Use of cordyceps sinensis in preparing drug for preventing and treating sepsis associated acute kidney injury

Abstract

Use of Cordyceps sinensis or a combination of Cordyceps sinensis and corticosteroid in preparing a drug for preventing or treating sepsis associated acute kidney injury. The drug prepared from the Cordyceps sinensis or the combination of the Cordyceps sinensis and the corticosteroid is used for preventing or treating sepsis associated acute kidney injury in a targeted manner, and the therapeutic effect of the drug can be fully exerted by improving mitochondrial energy metabolism in renal cells and regulating macrophage polarization in renal tissues, thereby effectively ameliorating renal dysfunction and damage caused by sepsis associated acute kidney injury, inhibiting expression and secretion of inflammatory cytokines, and providing a new drug choice for clinical prevention and treatment of sepsis associated acute kidney injury.

Description

Application of Cordyceps sinensis in the preparation of drugs for preventing and treating sepsis-related acute kidney injury Technical Field

[0001] The present invention belongs to the field of medicine and specifically relates to the use of Cordyceps sinensis in preparing a medicament for preventing and / or treating sepsis-related acute kidney injury. Background Art

[0002] Sepsis is a clinically complex disease characterized by organ dysfunction caused by an dysregulated response to infection, which is life-threatening. It is one of the main risk factors for death in patients with severe trauma, burns, shock or major surgery, and often affects multiple organs. There are approximately 30 million cases of sepsis each year worldwide, with a mortality rate as high as 40%. The kidney is an organ particularly susceptible to sepsis, and sepsis-associated acute kidney injury (S-AKI) is closely associated with the high mortality rate of patients with sepsis. Currently, most therapies for S-AKI are still reactive and non-specific, focusing on preventing secondary injuries and relying on the ability of clinicians to handle each case. Therefore, there is still a lack of effective and specific drugs for the prevention and treatment of S-AKI.

[0003] Cordyceps sinensis is a rare, naturally occurring Chinese medicinal herb. Besides its common components like sugar, fat, and crude fiber, it also contains active ingredients like mannitol, ergosterol, adenosine, cordycepic acid, cordycepin, and vitamin B12. Its nourishing and therapeutic properties surpass those of ginseng and deer antler. However, natural Cordyceps sinensis has a long growth period and is found in high-altitude alpine meadows. Its resources are scarce, difficult to collect, and extremely expensive.

[0004] Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd. exclusively produces Cordyceps sinensis mycelium, obtained through artificial fermentation, and its preparation, "Bailing Capsules" (containing fermented Cordyceps sinensis mycelium powder CS-C-Q80), a Class I new drug listed in the Pharmacopoeia of the People's Republic of China. Cordyceps sinensis powder (CS-C-Q80) is a dried powder of mycelium obtained through submerged fermentation of Hirsutella sinensis. Bailing Capsules are indicated for the adjunctive treatment of cough, asthma, hemoptysis, back pain, facial fatigue, and frequent nocturia caused by lung and kidney deficiency, as well as for the adjunctive treatment of chronic bronchitis and chronic renal insufficiency. Summary of the Invention

[0005] The present invention provides, on the one hand, the use of Cordyceps sinensis in the preparation of a medicament for preventing and / or treating sepsis-related acute kidney injury, and on the other hand, a therapeutic agent for treating sepsis-related acute kidney injury, a treatment method for sepsis-related acute kidney injury, and the use of a combination of corticosteroids and Cordyceps sinensis as a medicament for treating sepsis-related acute kidney injury.

[0006] Specifically, the present invention first provides the use of Cordyceps sinensis in the preparation of a drug for preventing and / or treating sepsis-related acute kidney injury, wherein the sepsis-related acute kidney injury is sepsis-related acute kidney injury characterized by high expression of any one of Cys-C, BUN, CRE, IL-6, TNF-α, and IL-1β proteins, and wherein the sepsis-related acute kidney injury is sepsis-related acute kidney injury characterized by impaired mitochondrial energy metabolism biological pathways in renal cells, and wherein the mitochondrial energy metabolism biological pathways in renal cells are mitochondrial oxidative phosphorylation and mitochondrial biogenesis in renal cells.

[0007] Furthermore, the Cordyceps sinensis has one or more of the following characteristics: improving the activity of the mitochondrial complex, mitochondrial membrane potential (MMP) and ATP production in mitochondria of kidney cells, and further, upregulating the expression of NADH-ubiquinone oxidoreductase chain, cytochrome c oxidase subunit, ATP6 and CYTB, which are related to mitochondrial electron transfer in kidney cells; upregulating the expression of complexes CⅠ-ND5, CⅡ-SDHC, CⅠ-ND6, CⅠ-ND7, CⅡ-SDHC, CⅢ-CYTB, CⅣ-MTCO2 and CⅤ-ATP6 proteins in mitochondria of kidney cells.

[0008] The sepsis-related acute kidney injury may also be sepsis-related acute kidney injury characterized by blocked polarization of renal M1 macrophages to M2 macrophages. The Cordyceps sinensis has one or more of the following properties: reducing blood levels of iNOS and IL-6; increasing blood levels of CD206 and Arg1; and upregulating TGF-β and IL-10 protein expression in lung tissue.

[0009] The cordyceps sinensis is obtained by artificially fermenting the asexual fungus of cordyceps sinensis. Specifically, the asexual fungus of cordyceps sinensis is obtained by fermenting Hirsutella sinensis. More specifically, the cordyceps sinensis is Bailing Capsule.

[0010] Another aspect of the present invention provides a method for treating sepsis-related acute kidney injury, comprising administering Cordyceps sinensis to a subject diagnosed with sepsis-related acute kidney injury.

[0011] The cordyceps sinensis is obtained by artificially fermenting the asexual fungus of cordyceps sinensis. Specifically, the asexual fungus of cordyceps sinensis is obtained by fermenting Hirsutella sinensis. More specifically, the cordyceps sinensis is Bailing Capsule.

[0012] The dosage of the cordyceps sinensis is 182-455 mg / Kg / d, 182-546 mg / Kg / d, 182-637 mg / Kg / d, 182-728 mg / Kg / d, 182-819 mg / Kg / d or 182-910 mg / Kg / d.

[0013] Another aspect of the present invention provides a therapeutic agent for sepsis-related acute kidney injury, comprising a corticosteroid and a second agent for use with the corticosteroid, wherein the second agent is selected from Cordyceps sinensis and / or its fermentation products. The therapeutic agent for sepsis-related acute kidney injury of the present invention can be administered to a subject with sepsis-related acute kidney injury in a therapeutically effective amount of the corticosteroid and the second agent for use with the corticosteroid, wherein the second agent is selected from Cordyceps sinensis and / or its fermentation products.

[0014] The second agent is provided in the form of fermented Cordyceps sinensis powder;

[0015] The corticosteroid is selected from the group consisting of prednisone, dexamethasone, triamcinolone, betamethasone, beclomethasone, flunisolide and fluticasone.

[0016] Furthermore, the sepsis-related acute kidney injury is sepsis-related acute kidney injury with high expression of any one of Cys-C, BUN, CRE, IL-6, TNF-α, and IL-1β.

[0017] Furthermore, the therapeutically effective amount refers to a target dosage of corticosteroids of 0.4-0.5 mg / Kg / d; the dosage of Cordyceps sinensis is 91-455 mg / Kg / d, 91-546 mg / Kg / d, 91-637 mg / Kg / d, 91-728 mg / Kg / d, 91-819 mg / Kg / d or 91-910 mg / Kg / d.

[0018] Furthermore, the therapeutically effective amount of the corticosteroid and the second agent used in combination with the corticosteroid can effectively reduce the high expression of any one of Cys-C, BUN, CRE, IL-6, TNF-α, and IL-1β in the subject.

[0019] The dosage form of the corticosteroid can be tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, pastes, effervescent tablets, pills, etc.

[0020] The dosage form of the second agent can be tablets, pills, granules, capsules, lozenges, syrups, emulsions, microemulsions, suspensions, injections, effervescent tablets, and the like.

[0021] Another aspect of the present invention provides a method for treating sepsis-related acute kidney injury, comprising administering a corticosteroid and a second agent used in combination with the corticosteroid to a subject diagnosed with sepsis-related acute kidney injury, wherein the second agent is selected from Cordyceps sinensis and / or a fermentation product thereof.

[0022] The dosage of the corticosteroid is 0.4-0.5 mg / Kg / d.

[0023] The dosage of the second agent is 91-455 mg / Kg / d, 91-546 mg / Kg / d, 91-637 mg / Kg / d, 91-728 mg / Kg / d, 91-819 mg / Kg / d or 91-910 mg / Kg / d.

[0024] Optionally, the subject has at least one alteration in Cys-C, BUN, CRE, IL-6, TNF-α, IL-1β protein.

[0025] Optionally, the subject has been diagnosed with sepsis-related acute kidney injury and has at least one alteration in Cys-C, BUN, CRE, IL-6, TNF-α, IL-1β protein.

[0026] Optionally, the subject has been diagnosed with sepsis-related acute kidney injury and has high expression of at least one of Cys-C, BUN, CRE, IL-6, TNF-α, IL-1β proteins.

[0027] The second agent is provided in the form of fermented Cordyceps sinensis powder. Optionally, the corticosteroid is selected from prednisone, dexamethasone, triamcinolone, betamethasone, beclomethasone, flunisolide and fluticasone.

[0028] The dosage form of the corticosteroid can be tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, pastes, effervescent tablets, pills, etc.

[0029] The corticosteroid can be administered by enteral administration (eg, oral) or parenteral administration (eg, intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intrarectal, etc.), preferably enteral administration.

[0030] The dosage form of the second agent can be tablets, pills, granules, capsules, lozenges, syrups, emulsions, microemulsions, suspensions, injections, effervescent tablets, and the like.

[0031] The second agent can be administered by enteral administration (eg, oral) or parenteral administration (eg, intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intrarectal, etc.), preferably enteral administration.

[0032] Another aspect of the present invention provides the use of a therapeutic agent for sepsis-related acute kidney injury in the preparation of a medicament, wherein the therapeutic agent for sepsis-related acute kidney injury comprises a corticosteroid and a second agent used in combination with the corticosteroid, wherein the second agent is selected from Cordyceps sinensis and / or its fermentation products.

[0033] Furthermore, the sepsis-related acute kidney injury is sepsis-related acute kidney injury with high expression of any one of Cys-C, BUN, CRE, IL-6, TNF-α, and IL-1β.

[0034] The cordyceps sinensis is provided in the form of fermented cordyceps sinensis powder;

[0035] The corticosteroid is selected from the group consisting of prednisone, dexamethasone, triamcinolone, betamethasone, beclomethasone, flunisolide and fluticasone.

[0036] In the present invention, Cordyceps sinensis powder or Cordyceps sinensis powder in combination with corticosteroids greatly improved the renal damage and inflammatory response of sepsis-related acute kidney injury model animals. The renal protective effect of Cordyceps sinensis powder or Cordyceps sinensis powder and corticosteroids on S-AKI and its mechanism are shown in Figure 6. Specifically: Cordyceps sinensis powder promotes mitochondrial energy metabolism of renal cells by improving mitochondrial oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in renal cells, and improves mitochondrial-mediated renal energy metabolism impairment. Furthermore, Cordyceps sinensis powder can not only significantly reduce the infiltration of macrophages in the kidneys, but also promote the polarization of M1 macrophages to M2 macrophages, which indicates that Cordyceps sinensis powder or in combination with corticosteroids can also help resist renal damage caused by sepsis by improving the infiltration and polarization state of macrophages in the kidneys, and inhibit the expression and secretion of inflammatory cytokines. Cordyceps sinensis powder is preferably in the form of Bailing capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0038] Figure 1. Schematic diagram showing the effects of Cordyceps sinensis on improving renal function and inhibiting inflammation in S-AKI mice. (A) Preparation of the S-AKI mouse model and the timing of BL and BL+DEX administration; (B) Representative images of hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) staining and IL-6 immunohistochemistry (IHC) staining in renal tissue (400×, scale bar: 50 μm); (CE) Serum renal function indicators Cys-C (C), BUN (D), and CRE (E) in S-AKI mice after Cordyceps sinensis or / and DEX treatment; (FH) Serum inflammatory cytokines IL-6 (F), TNF-α (G), and IL-1β (H) in S-AKI mice after Cordyceps sinensis or / and DEX treatment; All data are expressed as mean ± SEM (n = 10). ####p<0.0001 compared with the control group; *p<0.05, ***p<0.001, ****p<0.0001 compared with the model; &p<0.05, &&p<0.01 compared with Cordyceps-H+DEX.

[0039] Figure 2. Schematic diagram of transcriptomic analysis. Cordyceps sinensis reprograms genes involved in mitochondrial-mediated oxidative phosphorylation (OXPHOS) in S-AKI. (A) Violin plot shows the expression of all genes in each group; (B) Differential analysis shows the fold change of genes in the transcriptomic dataset; (C) Venn diagram shows the intersection of DEGs in each group; (D) Cluster analysis of DEGs in the Ctrl, Model, and BL groups based on mutation patterns; (E) GSEA enrichment results for the Model group relative to the Ctrl group; (F) GSEA enrichment results for the BL group relative to the Model group; (G) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results for downregulated and upregulated genes in the "Model vs. Ctrl" group. (H) GO enrichment analysis of DEGs in the "Model vs. Ctrl" and "BL vs. Model" groups; (I) Heat map shows DEGs associated with the OXPHOS pathway. All data are presented as mean ± SEM. ***p < 0.001.

[0040] Figure 3. Schematic diagram of proteomic analysis showing that Cordyceps sinensis promoted the expression of mitochondrial electron transport chain (ETC)-related proteins in the kidneys of S-AKI mice. (A) Correlation analysis of the abundance of three groups of proteins; (B) Volcano plot showing the DEPs in "Model vs. Ctrl"; (C) Volcano plot showing the DEGs in "BL vs. Model"; (D) Sankey plot showing the top 20 GO enrichment terms of upregulated proteins in "BL vs. Model"; (E) PPI analysis of the top 20 terms in the GO enrichment analysis. (F) Circular heatmap showing the expression changes of all quantified mitochondrial proteins.

[0041] Figure 4. Schematic diagram showing the analysis of the effect of Cordyceps sinensis on the expression and activity of mitochondrial complexes. (A-B) Protein expression levels of OXPHOS complexes in kidney tissues from the Ctrl, Model, and BL groups (n = 3); (C) Representative confocal immunofluorescence images of TOM20 and nuclear counterstaining (Hoechst) in kidney tissues from the Ctrl, Model, and BL groups. Scale bar, 20 μm; (D) Quantification of mitochondrial fluorescence intensity in immunofluorescence images (n = 3 mouse tissue sections); (E) MMPs in purified mitochondria from kidney tissue (n = 4); (F) ATP levels in kidney tissue (n = 4); (G-I) Activities of mitochondrial complexes I, II, and IV in purified mitochondria from kidney tissue (n = 4); (J) Summary of the effects of Cordyceps sinensis on mitochondrial biogenesis and activity. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0042] Figure 5. Schematic diagram showing the analysis of Cordyceps sinensis inhibiting renal inflammation in S-AKI mice by regulating macrophage polarization. (AB) Immunofluorescence staining of classical macrophage markers (CD206 for M2 type and iNOS for M1 type) in kidney tissue (scale bar = 50 μm). (CD) Representative blots of M2 markers (CD206 and Arg1) and M1 markers (iNOS and IL-6) in kidney tissue (n = 3). Protein expression was normalized to β-actin expression and expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; (E) Representative immunohistochemical images of TGF-β and IL-10 in kidney sections. Scale bar: 100 μm; (F) Quantitative analysis of TGF-β immunohistochemical staining in kidney sections (10 fields from 3 mice, n = 3); ****p < 0.0001 compared with the model; (G) Quantitative analysis of IL-10 immunohistochemical staining in kidney sections (10 fields from 3 mice, n = 3); ****p < 0.0001;

[0043] Figure 6 shows the renal protective effect of Cordyceps sinensis on LPS-induced S-AKI and its mechanism. Cordyceps sinensis treatment significantly alleviated LPS-induced renal dysfunction by promoting mitochondrial biogenesis, enhancing mitochondrial complex activity, improving renal energy metabolism reprogramming, and promoting M2 macrophage polarization. DETAILED DESCRIPTION

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0045] Unless otherwise specified, the materials and reagents used in the following examples are all common commercial products and can be purchased on the market.

[0046] The Bailing capsules used were produced by Hangzhou Sino-US Huadong Pharmaceutical Co., Ltd., dexamethasone (DEX) was purchased from Bidex Pharmaceutical Technology Co., Ltd. (Shanghai, China), lipopolysaccharide (LPS) was purchased from Sigma-Aldrich, Co., Ltd. (Missouri, USA), and ELISA kits for interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), blood urea nitrogen (BUN), creatinine (CRE), and cystatin C (Cys-C) were purchased from Shanghai ELISA Biotechnology Co., Ltd. (Shanghai, China).

[0047] In the present invention, fermented cordyceps sinensis powder refers to the dry powder of mycelium obtained by liquid fermentation culture of the asexual generation strain of the ergot fungus Cordyceps sinensis isolated from the fruiting body of Cordyceps sinensis. In conjunction with the context, fermented cordyceps sinensis powder is the dry powder of mycelium obtained by liquid fermentation culture of the asexual generation Chinese hairy spore (Hirsutella sinensis) mycelium strain isolated from the fruiting body of Tibetan Cordyceps sinensis. In an embodiment, the fermented cordyceps sinensis powder is derived from Bailing capsules. In the comparison of nucleosides, sugar alcohols, sterols, and amino acids that are rich in it and wild cordyceps sinensis, the fingerprints are consistent. Therefore, in terms of medicinal value, the two also have similar effects. Due to the high heavy metal content in wild cordyceps sinensis, the reaction conditions are accurately controlled by industrial fermentation process, which can ensure that the metal content in the fermented cordyceps sinensis powder meets safety standards and ensure that the quality of each batch of products is stable and controllable, eliminating consumer concerns. At the same time, its price is much lower than that of wild cordyceps sinensis.

[0048] In the present invention, "subject" is not limited and refers to a human or non-human mammal to whom the medicament of the present invention is administered, such as a mammal including cattle, horses, dogs, sheep, or cats. Preferably, the subject is a human. The subject includes patients (including humans and non-human mammals).

[0049] As used herein, "treating" means to slow, interrupt, arrest, control, stop, lessen, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0050] In the present invention, "prevention" refers to a method performed to prevent or delay the occurrence of a disease, disorder or symptom in the body.

[0051] In the present invention, "effective amount" refers to the amount or dosage of Cordyceps sinensis or therapeutic agent or drug of the present invention that provides the desired treatment or prevention after administration to a subject in single or multiple doses.

[0052] The present invention will be further described below by way of examples, which are not intended to further limit the present invention. Those skilled in the art will appreciate that any equivalent substitutions or corresponding improvements made to the present invention are still within the scope of protection of the present invention.

[0053] Example 1 Animal Experiment

[0054] In this example, sepsis-related acute kidney injury (S-AKI) was induced in mice by lipopolysaccharide (LPS). DEX is a classic corticosteroid with anti-inflammatory activity. The Cordyceps sinensis powder encapsulated in Bailing capsules was taken out and dissolved in a sterile 0.5% sodium carboxymethylcellulose solution (BL) for later use. C57BL / 6 mice (male, 6 weeks old) were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd. (Beijing, China) and adapted to standard conditions (constant temperature 22°C; ambient humidity 50%-60%; 12h / 12h light / dark cycle; free drinking water and standard rodent food) for 1 week before use. 60 mice were randomly divided into 6 groups, including:

[0055] 1) Control group: normal saline, referred to as Ctrl;

[0056] 2) Model group: LPS, 5 mg / kg (rat), referred to as Model;

[0057] 3) BL-L: 1 g / kg / d (rat) BL + 5 mg / kg (rat) LPS group;

[0058] 4) BL-H: 5 g / kg / d (rat) BL + 5 mg / kg (rat) LPS group;

[0059] 5) DEX: 5 mg / kg / d (rat) dexamethasone + 5 mg / kg (rat) LPS group;

[0060] 6) BL-H+DEX: 5 mg / kg / d (rat) dexamethasone + 5 g / kg / d (rat) BL + 5 mg / kg (rat) LPS group.

[0061] The low-dose group of Cordyceps sinensis (BL-L) was 1g / kg, equivalent to a human dose of 91mg / kg; the high-dose group of Cordyceps sinensis (BL-H) was 5g / kg, equivalent to a human dose of 455mg / Kg; BL and DEX were both administered orally by gavage, and LPS was injected intraperitoneally into mice.

[0062] Mice in the BL-L, BL-H, and BL-H+DEX groups were pretreated with BL by oral gavage once daily for 14 days before induction of systemic sepsis with LPS. Mice in the DEX and BL-H+DEX groups were gavaged with DEX 24 hours before induction of systemic sepsis with LPS. Mice in the control group received the same volume of saline. Except for the control group, mice in all other groups received an intraperitoneal injection of LPS 12 hours before euthanasia (see Figure 1A). Blood and kidney samples were collected after anesthesia for biochemical analysis, pathological evaluation, immunohistochemistry, transcriptomic sequencing, proteomics, and western blotting. A portion of the kidney was fixed with 4% paraformaldehyde, and the remainder was snap-frozen in liquid nitrogen and stored at −80°C.

[0063] Example 2 Detection and statistical analysis

[0064] 2.1 Determination of serum biochemical indicators

[0065] Early renal dysfunction in mice can be reflected by the levels of relevant biochemical indicators in serum. Serum Cys-C, BUN, and CRE levels were measured using the above-mentioned kits according to the manufacturer's protocol.

[0066] 2.2 Enzyme-linked immunosorbent assay (ELISA)

[0067] The systemic inflammatory state of mice was assessed by measuring the levels of related inflammatory factors in the serum. IL-6, TNF-α, and IL-1β levels were measured using the enzyme-linked immunosorbent assay kits provided by the manufacturer.

[0068] 2.3 Histological and immunohistochemical (IHC) observation of renal tissue

[0069] Kidney tissue was fixed with 4% paraformaldehyde, then embedded in paraffin and sliced. Hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) staining were performed according to the histological examination instructions provided by the manufacturer. Immunohistochemistry (IHC) staining was performed using IL-6 (Proteintech, China), TGF-β and IL-10 antibodies (Servicebio, China) to evaluate the expression of IL-6, TGF-β and IL-10 in kidney tissue. The sections were observed under an optical microscope (Nikon, Tokyo, Japan). Images were collected on an inverted fluorescence microscope (CKX53, Olympus, Japan) and analyzed by the configured MShot image analysis system.

[0070] 2.4 Immunofluorescence staining

[0071] Paraffin sections of renal tissue were deparaffinized and dehydrated, followed by antigen retrieval at 100°C for 5 minutes. Subsequently, the samples were permeabilized and blocked with bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.1% Tween-20. The sections were then incubated with primary antibodies (anti-TOM20, anti-F4 / 80, anti-CD206, and anti-iNOS) overnight at 4°C. Tissue sections were then stained with corresponding fluorescent secondary antibodies and Hoechst. Images were acquired and recorded using a confocal fluorescence microscope (Leica TBL SP8 SR, Germany), and the fluorescence intensity of the images was quantified using Image J software (version 1.50i).

[0072] 2.5 Transcriptomics and data analysis

[0073] Total mRNA was extracted from kidney samples (three samples per group) using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions. After enrichment with poly(A) template, the isolated total mRNA was sequenced on an Illumina Novaseq 6000 sequencer (Illumina) using PE150 reads. As reported in the literature (Chen et al., 2022), the quality of the raw sequencing data was controlled using fastp, and the reads were then aligned to the mouse reference genome mm10 using STAR (version 2.2.1). The reads were quantified using featureCounts (version 1.5.0). The data were then statistically analyzed and visualized using the R software package. Differentially expressed genes (DEGs) were analyzed using the "limma" R package (version 3.48.3). P values ​​were generated using an empirical Bayesian test model and adjusted using the Benjamini-Hochberg (BH) test. Genes with an absolute fold change (FC) ≥ 2 and an adjusted P (FDR) < 0.05 were considered significant DEGs. Gene Ontology (GO) analysis was performed using the "clusterprofiler" R package (version 3.18.1). DEGs were further visualized using the ggplot2 R package (version 3.3.5) based on their log2 (FC) and -log10 (FDR). Gene Set Enrichment Analysis (GSEA), KEGG pathway enrichment analysis, and GO functional enrichment analysis were performed on genes downregulated in the "Model vs. Con" group and upregulated in the "BL vs. Model" group, and the top 10 terms were visualized.

[0074] 2.6 Proteomics and data analysis

[0075] Kidney tissue (three samples per group) was lysed using RIPA lysis buffer and sonicated on ice. Samples were centrifuged (20,000 g, 4°C, 20 minutes) and the supernatant was collected. Protein concentration was determined using a BCA assay. 100 μg of protein was then reduced and alkylated with 20 mM dithiothreitol (DTT) and 50 mM iodoacetamide (IAA), respectively. The samples were then precipitated with pre-cooled acetone at -20°C for 30 minutes. The precipitate was collected by centrifugation (20,000 g, 4°C, 5 minutes) and dissolved in a buffer (8 M urea, 100 mM triethylamine bicarbonate (TEAB, pH 8.5)). The peptides were then slowly loaded onto a C18 desalting column and desalted by washing with 0.1% formic acid (FA) buffer. The eluate from each sample was collected after elution (0.1% formic acid, 60% acetonitrile) and lyophilized. Finally, the samples were redissolved in 0.1% formic acid and analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) (Thermo Orbitrap Fusion Lumos, USA). The mass spectrometry raw files were processed and analyzed by Proteome Discoverer2.4 (Thermo Scientific). Proteins with absolute FC ≥ 1.5 and P value (FDR) < 0.05 were defined as differentially expressed proteins (DEPs). The data were then visualized using the Bioladder website (https: / / www.bioladder.cn) and TBtools (Chen et al., 2020). KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis, GO analysis, and protein-protein interaction (PPI) analysis were performed using the bioinformatics platform (http: / / www.bioinformatiBL.com.cn).

[0076] 2.7 Immunoblotting analysis

[0077] Western immunoblotting confirmed the expression levels of proteins that were significantly altered between groups in the proteomic analysis. Kidney samples were lysed with ice-cold RIPA lysis buffer supplemented with protease inhibitors. Proteins were then concentrated and separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins from the gel were then electrotransferred to a polyvinylidene fluoride (PVDF) membrane at 120 V for 80 minutes. The membrane was then blocked with 5% bovine serum albumin (BSA) in 1× TBST containing 0.1% Tween-20 and incubated with primary antibodies (anti-ND5, anti-SDHC, anti-CYTB, anti-MTCO2, anti-ATP6, anti-β-actin, anti-CD206, anti-Arg1, anti-iNOS, and anti-IL-6) overnight at 4°C, followed by incubation with secondary antibodies for 1 hour at room temperature (RT). Protein bands were visualized using enzyme-linked chemiluminescence (ECL) (Thermo Fisher, USA). Protein band intensities were semi-quantified using Image J software and normalized to β-actin expression levels.

[0078] 2.8 Mitochondrial Function Analysis

[0079] Mitochondrial membrane potential (MMP) in renal tissue was measured using the JC-1 kit (Beyotime, China) according to the manufacturer's instructions. Briefly, fresh kidney tissue (100 mg) was collected from each mouse (four samples per group), and mitochondria were isolated and purified as quickly as possible using a Mitochondria Isolation Kit (Beyotime, China) according to the manufacturer's instructions. MMP was further expressed as red / green fluorescence intensity using a fluorescence microplate reader (PerkinElmer, USA). ATP content in fresh mouse kidney tissue was measured using a commercial assay kit (mblbio, China) according to the manufacturer's instructions.

[0080] 2.9 Enzymatic detection of mitochondrial complex activity

[0081] First, mitochondria were isolated from fresh kidney tissue using a mitochondrial isolation kit. Then, mitochondrial complex activity was assessed using a commercial kit from Shanghai ELISA Biotechnology Co., Ltd.

[0082] 2.10 Statistical Analysis

[0083] All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis of each group was performed using one-way analysis of variance and Tukey's post-hoc test. For two groups, significance was calculated using Student's t-test (two-tailed). P values ​​less than 0.05 were considered statistically significant. All data were analyzed using GraphPad Prism software (version 8.0).

[0084] Example 3 Test Results and Analysis

[0085] BL improves renal dysfunction and inflammatory response in S-AKI mice

[0086] Histopathological results of hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) staining revealed that LPS induced significant pathological damage in the mouse kidneys (Figure 1B). Most renal tubules were dilated and edematous, and tubular epithelial cells were commonly vacuolated, necrotic, and detached, with cast formation. Treatment with BL and / or dexamethasone effectively ameliorated this pathological damage. Furthermore, immunohistochemical staining revealed that BL and / or dexamethasone significantly inhibited the LPS-induced increase in the expression of the inflammatory cytokine IL-6 in the kidneys.

[0087] Serum cystatin C (Cys-C), blood urea nitrogen (BUN), and creatinine (CRE) are important indicators of renal function. As shown in Figures 1C-E, LPS severely impaired the renal function of mice, as manifested by increased levels of Cys-C, BUN, and CRE in the serum. BL and / or dexamethasone effectively reduced the levels of these indicators in the serum of S-AKI mice. In addition, BL and / or dexamethasone significantly reduced the levels of inflammatory factors IL-6 (interleukin-6), TNF-α (tumor necrosis factor-α), and IL-1β (interleukin-1β) in the serum of S-AKI mice, indicating that BL has anti-inflammatory activity against S-AKI (Figures 1F-H). These results indicate that BL can improve renal dysfunction and inflammation in S-AKI mice.

[0088] 3.2 Transcriptomics revealed that BL reprogrammed mitochondrial-mediated oxidative phosphorylation (OXPHOS) in the kidney

[0089] In transcriptomic analysis, Violin (Figure 2A) and heatmaps displayed the relative expression levels of genes in the transcriptomic dataset. Compared with the control group, the model group significantly upregulated gene expression levels, while BL treatment partially reversed LPS-induced gene expression changes, consistent with pathological observations. A scatter plot (Figure 2B) further depicted and highlighted differentially expressed genes (DEGs) with a fold change of more than 2 between the "model group vs. control" and "BL treatment group vs. model group." Compared with the control group, 1807 genes were upregulated and 1751 genes were downregulated in the model group. Comparing gene expression between the BL treatment group and the model group revealed 221 upregulated genes and 206 downregulated genes. Furthermore, analysis of the intersection of upregulated and downregulated genes, displayed in a Venn diagram (Figure 2C), revealed that in the "model group vs. control" group, 89 and 37 genes were repressed and activated, respectively, while in the "BL treatment group vs. model" group, these changes were reversed. The differentially expressed genes were divided into five clusters based on their expression patterns (Figure 2D). The genes in clusters 3 and 5 had a unique characteristic: BL could significantly restore the expression changes induced by LPS, suggesting that these genes are highly likely involved in the pathways by which BL repairs renal injury. Therefore, gene set enrichment analysis (GSEA) was performed on these genes to enrich for molecular pathways associated with the protective effects of BL (Figures 2E-F).

[0090] GSEA results showed that the oxidative phosphorylation (OXPHOS) process in the kidney was significantly downregulated in the "model group vs. control group" analysis (Figure 2E), but upregulated in the "BL treatment group vs. model group" analysis (Figure 2F). On the other hand, genes involved in the ribosome and endoplasmic reticulum protein processing pathways showed opposite change trends to genes enriched in the OXPHOS pathway. KEGG analysis of DEGs further indicated that OXPHOS and thermogenesis were the two pathways with the highest enrichment scores (Figure 2G). Functional enrichment analysis of DEGs was performed and compared between the "model group vs. control group" and "BL treatment group vs. model group" groups (Figure 2H). Consistent with the GSEA and KEGG results, mitochondrial-related energy metabolism processes such as oxidative phosphorylation, electron respiratory chain, and ATP biosynthesis were significantly downregulated after LPS induction, while BL treatment could rescue the affected processes in S-AKI. In addition, GO enrichment analysis of DEGs classified by molecular function and cellular component revealed that electron transport activity and respirosome were enriched, respectively. These two genes are also closely related to mitochondrial energy metabolism. The heat map further depicts the expression profile of the main genes related to OXPHOS (Figure 2I). BL partially restored the expression of these genes that were downregulated by LPS.

[0091] 3.3 Proteomic analysis showed that BL promoted the expression of mitochondrial electron transport chain (ETC)-related proteins in the kidneys of S-AKI mice

[0092] A total of 5,302 proteins were identified in the control, model, and BL-treated groups. Cluster analysis of protein abundance heatmaps revealed that the BL-treated group had a closer correlation with the control group than with the model group. Furthermore, correlation analysis among the three groups directly demonstrated that the BL-treated group was positively correlated with the control group in terms of protein abundance ratios, while the model group was negatively correlated with both the control group and the BL-treated group (Figure 3A). These results suggest that BL treatment can, to some extent, restore the proteome composition of S-AKI kidneys to a normal state. Differentially expressed proteins (DEPs) in the "model group vs. control" and "BL-treated group vs. model group" were displayed in a volcano plot, with a fold change > 2 and a p-value < 0.05 as critical values ​​(Figures 3B-C). 177 downregulated and 350 upregulated proteins were found in the "model group vs. control" group, and 345 downregulated and 197 upregulated proteins were found in the "BL-treated group vs. model" group. Proteins that were downregulated in the "model group vs. control group" and upregulated in the "BL-treated group vs. model group" were labeled with their protein names if they showed similar changes in the transcriptomics data. Respiratory complex proteins including the NADH-ubiquinone oxidoreductase chain (ND1, ND2, ND3, ND4, and ND5), cytochrome c oxidase subunits (COX1, COX2, and COX3), F-ATPase protein 6 (ATP6), and cytochrome b (CYTB) were highlighted, suggesting that BL may restore the expression of proteins related to mitochondrial electron transport in the kidney.

[0093] Gene ontology (GO) enrichment analysis was performed on the DEPs and visualized in a Sankey bubble plot (Figure 3D). The oxidative phosphorylation pathway was again preferentially enriched, and other energy metabolism biological processes including ATP synthesis, cellular respiration, mitochondrial respiratory chain complexes, and electron transport chain also had high enrichment priorities. In addition, most proteins involved in OXPHOS were associated with mitochondrial ETC function, indicating that the mitochondrial-mediated OXPHOS pathway is a key factor in BL's resistance to S-AKI. More detailed GO and KEGG enrichment analysis of DEPs further confirmed this result.

[0094] Protein-protein interaction (PPI) analysis of DEPs (Figure 3E) revealed robust and close interactions with mitochondrial proteins (such as ND1, ND2, COX1, etc.). The abundance of all quantified mitochondrial proteins was analyzed and compared using a circular heat map (Figure 3F). Most mitochondrial proteins, especially ETC-related proteins, were significantly downregulated in the "model group vs. control group," while their abundance was restored by BL administration.

[0095] BL increases the expression level and activity of mitochondrial complexes

[0096] Biochemical methods, including Western blotting (WB), immunofluorescence (IF), and enzyme activity assays, were used to examine the expression levels and activity of mitochondrial complex proteins and the integrity of mitochondria in renal tissue (Figure 4). Consistent with the transcriptomic and proteomic results, LPS significantly inhibited the expression levels of mitochondrial protein complex subunits, including complex I (CI-ND5), complex II (CI-SDHC), complex III (CI-ND6), complex IV (CI-ND7), complex III (CI-CYTB), complex IV (CI-MTCO2), and complex V (CI-ATP6), while BL restored the expression of mitochondrial complexes to varying degrees (see Figure 4A-B for examples).

[0097] Immunofluorescence staining of renal tissue using the mitochondrial marker TOM20 was performed. The results showed that the number of mitochondria in the renal proximal tubules of S-AKI mice was drastically reduced and their morphology was distorted, whereas BL treatment significantly improved this condition (Figures 4C-D), further demonstrating that BL can promote mitochondrial biogenesis and maintain their structural integrity. Mitochondrial membrane potential (MMP) is a key parameter of mitochondrial function. As shown in Figure 4E, MMP levels were lower in LPS-induced S-AKI mice compared with control mice. However, BL significantly increased MMP levels compared with model mice, partially reversing the LPS-induced decrease in MMP. Simultaneously, changes in ATP levels in renal tissue were similar among the three groups, with BL reversing the decrease in ATP in the S-AKI model (Figure 4F). Furthermore, after extracting mitochondria from renal tissue, the activity of mitochondrial complexes was assessed. The results showed that the activities of three complexes, I (Figure 4G), II (Figure 4I), and IV (Figure 4H), were inhibited after LPS induction and further restored to normal levels after BL treatment. BL increased the expression and activity of mitochondrial ETC proteins, promoted mitochondrial biogenesis and oxidative phosphorylation, and thus inhibited the damage of energy metabolism in S-AKI mice ( Figure 4J ).

[0098] BL inhibits renal inflammation in S-AKI mice by regulating macrophage polarization

[0099] Double immunofluorescence staining (Figure 5A-B) showed that BL could effectively alleviate LPS-induced macrophage infiltration, as evidenced by a decrease in F4 / 80 fluorescence signal after BL pretreatment in S-AKI mice. In addition, compared with the model group, the number of CD206+F4 / 80 macrophages in kidney tissue sections in the BL-treated group was significantly increased, while the number of iNOS+F4 / 80 macrophages was significantly decreased, suggesting that BL may improve renal inflammation by increasing the ratio of M2 / M1 macrophages.

[0100] Western blot results also showed that LPS significantly activated M1 macrophages in the kidney by increasing the expression of M1 macrophage producers iNOS and IL-6, while reducing the expression of M2 macrophage producers CD206 and Arg1. In contrast, BL promoted the transformation of M1 macrophages to M2 macrophages by reversing the LPS-induced changes in the expression of these biomarkers, confirming the anti-inflammatory ability of BL by regulating macrophage polarization (Figure 5C-D). Immunohistochemical staining confirmed that the expression levels of M2 macrophage-related anti-inflammatory factors (transforming growth factor-β (TGF-β) and IL-10) were significantly upregulated in the BL treatment group, even higher than those in the control group (Figure 5E-G).

[0101] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of Cordyceps sinensis in the preparation of a medicament for preventing and / or treating sepsis-related acute kidney injury.

2. The use according to claim 1, characterized in that The sepsis-related acute kidney injury is sepsis-related acute kidney injury with high expression of any one of Cys-C, BUN, CRE, IL-6, TNF-α, and IL-1β proteins.

3. The use according to any one of claims 1 to 2, characterized in that The sepsis-related acute kidney injury is a sepsis-related acute kidney injury in which the mitochondrial energy metabolism biological pathway of kidney cells is damaged.

4. The use according to claim 3, characterized in that The kidney cell mitochondrial energy metabolism biological pathway is kidney cell mitochondrial oxidative phosphorylation and mitochondrial biogenesis.

5. The use according to claim 4, characterized in that The cordyceps sinensis has one or more of the following characteristics: improving the activity of mitochondrial complexes, mitochondrial membrane potential (MMP) and the production of ATP in mitochondria of kidney cells.

6. The use according to claim 5, characterized in that The cordyceps sinensis has one or more of the following pathways: upregulating the expression of NADH-ubiquinone oxidoreductase chain, cytochrome c oxidase subunit, ATP6 and CYTB, which are related to electron transfer in kidney cell mitochondria; and upregulating the expression of complexes CⅠ-ND5, CⅡ-SDHC, CⅠ-ND6, CⅠ-ND7, CⅡ-SDHC, CⅢ-CYTB, CⅣ-MTCO2 and CⅤ-ATP6 proteins in kidney cell mitochondria.

7. The use according to any one of claims 1 to 2, characterized in that: The sepsis-related acute kidney injury is sepsis-related acute kidney injury in which the polarization of renal M1 macrophages to M2 macrophages is blocked.

8. The use according to claim 7, characterized in that The cordyceps sinensis has one or more of the following characteristics: reducing the content of iNOS and IL-6 in the blood; increasing the content of CD206 and Arg1 in the blood; and upregulating the protein expression of TGF-β and IL-10 in lung tissue.

9. The use according to any one of claims 1 to 8, characterized in that Cordyceps sinensis is obtained by artificially fermenting the asexual fungus of Cordyceps sinensis.

10. The use according to claim 9, characterized in that The asexual fungus of Cordyceps sinensis is obtained by fermentation of Hirsutella sinensis.

11. The use according to claim 10, characterized in that The cordyceps sinensis is Bailing capsule.

12. A therapeutic agent for sepsis-related acute kidney injury, characterized in that: The invention comprises a corticosteroid and a second agent used in combination with the corticosteroid, wherein the second agent is selected from Cordyceps sinensis and / or its fermentation products.

13. The therapeutic agent according to claim 12, characterized in that The second agent is provided in the form of fermented Cordyceps sinensis powder; Optionally, the corticosteroid is selected from prednisone, dexamethasone, triamcinolone, betamethasone, beclomethasone, flunisolide and fluticasone.

14. The therapeutic agent according to any one of claims 12 to 13, characterized in that The sepsis-related acute kidney injury is sepsis-related acute kidney injury with high expression of any one of Cys-C, BUN, CRE, IL-6, TNF-α, and IL-1β proteins.

15. A method for treating sepsis-related acute kidney injury, characterized in that: The method comprises administering a corticosteroid and a second agent selected from Cordyceps sinensis and / or a fermentation product thereof to a subject diagnosed with sepsis-related acute kidney injury.

16. The method according to claim 15, characterized in that The dosage of the corticosteroid is 0.4-0.5 mg / Kg / d; The dosage of the second agent is 91-455 mg / Kg / d, 91-546 mg / Kg / d, 91-637 mg / Kg / d, 91-728 mg / Kg / d, 91-819 mg / Kg / d or 91-910 mg / Kg / d; Optionally, the subject has at least one alteration in Cys-C, BUN, CRE, IL-6, TNF-α, IL-1β protein; Optionally, the subject has been diagnosed with sepsis-related acute kidney injury and has at least one alteration in Cys-C, BUN, CRE, IL-6, TNF-α, IL-1β protein; Optionally, the subject has been diagnosed with sepsis-related acute kidney injury and has high expression of at least one of Cys-C, BUN, CRE, IL-6, TNF-α, IL-1β proteins.

17. The method according to any one of claims 15 to 16, characterized in that: The second agent is provided in the form of fermented Cordyceps sinensis powder; Optionally, the corticosteroid is selected from prednisone, dexamethasone, triamcinolone, betamethasone, beclomethasone, flunisolide and fluticasone.

18. Use of a therapeutic agent for sepsis-related acute kidney injury in the preparation of a drug, characterized in that: The therapeutic agent for sepsis-related acute kidney injury contains a corticosteroid and a second agent used in combination with the corticosteroid, wherein the second agent is selected from Cordyceps sinensis and / or its fermentation product.

19. The use according to claim 18, characterized in that The sepsis-related acute kidney injury is sepsis-related acute kidney injury with high expression of any one of Cys-C, BUN, CRE, IL-6, TNF-α, and IL-1β proteins.

20. The use according to claim 19, characterized in that The cordyceps sinensis is provided in the form of fermented cordyceps sinensis powder; Optionally, the corticosteroid is selected from prednisone, dexamethasone, triamcinolone, betamethasone, beclomethasone, flunisolide and fluticasone.

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