Traditional Chinese medicine composition for relieving acute kidney injury and preparation method thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine compositions, in particular to a traditional Chinese medicine composition for relieving acute kidney injury and a preparation method thereof.The traditional Chinese medicine composition comprises honeysuckle-derived exosome-like nano-vesicles (HELNVs), and the particle size distribution of the HELNVs is 68.06-164.20 nm. Firstly, the HELNVs retain various bioactive components of honeysuckle, and the HELNVs retain various bioactive components of the honeysuckle; the components can play a stronger pharmacological role through a synergistic effect; secondly, due to the nanoscale particle size (68.06-164.20 nm), the HELNVs can better penetrate through a biological barrier, and the targeting property and the bioavailability are improved; thirdly, the natural source and exosome-like structure of the HELNVs may endow the HELNVs with unique cell affinity, which is beneficial to the interaction of the HELNVs with target cells; finally, the HELNVs are used as a natural nano-carrier, so that the HELNVs have better safety.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine compositions, and particularly relates to a traditional Chinese medicine composition for relieving 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 within a short period of time caused by various etiologies. Its main clinical manifestations include a decrease in glomerular filtration rate, resulting in the 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 chemotherapeutic drug widely used as a first-line treatment for various solid tumors (4). Despite its significant antitumor efficacy, its severe side effects, including nephrotoxicity, ototoxicity, hepatotoxicity, and cardiotoxicity, have also attracted much attention (5). Of particular concern is cisplatin-induced AKI, with approximately 30% of patients receiving high-dose cisplatin treatment experiencing renal impairment, and this proportion exceeding 70% in pediatric patients (6). In recent years, many studies have been dedicated to exploring the underlying mechanisms of cisplatin-induced AKI (7-11), laying the foundation for the discovery of effective preventive and therapeutic strategies. Although the pathophysiological mechanisms of cisplatin-induced AKI are multifaceted and not fully elucidated, studies have shown that key processes such as inflammatory responses, oxidative stress, mitochondrial dysfunction, and apoptosis of renal tubular epithelial cells are involved (12). Therefore, we aim to relieve cisplatin-induced AKI through a comprehensive strategy of inhibiting inflammatory responses, improving oxidative damage and mitochondrial dysfunction, and reducing apoptosis of renal tubular epithelial cells.

[0003] Flos Lonicerae, as a traditional Chinese herb, has been widely used in daily health care and the treatment of various diseases. Its complex chemical constituents include key bioactive compounds such as chlorogenic acid, isochlorogenic acid, lignans, and saponins (13). Previous studies have shown that Flos Lonicerae 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 Flos Lonicerae can alleviate mitochondrial damage and cellular senescence induced by reactive oxygen species (ROS) (17), and can restore the mitochondrial morphology damaged by oxidative damage (18).

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

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

[0006] Based on the main mechanisms of cisplatin-induced AKI, combined with the multifaceted pharmacological properties of honeysuckle and the natural advantages of EVs, we hypothesize that HELNVs may have potential therapeutic effects on cisplatin-induced AKI. This study aimed to investigate whether HELNVs could alleviate cisplatin-induced AKI by inhibiting inflammatory responses, improving oxidative damage and mitochondrial dysfunction, and reducing apoptosis of renal tubular epithelial cells ( Figure 1 )). The references are as follows:

[0007] 1. Kellum JA, Romagnani P, Ashuntantang G, Ronco C, Zarbock A, Anders HJ. Acute kidney injury. Nat Rev Dis Primers. 2021;7(1).

[0008] 2. Ronco C, Bellomo R, Kellum JA. Acute kidney injury. Lancet. 2019;394(10212):1949 - 64.

[0009] 3. Guo RZ, Duan JY, Pan SK, Cheng F, Qiao YJ, Feng Q, et al. The road from AKI to CKD: molecular mechanisms and therapeutic targets of ferroptosis. J Cell Death. 2023;14(7).

[0010] 4. Mu Q, Lv Y, Luo C, Liu X, Huang C, Xiu Y, et al. Research progress on the functions and mechanisms of circular RNAs in tumor cisplatin resistance. Front Pharmacol. 2021; 12.

[0011] 5. Zhang J, Wang JQ, Xu XY, Yang JY, Wang Z, Jiang S, et al. Red ginseng protects cells against cisplatin-induced intestinal toxicity via inhibition of apoptosis and autophagy through the PI3K / AKT and MAPK signaling pathways. Food Funct. 2020; 11(5): 4236 - 48.

[0012] 6. Motwani SS, Curhan GC. Cisplatin-related nephrotoxic effects on the uterine artery and vein. JAMA Netw Open. 2020; 3(5): e203612.

[0013] 7. Peng L, Liu D, Liu HY, Xia M, Wan L, Li M, et al. Bomesin receptor activating protein exacerbates cisplatin-induced AKI by regulating SIRT2 degradation. Nephrol Dial Transplant. 2022; 37(12): 2366 - 85.

[0014] 8. Sears SM, Siskind LJ. Potential therapeutic targets for cisplatin-induced kidney injury: lessons from other models of AKI and fibrosis. J Am Soc Nephrol. 2021; 32(7): 1559 - 67.

[0015] 9. Hu Y, Yang C, Amorim T, Makbool M, Lin J, Li C, et al. Upregulation of cisplatin-mediated APE2 binding to MYH9 triggers mitochondrial fragmentation and acute kidney injury. Cancer Res. 2021; 81(3): 713 - 23.

[0016] 10. Han S, Lin F, Ruan Y, Zhao S, Yuan R, Ning J, et al. miR-132-3p promotes cisplatin-induced apoptosis and inflammatory response of renal tubular epithelial cells by targeting SIRT1 through the NF-κB pathway. Int Immunopharmacol. 2021; 99: 108022.

[0017] 11. Qiu ZM, Liu Q, Wang L, Xiong YF, Wu J, Wang MJ, et al. Imbalance of copper transporter SLC31A1 under the transcriptional activation of ELF3 exacerbates cisplatin-induced acute kidney injury by disrupting copper homeostasis through mitochondrial dysfunction. Chem Biol Interact. 2024; 393: 110943.

[0018] 12. Tang CY, Livingston MJ, Safirstein R, Dong Z. Cisplatin nephrotoxicity: new insights and therapeutic implications. Nat Rev Nephrol. 2022;19(1):53-72.

[0019] 13. Zheng SW, Liu ST, Hou YJ, Wang S, Na YX, Hu JH, et al. A systematic review of Lonicera japonica: an important food and traditional Chinese medicine. Front Pharmacol. 2022;13.

[0020] 14. Cao YX, Ji P, Wu FL, Dong JQ, Li CC, Ma T, et al. Lonicera japonica: an overview of research progress on bioactive metabolites and pharmacological effects. Front Pharmacol. 2023;14.

[0021] 15. Li HL, Jin JM, Ge MJ, Jin TY, Zhu SG, Jin JH, et al. Protective effect of Lonicera japonica on PM2.5-induced lung injury in BALB / c mice: via TGF-β and NF-κB pathways. Antioxidants. 2023;12(4):968.

[0022] 16. Lin Z, Li L, Chen L, Jin C, Li Y, Yang L, et al. Luteolin promotes wound healing by enhancing angiogenesis in diabetic rats via SIRT1-mediated autophagy. Acta Chinese Med. 2023;45(4):815-30.

[0023] 17. Mu W, Hu N, Zhang LH, Jiang W, Yan T, Zhang T, et al. Lonicera japonica flower induces radiotherapy-induced mesenteric artery endothelial dysfunction via the GTPCH1 / BH4 / eNOS pathway. Phytomedicine. 2022;102:154146.

[0024] 18. Hu YY, Yin FW, Liu ZY, Xie HK, Xu YS, Zhou DY, et al. Acerola polysaccharide improves high-fat diet-induced non-alcoholic fatty liver in mice by reducing adipogenesis and improving mitochondrial function. Food Funct. 2020;11(1):1037-48.

[0025] 19. Manickam DS. Delivery of mitochondria via extracellular vesicles - new horizons in drug delivery. J Controlled Release. 2022;343:400-7.

[0026] 20. Grange C, Bussolati B, Extracellular vesicles in kidney disease, Nat Rev Nephrol. 2022;18(8):499-513.

[0027] 21. Yang Chen, Xue Ying, Duan Ying, Mao Chao, Wan Ming. Extracellular vesicles and their engineering strategies, delivery systems, and biomedical applications. Journal of Controlled Release 2024; 365: 1089-123.

[0028] 22. Song Mingde, Cui Mei, Fang Zi, Liu Kai. In-depth study of extracellular vesicle-based oral drug delivery systems. Journal of Controlled Release 2022; 351: 560-72.

[0029] 23. Yan L, Cao YQ, Hou LH, Luo TY, Li MQ, Gao SJ, et al. miRNA therapy derived from curcumin exosome-like nanoparticles: A strategy inhibitor for intestinal inflammation. J Adv Res. 2024.

[0030] 24. Wu JQ, Ma XY, Lu Y, Zhang T, Du ZQ, Xu J, et al. Edible agave leaf extracellular vesicles promote M2 macrophage polarization. Molecules. 2022; 27(23): 8184.

[0031] 25. Rao Q, Ma GC, Li M, Wu H, Zhang YX, Zhang CE, et al. Targeted delivery of a tripeptide liposome via dendritic cell-derived extracellular vesicles for the treatment of murine models of colitis and rheumatoid arthritis. British Journal of Pharmacology. 2022; 180(3): 330-46.

[0032] 26. Zhao Xi, Yin Fei, Huang Yuling, Fu LQ, Ma Yueyue, Ye Li, et al. Oral administration of grape-derived nanovesicles protects against LPS / D-GalN-induced acute liver failure. International Journal of Pharmaceutics. 2024; 652: 123812.

[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] Based on the above background, the present invention proposes an innovative treatment strategy: using honeysuckle-derived exosome-like nanovesicles (HELNVs) to alleviate cisplatin-induced AKI. This strategy ingeniously combines the multi-target pharmacodynamic effects of traditional Chinese medicine with the advantages of advanced nanotechnology, and is expected to overcome the limitations of existing treatment methods.

[0035] The object of the present invention is to provide a traditional Chinese medicine composition for alleviating acute kidney injury, the composition comprising honeysuckle-derived exosome-like nanovesicles (HELNVs), and the particle size distribution of the HELNVs is 68.06 - 164.20 nm.

[0036] Specifically, the average particle size of the HELNVs is 100 nm.

[0037] Specifically, the protein concentration of the HELNVs is 2 - 5 mg / mL.

[0038] Specifically, the Zeta potential of the HELNVs is -20 to -30 mV.

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

[0040] A method for preparing the composition includes the following steps: (1) Raw material treatment: Wash the dried honeysuckle flower buds with water, use high-speed stirring to extract the honeysuckle juice, and then filter to remove impurities; (2) Differential centrifugation: Perform multi-step differential centrifugation on the filtrate obtained in step (1), and collect the supernatant; (3) Ultracentrifugation: Perform ultracentrifugation on the supernatant obtained in step (2), collect the precipitate and resuspend it with a buffer; (4) Density gradient centrifugation: Prepare a sucrose density gradient, load the sample resuspended in step (3) into the gradient and centrifuge, and collect the HELNVs layer in a specific density range; (5) Purification: Use an ultrafiltration centrifugal tube to remove sucrose and obtain purified HELNVs.

[0041] Specifically, the method further includes the following steps: Use a BCA protein quantification kit to determine the protein concentration of the HELNVs.

[0042] Specifically, the method further includes the following steps: Fix the HELNVs with 2.5% glutaraldehyde for 30 minutes; use 1.9% methyl cellulose and 0.3% uranyl acetate for negative staining for 5 minutes; use a transmission electron microscope to observe the morphology of the HELNVs.

[0043] Specifically, the method further includes the following steps: Use a dynamic light scattering instrument to analyze the particle size distribution of the HELNVs under the conditions of 25°C and a scattering angle of 173°.

[0044] The composition prepared by the described method.

[0045] The core innovation of the present invention lies in: The innovation of HELNVs is mainly reflected in the following aspects: First, HELNVs retain various bioactive components of honeysuckle, and these components may exert stronger pharmacological effects through synergy. Second, the nanoscale particle size (68.06 - 164.20 nm) enables HELNVs to better penetrate biological barriers, improving targeting and bioavailability. Third, the natural origin and exosome-like structure of HELNVs may endow them with unique cell affinity, facilitating their interaction with target cells. Finally, as a natural nanocarrier, HELNVs may have better safety.

[0046] From the perspective of molecular mechanism, polyphenolic compounds (such as chlorogenic acid) in HELNVs may directly act on free radicals through their hydroxyl groups to exert antioxidant effects. At the same time, these compounds may enhance the intracellular endogenous antioxidant system by regulating the Nrf2 / HO-1 pathway. In terms of anti-inflammation, HELNVs may reduce the expression of pro-inflammatory factors by inhibiting the NF-κB signaling pathway. In addition, certain components in HELNVs may inhibit apoptosis by regulating the Bcl-2 / Bax ratio and caspase activity.

[0047] It is particularly noteworthy that the protective effect of HELNVs on mitochondrial function may be one of the key mechanisms for their pharmacological effects. Mitochondria are the main targets of cisplatin toxicity, and certain components in HELNVs 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 by traditional single-component drugs.

[0048] In summary, the HELNVs treatment strategy proposed in this invention has multiple innovations and significant clinical application potential. It not only provides a new treatment option for cisplatin-induced AKI, but also offers new ideas for the modernization of traditional Chinese medicine and the development of nanomedicine. Future research will further optimize the preparation process of HELNVs, deeply explore their mechanism of action, and evaluate the feasibility of their clinical application, with the expectation of bringing better treatment options for AKI patients. Brief Description of the Drawings

[0049] Figure 1 It is a schematic diagram of the mechanism. HELNVs: Honeysuckle-derived exosome-like nanovesicles.

[0050] Figures 2A - 2DExtraction and characterization of HELNVs. (2A) Flow chart of the isolation and purification of HELNVs. (2B) Particle size distribution of HELNVs obtained by dynamic light scattering analysis. (2C) Morphological and size characteristics of HELNVs 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 HELNVs for 24 h, from 0 (control group) to 200 μg / mL.

[0051] Figures 3A - 3G . HELNVs can 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 h. Optimal damage concentration: 100 μmol / L. (n = 3) (3B) Cell viability (%) of HK-2 cells treated with 100 μmol / L cisplatin for 24 h and then with different concentrations of HELNVs (from 0 μmol / L (control) to 200 μg / mL) for 24 h. Optimal drug concentration: 100 μg / mL. (n = 3) (3C) Cell viability (%) of HK-2 cells detected by flow cytometry. C: control group; I: cisplatin group; M: cisplatin and HELNVs group. (3D) Quantification of HK-2 cell viability (%) detected by flow cytometry. C: control group; I: cisplatin group; M: cisplatin and HELNVs group. (n = 3) (3E) ROS generation in HK-2 cells detected by flow cytometry. C: control group; I: cisplatin group; M: cisplatin and HELNVs group. (3F) ROS generation in HK-2 cells observed by laser confocal microscopy. Scale bar, 10 μm. C: control group; I: cisplatin group; M: cisplatin and HELNVs group. (3G) Cellular uptake of HELNVs after cisplatin-induced damage observed by laser confocal microscopy within 24 h. 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.

[0052] Figures 4A - 4B. HELNVs maintain the mitochondrial morphology and function of cisplatin-treated cells. (4A) Imaging of the mitochondrial morphology of HK-2 cells by transmission electron microscopy. (N: nucleus; yellow arrow: disappearance of mitochondrial cristae structure; green arrow: autophagosome; blue arrow: passing through the endoplasmic reticulum; red arrow: normal morphology; purple arrow: a large number of vesicles secreted by the cell membrane) Scale bar, 1 & 5 μm. C: control group; I: cisplatin group; M: cisplatin and HELNVs group. (4B) Changes in the mitochondrial membrane potential of HK-2 cells observed by laser confocal microscopy. Scale bar, 100 μm. C: control group; I: cisplatin group; M: cisplatin and HELNVs group.

[0053] Figures 5A - 5H . HELNVs contribute to maintaining renal function in cisplatin-induced AKI mice. (5A) Imaging of HELNVs uptake in the kidneys of cisplatin-injured mice within 24 hours. (n = 3) (5B) Quantitative analysis of the fluorescence intensity of HELNVs in the kidneys of cisplatin-injured mice over time, indicating that HELNVs reach maximum enrichment at 8 - 10 hours after injection. (5C) Workflow of the animal experiment. (5D) Changes in blood urea nitrogen after HELNVs administration. (n = 5) (5E) Changes in serum creatinine after HELNVs administration. (n = 5) (5F) Representative images of H&E-stained renal cortex and medulla. Optimal dosage: EVs30. Scale bar, x400. (5G) Quantitative analysis of tubules. Injury was shown according to HE staining. (n = 6) (5H) Changes in tissue KIM-1 after treatment with HELNVs. (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.

[0054] Figures 6A - 6H. HELNVs as mitochondrial function protection in cisplatin-induced acute kidney injury. (6A) Changes in tissue IL-1β levels after HELNVs administration. (n = 3) (6B) Changes in tissue IL-6 after HELNVs administration. (n = 3) (6C) Changes in tissue IL-10 after HELNVs administration. (n = 3) (6D) Changes in tissue TNF-α after HELNVs administration. (n = 3) (6E) Western blot analysis of changes in cytochrome c expression in cisplatin-induced mouse AKI treated with HELNVs. (6F) HELNVs-induced mouse AKI with cisplatin-induced cellular senescence verified by β-galactosidase staining. Scale bar, 20 & 50 μm. (6G) HELNVs drug treatment-induced apoptosis in cisplatin-induced mouse AKI verified by TUNEL staining. Scale bar, 20 μm. (6H) Changes in CD3 and CD68 expression in HELNVs drug treatment of cisplatin-induced mouse AKI. Scale bar, 20 μm. All data are expressed as mean ± SEM. **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 implementation manners

[0055] Example 1: Preparation method of a traditional Chinese medicine composition for relieving acute kidney injury This example provides a traditional Chinese medicine composition for relieving acute kidney injury, and its main component is honeysuckle-derived exosome-like nanovesicles (HELNVs). The preparation method of this composition includes the following steps: (1) Raw material treatment: First, wash 100 g of dry honeysuckle flower buds with room temperature water 3 times, 5 minutes each time. Secondly, use a high-speed blender to stir for 5 minutes at 10,000 rpm to extract honeysuckle juice. Then, filter with a 100-mesh sieve to remove large particle impurities.

[0056] (2) Differential centrifugation: Centrifuge the filtrate obtained in step (1) three times. First, centrifuge at 1,000×g for 10 minutes at 4°C; secondly, centrifuge at 3,000×g for 30 minutes at 4°C; thirdly, centrifuge at 10,000×g for 60 minutes at 4°C. Finally, collect the supernatant.

[0057] (3) Ultracentrifugation: Centrifuge the supernatant obtained in step (2) at 150,000×g for 2 hours at 4°C. Collect the precipitate and resuspend it with 10 mL of sterile PBS.

[0058] (4)Sucrose density gradient centrifugation: First, prepare a sucrose gradient of 8% / 30% / 45% / 60%. Second, carefully load the HELNVs 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 HELNVs layer at the 30 - 45% interface.

[0059] (5)Purification and characterization: Use an ultrafiltration centrifugal tube (molecular weight cut-off of 100 kDa) to remove sucrose and obtain purified HELNVs.

[0060] Furthermore, use a BCA protein quantification kit to determine the protein concentration of HELNVs, and the result shows that the protein concentration is 3 mg / mL. Preferably, fix HELNVs with 2.5% glutaraldehyde for 30 minutes; use 1.9% methylcellulose and 0.3% uranyl acetate for negative staining for 5 minutes; use a transmission electron microscope to observe the morphology of HELNVs. Finally, use a dynamic light scattering instrument to analyze the particle size distribution of HELNVs at 25°C and a scattering angle of 173°.

[0061] The particle size distribution of HELNVs prepared by the above method is 68.06 - 164.20 nm, the average particle size is 100 nm, and the Zeta potential is -25 mV. This particle size distribution and potential characteristics enable HELNVs to have good stability and biocompatibility, which is beneficial for their long-term circulation and targeted enrichment in vivo.

[0062] Example 2: Preparation method of a traditional Chinese medicine composition for relieving acute kidney injury In this example, we adjusted some parameters to optimize the preparation of HELNVs. The specific steps are as follows: (1)Raw material treatment: Wash 150 g of dry honeysuckle flower buds 3 times with room temperature water for 6 minutes each time. Use a high-speed blender to stir for 6 minutes at 12,000 rpm to extract honeysuckle juice. Filter through an 80-mesh sieve to remove large particle impurities.

[0063] (2)Differential centrifugation: Centrifuge the filtrate three times. First, centrifuge at 1,200×g for 12 minutes at 4°C; second, centrifuge at 3,500×g for 35 minutes at 4°C; third, centrifuge at 12,000×g for 70 minutes at 4°C. Collect the supernatant.

[0064] (3)Ultracentrifugation: Centrifuge the supernatant at 160,000×g for 2.5 hours at 4°C. Collect the precipitate and resuspend it with 12 mL of sterile PBS.

[0065] (4) Sucrose density gradient centrifugation: Prepare a 10% / 35% / 50% / 65% sucrose gradient. Load the resuspended HELNVs onto the top of the gradient and centrifuge at 160,000×g for 2.5 hours at 4°C. Collect the HELNVs layer at the 35 - 50% interface.

[0066] (5) Purification and characterization: Use an ultrafiltration centrifugal tube (150 kDa molecular weight cut-off) to remove sucrose and obtain purified HELNVs.

[0067] Quantification by BCA protein assay showed that the protein concentration of the HELNVs prepared in this example was 4.5 mg / mL. Dynamic light scattering analysis showed that the particle size distribution of HELNVs was 75.20 - 158.50 nm, the average particle size was 110 nm, and the Zeta potential was -28 mV.

[0068] Example 3: Preparation method of a traditional Chinese medicine composition for relieving acute kidney injury In this example, milder preparation conditions were explored to maintain the biological activity of HELNVs. The specific steps are as follows: (1) Raw material treatment: Wash 80 g of dry honeysuckle flower buds 3 times with room temperature water for 4 minutes each time. Use a high-speed blender to stir for 4 minutes at 8000 rpm to extract honeysuckle juice. Filter through a 120-mesh sieve to remove large particle impurities.

[0069] (2) Differential centrifugation: Centrifuge the filtrate three times. First, centrifuge at 800×g for 8 minutes at 4°C; second, centrifuge at 2,500×g for 25 minutes at 4°C; third, centrifuge at 8,000×g for 50 minutes at 4°C. Collect the supernatant.

[0070] (3) Ultracentrifugation: Centrifuge the supernatant at 140,000×g for 1.5 hours at 4°C. Collect the precipitate and resuspend it with 8 mL of sterile PBS.

[0071] (4) Sucrose density gradient centrifugation: Prepare a 6% / 25% / 40% / 55% sucrose gradient. Load the resuspended HELNVs onto the top of the gradient and centrifuge at 140,000×g for 1.5 hours at 4°C. Collect the HELNVs layer at the 25 - 40% interface.

[0072] (5) Purification and characterization: Use an ultrafiltration centrifugal tube (80 kDa molecular weight cut-off) to remove sucrose and obtain purified HELNVs.

[0073] The protein concentration of the HELNVs prepared in this example was 2.5 mg / mL. Dynamic light scattering analysis showed that the particle size distribution of HELNVs was 60.50 - 170.80 nm, the average particle size was 95 nm, and the Zeta potential was -22 mV.

[0074] Preferably, in the embodiments of the present invention, by adjusting the preparation parameters, we obtained HELNVs with different characteristics. These HELNVs showed varying degrees of anti-inflammatory and antioxidant capabilities in in vitro experiments. For example, after treating cisplatin-damaged HK-2 cells with the HELNVs prepared in Example 2 at a concentration of 100 μg / mL for 24 hours, the intracellular ROS level could be reduced by approximately 65%, and the expression of the inflammatory factor IL-6 was reduced by approximately 55%. This significant anti-inflammatory and antioxidant effect may be related to the relatively high protein concentration and moderate particle size of HELNVs, making them more easily taken up by cells and exert their functions.

[0075] Although the HELNVs prepared in Example 3 had a lower protein concentration, due to milder preparation conditions, they may have retained more bioactive components. When treating cisplatin-damaged HK-2 cells at the same concentration, these HELNVs could reduce the apoptosis rate by approximately 45% and increase the mitochondrial membrane potential (JC-1 red / green fluorescence ratio) by approximately 1.8-fold. This indicates that even at a lower concentration, the HELNVs prepared under mild conditions have significant cytoprotective effects, especially in maintaining mitochondrial function.

[0076] Through these examples, we not only demonstrated the tunability of the HELNVs preparation method but also preliminarily revealed the mechanisms by which HELNVs with different characteristics may play roles in alleviating acute kidney injury. This provides an important basis for further optimizing the HELNVs preparation process and developing more targeted treatment strategies for different types of kidney injury.

[0077] Next, the pharmacological effects of this traditional Chinese medicine composition (HELNVs) can be demonstrated through the following aspects: 1. Cell experiments: Using HK-2 cells (human renal tubular epithelial cell line) as an in vitro model, the protective effects of HELNVs were demonstrated through the following experiments: a) Cell viability detection: The CCK-8 method was used to detect cell viability. The results showed that treatment with 100 μg / mL of HELNVs could significantly increase the survival rate of cells damaged by cisplatin (100 μmol / L). This indicates that HELNVs have the effect of protecting renal tubular epithelial cells.

[0078] b) Apoptosis detection: Cell apoptosis was detected by flow cytometry using AnnexinV / PI double staining. The results showed that treatment with HELNVs could significantly reduce the apoptosis rate induced by cisplatin, demonstrating its anti-apoptotic effect.

[0079] c) ROS level detection: The intracellular ROS level was detected by flow cytometry and laser confocal microscopy using the DCFH-DA fluorescent probe. The results showed that HELNVs could significantly reduce the ROS level induced by cisplatin, indicating its antioxidant effect.

[0080] d) Mitochondrial function assessment: The mitochondrial membrane potential was evaluated by JC-1 staining. The results showed that treatment with HELNVs could restore the mitochondrial membrane potential of cisplatin-damaged cells, indicating its role in protecting mitochondrial function.

[0081] 2. Animal experiments: A cisplatin-induced acute kidney injury model was established using C57BL / 6 mice, and the therapeutic effect of HELNVs was evaluated by the following indicators: a) Renal function indicators: The serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured. The results showed that treatment with 30 mg / kg HELNVs could significantly reduce the elevated Scr and BUN levels induced by cisplatin, indicating that HELNVs could improve renal function.

[0082] b) Histopathological analysis: The pathological changes of renal tissues were evaluated by HE staining. The results showed that the tubular injury score in the HELNVs treatment group was significantly lower than that in the cisplatin group, demonstrating that HELNVs could reduce the pathological damage of renal tissues.

[0083] c) Renal injury markers: The expression level of KIM-1 in renal tissues was detected by ELISA method. The results showed that treatment with HELNVs could significantly reduce the expression of KIM-1, further confirming its role in protecting the kidneys.

[0084] d) Inflammatory factor detection: The levels of IL-1β, IL-6, TNF-α and IL-10 in renal tissues were detected by ELISA method. The results showed that HELNVs could reduce the expression of pro-inflammatory factors (IL-1β, IL-6, TNF-α), and at the same time increase the expression of the anti-inflammatory factor IL-10, demonstrating its anti-inflammatory effect.

[0085] e) Cellular senescence assessment: The cellular senescence was evaluated by β-galactosidase staining. The results showed that treatment with HELNVs could reduce the proportion of SA-β-gal positive cells, indicating that it could alleviate cellular senescence.

[0086] f) Apoptosis detection: The apoptosis of cells in tissues was evaluated using TUNEL staining. The results showed that HELNVs treatment could significantly reduce the number of TUNEL-positive cells, further confirming its anti-apoptotic effect.

[0087] g) Immune cell infiltration: The expressions of CD3 (a T cell marker) and CD68 (a macrophage marker) were detected by immunofluorescence staining. The results showed that HELNVs could reduce the infiltration of T cells and macrophages in the kidney tissue, indicating that it could regulate the immune response.

[0088] In summary, through cell and animal experiments, it was demonstrated that this traditional Chinese medicine composition (HELNVs) has multiple pharmacological effects, including antioxidant, anti-inflammatory, anti-apoptotic, protecting mitochondrial function, alleviating cellular senescence, and regulating the immune response, etc. These multi-faceted effects jointly alleviate cisplatin-induced acute kidney injury, providing an experimental basis for its potential clinical application.

[0089] Next, specific experimental data will be analyzed in depth to further clarify the role of HELNVs in alleviating cisplatin-induced acute kidney injury.

[0090] 1. Characterization of HELNVs: The experimental results showed that the particle size of the extracted HELNVs was mainly distributed between 68.06 - 164.20 nm, with an average particle size of about 100 nm. Transmission electron microscopy observation showed that HELNVs presented a round or cup-shaped structure. This nanoscale particle size distribution is conducive to the long-term circulation and targeted enrichment of HELNVs in vivo, providing a basis for its pharmacology.

[0091] 2. Analysis of cell experiment data: a) Cell viability: After treatment with cisplatin (100 μmol / L) for 24 hours, the survival rate of HK-2 cells decreased to about 50%. While treatment with 100 μg / mL HELNVs could increase the cell survival rate to about 80%, showing a significant cell protection effect.

[0092] b) Cell apoptosis: The results of flow cytometry showed that the proportion of late apoptotic cells (AnnexinV+ / PI+) in the cisplatin treatment group was about 30%, while HELNVs treatment could reduce this proportion to about 15%, reducing cell apoptosis by 50%.

[0093] c) ROS level: Analysis of DCFH-DA fluorescence intensity showed that cisplatin treatment increased the intracellular ROS level by about 3 times, while HELNVs treatment could reduce the ROS level to about 1.5 times that of the control group, showing a strong antioxidant capacity.

[0094] d) Mitochondrial function: The results of JC-1 staining showed that cisplatin treatment led to a decrease in the red / green fluorescence ratio by approximately 60%, while HELNVs treatment restored this ratio to about 80% of the control group, indicating a significant protective effect on mitochondrial function.

[0095] 3. Analysis of animal experiment data: a) Renal function indicators: Cisplatin treatment increased the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) in mice by approximately 4-fold and 3-fold, respectively. After 5 days of treatment with 30 mg / kg HELNVs, the levels of Scr and BUN decreased by approximately 50% and 40%, respectively, approaching normal levels.

[0096] b) Histopathology: The renal tubular injury score showed that the score of the cisplatin group was 3.5 ± 0.5, while that of the HELNVs treatment group decreased to 1.8 ± 0.3, and the degree of pathological injury was significantly reduced.

[0097] c) Renal injury markers: Cisplatin treatment increased the expression level of KIM-1 by approximately 5-fold, while HELNVs treatment reduced it to about 2-fold of the control group, further confirming the renal protective effect of HELNVs.

[0098] d) Inflammatory factors: ELISA results showed that HELNVs treatment reduced 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 strong anti-inflammatory effect.

[0099] e) Cellular senescence: β-Galactosidase staining showed that cisplatin treatment increased the proportion of SA-β-gal positive cells to approximately 40%, while HELNVs treatment reduced this proportion to approximately 15%, significantly alleviating cellular senescence.

[0100] f) Apoptosis: TUNEL staining results showed that the proportion of TUNEL positive cells in the cisplatin treatment group was approximately 25%, while HELNVs treatment reduced this proportion to approximately 8%, once again confirming its anti-apoptotic effect.

[0101] g) Immune cell infiltration: Immunofluorescence staining showed that HELNVs treatment reduced the numbers 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.

[0102] These experimental data comprehensively demonstrate the protective effect of HELNVs against cisplatin-induced acute kidney injury at multiple levels. HELNVs can not only directly protect renal tubular epithelial cells from the toxic effects of cisplatin, but also alleviate kidney injury by regulating oxidative stress, inflammatory response, and immune response.

[0103] Notably, the protective effect of HELNVs on mitochondrial function may be one of the key mechanisms underlying their pharmacological effects. Mitochondrial dysfunction is an important feature of cisplatin nephrotoxicity, and HELNVs can significantly improve mitochondrial membrane potential, which may be related to the antioxidant components (such as chlorogenic acid, isochlorogenic acid, etc.) contained therein.

[0104] In addition, the regulatory effect of HELNVs on the expression of inflammatory factors is also very significant. It can 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 its anti-inflammatory effect is superior to traditional anti-inflammatory drugs.

[0105] Finally, the inhibitory effect of HELNVs on immune cell infiltration suggests that it may alleviate kidney injury by regulating the immune microenvironment. This finding provides new ideas for the potential application of HELNVs in other inflammation-related kidney diseases.

[0106] Overall, these experimental data not only confirm the effectiveness of HELNVs in alleviating cisplatin-induced acute kidney injury, but also reveal its multi-target and multi-mechanism characteristics, providing a solid experimental basis for the further development and optimization of this novel therapeutic strategy.

[0107] The extraction and characterization of HELNVs of the present invention are as follows.

[0108] 1. HELNVs were prepared by a series of differential centrifugation, ultracentrifugation, and sucrose gradient density centrifugation ( Figure 2A ). The particle size distribution of HELNVs was measured using dynamic light scattering (DLS), and the results showed that the particle size mainly concentrated between about 68.06 - 164.20 nm ( Figure 2B ). The ultrastructural morphology analysis of the isolated and purified HELNVs under transmission electron microscopy (TEM) showed a round or cup-shaped appearance with disk-shaped or concave structural features. Given their structural similarity to exosomes secreted by mammalian cells, we speculate that HELNVs may have a tissue affinity similar to that of 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 HELNVs 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, HELNVs used within this concentration range were used for subsequent cell experiments.

[0109] 2. HELNVs can reduce oxidative damage and apoptosis in cisplatin-treated HK-2 cells.

[0110] To determine whether HELNVs could mitigate 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 for in vitro related cell experiments. To determine the optimal concentration of HELNVs for treatment, we tested the concentration of HELNVs from 0 - 200 μg / mL and found that after treatment with 100 μg / mL HELNVs, the cell viability increased significantly compared with cisplatin-treated HK-2 cells. ( Figure 3B ). Therefore, we used 100 μg / mL concentration of HELNVs as the optimal treatment concentration for subsequent in vitro experiments. Next, flow cytometry analysis showed that treatment with 100 μg / mL HELNVs significantly reduced the apoptosis of cisplatin-damaged HK-2 cells, showing a more obvious anti-apoptotic effect than the untreated cisplatin-damaged group. ( Figure 3C ). According to the quantitative analysis, it also showed that after treating cisplatin-damaged cells with 100 μg / mL concentration of HELNVs, the cell viability increased significantly ( Figure 3D ). These results indicate that HELNVs can alleviate cisplatin-induced apoptosis in HK-2 cells. Flow cytometry showed that HELNVs treatment led to a decrease in the ROS level in cisplatin-damaged cells, revealing the significant antioxidant effect of HELNVs. ( Figure 3E ), and this result was confirmed by laser confocal microscopy ( Figure 3F ). In addition, we also explored the targeted uptake of HELNVs by HK-2 cells after cisplatin damage. Time-course analysis showed that at 0, 2, 4, 6, 10, and 24 hours, the green fluorescence gradually increased, especially after the 6th hour, indicating that HELNVs were targeted and taken up in HK-2 cells ( Figure 3G ). This indicates that HELNVs have a targeted therapeutic effect on cisplatin-induced damage in HK-2 cells, providing promising evidence for subsequent in vivo studies.

[0111] 3. HELNVs maintain cisplatin-treated cells.

[0112] Although we have demonstrated in the previous section that HELNVs can alleviate cisplatin-induced AKI by reducing ROS generation and attenuating oxidative damage, we wished to further investigate whether HELNVs have potential protective effects on cisplatin-induced mitochondrial morphological changes. To explore this, in cisplatin-damaged HK-2 cells, we observed the morphology of mitochondria before and after HELNVs treatment using transmission electron microscopy (TEM). TEM analysis showed that the mitochondrial morphology of HK-2 cells treated with 100 μg / mL HELNVs was significantly restored, and the structure was more normalized than that of the untreated group. Autophagosomes were significantly reduced, and the number of vesicles secreted by the cells increased substantially ( Figure 4A ). In addition, we also observed the changes in mitochondrial membrane potential before and after HELNVs treatment using laser confocal microscopy. We found that after treatment with 100 μg / mL HELNVs, the green fluorescence was significantly weakened, while the red fluorescence was significantly enhanced, and the ratio of red to green fluorescence was significantly increased, compared with the control group and the cisplatin group ( Figure 4B ). Overall, HELNVs showed significant protective effects on cisplatin-induced mitochondrial morphological changes. Histological changes in HK-2 cells.

[0113] 4. HELNVs contribute to maintaining cisplatin-induced renal function in AKI mice.

[0114] Subsequently, we explored the targeted uptake of HELNVs in mouse kidney tissues after cisplatin injury. At 6 hours after injection, frozen kidney tissues began to show fluorescence, and the targeted uptake reached a peak at 8 to 10 hours within 24 hours ( Figure 5A &B). Therefore, we demonstrated that HELNVs showed targeted uptake in cisplatin-induced mouse kidney tissue injury, which was consistent with the results of our in vitro experiments ( Figure 3G ).

[0115] To determine whether the effect of HELNVs in alleviating cisplatin-induced AKI in vivo is the same as in vitro, we designed the following animal experimental protocol ( Figure 5C ). HELNVs were applied at doses of 30 mg / kg and 5 mg / kg to explore the optimal treatment concentration in in vivo experiments. After treating cisplatin-induced AKI mice with 30 mg / kg HELNVs, the levels of blood urea nitrogen and serum creatinine decreased significantly 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 HELNVs had more obvious improvements in pathological changes than the control group and the cisplatin group ( Figure 5FIn contrast, the renal pathology of mice in the 5 mg / mL HELNVs treatment group did not improve significantly, confirming that 30 mg / kg was 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, the level of KIM-1 in the bladder epithelial cells of mice was significantly reduced, and the level of KIM-1 in mice treated with 30mg / kg HELNVs was significantly reduced compared with the untreated group ( Figure 5H ). In conclusion, HELNVs can effectively alleviate cisplatin-induced AKI in mice, with 30 mg / kg being the optimal concentration.

[0116] 5. HELNVs can reduce cisplatin-induced inflammation and immune response in AKI mice.

[0117] 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 HELNVs, we detected the expression levels of IL-1β, IL-6, IL-10, and TNF-α in cisplatin-injured kidney tissues that were untreated and treated with HELNVs. Compared with the control and cisplatin groups, 30 mg / kg of HELNVs treatment significantly reduced the levels of these inflammatory markers ( Figure 6A -D), indicating that HELNVs 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 30 mg / kg HELNVs treatment compared with the control group. And the cisplatin group ( Figure 6E ). This suggests that HELNVs 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 HELNVs, the dark blue product, as a marker of SA-β-gal activity, was significantly reduced ( Figure 6F). This indicates that HELNVs reduce cellular senescence in cisplatin-induced AKI. To further confirm the anti-apoptotic effect of HELNVs, we performed TUNEL staining. Compared with the control group and the cisplatin group, the green fluorescence indicating apoptotic cells was significantly reduced in the HELNVs treatment group ( Figure 6G ), confirming that HELNVs inhibited apoptosis in cisplatin-induced AKI. CD3 is a biomarker of mature T lymphocytes, indicating the cellular immune status and participating in the regulation of various inflammatory and immune responses; CD68 is a biomarker of macrophages, which play an important role in macrophage phagocytosis, intracellular lysosomal metabolism, and extracellular interaction 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 HELNVs, compared with the control group and the cisplatin group, the expressions of CD3 and CD68 were significantly decreased ( Figure 6H ), which also indicated a significant reduction in the infiltration of T lymphocytes and macrophages in tissues. Therefore, HELNVs can alleviate cisplatin-induced AKI in mice by reducing the expressions of CD3 and CD68, inhibiting the infiltration of T lymphocytes and macrophages in tissues, thereby reducing inflammation and immune responses.

[0118] In summary, our study demonstrated that HELNVs can alleviate cisplatin-induced AKI by reducing inflammatory responses, inhibiting apoptosis, reducing oxidative damage, protecting mitochondrial function, and improving cellular senescence.

[0119] The above are only examples of the present invention and are not used to limit the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of the claims of the present invention.

Claims

1. A traditional Chinese medicine composition for relieving acute kidney injury, characterized in that, The composition comprises honeysuckle-derived exosome-like nanovesicles HELNVs, and the particle size distribution of the HELNVs is 68.06 - 164.20 nm.

2. The composition according to claim 1, characterized in that, The average particle size of the HELNVs is 100 nm.

3. The composition according to claim 1, characterized in that, The protein concentration of the HELNVs is 2 - 5 mg / mL.

4. The composition according to claim 1, wherein The Zeta potential of the HELNVs is -20 to -30 mV.

5. The composition according to any one of claims 1-4, characterized in that, The effective dose of the composition is 30 mg / kg.

6. A method for preparing a composition according to any one of claims 1-5, characterized in that, Comprising the following steps: (1) Raw material treatment: Wash the dried honeysuckle flower buds with water, use high-speed stirring to extract the honeysuckle juice, and then filter to remove impurities; (2) Differential centrifugation: Perform multi-step differential centrifugation on the filtrate obtained in step (1), and collect the supernatant; (3) Ultracentrifugation: Perform ultracentrifugation on the supernatant obtained in step (2), collect the precipitate and resuspend it with a buffer; (4) Density gradient centrifugation: Prepare a sucrose density gradient, load the sample resuspended in step (3) into the gradient and centrifuge, and collect the HELNVs layer in a specific density range; (5) Purification: Use an ultrafiltration centrifuge tube to remove sucrose to obtain purified HELNVs.

7. The method according to claim 6, wherein The method further comprises the following steps: Use a BCA protein quantification kit to determine the protein concentration of HELNVs.

8. The method according to claim 6, wherein The method further comprises the following steps: Fix the HELNVs with 2.5% glutaraldehyde for 30 minutes; negatively stain with 1.9% methylcellulose and 0.3% uranyl acetate for 5 minutes; observe the morphology of the HELNVs using a transmission electron microscope.

9. The method according to claim 6, wherein The method further comprises the following steps: Analyze the particle size distribution of the HELNVs using a dynamic light scattering instrument under the conditions of 25°C and a scattering angle of 173°.

Images