therapeutic strategy for renal transplant-induced AKI. In the case of AKI, accompanied by renal insufficiency, the oxidative phosphorylation of iron-containing mitochondria is unbalanced, and the level of reactive oxygen species (ROS) produced by cells exceeds the antioxidant capacity of cells, leading to lipid peroxidation. To a certain extent, the integrity of the plasma membrane can be damaged, which can trigger lipid peroxidation, manifested as divalent iron ions (Fe 2+ ) accumulate excessively in renal tubular epithelial cells (TEC), leading to TEC iron ptosis. Rhein, whose scientific name is "1,3,8-trihydroxy-6-methylanthraquinone", is a natural compound extracted from Chinese herbs such as rhubarb and knotweed, and has multiple pharmacological effects such as anti-inflammatory, antiviral, and anti-tumor. However, no application of rhein in the treatment of iron ptosis has been found.

[0003] PTEN-induced putative kinase 1 (PINK1) performs many important functions in cells and has attracted attention for its role in early-onset Parkinson's disease. In recent years, an increasing number of studies have found that PINK1 is involved in regulating a variety of cellular physiological and pathophysiological processes, among which the regulation of mitochondrial function is the most prominent. Loss of PINK1 accelerates the progression of ferroptosis, manifested by increased malondialdehyde (MDA) levels, decreased superoxide dismutase (SOD) and glutathione (GSH) levels, increased iron release, mitochondrial atrophy, and vesicle formation. However, the exact mechanism is unclear. Lysine acetylation may occur in proteins involved in transcription, translation, and pathways related to central metabolism and stress response. Although PINK1 inhibits glutathione peroxidase 4 (GPX4) degradation and mitochondrial damage, researchers have not yet clarified whether intramitochondrial acetylation of PINK1 is involved in regulating ferroptosis and preventing tubular epithelial cell damage.

[0004] PINK1 acetylation can be specifically regulated by SIRT3 (sirtuin 3). SIRT3 is the main deacetylase involved in mitochondrial protein deacetylation. The full-length 44-kD SIRT3 enzyme is inactive, but it becomes activated when it is transported to the mitochondria and cleaved into the 28-kD small SIRT3. Importantly, SIRT3 regulates various important non-histone substrates, including acetyl-CoA synthetase, manganese superoxide dismutase, and isocitrate dehydrogenase. Due to its key target effects, SIRT3 regulates a variety of pathophysiological functions, such as mitosis, macrophage polarization, and ferroptosis. Resveratrol improves intestinal ischemia-reperfusion injury by activating SIRT3 and reducing ferroptosis. Proanthocyanidin B2 can activate SIRT3 to deacetylate pyruvate kinase M2 (PKM2) and inhibit apoptosis. However, whether rhubarb can effectively regulate ferroptosis through SIRT3, the specific mechanism, and whether PINK1 serves as an anti-ferroptosis substrate of SIRT3 need further study. Summary of the invention

[0005] The purpose of the present invention is to provide a method for treating acute kidney injury by using rheumatoid arthritis extract in order to solve the above problems in the prior art.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] The invention discloses an application of rheumatoid arthritis in a drug for treating acute kidney injury, wherein the drug for treating acute kidney injury is a preparation for inhibiting renal iron ptosis.

[0008] The inhibition of renal ferroptosis refers to activating SIRT3, reducing the acetylation level of PINK1, upregulating the expression levels of glutathione peroxidase GPX4 and SLC7A11, reducing the expression level of transferrin TF, and inhibiting the level of lipid reactive oxygen species ROS.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention proposes the use of emodin in drugs for treating acute kidney injury, the main mechanism of which is that emodin activates SIRT3 in the kidney, upregulates the expression level of PINK1, and inhibits the acetylation level of PINK1. The SIRT3 / PINK1 pathway is a protective target for inhibiting renal ferroptosis, thereby restoring the expression level of ferroptosis-related proteins, thereby achieving the effect of treating acute kidney injury caused by iron poisoning. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The effect of different doses of rheumatoid arthritis on urea and creatinine levels in AKI mice.

[0012] Figure 2Effects of different doses of rhamnosus on the levels of IL-1β, IL-6, and TNF-α in AKI mice.

[0013] Figure 3 Effects of different doses of emodin on renal tubular injury score and PAS injury index in AKI mice.

[0014] Figure 4 The effect of different doses of emodin on the pathological morphology of renal tissue in AKI mice.

[0015] Figure 5 Effects of different doses of rhamnosus on the expression levels of PINK1, GPX4, SLC7A11, and TF in AKI mice.

[0016] Figure 6 This is the effect of different doses of rhein on the expression levels of GPX4, SLC7A11 and TF in HK-2 cells.

[0017] Figure 7 The effect of different doses of rhein on ROS levels in HK-2 cells.

[0018] Figure 8 The effect of different doses of rhein on the cell viability of HK-2 cells.

[0019] Fig. 9 This is the effect of PINK1 knockout on urea, creatinine, IL-1β, IL-6, and TNF-α in AKI mice.

[0020] Fig.10 This is the effect of PINK1 knockout on iron and GSH content in AKI mice.

[0021] Fig.11 This is the effect of PINK1 knockout on the expression levels of PINK1, GPX4, SLC7A11, and TF in AKI mice.

[0022] Fig.12 Effect of PINK1 knockout on ROS levels in HK-2 cells.

[0023] Fig.13 This is the effect of PINK1 knockout on cell viability and relative iron content of HK-2 cells.

[0024] Fig.14 This is the effect of PINK1 knockout on the expression levels of PINK1, GPX4, SLC7A11, and TF in HK-2 cells.

[0025] Fig.15 ROS levels in HK-2 cells after PINK1-FL / N / C-Myc plasmid transfection.

[0026] Fig.16 Erastin induces the acetylated PINK1 level in HK-2 cells.

[0027] Fig.17 The acetylated PINK1 level in HK-2 cells after rhamnosine intervention.

[0028] Fig.18 This is the effect of SIRT3 knockout on acetylated PINK1 and the expression levels of PINK1, GPX4, SLC7A11, and TF in AKI mice.

[0029] Fig.19 Effects of SIRT3 knockout on urea, creatinine, iron content, and GSH levels in AKI mice.

[0030] Fig. 20 Effect of SIRT3 knockout on the binding of PINK1 to TF in HK-2 cells.

[0031] Fig.21 Effect of SIRT3 knockout on ROS levels in HK-2 cells.

[0032] Fig. 22 This is the effect of SIRT3 knockout on cell viability and relative iron content of HK-2 cells. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] 1. Reagents

[0035] Rhein (E106693), Erastin (E424821), n-acetyl-l-methionine (N425321), and trichostatin A (T129665) were purchased from Aladdin, Shanghai, China. Primary antibodies against SIRT1 (8469), SIRT3 (2627), SIRT4 (24662), SIRT5 (8782), and SIRT7 (5360) were obtained from Cell Signaling Technology Inc (Beverly, MA, USA). Acetyl-lysine was from Abcam (Ab21623, Cambridge, UK). PINK1 was from Santa Cruz (SC-517353, Santa Cruz, USA). Other antibodies, including SLC7A11 (GB115276-100), TF (GB11637-100), GPX4 (GB124327-100), SIRT2 (GB112450), SIRT6 (GB115525), and β-actin (GB11001), were purchased from Servicebio, Inc. (Wuhan, China). PINK1 protein truncation and transferrin were constructed by Shanghai Jikai Company. HK-2 and human renal tubular cell lines were purchased from Wuhan Pricella Biotechnology Company (Wuhan, China).

[0036] 2. The therapeutic effect of different doses of rheumatoid arthritis on AKI

[0037] 2.1 Animal experimental models and drug treatment

[0038] Adult male C57BL / 6 mice, 8 weeks old, weighing 20±2g, were obtained from Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). All mice were randomly divided into groups. The LPS-induced AKI model group was treated with different doses of emodin (40mg, 80mg, 160mg) to ensure the blood drug concentration. Specifically, emodin was first given for 3 days and then LPS was injected intraperitoneally to induce AKI; the control group (Ctrl) mice were injected with an equal amount of 0.9% saline. After the experiment, the mice were killed and blood and kidney samples were collected for further analysis.

[0039] All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Ethics Committee of Hubei University of Chinese Medicine (NO: HUCMS202302007).

[0040] 2.2 Creatinine and urea levels

[0041] According to the manufacturer's instructions (E-BC-K188-M and E-BC-K183-M, Elabscience Biotechnology Co., Ltd.), the creatinine colorimetric assay kit and urea colorimetric assay kit were used to measure the creatinine and urea levels in mouse blood serum, respectively. The results of creatinine and urea assays are shown in Figure 1 shown.

[0042] 2.3 IL-1β (interleukin 1β), IL-6 (interleukin 6), TNF-α (tumor necrosis factor α) levels

[0043] According to the manufacturer's instructions (E-MSEL-M0003, E-HSEL-H0003 and E-EL-H0109, Elabscience Biotechnology Co., Ltd.), IL-1β, IL-6 and TNF-α assay kits were used to measure the levels of IL-1β, IL-6 and TNF-α in mouse blood serum, respectively. The results are shown in Figure 2 shown.

[0044] 2.4 Tubular injury score and PAS injury index determination

[0045] ImageJ software was used to assist in the determination of tubular injury scores and PAS injury index. Kidney sections of three mice were observed in each group and statistical analysis was performed. The results of tubular injury scores and PAS injury index were shown in Figure 2. Figure 3 .

[0046] 2.5 Histological analysis

[0047] Mouse kidney tissues were fixed and embedded in paraffin, and 4 μm sections were stained with hematoxylin and eosin (H&E staining), periodic acid-Schiff staining (PAS staining), and TUNEL, and the number of positive cells was quantitatively analyzed using ImageJ software. Figure 4 As shown, the arrows in the H&E staining images indicate damaged renal tubules and glomeruli, the arrows in the PAS staining images indicate lesions, and the arrows in the TUNEL staining images indicate positive cells.

[0048] 2.5 Results Analysis

[0049] from Figure 1-Figure 3 It can be seen that after treatment with emodin, renal function was significantly improved and inflammatory response was inhibited, which was specifically reflected in the inhibition of urea, creatinine, IL-1β, TNF-α, IL-6, tubular injury score, and PAS injury index (compared with the AKI group, *P<0.05, **P<0.01). Figure 4It can be seen that compared with the control group, HE staining showed that the AKI group mice showed tubular epithelial cell death, loss of brush borders, tubular dilatation and increased areas of tubular cast formation; PAS staining showed thickening of glomerular basement membrane and renal tubules; TUNEL staining showed a significant increase in the number of tunel-positive cells in the AKI group mice. In contrast, the above pathological changes in the renal tissue of the AKI group mice were improved after treatment with rhamnosine.

[0050] The above data indicate that 80 mg and 160 mg of rhamnosine can effectively improve LPS-induced AKI, improve systemic inflammatory response, and inhibit cell apoptosis in a step-dose gradient manner.

[0051] 3. Possible targets of emodin in the treatment of AKI

[0052] HK-2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, streptomycin (100 μg / ml) and penicillin (100 IU / ml). Erastin (ferroptosis inducer) was used to induce HK-2 cells to show ferroptosis phenotype, followed by intervention with different doses of emodin (10, 20, 40 g / mL).

[0053] 3.1 Effect of emodin on the expression levels of PINK1 and iron poisoning-related proteins in mouse kidney tissue

[0054] Western blot was used to detect the expression levels of PINK1 and iron poisoning-related proteins such as GPX4, SLC7A11, and TF in mouse kidney tissue. Figure 5 shown.

[0055] 3.2 Effects of rhein on the expression level of iron poisoning-related proteins, ROS, and cell viability in HK-2 cells

[0056] Western blot was used to detect the expression levels of iron poisoning-related proteins such as GPX4, SLC7A11, and TF in HK-2 cells. The test results were as follows Figure 6 Detection of ROS in HK-2 cells: stain with 200 μL 20 μM DCFDA at 37°C for 30 min, wash with PBS, add 200 μL Hoechst working solution to each well for 5 min, wash with PBS, and take images with a fluorescence microscope; the results are shown in Figure 7 Detection of HK-2 cell viability: The absorbance of each well was measured at 570 nm using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide (MTT) assay to quantitatively analyze cell viability; the test results are shown in Figure 8 shown.

[0057] 3.3 Results Analysis

[0058] from Figure 5 It can be seen that compared with the control group, the levels of PINK1, GPX4, and SLC7A11 in the AKI group were decreased, and the level of TF was increased. After treatment with rhamnosine, the expression levels of PINK1, GPX4, SLC7A11, TF and other iron poisoning-related proteins were significantly restored. Figure 6-Figure 8 It can be seen that in HK-2 cells, different doses of emodin significantly inhibited ROS levels, improved cell viability, and restored the expression levels of iron poisoning-related proteins such as PINK1, GPX4, SLC7A11, and TF.

[0059] 4. Role of PINK1 in the treatment of AKI with emodin

[0060] Mitochondrial proteins and iron poisoning-related proteins were analyzed, and PINK1 was selected as the key protein. To verify the key role of PINK1, PINK1 knockout mice (PINK1-) were first treated with rhamnosine for 3 days, and then LPS was intraperitoneally injected to induce AKI. The levels of urea, creatinine, IL-1β, IL-6, and TNF-α in the serum of the PINK1 knockout mouse AKI model, the iron content, GSH content, and the expression level of iron poisoning-related proteins in the kidney tissue were detected. The test results are as follows Figures 9 to 11 shown.

[0061] Erastin was used to induce ferroptosis in PINK1 knockout HK-2 cells, followed by intervention with Emdoin. The ROS level, cell viability, relative iron content, and iron poisoning-related protein expression levels of PINK1 knockout HK-2 cells were detected. The test results were as follows: Figure 12-14 shown.

[0062] from Figure 9-11 It can be seen that after PINK1 knockdown, the therapeutic effect of emodin on AKI in mice disappeared, and the restorative effects on iron content, GSH content, and GPX4, SLC7A11, and TF expression in the renal tissue of mice disappeared; similarly, Figure 12-14 It can be seen that the effects of rhamnosus on ROS levels, cell viability, relative iron content, and GPX4, SLC7A11, and TF expression levels in HK-2 cells were also significantly reduced by knocking down PINK1.

[0063] 5. Inhibitory effect of rhamnosine on PINK1 acetylation

[0064] IP-MS (immunoprecipitation-mass spectrometry) was used to identify the binding proteins of PINK1 after the intervention of rhamnosine, and it was found that the binding of PINK1 to TF was significantly weakened after the intervention of rhamnosine. Subsequently, immunoprecipitation was used to verify that rhamnosine inhibited the binding of PINK1 and TF. In order to verify the subcellular localization of PINK1 binding to TF, the protein truncation of PINK1 was constructed to obtain PINK1-FL (1-581aa), PINK1-N (mitochondrial part 1-155aa), and PINK1-C (cytoplasmic part 156-581aa). Subsequently, HK-2 cells were transfected with PINK1-FL / N / C-Myc plasmid and TF-HA plasmid, respectively, and ROS staining was performed after transfection. The ROS staining results are shown in the figure. Fig.15 As shown, it was found that the efficacy of rhamnosin was significantly abolished after transfection of PINK1-C plasmid; immunoprecipitation of Myc protein revealed that PINK1-FL and PINK1-N truncated proteins could bind to TF, indicating that the binding of PINK1 and TF occurred on the cytoplasmic side.

[0065] Acetyl-lysine immunoprecipitation experiment was performed on HK-2 cells. The experimental results are as follows Fig.16 , Fig.17 As shown, from Fig.16 It can be seen that the level of acetylated PINK1 in HK-2 cells increased significantly after Erastin treatment. Fig.17 It can be seen that the level of acetylated PINK1 was significantly reduced after intervention with rhamnosus, indicating that rhamnosus deacetylation of PINK1 may be a key strategy for inhibiting ferroptosis.

[0066] 6. Role of SIRT3 in the treatment of AKI with emodin

[0067] The expression of SIRT1-7 in the kidneys of mice in the CTRL group and the AKI group was detected, and it was found that SIRT1, SIRT3, and SIRT5 were significantly downregulated in AKI mice. After the intervention of emodin, the expression of SIRT3 was significantly upregulated. In order to verify the key role of SIRT3, SIRT3 knockout mice (SIRT3KO) were first treated with emodin for 3 days, and then LPS was intraperitoneally injected to induce AKI. The expression levels of acetylated PINK1 and iron poisoning-related proteins in the AKI model of SIRT3 knockout mice were detected. The test results are as follows Fig.18 shown; from Fig.18 It can be seen that in the absence of SIRT3, the deacetylation effect of emodin on PINK1 is weakened, but it has no effect on the expression of PINK1. The improvement effect of emodin on iron poisoning-related proteins such as GPX4, SLC7A11, and TF is weakened. The urea, creatinine, renal iron content, and GSH of the SIRT3 knockout mouse AKI model were detected. The test results are as follows Fig.19As shown in Figure 2, similarly, due to SIRT3 deficiency, rhamnosine had no significant effect on urea, creatinine, and Fe 2+ The therapeutic effect of GSH content was weakened.

[0068] Erastin was used to intervene SIRT3 knockout HK-2 cells to induce ferroptosis, followed by intervention with emodin. Immunofluorescence analysis was performed using anti-pink1 and anti-tf antibodies, and images were taken using a laser confocal microscope. The photographic results are shown in Fig. 20 As shown in Figure 2, it can be seen that emodin reduces the binding of PINK1 and TF, and this effect is weakened by knocking down SIRT3. The ROS, cell viability, and relative iron content of SIRT3 knockout HK-2 cells were detected. The test results are shown in Figure 2. Fig.21 , Fig. 22 As shown, the effects of emodin on ROS levels, relative iron content, and cell viability were abolished in HK-2 cells after SIRT3 deficiency.

Claims

1. The application of emodin in the treatment of acute kidney injury is characterized by: The drug for treating acute kidney injury is a preparation for inhibiting renal iron ptosis.

2. The use according to claim 1, characterized in that: The inhibition of renal ferroptosis refers to activating SIRT3, reducing the acetylation level of PINK1, upregulating the expression levels of glutathione peroxidase GPX4 and SLC7A11, reducing the expression level of transferrin TF, and inhibiting the level of lipid reactive oxygen species ROS.

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