Use of echinocandins for the preparation of a medicament for the treatment of sepsis

Abstract

The application belongs to the technical field of medicine, and mainly relates to application of echinocystic acid in preparation of a medicine for treating sepsis. A mouse sepsis model is established by using a cecal ligation and puncture (CLP) method, the influence of the echinocystic acid on sepsis mice and sepsis kidney injury is observed, and a human renal tubular epithelial cell (HK-2) is stimulated by using a lipopolysaccharide to evaluate the influence of the echinocystic acid on cell inflammation and apoptosis. The results show that the echinocystic acid can reduce the related symptoms of the sepsis mice, reduce kidney injury of the sepsis mice by inhibiting CLP-mediated kidney tissue inflammation and apoptosis, and improve the survival rate. Meanwhile, the echinocystic acid can effectively weaken LPS-induced inflammation and apoptosis of the HK-2 cells. It is shown that the echinocystic acid has a good application prospect in treatment of sepsis and sepsis-related acute kidney injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of cystic acid in the preparation of drugs for treating sepsis. Background Technology

[0002] Sepsis is characterized by a dysregulated host response to infection, leading to life-threatening organ dysfunction. Despite significant advances in treatment with antibiotics and life support, the mortality rate for sepsis patients remains high. Sepsis is a major cause of acute kidney injury in critically ill patients, with a high in-hospital mortality rate. Sepsis-associated-acute kidney injury (SA-AKI) often indicates a poorer prognosis for patients. Currently, the molecular mechanisms regulating this disease are not fully understood, there are no satisfactory therapeutic drugs in clinical practice, and breakthroughs in pathological mechanism research have not been achieved. Therefore, exploring new drug molecules for the treatment of sepsis and SA-AKI is an urgent problem to be solved.

[0003] Echinocystic acid (EA) is derived from a variety of medicinal plants, including Gleditsia sinensis, Gleditsia japonica, Gleditsia sinensis thorns, and Albizia julibrissin, and its sources are relatively widespread. Its structural formula is shown in formula (I). It possesses effective antioxidant, anti-inflammatory, and antitumor properties. However, there are currently no reports in this field regarding the application of echinocystic acid in the treatment of sepsis and SA-AKI.

[0004] Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the use of spiculic acid in the preparation of a drug for treating sepsis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of spiculosic acid in the preparation of drugs for treating sepsis.

[0008] Preferably, the drug can reduce serum inflammatory factor levels, improve sepsis-related organ function damage, and inhibit sepsis-related tissue damage.

[0009] This invention also provides the use of styracimenic acid in the preparation of drugs for treating sepsis-related organ dysfunction or tissue damage.

[0010] Preferably, the drug can inhibit colon shortening and improve liver and kidney function.

[0011] Preferably, the drug can inhibit sepsis-induced damage to the heart, liver, lungs, colon, and kidneys.

[0012] This invention also provides the use of spiculosic acid in the preparation of drugs for treating sepsis-related acute kidney injury.

[0013] Preferably, the drug can improve renal tissue function.

[0014] Preferably, the drug can inhibit apoptosis of renal tissue and renal cells and reduce inflammation of renal tissue and renal cells.

[0015] Preferably, the dosage form of the drug includes tablets, granules, capsules, pills, oral liquids, or injections.

[0016] Preferably, the drug also includes a pharmaceutically acceptable carrier.

[0017] The beneficial effects of this invention are:

[0018] This invention is the first to propose the application of spirochetal acid in the preparation of drugs for treating sepsis. A mouse sepsis model was established using the cecal ligation-puncture method (CLP), and the effects of spirochetal acid on sepsis-associated mice and sepsis-related kidney injury were observed. Lipopolysaccharide (LPS) stimulation of human kidney-2 (HK-2) cells was used to evaluate the effects of spirochetal acid on cellular inflammation and apoptosis. Results showed that spirochetal acid could alleviate the elevated levels of serum inflammatory factors, organ dysfunction, and tissue damage in sepsis-associated mice; and by inhibiting CLP-mediated renal tissue inflammation and apoptosis, it reduced kidney damage in sepsis-associated mice and improved their survival rate. Simultaneously, spirochetal acid treatment also effectively reduced LPS-stimulated inflammation and apoptosis in HK-2 cells. This indicates that spirochetal acid can effectively treat sepsis and sepsis-related acute kidney injury. Attached Figure Description

[0019] Figure 1 The therapeutic effect of EA on CLP-induced sepsis is shown in the figure below. A: Schematic diagram of the experimental process for the therapeutic effect of EA on CLP-induced sepsis; B: Survival rate of mice in each group; C: Body weight of mice in each group; D: MSS of each group; E: Serum IFN-γ level of each group; F: Serum IFN-α level of each group; G: Serum IL-6 level of each group; H: LDH level of each group; I: ALT level of each group; J: AST level of each group; K: Cr level of each group; L: BUN level of each group; M: Colon length of each group.

[0020] Figure 2 The therapeutic effect of EA on CLP-induced organ damage;

[0021] Figure 3The therapeutic effect of EA on CLP-induced renal tissue damage is shown in the following figures: A: magnified H&E staining images of renal tissue in each group; B: immunohistochemical staining images of IL-18 and NGAL in renal tissue in each group; C: expression level of NGAL mRNA in each group; D: expression level of KIM-1 mRNA in each group; E: expression level of MCP-1 mRNA in each group.

[0022] Figure 4 To illustrate the therapeutic effect of EA on CLP-induced renal apoptosis, A: apoptosis staining images of renal cells in each group, B: expression level of BAX / Bcl-2 mRNA in each group, C: expression level of related proteins in each group, and D: relative expression levels of BAX / Bcl-2 and Cleavedcaspase3 / β-Tubulin proteins in each group.

[0023] Figure 5 To illustrate the therapeutic effect of EA on LPS-induced apoptosis in HK-2 cells, A: Effect of EA on cell viability, B: Effect of EA on cell viability after LPS treatment, C: Expression level of BAX / Bcl-2 mRNA in each group, D: Expression level of related proteins in each group, E: Relative expression levels of BAX / Bcl-2 and Cleaved caspase3 / β-Tubulin proteins in each group.

[0024] Figure 6 For the therapeutic effect of EA on CLP-induced renal tissue inflammation, A: expression level of related proteins in each group, B: relative expression level of p-NF-κB p65 / NF-κB p65, NLRP3 / β-Tubulin, and IL-8 / β-Tubulin proteins in each group, C: expression ratio of iNOS, TNF-α, IL-6, and IL-1β mRNA in each group;

[0025] Figure 7 To evaluate the therapeutic effect of EA on LPS-induced renal cell inflammation, A: expression levels of related proteins in each group; B: relative expression levels of TLR4 / GAPDH, p-NF-κB p65 / NF-κB p65, and p-IKB / IKB proteins in each group; C: relative expression levels of NLRP3, ASC, Caspase1, IL-18, and IL-1β proteins relative to GAPDH in each group; D: expression level of TNF-α mRNA in each group; E: expression level of IL-6 mRNA in each group; F: expression level of IL-1β mRNA in each group; G: expression level of IL-8 mRNA in each group. Detailed Implementation

[0026] This invention provides the application of spiculosic acid in the preparation of drugs for treating sepsis.

[0027] In this invention, a mouse sepsis model was established using the cecum ligation and puncture (CLP) method, and human renal tubular epithelial cells (Humankidney-2, HK-2) were stimulated with lipopolysaccharide (LPS). After EA treatment, sepsis-related symptoms were alleviated, and organ dysfunction and tissue damage were inhibited, particularly inflammation and apoptosis of renal tissue and kidney cells. This effectively treats sepsis, sepsis-related organ dysfunction or tissue damage, especially sepsis-related acute kidney injury. This invention does not specifically limit the source of spirochetal acid; any commercially available product in the art can be used. The structural formula of the spirochetal acid is shown in Formula (I).

[0028]

[0029] In this invention, the drug can reduce serum inflammatory factor levels caused by sepsis, improve sepsis-related organ dysfunction, and inhibit sepsis-related tissue damage. Preferably, the serum inflammatory factors include TNF-α, IL-6, and IFN-γ.

[0030] This invention also provides the use of styracimenic acid in the preparation of drugs for treating sepsis-related organ dysfunction or tissue damage.

[0031] In this invention, the drug can inhibit colonic shortening and improve liver and kidney function to treat sepsis-related organ dysfunction. The drug can also inhibit sepsis-induced damage to the heart, liver, lungs, colon, and kidneys.

[0032] This invention also provides the use of spiculosic acid in the preparation of drugs for treating sepsis-related acute kidney injury.

[0033] In this invention, the drug can improve renal tissue function, inhibit renal tissue and renal cell apoptosis, and reduce renal tissue and renal cell inflammation to achieve the treatment of sepsis-related acute kidney injury.

[0034] In this invention, the dosage form of the drug includes, but is not limited to, tablets, granules, capsules, pills, oral liquids, or injections. The drug also includes a pharmaceutically acceptable carrier. This invention does not impose any particular limitation on the carrier component contained in the drug; commonly used pharmaceutical carriers in the art can be used.

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Unless otherwise specified, the following embodiments are all conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] Example 1

[0039] The therapeutic effect of EA on CLP-induced sepsis

[0040] I. Experimental Methods

[0041] Sepsis in mice was induced using a cecal ligation-perforation (CLP) model: C57BL / 6 mice (male, 6-8 weeks old, 20±2g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were placed under constant temperature and sterile conditions and given standard food and water (12-hour light-dark cycle). All experimental procedures were approved by the Institutional Animal Protection Committee of Nankai University (No.: 2022-SYDWLL-000586).

[0042] Mice were randomly divided into 5 groups of 8 mice each: a control group (sham-operated group), a CLP group, and 3 treatment groups: CLP+CS (20 mg / kg), CLP+EA (10 mg / kg), and CLP+EA (20 mg / kg). The CLP+CS group served as a positive control, where CS was cefpirome sulfate (20 mg / kg). Control mice were treated with saline. The CLP+CS (20 mg / kg), CLP+EA (10 mg / kg), and CLP+EA (20 mg / kg) groups were treated with the same volume of the drug via intraperitoneal injection at 2 h and 24 h after CLP induction.

[0043] Survival rate, body weight, and mouse sepsis score (MSS) of mice were recorded at 0, 12, 24, 36, and 48 h after CLP surgery.

[0044] Forty-eight hours after CLP surgery, blood was collected from mice anesthetized with tribromoethanol. The blood was centrifuged at 2500 rpm for 10 minutes, and then the serum levels of TNF-α, IFN-γ, IL-6, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN) were tested according to the manufacturer’s instructions using a commercially suitable kit.

[0045] Forty-eight hours after CLP, mice were rapidly euthanized by cervical dislocation. The colon was then removed and its length measured. Heart, liver, lung, and kidney tissues were removed and fixed together with the colon in 4% paraformaldehyde, embedded in paraffin, and sliced ​​into 5 μm thick sections using a microtome. The sections were stained with H&E, and the histological changes of organs in randomly selected histological fields were observed using an optical microscope to assess the role of EA in sepsis-induced tissue damage.

[0046] The experimental procedure is as follows Figure 1 As shown in Figure A.

[0047] II. Experimental Results

[0048] 1. Mouse survival rate: Administration of EA (10 and 20 mg / kg) to animals 2 hours and 24 hours after CLP significantly improved the survival rate of septic mice. Specifically, the 48-hour survival rate was 37.5% in the CLP group and 75% in the CLP+EA (10 and 20 mg / kg) groups. Figure 1 B).

[0049] 2. Mouse weight: e.g. Figure 1 As shown in C, compared with the control group, the CLP group had a significant weight loss. At 36 and 48 hours after the model was established, the weight gain of mice in the treatment group was greater than that in the CLP group. The effects of EA (10 and 20 mg / kg) were better than those of CS (20 mg / kg).

[0050] 3. Mouse sepsis score (MSS): The sepsis score (MSS) of mice in the CLP group was significantly higher than that of the control group, but in the groups treated with EA (10 and 20 mg / kg) and CS (20 mg / kg), the symptoms caused by sepsis were reduced (MSS at 12 and 24 hours: P<0.0001). Figure 1 D).

[0051] 4. Serum inflammatory factor levels: such as Figure 1 E, Figure 1 F and Figure 1 As shown in Figure G, TNF-α, IL-6, and IFN-γ levels were elevated in the CLP group, and decreased in a dose-dependent manner after EA treatment. We observed that low-dose EA (10 mg / kg) and CS (20 mg / kg) had similar effects, while high-dose EA (20 mg / kg) was more effective than both low-dose EA (10 mg / kg) and CS (20 mg / kg). This indicates that EA treatment can significantly reduce the elevation of serum inflammatory factor levels caused by sepsis.

[0052] 5. Organ damage: such as Figure 1 H, Figure 1 I, Figure 1 As shown in Figure J, at 48 hours, serum LDH, ALT, and AST were all significantly lower in the treatment group compared to the CLP group. Meanwhile, as... Figure 1 K and Figure 1 As shown in Figure L, serum creatinine (Cr) and blood urea nitrogen (BUN) levels at 48 hours were significantly increased in the CLP group and significantly decreased in the treatment group.

[0053] In sepsis, LDH, ALT, and AST levels are significantly elevated and positively correlated with disease severity and prognosis. Creatinine (Cr) and blood urea nitrogen (BUN) are commonly used as blood markers of renal function. Therefore, the above experimental results indicate that EA treatment can also improve liver and kidney dysfunction caused by sepsis.

[0054] The colon length of mice in the CLP group was shorter than that of mice in the control group, which may be related to enteritis induced by CLP modeling. This symptom was also relieved after EA treatment. Figure 1 M).

[0055] 6. Tissue damage: such as Figure 2 As shown, in the CLP group, there was mild disorder and loosening of cardiomyocytes; mild alveolar edema and leukocyte accumulation around the pulmonary arterioles; and extensive inflammatory infiltration in the liver, kidneys, and colon. All tissues showed some degree of structural damage. After EA intervention, these lesions were alleviated to some extent. This indicates that EA can inhibit sepsis-induced tissue damage.

[0056] Example 2

[0057] EA improves renal tissue function in septic mice

[0058] 1. Histological changes in the kidney:

[0059] Experimental Methods: Kidney H&E stained sections from Example 1 were used, and histological changes were further observed under an optical microscope within randomly selected histological fields of view. Figure 3 A).

[0060] Experimental results: such as Figure 3 As shown in Figure A, the kidney tissue exhibited extensive inflammatory infiltration and a degree of structural damage. After EA intervention, the aforementioned lesions were alleviated to some extent. This indicates that EA can inhibit sepsis-induced kidney tissue damage.

[0061] 2. Levels of IL-18 and NGAL:

[0062] IL-18 is associated with increased inflammatory infiltration and more severe kidney damage. Neutrophil gelatinase-associated lipocalin (NGAL) is a protein belonging to the lipocalin superfamily. Studies have shown that NGAL levels are significantly correlated with the severity of kidney function impairment.

[0063] Experimental Methods: The location and levels of IL-18 and NGAL were observed using IHC staining. Paraffin-embedded kidney sections (5 μm thick) from Example 1 were dewaxed, hydrated, and repaired, then incubated for 10 minutes each in endogenous peroxidase inhibitors and non-specific binding inhibitors. Subsequently, the sections were incubated overnight at 4°C with suitable primary antibodies (anti-IL-18 and anti-NGAL), followed by incubation with biotin-labeled secondary antibodies. Immunoreactivity was observed using diaminobenzidine, and cell nuclei were observed using hematoxylin staining. Figure 3 B) The immunoreactivity of the target protein was quantified using ImageJ software.

[0064] Experimental results: such as Figure 3 As shown in Figure B, 48 hours after CLP surgery in mice, renal tubular epithelial cells in the CLP group expressed high levels of IL-18 and NGAL, while CS and EA treatments reduced the levels of IL-18 and NGAL in kidney tissue. This indicates that EA treatment can alleviate sepsis-induced kidney tissue damage in mice.

[0065] 3. Determination of mRNA expression levels of NGAL, KIM-1, and MCP-1

[0066] MCP-1 is a chemokine. In some kidney disease models, blocking MCP-1 can alleviate the disease, suggesting that inhibiting MCP-1 is a promising strategy for treating patients with inflammatory kidney diseases. Kidney injury molecule-1 (KIM-1) is a type I transmembrane protein expressed on damaged proximal tubular epithelial cells. It is weakly expressed in normal kidney tissue and urine, but its expression is significantly enhanced in proximal tubular epithelial cells of the kidney during kidney injury, and is associated with the severity of kidney injury. Therefore, KIM-1 can serve as a highly sensitive and specific biomarker for diagnosing early kidney injury. Thus, to confirm the presence of kidney injury in septic AKI mice, we quantified the mRNA expression levels of NGAL, KIM-1, and MCP-1.

[0067] Experimental Methods: RNA was extracted from the kidney tissue in Example 1 using Trizol reagent (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. After determining the concentration, the RNA was reverse transcribed into cDNA using a reverse transcription kit (Yeasen, Shanghai, China). Then, quantitative PCR (qPCR) was performed using the SYBR Green quantitate CT RT-PCR kit (Yeasen, Shanghai, China) according to the manufacturer's instructions.

[0068] Experimental results: The mRNA expression of NGAL, KIM-1, and MCP-1 was significantly increased in septic mice (comparison between CLP group and sham-operated group: p<0.0001, p<0.001, p<0.0001). Compared with the CLP group, EA treatment significantly reduced the mRNA expression of NGAL, KIM-1, and MCP-1 (p<0.0001, p<0.001, p<0.0001), and this reduction was dose-dependent. Figure 3 C- Figure 3 E).

[0069] These results indicate that septic AKI mice exhibit kidney damage and increased inflammation levels, and EA treatment can suppress this response.

[0070] Example 3

[0071] EA inhibits CLP-induced renal apoptosis.

[0072] 1. Effects of EA stimulation on renal apoptosis

[0073] Renal apoptosis is an important cause of SA-AKI. In this embodiment, TUNEL staining was used to study the effect of EA stimulation on renal apoptosis.

[0074] Experimental method: Kidney tissue sections from Example 1 were taken and apoptosis was detected using a one-step TUNEL apoptosis detection kit (Shanghai Yisheng Biotechnology Co., Ltd.). After permeabilization with 0.5% Triton X-100 for 15 minutes, the tissue was measured by TUNEL in the dark at 37°C for 1 hour. Then, the kidney tissue was counterstained with DAPI.

[0075] Experimental results: such as Figure 4 As shown in Figure A, the number of TUNEL-positive cells in the kidney tissue of mice in the CLP+EA (10 mg / kg, 20 mg / kg) group was significantly reduced compared to the CLP group. The results indicate that EA significantly reduced the number of TUNEL-positive cells in the kidney tissue of CLP-treated mice.

[0076] 2. Determination of the BAX / Bcl-2 mRNA expression level ratio

[0077] Experimental method: Same as the experimental method under item 3 in Example 2.

[0078] Experimental Results: RT-qPCR analysis showed that, compared with the CLP group, EA treatment reduced the ratio of BAX (B-cell lymphoma protein 2-associated X protein, an apoptosis promoter) / Bcl-2 (B-cell lymphoma protein 2, an apoptosis inhibitor) mRNA levels in the kidney tissue of septic mice. Figure 4 B).

[0079] 3. Determination of expression levels of related proteins

[0080] Experimental Methods: Total protein was extracted from the kidney tissue in Example 1 using RIPA lysate (Beyotime, Shanghai, China). Protein concentration was determined using BCA reagent (Beyotime, Shanghai, China). Total protein was separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk for 2 hours at room temperature, the membrane was incubated overnight with primary antibody, followed by washing four times with TBS-T solution (8 minutes each time). The membrane was then incubated with secondary antibody for 2 hours, followed by washing four times with TBS-T solution (8 minutes each time). Next, the membrane was incubated with ECL luminescent liquid (Yeasen, Shanghai, China). Band density was analyzed using ImageJ 1.53e software.

[0081] Experimental results: EA can downregulate the protein levels of BAX and cleaved-caspase 3. Figure 4 C), and can also downregulate the relative expression levels of BAX / Bcl-2 and Cleaved caspase3 / β-Tubulin proteins. Figure 4 D).

[0082] The above data indicate that EA has an anti-apoptotic effect on the kidney tissue of septic AKI mice.

[0083] Example 4

[0084] EA inhibits LPS-induced apoptosis in kidney cells

[0085] To elucidate the role of EA in in vitro apoptosis, we cultured the human renal tubular epithelial cell line (HK-2 cells). HK-2 cells were obtained from ATCC. Cell culture conditions were: 90% DMEM / F12 (Dulbecco modified Eagle medium / nutrient mixture F-12) + 10% FBS, 5% CO2, 37°C.

[0086] 1. The role of EA in in vitro apoptosis

[0087] Experimental method: HK-2 cells were exposed to different concentrations of EA (0, 2.5, 5, 10 and 20 μM) for 24 hours.

[0088] Experimental results: No significant decrease in the viability of HK-2 cells was observed at the above concentrations. Figure 5 A).

[0089] 2. Effects of EA on LPS-induced apoptosis in HK-2 cells

[0090] Experimental Methods: The effect of different concentrations of EA on LPS-induced apoptosis in HK-2 cells was detected using the CCK-8 assay. Cells were seeded for 12-24 hours until good cell adhesion was achieved, after which the culture medium in each well was aspirated. The control group received drug-free medium, while the experimental groups received medium containing different concentrations of the drug. The cells were then incubated for 24 hours. 10 μL of CCK-8 solution was added to each well, and the plate was incubated for 0.5 hours. The 96-well plate was then removed and the absorbance was measured at 450 nm using a microplate reader.

[0091] Experimental results: such as Figure 5 As shown in Figure B, 8 μg / ml LPS successfully induced significant apoptosis in HK-2 cells, which was alleviated by 5 μM and 10 μM EA. The results indicate that 5 μM and 10 μM EA are non-cytotoxic and can inhibit LPS-induced apoptosis.

[0092] Based on the above experimental results, we selected 5μM and 10μM EA for further research.

[0093] 3. Determination of the BAX / Bcl-2 mRNA expression level ratio

[0094] Experimental methods: HK-2 cells were seeded into six-well plates. After cell attachment, EA (5 μM and 10 μM) was added to the culture dish and incubated for 1 hour. Subsequently, the cells were exposed to LPS (8 μg / ml) for 23 hours, and then total RNA was extracted for RT-qPCR analysis.

[0095] Experimental results: RT-qPCR analysis revealed that, compared to the LPS group, EA (5 μM and 10 μM) reduced the BAX / Bcl-2 mRNA level ratio in HK-2 cells. Figure 5 C).

[0096] 4. Determination of expression levels of related proteins

[0097] Experimental Methods: Cells were seeded into six-well plates. After cell attachment, EA was added to the culture dish and incubated for 1 hour. Subsequently, cells were exposed to LPS (8 μg / ml) for 30 minutes, and total protein was extracted from HK-2 cells using RIPA lysate (Beyotime, Shanghai, China). Protein concentration was determined using BCA reagent (Beyotime, Shanghai, China). Total protein was separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk at room temperature for 2 hours, the membrane was incubated overnight with primary antibody, followed by washing four times with TBS-T solution (8 minutes each time). The membrane was then incubated with secondary antibody for 2 hours, followed by washing four times with TBS-T solution (8 minutes each time). Finally, the membrane was incubated with ECL luminescent liquid (Yeasen, Shanghai, China). Band density was analyzed using ImageJ 1.53e software.

[0098] Experimental results: EA can downregulate the protein levels of BAX and cleaved caspase 3. Figure 5 D), and downregulated the relative expression levels of BAX / Bcl-2 and Cleaved caspase3 / β-Tubulin proteins (D). Figure 5 E).

[0099] The above results indicate that EA has an anti-LPS-induced apoptosis effect on HK-2 cells.

[0100] Example 5

[0101] The therapeutic effect of EA on CLP-induced renal tissue inflammation

[0102] Inflammatory infiltration in the kidneys can promote apoptosis of renal tubular epithelial cells and induce acute kidney injury (AKI). To evaluate whether EA can reverse CLP-induced inflammation, we used Western blotting to detect the protein expression of NF-κB p65, p-NF-κB p65, NLRP3, and IL-18.

[0103] 1. Determination of protein expression levels of NF-κB p65, p-NF-κB p65, NLRP3, and IL-18

[0104] Experimental method: Same as the experimental method under item 3 in Example 3.

[0105] Experimental results: Compared with the CLP group, EA pretreatment effectively attenuated the expression of p-NF-κB p65, NLRP3, and IL-18. Figure 6 A) and reduced the relative protein expression levels of p-NF-κB p65 / NF-κB p65, NLRP3 / β-Tubulin, and IL-8 / β-Tubulin.

[0106] 2. Determination of iNOS, TNF-α, IL-6 and IL-1β mRNA expression levels

[0107] Experimental method: Same as the experimental method under item 3 in Example 2.

[0108] Experimental results: CLP stimulation significantly increased the gene levels of iNOS, TNF-α, IL-6, and IL-1β in renal tissue, while EA decreased their levels. Figure 6 C).

[0109] These results indicate that EA can alleviate CLP-induced kidney inflammation in CLP-induced septic mice.

[0110] Example 6

[0111] The therapeutic effect of EA on LPS-induced renal cell inflammation

[0112] 1. Determination of expression levels of related proteins

[0113] Experimental methods: Cells were seeded into six-well plates. After cell attachment, EA was added to the culture dish and incubated for 1 hour. Subsequently, the cells were exposed to LPS (1 μg / ml) for 30 minutes, and then total protein was extracted for Western blot analysis.

[0114] Experimental results: Immunoblot analysis showed that LPS treatment significantly activated the NF-κB and NLRP3 signaling pathways, while pre-incubation with EA (5 μM and 10 μM) reduced LPS-triggered phosphorylation of NF-κB-p65 and IKB. Figure 7 A- Figure 7 C) It also inhibits the activation of downstream signals of NLRP3.

[0115] 2. Determination of mRNA expression levels of TNF-α, IL-6, IL-1β and IL-8

[0116] Experimental methods: HK-2 cells were seeded into six-well plates. After cell attachment, EA (5 μM and 10 μM) was added to the culture dish and incubated for 1 hour. Subsequently, the cells were exposed to LPS (1 μg / ml) for 23 hours, and then total RNA was extracted for RT-qPCR analysis.

[0117] Experimental results: 23 hours after LPS stimulation, EA could reverse the LPS-induced upregulation of TNF-α, IL-6, IL-1β and IL-8 mRNA abundance. Figure 7 D- Figure 7 G).

[0118] These results indicate that EA pretreatment reduced LPS-induced inflammatory responses in HK-2 cells.

[0119] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of spiculosic acid in the preparation of drugs for treating sepsis.

2. The application according to claim 1, characterized in that, The drug can reduce serum inflammatory factor levels, improve sepsis-related organ function damage, and inhibit sepsis-related tissue damage.

3. Application of cystic acid in the preparation of drugs for treating sepsis-related organ dysfunction or tissue damage.

4. The application according to claim 3, characterized in that, The drug can inhibit the shortening of colon length and improve liver and kidney function.

5. The application according to claim 3, characterized in that, The drug can inhibit sepsis-induced damage to the heart, liver, lungs, colon, and kidneys.

6. Application of cystic acid in the preparation of drugs for treating sepsis-related acute kidney injury.

7. The application according to claim 6, characterized in that, The drug can improve kidney tissue function.

8. The application according to claim 6, characterized in that, The drug can inhibit apoptosis of kidney tissue and kidney cells and reduce inflammation of kidney tissue and kidney cells.

9. The application according to any one of claims 1-8, characterized in that, The dosage forms of the drug include tablets, granules, capsules, pills, oral liquids, or injections.

10. The application according to claim 9, wherein the medicament further comprises a pharmaceutically acceptable carrier.

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