Application of aconite alkaloid in preparation of medicine for treating GSDMD related diseases

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of aconite alkaloid in preparation of a medicine for treating GSDMD related diseases. According to the application disclosed by the invention, research finds that mesaconitine (MC), hypaconitine (HC), benzoyl hypaconitine (BH) and benzoyl mesaconitine (BM) can be used for inhibiting pyroptosis induced by different agonists such as sodium urate crystals, lipopolysaccharide and nigericin; and activation of GSDMD protein can be inhibited in preparation of DSS-induced ulcerative colitis. Therefore, the aconitum alkaloid as an effective component is expected to be developed into a GSDMD inhibitor for inflammatory diseases such as ulcerative colitis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of aconitum alkaloids in preparing medicines for treating GSDMD-related diseases. Background Art

[0002] Pyroptosis is a newly discovered type of programmed cell death that has received increasing attention in recent years. When the body is exposed to infection, once the "danger signal" is recognized, the innate immune system will initiate a response that may trigger cell necrosis, apoptosis or pyroptosis to kill the invading microorganisms. As a lytic and inflammatory death that depends on inflammasomes and caspases, pyroptosis is characterized by plasma membrane rupture, cell swelling and lysis, which is mediated by the Gasdermin (GSDM) protein family. Finally, it leads to the secretion of proinflammatory cytokines such as IL-1β and IL-18, as well as the release of cellular contents. GSDMD plays a key role in pyroptosis. Therefore, pyroptosis is also defined as GSDM-mediated programmed necrotic cell death. Pyroptosis triggers strong inflammation by releasing inflammatory cytokines and danger molecules. Excessive pyroptosis can lead to various inflammatory diseases, especially age-related diseases, such as autoimmunity, autoinflammatory, chronic inflammatory and metabolic diseases, including gout, enteritis, sepsis, atherosclerosis, type II diabetes, and Alzheimer's disease (AD). Pyroptosis has both classical and non-classical pathways. After microbial infection, the classical pathway responds to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), while the non-classical pathway responds to intracellular lipopolysaccharide (LPS) of Gram-negative bacteria. The final step of pyroptosis requires the cleavage of GSDMD into N-terminus and C-terminus by Caspase1 and Caspase4 / 5 / 11 in the classical pathway. After cleavage, the N-terminus of GSDMD (GSDMD-N) forms a transmembrane pore that releases cytokines such as IL-1β and IL-18 and disrupts the regulation of ions and water, ultimately leading to strong inflammation and cell death. Since GSDMD is an effector of pyroptosis, inhibiting GSDMD is an effective strategy to inhibit inflammation.

[0003] Chuanwu, Caowu, and Fuzi are commonly used Chinese medicines in clinical practice. They have the effects of dispelling wind and dampness, warming the meridians and relieving pain. They are used for wind-cold dampness arthralgia, joint pain, cold pain in the heart and abdomen, cold hernia pain, and anesthesia and analgesia. They have a long history of medicinal use and high medicinal value. The main components of aconitine drugs are alkaloids, including monoester alkaloids, diester alkaloids, amino alcohol alkaloids and other types of compounds. Mesaconitine (MC), Hypoconitine (HC), Benzoylhypacoitine (BH), and Benzoylmesaconine (BM) are the main components of aconitine alkaloids. Modern pharmacological studies have shown that aconitine alkaloids can treat rheumatoid arthritis by inhibiting NF-κB and MAPk signaling pathways. In addition, aconitine alkaloids also have a good therapeutic effect on diseases such as neuropathic pain and hypertension, and can resist the occurrence of arrhythmias, providing options for better treatment of cardiovascular diseases. However, there are few studies on the effects of aconitum alkaloids on gout, atherosclerosis and other diseases, and there is no literature reporting that aconitum alkaloids can protect gout, atherosclerosis, Alzheimer's disease and other inflammatory diseases by inhibiting the GSDMD pathway. Therefore, aconitum alkaloids as potential therapeutic drugs for GSDMD-mediated cell pyroptosis-related diseases still have a gap in the existing technology and have good application prospects. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an application of aconitum alkaloids in the preparation of drugs for treating diseases related to GSDMD protein.

[0005] Furthermore, the aconite alkaloids exert their effects in the drug by inhibiting the activation of the GSDMD protein; specifically, by inhibiting the generation and activation of the NLRP3 inflammasome to improve the inflammatory response.

[0006] Furthermore, the aconite alkaloid is selected from at least one of mesaconitine (MC), hypaconitine (HC), benzoylhypaconitine (BH) and benzoylmeaconitine (BM).

[0007] Furthermore, the diseases associated with the GSDMD protein include gouty arthritis, ulcerative colitis, sepsis, atherosclerosis and Alzheimer's disease.

[0008] Furthermore, the gouty arthritis is induced by sodium urate crystals (MSU).

[0009] Furthermore, the ulcerative colitis is induced by DSS.

[0010] Furthermore, the sepsis is induced by LPS.

[0011] Furthermore, the atherosclerosis is induced by a high-fat diet.

[0012] Furthermore, the Alzheimer's disease is induced by the combination of D-galactose and β-amyloid protein.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] 1. Aconitum alkaloids have a strong inhibitory effect on GSDMD and are good GSDMD activation inhibitors.

[0015] 2. Aconitum alkaloids have strong pharmacological effects, indicating good medicinal prospects.

[0016] 3. Aconitum alkaloids have a wide range of sources, a simple preparation process, low preparation cost, and good drugability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing the effects of aconite alkaloids on GSDMD of BMDMs cells stimulated by MSU and Nigericin.

[0018] (A and E: Caspase-1 activity detection, B and F: ELISA detection of IL-1β, C and G: ELISA detection of NLRP3 level, D and H: ELISA detection of GSDMD level. Data are expressed as Mean ± SD, and statistical methods were used One-way ANOVA and unpaired Student's t test. Compared with the blank group, ### P<0.001; compared with the model group, *** P<0.001.)

[0019] Figure 2 This figure shows the effect of aconite alkaloids on NLRP3 inflammasome in MSU-induced gouty arthritis.

[0020] (A: toe swelling rate, BD: ELISA detection of IL-1β, IL-18 and NLRP3 levels. Data are expressed as Mean ± SD, and statistical methods were One-way ANOVA and unpaired Student's t test. Compared with the blank group, ### P<0.001; compared with the model group, *** P<0.001.)

[0021] Figure 3 A diagram showing the effect of aconitum alkaloids on NLRP3 inflammasome in DSS-induced colitis.

[0022] (A, B, D, E: ELISA detection of IL-1β and IL-6 levels in tissues and serum, C and F: NLRP3 levels in tissues and serum. Data are expressed as Mean ± SD, and statistical methods were One-way ANOVA and unpaired Student's t test. Compared with the blank group, ### P<0.001; compared with the model group, *** P<0.001.)

[0023] Figure 4 A diagram showing the effect of aconitum alkaloids on NLRP3 inflammasome in LPS-induced sepsis.

[0024] (AC: ELISA detection of IL-1β, IL-6 and NLRP3 levels in tissues, DF: mRNA levels of IL-1β, IL-6 and NLRP3 in tissues. Data are expressed as Mean ± SD, and the statistical methods were One-way ANOVA and unpaired Student's t test. Compared with the blank group, ### P<0.001; compared with the model group, ** P<0.01, *** P<0.001.)

[0025] Figure 5 This figure shows the effect of aconite alkaloids on NLRP3 inflammasome in high-fat diet-induced atherosclerosis.

[0026] (A: TC, B: TG, C: HDL-C, D: LDL-C, E and F: ELISA to detect serum IL-1β and NLRP3 levels. Data are expressed as Mean ± SD, and statistical methods were One-way ANOVA and unpaired Student's t test. Compared with the blank group, ### P<0.001; compared with the model group, *** P<0.001.)

[0027] Figure 6 This figure shows the effect of aconitum alkaloids on NLRP3 inflammasome in Alzheimer's disease induced by D-galactose + β-amyloid protein.

[0028] (A: escape latency time, B: number of times passing the original platform position, C: time staying in the original platform quadrant, DF: ELISA detection of IL-1β, IL-6 and NLRP3 levels in tissues. Data are expressed as Mean ± SD, and the statistical method was One-way ANOVA and unpaired Student's t test. Compared with the blank group, ### P<0.001; compared with the model group, ** P<0.01, *** P<0.001.) DETAILED DESCRIPTION

[0029] The applicant further describes the technical solution of the present invention in combination with specific embodiments and drawings below, but the scope of protection requested by the claims of the present invention is not limited to these embodiments.

[0030] M, nM, and μM appear after the Arabic numerals and refer to the units mol / L, nmol / L, and μmol / L.

[0031] 1. Instruments and Materials

[0032] 1.1 Instrument

[0033] Carbon dioxide incubator (Thermo Corporation, USA); microscope (Leica, Germany); clean bench (BioX, USA); electronic balance (METTLER TOLEDO, Switzerland); microplate reader (Bio Tek, USA); high-speed low-temperature centrifuge (Thermo Corporation, USA); toe volume meter (Chengdu Taimeng Software Co., Ltd.); Yuyue blood glucose meter 550 (Jiangsu Yuyue Medical Equipment Co., Ltd.); ABI 7500 real-time fluorescence quantitative PCR instrument (Applied Biosystems, USA).

[0034] 1.2 Materials

[0035] 1.2.1 Experimental animals

[0036] 6-8 week old SPF grade male C57BL / 6 mice, weight 20±2g, and adult SPF grade male SD rats, weight 200±20g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., and the experimental animal production license number is SCXK(Liao)2020-0001. 6-8 week old ApoE - / - Mice, weighing 20±2 g, were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0037] 1.2.2 Experimental cells

[0038] BMDMs were obtained from 6-8 week old C57BL / 6 mice.

[0039] 1.2.3 Main reagents

[0040] LPS, Nigericin, MSU, D-galactose, Aβ25-35, and DSS were all from Sigma-Aldrich, USA; Trizol kit was from TaKaRa, Japan; interleukin-1β (IL-1β), interleukin-18 (IL-18), and interleukin-6 (IL-6) ELISA kits were all from R&D systems, USA; Caspase-1 activity assay kit, NLRP3 inflammasome (NLRP3) ELISA kit, and GSDMD ELISA kit were all from Abcam; total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) kits were from Nanjing Jiancheng Biological Co., Ltd.; aconitine, hypaconitine, benzoylhypaconitine, and benzoylneaconitine were all from Shanghai Yuanye Biotechnology Co., Ltd.

[0041] 2. Experimental Methods

[0042] 2.1 BMDMs cell isolation and culture

[0043] 6-8 week old C57BL / 6 mice were killed by cervical dislocation and sterilized in 75% alcohol. The mice were placed in a clean bench, the femurs on both sides were cut and the attached muscles were peeled off. 2% FBS (prepared with 0.01M PBS) was drawn with a 1mL syringe to flush out the cells in the femur, blown evenly, and centrifuged. The mice were cultured in DMEM medium (containing 10% FBS, 1% penicillin / streptomycin, 25nM colony stimulating factor MCSF) and placed in CO 2 In a constant temperature incubator, differentiated BMDMs were obtained after 7 days of culture.

[0044] 2.2 BMDMs cell grouping and stimulation

[0045] 2.2.1 LPS+MSU stimulation

[0046] Take 5x10 differentiated BMDMs 4 Cells / well were inoculated in a 96-well plate and divided into 6 groups, with 6 replicate wells in each group. The specific operation is as follows:

[0047] (1) Blank group (Control): 200 μL Opti-MEM medium was added to each well and cultured for 3 h. Opti-MEM medium was then added to continue incubation for 6 h.

[0048] (2) Model group (MSU): 200 μL of Opti-MEM medium containing 200 ng / mL LPS was added to each well for stimulation for 3 h, then incubated in Opti-MEM medium for 30 min, and then Opti-MEM medium containing 200 μg / mL MSU was added for stimulation for 6 h.

[0049] (3) MSU+MC group: 200 μL of Opti-MEM medium containing 200 ng / mL LPS was added to each well for stimulation for 3 h, then incubated in Opti-MEM medium containing 10 μM MC for 30 min, and then Opti-MEM medium containing 200 μg / mL MSU was added for stimulation for 6 h.

[0050] (4) Hypoaconitine group (MSU+HC): 200 μL of Opti-MEM medium containing 200 ng / mL LPS was added to each well for stimulation for 3 h, then incubated in Opti-MEM medium containing 10 μM HC for 30 min, and then Opti-MEM medium containing 200 μg / mL MSU was added for stimulation for 6 h.

[0051] (5) MSU+BH group: 200 μL of Opti-MEM medium containing 200 ng / mL LPS was added to each well for stimulation for 3 h, then incubated in Opti-MEM medium containing 10 μM BH for 30 min, and then Opti-MEM medium containing 200 μg / mL MSU was added for stimulation for 6 h.

[0052] (6) MSU+BM group: 200 μL of Opti-MEM medium containing 200 ng / mL LPS was added to each well for stimulation for 3 h, then incubated in Opti-MEM medium containing 10 μM BM for 30 min, and then Opti-MEM medium containing 200 μg / mL MSU was added for stimulation for 6 h.

[0053] 2.2.2 LPS+Nigericin stimulation

[0054] Take 5x10 differentiated BMDMs 4 Cells / well were inoculated in a 96-well plate and divided into 6 groups, with 6 replicate wells in each group. The specific operation is as follows:

[0055] (1) Blank group (Control): 200 μL Opti-MEM medium was added to each well and cultured for 3 h. Opti-MEM medium was then added to continue incubation for 45 min.

[0056] (2) Model group (Nig): 200 μL of Opti-MEM medium containing 200 ng / mL LPS was added to each well for stimulation for 3 h, then incubated in Opti-MEM medium for 30 min, and then Opti-MEM medium containing 10 mM Nigericin was added for stimulation for 45 min.

[0057] (3) Nig+MC group: 200 μL of Opti-MEM medium containing 200 ng / mL LPS was added to each well for stimulation for 3 h, then incubated in Opti-MEM medium containing 10 μM MC for 30 min, and then Opti-MEM medium containing 10 mM Nigericin was added for stimulation for 45 min.

[0058] (4) Nig+HC group: 200 μL of Opti-MEM medium containing 200 ng / mL LPS was added to each well for stimulation for 3 h, then incubated in Opti-MEM medium containing 10 μM HC for 30 min, and then Opti-MEM medium containing 10 mM Nigericin was added for stimulation for 45 min.

[0059] (5) Nig+BH group: 200 μL of Opti-MEM medium containing 200 ng / mL LPS was added to each well for stimulation for 3 h, then incubated in Opti-MEM medium containing 10 μM BH for 30 min, and then Opti-MEM medium containing 10 mM Nigericin was added for stimulation for 45 min.

[0060] (6) Nig+BM group: 200 μL of Opti-MEM medium containing 200 ng / mL LPS was added to each well for stimulation for 3 h, then incubated in Opti-MEM medium containing 10 μM BM for 30 min, and then Opti-MEM medium containing 10 mM Nigericin was added for stimulation for 45 min.

[0061] 2.2.3 Caspase-1 activity assay

[0062] use 1 Inflammasome Assay kit was used to detect Caspase-1 in the cell supernatant of each group after MSU stimulation for 6h and Nigericin stimulation for 45min in 2.2, according to the instructions. Caspase-Glo 1 buffer, MG-132 and Z-WEHD fluorescein substrate were prepared into a 60μM fluorescence detection reagent and stored at -20℃. In a light-proof environment, 20μL of the cell supernatant to be tested was added to a 96-well plate, followed by 10μL of fluorescence detection reagent, and oscillated on an oscillator for 1min. Place at room temperature in a dark place for 50min and detect with an ELISA reader.

[0063] 2.2.4 ELISA determination of IL-1β, NLRP3, and GSDMD levels in cell supernatants

[0064] Take out the cell supernatant of each group after MSU stimulation for 6 hours and Nigericin stimulation for 45 minutes in 2.2 from -20℃, let it dissolve, and vortex to mix thoroughly. Take out the ELISA kit in advance, let it equilibrate at room temperature for 30 minutes, and perform the experiment according to the instructions, as follows:

[0065] (1) Dissolve the lyophilized powder first and dilute it into 8 concentrations in proportion to prepare the standard.

[0066] (2) Take out the pre-coated 96-well plate, add different concentrations of standards and samples, 50 μL per well, affix the sealing film, and place in a 37°C incubator for 90 min.

[0067] (3) Remove the well plate, discard the liquid in the wells, and dry on filter paper. Add 300 μL of diluted wash buffer to each well, wash on an oscillator for 1 min, and wash 5 times.

[0068] (4) Add the prepared enzyme reagent into the well plate, 100 μL per well, and place in a 37°C incubator for 30 min.

[0069] (5) Take out the plate and perform the same operation as (3). Add 100 μL of TMB colorimetric reagent to each well and place in a 37°C incubator for 15 min.

[0070] (6) Add 100 μL of stop reagent to each well and shake on an oscillator for 1 min. Detect the absorbance of each well with an ELISA reader at 450 nm.

[0071] 2.3 MSU-induced acute gouty arthritis model

[0072] 48 C57BL / 6 male mice were randomly divided into 6 groups, including blank group, model group, mesaconitine group, mesaconitine group, benzoyl mesaconitine group, and benzoyl mesaconitine group, with 8 mice in each group, and weighed and marked. The volume at 0.5 mm on the left hind limb ankle joint of the mouse was measured with a toe volume meter, and the measurement was repeated twice, and the average value was taken as the measurement value of 0h. Modeling method: The left hind ankle joint of the mouse was bent into a right angle to fully expose the gap between the ankle joint and the tibia and fibula. After local disinfection, 20 μL of 3.5% MSU needle crystal suspension was injected, and the blank control group was injected with an equal amount of normal saline. After injection of MSU, the hind ankle joint on the modeling side was observed. If there was obvious swelling, it indicated that the modeling was successful. 30 minutes after modeling, 20 μL of 10 μM corresponding drugs were injected into the ankle joints of mice in the aconitine group, hypaconitine group, benzoylhypaconitine group, and benzoylmeaconitine group.

[0073] (1) After modeling, the toe swelling volume of the test joint was measured at 3h, 6h, 9h, 12h, 24h, and 48h, and the swelling rate was calculated.

[0074] (2) After the relevant indexes of mice were tested, mice were killed by routine cervical dislocation, and joint effusion was extracted. 0.5 ml of saline was injected into the joint cavity with a syringe. The hind limbs of the mice were moderately flexed, extended, and rotated to mix the synovial fluid and saline. The lavage fluid was recovered by puncturing the outside of the trifurcation and centrifuged at 3000 r / min for 10 min. The supernatant was collected and the expression of inflammatory factors such as IL-1β, IL-18, and NLRP3 in the ankle joint fluid of each group of mice was detected by referring to the ELISA kit.

[0075] 2.4DSS-induced ulcerative colitis model

[0076] Forty-eight C57BL / 6 male mice were randomly divided into 6 groups: blank group, model group, mesaconitine group, mesaconitine group, benzoylmesaconitine group, and benzoylmesaconitine group, with 8 mice in each group. Modeling method: Except for the blank group drinking distilled water, the other groups freely drank 3% DSS solution (prepared with distilled water, prepared and used immediately), and replaced fresh DSS solution in time every day for 7 consecutive days to prepare the ulcerative colitis model. The occurrence of blood in the stool of mice indicated that the model was successfully established. Starting from the day of modeling, the mesaconitine group, mesaconitine group, benzoylmesaconitine group, and benzoylmesaconitine group were gavaged with 10μM of the corresponding drugs, and the administration volume was 1mL per 100g of mouse body weight. The blank group and model group were given an equal volume of 0.5% sodium carboxymethyl cellulose solution for 7 consecutive days.

[0077] (1) Record the weight changes of mice every day, observe the feces characteristics of mice (soft stool, mushy stool or watery stool), and detect fecal occult blood using the benzidine method. Complete the DAI score based on the changes in mouse feces and weight: 0 points: no weight loss, normal stool characteristics, no visible blood in the stool, and negative fecal occult blood; 1 point: 1% to 5% weight loss, loose stool, and positive fecal occult blood; 2 points: 5% to 10% weight loss, soft stool, and positive fecal occult blood; 3 points: 10% to 15% weight loss, mushy stool, and positive fecal occult blood; 4 points: >15% weight loss, watery stool, and visible blood in the stool.

[0078] (2) At the end of the experiment, mice in each group were killed, colon tissues were removed, and colon length was measured.

[0079] (3) After the experiment, the mice were anesthetized, blood was collected from the eye sockets, and the mixture was centrifuged at 3500 r / min for 100 min. The supernatant was collected, labeled according to the group, and placed in a -80°C refrigerator for later use. 20 mg of colon tissue was placed in a tissue homogenizer, 9 times the amount of PBS was added, and the mixture was ground on ice and centrifuged at 12000 r / min in a 4°C centrifuge for 20 min. The supernatant was collected and labeled according to the mouse group and placed at -80°C for later use. The levels of IL-1β, IL-6, and NLRP3 in serum and colon tissue homogenate supernatant were detected according to the instructions of the ELISA kit.

[0080] 2.5 Establishment of mouse sepsis model by LPS induction

[0081] 48 C57BL / 6 male mice were randomly divided into 6 groups: blank group, model group, meaconitine group, meaconitine group, benzoyl meaconitine group, and benzoyl meaconitine group, with 8 mice in each group. Modeling method: Except for the blank group, the other groups were injected with 200μL LPS (10mg / kg) by intraperitoneal injection, and the blank group was injected with the same volume of 0.9% saline. At the same time, the treatment group was given an intraperitoneal injection of 100μL of the corresponding group drug with a concentration of 10μM. The levels of proinflammatory cytokines (IL-1β, IL-6) in the serum of mice increased significantly, indicating that the modeling was successful.

[0082] (1) 20 hours after LPS injection, anesthetize mice, collect blood from the eye sockets, centrifuge at 3500r / min for 100min, collect the supernatant, label it according to the group, and place it in a -80℃ refrigerator for later use. After blood collection, the animals were killed, and 0.5mL PBS was injected into the left lung bronchus of each mouse. After repeated aspiration 3 times, BALF was collected, and the operation was repeated 3 times to ensure that the sample recovery rate exceeded 80% each time. Centrifuge at 12000r / min for 20min at 4℃. Divide the supernatant into small portions (0.1mL / portion) and store them at -80℃. Take the supernatant and label it according to the mouse group and place it at -80℃ for later use. Detect the levels of IL-1β, IL-6 and NLRP3 in BALF according to the instructions of the ELISA kit.

[0083] (2) Total RNA (1 μg) extracted from lung tissue using TRIzol reagent was reverse transcribed into cDNA using a cDNA reverse transcription kit. qRT-PCR was performed using GAPDH mRNA level as an internal reference to detect the expression of IL-1β, IL-6, and NLRP3 gene mRNA. IL-1β upstream primer: 5'-GGCAACTGTTCCTGAACTCAAC-3', downstream primer: 5'-CTGCGAGATTTGAAGCTGGATG-3'; IL-6 upstream primer: 5'-CTGCAAGAGACTTCCATCCA GT-3', downstream primer: 5'-GTTGGGAGTGGTATCCTCTGTG-3'; NLPR3 upstream primer: 5'-TTCT GGTATAGGGTCTGGAGCA-3', downstream primer: 5'-CTGAGGATATGATGGGAAGGGC-3'; GA PDH upstream primer: 5'-AATGGATTTGGACGCATTGGT-3', downstream primer: 5'-TTTGCACTGGT ACGTGTTGAT-3'. The reaction conditions of qRT-PCR were as follows: pre-denaturation at 95°C for 2 min; amplification cycle at 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min, for a total of 42 cycles; extension at 72°C for 5 min. Three replicates were performed for each sample, GAPDH was used as the internal reference primer, and the Ct value of the collected fluorescence signal was used to calculate the mRNA expression level of the relevant gene according to the relative quantitative method calculation formula.

[0084] 2.6 High-fat diet induces ApoE - / - Establishment of atherosclerosis model in mice

[0085] Eight 8-week-old WT mice were selected as the blank group, and 40 ApoE - / -Mice were randomly divided into a blank group, a model group, aconitine group, aconitine group, aconitine benzoyl aconitine group, and aconitine benzoyl aconitine group, with 8 mice in each group. Modeling method: The blank group was fed with ordinary feed, and the rest were given a high-fat diet (HFD). Ordinary feed (wheat flour 20%, raw bean cake 10%, corn flour 20%, standard flour 36%, salt 4%, eggshell powder 5%, egg 5%), high-fat diet (ordinary feed 82.3%, refined lard 10%, sugar 5%, cholesterol 2%, bile salt 0.5%, propylthiouracil 0.2%). After the mouse atherosclerosis model was successfully established by high-fat diet for 12 weeks, each drug group was intraperitoneally injected with 100 μL of the corresponding drug at a concentration of 10 μM every day for 6 consecutive weeks.

[0086] (1) In the sixth week of administration, mice were subjected to an insulin tolerance test (ITT). After fasting for 6 hours, 0.75 U / kg body weight of insulin was intraperitoneally injected. Blood glucose levels of mice were measured before insulin injection (0 min) and at 15, 30, 60, and 120 min after insulin injection.

[0087] (2) After 6 weeks of administration, mice were fasted for 12 hours, their eyeballs were removed and blood was collected. The blood was centrifuged at 3500 r / min for 15 minutes, and the serum was separated and stored at -80°C for later use. The levels of serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using an automatic microplate reader.

[0088] (3) The expression of IL-1β and NLRP3 inflammatory factors in the serum of each group of mice was detected by referring to the ELISA kit.

[0089] 2.7D-galactose + β-amyloid protein induced rats to establish Alzheimer's disease rat model

[0090] Forty-eight adult SD rats were randomly divided into 6 groups: blank group, model group, mesaconitine group, mesaconitine group, benzoylmesaconitine group, and benzoylmesaconitine group, with 8 rats in each group. D-galactose was injected intraperitoneally and aggregated Aβ was injected into the CA1 region of both hippocampi once. 25～35 Modeling method: Preparation of Alzheimer's disease rat model. Specific modeling method: Except for the blank group, all other groups were intraperitoneally injected with 150 mg / kg D-galactose for 6 consecutive weeks. In the 7th week, 4 nmol / L 0.9% saline-dissolved Aβ was injected into the bilateral hippocampus. 25～35The blank group was injected with an equal amount of saline subcutaneously for 6 consecutive weeks. In the 7th week, the equal amount of saline was injected bilaterally into the hippocampus. The model was verified by behavioral testing. After the model was successfully established, the drug-treated group was intraperitoneally injected with 100 μL of the corresponding drug at a concentration of 10 μM every day for 8 weeks.

[0091] (1) Each group of animals underwent Morris water maze behavioral tests on the 9th day of modeling. Positioning navigation test: The rats were placed in the water from two water entry points facing the pool wall, and the time it took to find the platform within 2 minutes (scape latency) and the swimming path were recorded. If the rats did not find the platform within 2 minutes, the latency was recorded as 120 seconds, and the rats were returned to the cage. Spatial exploration test: After the above tests were completed, the platform was removed and the rats were allowed to enter the water from any of the same water entry points. The swimming trajectory and time of the rats were recorded. The rats would cross the virtual platform within 2 minutes, and the frequency and the time they stayed in the platform quadrant were measured. The higher the recorded frequency and the longer the time they stayed in the platform, the stronger the learning and memory ability of the rats, and vice versa.

[0092] (2) The hippocampal tissue was ground with 0.9% saline at a ratio of 1:9 on ice and centrifuged at 12,000 r / min for 20 min in a 4°C centrifuge. The supernatant was placed at -80°C for later use. The levels of IL-1β, IL-6 and NLRP3 in the supernatant of the hippocampal tissue homogenate were detected according to the instructions of the ELISA kit.

[0093] 2.8 Statistics

[0094] Data are presented as Mean ± SD. When comparing the means of two groups, a two-tailed Student's t-test was used to determine significance. In the case of comparing multiple treatment groups or genotypes, one-way ANOVA and Tukey's test were used to calculate the significance between the means to calculate the P value. *P values ​​< 0.05 were considered statistically significant.

[0095] 3 Results

[0096] 3.1 Inhibitory effect of aconite alkaloids on GSDMD in BMDMs stimulated by MSU and Nigericin

[0097] To test the inhibitory effects of aconitum alkaloids on GSDMD, we used primary mouse bone marrow macrophages (BMDMs) to study their effects on caspase-1 activation, IL-1β secretion, and GSDMD. Figure 1ELISA was used to analyze the effects of aconitum alkaloids on Caspase-1 activation, IL-1β secretion, NLRP3 and GSDMD in LPS-activated BMDMs stimulated with MSU / Nigericin. The results showed that aconitum alkaloids could significantly inhibit Caspase-1, NLPR3 activation, IL-1β secretion, and the levels of NLRP3 and GSMDM (P < 0.001).

[0098] 3.2 Effects of aconite alkaloids on NLRP3 in MSU-induced gouty arthritis

[0099] MSU crystals are important inducers of gouty arthritis and are closely associated with NLRP3 inflammasome activation and mature IL-1β secretion. To evaluate the anti-inflammatory properties of aconitum alkaloids in vivo, we established a mouse model of acute gouty arthritis by injecting MSU crystals into the ankle joints of mice. MSU crystals can activate NLRP3 inflammasomes in synovial macrophages and induce ankle inflammation. Our results showed that aconitum alkaloids can effectively reduce ankle swelling ( Figure 2 A). ELISA results showed that aconite alkaloids could reduce the release of IL-1β and IL-18, and reduce the level of NLRP3 in ankle synovial macrophages ( Figure 2 BD). Aconite alkaloids can inhibit the generation and activation of NLRP3 inflammasomes, reduce the level of inflammatory factors, and more effectively alleviate inflammatory responses such as joint pain, swelling, and movement disorders in mice.

[0100] 3.3 Effects of aconite alkaloids on NLRP3 in DSS-induced ulcerative colitis

[0101] As shown in Table 1, compared with the blank group, the weight of DSS-induced mice decreased significantly after 7 days, while the groups treated with aconitum alkaloids significantly slowed down the weight loss of mice induced by DSS, and the colitis symptoms were milder. In addition, the DAI score (a comprehensive score representing weight loss, diarrhea, and rectal bleeding) of animals treated with aconitum alkaloids was also significantly reduced. Compared with the blank group, the colon length of the DSS group animals was significantly reduced, while the colon length of the aconitum alkaloid groups decreased less. Figure 3 The results showed that DSS induced an increase in the levels of IL-1β, IL-6, and NLRP3 in the serum and colon of mice, while the aconitine alkaloid treatment group significantly reduced the levels of IL-1β, IL-6, and NLRP3 in the serum and colon. Figure 3 AF). This indicates that aconitine alkaloids can improve the inflammatory state of colitis mice and inhibit the level of inflammation, and its mechanism of action is closely related to the regulation of NLRP3 inflammasome.

[0102] Table 1 Effects of aconite alkaloids on body weight, colon length and DAI score of mice with DSS-induced colitis.

[0103]

[0104] Note: Control: blank group, DSS: model group. Values ​​are expressed as mean ± SD. Statistical differences were calculated by one-way ANOVA and Tukey HSD test. Compared with blank: ### P<0.001; compared with the model: **P<0.01, ***P<0.001. 3.4 Effects of aconitum alkaloids on NLRP3 in LPS-induced sepsis

[0105] We examined the expression of inflammatory factors and NLRP3 in lung tissues and mRNA expression in LPS-induced sepsis. 20 hours after intraperitoneal injection of LPS, ELISA results showed that compared with the blank group, the expression of IL-1β, IL-6, and NLRP3 in the bronchoalveolar lavage fluid (BALF) of mice in the LPS group was significantly increased, while the expression of IL-1β, IL-6, and NLRP3 in the BALF of the aconitine alkaloid treatment group was significantly decreased ( Figure 4 AC). In addition, LPS injection significantly increased the mRNA levels of inflammatory genes in the lung tissue of mice, including IL-1β, IL-6, and NLRP3, while aconitine alkaloids inhibited the LPS-induced increase of these genes ( Figure 4 DF). This indicates that aconitine alkaloids can inhibit NLRP3 inflammasome and improve inflammatory response.

[0106] 3.5 Effects of aconite alkaloids on NLRP3 in high-fat diet-induced atherosclerosis

[0107] The ITT results showed (Table 2) that compared with the control group, the blood glucose concentration in the model group was significantly increased (P<0.05). Aconitine alkaloids can lower blood glucose. HC, BH, and BM had significant hypoglycemic effects at 15 minutes after insulin injection (P<0.05), and MC, HC, BH, and BM had significant effects at 120 minutes after insulin injection (P<0.05). After 6 weeks of drug intervention, compared with the blank group, the TC, TG, and LDL-C levels in the model group were significantly increased, and the HDL-C level was significantly decreased, and the differences were statistically significant (P<0.05). Compared with the model group, the TC, TG, and LDL-C levels in the aconitine alkaloids group were significantly decreased, and the HDL-C level was significantly increased, and the differences were statistically significant (P<0.05, Figure 5 AD). ELISA results showed that compared with the blank group, the levels of serum IL-1β and NLRP3 in the model group were significantly increased, and compared with the model group, the levels of IL-1β and NLRP3 in the aconitine alkaloid group were significantly decreased ( Figure 5E, F). The results showed that aconitine alkaloids can inhibit IL-1β secretion and the inflammatory response of atherosclerosis by regulating NLRP3 inflammasome, and have anti-inflammatory and anti-atherosclerotic effects.

[0108] Table 2 Effects of aconite alkaloids on glucose tolerance (ITT) in mice with high-fat diet-induced atherosclerosis.

[0109]

[0110] Note: Control: blank group, HFD: model group. Values ​​are expressed as mean ± SD. Statistical differences were calculated by one-way ANOVA and Tukey HSD test. Compared with blank: ### P<0.001; compared with the model group: *P<0.05, **P<0.01, ***P<0.001.

[0111] 3.6 Effects of aconite alkaloids on NLRP3 in Alzheimer's disease induced by D-galactose + β-amyloid protein

[0112] The results of the Morris water maze test showed that the blank group had significant differences (P<0.01) compared with the model group in terms of escape latency, the number of times the original platform position was passed, and the time the original platform quadrant was stayed. This indicated that the animals in the model group had obvious memory impairment and showed symptoms of dementia ( Figure 6 AC). The escape latency of the aconitum alkaloids treatment group was lower than that of the model group, and the number of times passing the original platform position and the time staying in the original platform quadrant were higher than those of the model group (P<0.05), and the symptoms of Alzheimer's disease were improved. ELISA results showed that the injection of D-galactose + β-amyloid protein in animals led to increased levels of IL-1β, IL-6 and NLRP3 in the hippocampus tissue of the brain, while the levels of IL-1β, IL-6 and NLRP3 in the aconitum alkaloids treatment group were significantly lower than those in the model group ( Figure 6 DF). This indicates that aconite alkaloids can improve the spatial learning and cognitive abilities of AD rats by inhibiting NLPR3 inflammasome.

Claims

1. Use of aconite alkaloids in the preparation of a drug for treating ulcerative colitis by targeting GSDMD protein, characterized in that: The aconitine alkaloid is selected from at least one of metaaconitine, hypaconitine, benzoylhypaconitine and benzoylmeaconitine.

2. The use according to claim 1, characterized in that The concentration of aconitum alkaloids in the drug for treating ulcerative colitis is 10 μM.

3. The use according to claim 1, characterized in that: The ulcerative colitis is induced by DSS.

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