Application of Ciliatoside A in preparation of medicine for treating neurodegenerative diseases

Abstract

The invention relates to the technical field of biological medicines, in particular to application of Ciliatoside A in preparation of a medicine for treating neurodegenerative diseases. Researches find that Ciliatoside A inhibits NLRP3 inflammasomes mediated by mitochondrial phagocytosis, reduces mitochondrial damage induced by oxidative stress, enables nerve cells to be less damaged by inflammatory substances, prevents or treats development of neurodegenerative diseases, and has a drug effect on treatment of Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for reducing neuroinflammation in Alzheimer's disease by inhibiting NLRP3 inflammasome mediated by mitophagy of Ciliatoside A. Background Art

[0002] Alzheimer's disease (AD) is a devastating neurodegenerative disorder and the leading cause of dementia worldwide. Characterized by cognitive decline, memory impairment, and progressive neuronal loss, AD not only places a significant burden on patients and their families but also poses a major challenge to public health systems. The precise mechanisms underlying AD pathogenesis remain elusive, and there is an urgent need to develop new approaches targeting key molecular pathways that contribute to disease onset and progression.

[0003] Ciliatoside A (CA) is a natural compound isolated from Herba Ciliae sylvestris. Previous studies have shown that it has multiple pharmacological effects, including antiviral and anti-inflammatory activities by regulating cellular autophagy. Summary of the Invention

[0004] The present invention aims to address the problem of a lack of effective drugs for treating neurodegenerative diseases in the prior art, which has plagued a large number of elderly people. The present invention provides a method for preparing a medicament using Ciliatoside A. In particular, the use of Ciliatoside A in preparing a medicament for treating neurodegenerative diseases is expected to significantly improve the quality of life of patients with neurodegenerative diseases, such as Alzheimer's disease.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention relates to an application of Ciliatoside A in preparing a medicine for treating neurodegenerative diseases.

[0007] The chemical formula of Ciliatoside A is C 36 H 40 O 19 , the chemical structure is:

[0008]

[0009] Furthermore, the neurodegenerative disease is Alzheimer's disease.

[0010] Ciliatoside A (hereinafter referred to as CA) can effectively inhibit the activation of NLRP3 inflammasome, reduce pyroptosis, and alleviate mitochondrial damage induced by oxidative stress. CA activates mitochondrial phagocytosis through the AMPK / ULK1 and PINK1 / Parkin pathways, which helps to reduce neuroinflammation. It makes nerve cells less vulnerable to damage by inflammatory substances, prevents or treats the development of neurodegenerative diseases, and is of special significance for the treatment of Alzheimer's disease. In Caenorhabditis elegans and 3xTg-AD mouse models, CA improved cognitive function, reduced Aβ pathology, and enhanced mitochondrial phagocytosis, highlighting its therapeutic potential for AD by targeting neuroinflammation and mitochondrial quality control. Therefore, CA can be used to prepare drugs for the treatment of neurodegenerative diseases, especially Alzheimer's disease.

[0011] Furthermore, Ciliatoside A exerts its effects through the following mechanisms: Ciliatoside A inhibits NLRP3 inflammasome activation, reduces pyroptosis, and alleviates H2O2-induced oxidative stress and mitochondrial damage. CA reduces NLRP3 inflammasome activation and pyroptosis in microglia. Ciliatoside A inhibits NLRP3 inflammasome activation and promotes mitochondrial phagocytosis in AD.

[0012] Furthermore, Ciliatoside A exerts its effects through the following mechanisms: Ciliatoside A activates mitophagy by enhancing the AMPK / ULK1 and PINK1 / Parkin signaling pathways, thereby promoting mitochondrial quality control and reducing the release of pro-inflammatory cytokines. CA activates mitophagy through the AMPK / ULK1 and PINK1 / Parkin pathways.

[0013] Furthermore, Ciliatoside A exerts its effects through the following mechanisms: Ciliatoside A can reduce Aβ-induced paralysis, lower ROS levels, and enhance autophagy. CA can improve cognitive ability, reduce Aβ plaques, and alleviate neuroinflammation in vivo.

[0014] Furthermore, Ciliatoside A exerts its effects by reducing amyloid plaques.

[0015] Furthermore, Ciliatoside A exerts its protective effect by inhibiting the activation of the NLRP3 inflammasome and promoting mitochondrial phagocytosis. CA simultaneously targets mitochondrial dysfunction and inflammasome activation in AD patients.

[0016] Furthermore, Ciliatoside A and other antioxidant drugs are used in combination to prepare medicines.

[0017] Furthermore, the dosage form of the drug is a solid preparation, a liquid preparation, a gel preparation, a semi-fluid preparation, an aerosol preparation, etc.

[0018] Furthermore, the medicine contains an effective dose of Ciliatoside A.

[0019] Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipient. The term "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable (i.e., does not cause unacceptable levels of undesirable biological effects or does not interact in a harmful manner).

[0020] "Pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil, sesame oil), and injectable organic esters such as ethyl oleate.

[0021] The carrier may be chosen to have proper fluidity and may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0022] In addition, when preparing pharmaceutical preparations, adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants may also be included. Preservatives can prevent the growth of microorganisms, for example, through various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, potassium sorbate, etc.

[0023] If the pharmaceutical preparation is a liquid preparation, it may also include isotonic agents such as sugars, sodium chloride, etc.

[0024] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0025] 1. The use of Ciliatoside A of the present invention in the preparation of a drug for treating neurodegenerative diseases utilizes CA to inhibit the activation of NLRP3 inflammasome, reduce pyroptosis, and alleviate mitochondrial damage induced by oxidative stress, thereby breaking the vicious cycle of mitochondrial dysfunction and neuroinflammation.

[0026] 2. The CA of the present invention activates mitochondrial phagocytosis through the AMPK / ULK1 and PINK1 / Parkin pathways, which helps reduce neuroinflammation, making nerve cells less susceptible to damage from inflammatory substances, preventing or treating the development of neurodegenerative diseases, and is of particular significance for the treatment of Alzheimer's syndrome.

[0027] 3. In Caenorhabditis elegans and 3xTg-AD mouse models, CA improved cognitive function, reduced Aβ pathology, and enhanced mitochondrial phagocytosis, highlighting its therapeutic potential for AD by targeting neuroinflammation and mitochondrial quality control. Therefore, CA can be used to prepare drugs for the treatment of neurodegenerative diseases, particularly Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the UHPLC-DAD-Q / TOF-MS / MS chromatogram of Ciliatoside A (purity>99%).

[0029] Figure 2 Figure 5 is the result of a BV-2 cell experiment. C shows the effect of different concentrations of LPS on BV-2 cell viability. D shows the effect of different concentrations of CA on BV-2 cell viability.

[0030] Figure 3 Results from a CA-protected BV-2 cell experiment. (E) shows BV-2 cell viability in the presence of LPS or various concentrations of CA (2.5, 5, and 10 μM). (F) shows representative bright-field images of BV-2 cells treated with LPS and various concentrations of CA. Magnification: 10×, Scale bar: 200 μm.

[0031] Figure 4 The results of quantitative analysis of protein grayscale values ​​are shown in Figures H, I, J, and K. These are the quantitative analysis results of protein grayscale values ​​for NLRP3, ASC, Caspase-1, pro-Caspase-1, IL-1β, and pro-IL-1β, respectively.

[0032] Figure 5 This is an experiment on the viability of BV-2 cells stimulated with LPS / NiG. A shows the effects of CA and MCC on the viability of BV-2 cells stimulated with LPS / NiG. B shows representative bright-field images showing the morphology of BV-2 cells stimulated with LPS / NiG, with or without CA or MCC. Magnification: 10×, scale bar: 200 μm.

[0033] Figure 6 Figure 2 is a mixed image. D is a quantification of flow cytometry results showing the percentages of viable and pyroptotic cells. E is an immunoblot analysis of GSDMD processing in LPS / NiG-stimulated BV-2 cells with or without CA or MCC. GAPDH was used as a loading control. Images of the original and unprocessed Western blots are available in the figure. F is a quantification of immunoblot bands for N-GSDMD relative to F-GSDMD.

[0034] Figure 7Figure 2 is the cell viability of PC-12 cells treated with CM. K is the cell viability of PC-12 cells treated with CM from BV-2 cells after various treatments. L is a representative Hoechst / PI staining of PC-12 cells treated with CM from BV-2 cells. Magnification: 10×, scale bar: 200 μm. M is the quantitative analysis of the PI / Hoechst ratio in PC-12 cells. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

[0035] Figure 8 Figure 2 is the cell viability of BV-2 cells treated with hydrogen peroxide. A shows the cell viability of BV-2 cells treated with different concentrations of H2O2. B shows the effects of CA and NAC (positive control) on the cell viability of H2O2-treated BV-2 cells.

[0036] Figure 9 Representative bright-field images show the morphological changes of BV-2 cells after H2O2 treatment, with or without co-treatment with CA or NAC. Magnification 10×, scale bar 200 μm.

[0037] Figure 10 Representative fluorescence images of BV-2 cells treated with H2O2 were stained with PI, DHE, and JC-1 to assess cell death, ROS production, and MMP. Magnification: 10× and 20×, scale bars: 200 μm and 100 μm.

[0038] Figure 11 (A) is a mixed graph. Among them, E is the quantification of PI-positive cells (PI / Hoechst ratio). F is the quantification of positive cells (DHE / Hoechst ratio). G is the quantitative analysis of the GFP / RFP ratio (MMP). I is the quantitative analysis of intracellular ROS levels in BV-2 cells. (K) Quantitative analysis of cell number in Q3 / Q2 (JC-1 red-green ratio). K is the quantitative statistical graph of Q3 / Q2.

[0039] Figure 12 Figure 5 Flow cytometric analysis of MMPs using JC-1 staining in BV-2 cells treated with H2O2 with or without co-treatment with CA or NAC.

[0040] Figure 13 A is a mixed plot. B is the quantification of LC3 puncta per cell. D is the quantification of the mCherry / GFP ratio.

[0041] Figure 14Figure 1 is a composite image. Figure E is a Western blot analysis of LC3-I and LC3-II protein expression in BV-2 cells treated with various concentrations of CA or Rapa (10 μM). GAPDH was used as a loading control. Images of the original and untreated Western blots are available in the figure. Figure F is a quantitative analysis of the LC3-II / LC3-I ratio. Figure G is a Western blot analysis of LC3-I and LC3-II protein expression in BV-2 cells treated with 10 μM CA or Rapa. GAPDH was used as a loading control. Images of the original and untreated Western blots are available in the figure. Figure H is a quantitative analysis of the LC3-II / LC3-I ratio.

[0042] Figure 15 is a mixed plot. J is the quantification of GFP-LC3 puncta per cell. L is the quantification of the LC3-II / LC3-I ratio.

[0043] Figure 16 The mCherry / GFP ratio was quantitatively analyzed. The data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

[0044] Figure 17 The values ​​are ratios for multiple signaling pathways. Among them, B is the quantitative value of the p-AMPK / AMPK ratio. C is the quantitative value of the p-mTOR / mTOR ratio. D is the quantitative value of the p-ULK1 / ULK ratio.

[0045] Figure 18 is the quantification of GFP-LC3 puncta per cell.

[0046] Figure 19 Figure 1 is a composite graph. (G) shows immunoblot analysis of LC3-I and LC3-II expression in BV-2 cells treated with CA or CCCP, with GAPDH used as a loading control. (H) shows quantitative analysis of the LC3-II / LC3-I ratio.

[0047] Figure 20 Figure 1 is a composite image. Figure 2 shows immunoblot analysis of PINK1 and p-Parkin expression in BV-2 cells treated with CA or CCCP. GAPDH was used as a loading control. Raw and untreated Western blot images are available in the figure. Figure 3 shows quantitative analysis of the PINK1 / GAPDH ratio. Figure 4 shows quantitative analysis of the p-Parkin 1 / GAPDH ratio. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

[0048] Figure 21Figure 1 is a mixed plot. Figure B shows the quantitative analysis of the PI / Hoechst ratio. Figure D shows the quantitative analysis of the NLRP3 / Hoechst ratio. Figure F shows the quantitative analysis of the NLRP3 / Hoechst ratio.

[0049] Figure 22 Confocal images show colocalization of LC3 (green, an autophagosome marker) and NLRP3 (red, an inflammasome marker) in BV-2 cells treated with LPS / NiG, with or without CA. Arrows indicate colocalization. Magnification: 63×, scale bar: 10 μm. Hoechst was used for nuclear staining (blue). Data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

[0050] Figure 23 Figure 1 is a mixed graph. Figure B is a quantitative analysis of the paralysis rate of CL4176 nematodes treated with different concentrations of CA. Figure C is a survival analysis of CL2006 nematodes treated with CA.

[0051] Figure 24 Figure 6 is a mixed graph. D shows the analysis of Aβ aggregation in CL4176 nematodes treated with CA at 15°C and 25°C (6E10) using β-actin as a loading control. E shows the quantitative analysis of Aβ aggregation in CL4176 nematodes treated with CA.

[0052] Figure 25 Figure 1 is a composite of the two images. G shows the quantification of Aβ deposition in CA-treated CL2331 nematodes. H shows the effect of CA treatment at different temperatures on the deceleration rate in CL2122 and CL2355 nematodes. J shows the quantification of DHE fluorescence intensity in CL4176 nematodes.

[0053] Figure 26 Figure 1 is a composite image. L is a quantitative analysis of GFP::LGG-1 puncta in DA2123 nematodes. M is an immunoblot analysis of GFP::LGG-1 and PE-GFP::LGG-1 levels in DA2123 nematodes treated with CA or Rapa. Raw and untreated Western blot images are available in the figure. N is a quantitative analysis of the PE-GFP::LGG-1 / GFP::LGG-1 ratio in DA2123 nematodes treated with and without CA.

[0054] Figure 27 This is a hybrid plot. Here, P is the quantitative analysis of the fluorescence intensity of GFP::p62. R is the quantitative analysis of the GFP / DsRed ratio.

[0055] Figure 28Mixed plots. T represents the quantification of paralysis rates in CL4176 nematodes treated with RNAi targeting anti-autophagy-related genes. V represents the quantification of paralysis rates in CL4176 nematodes treated with RNAi targeting anti-mitochondrial phagy-related genes. Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.00.

[0056] Figure 29 Figure 1 is a mixed graph. Figure B shows the average swimming speed of wild-type mice, 3xTg-AD mice, and CA-treated 3xTg-AD mice during the 6-day MWM test. Figure C shows the total swimming distance of wild-type mice, 3xTg-AD mice, and CA-treated 3xTg-AD mice during the 6-day MWM test. Figure D shows the escape latency of wild-type mice, 3xTg-AD mice, and CA-treated 3xTg-AD mice during the 6-day MWM test.

[0057] Figure 30 The images are mixed. F shows immunofluorescence staining of Iba1 (red, a microglial marker) and DAPI (blue) in the CA3, CA1, and DG regions of the hippocampus of wild-type mice, 3xTg-AD mice, and CA-treated 3xTg-AD mice. Magnification: 10×, scale bar: 200 μm. G shows immunofluorescence staining of Iba1 (red, a microglial marker) and DAPI (blue) in the CA3, CA1, and DG regions of the hippocampus of wild-type mice, 3xTg-AD mice, and CA-treated 3xTg-AD mice. Magnification: 10×, scale bar: 200 μm. H shows immunofluorescence staining of GFAP (green, an astrocyte marker) and DAPI (blue) in the CA3, CA1, and DG regions of the hippocampus of wild-type mice, 3xTg-AD mice, and CA-treated 3xTg-AD mice. Magnification: 10×, scale bar: 200 μm. DETAILED DESCRIPTION

[0058] The following research, combined with specific chemical reagents and cell experiments, investigates novel pharmaceutical uses of the compounds to help those skilled in the art better understand how the pharmaceutical uses of the present invention can achieve the desired therapeutic effect for neurodegenerative diseases. The relevant research content is merely an exemplary research plan, serving as one of the best possible solutions. It should not be construed as limiting the use of the following experimental methods. Any technical solution based on the same concept as the present invention shall be included in the scope of protection of this patent.

[0059] Percentages not otherwise specified in this disclosure are by weight. For solutions prepared from two liquids, percentages are given by volume. For solutions prepared from a solid solute dissolved in a liquid, percentages are given by weight and volume. If units are not specified, g / L is assumed; otherwise, the specified unit ratio shall prevail.

[0060] Chemicals, plasmids, and antibodies

[0061] Weigh an appropriate amount of Nine-Headed Lion Grass, add 10 times 75% ethanol, heat and condense under reflux for extraction for 2 hours, repeat three times, after extraction, combine the three extracts and use a rotary evaporator to evaporate to dryness, collect the evaporated drug to obtain the total extract of Nine-Headed Lion Grass. Use a silica gel chromatography column, dissolve it with a mixed solution of dichloromethane and methanol = 1:1, and then apply it to the chromatography column. Use a 40v%-80v% ethanol aqueous solution for gradient elution, 500mL as a group, and collect each fraction. Then use a reversed phase C18 column for further separation and purification, column temperature 30 ° C, mobile phase methanol and 0.1% formic acid aqueous solution (80:20, v / v), flow rate 4mL / min, collect the main peak corresponding to CA, and use a rotary evaporator to evaporate to dryness to obtain Ciliatoside A (CA).

[0062] The chemical formula of Ciliatoside A was identified as C 36 H 40 O 19 , CA (purity>98%), the chemical structure is as follows. The relevant extraction and purification methods are the same as those reported in existing literature and the same as compound CA disclosed in Chinese patent CN113304165A, and will not be described in detail.

[0063]

[0064] N-Acetylcysteine ​​(NAC, A9165), Hoechst 33342 (B2261), propidium iodide (PI, P4170), dihydroethidium (DHE, 37291), lipopolysaccharide (LPS, L4130), and JC-1 (T4069) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Compound C (CC, T6146), bafilomycin A1 (Baf, T6740), carbonyl cyanide m-chlorophenylhydrazone (CCCP, T7081), quizartinib (AC220, T2066), 3-methyladenine (3-MA, T1879), rapamycin (Rapa, T1537), uridine A (UA, T7174), nigericin (Nig, T3092), and MCC950 (MCC, T3701) were obtained from TargetMol (Boston, MA, USA). YO-PRO-1 (Y3603) and ethidium homodimer-1 (EthD-1, E1169) were purchased from ThermoFisher (Waltham, MA, USA). Antibodies against mTOR (2983S), p-AMPK (2535L), and IL-1β (12426S) were obtained from Cell Signaling Technology (CST, Beverly, MA, USA). Antibodies against p-mTOR (67778-1-Ig), AMPK (66536-1-Ig), LC3 (14600-1-AP), ASC (10500-1-AP), caspase-1 (22915-1-AP), GSDMD (20770-1-AP), and GFAP (16825-1-AP) were obtained from Proteintech (Wuhan, China). Antibody against NLRP3 (ab270449) was obtained from Abcam (Cambridge, MA, USA), and antibody against Iba-1 (bs-1363R) was obtained from Bioss (Beijing, China). The specificity of these antibodies was confirmed by their respective suppliers. Plasmids pEGFP-LC3 (Addgene #21073) and tf-LC3 (Addgene #21074) were generously provided by Prof. Yoshimorita Matsu from Osaka University, Japan. Plasmids pEGFP-N1-NLRP3 (PPL00151-2a), pEGFP-N1-Caspase-1 (PPL00392-2e), pLVX-AcGFP1-N1-GSDMD- (PPL00857-4b), and mCherry-ASC (PPL01752-2b) were from the Public Protein / Plasmid Library (PPL, Nanjing, China).

[0065] Cell culture

[0066] BV-2 microglial cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in an incubator containing 5% CO2 at 37°C. PC-12 cells, derived from rat pheochromocytoma, were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin under the same conditions.

[0067] CCK-8 assay

[0068] Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) kit (GlpBio, USA). BV-2 and PC-12 cells were seeded in 96-well plates at a density of 5,000 cells per well and allowed to adhere overnight. After treatment at the designated time points, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for an additional 2 hours. The absorbance was measured at a wavelength of 450 nm using a microplate reader. The percentage of viable cells relative to the control group was calculated.

[0069] Cell transfection

[0070] BV-2 microglia cells were transfected using Lipomaster 3000 transfection reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Briefly, cells were plated at 1 × 10 5 Cells were seeded at a density of 100 μg / mL per well of a 6-well plate and allowed to grow for 24 hours. A transfection mixture of the relevant plasmid and Lipomaster 3000 transfection reagent was prepared in Opti-MEM medium and added to the cells for a 6-hour incubation. Following incubation, the medium was replaced with complete DMEM supplemented with 10% fetal bovine serum before further experimental treatments.

[0071] Annexin V-FITC / PI apoptosis detection

[0072] To evaluate the apoptosis and pyroptosis rates of LPS / Nig-induced BV-2 cells under CA and MCC950 treatment, Annexin V-FITC / PI apoptosis assay was performed, referring to Teng JF, Mei QB, Zhou XG, et al.: PolyphyllinVI Induces Caspase-1-Mediated Pyroptosis via the Induction of ROS / NF-κB / NLRP3 / GSDMD Signal Axis in Non-Small Cell Lung Cancer. Cancers (Basel) 122020. After treatment, the cells were collected and washed twice with phosphate-buffered saline (PBS). The cells were then resuspended in 1× binding buffer at a concentration of 1×10 6 Annexin V-FITC and PI were added to the cell suspension and incubated in the dark at room temperature for 15 minutes. The percentages of apoptotic (Annexin V+ / PI-) and pyroptotic (Annexin V+ / PI+) cells were quantitatively analyzed by flow cytometry using FlowJo. TM Data were analyzed using BD Biosciences v10 software (BD Biosciences, CA, USA).

[0073] YO-PRO-1 / EthD-1 staining

[0074] To assess the permeability of membrane pores associated with GSDMD (Gasdermin D) and evaluate pyroptosis in BV-2 cells, YO-PRO-1 and EthD-1 staining experiments were performed, as described in Han X, Xu T, Fang Q, et al.: Quercetinhinders microglial activation to alleviate neurotoxicity via the interplay between NLRP3 inflammasome and mitophagy. Redox Biol 44:102010, 2021. After treatment with CA and MCC, cells were washed with PBS and incubated with a mixture of YO-PRO-1 (1 μM) and EthD-1 (2 μM) for 30 minutes at room temperature in the dark. YO-PRO-1 can permeate the membrane pores formed by GSDMD, indicating that cells undergo pyroptosis, whereas EthD-1 does not readily penetrate these pores. After staining, cells were washed with PBS and analyzed under a fluorescence microscope. The percentage of YO-PRO-1-positive cells was quantified to assess the occurrence of pyroptosis.

[0075] Hoechst / PI staining

[0076] Hoechst 33342 and PI staining were used to detect cell death in BV-2 or PC-12 cells, according to Guo M, Zhu F, Qiu W, et al.: High-throughput screening for amyloid-βbinding natural small-molecules based on the combinational use of biolayer interferometry and UHPLC-DAD-Q / TOF-MS / MS. Acta Pharm Sin B 12: 1723-1739, 2022. After treatment, the cells were washed with phosphate buffered saline (PBS, pH = 7.4) and incubated with Hoechst 33342 (5 μg / mL) and PI (10 μg / mL) at 37°C in the dark for 20 minutes. After staining, the cells were washed with PBS and observed under a fluorescence microscope. Hoechst 33342 labels all cell nuclei with blue fluorescence, while PI specifically stains cells with damaged cell membranes, showing red fluorescence. The analysis involved counting the number of blue and red nuclei in randomly selected fields using ImageJ software to quantify the percentage of live (blue) and dead (red) cells.

[0077] GFP-LC3 puncta formation

[0078] In BV-2 cells transiently transfected with pEGFP-LC3, the formation of GFP-LC3 spots was quantitatively detected to assess the level of autophagy, according to Zhou XG, Qiu WQ, Yu L, et al.: Targeting microglial autophagic degradation of the NLRP3 inflammasome for identification of thonningianin A in Alzheimer's disease. Inflamm Regen 42:25, 2022. After treatment with CA and Rapa, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and then washed with PBS. Images of GFP-LC3 spots were captured using a confocal microscope (LSM980, Zeiss) at a magnification of 63 times. For quantification, images were analyzed using ImageJ software, and the number of GFP-LC3 spots per cell was counted in randomly selected fields of view (at least 10 fields per condition). Spots were defined as distinct bright GFP fluorescent spots within the cytoplasm. The average number of spots per cell in each treatment group was calculated to provide a quantitative measure of autophagic activity.

[0079] 1.9. GFP-RFP-LC3 Fluorescence Analysis

[0080] GFP-RFP-LC3 fluorescence analysis was used to assess autophagic flux in BV-2 cells transiently transfected with tf-LC3 plasmids. Reference: Qiu WQ, Pan R, Tang Y, et al.: Lychee seed polyphenol inhibits Aβ-induced activation of NLRP3 inflammasome via the LRP1 / AMPK mediated autophagy induction. Biomed Pharmacother 130:110575, 2020. After treatment with CA and Rapa, cells were fixed with 4% paraformaldehyde and washed with PBS. Fluorescent images of cells were captured at 63x magnification using a confocal microscope. The ratio of mCherry (RFP) to GFP fluorescence was quantified using ImageJ software. The presence of GFP and mCherry signals indicates the formation of autophagosomes, while the absence of GFP fluorescence and retained mCherry fluorescence indicates the formation of autolysosomes. The mCherry / GFP ratio was calculated for each treatment group to provide a quantitative measure of autophagic flux.

[0081] Mito-QC analysis

[0082] Mito-QC expression and analysis were performed in transiently transfected BV-2 cells and stably transfected HEK293T cells to assess mitophagy. After treatment with CA and CCCP, cells were fixed with 4 wt% paraformaldehyde and washed with PBS. Images were captured at 10x magnification using a fluorescence microscope. mCherry and GFP fluorescence signals were analyzed using ImageJ software. The mCherry / GFP fluorescence ratio was calculated, with an increase in this ratio indicating enhanced mitophagy activity.

[0083] 1.11. DHE staining of BV-2 cells

[0084] Dihydroethidium (DHE) staining was used to detect ROS levels in H2O2-induced BV-2 cells. After treatment with N-acetylcysteine ​​(NAC) and Ciliatoside A (CA), the cells were washed with PBS and then incubated with DHE (10 μM) and Hochest (5 μg / mL) at 37°C in the dark for 30 minutes. After staining, the cells were washed again with PBS and fixed with 4% paraformaldehyde. Fluorescence images were captured using a fluorescence microscope to observe the generation of ROS. DHE produces red fluorescence after oxidation. Fluorescence intensity was quantified using ImageJ software.

[0085] 1.12. JC-1 staining

[0086] JC-1 staining was used to measure the mitochondrial membrane potential (MMP) in BV-2 cells induced by H2O2. After treatment with NAC and CA, the cells were washed with PBS and incubated with JC-1 (5 μg / mL) at 37°C for 30 minutes. After incubation, the cells were washed again with PBS and fixed with 4% paraformaldehyde. The cells were then imaged using a fluorescence microscope. Aggregated JC-1 in healthy mitochondria emits red fluorescence, while JC-1 monomers present in depolarized mitochondria emit green fluorescence. The red / green fluorescence ratio was calculated using ImageJ software to quantify changes in mitochondrial membrane potential after treatment.

[0087] 1.13. Flow cytometry detection of ROS

[0088] Flow cytometry was used to quantify ROS levels in H2O2-induced BV-2 cells. After treatment with NAC and CA, cells were harvested, washed with PBS, and incubated with DHE (10 μM) for 30 minutes at 37°C in the dark. After incubation, cells were washed to remove excess dye. Samples were analyzed using flow cytometry, and fluorescence intensity was measured to determine ROS levels. Data were analyzed using FlowJo software (BD Biosciences, CA, USA).

[0089] 1.14. Detection of MMPs by flow cytometry

[0090] Flow cytometry was used to assess MMP levels in H2O2-induced BV-2 cells. After treatment with CA and NAC, cells were harvested, washed with PBS, and incubated at 37°C for 30 minutes using JC-1 according to the manufacturer's instructions. After incubation, cells were washed to remove excess dye. Fluorescence intensity analysis of samples was performed using a flow cytometer, with high MMP levels indicated by red fluorescence and low MMP levels indicated by green fluorescence. Data analysis was performed using FlowJo software to quantify changes in MMP levels after treatment.

[0091] 1.15. Confocal microscopy of mitochondria

[0092] Confocal microscopy was used to investigate mitochondrial morphological changes in BV-2 cells induced by H₂O₂ and treated with NAC and CA. Following treatment, cells were washed with PBS and stained with a mitochondrial-specific dye, such as MitoTracker Red CMXRos, for 30 minutes at 37°C according to the manufacturer's instructions. Following staining, cells were washed again and fixed with 4% paraformaldehyde. Images were taken using a confocal microscope at 63x magnification. Mitochondrial morphology was analyzed based on the intensity and distribution of the fluorescent signal to assess changes in mitochondria following treatment.

[0093] 1.16. Aβ1-42 Preparations

[0094] Aβ1-42 peptide (Zhongpeptide Biochemical Co., Ltd., Shanghai, China) was prepared by dissolving lyophilized Aβ1-42 in DMSO to generate a stock solution at a concentration of 1 mM. The solution was then diluted with PBS to the desired working concentration (10 μM). The peptides were incubated at 37°C for 24 hours to allow aggregation before use in experiments. The aggregated Aβ1-42 solution was briefly vortexed before processing to ensure homogeneity.

[0095] 1.17. Caenorhabditis elegans (C. elegans) experiments

[0096] Caenorhabditis elegans (C. elegans) strains, including CL4176, CL2006, CL2331, CL2122, CL2355, BC12921, DA2123, and IR1631, were obtained from the Caenorhabditis elegans Genetic Center (CGC, USA). They were cultured on Nematode Growth Medium (NGM) agar plates at 20°C with a standard food source of Escherichia coli OP50. For synchronization, adult nematodes were treated with bleach to isolate eggs, which were then hatched in M9 buffer to produce L1 larvae. Synchronized larvae were then transferred to NGM plates for further growth and processing.

[0097] 1.18. Paralysis Measurement

[0098] Paralysis was measured in C. elegans (C. elegans) lines CL4176 and CL2006, which express human Aβ1-42 to model neurodegenerative paralysis, according to the method in Zhu FD, Chen X, Yu L, et al.: Targeting autophagy to discover the Piper wallichii petroleum ether fraction exhibiting antiaging and anti-Alzheimer's disease effects in Caenorhabditis elegans. Phytomedicine 117:1549-16, 2023. For CL4176, nematodes were cultured at 16°C until the L3 / L4 larval stage, after which the temperature was raised to 25°C to induce Aβ1-42 expression and trigger paralysis. Nematodes were observed every 2-4 hours using a stereomicroscope. Paralysis was defined as the inability to move in response to gentle stimulation with a platinum wire. Paralyzed nematodes were recorded and photographed. The percentage of paralyzed nematodes was calculated at each time point. For the CL2006 strain, which continuously expresses Aβ1-42, paralysis rates were assessed daily starting at the L4 stage, with the number of paralyzed worms recorded daily for several consecutive days. The progression of paralysis was monitored by calculating the percentage of paralysis at each time point. Data analysis included comparisons of paralysis rates between the control and CA-treated groups in both strains.

[0099] 1.19. Measurement of Aβ3-42 Aggregation

[0100] Aβ3-42 aggregation was assessed in the CL2331 Caenorhabditis elegans strain, which expresses temperature-sensitive GFP-tagged Aβ3-42 in body wall muscle cells, as described in Zhu FD and Chen X (2023). CA-treated and untreated CL2331 worms were shifted from 16°C to 23°C to induce Aβ3-42 aggregation until day 2 of adulthood. After incubation, the worms were mounted on slides, and fluorescence images were captured using a fluorescence microscope. Images were analyzed to quantify Aβ deposits in the anterior region of each worm. At least 20 worms were examined per group for statistical analysis.

[0101] 1.20. DHE Staining of Caenorhabditis elegans

[0102] ROS levels were assessed using DHE staining in the CL4176 C. elegans line, following the method described in Yong Y, Yan L, Wei J, et al.: A novel ferroptosis inhibitor, Thonningianin A, improves Alzheimer's disease by activating GPX4. Theranostics 14:6161-6184, 2024. After treatment with or without CA, nematodes were incubated with 10 μM DHE dye for 30 minutes in the dark at 20°C. After incubation, the nematodes were mounted on slides and imaged using a fluorescence microscope. Fluorescence intensity, an indicator of ROS levels, was quantified using ImageJ software. At least 20 nematodes per group were examined for statistical analysis.

[0103] Immunoblot analysis

[0104] To assess protein expression levels, BV-2 cells and DA2123 nematodes treated with different conditions were lysed in RIPA buffer containing protease and phosphatase inhibitors. TM Protein concentration was determined using a Bradford protein assay kit (Bio-Rad, USA). Equal amounts of protein (30 μg) were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder for 1 hour at room temperature and then incubated overnight at 4°C with specific primary antibodies (including NLRP3, caspase-1, and β-actin). After washing, the membrane was incubated with appropriate horseradish peroxidase (HRP)-labeled secondary antibodies for 1 hour at room temperature. Protein bands were visualized using ECL detection reagents and a ChemiDoc MP imaging system (Bio-Rad, USA). Band intensities were quantified using ImageJ software (NIH, USA).

[0105] 1.22. Determination of C. elegans autophagy capacity

[0106] Autophagy was measured in C. elegans using strains DA2123 and BC12921, treated with CA and Rapa, as described in Zhu FD and Chen X (2023). For the DA2123 strain expressing GFP::LGG-1, autophagy-related fluorescence was visualized using fluorescence microscopy, and whole-worm protein extracts were analyzed by immunoblotting to detect GFP::LGG-1 levels. The number of GFP::LGG-1 puncta was quantified using ImageJ software to assess autophagosome formation. For the BC12921 strain, p62 expression was assessed by fluorescence imaging, and the fluorescence intensity in the anterior region was quantified using ImageJ software. At least 20 worms were analyzed for each experiment.

[0107] 1.23. Measurement of Mitophagy in Caenorhabditis elegans

[0108] Mitophagy was assessed in IR1631 nematodes, which express the mitochondrial marker TOMM-20::Rosella, a dual fluorescent reporter gene with pH-sensitive GFP and pH-insensitive DsRed (Wu AG, Yong YY, He CL, et al.: Novel 18-norspirostane steroidal saponins: Extending lifespan and mitigating neurodegeneration through promotion of mitophagy and mitochondrial biogenesis in Caenorhabditis elegans. Mech Ageing Dev 218:111901, 2024). After treatment with CA and UA (a mitophagy inducer), nematodes were imaged using confocal microscopy to capture GFP and DsRed fluorescence. The GFP / DsRed fluorescence ratio was calculated using ImageJ software; a decrease in this ratio indicates mitophagy, as GFP fluorescence decreases in the acidic environment of lysosomes, while DsRed fluorescence remains stable. At least 20 nematodes were analyzed per group.

[0109] RNA interference

[0110] RNA interference (RNAi) was used to knock down autophagy- and mitophagy-related genes in CL4176 nematodes, following the same method described in Zhu FD and Chen X (2023). Nematodes were fed E. coli HT115 bacteria expressing RNAi constructs targeting the genes unc-51, bec-1, lgg-1, vps-34, dct-1, pdr-1, and PINK-1. Synchronized L1 larvae were placed on RNAi plates and treated with or without calcium until they reached the L3 / L4 stage. At this stage, the temperature was shifted from 16°C to 25°C to induce Aβ expression and trigger paralysis.

[0111] 1.25. Animal and Drug Management

[0112] This study used 6-month-old male 3xTgAD mice. Mice were maintained in a temperature-controlled environment with a 12-h light / dark cycle. CA was administered intraperitoneally at doses of 0.5, 1, and 2 mg / kg in ultrapure water. Male B6;129 mice of the same age served as a normal control group, while 3xTgAD mice fed an ultrapure water diet served as a model group. All experimental procedures were approved by the Animal Ethics Committee of Southwest Medical University (Ethics Approval Number: 20230226-007).

[0113] 1.26. Morris water maze test

[0114] To assess the spatial learning and memory abilities of 3xTgAD mice, a water maze test was performed (reference Xiong R, Zhou XG, Tang Y, et al.: Lychee seed polyphenol protects the blood-brain barrier through inhibiting Aβ(25-35)-induced NLRP3 inflammasome activationvia the AMPK / mTOR / ULK1-mediated autophagy in bEnd.3 cells and APP / PS1 mice. Phytother Res 35:954-973, 2021.) The maze consisted of a 120-cm-diameter circular pool filled with opaque water maintained at 22±1°C and marked with spatial cues around the pool. Mice were trained for six consecutive days, with four trials per day. In each trial, a hidden platform with a diameter of 10 cm was submerged 1 cm below the water surface. The mice had 60 seconds to find the platform, and if they failed to find it, they were gently guided to the platform. The time it took the mice to reach the platform (latency) and the distance they swam were recorded. Following training, a probe trial was conducted on day seven, during which the platform was removed and the time the mice spent in the target quadrant was measured to assess memory retention. All behavioral testing was performed during the light cycle to minimize variability.

[0115] Immunohistochemistry

[0116] Immunohistochemistry was used to examine Aβ deposition in the brains of 3xTgAD mice. (Yu L, Wang H, Yao Q, et al.: Thonningianin A from Penthorumchinense Pursh as a targeted inhibitor of Alzheimer's disease-related β-amyloid and Tau proteins. Phytother Res 38:4815-4831, 2024) Following euthanasia, the brains were rapidly removed, fixed in 4% paraformaldehyde, and embedded in paraffin. Serial sections (5 μm thick) were cut using a microtome and mounted on glass slides. After deparaffinization and rehydration, sections underwent antigen retrieval with citrate buffer. The slides were then incubated with a primary antibody specific for Aβ overnight at 4°C. After washing with PBS, sections were incubated with a biotinylated secondary antibody, followed by incubation with avidin-biotin complex and DAB substrate to visualize the antigen-antibody complex. Finally, sections were counterstained with hematoxylin, dehydrated, and mounted. Images of immunohistochemical sections were captured using a digital microscope equipped with a high-resolution camera.

[0117] Immunofluorescence

[0118] Immunofluorescence staining was used to examine the expression of key proteins associated with NLRP3 inflammasome activation and mitophagy in BV-2 cells and brain tissue samples, following the same methods as described in reference 1.27 by Yu L and Wang H (2024). LPS / NiG-induced BV-2 cells were cultured on glass coverslips and treated with CA for 24 hours. After fixation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100, then blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature. Primary antibodies against NLRP3 and LC3 were incubated overnight at 4°C. After extensive washing, cells were incubated with appropriate fluorescent-conjugated secondary antibodies for 1 hour at room temperature. For brain tissue, sections were prepared and a similar staining procedure was applied. Cell nuclei were stained with DAPI. Images were captured using a confocal or fluorescence microscope, and fluorescence intensity was analyzed to quantify target protein expression levels.

[0119] Data Analysis

[0120] Statistical analysis was performed using GraphPad Prism software (version 9.0). Data are presented as mean ± standard deviation (SD). Differences between groups were analyzed using one-way analysis of variance (ANOVA), and multiple comparisons were performed using Tukey's post hoc test. A p value less than 0.05 was considered statistically significant. In addition, the Shapiro-Wilk test was used to assess the normality of the data.

[0121] 2. Experimental Results

[0122] CA inhibits NLRP3 inflammasome activation in LPS / NiG-stimulated BV-2 cells

[0123] UHPLC-DAD-Q / TOF-MS / MS analysis evaluated the purity and structure of CA and determined that its purity was greater than 99%. Figure 1 The effect of LPS on the viability of BV-2 cells was determined, and the results showed that LPS at 2.5 μM significantly promoted the proliferation of BV-2 cells. Figure 2 As shown in C. Then, the effect of CA on cell viability was detected in different concentration ranges, and it was found that CA had no toxicity to BV-2 cells at a concentration of 12.5 μM. Figure 2 As shown in D. In addition, when LPS stimulated BV-2 cells, the proliferation of BV-2 cells was significantly inhibited and the morphological changes were restored under increasing CA concentrations (2.5, 5, and 10 μM). Figure 3 As shown in Figure EF. To investigate the effect of CA on NLRP3 inflammasome activation, BV-2 cells were stimulated with LPS / Nig to induce inflammasome activation, with or without CA or the NLRP3 inhibitor MCC950 as a control. Immunoblotting analysis showed that LPS / Nig treatment significantly upregulated the expression of NLRP3, ASC, caspase-1 (p21), and IL-1β (p17) proteins, while CA treatment significantly reduced the expression of these inflammasome-related proteins in a dose-dependent manner, similar to MCC950. Figure 4 As shown in Figure HIJK. Fluorescence analysis further showed that CA reduced the fluorescence intensity of NLRP3, caspase-1, and ASC spots in BV-2 cells stimulated by LPS / Nig, indicating that inflammasome activation was reduced. This indicates that CA can effectively inhibit the activation of NLRP3 inflammasome in BV-2 cells stimulated by LPS / Nig.

[0124] CA inhibits LPS / Nig-induced pyroptosis in BV-2 cells

[0125] Pyroptosis is a form of inflammatory cell death associated with neuroinflammation in microglia. To determine whether CA inhibits LPS / Nig-induced pyroptosis in BV-2 cells, cell viability assays were performed and morphological changes were assessed. CCK-8 results showed that LPS / Nig stimulation significantly reduced cell viability, while co-treatment with CA or MCC950 effectively restored cell viability in a dose-dependent manner. Figure 5As shown in middle A. Bright field microscopy showed that CA and MCC950 treatment could alleviate the morphological changes of pyroptosis induced by LPS / nig. Figure 5 As shown in Figure B. Flow cytometric analysis using Annexin V-FITC and PI staining showed that the proportion of pyroptotic cells increased after LPS / Nig treatment, while co-treatment with CA and MCC950 significantly reduced pyroptosis and increased the proportion of viable cells. Figure 6 In addition, immunoblot analysis showed that LPS / Nig stimulation led to an increase in the proportion of GSDMD cleaved into its active N-terminal form (N-GSDMD), while CA and MCC950 treatment significantly inhibited this cleavage. Figure 6 As shown in Figure EF. Immunofluorescence staining of GSDMD further confirmed this protective effect of CA, showing that GSDMD expression on the cell membrane of CA-treated cells was reduced compared with that in the LPS / Nig group. In addition, Yo-Pro-1 and EthD-I staining showed that the membrane permeability of the LPS / Nig group was increased, which is a hallmark of cell pyroptosis, while CA or MCC950 treatment significantly reduced the membrane permeability. To evaluate the effects of pyroptosis-inducing factors on neuronal cells, we collected conditioned medium (CM) from BV-2 cells treated with different methods and added it to cultured PC-12 cells. Cell viability analysis showed that the conditioned medium of BV-2 cells treated with LPS / Nig significantly reduced the viability of PC-12 cells, while the conditioned medium of BV-2 cells treated with CA- or MCC950 restored the viability of PC-12 cells, Figure 7 As shown in middle K. PI staining also showed that PC-12 cells treated with conditioned medium from LPS / NiG-stimulated BV-2 cells showed increased cell death, and CA and MCC950 treatment alleviated this trend. Figure 7 Taken together, these results indicate that CA effectively inhibited LPS / Nig-induced pyroptosis in BV-2 cells and reduced its cytotoxic effects on neuronal PC-12 cells.

[0126] CA inhibits H2O2-induced mitochondrial damage and cell death in BV-2 cells

[0127] Mitochondrial damage and ROS accumulation are important factors in the activation of NLRP3 inflammasomes, leading to neuroinflammation. To explore whether CA can alleviate mitochondrial damage induced by oxidative stress and inhibit the corresponding cell death, BV-2 cells were exposed to H2O2 and then subjected to cell viability assays. The results showed that H2O2 treatment significantly reduced cell viability in a dose-dependent manner. Figure 8 As shown in A. However, co-treatment with CA or NAC significantly increased cell viability. Figure 8As shown in Figure B. Bright field microscopy images confirmed that CA or NAC treatment could alleviate H2O2-induced morphological damage. Figure 9 As shown. To further investigate the protective effects of CA, we used various detection methods to evaluate cell death, ROS generation, and mitochondrial status. Fluorescence staining showed that the apoptotic ratio and DHE staining of cells treated with H2O2 increased, indicating increased cell death and ROS levels, respectively. JC-1 staining also showed that the mitochondrial membrane potential decreased in the H2O2 group, and that CA or NAC treatment significantly reduced the apoptotic ratio and ROS levels, while restoring the mitochondrial membrane potential, indicating that cell death was reduced, ROS levels were reduced, and mitochondrial integrity was improved. Figure 10 D and Figure 11 As shown in Figure EFG. Flow cytometric analysis of DHE staining showed that ROS in H2O2 increased significantly, and CA or NAC treatment effectively reduced the ROS in cells. Figure 11 As shown in Figure 1. Flow cytometric analysis of MMPs using JC-1 staining also showed that CA or NAC alleviated mitochondrial depolarization in H2O2-treated BV-2 cells. Figure 11 In K and, Figure 12 Finally, confocal microscopy images of mitochondrial morphology showed that H2O2 treatment induced mitochondrial fragmentation, while CA or NAC treatment significantly reduced this fragmentation. These results together indicate that CA effectively alleviates H2O2-induced oxidative stress, mitochondrial damage, and cell death.

[0128] 2.4. CA activates mitophagy in BV-2 cells.

[0129] Mitophagy is a selective autophagy that plays a key role in maintaining mitochondrial quality and cellular homeostasis. To investigate whether CA can activate mitophagy in BV-2 cells, fluorescence images showed that CA treatment significantly increased the formation of GFP-LC3 puncta in a dose-dependent manner compared with the control group. Figure 13 As shown in B. GFP-mCherry-LC3 double labeling experiments can monitor autophagic flux, and the results show that the autophagic flux increases at different time points after CA treatment. Figure 13 As shown in D. Immunoblot analysis showed that CA treatment increased the accumulation of LC3-II, indicating increased autophagosome formation. After CA treatment, the LC3-II / LC3-I ratio was observed to increase with both dose and time. Figure 14 To further verify the induction of autophagy, BV-2 cells were treated with CA in the presence or absence of the autophagy inhibitor Baf. Fluorescence images of GFP-LC3 showed that the number of LC3 puncta increased in the CA-treated group, and Baf co-treatment further enhanced this effect, indicating that autophagy activity was enhanced. Figure 15As shown in middle J. Immunoblot images also showed that LC3-II levels increased after CA and Baf treatment, further confirming the role of inducing autophagy. Figure 15 Figure 1. Mitophagy was specifically assessed using Mito-QC. In HEK293T cells stably expressing Mito-QC, CA treatment induced mitophagy, as evidenced by an increase in the mCherry / GFP ratio in cells treated with 2.5, 5, and 10 μM CA, similar to the effect observed with CCCP, a known inducer of mitophagy. Figure 16 As shown in Figure O. In BV-2 cells transfected with Mito-QC plasmid, 10 μM CA treatment also enhanced mitochondrial autophagy, as shown by an increase in the mCherry / GFP ratio, similar to the CCCP treatment group. Figure 16 Shown in P. Together, these findings indicate that CA activates mitophagy in BV-2 cells.

[0130] 2.5.CA enhances mitophagy by activating AMPK / ULK1 and PINK1 / Parkin signaling pathways in BV-2 cells.

[0131] To further investigate the mechanism of CA-induced mitophagy, we examined the roles of the AMPK / ULK1 and PINK1 / Parkin signaling pathways in BV-2 cells. Immunoblotting analysis showed that CA treatment significantly increased the phosphorylation of AMPK and ULK1, but did not inhibit the phosphorylation of mTOR. Figure 17 To confirm the role of AMPK activation in CA-induced mitophagy, BV-2 cells were treated with CA in the presence or absence of the AMPK inhibitor CC. Fluorescence images showed that CA treatment significantly increased the formation of GFP-LC3 puncta, while co-treatment with CC attenuated the formation of GFP-LC3 puncta, indicating that AMPK activation is crucial for CA-induced autophagy. Figure 18 These results were confirmed by immunoblot analysis of LC3, which showed that CC co-treatment significantly reduced CA-induced LC3-II accumulation. Figure 19 We also evaluated the involvement of the PINK1 / Parkin pathway in CA-induced mitophagy. Immunoblotting analysis showed that CA treatment increased PINK1 expression and Parkin phosphorylation in a dose-dependent manner, similar to the effects of CCCP, a positive control for mitophagy induction. Figure 20 These results indicate that CA enhances mitophagy in BV-2 cells by activating the AMPK / ULK1 pathway and the PINK1 / Parkin signaling pathway.

[0132] CA inhibits NLRP3 expression and pyroptosis in BV-2 cells via mitophagy

[0133] To further investigate whether CA could inhibit NLRP3 inflammasome activation and pyroptosis in BV-2 cells through mitophagy, we used autophagy / mitophagy inhibitors, including Baf and AC220. In BV-2 cells stimulated with H2O2 or Aβ1-42, the presence of AC220, a Parkin inhibitor, reduced the cytotoxicity of CA against H2O2 (Figure S1). Similarly, Baf attenuated the protective effect of CA against LPS / Nig-induced BV-2 cell death (Figure S2). In addition, Hoechst / PI staining also showed that co-treatment with CA reduced LPS / Nig-induced cell death, while Baf attenuated this protective effect. Figure 21 As shown in Figure B. Fluorescence analysis showed that treatment with H2O2 or Aβ1-42 significantly increased the expression of NLRP3 in BV-2 cells, while co-treatment with CA effectively reduced the expression of NLRP3. However, the addition of Baf partially reversed the inhibitory effect of CA, indicating that mitophagy is involved in the regulation of NLRP3 expression. Figure 21 In addition, confocal microscopy images further revealed the colocalization of LC3 (an autophagosome marker) and NLRP3 in BV-2 cells treated with LPS / Nig. In the presence of CA, the colocalization of LC3 and NLRP3 increased, indicating that mitophagy was induced, leading to the degradation of NLRP3. Figure 22 Taken together, these results indicate that CA inhibits NLRP3 inflammasome activation and pyroptosis in BV-2 cells by inducing mitophagy.

[0134] CA exerts neuroprotective effects in the Alzheimer's disease model Caenorhabditis elegans through mitochondrial autophagy

[0135] To investigate whether CA exerts a neuroprotective effect in the AD model Caenorhabditis elegans through mitochondrial autophagy, we used various transgenic Caenorhabditis elegans strains expressing human Aβ. In CL4176 nematodes, which express temperature-induced Aβ1-42 in muscle cells, treatment with different concentrations of CA (50, 100, 150, and 200 μM) significantly reduced the paralysis rate of nematodes, indicating a neuroprotective effect. Figure 23 As shown in Figure B. Survival analysis of CL2006 nematodes that continuously express Aβ3-42 showed that CA treatment significantly extended lifespan compared with the control group. Figure 23 As shown in C. Immunoblot analysis showed that at 25°C, CA treatment significantly reduced the level of Aβ aggregation in CL4176 nematodes. Figure 24As shown in Figures DE. In CL2331 nematodes, CA treatment significantly reduced Aβ deposition. Figure 25 As shown in G. Deceleration rate experiments in CL2122 and CL2355 nematodes showed that CA improved the movement rate, showing a reduction in Aβ-induced toxicity. Figure 25 As shown in Figure H. DHE staining in CL4176 nematodes showed that CA significantly reduced the level of ROS, indicating an antioxidant effect. Figure 25 As shown in J.

[0136] To investigate the role of autophagy in the neuroprotective effects of CA, we analyzed GFP::LGG-1 puncta in DA2123 nematodes. CA treatment significantly increased the number of GFP::LGG-1 puncta, indicating enhanced autophagosome formation, similar to the effect of Rapa. Figure 26 As shown in middle L. Western blot analysis of GFP::LGG-1 and PE-GFP::LGG-1 further confirmed that CA treatment increased autophagy activity. Figure 26 As shown in MN. In BC12921 nematodes expressing GFP::p62, CA treatment significantly reduced the expression of GFP::p62, indicating that autophagic flux was enhanced. Figure 27 As shown in P.

[0137] Furthermore, in IR1631 worms expressing DsRed and GFP to monitor mitophagy, CA significantly increased the GFP / DsRed ratio, indicating enhanced mitophagy, similar to the effect of UA. Figure 27 To further validate the role of autophagy and mitophagy in the neuroprotective effects of CA, we used RNAi to knock down autophagy and mitophagy-related genes in CL4176 nematodes. When autophagy-related genes including unc-51, bec-1, lgg-1, and vps-34 were knocked down, the protective effect of CA against paralysis was abolished, indicating that autophagy is required for the neuroprotective effects of CA. Figure 28 Similarly, knockdown of mitophagy-related genes including dct-1, pdr-1, and pink-1 also weakened the protective effect of CA, further supporting the role of mitophagy in CA-mediated neuroprotection. Figure 28 As shown in Figure 5. Taken together, these results indicate that CA exerts neuroprotective effects in the AD model of Caenorhabditis elegans by enhancing mitophagy.

[0138] CA exerts neuroprotective effects in 3xTg-AD mice via mitochondrial autophagy

[0139] Based on our previous findings of CA neuroprotection in cells and nematode models, we evaluated its effects in vivo using 3xTg-AD mice, a well-established AD model. To assess spatial learning and memory abilities, the Morris water maze test was performed. Representative swimming paths showed that untreated 3xTg-AD mice exhibited an irregular swimming pattern, indicating impaired learning ability, while CA-treated 3xTg-AD mice exhibited an improved swimming pattern, similar to wild-type (WT) mice. The average swimming speeds of the groups were similar, indicating no changes in motor ability, Figure 29 As shown in middle B. CA treatment significantly reduced the total swimming distance and escape latency during training days, indicating that CA improved the learning and memory abilities of 3xTg-AD mice. Figure 29 As shown in Figures CD. To determine the effect of CA on Aβ pathology, we performed IHC staining of Aβ plaques in the hippocampus. Untreated 3xTg-AD mice exhibited a high density of Aβ plaques, while CA treatment significantly reduced the deposition of Aβ plaques. These findings indicate that CA alleviated Aβ aggregation in 3xTg-AD mice. We further assessed neuroinflammation and neuronal integrity in the hippocampus. Immunofluorescence staining of Iba1 (a microglial marker) showed that untreated 3xTg-AD mice had increased microglial activation in the CA3, CA1, and dentate gyrus (DG) regions of the hippocampus compared with wild-type mice. CA treatment reduced microglial activation, indicating an anti-inflammatory effect, Figure 30 As shown in Figure F. Similarly, immunofluorescence staining of GFAP (astrocyte marker) showed that CA reduced astrocyte activation in 3xTg-AD mice. Figure 30 NeuN staining showed that CA treatment protected neuronal integrity in the hippocampus compared with untreated 3xTg-AD mice, as shown by an increase in NeuN-positive cells. Figure 30 As shown in Figure H. To determine whether the neuroprotective effect of CA involves mitophagy, we performed immunofluorescence staining of NLRP3, LC3 (an autophagosome marker), and PINK1 (a mitophagy marker). Colocalization analysis of NLRP3 and LC3 showed that the colocalization of LC3 and NLRP3 increased in CA-treated 3xTg-AD mice compared with untreated 3xTg-AD mice, indicating enhanced clearance of NLRP3 inflammasomes mediated by mitophagy. In addition, CA-treated 3xTg-AD mice showed increased PINK1 expression, indicating enhanced mitophagy compared with untreated 3xTg-AD mice. These results suggest that CA exerts a neuroprotective effect in 3xTg-AD mice by enhancing mitophagy.

[0140] 3. Discussion of Results

[0141] Neuroinflammation is a key factor in the progression of Alzheimer's disease (AD) and is primarily driven by activation of the NLRP3 inflammasome. Previous studies have established a link between mitochondrial dysfunction, increased reactive oxygen species (ROS) production, and NLRP3 inflammasome activation, creating a vicious cycle that exacerbates AD pathology. Despite significant progress in understanding these mechanisms, therapeutic interventions targeting NLRP3 inflammasome inhibition and mitochondrial quality control remain limited. Autophagy, particularly mitophagy, is crucial for maintaining mitochondrial health, but the potential of mitophagy activators to alleviate AD-associated neuroinflammation by inhibiting the NLRP3 inflammasome remains underexplored. This study provides strong evidence that CA effectively suppresses neuroinflammation in AD models through mitophagy-mediated modulation of the NLRP3 inflammasome. Results from BV-2 cells, Caenorhabditis elegans, and 3xTg-AD mice consistently demonstrate the beneficial effects of CA in reducing neuroinflammation, promoting mitophagy, and alleviating disease pathology, highlighting its therapeutic potential in AD.

[0142] In LPS / Nig-stimulated BV-2 cells, CA significantly inhibited NLRP3 inflammasome activation and reduced pyroptosis, a form of inflammatory cell death associated with AD pathology. The reduction of key inflammasome components (including NLRP3, ASC, caspase-1, and IL-1β) suggests that CA can directly interfere with the signaling cascade that leads to inflammasome activation. In addition, CA-mediated inhibition of GSDMD cleavage and reduced cell membrane permeability further suggest that CA can alleviate pyroptosis, a major contributor to neuroinflammation. These findings are consistent with previous studies that have shown that NLRP3 inflammasome activation and pyroptosis play a role in microglia-mediated neuroinflammation, highlighting the importance of targeting these processes for AD treatment.

[0143] In addition to its effects on microglial inflammasome activation, CA has also been shown to significantly influence mitochondrial function, oxidative stress, and autophagy regulation, all of which are key factors in the pathogenesis of AD. In the current study, CA effectively reduced H2O2-induced mitochondrial depolarization and ROS generation. By maintaining mitochondrial function and promoting the clearance of damaged mitochondria through mitophagy, CA may play a multifaceted protective role in AD.

[0144] CA effectively activates mitophagy in BV-2 cells, as evidenced by increased LC3 puncta formation, enhanced autophagic flux, and increased expression of mitophagy-specific markers. CA promotes mitophagy through a dual mechanism, activating both the AMPK / ULK1 and PINK1 / Parkin pathways. ULK1, a downstream target of AMPK, is essential for autophagosome initiation and plays a key role in the early stages of autophagy. In this study, CA increased the phosphorylation of both AMPK and ULK1, suggesting that it initiates mitophagy by activating energy-sensing pathways. Furthermore, CA activated the PINK1 / Parkin pathway, which is involved in recognizing and marking damaged mitochondria for autophagic degradation. Inhibition of mitophagy by the AMPK inhibitor CC and the Parkin inhibitor AC220 further confirmed the involvement of these pathways in CA-mediated mitophagy induction. Furthermore, the increased PINK1 expression and Parkin phosphorylation observed in CA-treated cells suggest that CA enhances the recognition and clearance of damaged mitochondria, thereby reducing mitochondrial ROS production and preventing further activation of the NLRP3 inflammasome.

[0145] This was further validated in Caenorhabditis elegans and 3xTg-AD mouse models, where CA treatment not only reduced Aβ aggregation but also improved behavioral performance. Similarly, in 3xTg-AD mice, CA treatment significantly reduced Aβ plaque burden, microglial activation, and astrocyte reactivity, and improved cognitive performance. The reduction in neuroinflammation and the preservation of neuronal integrity suggest that CA has broad-spectrum anti-inflammatory and neuroprotective effects in AD.

[0146] In addition to its effects on mitophagy and inflammasome regulation, CA exhibits significant antioxidant properties. The reduction in ROS levels observed in both in vivo and in vitro models suggests that CA may have direct antioxidant effects in addition to its role in maintaining mitochondrial function. Oxidative stress is a major factor in the pathogenesis of AD. CA's ability to reduce oxidative stress through both direct antioxidant effects and by enhancing mitochondrial quality control mechanisms suggests a potential dual mechanism of action in combating oxidative damage in AD.

[0147] CA may inhibit the NLRP3 inflammasome by directly interacting with various domains of the NLRP3 protein. Molecular docking analysis and biolayer interferometry (BLI) results indicate that CA binds to key NLRP3 domains, including the pyrin domain (PYD), the NACHT domain, and the LRR domain. These interactions involve key residues such as PRO-90 and LYS-91 in the PYD; CYS-463, PHE-502, ASN-506, and LEU-507 in the NACHT domain; and TYR-859 and LYS-886 in the LRR domain. CA binding to these domains may stabilize the inactive conformation of NLRP3, preventing its activation and subsequent inflammasome assembly. Notably, CA exhibited the strongest binding affinity to the LRR domain (KD = 160 μM), followed by NACHT (KD = 169 μM) and PYD domain (KD = 285 μM), suggesting that its primary mechanism of action may involve interfering with conformational changes required for inflammasome activation.

[0148] In summary, this study provides strong evidence that CA exerts neuroprotective effects in AD by regulating mitophagy and inhibiting NLRP3 inflammasome activation. Dual activation of the AMPK / ULK1 and PINK1 / Parkin pathways highlights the ability of CA to enhance mitochondrial quality control, thereby reducing neuroinflammation and neuronal damage.

Claims

1. Use of Ciliatoside A in preparing medicines, characterized in that: The drug is a drug for treating neurodegenerative diseases; the chemical structural formula of Ciliatoside A is: 。 2. The use of Ciliatoside A in the preparation of medicines according to claim 1, characterized in that: The neurodegenerative disease is Alzheimer's disease.

3. The use of Ciliatoside A in the preparation of medicines according to claim 1, characterized in that: The Ciliatoside A works through the following mechanism: Ciliatoside A can inhibit NLRP3 inflammasome activation, reduce pyroptosis, and alleviate H2O2-induced oxidative stress and mitochondrial damage.

4. The use of Ciliatoside A in the preparation of medicines according to claim 1, characterized in that: Ciliatoside A works through the following mechanism: Ciliatoside A activates mitochondrial phagocytosis by enhancing the AMPK / ULK1 and PINK1 / Parkin signaling pathways, thereby promoting mitochondrial quality control and reducing the release of pro-inflammatory cytokines.

5. The use of Ciliatoside A in the preparation of medicines according to claim 1, characterized in that: The Ciliatoside A works through the following mechanisms; Ciliatoside A can reduce Aβ-induced paralysis, lower ROS levels, and enhance autophagy.

6. The use of Ciliatoside A in the preparation of medicines according to claim 1, characterized in that: Ciliatoside A works by reducing amyloid plaques.

7. The use of Ciliatoside A in the preparation of medicines according to claim 1, characterized in that: Ciliatoside A exerts its protective effect by inhibiting the activation of NLRP3 inflammasome and promoting mitochondrial phagocytosis.

8. The use of Ciliatoside A in the preparation of medicines according to claim 1, characterized in that: Ciliatoside A and other antioxidant drugs are used in combination for preparing medicines.

9. The use of Ciliatoside A in the preparation of medicines according to claim 1, characterized in that: The dosage form of the drug is any one of a solid preparation, a liquid preparation, a gel preparation, a semi-fluid preparation, and an aerosol preparation.

10. The use of Ciliatoside A in the preparation of medicines according to claim 1, characterized in that: The medicine contains Ciliatoside A in an effective dose.

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