Application of hydrated oxidized imperatorin in the preparation of drugs for the prevention and / or treatment of acute kidney injury

Abstract

This invention discloses the application of hydrated oxidized imperatorin in the preparation of drugs for the prevention and / or treatment of acute kidney injury. Hydrated oxidized imperatorin can effectively alleviate septic acute kidney injury and cisplatin-induced acute kidney injury, and its mechanism is related to improving the inflammatory microenvironment and reducing renal tubular epithelial cell damage. In an in vitro human renal proximal tubular epithelial cell model (HK2 cells), hydrated oxidized imperatorin can significantly inhibit cisplatin-induced cell damage, reduce the mRNA expression level of kidney injury molecules, and promote cell viability recovery in a dose-dependent manner.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, and more specifically relates to a new application of hydrated oxidized imperatorin in the preparation of drugs for treating acute kidney injury. Background Technology

[0002] Septic acute kidney injury (S-AKI) and drug-induced acute kidney injury (D-AKI) are the two most common subtypes of AKI in clinical practice, accounting for 50% of complications in critically ill patients and 20-30% in patients undergoing chemotherapy for cancer, respectively, and both lack specific treatments. The core pathological mechanism of S-AKI involves dysregulation of systemic and local renal inflammatory responses, mitochondrial dysfunction, and metabolic reprogramming. Among these, M1 polarization of renal macrophages, by activating the oxidative stress-inflammation pathway and remodeling the inflammatory microenvironment, exerts cytotoxic effects on adjacent renal tubular epithelial cells, and is a key step driving the progression of kidney injury. Cisplatin- and other chemotherapy-induced AKI, on the other hand, is centered on direct renal tubular epithelial cell cytotoxicity, leading to a rapid decline in renal function through DNA cross-linking, mitochondrial damage, and secondary inflammatory responses. Although the initiating factors of the two types of AKI differ—S-AKI is triggered by pathogen-associated molecular patterns, such as cecal ligation perforation (CLP), leading to a systemic inflammatory storm, while cisplatin-based AKI is dominated by direct drug cytotoxicity—both converge on a common pathway in their pathological progression: renal tubular epithelial cell damage, macrophage overactivation, and imbalance of the inflammatory microenvironment. Currently, there are no specific therapeutic drugs for these AKI subtypes in clinical practice; therefore, finding drugs that can simultaneously intervene in multiple targets of inflammation and cell damage is of great significance.

[0003] Oxypeucedanin hydrate (CAS No.: 2643-85-8) is a natural furanocoumarin compound with the molecular formula C2. 16 H 16 O6 has a molecular weight of 304.29 and a chemical structure of 4-[(2R)-2,3-dihydroxy-3-methylbutoxy]furan[3,2-g]chromene-7-one.

[0004]

[0005] Hydrated oxidized imperatorin.

[0006] This compound is widely found in various Apiaceae medicinal plants, including Peucedanum praeruptorum, Peucedanum dahurica, Angelica dahurica, and Peucedanum apaense, as well as in large quantities in Rutaceae plant, Ligusticum chuanxiong. Numerous studies have shown that hydrated oxypenicillin possesses significant antioxidant, antifungal, and calcium antagonistic activities, but there are currently no reports on its use in the treatment of acute kidney injury (AKI). Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide the use of hydrated oxidized imperatorin in the preparation of a drug for treating acute kidney injury.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions: On one hand, this invention provides the use of hydrated oxidized imperatorin in the preparation of medicaments for the prevention and / or treatment of acute kidney injury. The structural formula of hydrated oxidized imperatorin is: .

[0009] The acute kidney injury mentioned is septic acute kidney injury.

[0010] The septic acute kidney injury includes those induced by Gram-negative bacterial infection or lipopolysaccharide, or by cecal ligation and perforation surgery.

[0011] The acute kidney injury mentioned is drug-induced acute kidney injury.

[0012] The drug-induced acute kidney injury includes kidney injury induced by cisplatin.

[0013] The hydrated oxytocin can prevent or treat acute kidney injury by inhibiting inflammatory responses and / or reducing damage to renal tubular epithelial cells.

[0014] The inhibition of the inflammatory response includes inhibiting macrophage polarization toward the M1 type and / or reducing the levels of inflammatory factors.

[0015] The inflammatory factors include one or more of tumor necrosis factor-α, interleukin-1β, and interleukin-6.

[0016] The in vivo dosage of the hydrated oxidized imperatorin is 20-40 mg / kg.

[0017] The dosage form of the drug is injection, tablet, capsule or suspension.

[0018] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of acute kidney injury, comprising an effective amount of hydrated oxypereugenol as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

[0019] The acute kidney injury referred to is either septic acute kidney injury or drug-induced acute kidney injury.

[0020] This invention offers the following advantages over existing technologies: It is the first to disclose the use of hydrated oxidized imperatorin as a drug for treating septic acute kidney injury. Hydrated oxidized imperatorin exhibits significant protective effects against CLP-induced septic acute kidney injury models, significantly reducing serum creatinine, blood urea nitrogen, and cystatin C levels, and alleviating renal tissue inflammation and tubular damage. In an in vitro macrophage / renal tubular epithelial cell co-culture system (human cell system: PBMC / HK2), hydrated oxidized imperatorin significantly reduces the levels of inflammatory factors in the supernatant of the co-culture system and decreases the expression of KIM1 in renal tubular epithelial cells. Its mechanism of action is related to hydrated oxidized imperatorin's inhibition of macrophage M1 polarization, reduction of inflammatory factor levels, and alleviation of renal tubular epithelial cell damage, showing broad application prospects and providing a new drug option for the treatment of septic acute kidney injury.

[0021] The drug-induced acute kidney injury was induced by cisplatin.

[0022] This invention also discloses for the first time the use of hydrated oxidized imperatorin as a medicament for treating drug-induced acute kidney injury (DIK), which is induced by cisplatin. Hydrated oxidized imperatorin exhibits significant protective effects against cisplatin-induced DIK, significantly reducing serum creatinine, blood urea nitrogen, and cystatin C levels, alleviating the release of the renal inflammatory factor IL-6, and reducing renal tubular epithelial cell damage. In an in vitro human renal proximal tubular epithelial cell (HK2 cell) model, hydrated oxidized imperatorin significantly inhibited cisplatin-induced cell damage, reduced the mRNA expression level of kidney injury molecule 1 (KIM-1), and promoted cell viability recovery in a dose-dependent manner. Its mechanism of action is related to hydrated oxidized imperatorin's inhibition of inflammatory responses, reduction of renal proximal tubular epithelial cell apoptosis, and promotion of cell proliferation. It shows broad application prospects and provides a new drug option for the treatment of drug-induced DIK caused by cisplatin and other chemotherapy drugs. Attached Figure Description

[0023] Figure 1Effects of hydrated oxidized imperatorin on renal function indicators and pathological damage in CLP-induced septic acute kidney injury in mice. AC: Effects of hydrated oxidized imperatorin on serum urea nitrogen (A), creatinine (B), and cystatin C (C) levels in mice with CLP-induced septic acute kidney injury. DF: Effects of hydrated oxidized imperatorin on IL-1β (D), TNF-α (E), and IL-6 (F) levels in renal tissue homogenate from mice with CLP-induced septic acute kidney injury. G: Effects of hydrated oxidized imperatorin on KIM1 mRNA levels in renal tissue from mice with CLP-induced septic acute kidney injury.

[0024] Figure 2 : Protective effect of hydrated oxidized imperatorin on cisplatin-induced acute kidney injury in mice. AC: Effects of hydrated oxidized imperatorin on serum urea nitrogen (A), creatinine (B), and cystatin C (C) levels in mice with cisplatin-induced acute kidney injury. DF: Effects of hydrated oxidized imperatorin on TNF-α (D), IL-1β (E), and IL-6 (F) levels in renal tissue homogenate from mice with cisplatin-induced acute kidney injury. G: Effects of hydrated oxidized imperatorin on KIM1 mRNA levels in renal tissue from mice with cisplatin-induced acute kidney injury.

[0025] Figure 3 Effects of hydrated oxidized imperatorin on the release of inflammatory mediators in PBMC cells induced by LPS in a PBMC / HK2 co-culture system and its influence on HK2 damage markers. A: Schematic diagram of the PBMC / HK2 Transwell co-culture system. B: Effect of hydrated oxidized imperatorin on PBMC survival rate. C: Effect of hydrated oxidized imperatorin on CD86 mRNA levels in LPS-induced PBMCs. D, E: Effects of hydrated oxidized imperatorin on LDH (D) and IL-6 (E) levels in the supernatant of the LPS-induced PBMC / HK2 co-culture system. F: Effect of hydrated oxidized imperatorin on KIM1 mRNA levels in HK2 cells in the LPS-induced PBMC / HK2 co-culture system.

[0026] Figure 4 Effects of hydrated oxidized imperatorin on HK2 cell survival.

[0027] Figure 5 Effects of hydrated oxidized imperatorin on cisplatin-induced HK2 cell damage, specifically the effects of AC:hydrated oxidized imperatorin on the levels of KIM1, MCP-1, and IL-6 mRNA in cisplatin-induced HK2 cells. Detailed Implementation

[0028] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0029] The RT-qPCR, ELISA, and CCK-8 methods used in the examples are all conventional experimental methods.

[0030] Consumables and reagents: Hydrated oxidized imperatorin (purity >98%, CAS: 2643-85-8, purchased from Chengdu Efa Biotechnology Co., Ltd.); Lipopolysaccharide (LPS, Sigma); Cisplatin (CAS: 15663-27-1, Yisheng Biotechnology (Shanghai) Co., Ltd.); Creatinine assay kit and urea nitrogen assay kit purchased from Nanjing Jiancheng Bioengineering Institute; KIM1 antibody purchased from Proteintech; Surgical instruments for cecal ligation and perforation.

[0031] Cells: Human renal proximal tubule epithelial cells (HK-2), purchased from the American Type Culture Collection Center (ATCC); peripheral blood mononuclear cells (PBMCs), purchased from Nanjing Runyan Biotechnology Co., Ltd.

[0032] Animals: C57BL / 6 mice, 6-8 weeks old, weighing 22±2 g, acclimatized for 1 week.

[0033] Table 1: List of English-Chinese Abbreviations

[0034] Example 1: Protective effect of hydrated oxidized imperatorin against CLP-induced septic acute kidney injury in mice C57BL / 6 mice were randomly divided into four groups: a sham-operated group (Sham), a CLP model group, a low-dose CLP + hydrated oxyperturbinate group (20 mg / kg), a high-dose CLP + hydrated oxyperturbinate group (40 mg / kg), and a CLP + dexamethasone positive control group (5 mg / kg), with eight mice in each group. In the CLP model group and the drug-treated group, a sepsis model was established using the cecal ligation and perforation method: after anesthesia, the mice underwent abdominal incision along the midline to expose the cecum. The cecum was ligated at the distal half with 4-0 silk suture, and a 21G needle was used to puncture it once. A small amount of feces was gently squeezed out, the cecum was returned to its original position, and the abdominal wall was sutured. In the sham-operated group, only the cecum was opened, turned over, and then sutured. The drugs were administered intraperitoneally immediately after surgery and at 6 and 12 hours post-surgery.

[0035] Serum and kidney tissue were collected under anesthesia 24 hours postoperatively. Serum creatinine (CRE), blood urea nitrogen (BUN), and cystatin C (Cys C) levels were measured according to the kit instructions. Total RNA was extracted from kidney tissue, reverse transcribed into cDNA, and the mRNA expression level of kidney injury molecule-1 (KIM-1) was detected by RT-qPCR. Primers were synthesized by Anhui General Company, and the primer sequences are as follows. mouse KIM1: Forward: SEQ ID NO:1; mouse Reverse: SEQ ID NO:2; mouse / human GAPDH: Forward: SEQ ID NO:3; mouse / human Reverse: SEQ ID NO:4.

[0036] The results are as follows Figure 1 As shown: Compared with the Sham group, the CLP model group had significantly increased serum CRE, BUN, and Cys C levels, and significantly increased KIM-1 mRNA and protein expression levels in kidney tissue, indicating severe pathological damage to the kidney tissue. Compared with the CLP model group, the hydrated oxidized imperatorin treatment group had significantly decreased BUN, CRE, and Cys C levels. Figure 1 In the middle jiao (AC), the levels of interleukin-1β (IL-1β), the inflammatory cytokine tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in renal tissue homogenate decreased. Figure 1 In addition to DF), the expression of kidney injury molecule-1 (KIM-1) decreased in a dose-dependent manner. Figure 1 (G). The above results indicate that hydrated oxidized imperatorin can alleviate acute kidney injury caused by sepsis, and its mechanism may be related to reducing renal tissue inflammation and renal tubular damage.

[0037] Example 2: The alleviating effect of hydrated oxidized imperatorin on a cisplatin-induced acute kidney injury model Six- to eight-week-old C57BL / 6 mice were acclimatized for one week and then randomly divided into four groups: a control group (NC), a model group (Cisplatin), a low-dose hydrated imperatorin group (Cisplatin + HO 20 mg / kg), a high-dose hydrated imperatorin group (Cisplatin + HO 40 mg / kg), and a positive control group (Cisplatin + DEX 5 mg / kg), with eight mice in each group. Acute kidney injury models were established by intraperitoneal injection of 20 mg / kg cisplatin into the model group, the low- and high-dose hydrated imperatorin groups, and the positive control group. Simultaneously with model establishment, the low- and high-dose hydrated imperatorin groups were intraperitoneally injected with 20 mg / kg and 40 mg / kg hydrated imperatorin, respectively, while the positive control group was intraperitoneally injected with 5 mg / kg dexamethasone. The control group was injected with an equal volume of solvent, and these treatments were administered for three consecutive days.

[0038] Three days later, blood samples and kidney tissue were collected from mice under anesthesia. The levels of IL-1β, TNF-α, and IL-6 in the kidney tissue homogenate were detected using enzyme-linked immunosorbent assay (ELISA). Total RNA was extracted from the kidney tissue, reverse transcribed, and the mRNA expression level of KIM-1 was detected by RT-qPCR. Primers were synthesized by Anhui General Machinery Co., Ltd., and the primer sequences were the same as in Example 1.

[0039] The results are as follows Figure 2 As shown: Compared with the control group, the cisplatin model group showed significantly increased serum BUN, CRE, and Cys C levels, significantly increased levels of inflammatory factors in renal tissue homogenate, and significantly increased KIM-1 mRNA expression levels in renal tissue. Compared with the model group, hydrated oxidized imperatorin at 20 mg / kg and 40 mg / kg significantly reduced the above-mentioned serum biochemical indicators BUN, CRE, and Cys C. Figure 2 The levels of inflammatory factors (TNF-α, interleukin-1β, and interleukin-6) in renal tissue homogenate were also measured. Figure 2 (DF), and showed dose-dependent inhibition of KIM-1 mRNA expression ( Figure 2 (G). The results confirmed that hydrated oxidized imperatorin has a protective effect against cisplatin-induced acute kidney injury.

[0040] Example 3: Establishment of PBMC / HK2 Transwell co-culture system and intervention of hydrated oxidized imperatorin A 24-well Transwell co-culture system was used. PBMCs induced by m-CSF for 7 days were cultured at 1×10⁻⁶. 6 The cells were seeded at a density of 10 cells / well in the upper chamber; HK2 cells were simultaneously seeded at a rate of 2 × 10⁶ cells / well. 5Cells were seeded at a density of 1 cell per well in the lower 24-well culture plate and cultured in DMEM / F12 complete medium. After both cell lines were stably adhered, the upper chamber containing PBMCs was transferred to the lower culture plate containing HK2 to establish a non-contact co-culture system (e.g., ...). Figure 3 (As shown in A).

[0041] Cytotoxicity assay of hydrated oxidized imperatorin on PBMCs (CCK-8 assay) The cytotoxicity of hydrated oxidized imperatorin to m-CSF-induced differentiation of PBMCs was detected using the CCK-8 assay. PBMCs were divided into groups of 4 × 10⁶ cells / year. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and induced to differentiate for 7 days in RPMI 1640 complete medium containing 50 ng / mL m-CSF. Experimental groups included: NC group (normal control group, containing 0.1% DMSO) and hydrated oxidized imperatorin groups (10, 20, 40, 80, 160 μM). After 24 h of drug treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37 ℃ for 4 h. The absorbance (OD value) at 450 nm was measured using a microplate reader, and cell viability was calculated. Results are as follows: Figure 3 As shown in Figure B, hydrated oxidized imperatorin showed no significant cytotoxicity to PBMCs in the concentration range of 10–160 μM (cell viability >95%), suggesting that this concentration range can be used for subsequent co-culture experiments.

[0042] After establishing the co-culture system, the cells were randomly divided into the following 5 groups (n=4): ① NC group (normal control group, containing 0.1% DMSO); ② LPS group (1 μg / mL LPS); ③ LPS + low-dose hydrated imperatorin group (LPS + 10 μM); ④ LPS + medium-dose hydrated imperatorin group (LPS + 20 μM); ⑤ LPS + high-dose hydrated imperatorin group (LPS + 40 μM). After 1 h of LPS stimulation, the appropriate concentration of hydrated imperatorin was added to the upper chamber, and co-culture continued for 24 h. After the culture was completed, HK2 cells were collected from the lower culture plate, RNA was extracted, and the transcription level of KIM1 mRNA was detected by RT-qPCR after reverse transcription. Simultaneously, the supernatant of the lower culture medium was collected, and the IL-6 content was detected by ELISA.

[0043] The results are as follows Figure 3 As shown in CF: Compared with the NC group, the LPS group showed increased CD86 mRNA levels in PBMCs, significantly increased IL-6 and LDH levels in the co-culture supernatant, and significantly increased KIM-1 protein transcription levels in HK2. Compared with the LPS group, each dose group of hydrated oxidized imperatorin (10, 20, 40 μM) decreased the CD86 mRNA level, an M1 polarization marker, in PBMCs in a concentration-dependent manner. Figure 3(C), while reducing the levels of inflammatory factors LDH and IL-6 in the supernatant ( Figure 3 Transcriptional expression of KIM-1 in D, E) and HK2 cells ( Figure 3 (F). The above results indicate that hydrated oxidized imperatorin can alleviate renal tubular epithelial cell damage by inhibiting PBMC-mediated inflammatory responses.

[0044] Example 4: Effect of hydrated oxidized imperatorin on cisplatin-induced HK-2 cell damage Human renal proximal tubular epithelial cells (HK-2) in the logarithmic growth phase were used at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured in DMEM / F12 medium containing 10% fetal bovine serum for 24 h at 37 ℃ in a 5% CO2 incubator. After cell attachment, cells were randomly divided into the following groups: blank control group: cultured in normal medium; cisplatin model group: treated with 20 μM cisplatin for 24 h; low-dose hydrated imperatorin group: 20 μM cisplatin + 10 μM hydrated imperatorin; medium-dose hydrated imperatorin group: 20 μM cisplatin + 20 μM hydrated imperatorin; high-dose hydrated imperatorin group: 20 μM cisplatin + 40 μM hydrated imperatorin.

[0045] Cell viability assay: The CCK-8 assay was used. After 24 h of cell treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 2 h. The absorbance (OD) at 450 nm was measured using a microplate reader. Cell viability (%) = (OD value of the drug treatment group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%. Detection of renal injury markers: After 24 h of cell treatment, total RNA was extracted from each group and reverse transcribed into cDNA. Using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal control, RT-qPCR was performed to detect the expression level of KIM-1 mRNA using the SYBR Green assay. Primers were synthesized by Anhui General Company, and the primer sequences are as follows. human MCP-1, forward,: SEQ ID NO:5; human MCP-1, reverse,: SEQ ID NO:6; human KIM1 Forward: SEQ ID NO:7; human KIM1 Reverse: SEQ ID NO:8; mouse / human GAPDH: Forward: SEQ ID NO:9; mouse / human GAPDH Reverse: SEQ ID NO:10.

[0046] The results are as follows Figure 4 As shown: Compared with the blank control group, cell viability in the cisplatin model group was significantly reduced to approximately 45%, and KIM-1 mRNA expression level was significantly increased. Compared with the cisplatin model group, cell viability increased in a dose-dependent manner in each dose group of hydrated oxidized imperatorin. Figure 4 KIM-1 mRNA expression levels decreased in a dose-dependent manner. Figure 5 (A) suggests that the levels of MCP-1 and IL-6 in HK2 cells were significantly reduced after treatment with hydrated oxidized imperatorin. Figure 5 (B, C) The above results suggest that hydrated oxidized imperatorin has a significant protective effect against cisplatin-induced HK-2 cell damage.

Claims

1. The use of hydrated oxypereugenol in the preparation of drugs for the prevention and / or treatment of acute kidney injury, characterized in that, The structural formula of hydrated oxidized imperatorin is: .

2. The application according to claim 1, characterized in that, The acute kidney injury mentioned is septic acute kidney injury.

3. The application according to claim 2, characterized in that, The septic acute kidney injury includes those induced by Gram-negative bacterial infection or lipopolysaccharide (LPS), or by cecal ligation and perforation (CLP) surgery.

4. The application according to claim 1, characterized in that, The acute kidney injury mentioned is drug-induced acute kidney injury.

5. The application according to claim 4, characterized in that, The drug-induced acute kidney injury includes kidney injury induced by cisplatin.

6. The application according to any one of claims 1-5, characterized in that, The hydrated oxytocin can prevent or treat acute kidney injury by inhibiting inflammatory responses and / or reducing damage to renal tubular epithelial cells.

7. The application according to claim 6, characterized in that, The inhibition of the inflammatory response includes inhibiting macrophage polarization toward the M1 type and / or reducing the levels of inflammatory factors.

8. The application according to claim 7, characterized in that, The inflammatory factors include one or more of tumor necrosis factor-α, interleukin-1β, and interleukin-6.

9. The application according to claim 1, characterized in that, The in vivo dosage of the hydrated oxidized imperatorin is 20-40 mg / kg.

10. The application according to claim 9, characterized in that, The dosage form of the drug is injection, tablet, capsule or suspension.

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