Use of lice extract in preparations for treating Parkinson's disease

Abstract

The present invention discloses the use of CFE extract in a preparation for treating Parkinson's disease; reveals the broad prospects of CFE for neuroprotection and treatment of neurological diseases, gives a research mechanism and conclusion, provides technical inspiration for the use of CFE in neuroprotective preparations, provides valuable insights for developing new treatment strategies for Parkinson's disease, and plays a positive role in expanding PD treatment plans, enriching treatment methods, and improving treatment effects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the use of extracts of Lappula echinata in the preparation of a medicament for treating Parkinson's disease. Background Art

[0002] Neurodegenerative diseases are a major threat to human health. These diseases are pathophysiologically diverse, with some causing memory and cognitive impairment, while others affect a person's ability to move, speak, or even breathe. Parkinson's disease (PD) is a complex neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the brain. This loss leads to motor disorders such as tremors, bradykinesia, and postural instability, along with non-motor symptoms such as cognitive decline and psychological disorders. Despite extensive research on PD treatment drugs, no effective PD treatment methods or drugs have been found. Therefore, there is an urgent need to continue researching PD treatment-related drugs and methods.

[0003] The pathogenesis of PD involves complex molecular processes, among which the aggregation of α-synuclein and subsequent Lewy body formation are a prominent hallmark. These abnormal protein depositions trigger a series of events, including mitochondrial dysfunction, oxidative stress, impaired protein degradation pathways, and neuroinflammation. In addition, new research has found that environmental factors (including pesticides, heavy metals, and mitochondrial toxins) are involved in the pathogenesis of PD, highlighting the intricate interplay between genetic susceptibility and external triggers. The use of precise cell and animal models has proven to be important for elucidating the underlying complex mechanisms of PD. Studying in vitro and in vivo models overexpressing α-synuclein or using neurotoxic agents is conducive to the research and development of PD-related neurodegenerative and potential neuroprotective drugs.

[0004] With the in-depth study of plant-derived natural products, the potential therapeutic value of plant-derived natural products against various diseases (including neurodegenerative diseases) has been discovered and attracted much attention. To date, many natural products have been reported to have neuroprotective effects on PD cell and animal models. Lappula echinata is a herb traditionally used in East Asian medicine and has been reported to have various pharmacological activities, including antioxidant, anti-parasitic, anti-inflammatory, and anti-tumor effects. However, the neuroprotective effect of Lappula echinata and its role in PD models have not been widely studied and clarified. Summary of the Invention

[0005] The inventors first studied the effect of extracts of Lappula echinata (CFE) on PD cell and Caenorhabditis elegans (C. elegans) models through a large number of experiments.

[0006] The inventors found that when CFE was used to treat PD cell models, CFE had a significant effect in protecting PC-12 cells from H 2 O 2 The effect of 6-OHDA-induced cytotoxicity was shown by a significant increase in cell survival rate and a significant decrease in apoptosis rate. 2 O 2 or 6-OHDA-induced cytotoxicity in PC-12 cells, indicating that CFE has the potential to protect 2 O 2 And / or use of 6-OHDA cytotoxicity protection preparations.

[0007] Furthermore, when CFE was used to treat the PD cell model, CFE significantly inhibited the production of reactive oxygen free radicals in the cell model and restored the mitochondrial membrane potential; it can be seen that CFE can act as a mitochondrial protective agent, that is, CFE has the use of being a mitochondrial protective agent.

[0008] Furthermore, when CFE was used to treat the Caenorhabditis elegans model, CFE protected the 6-OHDA-induced dopaminergic neuron degeneration, prolonged lifespan, delayed the phenotype associated with aging, and enhanced stress resistance; it can be seen that CFE can significantly inhibit the degeneration of dopaminergic neurons, and CFE has a use as a preparation for protecting dopaminergic neuron degeneration.

[0009] The inventors also found that when CFE was used to treat PD cell models, CFE significantly reduced the expression of α-synuclein in PC-12 cell lines (WT, A53T, A30P or E46K) that widely expressed α-synuclein; when CFE was used to treat the Caenorhabditis elegans model, CFE also improved the movement disorders caused by α-synuclein; it can be seen that CFE can significantly inhibit the expression of α-synuclein, that is, CFE has the use of a preparation for inhibiting the expression of α-synuclein in cells.

[0010] Furthermore, when CFE was used to treat NL5901, BZ555 and N2 Caenorhabditis elegans, CFE showed a protective effect on nerves; at the same time, further, the neuroprotective effect of CFE may involve the regulation of MAPK signaling pathways, including ERK, JNK and p38, and further, the interference of these pathways weakened the neuroprotective effect of CFE in vitro and in vivo; it can be seen that CFE has a protective effect on nerves, that is, CFE has a use as a preparation with neuroprotective properties.

[0011] In addition, mitochondrial dysfunction, α-synuclein aggregation, environmental toxins, and glial cell activation serve as common clues linking various pathological features of Parkinson's disease. 2O 2 In the case of the cytotoxicity of 5-hydroxy-6-OHDA and the effect of inhibiting the expression of α-synuclein in cells, it can be confirmed that CFE has a good effect in protecting mitochondria, avoiding neuronal degeneration and inhibiting the expression of α-synuclein, that is, it is proved that CFE has a potential therapeutic effect on Parkinson's disease and is useful as a preparation for treating Parkinson's disease.

[0012] Among them, preferably, the preparation is a tablet, capsule, granule, syrup, suspension, solution, dispersion, sustained-release preparation for oral administration, intravenous injection preparation or subcutaneous injection preparation; a variety of CFE preparation types are conducive to the expansion of PD treatment options, enriching treatment methods and achieving better treatment effects.

[0013] Preferably, the extract of the Artemisia selengensis is obtained by crushing the Artemisia selengensis fruit and then extracting it with an ethanol aqueous solution.

[0014] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0015] 1. This invention reveals the broad prospects of CFE for neuroprotection and treatment of neurological diseases, and gives the research mechanism and conclusion, providing technical inspiration for the use of CFE in neuroprotective preparations.

[0016] 2. The present invention further reveals the potential therapeutic value of CFE as a neuroprotectant for Parkinson's disease, provides valuable insights into the development of new treatment strategies for Parkinson's disease, and plays a positive role in expanding PD treatment options, enriching treatment methods, and improving treatment effects.

[0017] 3. The present invention points out the direction for the research and development of PD therapeutic preparations, which is of great significance for shortening the development and application time of PD therapeutic preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The UHPLC-DAD-Q / TOF-MS / MS analysis of the crane lice extract in Experimental Example 1 (A is a representative total ion chromatogram (TIC) of CFE in positive ion mode; B is a representative UHPLC-DAD chromatogram of CFE in the range of 190-600 nm);

[0019] Figure 2 The results of Experimental Examples 2 and 3 show that the extract of the louse extract protects PC-12 cells from H 2 O 2 Figure 2 shows the experimental results of induced cytotoxicity (A is the dried Tianmingjing fruit and its source plants; B is the viability of PC-12 cells after treatment with different concentrations of CFE; C is H 2 O 2After treatment, the survival rate of PC-12 cells in the presence or absence of CFE and NAC; D is H 2 O 2 Typical images of DHE staining, JC-1 staining, and Hoechst / PI staining captured by fluorescence microscopy in PC-12 cells in the presence or absence of CFE and NAC after treatment, magnification: 20x, scale bar: 100 μm. Meanwhile, cell apoptosis was detected by flow cytometry using FITC / PE apoptosis detection kit; EH are DHE / DAPI ratio, JC-1 monomer / aggregate ratio, PI / Hoechst ratio, and cell apoptosis ratio in PC-12 cells);

[0020] Figure 3 The results of Experimental Examples 2 and 3 show that the extract of the louse flea protects PC-12 cells from H 2 O 2 The experimental results of induced cytotoxicity (A is different concentrations of H 2 O 2 The survival rate of PC-12 cells after treatment; B is the survival rate of PC-12 cells after treatment with different concentrations of H 2 O 2 Representative images of PC-12 cell morphology after treatment; C is a representative image of flow cytometry analysis using DHE reagent; D is the intensity of DHE in PC-12 cells);

[0021] Figure 4 The experimental results of the crane lice extract protecting PC-12 cells from 6-OHDA-induced cytotoxicity in Experimental Example 4 are shown in Figure 4 (A is the survival rate of PC-12 cells after treatment with different concentrations of 6-OHDA; B is a representative image of the morphology of PC-12 cells after treatment with different concentrations of 6-OHDA; C is a representative image of flow cytometric analysis using JC-1 reagent; D is the ratio of JC-1 green and red fluorescence intensities in PC-12 cells);

[0022] Figure 5Figure 4 shows the experimental results of the protection of PC-12 cells from 6-OHDA-induced cytotoxicity by the extract of the crane lice. (A is the survival rate of PC-12 cells after treatment with CFE and NAC in the presence or absence of 6-OHDA; B is the DHE fluorescence intensity of PC-12 cells after treatment with CFE and NAC in the presence or absence of 6-OHDA; C is the generation of ROS in PC-12 cells; D is a typical image of DHE staining, JC-1 staining and Hoechst / PI staining captured by fluorescence microscopy of PC-12 cells after treatment with CFE and NAC in the presence or absence of 6-OHDA. Magnification: 20 times, scale: 100 μm. At the same time, cell apoptosis was detected by flow cytometry using a FITC / PE apoptosis detection kit; EH are the DHE / DAPI ratio, JC-1 monomer / aggregate ratio, PI / Hoechst ratio and cell apoptosis ratio in PC-12 cells; I is the Western blotting in PC-12 cells induced by 6-OHDA. Blotting was used to detect p-ERK, ERK, p-JNK, JNK, p-p38, p38, and β-actin in the presence or absence of CFE; JK is the ratio of p-ERK / ERK, p-JNK / JNK, and p-p38 / p38);

[0023] Figure 6Figure 5 is the experimental result of reducing the expression of α-synuclein in PC-12 cells by the CFE extract in Experimental Example 5 (A is a typical image captured by fluorescence microscopy in PC-12 cells transfected with EGFP-α-synuclein-WT, EGFP-α-synuclein-A53T, EGFP-α-synuclein-A30P or EGFP-α-synuclein-E46K after CFE treatment. Magnification: 20 times, scale: 100 μm; BE is the GFP / DAPI ratio in PC-12 cells. FI is the percentage of GFP signal in PC-12 cells detected by flow cytometry, see Figure S8 for details. (JM) By Western Blotting was used to detect the expression of GFP and GAPDH in PC-12 cells transfected with EGFP-α-synuclein-WT, EGFP-α-synuclein-A53T, EGFP-α-synuclein-A30P or EGFP-α-synuclein-E46K after CFE treatment. The original images of Western blotting are shown in Figure S10, and the protein molecular weight markers are annotated; NQ is the ratio of GFP / GAPDH. R is DHE staining of PC-12 cells transfected with pHM6-α-synuclein-A53T by CFE and treated in the presence or absence of CFE. Magnification: 20 times, scale: 100 μm. S is the ratio of DHE / DAPI in PC-12 cells);

[0024] Figure 7 The figure is a flow cytometric analysis result of GFP signal in PC-12 cells transfected with different GFP-α-synuclein in Experimental Example 5;

[0025] Figure 8The results of the experiment in which CFE inhibited 6-OHDA-induced PC-12 cell death in Experimental Example 6. Figure (A) is the expression of p-ERK, ERK, p-JNK, JNK, p-p38, p38 and β-actin in PC-12 cells in the presence or absence of CFE detected by Western blotting. Figure (B) is the ratio of p-ERK / ERK, p-JNK / JNK and p-p38 / p38. Figure (E) is the expression of p-JNK, JNK, p-p38, p38 and β-actin in the presence or absence of CFE detected in PC-12 cells treated with 6-OHDA detected by Western blotting. Figure (F) is the ratio of p-JNK / JNK and p-p38 / p38. Figure (H) is the survival rate of PC-12 cells treated with CFE and SCH in the presence or absence of 6-OHDA. I is a typical image captured by fluorescence microscopy of Hoechst / PI staining in PC-12 cells treated with CFE and SCH in the presence or absence of 6-OHDA. Magnification: 20 times, scale bar: 100 μm. J is the ratio of Hoechst / PI);

[0026] Fig. 9 The experimental results of the crane lice extract inhibiting the expression of α-syn in the nematode NL5901 in Experimental Example 7-1 are shown in Figure 1 (A is a typical image captured by fluorescence microscopy in the muscle wall of the NL5901 nematode transfected with GFP-α-synuclein after CFE treatment. Magnification: 10 times, scale: 250 μm. B is the intensity of GFP in the NL5901 nematode. C is the number of body bends of the NL5901 nematode on the 5th and 10th days of CFE treatment. D is a typical image of DHE staining of the NL5901 nematode after CFE treatment. Magnification: 10 times, scale: 250 μm. E is the intensity of DHE in the NL5901 nematode);

[0027] Fig.10 The results of the experiment in Experimental Example 7-2 in which CFE inhibits the degeneration of dopaminergic neurons in the BZ555 strain (A is the GFP expression of dopaminergic neurons in 6-OHDA-treated BZ555 nematodes in the presence or absence of CFE and L-Dopa. Magnification: 20 times, scale bar: 100 μm. B is the intensity of GFP in BZ555 nematodes. C is the deceleration rate of 6-OHDA-treated BZ555 nematodes after CFE and L-Dopa treatment. D is a typical DHE staining image of BZ555 nematodes treated with CFE and L-Dopa in the presence or absence of 6-OHDA captured by fluorescence microscopy. Magnification: 20 times, scale bar: 100 μm. E is the intensity of DHE in BZ555 nematodes);

[0028] Fig.11 The extract of the crane lice in Experimental Example 7-3 delayed the growth of wild-type nematodes N 2 Figure (A) shows the results of the aging experiment of N 2 Survival curve of nematodes after CFE treatment. B is the survival curve of N. elegans treated with CFE on the 5th and 10th days. 2 Image of fat pigment accumulation in nematodes; C is N 2 The intensity of blue fluorescence of fat pigments in nematodes. D is the intensity of blue fluorescence of fat pigments in nematodes treated with CFE on the 5th and 10th days. 2 The number of body bends of nematodes. E is the number of N treated with CFE on the 5th and 10th days. 2 Pumping rate of nematodes. F is the pumping rate of N treated with CFE on the 1st, 3rd and 5th days. 2 G is the length of N treated with CFE on the 1st, 2nd, 3rd, 4th and 5th day. 2 nematode reproductive size);

[0029] Fig.12 is the CFE enhancement N in Experimental Example 7-4 2 The results of the stress resistance experiment on nematodes (A is the results of the CFE-treated N. 2 Survival curve of nematodes. B is the survival curve of H elegans treated with CFE and NAC. 2 O 2 Processed N 2 Survival curve of nematodes. C is the survival curve of H elegans treated with CFE and NAC. 2 O 2 Processed N 2 Typical images of DHE staining in C. elegans. Magnification: 10x, scale bar: 200 μm. E is N 2 The intensity of DHE in nematodes. C is the expression of GFP-sod-3 in CF1553 nematodes treated with CFE; D is the expression of GFP-gst-4 in CL2166 nematodes treated with CFE. F is the expression of GFP-sod-3 in CF1553 nematodes. G is the expression of GFP-gst-4 in CL2166 nematodes. H is the expression of N-terminal nematodes treated with PA14 treated with CFE. 2 elegans survival curve);

[0030] Fig.13The figures are the experimental results of CFE activating nematode mpk-1 in Experimental Example 7-5 (A is the expression of GFP-α-synuclein in NL5901 nematodes raised in HT115 or mpk-1 RNAi bacteria after CFE treatment. Magnification: 10x, scale: 200μm. B is the intensity of GFP in NL5901 nematodes. C is the GFP expression of dopaminergic neurons in BZ555 nematodes treated with 6-OHDA in the presence or absence of CFE and L-Dopa. Magnification: 20x, scale: 100μm. D is the intensity of GFP in BZ555 nematodes). DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] English-Chinese abbreviation comparison table

[0034]

[0035] Example:

[0036] 1. Experimental Materials

[0037] DMEM basal medium; FBS; trypsin cell digestion solution; penicillin-streptomycin-gentamicin solution (100X); PBS powder; DMSO; Hoechst solution; PI solution; DAPI solution; DHE staining solution; RIPA buffer (10X); ECL luminescent solution (high sensitivity); ECL luminescent solution (ultrasensitive); Striping buffer; 4% paraformaldehyde fixative; primary antibody diluent; secondary antibody diluent; GAPDH; GFP; Caspase-3; Caspase-9; Bax; Bcl-2; PARP; ERK; p-ERK; JNK; p-JNK; p38 MAPK; p-p38 MAPK; α-actin; three-color pre-stained marker; Bradford protein assay solution; protein buffer loading buffer; SDS; Tris; glycine; Tween 20; rapid cell freezing solution; Opti-MEM; Lipo2000; one-step PAGE gel rapid preparation kit (12.5%); one-step PAGE gel rapid preparation kit (10%); PVDF membrane (Merck Milipor); plasmid EGFP-alphasynuclein-WT; plasmid EGFP-alphasynuclein-A30P; plasmid EGFP-alphasynuclein-E46K; plasmid EGFP-alphasynuclein-A53T; plasmid pHM6-alphasynuclein-A53T; mitochondrial membrane potential detection kit (JC-1); Annexin V Alexa Fluor488 / PI apoptosis detection; kit; agar powder; peptone; sodium chloride; sodium hypochlorite; anhydrous dipotassium hydrogen phosphate; potassium dihydrogen phosphate; disodium hydrogen phosphate dodecahydrate; sodium hydroxide; anhydrous ethanol; tetramisole hydrochloride; 5-fluoro-2'-deoxyurea nucleoside; ampicillin sodium; cholesterol; anhydrous magnesium sulfate; anhydrous calcium chloride; glycerol; L-dopa; L-ascorbic acid; 6-hydroxydopamine; isopropyl-β-D-thiogalactoside.

[0038] Extract of Psoralea corylifolia: Dried Psoralea corylifolia was purchased from Beijing Tong Ren Tang Co., Ltd. (Beijing, China); Preparation method: The dried fruits were ground into fine powder and subjected to ultrasonic-assisted extraction using 75% ethanol. The extract was then filtered, concentrated under reduced pressure, and then freeze-dried to obtain a powder form. The extract was stored at -20°C until further use.

[0039] Caenorhabditis elegans: The Caenorhabditis elegans used in the examples were all from the Caenorhabditis elegans Genetics Center (CGC). Strain information is described in detail at https: / / wormbase.org / . Caenorhabditis elegans was maintained on Nematode Growth Medium (NGM) agarose plates with Escherichia coli (E. coli) OP50 as a food source. In order to obtain a synchronized population of nematodes, adult hermaphroditic nematodes laid eggs on NGM plates for 4 hours. Subsequently, the adult nematodes were removed and the eggs in the plates hatched to the desired developmental stage. Caenorhabditis elegans type: N 2 (wild type); NL5901 pkIs2386[unc-54p::alpha-synnuclein::YFP+unc-119(+)]; BZ555 egIs1[Pdat-1::gfp].

[0040] 2. Experimental instruments

[0041] Analytical balance; flow cytometer; TS2R-inverted fluorescence microscope; clean bench; pipette; desktop high-speed low-temperature centrifuge; constant temperature and humidity incubator; high-temperature and high-pressure steam sterilizer; fully automatic chemiluminescence imaging analysis system; electrophoresis instrument; membrane transfer instrument; multifunctional microplate reader; upright fluorescence microscope (Leica / DM6B); stereo fluorescence microscope (Leica / M205FA).

[0042] 3. Solution preparation

[0043] Preparation of the extract of the schizontid: First, dry and crush the purchased schizontid, weigh 25g of the schizontid powder, soak it in 200mL75% ethanol for 24 hours, and then use an ultrasonic instrument to sonicate twice, each time for 1.5 hours. Filter to remove the drug residue, put the collected drug soaking liquid into an evaporating dish, and place it in a constant temperature water bath until it is evaporated to dryness. Scrape the dry extract from the wall of the evaporating dish, weigh 200mg of the dry extract of the schizontid, add 1mL of DMSO to prepare a mother liquor of the schizontid extract (200mg / mL), and store it in a -20℃ refrigerator.

[0044] 6-OHDA: Weigh an appropriate amount of 6-OHDA powder, add an appropriate amount of DMSO according to the calculated results to obtain a 6-OHDA solution with a concentration of 200 mM. Pipette 100 μL of 6-OHDA solution into centrifuge tubes, keep one tube in a -20°C refrigerator for use, and store the rest in a -80°C refrigerator.

[0045] 10X transfer buffer: weigh 30.3 g of Tris and 151.1 g of glycine, then add pure water to dilute to 1000 mL, sonicate to completely dissolve, prepare 10X transfer buffer, and store at 4°C away from light.

[0046] 5X electrophoresis buffer: Weigh 15.1 g of Tris, 94 g of glycine and 5 g of SDS, then add pure water to dilute to 1000 mL, sonicate to completely dissolve, and prepare 5X electrophoresis buffer. Store at 4°C away from light.

[0047] 1X transfer solution: Take 100mL of 10X transfer solution, 200mL of methanol, and 700mL of ultrapure water and mix to obtain 1L of 1X transfer solution. 1X electrophoresis solution: Take 200mL of 5X electrophoresis solution and 800mL of pure water and mix to obtain 1L of 1X electrophoresis solution.

[0048] PBST: Take a packet of PBS powder, add 2L of pure water, mix by ultrasonic, then add 2mL of Tween 20, mix well to get 2L of PBST solution.

[0049] Complete culture medium: Take 20 mL of fetal bovine serum, 178 mL of DMEM basal culture medium, and 2 mL of penicillin-streptomycin-gentamicin solution (100X), mix well to obtain 200 mL of DMEM complete culture medium.

[0050] Preparation of antibodies: Use primary antibody diluent or secondary antibody diluent according to the dilution ratio recommended by the antibody company.

[0051] Preparation of NGM solid culture medium: Taking 400mL system as an example, weigh 1.2g NaCl powder, 7.0g agar powder, 1.0g peptone, add 400mL ultrapure water, shake evenly and put into high-temperature steam sterilizer. After sterilization, add 400μL 5% cholesterol solution, 400μL CaCl 2 Solution, 400 μL MgSO 4 Solution, 10mLK 2 HPO 4 / KH 2 PO 4 Solution, gently shake to mix. Before the NGM medium solidifies, quickly transfer it to a 90mm or 35mm culture dish in the clean bench. After the transfer is completed, let the culture dish stand for 2 hours to completely solidify. The culture dish can be wrapped with plastic wrap and stored in a 4℃ refrigerator or at room temperature. When needed, just take out the corresponding number of culture dishes.

[0052] Preparation of LB medium: Taking 200mL system as an example, weigh 1.0g NaCl powder, 1.0g yeast powder, 2.0g peptone, add 200mL ultrapure water, shake evenly and put into high-temperature steam sterilizer. After sterilization, 200mL LB liquid medium is obtained. If LB solid medium is required, add 1.5g solid medium to every 100mL LB liquid medium before sterilization.

[0053] Preparation of M9 buffer: Taking 400mL system as an example, weigh 2.0g NaCl powder, Na 2 HPO 4 12H 2 O powder 6.05g, KH 2 PO 4 1.2g of powder, add 400mL of ultrapure water, shake evenly and put into high-temperature steam sterilizer. After sterilization, add 400μLMgSO 4 The solution is OK.

[0054] MgSO 4 Solution preparation: Taking 100mL system as an example, weigh 12.0g of anhydrous magnesium sulfate powder, add 100mL of ultrapure water, shake evenly and put into a high-temperature steam sterilizer. After sterilization, MgSO 4 Solution, concentration is 1 mol / L.

[0055] CaCl 2 Solution preparation: Taking 100mL system as an example, weigh 11.098g of anhydrous calcium chloride powder, add 100mL of ultrapure water, shake evenly and put into a high-temperature steam sterilizer. After sterilization, you will get CaCl 2 Solution, concentration is 1 mol / L.

[0056] K 2 HPO 4 / KH 2 PO 4 Solution preparation: Taking 400mL system as an example, weigh K 2 HPO 4 Powder 14.24g, KH 2 PO 4 Powder 43.32g, add 400mL ultrapure water, shake evenly and put into high-temperature steam sterilizer. After sterilization, K 2 HPO 4 / KH 2 PO 4 Solution, concentration is 1 mol / L.

[0057] Preparation of NaOH solution: Taking 100mL system as an example, weigh 20g of sodium hydroxide powder, add 100mL of ultrapure water, shake evenly and put into high-temperature steam sterilizer. After sterilization, NaOH solution with a concentration of 5mol / L is obtained.

[0058] Preparation of FUDR solution: Taking 50mL system as an example, weigh 250mg of FUDR powder, add 50mL of ultrapure water, shake evenly and put into high-temperature steam sterilizer. After sterilization, FUDR solution is obtained with a concentration of 5g / L.

[0059] Preparation of IPTG solution: Taking 20mL system as an example, weigh 4.7662g of IPTG powder, add 20mL of ultrapure water, shake evenly and put into high-temperature steam sterilizer. After sterilization, IPTG solution is obtained with a concentration of 100mol / L.

[0060] Preparation of S buffer: Taking 200mL system as an example, weigh 1.2g NaCl powder, K 2 HPO 4 Powder 1.2g, KH2PO4 powder 0.22g, add ultrapure water 20mL, shake evenly and put into high temperature steam sterilizer. After sterilization, it is ready. Preparation of S buffer + glycerol: Taking 200mL system as an example, the preparation process of S buffer is the same as before, and 53.2mL glycerol can be added before sterilization.

[0061] Preparation of nematode freezing solution: Taking 400mL system as an example, add 280mL S buffer + glycerol solution, 32mL DMSO, and 88mL sterilized ultrapure water and mix well.

[0062] Preparation of nematode lysis solution: Mix equal volumes of NaOH solution and sodium hypochlorite solution.

[0063] 4. Experimental methods

[0064] (1)UHPLC-DAD-Q / TOF-MS / MS analysis

[0065] CFE component identification was performed using a Shimadzu UHPLC system located in Kyoto, Japan. The system consisted of an LC-3AD solvent dispenser, a SIL30ACXR autosampler, a CTO-30AC column oven, a DGU-20A3 degasser, and a CBM-20A controller. An Agilent UHPLC Zorbax EcLipse Plus C18 column (100 mm x 2.1 mm, 1.8 μm) was used for sample separation at a flow rate of 0.3 mL / min. The column temperature was maintained at 40 °C throughout the analysis. The mobile phase consisted of an aqueous solution containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The elution gradient program was as follows: from 0 to 15 min, solvent B was gradually increased from 10% to 95%; from 15.01 to 19 min, solvent B was linearly increased from 95% to 10%. Mass spectrometry analysis was performed using an AB SCIEX triple TOF X500R mass spectrometer equipped with a Duo Spray ion source. Negative electrospray ionization (ESI) mode was used with the following parameters: ion spray voltage was set to -4500 V, curtain gas was maintained at 35 psi, ion source temperature was set to 550 °C, deagglomeration potential (DP) was set to -100 V, nebulizer gas (GS1) was set to 55 psi, and heating gas (GS2) was set to 55 psi. The MS scan range was set from 50 to 1600 Da. PeakView 1.4 software was used for data processing and interpretation.

[0066] (2) Cell culture

[0067] Cryopreservation of cells: After digesting and collecting cells in good growth condition, add an appropriate amount of rapid freezing solution, divide into cryopreservation tubes, and place in a -80℃ refrigerator. After 24 hours, transfer to a liquid nitrogen tank.

[0068] Cell recovery: Take out the target cryopreservation tube from the liquid nitrogen tank, place it in a 37℃ constant temperature water bath for rapid thawing, add an appropriate amount of DMEM complete medium, mix well, centrifuge, and transfer to a culture bottle.

[0069] Cell passaging: When the cell fusion rate reaches about 80%, it is necessary to pass the cells. After digestion, transfer the cells to 2 or 3 new culture flasks according to experimental needs and continue to culture.

[0070] Cell plating: When the cells are growing well, plate according to the experimental requirements. Generally, for a 96-well plate, 100 μL of complete medium is added to each well, and the number of cells is 3,000-5,000; for a 12-well plate, 1 mL of complete medium is added to each well, and the number of cells is 80,000-100,000; for a 6-well plate, 2 mL of complete medium is added to each well, and the number of cells is 150,000-200,000.

[0071] (3) Cell administration

[0072] About 24 hours after the plate was inoculated, the cells were observed under a microscope to grow normally and in appropriate numbers, and the cells could be administered. The specific operation is to prepare the corresponding concentration of the lice extract solution with DMEM complete medium according to the experimental requirements, aspirate and discard the medium in the corresponding wells, and add the prepared lice extract working solution respectively. Cover the well plate with a lid, observe under a microscope and find no obvious abnormalities, then place it in a 37°C incubator and continue to culture until the detection time.

[0073] (4) Cell transfection

[0074] About 24 hours after the plate was inoculated, the cells were observed under the microscope and grew normally. The number of cells was appropriate, and the cells could be transfected. Taking the transfection of each well of the 6-well plate as an example, two ep tubes were prepared in the clean bench, divided into tube A and tube B. 300μL Opti-MEM was added to both tubes at the same time, an appropriate volume of plasmid solution (1.5μg plasmid per well) was added to tube A, and a transfection reagent twice the volume of the plasmid mass of tube A was added to tube B. The ep tube was gently flicked with a finger to mix it and let it stand for 10 minutes. After standing, the liquid in tube B was added to tube A, and the ep tube was gently flicked with a finger to mix it and let it stand for 15 minutes. During this period, a 15mL centrifuge tube was taken and 11.4mL Opti-MEM culture medium was added. After standing, all the liquid in tube A was aspirated into the 15mL centrifuge tube and mixed. 2mL of Opti-MEM culture medium containing plasmid was added to each well of the 6-well plate. Place it in a 37℃ incubator and culture it for 16-24 hours. The operation and reagent usage ratios for 96-well and 12-well plates are the same, just recalculate the system.

[0075] (5) MTT assay

[0076] Take a 96-well plate as an example. Add 10 μL of the prepared MTT solution to each well and place it in the incubator for 2-4 hours. Discard the original culture medium and add 150 μL of DMSO solution to each well. Place it on a shaker and shake for ten minutes. Use an enzyme-labeled instrument to measure at a wavelength of 570 nm to obtain the corresponding OD value.

[0077] (6) DHE staining

[0078] Add 1 μL DHE staining solution to 1 mL DMEM complete medium to obtain DHE staining working solution. Prepare an appropriate amount of DHE staining working solution according to experimental requirements. In the clean bench, aspirate the medium in the corresponding wells of the cells to be stained, add the corresponding DHE staining working solution, and incubate in the incubator for 1 hour. After the incubation is completed, discard the original medium, add a certain amount of DAPI solution, and leave it at room temperature for 15 minutes. After DAPI staining is completed, discard the DAPI solution in the well, add an appropriate amount of DMEM complete medium, and take photos in the red fluorescence channel and blue fluorescence channel under a fluorescence microscope with the same field of view, with a shooting magnification of 10 times or 20 times. The shooting conditions of the red channel and the blue channel are kept consistent to facilitate subsequent quantitative analysis.

[0079] (7) Hoechst 33342 / PI staining

[0080] Add 1 μL Hoechst stock solution to 1 mL DMEM complete medium to obtain Hoechst staining working solution, and add 1 μL PI stock solution to 1 mL DMEM complete medium to obtain PI staining working solution. In the clean bench, discard the medium, add the corresponding volume of Hoechst staining working solution, and place it in the incubator for 15 minutes. Discard the Hoechst staining working solution, add the corresponding volume of PI staining working solution, and place it in the incubator for 15 minutes. Discard the PI staining working solution, add the corresponding amount of DMEM complete medium, and take photos in the red fluorescence channel and the blue fluorescence channel under the fluorescence microscope with the same field of view, with a shooting magnification of 10 times. The shooting conditions of the red channel and the blue channel should be kept consistent for subsequent quantitative analysis.

[0081] (8) Mitochondrial membrane potential staining

[0082] In the clean bench, take one well in a 6-well plate as an example, discard the culture medium, add an appropriate amount of PBS to wash 3 times, add 1mL DMEM complete culture medium, then add 1mL JC-1 staining working solution, mix well, and incubate in the incubator for 20 minutes. After incubation, discard the staining solution, wash twice with staining buffer, and then add 2mL DMEM complete culture medium. Take photos in the red fluorescence channel and green fluorescence channel with the same field of view under a fluorescence microscope, and the shooting magnification is 20 times. The shooting conditions of the red channel and the green channel should be kept consistent for subsequent quantitative analysis.

[0083] (9) Flow cytometry

[0084] Apoptosis assay: Digest the cells in the well plate and collect them in centrifuge tubes. Add 500μL of pre-diluted 1X binding buffer to each ep tube, add 5μL Annexin V / Alexa Fluor 488 in sequence, and then add 5μL PI solution. Incubate at room temperature in the dark for 10 minutes. These are used as test tubes. The other conditions are the same. One tube of cells should only add 5μL Annexin V / Alexa Fluor 488, and the other tube of cells should only add 5μL PI solution to serve as a single staining tube; a blank tube without any dye is used for subsequent voltage adjustment. Use a 100-mesh nylon mesh to filter into a flow tube and immediately test on the machine. After plasmid transfection, the fluorescence intensity of the target protein is measured: the experimental groups are set as Ctrl group, transfected plasmid group, and transfected plasmid + corresponding concentration (50, 75, 100μg / mL) of the lice working solution group. After plasmid transfection for 16-24 hours, the original culture medium is discarded. DMEM complete medium was used to prepare the working solution of the lice extract at a concentration of 50, 75, and 100 μg / mL, and added to the corresponding wells. The Ctrl group and the transfected plasmid group were each added with an equal amount of DMEM complete medium. After 24 hours of administration, the test was performed, and the digestion, centrifugation, cell collection and filtration operations were the same as those for the apoptosis assay. After being transferred to the flow tube, it was immediately tested on the machine.

[0085] ROS assay: The experimental group settings are the same as those for apoptosis assay, and the staining process is the same as (7). After incubation, the assay is performed. The digestion, centrifugation, cell collection process, and filtration operations are the same as those for apoptosis assay. After transfer to the flow cytometer, the assay is performed immediately. Mitochondrial membrane potential assay: The experimental group settings are the same as those for apoptosis assay, and the staining process is the same as (8). After incubation, the assay is performed. The digestion, centrifugation, cell collection, and filtration operations are the same as those for apoptosis assay. After transfer to the flow cytometer, the assay is performed immediately.

[0086] (10) Morphological photography

[0087] The experimental group setting is the same as the group setting for apoptosis determination. After the cells are modeled, treated with drugs, etc., they are photographed under a microscope in the bright field at a magnification of 10 times. The shooting conditions should be kept consistent.

[0088] (11) Western blot detection

[0089] Preparation of protein samples: After a certain period of time, cells can be added with lysis buffer to extract proteins after specific operations such as drug administration or transfection. The specific operation is to aspirate the culture medium in the 6-well plate or 12-well plate, add an appropriate amount of PBS, discard the PBS, and repeat 3 times. Prepare a tray in advance and fill the tray with crushed ice. Place the 6-well plate or 12-well plate on a tray filled with crushed ice, and add an appropriate amount of lysis buffer to each well according to the number of cells. Generally, 60-100μL of lysis buffer is added to each well of the 6-well plate, and 30-50μL of lysis buffer is added to each well of the 12-well plate. Transfer the 6-well plate or 12-well plate to a -80℃ refrigerator. After 1 hour, take out the well plate and place it on a tray filled with crushed ice. At this time, the low-temperature centrifuge can be turned on for precooling. Use a cell scraper to quickly scrape the bottom of the well, and quickly transfer the liquid obtained from each well to the ep tube in the ice box. After scraping each well, the cell scraper should be cleaned with PBS before scraping other wells. After all the proteins in all wells are scraped, all ep tubes are placed in a low-temperature centrifuge precooled to 4°C, and the centrifugation conditions are 12000r / min for 10min. After centrifugation, the supernatant is transferred to a new centrifuge tube. At the same time, Bradford protein assay solution is added to the 96-well plate according to the number of samples, 300μL assay solution is added to each well, and 2μL of the transferred liquid obtained in the previous step is added to each well. One more well needs to be left to add 2μL protein assay solution as a control. Use an enzyme marker to measure at a wavelength of 590nm to obtain the corresponding value. According to the protein standard, the protein concentration calculation formula is obtained, and the value measured by the enzyme marker is substituted into the formula to calculate the concentration of each sample, and the protein is quantified to 30μg, and the corresponding loading amount of each sample is calculated. The volume of each sample minus 2μL is the actual volume now, and the corresponding volume of loading buffer (6X) is added, that is, 10μL loading buffer is added to every 50μL sample. After adding loading buffer to all samples, place them on a vortex machine and vortex quickly for 5 seconds to mix the samples and loading buffer. Open the metal bath in advance and set the conditions to 95℃ for 10 minutes. Put the vortexed samples into the metal bath and click the "Run" button to start the program. After ten minutes, put the ep tubes in the metal bath into an ice box and quickly transfer them to a -20℃ or -80℃ refrigerator for rapid cooling. Take out the samples when you need them.

[0090] Electrophoresis: Select the appropriate gel concentration according to the molecular weight of the target protein, and determine the number of gels to be configured according to the specific experiment. Take out the One-Step PAGE Gel Fast Preparation Kit from the 4°C refrigerator and complete the assembly of the long glass plate, short glass plate, gel frame and gel casting rack. Mix the appropriate amount of lower gel solution and lower gel buffer, and at the same time, mix the appropriate amount of upper gel solution and upper gel buffer. First inject the lower gel mixture between the short glass plate and the long glass plate, and then gently inject the upper gel mixture between the short glass plate and the long glass plate, and insert the electrophoresis comb between the glass plates at an angle to avoid the generation of bubbles. Let it stand for 30 minutes and wait for the gel to solidify. After gelation, take the glass plate out of the gel casting system and put it into the buffer tank. Add enough electrophoresis solution to the buffer tank until the long glass plate is covered. At the same time, add enough electrophoresis solution to the electrophoresis tank until the indicated position. Gently pull out the electrophoresis comb between the two glass plates, and add the samples to the corresponding channels in turn according to the calculated loading volume. After all the samples are added, add 5μL marker to the channels on the left and right of the samples. Cover the lid of the electrophoresis tank, open the electrophoresis instrument, connect the instrument, set the voltage to 150v, click the "RUN" button, and start electrophoresis. Stop electrophoresis when you see the marker running to 4 / 5 of the entire lower gel.

[0091] Transfer: After the electrophoresis stops, quickly remove the glass plate and place it in a tray filled with transfer solution. Cut the PVDF membrane to a suitable size and activate it with methanol for 2-5 minutes. Use a gel opener to gently pry open the two glass plates, cut off the excess gel according to the experimental requirements, and place the activated PVDF membrane in a tray filled with transfer solution. Place the sponge pad, transfer filter paper, and gel on the filter paper on the black side of the transfer clip in turn, and gently roll the gel with a roller to drive out bubbles. Place the PVDF membrane placed in the tray on the gel. When placing it, it should be slowly lowered from one side of the gel until it is fully attached. Use a roller to gently roll on the PVDF membrane to eliminate bubbles, then place the transfer filter paper and sponge pad on the PVDF membrane in turn, close the transfer clip, and place it in the transfer tank. Pour the transfer solution in the tray to the indicator line in the transfer tank, add the matching ice box, and place the transfer tank in a basin filled with crushed ice. Cover the transfer tank, connect the electrophoresis instrument, set the condition to 250mA, and determine the transfer time according to the molecular weight of the target band.

[0092] Blocking and antibody incubation: After the transfer is completed, the strips are removed and blocked with 5% skim milk for 1 hour. After the blocking is completed, the blocking solution is discarded, and a sufficient amount of PBST solution is added and washed three times. After the washing is completed, an appropriate amount of primary antibody is added and placed in a 4°C refrigerator for incubation overnight. The next day, the primary antibody is recovered to the corresponding 15mL centrifuge tube and washed 3 times with PBST solution. After repeating the washing three times, the corresponding secondary antibody is added, incubated on a shaker for 1 hour, the secondary antibody is recovered to the corresponding centrifuge tube, and PBST is repeatedly washed three times.

[0093] Development: Choose to use high-sensitivity or ultra-sensitive luminescent liquid according to the strength of the target band. Mix ECL luminescent liquid A and luminescent liquid B in a small aluminum box, clamp the target band and place it in the small aluminum box, wait for the reaction for 2-3 minutes, clamp the band in the darkroom of the developer, first select the color mode, and shoot the marker. Then select the chemiluminescence mode, expose the target band, and finally overlap the two bands to obtain an image.

[0094] (12) Recovery and cryopreservation of nematodes

[0095] Resuscitation of nematodes: Take out the cryovials from the -80℃ refrigerator or liquid nitrogen tank, prepare NGM solid culture medium containing 1000uL OP50 bacterial solution in advance, and after the liquid in the cryovials melts, quickly place it in a centrifuge for centrifugation at 3500r / min for 2min. Pipette out an appropriate amount of supernatant and discard it, leaving about 200μL of liquid in the cryovials. Pipette out the remaining liquid and transfer it to the prepared NGM culture medium containing bacteria, place it in a clean bench and blow it dry, and place it in an incubator at different temperatures according to the characteristics of the nematodes for cultivation, and observe it daily to ensure that it is in good growth and not starving.

[0096] Cryopreservation of nematodes: Transfer about 1000 synchronized nematodes to NGM solid medium (the diameter of the culture dish is about 90 mm) containing 1000 μL OP50 bacterial solution and culture normally. When the nematodes lay eggs in large quantities, do not add new OP50 bacterial solution, causing more nematodes to enter the dauer stage.

[0097] After observing that most of the nematodes in the culture dish have entered the dauer stage, rinse the culture dish with M9 buffer and centrifuge at 3500r / min for 2min. Repeat the rinse three times, discard as much supernatant as possible, add 3-5mL of nematode freezing solution, mix well, and mark the name of the nematode and the freezing time on the cryopreservation tube. Place the cryopreservation tube in a programmed cooling cryopreservation box and transfer it to a -80℃ refrigerator. After 24 hours, take it out of the cryopreservation box and transfer it to a liquid nitrogen tank.

[0098] (13) Cultivation and administration of nematodes

[0099] Normal culture of nematodes: Generally speaking, add 150μLO OP50 / PA14 / RNAi bacterial solution to a 35mm culture dish, and add 1mL bacterial solution to a 90mm culture dish, and blow dry in a clean bench. Store in a 4℃ refrigerator. After counting the synchronized nematodes, calculate the required volume of worm solution according to the purpose of the experiment. Generally speaking, 100-200 nematodes are cultured in a 35mm culture dish, and 1000 nematodes are cultured in a 90mm culture dish. Place the corresponding volume of worm solution in the aforementioned culture dish containing bacteria, blow dry the liquid, and culture it in an incubator with a suitable temperature according to the growth characteristics of the nematodes. Observe daily to ensure that there is no lack of bacterial solution during the growth cycle of the nematodes. If there is a lack of bacterial solution, replenish it in time.

[0100] Dosing of nematodes: Calculate the required volume of drug stock solution according to the working concentration of the drug, add the stock solution to the corresponding volume of sterilized ultrapure water, shake and mix to obtain the working solution of the drug. Generally speaking, add 400μL of the working solution of the drug to a 35mm culture dish, and add 3mL of the working solution of the drug to a 90mm culture dish, and blow dry in an ultra-clean workbench. Then add the corresponding volume of OP50 bacterial solution and blow dry in an ultra-clean workbench. When dosing is required, transfer the nematodes to the culture dish containing the drug.

[0101] (14) Synchronization of nematodes

[0102] After normal culture and well-growing nematodes enter the egg-laying period, synchronous operations can be performed. In the clean bench, add an appropriate amount of sterilized ultrapure water to the nematodes in the culture dish, gently shake the culture dish to make the ultrapure water fully contact every part of the culture dish, and use a pipette to collect the worm liquid in a 15mL centrifuge tube. The worm liquid should be controlled at 8mL at the end. If it exceeds 8mL, let the centrifuge tube stand and use a pipette to suck out the upper liquid after the nematodes settle. If it is less than 8mL, use a pipette to make it up to 8mL. Then add 2mL of nematode lysis solution to the centrifuge tube and cover the centrifuge tube cap. Place the centrifuge tube flat under a stereo microscope to observe the lysis of the nematodes. When most of the nematode bodies are broken and the eggs are fully exposed, quickly place the centrifuge tube in a centrifuge for centrifugation at 3500r / min for 2min. After the first centrifugation is completed, quickly pour out the supernatant to about 1mL in the clean bench, add sterilized ultrapure water to 10mL, and perform a second centrifugation under the same conditions. After the second centrifugation, perform the third centrifugation under the same conditions. After the third centrifugation, discard the supernatant, add M9 buffer to 10mL, and place in the incubator for overnight culture. Observe the nematode incubation under the microscope the next day. If synchronization is successful, if you need to continue to culture nematodes or administer drugs, place the centrifuge tube on ice for 5 minutes. After the ice bath, place it in a centrifuge for centrifugation at 4000r / min for 10 minutes. After the centrifugation is completed, discard the supernatant. It is appropriate to have about 1-2mL of worm liquid left. Gently shake to mix, pipette 10μL of worm liquid onto the slide, and count the number of nematodes in 10μL of worm liquid under the microscope.

[0103] (15) RNAi interference in nematodes

[0104] The RNAi interference (RNAi) effect is achieved by feeding RNAi bacteria to nematodes. The specific operation is to select a monoclonal colony with good growth from the LB solid culture medium, pick it into a conical flask containing LB liquid culture medium (containing 0.1% ampicillin), and place it on a shaker for expansion culture at 37°C, 160r / min, and culture for 12 hours. After 12 hours, take out the conical flask from the shaker, add the corresponding volume of IPTG solution (IPTG solution: LB liquid culture medium = 1:1000) to the conical flask to induce the synthesis of dsRNA, and place it on the shaker again for culture at 37°C, 80r / min, and culture for 8 hours.

[0105] (16) Detection of α-syn expression

[0106] The nematode PD model NL5901 was selected to take photos under a fluorescence microscope to detect the content of α-syn. The synchronized nematodes L4 stage NL5901 nematodes were cultured until the 5th or 10th day, 30 μL of 2% tetramisole hydrochloride was added to the center of the coverslip, 20 nematodes were randomly picked with a nematode pick and placed in tetramisole hydrochloride, and the coverslip was covered to avoid the generation of bubbles as much as possible. After all the nematodes were paralyzed and there were no obvious signs of activity, they were placed under the green channel of an upright fluorescence microscope and photographed at a magnification of 10 times. The shooting conditions should be kept consistent for subsequent quantitative analysis.

[0107] (17) Observation of the number of body bends

[0108] Counting the number of body swings of nematodes under a stereomicroscope reflects the nematode's locomotion ability. The synchronized L4 nematodes were cultured until the 5th or 10th day, 1 mL of M9 buffer was added to a 35 mm culture dish, and an appropriate amount of nematodes was picked up with a nematode picker. After waiting for 2 minutes, the number of body swings of the nematodes within 20 seconds was observed and recorded under a stereomicroscope.

[0109] (18) DA neuron damage detection

[0110] The nematode BZ555 was photographed under a fluorescence microscope to detect the DA neurons. The specific operation is as follows: Use M9 buffer to rinse the L3 stage BZ555 nematodes in the culture dish into a centrifuge tube and centrifuge at 3500r / min for 2min. Repeat three times, discard the supernatant, and add 60μL S buffer. In another centrifuge tube, add 50μL 6-OHDA (1mol / L) solution, 50μL VC solution, 50μL OP50 bacterial solution, 750μL S buffer, and finally add 50μL of the worm solution in the previous step, and shake gently to mix. Place in a 20℃ incubator for induction for 1 hour, shaking every 5 minutes during the period. After the induction is completed, add an appropriate amount of M9 buffer and centrifuge. Repeat the washing until the liquid in the centrifuge tube is clear and transparent, and finally discard the supernatant, leaving 200μL buffer, and take 10μL to count. Calculate the number of nematodes and the volume of worm solution required for each group according to the purpose of the experiment. After transferring it to a culture dish containing drugs and bacteria, culture it to the L4 stage, transfer it to NGM medium containing FUDR, and continue to culture it for 48 hours. After the culture is completed, prepare the slides and take photos under the green channel of an upright fluorescence microscope at a magnification of 20 times. Note that the shooting conditions should be kept consistent to facilitate subsequent quantitative analysis.

[0111] (19) Observation of food perception behavior

[0112] After the nematode BZ555 was cultured, the nematodes of each group were randomly picked with a nematode picker and placed on the sterile and bacterial NGM culture media. After waiting for 2 minutes, the number of movements of the nematodes within 20 seconds was observed under a microscope and recorded. The rate of decrease in the nematode food perception was calculated using the following formula:

[0113] Slowing rate=(N 线虫在无菌条件下的运动次数 -N 线虫在有菌条件下的运动次数 ) / N 线虫在无菌条件下的运动次数 *100%

[0114] (20) Observation of pharyngeal pumping rate

[0115] N 2 On the 5th or 10th day after adulthood, transfer them to new NGM medium, wait for 5 minutes, and observe and record the number of pharyngeal pumps within 20 seconds under a stereomicroscope.

[0116] (21) Detection of body length and egg laying amount

[0117] Body length measurement: N after synchronization 2 After the nematodes reach the L4 stage, prepare slides and randomly select an appropriate number of nematodes to photograph under an upright microscope in the bright field at a magnification of 10 times. The photographing conditions should be kept consistent to facilitate subsequent quantitative analysis. Then photograph every other day until the 5th day after the first photograph.

[0118] Egg laying: N after synchronization 2 After the nematodes reach the L4 stage, pick an appropriate amount of nematodes on a new NGM medium. Only one nematode is picked from each culture dish. 20 nematodes are picked from each group and marked. This is recorded as the first day of the egg-laying experiment. After 24 hours, each nematode is transferred to a new NGM medium and the egg-laying amount of the nematodes in each culture dish is counted until the nematodes lose their reproductive ability.

[0119] (22) Lifespan test

[0120] Synchronized L4 N 2 The nematodes were transferred to new NGM medium containing FUDR, which was recorded as day 0 of the lifespan experiment. For the first 10 days, the nematode picker was used to transfer the nematodes to new NGM medium containing FUDR every day. After the 10th day, the number of dead nematodes in each culture dish was counted every day until all nematodes died. Use the picker to touch the worm body. If there is no reaction on its head or body, it is recorded as dead. Nematodes that crawled away from the culture medium and died, nematodes that died due to bagworms, and nematodes that burrowed into the agar were removed.

[0121] (23) Detection of lipofuscin

[0122] On the 5th and 10th days of the lifespan experiment, slices were prepared and a suitable number of nematodes were randomly picked from each group using a picking needle and photographed under the blue channel of a fluorescent microscope at a magnification of 10 times. The photographing conditions should be maintained consistently to facilitate subsequent quantitative analysis.

[0123] (24) Adversity resistance experiment

[0124] Oxidative stress experiment: On the 5th day of the lifespan experiment, nematodes were transferred to a medium containing or not containing H 2 O 2 The number of dead cells was observed and recorded every hour until all the cells died.

[0125] Heat stress experiment: On the fifth day of the lifespan experiment, the nematodes were transferred from the 20°C incubator to the 35°C incubator, and the number of deaths was observed and recorded every hour until all of them died.

[0126] Anti-pathogen experiment: On the 5th day of the lifespan experiment, the nematodes were transferred to NGM medium containing or not containing PA14, and the number of deaths was observed and recorded every day until all of them died.

[0127] (25) Detection of ROS

[0128] DHE staining was used to reflect the level of ROS in nematodes.

[0129] ROS determination of nematodes NL5901 and BZ555: After the nematodes are cultured for an appropriate time, rinse the nematodes in the culture dish with M9 buffer and collect them in a 1.5mL centrifuge tube. Rinse three times, discard the supernatant, add 100μL M9 buffer to each centrifuge tube, add 2μL DHE staining solution, shake gently to mix, place in a 20℃ incubator for staining for 1 hour, and shake the centrifuge tube every 5 minutes. After staining, add an appropriate amount of M9 buffer and centrifuge. Repeat three times, and finally discard the supernatant, leaving 50μL buffer. Prepare slides and take photos under the red channel of an upright fluorescence microscope. Note that the shooting conditions should be kept consistent for subsequent quantitative analysis.

[0130] Nematode N 2 ROS determination: L4 stage after synchronization 2 elegans, transferred to a 2 O 2 or does not contain H 2 O 2On NGM medium, culture normally in a 20℃ incubator for 24 hours, rinse the nematodes in the culture dish with M9 buffer and collect them in a 1.5mL centrifuge tube. Rinse three times, discard the supernatant, add 100μL M9 buffer to each centrifuge tube, then add 2μLDHE staining solution, shake gently to mix, place in a 20℃ incubator for staining for 1 hour, and shake the centrifuge tube every 5 minutes. After staining, add an appropriate amount of M9 buffer and centrifuge. Repeat three times, and finally discard the supernatant, leaving 50μL buffer. Prepare slides and take photos under the red channel of an upright fluorescence microscope. Note that the shooting conditions should be kept consistent to facilitate subsequent quantitative analysis.

[0131] (26) Detection of sod-3 and gst-4

[0132] After synchronization, CF1553 nematodes or CL2166 nematodes cultured to the L4 stage were transferred to NGM medium containing FUDR for 72 hours, and the nematodes were picked onto a glass slide and sliced. The green fluorescence was observed using a Leica upright fluorescence microscope. Note that the shooting conditions should be kept consistent for subsequent quantitative analysis.

[0133] (27) Data processing and result analysis

[0134] Image J was used to quantitatively analyze all fluorescent images. GraphPad Prism8 software was used to perform statistical analysis and plot the experimental data. Mean ± SEM was used to represent the experimental results. The differences between the two groups of experimental data were statistically tested using t-test. The differences between three or more groups were statistically tested using one-way ANOVA. p < 0.05 indicated significant differences. All experiments were performed in parallel 3 times.

[0135] All survival rate experiments used the Kaplan-Meier method in GraphPad Prism software to generate survival curves, and the log-rank method was used to compare the differences between groups. p<0.05 indicated a significant difference. All experiments were performed in parallel 3 times. Experimental Example 1: Analysis of the components of the extract of the crane louse

[0136] UHPLC-DAD-Q / TOF-MS / MS was used to analyze the components of the extract of the crane lice. The chromatogram is shown in Figure 1 shown.

[0137] Experimental Example 2: Effect of the Extract of Crane Louse on the Viability of PC-12 Cells

[0138] The MTT method was used to detect the cell viability of PC-12 cells after being treated with a series of concentration gradients of the extract of the schizont (3.90, 7.81, 15.65, 31.25, 62.5, 125, 250, 500, 1000 μg / mL) for 24 hours. Figure 2 As shown in B, when the concentration of the crane louse extract is lower than 125 μg / mL, it has little effect on cell viability; when the concentration of the crane louse extract is higher than 250 μg / mL, it exhibits greater cytotoxicity.

[0139] Experimental Example 3: Effect of the extract of lice on H 2 O 2 Experimental study on the protective effect of induced cytotoxicity in PC-12 cells

[0140] The MTT assay was used to detect the effect of H 2 O 2 The cell viability of PC-12 cells was measured by pre-saturation with 50, 75, 100 μg / mL of tricholoma (50, 75, 100 μg / mL) for 8 h, and then HO was used to 2 O 2 (200 μM) for 24 h, the experimental groups were set as Ctrl group, H 2 O 2 Group, H 2 O 2 + lice (50, 75, 100 μg / mL) group, H 2 O 2 +NAC group (10 mM);

[0141] The bright field microscopy showed that the extract of the crane lice had an effect on H 2 O 2 The morphological changes of PC12 cells were induced by pre-saturation with 50, 75, 100 μg / mL of tricholoma. 2 O 2 (200 μM) for 24 h, the experimental groups were set as Ctrl group, H 2 O 2 Group, H 2 O 2 + lice (50, 75, 100 μg / mL) group, H 2 O 2 +NAC group (10 mM);

[0142] Hoechst33342 / PI staining and flow cytometry revealed that the extract from the crane tick significantly rescued the H 2 O 2 To induce apoptosis of PC12 cells, the cells were pre-saturated with 50, 75, 100 μg / mL of chloramphenicol for 8 h, and then hydrated with H 2 O2 (200 μM) for 24 h, the experimental groups were set as Ctrl group, H 2 O 2 Group, H 2 O 2 + lice (50, 75, 100 μg / mL) group, H 2 O 2 +NAC group (10 mM);

[0143] DHE staining showed that the extract of the crane lice significantly reduced H 2 O 2 The PC-12 cells were pre-saturated with 50, 75, and 100 μg / mL of H2O for 8 h. 2 O 2 (200 μM) for 24 h, the experimental groups were set as Ctrl group, H 2 O 2 Group, H 2 O 2 + lice (50, 75, 100 μg / mL) group, H 2 O 2 +NAC group (10 mM);

[0144] Mitochondrial membrane potential staining showed that the extract of the crane tick inhibited H 2 O 2 Induced mitochondrial damage in PC-12 cells, the experimental groups were set as Ctrl group, H 2 O 2 Group, H 2 O 2 + lice (50, 75, 100 μg / mL) group, H 2 O 2 +NAC group (10 mM).

[0145] The results are as follows Figure 2 , 3 shown.

[0146] Analysis of the experimental results shows that H 2 O 2 The cell viability was significantly reduced, and after administration of C. rapae extract (50, 75, 100 μg / mL), the cell viability was significantly improved. CFE dose-dependently improved cell morphology, increased cell survival rate, and reduced H 2 O 2 induced cell death in PC-12 cells; CFE significantly restored mitochondrial membrane potential (MMP); significantly reduced H 2 O 2 In summary, CFE inhibits H-induced apoptosis by inhibiting mitochondria-mediated apoptosis.2 O 2 The induced cytotoxicity of PC-12 cells produced a protective effect.

[0147] Experimental Example 4: Effect of the extract of lice on H 2 O 2 Experimental study on the protective effect of 6-OHDA-induced cell cytotoxicity

[0148] MTT method was used to detect the cell viability of PC-12 cells after administration of 6-OHDA in the presence or absence of lice extract. The cells were pre-saturated with lice extract (50, 75, 100 μg / mL) for 8 hours, and then 6-OHDA (125 μM) was used to model for 24 hours. The experimental groups were set as Ctrl group, 6-OHDA group, 6-OHDA+lice (50, 75, 100 μg / mL) group, and 6-OHDA+NAC (10 mM) group.

[0149] Bright field microscopy was used to photograph the effect of the pheasant extract on the morphological changes of PC12 cells caused by 6-OHDA. The pheasant extract (50, 75, 100 μg / mL) was pre-saturated for 8 hours, and then 6-OHDA (125 μM) was used to model for 24 hours. The experimental groups were set as Ctrl group, 6-OHDA group, 6-OHDA+pheasant (50, 75, 100 μg / mL) group, and 6-OHDA+NAC (10 mM) group.

[0150] Hoechst33342 / PI staining and flow cytometry revealed that the extract of the crane lice could significantly rescue the apoptosis of PC-12 cells induced by 6-OHDA. The experimental groups were Ctrl group, 6-OHDA group, 6-OHDA+crane lice (50, 75, 100 μg / mL) group, and 6-OHDA+NAC (10 mM) group.

[0151] Western Blot was used to detect the changes of apoptosis-related proteins in PC-12 cells induced by 6-OHDA in the presence or absence of B. raptoris extract. The experimental groups were set as Ctrl group, 6-OHDA group, 6-OHDA+B. raptoris (50, 75, 100 μg / mL) group, and 6-OHDA+NAC (10 mM) group.

[0152] DHE staining and flow cytometry showed that the crane foot bark extract significantly reduced the ROS level of PC-12 cells induced by 6-OHDA. The experimental groups were set as Ctrl group, 6-OHDA group, 6-OHDA+crane foot bark (50, 75, 100 μg / mL) group, and 6-OHDA+NAC (10 mM) group.

[0153] The results are as follows Figure 4 , 5shown.

[0154] Analysis of the experimental results showed that CFE significantly increased cell survival, reduced cell death, and improved the cell morphology of 6-OHDA-induced PC-12 cells. CFE significantly reduced the ratio of JC-1 monomers to JC-1 aggregates, indicating that the MMP of 6-OHDA-induced PC-12 cells was restored. CFE reduced ROS production, as manifested by a decrease in DHE intensity. In addition, CFE inhibited 6-OHDA-induced apoptosis of PC-12 cells. The results showed that the apoptosis rate of 6-OHDA-induced PC-12 cells was reduced, and the expression of apoptosis-related proteins was also reduced. In summary, CFE has a protective effect on 6-OHDA-induced cytotoxicity of PC-12 cells.

[0155] Experimental Example 5: Experiment on the inhibition of α-syn expression by extract from crane lice

[0156] Fluorescence imaging was used to show that the crane louse extract inhibited the expression of α-syn: PC12 cells were transfected with EGFP-α-syn-WT, EGFP-α-syn-A53T, EGFP-α-synA30P, and EGFP-α-syn-E46K plasmids 16-24 hours later, and then treated with crane louse extract (50, 75, and 100 μg / mL) for 24 hours. The cells were stained with DAPI staining solution, and the expression of α-syn was indirectly judged by the ratio of green fluorescence to blue fluorescence.

[0157] Flow cytometry showed that crane louse extract inhibited the expression of α-syn: PC12 cells were transfected with EGFP-α-syn-WT, EGFP-α-syn-A53T, EGFP-α-synA30P, and EGFP-α-syn-E46K plasmids for 16-24 hours, and then given crane louse extract (50, 75, 100 μg / mL) for 24 hours. The cells were collected and their fluorescence intensity was detected by flow cytometry, and the channel was set to FITC.

[0158] WB results revealed that crane louse extract inhibited the expression of α-syn: PC12 cells were transfected with EGFP-α-syn-WT, EGFP-α-syn-A53T, EGFP-α-synA30P, and EGFP-α-syn-E46K plasmids for 16-24 hours, and then given crane louse extract (50, 75, 100 μg / mL) for 24 hours. Lysate was added to prepare protein samples, and GFP expression was detected by WB.

[0159] The extract of slugs reduces the release of ROS by PC12 cells induced by α-syn: DHE staining shows the ROS level of PC12 cells after transfection with α-syn. PC12 cells were transfected with pHM6-alphasynuclein-A53T plasmid for 16-24 hours, and then treated with slug extract (50, 75, 100 μg / mL) for 24 hours, incubated with DHE staining solution for 1 hour, and then incubated with DAPI staining solution for 15 minutes.

[0160] The results are as follows Figure 6 , 7 shown.

[0161] Analysis of the experimental results showed that CFE significantly reduced the intensity of GFP, reflecting the expression of EGFP-α-synuclein-WT, EGFP-α-synuclein-A53T, EGFP-α-synuclein-A30P, and EGFP-α-synuclein-E46K. Flow cytometry analysis showed that CFE reduced the percentage of cells with GFP signals in transfected PC-12 cells, suggesting that CFE reduced the expression of α-synuclein. In addition, in transfected PC-12 cells treated with CFE, GFP expression was significantly reduced, and decreased with increasing CFE concentrations. In addition, CFE significantly reduced the intensity of DHE in cells, indicating a reduction in ROS production. In summary, CFE reduced the expression of α-synuclein and its induced ROS production.

[0162] Experimental Example 6: Experiment on the mechanism of inhibition of 6-OHDA-induced cell death by scutellaria baicalensis extract

[0163] Western Blot was used to detect the changes in the expression of ERK, JNK and p38 MAPK caused by the extract of the crane lice. The groups were set as Ctrl group, 6-OHDA group, and 6-OHDA + crane lice extract group (50, 75, 100 μg / mL).

[0164] The ERK inhibitor SCH772984 was used to verify whether the neuroprotective effect of the crane lice was related to the upregulation of ERK. The groups were set as Ctrl group, 6-OHDA group, 6-OHDA + crane lice extract group (100 μg / mL), and 6-OHDA + crane lice extract + SCH group;

[0165] The expressions of related proteins in the 6-OHDA model were detected, and the groups were set as Ctrl group, 6-OHDA group, and 6-OHDA+Hydrocchi extract group (50, 75, 100 μg / mL).

[0166] The results are as follows Figure 8 shown.

[0167] Analysis of the experimental results showed that CFE dose-dependently upregulated the ERK signaling pathway and downregulated the JNK and p38 signaling pathways. In 6-OHDA-induced PC-12 cells, the ratios of p-JNK / JNK and p-p38 / p38 increased significantly, and CFE treatment successfully reversed these increasing trends. CFE exerted a neuroprotective effect in 6-OHDA-induced PC-12 cells by regulating the MAPK signaling pathway. In addition, CFE offset the effects of SCH772984 on improving cell survival and inhibiting cell death. In summary, CFE exerts a neuroprotective effect in 6-OHDA-induced PC-12 cells by regulating the MAPK signaling pathway.

[0168] Experimental Example 7: Experiment on the improvement of pathological and behavioral functions of PD model nematode NL5901 by extract from lice

[0169] (1) Experiment on the inhibition of α-syn expression in nematode NL5901 by extract from lice

[0170] The intensity of fluorescence was used to characterize the content of α-syn. The experimental groups were set as Ctrl group and Gomphotherium extract group (100, 200, 400, 800 μg / mL).

[0171] The results are as follows Fig. 9 shown.

[0172] Analysis of the experimental results showed that different concentrations of Broomrape lice extracts could reduce the expression of α-syn, among which 200 μg / mL of Broomrape lice extract had the best effect.

[0173] (2) Experiments on the inhibition of dopaminergic neuron degeneration in the BZ555 strain by CFE

[0174] The changes in fluorescence intensity and the morphology of the labeled neurons were observed visually under a fluorescence microscope to characterize the damage and repair of DA neurons. The experimental groups were set as Ctrl group, 6-OHDA group, 6-OHDA+Hydrocoposphagnum extract group (200 μg / mL), and 6-OHDA+L-DA group (2 mM).

[0175] DHE staining was used to detect the ROS level of nematode BZ555 after 6-OHDA induction. The experimental groups were set as Ctrl group, 6-OHDA group, 6-OHDA+Hydrocoposphagnum extract group (200 μg / mL), and 6-OHDA+L-DA group (2 mM);

[0176] The food perception behavior of BZ555 was tested to reflect the damage of DA neurons caused by 6-OHDA, and the groups were set as Ctrl group, 6-OHDA group, 6-OHDA+ lice extract group (200 μg / mL), and 6-OHDA+ L-DA group (2 mM);

[0177] The results are as follows Fig.10 shown.

[0178] Analysis of the experimental results showed that treatment with CFE or L-Dopa successfully restored the expression of GFP, indicating that CFE and L-Dopa can rescue the dopaminergic neurons of BZ555 nematodes induced by 6-OHDA. CFE and L-Dopa significantly increased the deceleration rate of BZ555 nematodes induced by 6-OHDA. In 6-OHDA-induced BZ555 nematodes, the fluorescence intensity increased significantly, while CFE or L-Dopa treatment significantly inhibited the DHE intensity. In summary, CFE inhibited the degeneration of dopaminergic neurons in BZ555 nematodes.

[0179] (3) The extract of the crane lice delayed the inhibition of wild-type nematode N 2 Experiments on aging

[0180] C. elegans was cultured in NGM medium with or without lice extract at 20°C. 2 , observed the effect of the extract of the crane tick on the life span of nematodes;

[0181] The intensity of the fluorescence under the microscope can be used to visually display the aging of nematodes. 2 The fluorescence intensity at the 5th and 10th day after adulthood, the experimental groups were set as Ctrl group and crane tick extract group (200 μg / mL);

[0182] Detection of nematode N 2 The number of body bending on the 5th and 10th days, the experimental groups were set as Ctrl group and T. raptoris extract group (200 μg / mL);

[0183] Detection of nematode N 2 The body length on the 1st, 3rd, and 5th day after the start of the L4 stage was measured. The experimental groups were set as the Ctrl group and the Artemisia argyi extract group (200 μg / mL).

[0184] The results are as follows Fig.11 shown.

[0185] Analysis of the experimental results shows that CFE significantly prolongs N 2The lifespan and survival rate of nematodes increased. CFE treatment significantly reduced the intensity of blue fluorescence. CFE treatment improved movement ability, and the number of body bends increased within 20 seconds. CFE treatment significantly increased the pharyngeal pumping rate of nematodes on the 5th and 10th days. In addition, CFE significantly increased N 2 The body length and reproduction of nematodes indicate that CFE has non-reproductive toxicity and can promote the growth and development of nematodes. In summary, CFE extends the lifespan of nematodes and promotes healthy aging.

[0186] (4) CFE Enhanced N 2 Experiment on the stress resistance of nematodes

[0187] elegans N 2 The survival rate under the conditions of high temperature, strong oxidation and the presence of pathogens (PA14), the experimental groups were set as Ctrl group and Crane louse extract group (200μg / mL);

[0188] The fluorescent strain CF1553 of nematodes was used to detect ROS levels; the fluorescent strain CL2166 of nematodes was used to detect its ability to resist oxidative stress.

[0189] The results are as follows Fig.12 shown.

[0190] Analysis of the experimental results showed that the treatments of CFE and NAC significantly increased the 2 Survival rate of nematodes. CFE and NAC reduced N 2 elegans. CFE significantly increased the GFP intensity of sod-3 in the CF1553 strain and the GFP intensity of gst-4 in the CL2166 strain, reflecting the increased expression of antioxidant genes. Therefore, CFE exhibits an antioxidant effect in nematodes. In addition, CFE treatment can increase N 2 The survival rate of nematodes under PA14 treatment indicated that CFE played a role in resisting pathogenic bacteria. 2 Stress resistance of nematodes.

[0191] (5) Experiments on activating nematode mpk-1 by CFE

[0192] Feeding nematodes with NL5901 mpk-1RNAi bacteria reduced the expression of mpk-1. HT115 was the vector control of mpk-1RNAi bacteria. The experimental groups were set as HT115 group, HT115+Gastropoda excerpt group (200μg / mL), mpk-1 group, and mpk-1+Gastropoda excerpt group (200μg / mL);

[0193] Feeding nematodes with BZ555 mpk-1 RNAi bacteria reduced the expression of mpk-1. The experimental groups were set as HT115 group, HT115+6-OHDA group, HT115+6-OHDA+Cranberry extract group (200μg / mL), HT115+6-OHDA+L-DA group (2mM), mpk-1+6-OHDA group, and mpk-1+6-OHDA+Cranberry extract group (200μg / mL).

[0194] The results are as follows Fig.13 shown.

[0195] Analysis of the experimental results showed that CFE and L-Dopa treatment significantly restored the survival rate of dopaminergic neurons in BZ555 nematodes induced by 6-OHDA, while mpk-1 RNAi bacterial treatment weakened the protective effect of CFE on dopaminergic neurons. In summary, CFE inhibits the expression of α-synuclein and the degeneration of dopaminergic neurons by activating mpk-1.

[0196] It can be seen that the extract of the sphaerocephalus sphaerocephalus showed good anti-Parkinson's disease and anti-aging effects in the Caenorhabditis elegans model. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a louse extract in preparing a preparation for treating Parkinson's disease, wherein the louse extract is obtained by crushing louse fruits and then extracting them with an ethanol aqueous solution.

2. The use according to claim 1, It is characterized in that Use in the preparation of a cytoprotective agent against the cytotoxicity of H 2 O 2 and / or 6-OHDA.

3. The use according to claim 1, It is characterized in that Use in preparing a preparation for inhibiting dopaminergic neuron degeneration.

4. The use according to claim 1, It is characterized in that Use in preparing mitochondrial protection preparations.

5. The use according to claim 1, It is characterized in that Use in preparing a preparation for inhibiting α-synuclein expression in cells.

6. The use according to claim 1, It is characterized in that Use in preparing preparations with neuroprotective properties.

7. The use according to any one of claims 1 to 6, It is characterized in that The preparation is one of tablets, capsules, granules, syrups, suspensions, solutions, dispersions or sustained-release preparations for oral administration.

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