Use of linoleyl acetate in the prevention and treatment of kidney disease and inflammatory diseases

Abstract

This invention discloses the application of linoleyl acetate in the prevention and treatment of kidney disease and inflammatory diseases, belonging to the field of biomedical technology. Linoleyl acetate can significantly reduce serum creatinine levels in animal models of kidney injury, and significantly alleviate kidney damage; therefore, it has good application prospects for the prevention and / or treatment of kidney disease. Linoleyl acetate also has good anti-inflammatory effects and can be used to treat various inflammatory diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of linoleic acid acetate in the prevention and treatment of kidney disease and inflammatory diseases. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, leading to multiple organ dysfunction, among which acute kidney injury (AKI) is one of the most common and serious complications. The pathogenesis of sepsis-associated acute kidney injury (SA-AKI) is complex, involving multiple pathophysiological processes such as inflammatory response, microcirculatory disturbances, oxidative stress, and apoptosis. Current clinical treatments for SA-AKI mainly include anti-infection therapy, fluid resuscitation, the use of vasoactive drugs, and renal replacement therapy. However, because the pathological mechanisms of SA-AKI are not fully understood, existing treatments often fail to effectively reverse kidney damage, resulting in poor patient prognosis.

[0003] Currently, dexamethasone, as a glucocorticoid, is widely used in clinical practice for anti-inflammatory and immunosuppressive treatments. In the treatment of sepsis-associated acute kidney injury (SA-AKI), dexamethasone is often used to suppress excessive inflammatory responses and improve microcirculatory disturbances. Existing research indicates that the pathogenesis of SA-AKI involves multiple pathophysiological processes, including cytokine storms, endothelial cell damage, and microthrombus formation, and glucocorticoids can alleviate these damages by downregulating inflammatory pathways such as NF-κB. However, in clinical practice, significant individual differences have been observed in the treatment of SA-AKI with dexamethasone. Some patients do not respond well to standard-dose treatment, and long-term use may lead to adverse reactions such as glucose metabolism disorders and increased risk of infection. Furthermore, because SA-AKI patients often have multiple organ dysfunction, changes in pharmacokinetics may affect the maintenance of effective plasma concentrations of dexamethasone, posing a challenge to optimizing the dosing regimen.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of linoleic acid acetate in the prevention and treatment of kidney disease and inflammatory diseases, thereby providing a new drug option for kidney disease and inflammatory diseases.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides the use of linoleic acid acetate in the preparation of medicaments for the prevention and / or treatment of kidney disease.

[0007] Secondly, the present invention also provides the use of linoleic acid acetate in the preparation of medicaments for the prevention and / or treatment of inflammation or inflammatory diseases.

[0008] The present invention has the following beneficial effects: This invention extracts an active substance from Cordyceps sinensis that reduces serum creatinine levels in sepsis-associated acute kidney injury. After separation and analysis, the substance was identified as linoleic acid acetate. Oral administration of this substance to a mouse model of sepsis-associated acute kidney injury significantly reduced serum creatinine levels and markedly alleviated kidney damage. Therefore, it shows promising application potential in the prevention and / or treatment of kidney diseases.

[0009] In inflammation models, the administration of linoleyl acetate can effectively inhibit the inflammatory response in the tested inflammatory animals, demonstrating a good anti-inflammatory effect. Therefore, linoleyl acetate has good application prospects for the prevention and / or treatment of inflammation or inflammatory diseases. Attached Figure Description

[0010] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 Figure 1 shows the experimental results of using Cordyceps sinensis extract to alleviate lipopolysaccharide (LPS)-induced acute kidney injury (AKI) in mice with sepsis; (A) Schematic diagram of the experimental procedure: intraperitoneal injection of LPS (106 mg / kg) induced sepsis AKI, followed by gavage administration of Cordyceps sinensis extract (5 g / kg) or intraperitoneal injection of dexamethasone (5 mg / kg, positive control), and assessment of renal function and renal pathological changes; (B) Serum creatinine (sCr) levels in each group. The sCr level in the LPS group was significantly higher than that in the normal control group; after treatment with Cordyceps sinensis extract or dexamethasone, the sCr level was significantly lower. The extract alone did not cause significant changes in sCr; data are expressed as mean ± standard error; (C) HE staining of kidney tissue (scale bar = 50 µm); (D) PAS staining of kidney tissue (scale bar = 50 µm); Figure 2 LC-MS analysis of chemical components of Cordyceps sinensis extract: (A) Base peak chromatogram (BPC) of the sample in positive and negative ion modes, showing the continuous distribution of the strongest mass spectrometry signal at each time point; (B) Total ion chromatogram (TIC), reflecting the change of total ion intensity over time within the selected mass range; Compared with TIC, BPC mode can effectively eliminate chemical background noise and highlight the target peak; The results of both are consistent, verifying the reliability of the detection. Figure 3 The classification and statistical analysis of chemical components in Cordyceps sinensis extract; Figure 4The results of screening and identification of active ingredients targeting the kidney in Cordyceps sinensis extract are shown in the figure. Based on the screening criterion of fold difference (FC) ≥ 2.0 between the treatment group and the control group, a total of 11 candidate ingredients were obtained. The chemical structural formulas of the 11 ingredients are shown. Figure 5 This is a secondary mass spectrum of the active components of Cordyceps sinensis extract in the kidney; Figure 6 The graph shows the serum creatinine detection results of different experimental groups in Example 2; Figure 7 The images show the results of HE staining and PAS staining of paraffin sections of kidney tissue from different experimental groups in Example 3 under a light microscope. Figure 8 Figure showing the experimental results of Linoleyl acetate administration in inhibiting inflammation; Figure 9 A statistical graph showing the activity of LA on TCMK cells; Figure 10 Mass spectra of the drug, control group, and drug-only group kidneys, identified by LC-MS, for 11 renal target components. Chemical structural formulas and adduct ion information (positive / negative ion mode) of the 11 candidate components are shown. High-selectivity extraction ion chromatogram mode was used for targeted extraction based on accurate mass-to-charge ratio, and 11 components were successfully identified in the kidney samples, with retention times ranging from 0.83 to 35.85 min. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0013] Linoleyl alcohol acetate, chemically named 9,12-octadecadien-1-ol acetate, belongs to the acetate esters of unsaturated fatty alcohols. The molecular formula of this compound is C1. 20 H 36 O2, with a molecular weight of 308.50 and CAS number 5999-95-1.

[0014] In a first aspect, the present invention provides the use of linoleic acid acetate in the preparation of medicaments for the prevention and / or treatment of kidney disease.

[0015] Based on an LPS-induced AKI model, the inventors administered Cordyceps sinensis extract and identified the active substance in the Cordyceps sinensis extract that can reduce serum creatinine levels in sepsis-related acute kidney injury—linoleyl acetate—through LC-MS analysis and identification.

[0016] Linoleyl acetate can inhibit serum creatinine (SCr) levels in a sepsis-associated acute kidney injury model and significantly reduce kidney damage. HE and PAS staining results showed that after administration of linoleyl acetate, acute kidney injury phenotypes such as brush border shedding, vacuolar degeneration, and inflammatory cell infiltration of renal tubular epithelial cells in the sepsis-associated acute kidney injury model were significantly reduced.

[0017] In a preferred embodiment of the present invention, the kidney disease is selected from kidney injury, glomerulonephritis, polycystic kidney disease, kidney stones, pyelonephritis, IgA nephropathy, lupus nephritis, or interstitial nephritis.

[0018] In a preferred embodiment of the present invention, kidney injury is selected from acute kidney disease or chronic kidney disease.

[0019] Acute kidney disease is selected from sepsis-related acute kidney injury, acute tubular necrosis, acute interstitial nephritis, prerenal azotemia, postrenal obstruction, or renal vascular disease.

[0020] In a preferred embodiment of the present invention, the drug is administered orally.

[0021] In a preferred embodiment of the present invention, the drug also includes pharmaceutically acceptable excipients.

[0022] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient is selected from at least one of surfactants, binders, stabilizers, pH adjusters, protectants, excipients, disintegrants, lubricants, suspending agents, dispersants, and diluents.

[0023] Examples include: surfactants such as Tween 20 and Tween 80; pH adjusters such as disodium hydrogen phosphate and dipotassium hydrogen phosphate; excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylene pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, hydrolyzed starch, carboxymethyl cellulose, calcium carboxymethyl cellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, and calcium citrate; and lubricants such as magnesium stearate, talc, and lauryl sulfate. Sodium benzoate; flavorings, such as citric acid, menthol, glycine, orange powder; preservatives, such as sodium benzoate, sodium bisulfite, parabens (e.g., methylparaben, ethylparaben, propylparaben, butylparaben); stabilizers, such as citric acid, sodium citrate, acetic acid, and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0024] Secondly, the present invention also provides the use of linoleic acid acetate in the preparation of medicaments for the prevention and / or treatment of inflammation or inflammatory diseases.

[0025] Linoleyl acetate can effectively inhibit the occurrence of inflammation and suppress the level of inflammatory factors. After administration, it can play a good anti-inflammatory role. Therefore, linoleyl acetate has good application prospects for the prevention and / or treatment of inflammation or inflammatory diseases.

[0026] In a preferred embodiment of the present invention, inflammation or inflammatory diseases are selected from: rheumatic diseases, skin diseases, respiratory diseases, ophthalmic diseases, allergic reactions, autoimmune diseases, or nervous system diseases.

[0027] In a preferred embodiment of the present invention, the rheumatic disease is selected from rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriatic arthritis, gouty arthritis, or osteoarthritis.

[0028] The skin diseases are selected from eczema, psoriasis, contact dermatitis, atopic dermatitis, urticaria, seborrheic dermatitis, or lichen planus; Respiratory diseases are selected from asthma or chronic obstructive pulmonary disease, acute bronchitis, chronic bronchitis, pneumonia, or pulmonary fibrosis; The ophthalmological diseases are selected from conjunctivitis, iritis, keratitis, scleritis, retinal vasculitis, or dry eye syndrome; Allergic reactions can be selected from allergic rhinitis, urticaria, allergic asthma, food allergy, drug allergy, or insect bite allergy; Autoimmune diseases are selected from multiple sclerosis, Crohn's disease, ulcerative colitis, autoimmune thyroiditis, type 1 diabetes, or systemic sclerosis; Neurological disorders are selected from cerebral edema, multiple sclerosis, Guillain-Barré syndrome, autoimmune encephalitis, or neuromyelitis optica spectrum disorders.

[0029] In a preferred embodiment of the present invention, the drug further includes pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are selected from at least one of surfactants, binders, stabilizers, pH adjusters, protectants, excipients, disintegrants, lubricants, suspending agents, dispersants, and diluents.

[0030] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0031] Example 1 This embodiment involves screening for the active compound linoleyl acetate, specifically screening for active compounds from Cordyceps extract.

[0032] 1. AKI mice were administered Cordyceps sinensis extract (60 mice in total, n=10 per group).

[0033] To evaluate the in vivo renal protective activity of Cordyceps sinensis extract, an acute kidney injury model induced by lipopolysaccharide (LPS) was established in C57BL / 6J mice. Sixty male SPF-grade C57BL / 6J mice (Chengdu Yaokang Biotechnology Co., Ltd.), 7 weeks old and weighing 18–22 g at arrival, were purchased and randomly assigned to 6 groups. They were acclimatized for one week within a barrier system. Temperature and humidity were controlled, with a 12-hour light-dark cycle, and free access to food and water.

[0034] This study established an LPS-induced AKI model, with six groups: a control group (Ctrl), a high-dose Cordyceps sinensis extract control group (Ctrl CS High, 5 g / kg), a model group (LPS, 6 mg / kg), a low-dose Cordyceps sinensis extract treatment group (LPS CS Low, 1 g / kg), a high-dose Cordyceps sinensis extract treatment group (LPS CS High, 5 g / kg), and a positive control group (LPS DEX, 5 mg / kg). Cordyceps sinensis extract was prepared daily with freshly prepared saline suspensions at the required concentrations. Each group received the corresponding dose of Cordyceps sinensis extract suspension via gavage daily. The Ctrl, LPS, and LPS-DEX groups received an equal volume of saline (10 mL / kg) via gavage daily for 7 consecutive days. On day 7, after gavage, the LPS group, LPS-CS-Low group, LPS-CS-High group, and LPS-DEX group were intraperitoneally injected with LPS (10 mg / kg, Sigma-Aldrich, catalog number L2630-100MG, E. coli O111:B4, Source: 0000322625) to establish the mouse model. The Ctrl group and Ctrl-CS-High group were intraperitoneally injected with an equal volume of physiological saline. One hour after LPS injection, the LPS-DEX group was intraperitoneally injected with dexamethasone (Selleck, catalog number S5956) 5 mg / kg. Twenty-four hours after LPS injection, mice were anesthetized by intraperitoneal injection of 100 μL (10 mg / ml) of sodium pentobarbital, blood was collected by enucleation, and the mice were euthanized by cervical dislocation and kidneys were quickly harvested. After the blood samples were allowed to stand at room temperature for 2 hours, they were centrifuged at 3000 ×g for 20 minutes at 4°C. The supernatant was collected and centrifuged again under the same conditions. The resulting serum was stored at -80°C. Serum creatinine was measured using the Nanjing Jiancheng Creatinine (Cr) Assay Kit (sarcosine oxidase method) (microplate method) (C011-2-1) according to the manufacturer's instructions. After peeling the capsule from the left kidney, half of the kidney tissue was removed and fixed in 4% paraformaldehyde for 48 hours. Subsequent HE and PAS staining were performed for pathological observation. The remaining half of the left kidney was used for seq-RNA sequencing. The right kidney was freshly frozen for subsequent molecular analysis.

[0035] To obtain the important components of Cordyceps sinensis extract in the kidney, kidney tissue from animals treated with Cordyceps sinensis extract was analyzed and identified by LC-MS to obtain the components of Cordyceps sinensis extract in the kidney.

[0036] 2. Sample pretreatment and instrumental analysis conditions Kidney tissue samples (50 mg) and Cordyceps sinensis extract (100 mg) were extracted using methanol-water solution (V:V=4:1, containing mixed internal standards (see table below for specific internal standards), 2 μg / mL). Tissue samples were added to 300 μL of methanol-water solution, subjected to low-temperature grinding (60 Hz, 2 min), ultrasonic extraction for 10 min, and incubated overnight at -40 ℃ before centrifugation to collect the supernatant. Drug samples were added to 1 mL of methanol-water solution, subjected to vortexing for 1 min, low-temperature grinding, and ultrasonic extraction for 60 min before centrifugation to collect the supernatant. All samples were stored at -80 ℃ until analysis.

[0037]

[0038] Analysis was performed using an ultra-high performance liquid chromatograph (ACQUITY UPLC I-Class, Waters) and a high-resolution mass spectrometer (Thermo-Orbitrap-QE HF, Thermo). Chromatographic separation was performed using an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) at a column temperature of 45 ℃. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B), with a flow rate of 0.35 mL / min and an injection volume of 2 μL. The gradient elution program was as follows: 0.0–3.0 min, 100% A; 3.0–18.5 min, A linearly decreased from 100% to 80%; 18.5–20.0 min, A linearly decreased from 80% to 65%; 20.0–26.0 min, A linearly decreased from 65% to 60%; 26.0–35.0 min, A linearly decreased from 60% to 5%; 35.0–38.0 min, maintained at 5% A; 38.0–38.1 min, A rapidly increased from 5% to 100%; 38.1–40.0 min, maintained at 100% A to equilibrate the column.

[0039] Mass spectrometry was performed using a heated electrospray ionization (HESI) source, acquiring data in both positive and negative ion modes using a data-dependent acquisition (DDA, Top 10) mode. Ion source parameters were: spray voltage 3800 V for positive ions / -3200 V for negative ions; ion transfer tube temperature 320 °C; auxiliary gas heating temperature 350 °C; sheath gas flow rate 35 Arb; auxiliary gas flow rate 8 Arb; S-lensRF level 50. The full scan resolution was 60,000, the secondary mass spectrometry resolution was 15,000, and the mass scan range was 100–1500 m / z, using step collision energies (NCE 10, 20, 40 eV).

[0040] Base Peak Chromatogram (BPC) of each sample in positive and negative ion modes are as follows:Figure 2 As shown in Figure A (N=3 biological replicates), the continuous distribution of the strongest mass spectrometry signal at each time point is illustrated. The total ion chromatogram (TIC) is shown below. Figure 2 As shown in Figure B, the total ionic strength within the selected mass range changes over time. The figure also presents chromatograms of the kidney from the blank control group, the kidney from the high-dose Cordyceps sinensis extract group, and the Cordyceps sinensis extract itself.

[0041] 3. Compound Identification and Classification Statistics The raw data were processed by XCMS v4.5.1 software for peak identification, retention time correction and integration. The compounds were then identified by comparison with the LuMet-TCM standard database (5000+ standards) and the Herb public database. The identification was based on three dimensions: (1) the retention time error between the standard and the database was within ±0.3 min (40 min gradient error within ±0.5 min); (2) the first-order mass-to-charge ratio (m / z) error was ≤10 ppm (database); and (3) the matching degree between the measured second-order fragment spectrum and the MS2 spectrum of the standard. Based on the above process, the identification of the relevant chemical components of Cordyceps sinensis extract and Cordyceps sinensis extract in kidney tissue was completed.

[0042] 4. Screening and chemical structure characterization of potential target prototype components.

[0043] To identify the prototypical components in Cordyceps sinensis extract that may be absorbed into the kidneys, a peak area ratio (Fold Change, FC) ≥ 2.0 was used as the criterion, combined with the qualitative results from step 3, to screen potential target prototypical components. To verify their structural accuracy, the extracted ion chromatogram (EIC) peaks of each compound were extracted, and their secondary fragment ions were analyzed. The secondary mass spectrometry mirror images of the 11 target components are summarized in [the table / document / reference]. Figure 4 The figure shows the m / z values ​​of the main fragment ions and the fragment structures derived by the MetaFrag software. The measured spectra are consistent with the standard spectra in the database in terms of the main fragmentation pathways, further supporting the identification results. Among them, no effective secondary fragment information was obtained for 3 compounds in DDA acquisition mode. Figure 4 (marked as "NoMS2"), its identification mainly relies on retention time and first-order quality precision.

[0044] (1) Sample preparation Kidney tissue: The sample stored at -80°C was removed, thawed at room temperature, and 50 mg of sample was weighed into a 1.5 mL EP tube. Two small steel beads were added. 300 μL of methanol-acetonitrile solution (V:V=2:1, containing internal standard L-2-chlorophenylalanine, 2 μg / mL) was added, and the sample was pre-cooled at -40°C for 2 min and then ground (60 Hz, 2 min). Subsequently, it was extracted by sonication in an ice-water bath for 10 min and incubated overnight at -40°C. The next day, it was centrifuged for 10 min (12000 rpm, 4°C), and 120 μL of the supernatant was transferred into an LC-MS vial with a support liner and stored at -80°C until liquid chromatography-mass spectrometry (LC-MS) analysis.

[0045] Traditional Chinese Medicine: Weigh approximately 100 mg of sample into a 1.5 mL centrifuge tube, add 1 mL of methanol-water mixture (methanol:water = 7:3 (v / v), containing internal standard L-2-chlorophenylalanine, 2 μg / mL), vortex for 1 min, add steel beads, pre-cool at -40℃ for 2 min, then grind in a grinder (60 Hz, 2 min). Extract by sonication in an ice-water bath for 60 min, then centrifuge for 10 min (12000 rpm, 4℃). Take 120 μL of the supernatant and transfer it to an LC-MS vial with an inner liner for analysis.

[0046] (2) Liquid chromatography-tandem mass spectrometry analysis Metabolomics data analysis was performed by Shanghai Luminbio Biotechnology Co., Ltd. (Shanghai, China). Metabolic profile analysis was conducted using an ACQUITY UPLC I-Class plus (Waters Corporation, Milford, USA) Q-Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a heated electrospray ionization source, in both ESI positive and negative ion modes. Both positive and negative ion modes used an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm). The gradient elution system consisted of (A) water (containing 0.1% formic acid, v / v) and (B) acetonitrile, using the following gradients for separation: 0–3 min, 0% B; 3–18.5 min, 0–20% B; 18.5–20 min, 20–35% B; 20–26 min, 35–40% B; 26–35 min, 40–95% B; 35–38 min, 95% B; 38–38.1 min, 95–0% B; 38.1–40 min, 0% B. The flow rate was 0.35 mL / min, and the column temperature was 45 °C. All samples were maintained at 4 °C during the analysis. The injection volume was 2 μL.

[0047] Mass spectrometry detection parameters: mass range 100~1500 m / z. Resolution of full MS scan: 60,000; resolution of HCD MS / MS scan: 15,000. Collision energy settings: 10, 20, and 40 eV. Spray voltage: 3800 V (+) and 3200 V (-); sheath flow rate: 35 arbitrary units; auxiliary gas flow rate: 8 arbitrary units; capillary temperature: 320°C; auxiliary gas heater temperature: 350°C; S-lens RF level: 50.

[0048] (3) Data processing and analysis Raw LC-MS data were processed using XCMS V4.5.1 software, including baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization. The key parameters were a precursor tolerance of 5 ppm and a product tolerance of 20 ppm. Compounds were identified using a custom LuMet-TCM library and the Herb database, based on precise mass-to-charge ratios (m / z), secondary fragments, and isotopic distributions.

[0049] For substances identified in the XCMS database, those with a total score greater than 40 were retained. Metabolites with an FC value ≥2 from both the treatment group (treated serum) and the control group (blank serum) were considered as blood-entering components. After merging and deduplicating substances in both positive and negative ion modes, the total relative peak area of ​​the metabolites was set to 100% to obtain a qualitative and quantitative results data matrix. This matrix contained all information available for analysis extracted from the raw data, and subsequent analyses were based on this matrix. EIC diagrams and MS2 mirror diagrams with secondary fragment structure annotations were plotted for each identified traditional Chinese medicine component. In addition, pie charts were plotted based on the number of all identified traditional Chinese medicine components under each chemical classification category.

[0050] To further ensure the accuracy of the identification results, this embodiment extracts the EIC spectrum and secondary mass spectrum of each component in the data matrix table, labels the fragment information and annotates the fragment structure.

[0051] The assessment is based on three reference dimensions: The retention time error between the retention time and the standard retention time in the database is within ± 0.3 min; the first molecular weight error is within 10 ppm; comparison between the measured MS2 and the standard MS2.

[0052] Table 1. Components of Cordyceps sinensis extract by LC-MS

[0053] M0009 in Table 1 is linoleyl acetate.

[0054] If there is no information on secondary fragment peaks, the identification results should be confirmed by referring to the retention time and primary molecular weight error dimensions of the standard comparison.

[0055] 5. Experimental Results (1) The experimental flowchart of the effect of Cordyceps sinensis extract on lipopolysaccharide (LPS) induced acute kidney injury in mice with sepsis is shown in the figure. Figure 1 As shown in A, the serum creatinine (sCr) levels of each group were referenced. Figure 1 As shown in B. The results showed that sCr was significantly higher in the LPS group than in the normal control group; sCr was significantly lower after treatment with Cordyceps sinensis extract or dexamethasone. The extract alone did not cause significant changes in sCr. HE staining results of kidney tissue showed ( Figure 1 LPS (C) causes diffuse swelling, vacuolar degeneration, inflammatory cell infiltration, and luminal dilation of renal tubular epithelial cells; the above lesions were significantly reduced after treatment with Cordyceps sinensis extract.

[0056] PAS staining results of kidney tissue showed ( Figure 1 (D in the text). LPS causes renal tubular dilation, epithelial cell vacuolation, and loss of the proximal tubular brush border; Cordyceps sinensis extract significantly improves these pathological changes.

[0057] (2) LC-MS analysis results of chemical components of Cordyceps sinensis extract: Reference Figure 2 As shown, compared with the total ion chromatogram (TIC), the BPC mode can effectively eliminate chemical background noise and highlight the target peak in both positive and negative ion modes; the results of both are consistent, verifying the reliability of the detection.

[0058] Figure 3 The classification and statistical results of chemical components in Cordyceps sinensis extract showed that LC-MS identified a total of 730 components, mainly including: terpenes (100), amino acids, peptides and their derivatives (88), organic heterocyclic compounds (85), fatty acyls (72), sugars and glycosides (58), phenols (40), alkaloids (32), phenylpropanoids (30), lipids (27), flavonoids (21), steroids (19), nucleotides and their derivatives (16), indole and its derivatives (12), pyridine and its derivatives (10), organic acids and their derivatives (9), imidazole pyrimidines (7), carboxylic acids and their derivatives (5), glycerides (3), pteridine and its derivatives (2), glycerophospholipids (1), tetrapyrrole and its derivatives (1), and other components not clearly classified (92).

[0059] (3) Screening and identification of active ingredients targeting the kidney in Cordyceps sinensis extract: Figure 4The results showed that, based on the screening criterion of a fold change (FC) ≥ 2.0 between the treatment group and the control group, a total of 11 candidate components were obtained. The distribution by chemical category was as follows: amino acids, peptides and their derivatives (2, 18.2%), alkaloids (2, 18.2%), fatty acyls (2, 18.2%), indole and its derivatives (1, 9.1%), flavonoids (1, 9.1%), organic heterocyclic compounds (1, 9.1%), carboxylic acids and their derivatives (1, 9.1%), and others (1, 9.1%).

[0060] Chemical structural formulas of 11 candidate components (reference) Figure 4 As shown.

[0061] Figure 10 The results showed that, using a highly selective extraction ion chromatogram mode and targeted extraction based on accurate mass-to-charge ratio, 11 components were successfully identified in the kidney sample, with retention times ranging from 0.83 to 35.85 min. These components included: arecoline (0.83 min, m / z 142.0865, positive ion), L-lysine (2.19 min, m / z 219.1339, positive ion), acrylic acid (2.21 min, m / z 117.0182, negative ion), pyromeconic acid (2.4 min, m / z 157.0138, negative ion), alanylleucine (6.61 min, m / z 203.1390, positive ion), 1-tyrosine betaine (7.2 min, m / z 262.0838, positive ion), and indole-3-lactic acid (15.9 min, m / z 15.85). The extracted ion chromatogram peaks showed that the above components were clearly identifiable in the kidneys, and their retention times were completely consistent with those of the standards. These included genistein (21.94 min, m / z 269.0457, negative ion), cytochalasin N (27.39 min, m / z 492.2726, negative ion), 1-linoleic acid glyceride (33.71 min, m / z 355.2836, positive ion), and linoleyl acetate (35.85 min, m / z 353.2701, negative ion).

[0062] To verify the structural accuracy of the target components, secondary mass spectrometry analysis was performed on each candidate compound. Figure 5 The main fragment ion peaks and their relative abundances of the parent ion were recorded and compared with secondary spectra in the standard database. The reliability of component identification was confirmed by the consistency of fragment ions. Three compounds did not obtain effective secondary fragment information in DDA acquisition mode. Figure 5The identification of fragments marked "no MS2" relies primarily on retention time and first-order precise mass number. In the fragment structure annotation, the difference between the fragment m / z and the measured m / z is calculated based on the number of protons and the mass shift of adduct ions. (A)~(K) are, in order: indole-3-lactic acid (A), alanylleucine (B), genistein (C), pyromeconic acid (D), arecoline (E), cytochalasin N (F), acrylic acid (G), L-lysine (H), linoleic acid (I), 1-tyrosine betaine (J), and 1-linoleic acid glyceride (K) secondary mass spectra.

[0063] Example 2 This embodiment tests serum creatinine (SCr) levels after administration of linoleic acid acetate to a sepsis-associated acute kidney injury model.

[0064] 1. Raw materials, reagents and compounds The active compound used in this invention is linoleyl acetate (#Y20628, ACMEC), chemically named 9,12-octadecadien-1-ol acetate, which belongs to the acetate esters of unsaturated fatty alcohols. The molecular formula of this compound is C1. 20 H 36 O2, with a molecular weight of 308.50 and CAS number 5999-95-1.

[0065] Its structural formula is as follows: .

[0066] The drug used for animal model establishment was LPS (L2630, Lot 0000322625, SIGMA, CAS:93572-42-0). The drug used for positive control was Dexamethasone (S1322, Selleck, CAS:50-02-2).

[0067] 2. Serum creatinine test In this embodiment, a prophylactic administration strategy was adopted for Linoleyl acetate: Linoleyl acetate was administered by gavage for 7 consecutive days before LPS induction to establish a protective effect.

[0068] Dosage: The oral dose of Linoleyl acetate is 40-80 mg / kg / day. The administration volume is usually 0.1 mL / 10g body weight, and the drug should be prepared to an appropriate concentration according to the dosage and volume.

[0069] Dosing cycle: Pre-administer by gavage for 7 days. After the last dose, perform LPS intraperitoneal injection to establish the model.

[0070] To ensure the reliability of the experimental conclusions, the following experimental groups were set up (10 animals in each group): (1) Blank control group (Ctrl): normal saline was administered by gavage daily, and normal saline was injected into the peritoneum on the seventh day. The animals were sacrificed and the samples were collected 24 hours later. (2) Model group (LPS): physiological saline was administered by gavage daily, and LPS solution was injected into the peritoneum on the seventh day. The animals were sacrificed and the samples were collected 24 hours later. (3) Low-dose administration model group (LPS-LA-L): Linoleyl acetate 40 mg / kg was administered by gavage daily, LPS was injected intraperitoneally on the seventh day, and the animals were sacrificed 24 hours later for sampling; (4) High-dose administration model group (LPS-LA-H): Linoleyl acetate 80 mg / kg was administered by gavage daily, and LPS was injected intraperitoneally on the seventh day. The animals were sacrificed 24 hours later for tissue collection. (5) Drug control group (LA-H): Linoleyl acetate (e.g., 80 mg / kg) was administered by gavage daily + normal saline was injected intraperitoneally on the seventh day. The patient was sacrificed 24 hours later to collect samples to evaluate the effect of the drug on the kidneys. (6) Positive control group (LPS-DEX): Daily oral administration of normal saline, intraperitoneal injection of dexamethasone 1 hour after LPS injection on the seventh day, sacrificed 24 hours after LPS injection for tissue collection to confirm the model’s response to protective intervention.

[0071] The specific experimental steps and process parameters for serum creatinine detection are as follows: SPF-grade male C57BL / 6J mice, 7 weeks old (age at arrival, 8 weeks after acclimatization), weighing 17.5-21.2g, were used. The animals were ear-tagged and acclimatized in a standard barrier environment for one week, with free access to food and water.

[0072] During the administration period, mice were administered via gavage at 10:00 AM daily in the same order. Mice were weighed and their weight recorded before gavage to adjust the daily dosage. The mice were observed after administration. Linoleyl acetate was freshly prepared with physiological saline to the required concentration, dispersed thoroughly by vortexing or sonication, and used immediately. Administered via gavage for 7 consecutive days.

[0073] On day 7, after all gavage was completed, each group received an intraperitoneal injection of LPS (6 mg / kg) or an equal volume of saline, as designed. Animals were returned to their original cages after injection and given normal water and feed. One hour later, the positive control group received an intraperitoneal injection of dexamethasone. The sepsis-associated acute kidney injury model in this embodiment was established by intraperitoneal injection of lipopolysaccharide (LPS, derived from *Escherichia coli*, serotype O111:B4). LPS was dissolved in sterile saline, and the injection dose was 6 mg / kg body weight.

[0074] Samples were collected 24 hours after intraperitoneal injection of LPS (day 8). Blood was collected by enucleation, and the blood was allowed to stand at room temperature for 1 hour (avoiding vibration to prevent hemolysis). The serum was then separated twice by centrifugation at 3000 g for 20 min at 4°C and stored at -80°C for renal function index detection.

[0075] Immediately after blood collection, both kidneys were dissected. The left kidney was coronally dissected: half of the kidney tissue was fixed in 4% paraformaldehyde for paraffin embedding, sectioning, and H&E and PAS staining for pathological observation; the other half was embedded in OCT and then flash-frozen in liquid nitrogen to prepare frozen sections for later use. The right kidney was divided into three portions, flash-frozen directly in liquid nitrogen, and then stored at -80°C for molecular marker detection such as Western blot and real-time quantitative PCR.

[0076] Serum creatinine (SCr) was measured using the Amplex Red Creatinine Assay Kit (e.g., Beyotime S0291S). Serum creatinine undergoes a cascade enzymatic hydrolysis involving creatinine enzyme, creatine oxidase, and creatine oxidase, ultimately producing hydrogen peroxide. Hydrogen peroxide reacts 1:1 with the Amplex Red probe under the catalysis of horseradish peroxidase to generate halogenated creatinine, the fluorescence intensity of which is directly proportional to the creatinine concentration. Following the kit instructions, creatinine standard (5 mM) was diluted to 100 μM with Creatinine Assay Buffer, and then 0, 2, 5, 10, 20, 50, and 100 μM standard solutions were prepared sequentially. 20 μL / well was added to a black 96-well plate. 10 μL of serum sample was added to each well, and the volume was brought to 20 μL with Creatinine Assay Buffer. Prepare the following working solution (80 μL per well): Creatinine Assay Buffer 74 μL + Amplex Red 2 μL + Enzyme Solution A 2 μL + Enzyme Solution B 2 μL. Add 80 μL of working solution to each well, incubate at 37°C in the dark for 30 min, and then measure the fluorescence intensity using a multi-function detector at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Substitute the values ​​into the standard curve to obtain the creatinine value for each well.

[0077] Twenty-four hours after LPS injection, tissue analysis revealed a significant increase in serum creatinine (SCr), and HE and PAS pathological staining showed typical signs of acute kidney injury, such as brush border shedding of renal tubular epithelial cells, vacuolar degeneration, and inflammatory cell infiltration, proving the successful establishment of the model.

[0078] Figure 6Experimental results showed that the creatinine concentration levels in mouse models treated with linoleyl acetate were comparable to those treated with DEX. Compared with the model group, serum creatinine levels in mouse models significantly decreased after treatment with both low-dose (40 mg / kg) and high-dose (80 mg / kg) linoleyl acetate, indicating that linoleyl acetate can significantly reduce serum creatinine levels. There was no significant difference in serum creatinine levels between the different concentrations administered to the model groups. In animals that did not undergo LPS modeling, serum creatinine levels showed no significant difference from the control group after administration of linoleyl acetate.

[0079] Example 3 In this embodiment, HE and PAS staining were performed on each experimental group in Example 2.

[0080] Under a light microscope, HE staining of paraffin sections of kidney tissue showed that cell nuclei were blue-purple, while the cytoplasm and extracellular matrix were pink. PAS staining showed that positive substances such as glycogen and basement membrane appeared purplish-red. This allowed for the assessment of renal tubular epithelial damage changes, including brush border loss, vacuolar degeneration, cast formation, epithelial necrosis and shedding, and interstitial inflammatory cell infiltration (including brush border loss, cast formation, epithelial cell necrosis and shedding, interstitial congestion and edema).

[0081] Figure 7 The results showed that kidney damage was observed in both HE and PAS staining of the pathological sections in the LPS group. Kidney damage was significantly reduced in different concentrations of Linoleyl acetate groups compared to the LPS group. No significant damage was observed in the Linoleyl acetate group (LA-H group) that was administered alone.

[0082] Example 4 An experiment on the inhibition of inflammation by Linoleyl acetate administration. This example sequenced the experimental groups from Example 2. The raw RNA-seq counts from the sequencing results were transformed using DESeq2 variance stabilization transformation (VST) for visualization. In the animal experiments using Linoleyl acetate, the kidneys of each experimental group in Example 2 (except the LPS+DEX group) were homogenized using RIPA lysis buffer (strong) (Biosharp, BL204A) and lysed on ice for 10 min. Then, they were centrifuged at 120,000 rpm for 20 min at 4°C in a pre-chilled centrifuge. After centrifugation, the supernatant was transferred to a new 1.5 mL eptub. Protein was quantified using BCA (targetmol, BL521S). After balancing, the protein was diluted according to the BCA quantitative dilution factor, and 1 / 4 volume of 5× loading buffer was added. The mixture was incubated in a metal bath at 100°C for 5 min. After the metal bath, the mixture was aliquoted and stored at -80°C for subsequent Western blot analysis. 1.5 mm thick gels were prepared using a 10% PAGE gel rapid preparation kit (BAIHE, PG112). The electrophoresis buffer was an aqueous solution containing 3.03 g Tris, 14.4 g glycine, and 1 g SDS per liter. The transfer buffer contained 3.03 g Tris, 14.4 g glycine, 200 ml methanol, and 800 ml ultrapure water per liter. The electrophoresis program was 80 V for 30 min, then increased to 120 V to the bottom. PVDF membranes (Merck, IPVH00010) were activated with methanol and used for transfer. The transfer conditions were 100 V wet transfer for 60 min. After transfer, the membranes were blocked at room temperature for 1 h in 5% BSA / TBST (BSA was Albumin Fraction V, BIOFROXX 4240GR100; 1×TBST was diluted from 10×TBST, Servicebio G0004). The antibody was then incubated overnight with primary antibodies: Phospho-NF-KB p65 (Ser536) Rabbit pAb (positive energy, 310013) (dilution 1:1000) and NF-KB p65Rabbit pA (positive energy, 310099) (dilution 1:1000). The internal control was β-actin (Zen Bioscience, 700068, HRP directly labeled, dilution 1:10000). Secondary antibody incubation was performed using HRP-labeled goat anti-rabbit IgG (ZENBIO, 511203, dilution 1:5000) at room temperature for 1 hour. Since the β-actin antibody contains HRP, no secondary antibody was needed; incubation at room temperature for 1 hour was also performed.After incubation with the secondary antibody, wash the membrane three times for 10 min each time with 1×TBST, add chemiluminescent developing agent (Zen Bioscience, #17046), and expose and image using a chemiluminescent gel imaging system (Guangzhou Guangyi Biotechnology Co., Ltd., OI600).

[0083] Figure 8 The results showed that, according to the mRNA sequencing results of the kidneys of mice in each group, the expression of inflammatory factors IL6 and iNOS was increased in the LPS group. The expression of inflammatory factors IL6 and iNOS was significantly reduced in the different concentrations of Linoleyl acetate administration groups compared to the LPS group. No significant changes were observed in the expression of inflammatory factors IL6 and iNOS in the Linoleyl acetate-only group (LA-H group). The Western blot (WB) results of the kidneys of mice in each group showed that, according to the LPS group, the expression of NFKB phosphorylated p65 was increased. The expression of NFKB phosphorylated p65 was significantly reduced in the different concentrations of Linoleyl acetate administration groups compared to the LPS group. No significant changes were observed in the expression of NFKB phosphorylated p65 in the Linoleyl acetate-only group (LA-H group).

[0084] Example 5 In this embodiment, the CCK-8 assay was used to detect the activity of LA at concentrations from 0 to 320 μM against TCMK cells. During the logarithmic growth phase, 1 × 10⁻⁶ μM LA was added to TCMK cells. 4 TCMK cells were seeded into 96-well plates. Cells were then treated with 0, 2.5, 5, 10, 20, 40, 80, 160, and 320 µM linolenic acid acetate for 24 hours. After treatment, 10 µL of CCK-8 solution (catalog number #CK04-500, Dojindo, Japan) was added to each well. After incubation for 4 hours, the optical density was measured at 450 nm using a microplate reader (SpectraMax ABS, Molecular Devices, USA). Results are as follows. Figure 9 As shown, LA at concentrations from 0 to 320 μM is non-toxic to TCMK cells and does not affect cell survival.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of linoleyl acetate in the preparation of drugs for the prevention and / or treatment of kidney disease.

2. The application according to claim 1, characterized in that, The kidney diseases mentioned are selected from kidney injury, glomerulonephritis, polycystic kidney disease, kidney stones, pyelonephritis, IgA nephropathy, lupus nephritis, or interstitial nephritis.

3. The application according to claim 2, characterized in that, The kidney injury is selected from acute kidney disease or chronic kidney disease; The acute kidney disease is selected from sepsis-related acute kidney injury, acute tubular necrosis, acute interstitial nephritis, prerenal azotemia, postrenal obstruction, or renal vascular disease.

4. The application according to any one of claims 1-3, characterized in that, The drug is administered orally.

5. The application according to any one of claims 1-3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of surfactants, binders, stabilizers, pH adjusters, protectants, excipients, disintegrants, lubricants, suspending agents, dispersants, and diluents.

7. Use of linoleyl acetate in the preparation of drugs for the prevention and / or treatment of inflammation or inflammatory diseases.

8. The application according to claim 7, characterized in that, The inflammation or inflammatory disease mentioned is selected from: rheumatic diseases, skin diseases, respiratory diseases, ophthalmic diseases, allergic reactions, autoimmune diseases, or nervous system diseases.

9. The application according to claim 8, characterized in that, The rheumatic diseases mentioned are selected from rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriatic arthritis, gouty arthritis, or osteoarthritis; The skin disease is selected from eczema, psoriasis, contact dermatitis, atopic dermatitis, urticaria, seborrheic dermatitis, or lichen planus; The respiratory diseases mentioned are selected from asthma or chronic obstructive pulmonary disease, acute bronchitis, chronic bronchitis, pneumonia or pulmonary fibrosis. The ophthalmological diseases mentioned are selected from conjunctivitis, iritis, keratitis, scleritis, retinal vasculitis, or dry eye syndrome; The allergic reaction is selected from allergic rhinitis, urticaria, allergic asthma, food allergy, drug allergy, or insect bite allergy; The autoimmune diseases mentioned are selected from multiple sclerosis, Crohn's disease, ulcerative colitis, autoimmune thyroiditis, type 1 diabetes, or systemic sclerosis. The neurological diseases mentioned are selected from cerebral edema, multiple sclerosis, Guillain-Barré syndrome, autoimmune encephalitis, or neuromyelitis optica spectrum disorders.

10. The application according to claim 8 or 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

Images