Application of chrysophanol in preparation of medicine for treating sepsis acute kidney injury

Abstract

The invention provides an application of chrysophanol in preparation of a medicine for treating sepsis acute kidney injury, and relates to the technical field of biomedicine.The chrysophanol is a main effective component in traditional Chinese medicine rheum officinale and has the advantages of being high in safety, high in economical efficiency and the like. According to the application disclosed by the invention, the chrysophanol is found to have relatively good improvement and treatment effects on the acute kidney injury caused by sepsis for the first time, and the chrysophanol can effectively improve symptoms of the acute kidney injury caused by sepsis and protect kidney functions through multiple mechanisms of reducing inflammatory factors and oxidative stress levels, enhancing cell viability and energy metabolism, inhibiting cell apoptosis and the like. Based on the advantages of high safety, strong economy and the like, chrysophanol is expected to become an important innovative drug in treatment of sepsis acute kidney injury, provides a new treatment strategy for clinic, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of chrysophanol in preparing a medicine for treating sepsis-induced acute kidney injury. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome triggered by infection, often accompanied by multiple organ failure and, in severe cases, life-threatening. As one of the organs most susceptible to sepsis, the kidney plays a crucial role in the development and progression of sepsis. Sepsis-associated acute kidney injury (SA-AKI) is associated with a high morbidity and mortality rate, with approximately 40%-50% of sepsis patients developing SA-AKI. Compared with other types of renal injury, SA-AKI has a significantly higher mortality rate and a poorer prognosis. The defining characteristic of SA-AKI is the uncontrolled systemic inflammatory cascade triggered by infection. Its core mechanism involves the release of pathogen-associated molecular patterns (PAMs) and damage-associated molecular patterns (DAMs), which activate immune cells and trigger a "cytokine storm." This process promotes the massive release of proinflammatory cytokines and chemokines, exacerbating inflammatory infiltration in renal tissue and leading to apoptosis of renal tubular epithelial cells, endothelial barrier disruption, and mitochondrial dysfunction. The inflammatory cascade also triggers oxidative stress and impaired tubular energy metabolism, further exacerbating cellular damage. Currently, there is no effective drug to prevent or treat SA-AKI. Related targeted drugs have limited efficacy and serious toxic and side effects in clinical trials. Therefore, it is urgent to find a safe and effective drug for the treatment of SA-AKI.

[0003] Currently, the treatment of SA-AKI primarily relies on supportive therapies, such as continuous renal replacement therapy (CRRT) and kidney transplantation, but these approaches have limitations. While CRRT can provide temporary renal replacement in the acute phase of sepsis, it primarily targets patients in the end stages of renal failure and lacks early intervention in the pathological process. Furthermore, kidney transplantation is only suitable for patients with end-stage renal failure and carries the risk of rejection. Therefore, existing treatments are unable to prevent or mitigate the onset and progression of SA-AKI.

[0004] Compared with the single targeted therapy of modern medicine, the multi-target effect of traditional Chinese medicine gives it a unique advantage in the treatment of SA-AKI. Chrysophanol is the most abundant free anthraquinone compound in rhubarb, with high bioavailability. Compared with other free anthraquinone compounds (such as emodin and rhubarb A), it has less toxicity to the liver and kidneys. In addition, chrysophanol is the main active ingredient in the traditional Chinese medicine rhubarb. As the most abundant free anthraquinone compound in rhubarb, it has less toxicity to the liver and kidneys than other free anthraquinones, has higher bioavailability, and has multiple pharmacological effects such as anti-inflammatory, anti-cancer, anti-tumor, and myocardial protection. However, there are currently no reports on the pharmacological effects of chrysophanol on septic acute kidney injury. Therefore, the present invention proposes the use of chrysophanol in the preparation of a drug for treating septic acute kidney injury to solve the problems existing in the prior art. Summary of the Invention

[0005] In response to the above problems, the purpose of the present invention is to propose an application of chrysophanol in the preparation of a drug for treating acute kidney injury due to sepsis. The application of chrysophanol in the preparation of a drug for treating acute kidney injury due to sepsis provides a new approach for treating acute kidney injury due to sepsis and can solve the problems existing in the prior art.

[0006] To achieve the purpose of the present invention, the present invention is implemented through the following technical solution: an application of chrysophanol in the preparation of a medicine for treating sepsis-induced acute kidney injury.

[0007] The further improvement is that the CAS number of the chrysophanol is 481-74-3, and the molecular formula is C 15 H 10 O4, molecular weight 254.24, its molecular structure is as follows:

[0008]

[0009] A further improvement is that the use comprises inhibiting sepsis-induced acute kidney injury in an in vivo model, wherein the in vivo model is a mouse model induced by cecal ligation and puncture.

[0010] A further improvement is that the inhibition of sepsis-induced acute kidney injury in the in vivo model includes one or more of improving kidney tissue structural damage, reducing the level of oxidative stress in the mouse kidney, inhibiting inflammatory response and reducing the expression level of kidney apoptosis protein.

[0011] A further improvement is that the application also includes inhibiting sepsis-induced acute kidney injury in an in vitro model, wherein the in vitro model is a lipopolysaccharide-induced renal tubular epithelial cell model.

[0012] A further improvement is that the inhibition of sepsis-induced acute kidney injury in the in vitro model includes one or more of enhancing the activity of renal tubular epithelial cells, reducing their apoptosis level and expression of apoptosis-related proteins, and reducing inflammatory response.

[0013] Animal experiments have shown that the drug chrysophanol of the present invention can improve kidney tissue structure damage and reduce renal function indicators such as Cr and BUN after chrysophanol treatment.

[0014] Animal experiments have shown that the drug chrysophanol of the present invention can increase the ATP level of kidney tissue energy metabolism and improve the damaged structure of mitochondria after chrysophanol treatment.

[0015] The drug chrysophanol of the present invention has been verified by cell experiments. After treatment with chrysophanol, the activity of renal tubular epithelial cells is improved.

[0016] The drug chrysophanol of the present invention has been verified by in vivo and in vitro experiments. After chrysophanol treatment, oxidative stress (MDA, SOD, ROS, GSH) is improved, pro-inflammatory factors such as IL-6 and TNF-α are reduced, inflammatory response is alleviated, and cell apoptosis level is reduced.

[0017] The above results indicate that the drug chrysophanol of the present invention has the function of alleviating acute kidney injury caused by sepsis.

[0018] A pharmaceutical composition for treating acute kidney injury caused by sepsis, comprising an effective therapeutic dose of the active ingredient chrysophanol and a pharmaceutically acceptable carrier, wherein the carrier is selected from at least one of a diluent, a binder, a disintegrant, a lubricant, a solvent, a solubilizer, a preservative or an antioxidant. The pharmaceutical composition is in the form of a suspension, an emulsion, a tablet, a capsule, a granule, an oral solution or an injection.

[0019] When the dosage form is a tablet or capsule, the carrier includes a diluent, a disintegrant and a lubricant; and when the dosage form is an injection, the carrier includes a solvent, a solubilizer, an osmotic pressure regulator and an antioxidant.

[0020] The beneficial effects of the present invention are as follows: the present invention proposes a novel drug, chrysophanol, for treating acute kidney injury caused by sepsis. Chrysophanol is the main active ingredient in the traditional Chinese medicine rhubarb and has the characteristics of high safety and strong economy. The present invention has discovered for the first time that chrysophanol has a good improvement and therapeutic effect on acute kidney injury caused by sepsis. Through multiple mechanisms such as reducing the levels of inflammatory factors and oxidative stress, enhancing cell viability and energy metabolism, and inhibiting cell apoptosis, chrysophanol can effectively improve the symptoms of acute kidney injury caused by sepsis and protect kidney function. Based on its advantages of high safety and strong economy, chrysophanol is expected to become an important innovative drug in the treatment of acute kidney injury caused by sepsis, providing a new treatment strategy for the clinic and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram comparing the results of chrysophanol improving kidney tissue structure damage and renal function levels in mice with acute kidney injury caused by sepsis in Example 1 of the present invention.

[0022] Figure 2 This is a schematic diagram comparing the results of chrysophanol improving energy metabolism and mitochondrial damage in mice with acute kidney injury caused by sepsis in Example 2 of the present invention.

[0023] Figure 3 This is a schematic diagram comparing the results of chrysophanol improving the activity of renal tubular epithelial cells in septic acute kidney injury according to Example 3 of the present invention.

[0024] Figure 4 3. This is a schematic diagram showing the comparative results of chrysophanol reducing the oxidative stress level in the sepsis acute kidney injury model according to Example 4 of the present invention.

[0025] Figure 5 This is a schematic diagram comparing the results of chrysophanol inhibiting inflammatory response in a sepsis-induced acute kidney injury model according to Example 5 of the present invention.

[0026] Figure 6 This is a schematic diagram comparing the results of chrysophanol in Example 6 of the present invention in reducing the apoptosis level of renal tubular epithelial cells. DETAILED DESCRIPTION

[0027] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the examples. The examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0028] In the following examples, chrysophanol can be obtained by, but is not limited to, chemical isolation or synthesis, or commercial purchase. Statistical analysis was performed using SPSS 20.0 software. Data are presented as mean ± standard deviation. Comparisons between multiple groups were performed using one-way analysis of variance. "ns" indicates no significant difference between groups. "***" indicates P < 0.001, "**" indicates P < 0.01, and "*" indicates P < 0.05. All data are presented as mean ± standard deviation (n = 3). GraphPad 9.3 was used to plot the results.

[0029] Example 1

[0030] according to Figure 1 As shown in the results, it was found that chrysophanol can significantly improve the renal tissue structure damage in mice with acute kidney injury caused by sepsis, reduce the degeneration, shedding and necrosis of renal tubular epithelial cells, significantly reduce the expression levels of renal function indicators such as creatinine and urea nitrogen, and alleviate renal damage. Figure 1In the figure, A is the pathological changes of renal tissues of mice in different groups stained with HE; B is the renal injury score; C is the level of serum creatinine (Cr) of mice in each group; D is the level of serum urea nitrogen (BUN) of mice in each group.

[0031] The steps are as follows:

[0032] Step 1. Animal model construction: Male healthy C57BL6 / J mice were randomly divided into 4 groups: blank control group, sham operation group, sepsis acute kidney injury group, and chrysophanol treatment group. The cecal ligation and puncture (CLP) method was used to establish a sepsis acute kidney injury model. Specifically, the blank control group mice did not receive any treatment; the sham operation group mice were opened and the cecum was pulled out of the abdominal cavity after the abdominal cavity was found, and then the cecum was immediately returned to the abdominal cavity without ligation and puncture; the sepsis group mice were prepared with a sepsis mouse model according to the above-mentioned cecal ligation and puncture method; the chrysophanol treatment group was intraperitoneally injected with chrysophanol CHR (30 mg / kg) after the sepsis model was prepared. Among them, the sham operation group and the sepsis group were intraperitoneally injected with equal volumes of normal saline after the model was prepared.

[0033] Step 2: Sampling: After modeling, each group of mice was sacrificed 24 hours after surgery. To begin sampling, specifically, to obtain blood samples, the mice's whiskers were trimmed, their eyeballs were pulled up to protrude, and then their eyeballs were quickly removed with forceps. The blood was collected and allowed to stand at room temperature for 1 hour. The blood samples were then centrifuged, labeled, and stored at -80°C.

[0034] To obtain tissue specimens, the mouse was fixed on the operating table, the skin was prepared and disinfected, and the abdomen was cut open to expose the surgical field. The kidneys of the mouse were removed. Some kidney specimens were fixed in 4% neutral paraformaldehyde for 24-48 hours and then stained with HE.

[0035] Step 3: HE staining of renal tissue: Dewax, stain, dehydrate, and mount the paraffin sections. Three randomly selected fields of view on the HE sections were scored for renal tubular injury.

[0036] Step 4. Creatinine, urea nitrogen and other renal function tests: After the model of each group of mice is successfully established, serum samples prepared in advance are taken for the detection of renal function indicators (creatinine Cr, urea nitrogen BUN), and the absorbance value is measured using an enzyme labeling instrument.

[0037] The results show that:

[0038] (1) HE staining of renal tissue: There was no abnormality in the renal tissue structure of mice in the blank control group and the sham operation group; the renal tissue damage of mice in the sepsis acute kidney injury group was obvious, manifested by obvious degeneration, shedding and necrosis of renal tubular epithelial cells; the renal tissue damage of mice in the chrysophanol treatment group was improved, and only partial degeneration of renal tubular epithelial cells was observed.

[0039] (2) Renal function test: The levels of Cr and BUN in the serum of mice in the sepsis acute kidney injury group were significantly higher than those in the blank control group and the sham operation group; while the levels of Cr and BUN in the chrysophanol treatment group were significantly reduced.

[0040] The above results indicate that chrysophanol can effectively improve renal tissue structural damage and renal function levels in mice with acute kidney injury caused by sepsis.

[0041] Example 2

[0042] according to Figure 2 As shown in the results, it was found that chrysophanol could significantly improve the ATP level in the kidney tissue of mice with acute kidney injury caused by sepsis, reduce the swelling of mitochondria, and the damage to the mitochondrial double membrane and mitochondrial cristae structure. Figure 2 A shows the ATP level in the kidney tissue of each group of mice; B shows the mitochondrial ultrastructure in the kidney tissue of each group of mice. The steps are as follows:

[0043] Step 1. ATP detection: After the model is successfully established, the kidney tissue is collected and mashed with physiological saline. The supernatant is collected by centrifugation and the ATP content in the homogenate of the experimental mouse kidney tissue is calculated based on the absorbance value.

[0044] Step 2: Mitochondrial transmission electron microscopy: Fix, dehydrate, embed, and stain the mouse kidney tissue, and finally photograph it under a transmission electron microscope.

[0045] The results show that:

[0046] (1) ATP detection: The ATP level in the kidney tissue of mice in the sepsis acute kidney injury group was significantly lower than that in the blank control group and the sham operation group; while the ATP level in the chrysophanol treatment group was significantly increased.

[0047] (2) Transmission electron microscopy results: The mitochondria in the blank control group and the sham operation group had uniform morphology, and the mitochondrial organelle structure was clear and complete; the number of mitochondria in the sepsis acute kidney injury group decreased, the morphology was strange, and there was vacuolation, and the double-layer membrane organelle structure was severely dissolved and damaged; the mitochondria in the chrysophanol treatment group showed varying degrees of damage, but from an overall analysis, the degree of mitochondrial swelling, the degree of damage to the mitochondrial double membrane and the mitochondrial cristae structure were lighter than those in the disease group.

[0048] The above results show that chrysophanol can significantly improve the energy metabolism level of mouse kidney tissue and improve the ultrastructural damage of mitochondria in mouse kidney.

[0049] Example 3

[0050] according to Figure 3 As shown in the results, it was found that chrysophanol could significantly enhance the viability of renal tubular epithelial cells in the sepsis acute kidney injury model. Figure 3A is the screening of the optimal concentration of chrysophanol; B is the effect of chrysophanol on the viability of renal tubular epithelial cells in each group. The steps are as follows:

[0051] Step 1, cell culture: In the present invention, human renal tubular epithelial cells (HK-2) are selected, and the culture medium is a low-glucose standard DMEM medium, 10% sterile serum and 1% penicillin and streptomycin mixture are added, and cultured in a constant temperature cell culture incubator at 37°C and 5% CO2.

[0052] Step 2: Model establishment: HK-2 cells were treated with lipopolysaccharide (10 μg / mL LPS) for about 24 hours to construct a cell model of sepsis-induced acute kidney injury, and the cells were divided into a control group, a sepsis-induced acute kidney injury group, and a chrysophanol-treated group.

[0053] Step 3. Drug concentration screening and cell viability detection: Set a concentration gradient of 0.1μM, 1μM, 5μM, 7.5μM, 10μM, 15μM and 20μM chrysophanol to intervene in the HK-2 cells of the sepsis acute kidney injury model respectively. The CCK-8 method was used to screen the optimal concentration of chrysophanol, and the cells were treated with this concentration to detect the difference in cell viability before and after chrysophanol treatment.

[0054] The results showed that cell viability was optimal when the concentration of chrysophanol was 10 μM; and at this concentration, the cell viability in the chrysophanol treatment group was significantly higher than that in the septic acute kidney injury group.

[0055] Example 4

[0056] according to Figure 4 As shown in the results, it was found that chrysophanol can reduce the level of oxidative stress in sepsis-induced acute kidney injury in vitro and in vivo. Figure 4 A is the level of malondialdehyde (MDA) in the serum of mice in each group; B is the level of superoxide dismutase (SOD) in the serum of mice in each group; C is the level of glutathione (GSH) in renal tubular epithelial cells in each group; D is the level of malondialdehyde (MDA) in renal tubular epithelial cells in each group; E is the level of reactive oxygen species (ROS) in renal tubular epithelial cells in each group and their statistical differences. The steps are as follows:

[0057] Step 1. Malondialdehyde, superoxide dismutase and glutathione (MDA, SOD, GSH) detection: collect mouse serum or cell lysate, detect the absorbance value of each group, and detect the MDA, SOD and GSH levels of each group of mice or cells.

[0058] Step 2: Reactive oxygen species (ROS) detection: The treated cells were loaded with Iris fluorescent probes, incubated for 30 minutes, and then photographed using a fluorescence microscope.

[0059] The results show that:

[0060] (1) Malondialdehyde, superoxide dismutase and glutathione (MDA, SOD, GSH) detection: Compared with the sepsis acute kidney injury group, the MDA level in the chrysophanol treatment group was significantly decreased, and the SOD and GSH levels were significantly increased.

[0061] (2) Reactive oxygen species (ROS) detection: ROS in the septic acute kidney injury group was significantly higher than that in the control group, and ROS showed a downward trend after treatment with chrysophanol.

[0062] The above results indicate that chrysophanol reduces the level of oxidative stress in the sepsis-induced acute kidney injury model.

[0063] Example 5

[0064] according to Figure 5 As shown, it can be found that chrysophanol can reduce the expression levels of inflammatory cytokines IL-6 and TNF-α in vivo and in vitro, inhibit the inflammatory response, Figure 5 A is the level of IL-6 in the serum of each group of mice; B is the level of TNF-α in the serum of each group of mice; C is the level of IL-6 in the supernatant of renal tubular epithelial cells of each group; D is the level of TNF-α in the supernatant of renal tubular epithelial cells of each group. The steps are as follows:

[0065] ELISA (immunoenzyme-linked adsorption assay) detection of inflammatory factors (TNF-α, IL-6): serum or supernatant of mice and cells in each group was collected to detect the expression of pro-inflammatory factors TNF-α and IL-6, and the level of inflammatory response was analyzed.

[0066] The results showed that the levels of proinflammatory factors TNF-α and IL-6 were significantly increased in the septic acute kidney injury group, but after treatment with chrysophanol, the levels of proinflammatory factors were significantly reduced.

[0067] The above results show that chrysophanol significantly inhibits the inflammatory response in septic acute kidney injury and reduces the expression level of proinflammatory factors.

[0068] Example 6

[0069] according to Figure 6 As shown in the results, it was found that chrysophanol can reduce the apoptosis level of renal tubular epithelial cells in vivo and in vitro. Figure 6 A shows the expression of apoptosis-related proteins in the kidney tissue of mice in each group; B shows the morphological changes of apoptosis in renal tubular epithelial cells in each group, and the steps are as follows:

[0070] Step 1. Western Blot detection of apoptosis-related protein expression: fully grind and lyse the kidney tissue of each group of mice, extract tissue protein, separate 50 μg of protein on 10% SDS-PAGE gel electrophoresis, and electroblot onto a PVDF membrane. The following steps were performed in sequence: blocking - primary antibody incubation - washing - secondary antibody incubation - washing - chemiluminescence development, and the expression of apoptosis proteins in the kidneys of each group of mice was photographed and analyzed.

[0071] Step 2: Hoechst 33258 apoptosis morphology staining: Each group of HK-2 cells was fixed with paraformaldehyde for 30 minutes, and stained with Hoechst 33258 staining solution at room temperature in the dark for 5 minutes. The staining solution was discarded and the cells were gently rinsed. Finally, a small amount of anti-fluorescence quenching mounting solution was added and the cells were photographed and observed under a fluorescence microscope.

[0072] The results show that:

[0073] (1) Western Blot analysis of apoptosis-related protein expression: The expression level of the pro-apoptotic protein BAX in the kidney tissue of mice in the septic acute kidney injury group was significantly higher than that in the blank control group and the sham operation group, while the anti-apoptotic protein Bcl-2 was significantly decreased; while the expression level of the pro-apoptotic protein BAX in the chrysophanol treatment group was significantly decreased, while the expression level of the anti-apoptotic protein Bcl-2 was significantly increased.

[0074] (2) Hoechst33258 apoptosis morphological staining: cells in the control group showed uniformly diffused blue fluorescence; cells in the sepsis acute kidney injury group showed obvious apoptosis characteristics, abnormal cell morphology, increased cell fragments, and densely stained granular fluorescence in the cell nucleus; while the apoptotic cells in the chrysophanol treatment group were significantly reduced, indicating that chrysophanol can inhibit cell apoptosis in sepsis acute kidney injury.

[0075] The above results indicate that chrysophanol can inhibit cell apoptosis in septic acute kidney injury.

[0076] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above-described embodiments. The above-described embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the framework and scope of application of the present invention. Such changes and improvements are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A use of chrysophanol in the preparation of a medicine for treating acute kidney injury caused by sepsis.

2. The use of chrysophanol according to claim 1 in the preparation of a medicament for treating sepsis-induced acute kidney injury, characterized in that: The molecular structural formula of chrysophanol is shown below:

3. The use of chrysophanol according to claim 1 in preparing a medicament for treating acute kidney injury caused by sepsis, characterized in that: The application includes inhibiting sepsis-induced acute kidney injury in an in vivo model, which is a mouse model induced by cecal ligation and puncture.

4. The use of chrysophanol according to claim 3 in the preparation of a medicament for treating acute kidney injury caused by sepsis, characterized in that: Inhibiting sepsis-induced acute kidney injury in the in vivo model includes one or more of improving kidney tissue structural damage, reducing the oxidative stress level in the mouse kidney, inhibiting inflammatory response, and reducing the expression level of kidney apoptosis protein.

5. The use of chrysophanol according to claim 1 in preparing a medicament for treating acute kidney injury caused by sepsis, characterized in that: The application also includes inhibiting sepsis-induced acute kidney injury in an in vitro model, wherein the in vitro model is a lipopolysaccharide-induced renal tubular epithelial cell model.

6. Use of chrysophanol according to claim 5 in preparing a medicament for treating acute kidney injury caused by sepsis, characterized in that: Inhibiting sepsis-induced acute kidney injury in the in vitro model includes one or more of enhancing the viability of renal tubular epithelial cells, reducing their apoptosis level and expression of apoptosis-related proteins, and reducing inflammatory response.

7. A pharmaceutical composition for treating acute kidney injury caused by sepsis, characterized in that: The invention comprises an effective therapeutic dose of chrysophanol and a pharmaceutically acceptable carrier.

8. A pharmaceutical composition for treating sepsis-induced acute kidney injury according to claim 7, characterized in that: The dosage form of the pharmaceutical composition is injection, tablet, capsule, oral solution, suspension or granule.

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