A honeysuckle-derived exosome-like nanovesicle for alleviating acute kidney injury and a preparation method thereof

Abstract

The present invention relates to the technical field of traditional Chinese medicine compositions, and in particular to a honeysuckle-derived exosome-like nanovesicle for alleviating acute kidney injury and a preparation method thereof. The honeysuckle-derived exosome-like nanovesicle has a particle size distribution of 68.06-164.20 nm. First, the honeysuckle-derived exosome-like nanovesicle retains multiple bioactive components of honeysuckle, and these components may exert stronger pharmacological effects through synergistic effects; second, the nanoscale particle size enables the honeysuckle-derived exosome-like nanovesicle to better penetrate biological barriers, improving targeting and bioavailability; third, the natural origin and exosome-like structure of the honeysuckle-derived exosome-like nanovesicle may give it unique cell affinity, which is beneficial for its interaction with target cells; finally, as a natural nanocarrier, the honeysuckle-derived exosome-like nanovesicle has better safety.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine compositions, and in particular to honeysuckle-derived exosome-like nanovesicles for alleviating acute kidney injury and a preparation method thereof. Background Art

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function over a short period of time due to a variety of causes. Its main clinical manifestations include decreased glomerular filtration rate, resulting in retention of nitrogenous products such as creatinine and urea nitrogen, as well as disturbances in water, electrolyte, and acid-base balance (1). AKI is closely associated with increased hospitalization rates, morbidity, and mortality (2). In addition, AKI often increases the risk of progression to chronic kidney disease (CKD) and end-stage renal disease (3). Cisplatin is a platinum-based chemotherapy drug that is widely used as a first-line treatment for a variety of solid tumors (4). Despite its significant anti-tumor efficacy, its serious side effects (including nephrotoxicity, ototoxicity, hepatotoxicity, and cardiotoxicity) have also attracted much attention (5). Cisplatin-induced AKI is particularly noteworthy. Approximately 30% of patients receiving high-dose cisplatin treatment experience renal impairment, and this proportion can exceed 70% in pediatric patients (6). In recent years, many studies have been devoted to exploring the potential mechanisms of cisplatin-induced AKI (7-11), laying the foundation for the discovery of effective prevention and treatment strategies. Although the pathophysiological mechanisms of cisplatin-induced AKI are multifaceted and have not yet been fully elucidated, studies have shown that key processes such as inflammation, oxidative stress, mitochondrial dysfunction, and renal tubular epithelial cell apoptosis are involved (12). Therefore, we aimed to alleviate cisplatin-induced AKI through a comprehensive strategy of inhibiting inflammation, improving oxidative damage and mitochondrial dysfunction, and reducing renal tubular epithelial cell apoptosis.

[0003] Honeysuckle, as a traditional Chinese herbal medicine, has been widely used in daily health care and the treatment of various diseases. Its complex chemical composition includes key bioactive compounds such as chlorogenic acid, isochlorogenic acid, lignans, and saponins (13). Previous studies have shown that honeysuckle has anti-inflammatory, antioxidant, anti-apoptotic, and mitochondrial protective properties, and can improve inflammatory responses, oxidative damage, apoptosis activation, and mitochondrial dysfunction under different pathological conditions (14-16). In addition, studies have confirmed that honeysuckle can alleviate reactive oxygen species (ROS)-induced mitochondrial damage and cellular senescence (17) and restore mitochondrial morphology destroyed by oxidative damage (18).

[0004] Extracellular vesicles (EVs) (e.g., exosomes and microvesicles) are tiny membrane particles (40 to 1000 nm in size) that are secreted by cells in a constitutive or inducible manner and can be isolated from the fresh juices of a variety of edible plants (19). EVs act as intercellular messengers in the body, transporting nucleic acids and proteins to distant or neighboring cells (20). Recent studies have shown that EVs have the potential to serve as stable and effective nanocarriers in a variety of therapeutic scenarios, from chemotherapy to gene therapy (21). Compared with existing delivery systems, the natural origin of EVs gives them significant advantages: enhanced biocompatibility, reduced immunogenicity, and the ability to evade phagocytic clearance, thereby extending circulation time in the body and improving targeting and bioavailability (22).

[0005] Plant-derived exosome-like nanovesicles (PELNVs) have been shown to be enriched in various cell types, including intestinal stem cells (23), macrophages (24), dendritic cells (25), and hepatocytes (26), as novel therapeutic adjuvants or delivery vehicles. In addition, PELNVs exhibit enhanced ability to resist inflammatory infiltration and modulate immune responses (27). Based on these properties of PELNVs, we speculate that EVs may also be targeted and enriched in renal tissue and have the potential to be used as combination therapeutics for the treatment of renal diseases, including cisplatin-induced acute kidney injury.

[0006] Based on the main mechanism of cisplatin-induced AKI, combined with the multifaceted pharmacological properties of honeysuckle and the natural advantages of EVs, we hypothesized that honeysuckle-derived exosome-like nanovesicles may have potential therapeutic effects on cisplatin-induced AKI. This study aimed to investigate whether honeysuckle-derived exosome-like nanovesicles can alleviate cisplatin-induced AKI by inhibiting inflammatory response, improving oxidative damage and mitochondrial dysfunction, and reducing renal tubular epithelial cell apoptosis ( Figure 1 ). References are as follows:

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[0033] 27. Zhang ZY, Yu Y, Zhu GX, Zeng LT, Xu SF, Cheng HY, et al. New roles of plant-derived exosome-like nanoparticles in immunomodulation and periodontitis treatment. Front Immunol. 2022;13. Summary of the Invention

[0034] Against this backdrop, the present invention proposes an innovative therapeutic strategy: utilizing honeysuckle-derived exosome-like nanovesicles (LOX) to alleviate cisplatin-induced AKI. This strategy cleverly combines the multi-target efficacy of traditional Chinese medicine with the advantages of advanced nanotechnology, potentially overcoming the limitations of existing treatments.

[0035] The object of the present invention is to provide a honeysuckle-derived exosome-like nanovesicle for alleviating acute kidney injury, wherein the particle size distribution of the honeysuckle-derived exosome-like nanovesicle is 68.06-164.20 nm.

[0036] Specifically, the average particle size of the honeysuckle-derived exosome-like nanovesicles is 100 nm.

[0037] Specifically, the protein concentration of the honeysuckle-derived exosome-like nanovesicles is 2-5 mg / mL.

[0038] Specifically, the zeta potential of the honeysuckle-derived exosome-like nanovesicles is -20 to -30 mV.

[0039] Specifically, the effective dose of the composition is 30 mg / kg.

[0040] A method for preparing the honeysuckle-derived exosome-like nanovesicles comprises the following steps:

[0041] (1) Raw material processing: Wash the dried honeysuckle buds with water, extract the honeysuckle juice using high-speed stirring, and then filter to remove impurities;

[0042] (2) Differential centrifugation: The filtrate obtained in step (1) is subjected to multiple steps of differential centrifugation, and the supernatant is collected;

[0043] (3) Ultracentrifugation: The supernatant obtained in step (2) was subjected to ultracentrifugation, and the precipitate was collected and resuspended in buffer;

[0044] (4) Density gradient centrifugation: Prepare a sucrose density gradient, load the resuspended sample in step (3) into the gradient and centrifuge to collect the honeysuckle-derived exosome-like nanovesicle layer in a specific density range;

[0045] (5) Purification: Use ultrafiltration centrifuge tubes to remove sucrose and obtain purified honeysuckle-derived exosome-like nanovesicles.

[0046] Specifically, the method further comprises the following steps:

[0047] The protein concentration of honeysuckle-derived exosome-like nanovesicles was determined using a BCA protein quantification kit.

[0048] Specifically, the method further comprises the following steps:

[0049] The honeysuckle-derived exosome-like nanovesicles were fixed with 2.5% glutaraldehyde for 30 minutes and negatively stained with 1.9% methylcellulose and 0.3% uranyl acetate for 5 minutes. The morphology of the honeysuckle-derived exosome-like nanovesicles was observed using transmission electron microscopy.

[0050] Specifically, the method further comprises the following steps:

[0051] The particle size distribution of honeysuckle-derived exosome-like nanovesicles was analyzed using a dynamic light scattering instrument at 25°C and a scattering angle of 173°.

[0052] The core innovation of the present invention is:

[0053] The innovative nature of honeysuckle-derived exosome-like nanovesicles is primarily reflected in the following aspects: First, they retain multiple bioactive components of honeysuckle, which may exert a synergistic effect and enhance pharmacological effects. Second, their nanoscale particle size (68.06-164.20 nm) enables them to better penetrate biological barriers, improving targeting and bioavailability. Third, their natural origin and exosome-like structure may confer unique cell affinity, facilitating their interaction with target cells. Finally, as a natural nanocarrier, honeysuckle-derived exosome-like nanovesicles may have improved safety.

[0054] From a molecular mechanistic perspective, polyphenolic compounds (such as chlorogenic acid) in honeysuckle-derived exosome-like nanovesicles may exert antioxidant effects through direct interactions of their hydroxyl groups with free radicals. Furthermore, these compounds may enhance the cellular endogenous antioxidant system by regulating the Nrf2 / HO-1 pathway. Regarding anti-inflammatory properties, honeysuckle-derived exosome-like nanovesicles may reduce the expression of proinflammatory cytokines by inhibiting the NF-κB signaling pathway. Furthermore, certain components of honeysuckle-derived exosome-like nanovesicles may inhibit cell apoptosis by regulating the Bcl-2 / Bax ratio and caspase activity.

[0055] Of particular note, the protective effect of honeysuckle-derived exosome-like nanovesicles on mitochondrial function may be one of the key mechanisms of their pharmacological action. Mitochondria are the primary target of cisplatin toxicity, and certain components of honeysuckle-derived exosome-like nanovesicles may alleviate cisplatin-induced mitochondrial dysfunction by maintaining mitochondrial membrane potential, reducing ROS production, and protecting mitochondrial DNA. This comprehensive protective effect on mitochondria is difficult to achieve with traditional single-ingredient drugs.

[0056] In summary, the proposed honeysuckle-derived exosome-like nanovesicles therapeutic strategy possesses multiple innovations and significant clinical potential. It not only provides a new treatment option for cisplatin-induced AKI but also offers new insights into the modernization of traditional Chinese medicine and the development of nanomedicines. Future research will further optimize the preparation process of honeysuckle-derived exosome-like nanovesicles, further explore their mechanism of action, and evaluate their feasibility for clinical application, aiming to provide better treatment options for patients with AKI. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1Schematic diagram of the mechanism. Exosome-like nanovesicles derived from honeysuckle: Exosome-like nanovesicles derived from honeysuckle.

[0058] Figures 2A-2D Extraction and characterization of honeysuckle-derived exosome-like nanovesicles. (2A) Flowchart for the isolation and purification of honeysuckle-derived exosome-like nanovesicles. (2B) Particle size distribution of honeysuckle-derived exosome-like nanovesicles as determined by dynamic light scattering analysis. (2C) Morphological and size characteristics of honeysuckle-derived exosome-like nanovesicles as observed by transmission electron microscopy (TEM) after sucrose density gradient centrifugation. Scale bar: 200 nm. (2D) Survival rate (%) of HK-2 cells after treatment with different concentrations of honeysuckle-derived exosome-like nanovesicles, ranging from 0 (control) to 200 μg / mL, for 24 hours.

[0059] Figures 3A-3G Honeysuckle-derived exosome-like nanovesicles reduce oxidative damage and apoptosis in cisplatin-treated HK-2 cells. (3A) Cell viability (%) of HK-2 cells after treatment with different concentrations of cisplatin (from 0 μmol / L (control) to 100 μmol / L) for 24 hours. Optimal damaging concentration: 100 μmol / L. (n=3). (3B) Cell viability (%) of HK-2 cells after treatment with 100 μmol / L cisplatin for 24 hours and then with different concentrations of honeysuckle-derived exosome-like nanovesicles (from 0 μmol / L (control) to 200 μg / mL) for 24 hours. Optimal drug concentration: 100 μg / mL. (n=3). (3C) Cell viability (%) of HK-2 cells as assessed by flow cytometry. C: control group; I: cisplatin group; M: cisplatin and honeysuckle-derived exosome-like nanovesicle group. (3D) Quantification of HK-2 cell viability (%) as assessed by flow cytometry. C: Control group; I: Cisplatin group; M: Cisplatin and honeysuckle-derived exosome-like nanovesicles group. (n = 3) (3E) ROS production in HK-2 cells as assessed by flow cytometry. C: Control group; I: Cisplatin group; M: Cisplatin and honeysuckle-derived exosome-like nanovesicles group. (3F) ROS production in HK-2 cells as assessed by laser confocal microscopy. Scale bar, 10 μm. C: Control group; I: Cisplatin group; M: Cisplatin and honeysuckle-derived exosome-like nanovesicles group. (3G) Cellular uptake of honeysuckle-derived exosome-like nanovesicles after cisplatin-induced injury as assessed by laser confocal microscopy over 24 hours. Scale bar, 100 μm. All data are expressed as means ± SEM (n = 3). ***P<0.001, ****P<0.0001 compared with the control group; #P<0.05, ###P<0.001, ####P<0.0001 compared with the cisplatin group.

[0060] Figures 4A-4B Honeysuckle-derived exosome-like nanovesicles maintain mitochondrial morphology and function in cisplatin-treated cells. (4A) Transmission electron microscopy imaging of mitochondrial morphology in HK-2 cells. (N: nucleus; yellow arrow: loss of mitochondrial cristae; green arrow: autophagosome; blue arrow: traversal of the endoplasmic reticulum; red arrow: normal morphology; purple arrow: numerous vesicles secreted from the cell membrane). Scale bars, 1 and 5 μm. C: control group; I: cisplatin group; M: cisplatin and honeysuckle-derived exosome-like nanovesicle group. (4B) Changes in mitochondrial membrane potential in HK-2 cells observed by laser confocal microscopy. Scale bar, 100 μm. C: control group; I: cisplatin group; M: cisplatin and honeysuckle-derived exosome-like nanovesicle group.

[0061] Figures 5A-5G Honeysuckle-derived exosome-like nanovesicles help maintain renal function in mice with cisplatin-induced AKI. (5A) Imaging of honeysuckle-derived exosome-like nanovesicle uptake in the kidneys of cisplatin-injured mice over 24 hours. (n = 3) (5B) Quantitative analysis of the fluorescence intensity of honeysuckle-derived exosome-like nanovesicles over time in the kidneys of cisplatin-injured mice, demonstrating that honeysuckle-derived exosome-like nanovesicles reach maximum enrichment 8-10 hours after injection. (5C) Flowchart of the animal experiment. (5D) Changes in blood urea nitrogen after administration of honeysuckle-derived exosome-like nanovesicles. (n = 5) (E) Changes in serum creatinine after administration of honeysuckle-derived exosome-like nanovesicles. (n = 5) (F) Representative images of the renal cortex and medulla stained with H&E. Optimal administration dose: EVs 30. Scale bar, x400. (G) Quantitative analysis of tubules. Damage is indicated by H&E staining. (n = 6) (H) Changes in tissue KIM-1 expression after treatment with honeysuckle-derived exosome-like nanovesicles (n = 3). All data are expressed as means ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 compared with the control group; ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared with the cisplatin group.

[0062] Figures 6A-6HHoneysuckle-derived exosome-like nanovesicles as a protective agent for mitochondrial function in cisplatin-induced acute kidney injury. (6A) Changes in tissue IL-1β levels after administration of honeysuckle-derived exosome-like nanovesicles. (n=3) (6B) Changes in tissue IL-6 levels after administration of honeysuckle-derived exosome-like nanovesicles. (n=3) (6C) Changes in tissue IL-10 levels after administration of honeysuckle-derived exosome-like nanovesicles. (n=3) (6D) Changes in tissue TNF-α levels after administration of honeysuckle-derived exosome-like nanovesicles. (n=3) (6E) Western blot analysis of changes in cytochrome c expression in cisplatin-induced AKI mice treated with honeysuckle-derived exosome-like nanovesicles. (6F) Cisplatin-induced cellular senescence in honeysuckle-derived exosome-like nanovesicle-induced AKI mice verified by β-galactosidase staining. Scale bars, 20 & 50 μm. (6G) Apoptosis in cisplatin-induced AKI in mice treated with honeysuckle-derived exosome-like nanovesicles, validated by TUNEL staining. Scale bar, 20 μm. (6H) Changes in CD3 and CD68 expression in cisplatin-induced AKI in mice treated with honeysuckle-derived exosome-like nanovesicles. Scale bar, 20 μm. All data are expressed as mean ± standard error. **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the control group; ##P < 0.01, ###P < 0.001 compared with the control group. DETAILED DESCRIPTION

[0063] Example 1: A method for preparing honeysuckle-derived exosome-like nanovesicles for alleviating acute kidney injury

[0064] This embodiment provides a honeysuckle-derived exosome-like nanovesicle for alleviating acute kidney injury, wherein the main component is honeysuckle-derived exosome-like nanovesicle (honeysuckle-derived exosome-like nanovesicle). The preparation method of the composition comprises the following steps:

[0065] (1) Raw material processing: First, 100 g of dried honeysuckle buds were washed three times with room temperature water for 5 minutes each time. Second, the honeysuckle juice was extracted by stirring at 10,000 rpm in a high-speed blender for 5 minutes. Then, the juice was filtered through a 100-mesh sieve to remove large particles of impurities.

[0066] (2) Differential centrifugation: The filtrate obtained in step (1) was centrifuged three times. First, centrifuge at 1,000 × g for 10 minutes at 4°C; second, centrifuge at 3,000 × g for 30 minutes at 4°C; and third, centrifuge at 10,000 × g for 60 minutes at 4°C. Finally, collect the supernatant.

[0067] (3) Ultracentrifugation: Centrifuge the supernatant obtained in step (2) at 150,000 × g for 2 h at 4°C. Collect the precipitate and resuspend it in 10 mL of sterile PBS.

[0068] (4) Sucrose density gradient centrifugation: First, prepare an 8% / 30% / 45% / 60% sucrose gradient. Next, carefully load the honeysuckle-derived exosome-like nanovesicles resuspended in step (3) onto the top of the gradient. Then, centrifuge at 150,000 × g for 2 hours at 4°C. Finally, collect the honeysuckle-derived exosome-like nanovesicle layer at the 30-45% interface.

[0069] (5) Purification and characterization: Sucrose was removed using ultrafiltration centrifuge tubes (100 kDa molecular weight cutoff) to obtain purified honeysuckle-derived exosome-like nanovesicles.

[0070] Furthermore, the protein concentration of the honeysuckle-derived exosome-like nanovesicles was measured using a BCA protein quantification kit, which showed a protein concentration of 3 mg / mL. Preferably, the honeysuckle-derived exosome-like nanovesicles were fixed with 2.5% glutaraldehyde for 30 minutes, negatively stained with 1.9% methylcellulose and 0.3% uranyl acetate for 5 minutes, and the morphology of the honeysuckle-derived exosome-like nanovesicles was observed using transmission electron microscopy. Finally, the particle size distribution of the honeysuckle-derived exosome-like nanovesicles was analyzed using dynamic light scattering at 25°C and a scattering angle of 173°.

[0071] The honeysuckle-derived exosome-like nanovesicles prepared by the above method had a particle size distribution of 68.06-164.20 nm, an average particle size of 100 nm, and a zeta potential of -25 mV. This particle size distribution and potential characteristics give the honeysuckle-derived exosome-like nanovesicles excellent stability and biocompatibility, facilitating their long-term circulation and targeted enrichment in the body.

[0072] Example 2: A method for preparing honeysuckle-derived exosome-like nanovesicles for alleviating acute kidney injury

[0073] In this example, we adjusted some parameters to optimize the preparation of honeysuckle-derived exosome-like nanovesicles. The specific steps are as follows:

[0074] (1) Raw material processing: Wash 150 g of dried honeysuckle buds with room temperature water three times for 6 minutes each time. Use a high-speed blender at 12,000 rpm for 6 minutes to extract the honeysuckle juice. Filter through an 80-mesh sieve to remove large particles.

[0075] (2) Differential centrifugation: The filtrate was centrifuged three times. First, at 1,200 × g for 12 minutes at 4°C; second, at 3,500 × g for 35 minutes at 4°C; and third, at 12,000 × g for 70 minutes at 4°C. The supernatant was collected.

[0076] (3) Ultracentrifugation: Centrifuge the supernatant at 160,000 × g for 2.5 hours at 4°C. Collect the precipitate and resuspend it in 12 mL of sterile PBS.

[0077] (4) Sucrose density gradient centrifugation: Prepare a 10% / 35% / 50% / 65% sucrose gradient. Load the resuspended honeysuckle-derived exosome-like nanovesicles onto the top of the gradient and centrifuge at 160,000 × g for 2.5 hours at 4°C. Collect the honeysuckle-derived exosome-like nanovesicle layer at the 35-50% interface.

[0078] (5) Purification and characterization: Sucrose was removed using an ultrafiltration centrifuge tube (150 kDa molecular weight cutoff) to obtain purified honeysuckle-derived exosome-like nanovesicles.

[0079] BCA protein quantification revealed a protein concentration of 4.5 mg / mL for the honeysuckle-derived exosome-like nanovesicles prepared in this example. Dynamic light scattering analysis revealed a particle size distribution of 75.20-158.50 nm, with an average particle size of 110 nm and a zeta potential of -28 mV.

[0080] Example 3: A method for preparing honeysuckle-derived exosome-like nanovesicles for alleviating acute kidney injury

[0081] This example explores milder preparation conditions to maintain the biological activity of honeysuckle-derived exosome-like nanovesicles. The specific steps are as follows:

[0082] (1) Raw material processing: 80 g of dried honeysuckle buds were washed three times with room temperature water for 4 minutes each time. The honeysuckle juice was extracted using a high-speed blender at 8000 rpm for 4 minutes. The juice was filtered through a 120-mesh sieve to remove large particles.

[0083] (2) Differential centrifugation: The filtrate was centrifuged three times. First, at 800 × g for 8 minutes at 4°C; second, at 2,500 × g for 25 minutes at 4°C; and third, at 8,000 × g for 50 minutes at 4°C. The supernatant was collected.

[0084] (3) Ultracentrifugation: Centrifuge the supernatant at 140,000 × g for 1.5 hours at 4°C. Collect the precipitate and resuspend it in 8 mL of sterile PBS.

[0085] (4) Sucrose density gradient centrifugation: Prepare a 6% / 25% / 40% / 55% sucrose gradient. Load the resuspended honeysuckle-derived exosome-like nanovesicles onto the top of the gradient and centrifuge at 140,000 × g for 1.5 hours at 4°C. Collect the honeysuckle-derived exosome-like nanovesicle layer at the 25-40% interface.

[0086] (5) Purification and characterization: Sucrose was removed using ultrafiltration centrifuge tubes (80 kDa molecular weight cutoff) to obtain purified honeysuckle-derived exosome-like nanovesicles.

[0087] The protein concentration of the honeysuckle-derived exosome-like nanovesicles prepared in this example was 2.5 mg / mL. Dynamic light scattering analysis showed that the honeysuckle-derived exosome-like nanovesicles had a particle size distribution of 60.50-170.80 nm, an average particle size of 95 nm, and a zeta potential of -22 mV.

[0088] Preferably, in the examples of the present invention, by adjusting the preparation parameters, we obtained honeysuckle-derived exosome-like nanovesicles with different properties. These honeysuckle-derived exosome-like nanovesicles exhibited varying degrees of anti-inflammatory and antioxidant capabilities in in vitro experiments. For example, after treating cisplatin-damaged HK-2 cells at a concentration of 100 μg / mL for 24 hours, the honeysuckle-derived exosome-like nanovesicles prepared in Example 2 reduced intracellular ROS levels by approximately 65% and the expression of the inflammatory factor IL-6 by approximately 55%. This significant anti-inflammatory and antioxidant effect may be related to the high protein concentration and moderate particle size of the honeysuckle-derived exosome-like nanovesicles, making them more easily taken up by cells and exerting their effects.

[0089] Although the honeysuckle-derived exosome-like nanovesicles prepared in Example 3 have a lower protein concentration, they likely retain more bioactive components due to the milder preparation conditions. When treated with cisplatin at the same concentration in HK-2 cells, these honeysuckle-derived exosome-like nanovesicles reduced the apoptotic rate by approximately 45% and increased mitochondrial membrane potential (JC-1 red-green fluorescence ratio) by approximately 1.8-fold. This demonstrates that even at lower concentrations, the mildly prepared honeysuckle-derived exosome-like nanovesicles exhibit significant cytoprotective effects, particularly in maintaining mitochondrial function.

[0090] Through these examples, we not only demonstrate the tunability of the preparation method for honeysuckle-derived exosome-like nanovesicles, but also preliminarily reveal the mechanisms by which honeysuckle-derived exosome-like nanovesicles with different characteristics may play a role in alleviating acute kidney injury. This provides important evidence for further optimizing the preparation process of honeysuckle-derived exosome-like nanovesicles and developing more targeted treatment strategies for different types of renal injury.

[0091] Next, the pharmacological effects of this traditional Chinese medicine composition (exosome-like nanovesicles derived from honeysuckle) can be demonstrated through the following aspects:

[0092] 1. Cell experiments:

[0093] Using HK-2 cells (a human renal tubular epithelial cell line) as an in vitro model, the protective effects of honeysuckle-derived exosome-like nanovesicles were demonstrated by the following experiments:

[0094] a) Cell viability assay:

[0095] Cell viability was assessed using the CCK-8 assay. The results showed that treatment with 100 μg / mL of honeysuckle-derived exosome-like nanovesicles significantly increased the survival rate of cells damaged by cisplatin (100 μmol / L). This suggests that honeysuckle-derived exosome-like nanovesicles have a protective effect on renal tubular epithelial cells.

[0096] b) Apoptosis detection:

[0097] Cell apoptosis was detected by flow cytometry using Annexin V / PI double staining. The results showed that treatment with honeysuckle-derived exosome-like nanovesicles significantly reduced the rate of cell apoptosis induced by cisplatin, demonstrating its anti-apoptotic effect.

[0098] c) ROS level detection:

[0099] Intracellular ROS levels were detected by flow cytometry and laser confocal microscopy using a DCFH-DA fluorescent probe. The results showed that honeysuckle-derived exosome-like nanovesicles significantly reduced cisplatin-induced ROS levels, indicating their antioxidant effect.

[0100] d) Assessment of mitochondrial function:

[0101] Mitochondrial membrane potential was assessed by JC-1 staining. The results showed that treatment with honeysuckle-derived exosome-like nanovesicles restored the mitochondrial membrane potential of cisplatin-injured cells, indicating that it has a protective effect on mitochondrial function.

[0102] 2. Animal Experiments

[0103] A cisplatin-induced acute kidney injury model was established in C57BL / 6 mice, and the therapeutic effects of honeysuckle-derived exosome-like nanovesicles were evaluated using the following indicators:

[0104] a) Renal function indicators:

[0105] Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured. The results showed that treatment with 30 mg / kg of honeysuckle-derived exosome-like nanovesicles significantly reduced the increase in Scr and BUN levels induced by cisplatin, indicating that honeysuckle-derived exosome-like nanovesicles can improve renal function.

[0106] b) Histopathological analysis:

[0107] HE staining was used to evaluate the pathological changes in renal tissue. The results showed that the renal tubular injury score in the honeysuckle-derived exosome-like nanovesicle treatment group was significantly lower than that in the cisplatin group, demonstrating that honeysuckle-derived exosome-like nanovesicles can alleviate the pathological damage of renal tissue.

[0108] c) Kidney injury markers:

[0109] The expression level of KIM-1 in renal tissue was detected using ELISA. The results showed that treatment with honeysuckle-derived exosome-like nanovesicles significantly reduced the expression of KIM-1, further confirming its renal protective effect.

[0110] d) Inflammatory factor detection:

[0111] ELISA was used to measure the levels of IL-1β, IL-6, TNF-α, and IL-10 in renal tissue. The results showed that honeysuckle-derived exosome-like nanovesicles could reduce the expression of pro-inflammatory factors (IL-1β, IL-6, TNF-α) and increase the expression of the anti-inflammatory factor IL-10, demonstrating its anti-inflammatory effect.

[0112] e) Assessment of cellular senescence:

[0113] Cell senescence was assessed by β-galactosidase staining. The results showed that treatment with honeysuckle-derived exosome-like nanovesicles reduced the proportion of SA-β-gal-positive cells, indicating that it can alleviate cell senescence.

[0114] f) Apoptosis detection:

[0115] TUNEL staining was used to assess cell apoptosis in tissues. The results showed that treatment with honeysuckle-derived exosome-like nanovesicles significantly reduced the number of TUNEL-positive cells, further confirming their anti-apoptotic effect.

[0116] g) Immune cell infiltration:

[0117] Immunofluorescence staining was used to detect the expression of CD3 (T cell marker) and CD68 (macrophage marker). The results showed that honeysuckle-derived exosome-like nanovesicles could reduce the infiltration of T cells and macrophages in renal tissue, indicating that they can regulate immune responses.

[0118] In summary, cell and animal studies have demonstrated that this traditional Chinese medicine composition (exosome-like nanovesicles derived from honeysuckle) exhibits multiple pharmacological effects, including antioxidant, anti-inflammatory, anti-apoptotic, mitochondrial protection, reduced cellular senescence, and immune response regulation. These multiple effects collectively alleviate cisplatin-induced acute kidney injury, providing experimental evidence for its potential clinical application.

[0119] Next, we will conduct an in-depth analysis of the specific experimental data to further elucidate the role of honeysuckle-derived exosome-like nanovesicles in alleviating cisplatin-induced acute kidney injury.

[0120] 1. Characterization of Honeysuckle-derived Exosome-like Nanovesicles:

[0121] Experimental results showed that the particle size of the extracted honeysuckle-derived exosome-like nanovesicles ranged from 68.06 to 164.20 nm, with an average size of approximately 100 nm. Transmission electron microscopy revealed that the honeysuckle-derived exosome-like nanovesicles exhibited round or cup-shaped structures. This nanoscale size distribution facilitates the long-term circulation and targeted enrichment of honeysuckle-derived exosome-like nanovesicles in the body, providing a basis for their pharmacological effects.

[0122] 2. Cell experiment data analysis:

[0123] a) Cell viability:

[0124] After 24 hours of cisplatin treatment (100 μmol / L), the viability of HK-2 cells dropped to approximately 50%. However, treatment with 100 μg / mL of honeysuckle-derived exosome-like nanovesicles increased the cell viability to approximately 80%, demonstrating a significant cytoprotective effect.

[0125] b) Apoptosis:

[0126] Flow cytometry results showed that the proportion of late apoptotic cells (AnnexinV+ / PI+) in the cisplatin-treated group was approximately 30%, while treatment with honeysuckle-derived exosome-like nanovesicles reduced this proportion to approximately 15%, reducing cell apoptosis by 50%.

[0127] c) ROS levels:

[0128] DCFH-DA fluorescence intensity analysis showed that cisplatin treatment increased the intracellular ROS level by about 3 times, while treatment with honeysuckle-derived exosome-like nanovesicles reduced the ROS level to about 1.5 times that of the control group, demonstrating a strong antioxidant capacity.

[0129] d) Mitochondrial function:

[0130] JC-1 staining results showed that cisplatin treatment caused the red / green fluorescence ratio to decrease by about 60%, while treatment with honeysuckle-derived exosome-like nanovesicles could restore this ratio to about 80% of the control group, indicating that it has a significant protective effect on mitochondrial function.

[0131] 3. Animal Experiment Data Analysis:

[0132] a) Renal function indicators:

[0133] Cisplatin treatment increased serum creatinine (Scr) and blood urea nitrogen (BUN) levels in mice by approximately 4-fold and 3-fold, respectively. After 5 days of treatment with 30 mg / kg of honeysuckle-derived exosome-like nanovesicles, Scr and BUN levels decreased by approximately 50% and 40%, respectively, approaching normal levels.

[0134] b) Histopathology:

[0135] The renal tubular injury score showed that the score in the cisplatin group was 3.5±0.5, while that in the honeysuckle-derived exosome-like nanovesicle treatment group dropped to 1.8±0.3, and the degree of pathological damage was significantly alleviated.

[0136] c) Kidney injury markers:

[0137] Cisplatin treatment increased the KIM-1 expression level by about 5 times, while treatment with honeysuckle-derived exosome-like nanovesicles reduced it to about 2 times that of the control group, further confirming the renal protective effect of honeysuckle-derived exosome-like nanovesicles.

[0138] d) Inflammatory factors:

[0139] ELISA results showed that honeysuckle-derived exosome-like nanovesicles treatment could reduce the levels of IL-1β, IL-6 and TNF-α by approximately 40%, 50% and 45%, respectively, while increasing the level of the anti-inflammatory factor IL-10 by approximately 80%, demonstrating a powerful anti-inflammatory effect.

[0140] e) Cellular Senescence:

[0141] β-galactosidase staining showed that cisplatin treatment increased the proportion of SA-β-gal-positive cells to approximately 40%, while treatment with honeysuckle-derived exosome-like nanovesicles reduced this proportion to approximately 15%, significantly alleviating cell senescence.

[0142] f) Apoptosis:

[0143] TUNEL staining results showed that the proportion of TUNEL-positive cells in the cisplatin-treated group was approximately 25%, while treatment with honeysuckle-derived exosome-like nanovesicles reduced this proportion to approximately 8%, again confirming its anti-apoptotic effect.

[0144] g) Immune cell infiltration:

[0145] Immunofluorescence staining showed that treatment with honeysuckle-derived exosome-like nanovesicles could reduce the number of CD3- and CD68-positive cells by approximately 60% and 55%, respectively, indicating that it can effectively inhibit the infiltration of T cells and macrophages and regulate the immune response.

[0146] These experimental data comprehensively demonstrate the protective effects of honeysuckle-derived exosome-like nanovesicles against cisplatin-induced acute kidney injury at multiple levels. Honeysuckle-derived exosome-like nanovesicles not only directly protect renal tubular epithelial cells from the toxic effects of cisplatin but also alleviate renal injury by regulating oxidative stress, inflammatory responses, and immune responses.

[0147] Of particular note, the protective effect of honeysuckle-derived exosome-like nanovesicles on mitochondrial function may be one of the key mechanisms of their pharmacological effects. Mitochondrial dysfunction is a key feature of cisplatin nephrotoxicity, and honeysuckle-derived exosome-like nanovesicles can significantly improve mitochondrial membrane potential, which may be related to the antioxidant components contained in them (such as chlorogenic acid and isochlorogenic acid).

[0148] Furthermore, honeysuckle-derived exosome-like nanovesicles significantly regulate the expression of inflammatory factors. They not only reduce the levels of pro-inflammatory factors but also increase the expression of the anti-inflammatory factor IL-10. This bidirectional regulation may be one of the reasons why their anti-inflammatory effects are superior to those of traditional anti-inflammatory drugs.

[0149] Finally, the inhibitory effect of honeysuckle-derived exosome-like nanovesicles on immune cell infiltration suggests that it may alleviate renal injury by regulating the immune microenvironment. This finding provides new ideas for the potential application of honeysuckle-derived exosome-like nanovesicles in other inflammation-related kidney diseases.

[0150] In general, these experimental data not only confirm the effectiveness of honeysuckle-derived exosome-like nanovesicles in alleviating cisplatin-induced acute kidney injury, but also reveal its multi-target and multi-mechanism characteristics, providing a solid experimental basis for further development and optimization of this new therapeutic strategy.

[0151] The extraction and characterization of the honeysuckle-derived exosome-like nanovesicles of the present invention are as follows.

[0152] 1. Exosome-like nanovesicles derived from honeysuckle were prepared by a series of differential centrifugation, ultracentrifugation and sucrose gradient density centrifugation ( Figure 2A The particle size distribution of honeysuckle-derived exosome-like nanovesicles was measured using dynamic light scattering (DLS), and the results showed that the particle size was mainly concentrated between 68.06 and 164.20 nm ( Figure 2BThe isolated and purified honeysuckle-derived exosome-like nanovesicles showed round or cup-shaped ultrastructural morphology under transmission electron microscopy (TEM), with disc-shaped or concave structural features. Given their structural similarities with exosomes secreted by mammalian cells, we speculate that honeysuckle-derived exosome-like nanovesicles may have similar tissue affinity as mammalian exosomes ( Figure 2C Since cisplatin-induced AKI mainly occurs in the epithelial cells of the renal proximal tubules, we selected HK-2 cells as the cell line for in vitro experiments. To verify the biosafety of honeysuckle-derived exosome-like nanovesicles and confirm their natural safety, we observed that the survival rate of HK-2 cells exceeded 85% within the concentration range of 0-200 μg / mL ( Figure 2D Therefore, the honeysuckle-derived exosome-like nanovesicles used in this concentration range were used in subsequent cell experiments.

[0153] 2. Honeysuckle-derived exosome-like nanovesicles can reduce oxidative damage and apoptosis in HK-2 cells after cisplatin treatment.

[0154] To determine whether honeysuckle-derived exosome-like nanovesicles can alleviate cisplatin-induced apoptosis in HK-2 cells, we first found that when treated with 100 μmol / L cisplatin, HK-2 cells reached the LD50 ( Figure 3A ). Therefore, 100 μmol / L was selected as the optimal damage concentration in in vitro related cell experiments. In order to determine the optimal concentration of honeysuckle-derived exosome-like nanovesicles for treatment, we tested the concentration of honeysuckle-derived exosome-like nanovesicles from 0-200 μg / mL and found that after treatment with 100 μg / mL honeysuckle-derived exosome-like nanovesicles, cell viability was significantly increased compared with cisplatin-treated HK-2 cells. ( Figure 3B ). Therefore, we used 100 μg / mL of honeysuckle-derived exosome-like nanovesicles as the optimal therapeutic concentration for subsequent in vitro experiments. Next, flow cytometry analysis showed that treatment with 100 μg / mL of honeysuckle-derived exosome-like nanovesicles significantly reduced the apoptosis of HK-2 cells damaged by cisplatin, showing a more obvious anti-apoptotic effect than the untreated cisplatin-damaged group. ( Figure 3C Quantitative analysis also showed that after treating cisplatin-damaged cells with 100 μg / mL of honeysuckle-derived exosome-like nanovesicles, cell viability was significantly increased ( Figure 3D ). These results indicate that honeysuckle-derived exosome-like nanovesicles can alleviate cisplatin-induced apoptosis in HK-2 cells. Flow cytometry showed that honeysuckle-derived exosome-like nanovesicle treatment led to a decrease in ROS levels in cisplatin-damaged cells, revealing the significant antioxidant effect of honeysuckle-derived exosome-like nanovesicles. ( Figure 3E ), and this result was confirmed by laser confocal microscopy ( Figure 3F ). In addition, we also explored the targeted uptake of honeysuckle-derived exosome-like nanovesicles by HK-2 cells after cisplatin injury. Time course analysis showed that green fluorescence gradually increased at 0, 2, 4, 6, 10, and 24 hours, especially after the 6th hour, indicating that honeysuckle-derived exosome-like nanovesicles were targeted for uptake in HK-2 cells ( Figure 3G This indicates that honeysuckle-derived exosome-like nanovesicles have a targeted therapeutic effect on cisplatin-induced HK-2 cell injury, providing promising evidence for subsequent in vivo studies.

[0155] 3. Honeysuckle-derived exosome-like nanovesicles maintain cisplatin-treated cells.

[0156] Although we have demonstrated in the previous section that honeysuckle-derived exosome-like nanovesicles can alleviate cisplatin-induced AKI by reducing ROS generation and attenuating g-oxidative damage, we wanted to further investigate whether honeysuckle-derived exosome-like nanovesicles have a potential protective effect against cisplatin-induced mitochondrial morphological changes. To explore this, we used transmission electron microscopy (TEM) to observe the morphology of mitochondria before and after treatment with honeysuckle-derived exosome-like nanovesicles in cisplatin-injured HK-2 cells. TEM analysis showed that the mitochondrial morphology of HK-2 cells treated with 100 μg / mL honeysuckle-derived exosome-like nanovesicles was significantly restored, and the structure was more normalized than that of the untreated group. Autophagic vesicles were significantly reduced, and the number of vesicles secreted by cells increased significantly ( Figure 4A ). In addition, we also used laser confocal microscopy to observe the changes in mitochondrial membrane potential before and after treatment with honeysuckle-derived exosome-like nanovesicles. We found that after treatment with 100 μg / mL honeysuckle-derived exosome-like nanovesicles, green fluorescence was significantly weakened, while red fluorescence was significantly enhanced, and the ratio of red to green fluorescence was significantly increased compared with the control group and cisplatin group ( Figure 4B Overall, honeysuckle-derived exosome-like nanovesicles exhibited significant protective effects against cisplatin-induced mitochondrial morphological changes and histological changes in HK-2 cells.

[0157] 4. HELNV helps maintain renal function in cisplatin-induced AKI mice.

[0158] We then explored the targeted uptake of honeysuckle-derived exosome-like nanovesicles in mouse kidney tissue following cisplatin injury. Six hours after injection, frozen kidney tissue began to show fluorescence, and targeted uptake peaked between 8 and 10 hours within 24 hours ( Figure 5A&B). Therefore, we demonstrated that honeysuckle-derived exosome-like nanovesicles exhibited targeted uptake in cisplatin-induced renal tissue injury in mice, which was consistent with our in vitro experimental results ( Figure 3G ).

[0159] To determine whether the effect of honeysuckle-derived exosome-like nanovesicles in alleviating cisplatin-induced AKI in vivo is the same as in vitro, we designed the following animal experimental protocol ( Figure 5C ). Honeysuckle-derived exosome-like nanovesicles were applied at doses of 30 mg / kg and 5 mg / kg to explore the optimal therapeutic concentration for in vivo experiments. After treatment of cisplatin-induced AKI mice with 30 mg / kg of honeysuckle-derived exosome-like nanovesicles, urea nitrogen and serum creatinine levels were significantly decreased compared with the untreated group ( Figure 5D &E). In addition, histopathological analysis by hematoxylin and eosin (H&E) staining showed that the kidney tissues of mice treated with 30 mg / kg of honeysuckle-derived exosome-like nanovesicles showed more significant improvements in pathological changes than those in the control and cisplatin groups ( Figure 5F In contrast, the renal pathology of mice treated with 5 mg / ml of honeysuckle-derived exosome-like nanovesicles did not improve significantly, confirming that 30 mg / kg is the optimal therapeutic concentration for animal studies. Quantitative analysis of H&E staining further supported this conclusion ( Figure 5G Kidney injury molecule-1 (KIM-1) is a reliable biomarker for renal injury and recovery, and its expression is significantly enhanced in the proximal tubules. After renal injury, KIM-1 levels were significantly reduced in the bladder epithelial cells of mice, and KIM-1 levels were significantly reduced in mice treated with 30 mg / kg of honeysuckle-derived exosome-like nanovesicles compared to the untreated group ( Figure 5H In conclusion, honeysuckle-derived exosome-like nanovesicles can effectively alleviate cisplatin-induced AKI in mice, with 30 mg / kg being the optimal concentration.

[0160] 5. Honeysuckle-derived exosome-like nanovesicles can reduce cisplatin-induced inflammation and immune response in AKI mice.

[0161] IL-10 is considered an anti-inflammatory and immunosuppressive factor that plays a role in regulating cell growth, differentiation, and inflammatory and immune responses. Cisplatin-induced AKI expression was significantly reduced in the proximal tubular epithelial cells of the injured kidney, making it a good marker for evaluating the relief of AKI. To investigate the anti-inflammatory activity of honeysuckle-derived exosome-like nanovesicles, we examined the expression levels of IL-1β, IL-6, IL-10, and TNF-α in cisplatin-injured renal tissues that were untreated and treated with honeysuckle-derived exosome-like nanovesicles. Compared with the control and cisplatin groups, treatment with 30 mg / kg of honeysuckle-derived exosome-like nanovesicles significantly reduced the levels of these inflammatory markers ( Figure 6A -D), indicating that honeysuckle-derived exosome-like nanovesicles alleviated the inflammatory response in cisplatin-induced AKI. In cisplatin-induced mitochondrial dysfunction, excessive ROS generation triggers apoptosis through the cytochrome oxidase pathway, in which the release of cytochrome c activates the apoptotic pathway. Western blot analysis showed that cytochrome c expression was significantly reduced after treatment with 30 mg / kg honeysuckle-derived exosome-like nanovesicles compared with the control group. And the cisplatin group ( Figure 6E ). This suggests that honeysuckle-derived exosome-like nanovesicles inhibit apoptosis by reducing the release of cytochrome c, thereby alleviating AKI. Cellular senescence also plays an important role in the development of AKI. The product associated with β-galactosidase (SA-β-gal) activity, which appears dark blue when catalyzed by β-galactosidase, is widely used to detect senescent cells, and SA-β-gal levels are significantly reduced after cisplatin-induced AKI. We found that in mice treated with 30 mg / kg honeysuckle-derived exosome-like nanovesicles, the dark blue product, a marker of SA-β-gal activity, was significantly reduced ( Figure 6F ). This indicates that honeysuckle-derived exosome-like nanovesicles reduce cell senescence in cisplatin-induced AKI. To further confirm the anti-apoptotic effect of honeysuckle-derived exosome-like nanovesicles, we performed TUNEL staining. Compared with the control group and the cisplatin group, the green fluorescence indicating apoptotic cells in the honeysuckle-derived exosome-like nanovesicle-treated group was significantly reduced ( Figure 6G), which confirmed that honeysuckle-derived exosome-like nanovesicles inhibited apoptosis in cisplatin-induced AKI. CD3 is a biomarker of mature T lymphocytes, indicating the state of cellular immunity and involved in regulating various inflammatory and immune responses; CD68 is a biomarker of macrophages, which play an important role in macrophage phagocytosis, intracellular lysosomal metabolism, and extracellular interactions with pathogens. Therefore, we measured the expression levels of CD3 and CD68 to illustrate the infiltration status of T lymphocytes and macrophages in tissues. We found that after treatment with 30 mg / kg of honeysuckle-derived exosome-like nanovesicles, the expression of CD3 and CD68 was significantly reduced compared with the control group and cisplatin group ( Figure 6H ), which also showed that the infiltration of T lymphocytes and macrophages in tissues was significantly reduced. Therefore, honeysuckle-derived exosome-like nanovesicles can inhibit the infiltration of T lymphocytes and macrophages in tissues by reducing the expression of CD3 and CD68, thereby alleviating cisplatin-induced AKI in mice and reducing inflammation and immune responses.

[0162] In conclusion, our study demonstrated that honeysuckle-derived exosome-like nanovesicles could alleviate cisplatin-induced AKI by alleviating inflammatory response, inhibiting cell apoptosis, reducing oxidative damage, protecting mitochondrial function, and improving cell senescence.

[0163] The foregoing is merely an embodiment of the present invention and is not intended to limit the present invention. It will be apparent to those skilled in the art that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are intended to be included within the scope of the claims of the present invention.

Claims

1. A honeysuckle-derived exosome-like nanovesicle for alleviating acute kidney injury, characterized in that: The particle size distribution of the honeysuckle-derived exosome-like nanovesicles is 68.06-164.20 nm; The method for preparing the honeysuckle-derived exosome-like nanovesicles is characterized by comprising the following steps: (1) Raw material processing: Wash the dried honeysuckle buds with water, extract the honeysuckle juice using high-speed stirring, and then filter to remove impurities; (2) Differential centrifugation: The filtrate obtained in step (1) is subjected to multiple steps of differential centrifugation, and the supernatant is collected; (3) Ultracentrifugation: The supernatant obtained in step (2) was subjected to ultracentrifugation, and the precipitate was collected and resuspended in buffer; (4) Density gradient centrifugation: Prepare a sucrose density gradient, load the resuspended sample in step (3) into the gradient and centrifuge to collect the honeysuckle-derived exosome-like nanovesicle layer in a specific density range; (5) Purification: Use ultrafiltration centrifuge tubes to remove sucrose and obtain purified honeysuckle-derived exosome-like nanovesicles.

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