CN115192574A

CN115192574A - Application of aconite alkaloids in preparing medicine for treating GSDMD related diseases

Info

Publication number: CN115192574A
Application number: CN202210519275.0A
Authority: CN
Inventors: 陈旅翼; 刘鹏; 张仲云
Original assignee: Zhenjiang Zhaoxu Jisheng Biotechnology Co ltd
Current assignee: Zhenjiang Zhaoxu Jisheng Biotechnology Co ltd
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2022-10-18
Anticipated expiration: 2042-05-12
Also published as: CN115192574B

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of aconite alkaloids in preparation of medicines for treating GSDMD (glutathione S-activated Diamond disease) related diseases. According to the invention, researches show that Mesaconitine (MC), hypaconitine (HC), benzoylhypaconine (BH) and Benzoylmesaconine (BM) can inhibit cell apoptosis induced by different agonists such as sodium urate crystal, lipopolysaccharide, nigericin and the like; can inhibit the activation of GSDMD protein in the preparation of models of gouty arthritis induced by MSU, ulcerative colitis induced by DSS, sepsis induced by LPS, atherosclerosis induced by high fat diet and senile dementia induced by D-galactose + beta-amyloid protein. Therefore, the aconite alkaloids as the active ingredient are expected to be developed into GSDMD inhibitors for inflammatory diseases such as gouty arthritis, ulcerative colitis, sepsis, atherosclerosis, senile dementia and the like.

Description

Application of aconite alkaloids in preparing medicine for treating GSDMD related diseases

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of aconite alkaloids in preparation of medicines for treating GSDMD (glutathione S-methyl diphosphate) related diseases.

Background

Apoptosis is a newly discovered type of programmed cell death and has received increasing attention in recent years. When the body is exposed to infection, once the "danger signal" is recognized, the innate immune system initiates a response that may lead to necrosis, apoptosis, or scorching of cells to kill the invading microorganisms. Cell apoptosis, which is inflammatory body and caspase-dependent lytic and inflammatory death, is characterized by plasma membrane rupture, cell swelling and lysis, mediated by the Gasdermin (GSDM) family of proteins. Finally, it leads to the secretion of pro-inflammatory factors such as IL-1. Beta. And IL-18, and the release of cellular contents. GSDMD plays a critical role in cell apoptosis. Thus, cellular apoptosis is also defined as GSDM-mediated apoptosis of necrotic cells. Pyro-death initiates intense inflammation by releasing inflammatory cytokines and danger molecules. Excessive cell apoptosis can lead to a variety of inflammatory diseases, particularly age-related diseases such as autoimmunity, autoinflammation, chronic inflammation and metabolic diseases including gout, enteritis, sepsis, atherosclerosis, type II diabetes, alzheimer's Disease (AD). There are typical and atypical pathways for coke death. Following 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 Lipopolysaccharides (LPS) of gram-negative bacteria. The final step of apoptosis requires cleavage of GSDMD into N-and C-termini by Caspase1 and Caspase4/5/11 in the classical pathway, after which the N-terminus of GSDMD (GSDMDM-N) forms a transmembrane pore which releases cytokines such as IL-1 β and IL-18 and disrupts regulation of ions and water, ultimately leading to strong inflammation and cell death. Since GSDMD is an effector of apoptosis, inhibition of GSDMD is a potent strategy to inhibit inflammation.

The common monkshood mother root, the kusnezoff monkshood root and the monkshood are common clinical traditional Chinese medicines, have the effects of dispelling wind and eliminating dampness and warming channels and relieving pain, are used for treating wind-cold-dampness arthralgia, cold pain in heart and abdomen, cold hernia pain, anesthesia and pain, have long medicinal history and high medicinal value. The main component of aconitine medicine is alkaloid, including monoester alkaloid, diester alkaloid, ammonia alcohol alkaloid and other compounds. Mesaconine (MC), hypaconine (HC), benzoylhypaconine (BH), benzoylmesaconine (BM) are the main components of aconitine. Modern pharmacological studies show that aconite alkaloids can treat rheumatoid arthritis by inhibiting NF-kB and MAPK signal pathways, and aconite alkaloids have good therapeutic effects on diseases such as neuropathic pain and hypertension, can resist arrhythmia, and provide a choice for better treating cardiovascular diseases. However, few research reports on aconite alkaloids for diseases such as gout, atherosclerosis and the like exist at present, and no literature reports that aconite alkaloids can play a role in protecting various inflammatory diseases such as gout, atherosclerosis, alzheimer disease and the like by inhibiting a GSDMD (glutathione S-methyl diphosphate) pathway. Therefore, the aconite alkaloids as potential therapeutic drugs for treating GSDMD mediated cell apoptosis related diseases still have blank in the prior art and have good application prospect.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide the application of the aconite alkaloids in preparing the medicines for treating the diseases related to the GSDMD protein.

Further, the aconite alkaloids play a role in medicines by inhibiting the activation of GSDMD protein; specifically by inhibiting the production and activation of NLRP3 inflammasome to ameliorate the inflammatory response.

Further, the aconite alkaloid is at least one selected from Mesaconine (MC), hypaconitine (HC), benzoylhypaconine (BH) and Benzoylmesaconine (BM).

Further, the diseases related to the GSDMD protein comprise gouty arthritis, ulcerative colitis, sepsis, atherosclerosis, senile dementia and the like.

Further, the gouty arthritis is induced to be pathogenic by sodium urate crystal (MSU).

Further, the ulcerative colitis is pathogenic induced by DSS.

Further, the sepsis is pathogenic by LPS induction.

Further, the atherosclerosis is induced by high fat diet.

Further, the senile dementia is caused by the combined induction of D-galactose and beta-amyloid.

Compared with the prior art, the invention has the advantages and beneficial effects as follows:

1. the aconite alkaloids have strong inhibitory effect on GSDMD, and are good GSDMD activation inhibitors.

2. The aconite alkaloids have strong pharmacological action, indicating good medicinal prospect.

3. The aconite alkaloids have wide source, simple preparation process, low preparation cost and good drug properties.

Drawings

FIG. 1 is a graph showing the effect of aconite alkaloids on GSDMDM cells of BMDMs under the stimulation of MSU and Nigericin.

(A and E: caspase-1 activity assay, B and F: ELISA for IL-1. Beta., C and G: ELISA for NLRP3 level, D and H: ELISA for GSDMDM level data are expressed as Means + -SD, statistical methods using One-way ANOVA and unpaired student t-test, compare to blank, ^### P<0.001; in comparison to the set of models, ^*** P<0.001。)

FIG. 2 is a graph of the effect of aconitine alkaloids on NLRP3 inflammasome in MSU-induced gouty arthritis.

(A: toe swelling Rate, B-D: ELISA for IL-1. Beta., IL-18 and NLRP3 levels data are expressed as Means + -SD, and statistical methods were performed using One-wayANOVA and notPaired student t-test. In comparison to the blank set, the data is, ^### P<0.001; in comparison to the set of models, ^*** P<0.001。)

FIG. 3 is a graph of the effect of aconitine alkaloids on NLRP3 inflammasome in DSS-induced colitis.

(A, B, D, E: ELISA for IL-1. Beta. And IL-6 levels in tissues and serum, C and F: NLRP3 levels in tissues and serum. Data are expressed as Means + -SD, statistical methods using One-way ANOVA and unpaired student's t-test, compare to blank, ^### P<0.001; in comparison to the set of models, ^*** P<0.001。)

FIG. 4 is a graph of the effect of aconitine alkaloids on the NLRP3 inflammasome in LPS-induced sepsis.

(A-C: ELISA for IL-1 β, IL-6 and NLRP3 levels in tissues, D-F: mRNA levels for IL-1 β, IL-6 and NLRP3 in tissues. Data are expressed as Means + -SD, statistical methods using One-wayANOVA and unpaired student t-test, compare to blank, ^### P<0.001; in comparison to the set of models, ^** P<0.01， ^*** P<0.001。)

FIG. 5 is a graph of the effect of aconitine alkaloids on NLRP3 inflammasome in high fat diet induced atherosclerosis.

(A: TC, B: TG, C: HDL-C, D: LDL-C, E and F: ELISA for IL-1 β and NLRP3 levels in serum data expressed as Means + -SD, statistical methods using One-wayANOVA and unpaired student t-test, comparison to blank, ^### P<0.001; in comparison to the set of models, ^*** P<0.001。)

FIG. 6 is a graph of the effect of aconitine alkaloids on NLRP3 inflammasome in D-galactose + beta-amyloid induced Alzheimer's disease.

(A: escape latency time, B: number of passes over the position of the original platform, C: time of stay of quadrant of the original platform, D-F: ELISA for detecting the levels of IL-1 β, IL-6 and NLRP3 in the tissues, expressed as Means + -SD, statistical methods using One-way ANOVA and unpaired student's t-test, compare to the blank group, ^### P<0.001; in comparison to the set of models, ^** P<0.01， ^*** P<0.001。)

Detailed Description

The following embodiments and drawings are used to further describe the technical solution of the present invention, but the claimed scope of the present invention is not limited to these embodiments.

M, nM, μ M appear after the Arabic numerals and refer to the units mol/L, nmol/L, μmol/L.

1. Apparatus and materials

1.1 instruments

Carbon dioxide incubator (Thermo corporation, usa); microscope (Leica, germany); clean bench (bio x corporation, usa); electronic balance (METTLER TOLEDO, switzerland); microplate reader (Bio Tek, usa); high speed cryogenic centrifuges (Thermo corporation, usa); toe volume measurement (chengdotai alli software limited); fish jumping blood glucose meter 550 (Jiangsu fish jumping medical devices, inc.); ABI 7500 real-time fluorescence quantitative PCR instrument (applied biosystems, USA).

1.2 materials

1.2.1 Experimental animals

SPF level male C57BL/6 mice of 6-8 weeks old, the body mass is 20 +/-2 g, adult SPF level male SD rats, the body mass is 200 +/-20 g, the male SD rats are purchased from Liaoning Biotechnology GmbH, and the production license number of the experimental animal is SCXK (Liao) 2020-0001. ApoE 6-8 weeks old ^-/- A mouse, 20 + -2 g in physical mass, was provided by Beijing Wintolite laboratory animal technology, inc.

1.2.2 Experimental cells

BMDMs were taken from 6-8 week old C57BL/6 mice.

1.2.3 Primary reagents

LPS, nigericin, MSU, D-galactose, abeta 25-35, DSS are all from Sigma-Aldrich, USA; trizol kit is from TaKaRa, japan; interleukin-1 beta (IL-1 beta), interleukin-18 (IL-18), interleukin-6 (IL-6) ELISA kits were from R & D systems, USA; caspase-1 activity assay kit, NLRP3 inflammasome (NLRP 3) ELISA kit and GSDMD ELISA kit are all from Abcam company; total Cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) kit is from Nanjing Biotechnology Ltd; sinaconitine, hypaconitine, benzoylhypaconine, and benzoylmesaconine are all from Shanghai-sourced leaf Biotech limited.

2. Experimental methods

2.1 BMDMs cell isolation and culture

6-8 week old C57BL/6 mice were sacrificed by cervical dislocation and sterilized in 75% alcohol. The mouse was placed in a clean bench, the femur on both sides was cut off and the adhered muscle was peeled off, the intrafemoral cells were flushed out by sucking 2% FBS (0.01 MPBS formulation) with a 1mL syringe, pipetting uniformly, and centrifuging. It was cultured in DMEM medium (containing 10% FBS,1% penicillin/streptomycin, 25nM colony stimulating factor MCSF) and placed in CO ₂ A constant temperature incubator. After 7 days of culture, differentiated BMDMs cells were obtained.

2.2 BMDMs cell grouping and stimulation

2.2.1 LPS + MSU stimulation

Taking differentiated BMDMs at 5x10 ⁴ Individual cells/well were seeded in 96-well plates, which were divided into 6 groups of 6 replicates. The specific operation is as follows:

(1) Blank group (Control): 200 μ of LOpti-MEM medium was added to each well, incubated for 3h, and supplemented with Opti-MEM medium for additional incubation for 6h.

(2) Model group (MSU): after adding 200. Mu.L of Opti-MEM medium containing 200ng/mL LPS per well for 3h of stimulation, the wells were incubated in Opti-MEM medium for 30min, and then added Opti-MEM medium containing 200. Mu.g/mL MSU for 6h of stimulation.

(3) Neoaconitine group (MSU + MC): after adding 200. Mu.L of Opti-MEM medium containing 200ng/mL LPS per well for 3h of stimulation, incubation was performed for 30min in Opti-MEM medium containing 10. Mu.M MC followed by adding Opti-MEM medium containing 200. Mu.g/mL MSU for 6h of stimulation.

(4) Hypaconitine group (MSU + HC): after adding 200. Mu.L of Opti-MEM medium containing 200ng/mL LPS per well for 3h, incubation was performed for 30min in Opti-MEM medium containing 10. Mu.M HC, followed by addition of Opti-MEM medium containing 200. Mu.g/mL MSU for 6h.

(5) Benzoylhypaconine group (MSU + BH): after adding 200. Mu.L of Opti-MEM medium containing 200ng/mL LPS per well for 3h of stimulation, incubation was performed in Opti-MEM medium containing 10. Mu.M BH for 30min, followed by adding Opti-MEM medium containing 200. Mu.g/mL MSU for 6h of stimulation.

(6) Benzoylmesaconine group (MSU + BM): after adding 200. Mu.L of Opti-MEM medium containing 200ng/mL LPS per well for 3h, incubation was performed for 30min in Opti-MEM medium containing 10. Mu.M BM, followed by adding Opti-MEM medium containing 200. Mu.g/mL MSU for 6h.

2.2.2 LPS + Nigericin stimulation

(1) Blank group (Control): 200 μ of LOpti-MEM medium was added to each well, incubated for 3h, and supplemented with Opti-MEM medium for further incubation for 45min.

(2) Model group (Nig): after adding 200 μ L of Opti-MEM medium containing 200ng/mL LPS to each well for 3h, the wells were incubated in Opti-MEM medium for 30min, and then added with Opti-MEM medium containing 10mM Nigericin for 45min.

(3) Sinoaconitine group (Nig + MC): after adding 200. Mu.L of Opti-MEM medium containing 200ng/mL LPS per well for 3h, incubation was performed for 30min in Opti-MEM medium containing 10. Mu.M MC followed by adding Opti-MEM medium containing 10mM Nigericin for 45min.

(4) Hypaconitine group (Nig + HC): after adding 200. Mu.L of Opti-MEM medium containing 200ng/mL LPS per well for 3h, incubation was performed for 30min in Opti-MEM medium containing 10. Mu.M HC, followed by adding Opti-MEM medium containing 10mM nigericin for 45min.

(5) Benzoylhypaconine group (Nig + BH): after stimulating for 3h by adding 200. Mu.L of Opti-MEM medium containing 200ng/mL LPS to each well, the wells were incubated for 30min in Opti-MEM medium containing 10. Mu.M BH, and then stimulated for 45min by adding Opti-MEM medium containing 10mM Nigericin.

(6) Benzoylmesaconine group (Nig + BM): after adding 200. Mu.L of Opti-MEM medium containing 200ng/mL LPS per well for 3h, incubation was performed for 30min in Opti-MEM medium containing 10. Mu.M BM, followed by adding Opti-MEM medium containing 10mM Nigericin for 45min.

2.2.3 Caspase-1 Activity assay

Using Caspase-

The 1 Infmasome Assay kit detects Caspase-1 in the supernatant of each group of cells after 6h of MSU stimulation and 45min of Nigericin stimulation in 2.2 according to the instruction. Caspase-Glo 1 buffer solution, MG-132 and Z-WEHD fluorescein substrate are prepared into a fluorescence detection reagent with the concentration of 60 mu M, and the fluorescence detection reagent is stored at the temperature of minus 20 ℃. In a dark environment, 20. Mu.L of cell supernatant to be detected is added into a 96-well plate, and then 10. Mu.L of fluorescence detection reagent is added, and the mixture is shaken for 1min on a shaker. Standing at room temperature in dark for 50min, and detecting with enzyme labeling instrument.

2.2.4 ELISA (enzyme-Linked immuno sorbent assay) for determining IL-1 beta, NLRP3 and GSDMD contents of cell supernatant

Taking out cell supernatants of each group after MSU stimulation for 6h and Nigericin stimulation for 45min in 2.2 from-20 ℃, dissolving, and sufficiently and uniformly mixing by vortex. The ELISA kit was taken out in advance, equilibrated at room temperature for 30min, and tested as indicated in the specification, as follows:

(1) The freeze-dried powder is dissolved and diluted into 8 concentrations according to the proportion to prepare a standard substance.

(2) And (3) taking out the pre-coated 96-well plate, adding standard substances and samples with different concentrations, attaching a sealing plate film to each well by 50 mu L, and putting the plate film into a 37 ℃ incubator for incubation for 90min.

(3) The well plate was removed, the well liquid was discarded and dried on filter paper. Add diluted wash buffer, 300. Mu.L per well, rinse for 1min on shaker, 5 times.

(4) The prepared enzyme reagent is added into the pore plate, each pore is 100 mu L, and the mixture is put into an incubator at 37 ℃ for incubation for 30min.

(5) The well plate was removed and the same procedure as in (3) was carried out. Adding TMB color reagent, 100 μ L per well, and incubating in 37 deg.C incubator for 15min.

(6) Stop reagent was added at 100. Mu.L per well and shaken for 1min on a shaker. The plate was then examined for absorbance at 450nm using a microplate reader.

2.3 MSU-induced acute gouty arthritis model

Taking 48C 57BL/6 male mice, randomly dividing the animals into 6 groups, a blank group, a model group, a mesaconine group, a hypaconitine group, a benzoylhypaconine group and a benzoylmesaconine group, wherein each group comprises 8 animals, and weighing and marking the animals. The volume of the left hind limb ankle joint of the mouse at the position of 0.5mm is measured by a toe volume measuring instrument, the measurement is repeated for 2 times, and the average value is taken as the measured value of 0 h. The molding method comprises the following steps: the left posterior ankle of the mouse was bent at a right angle to sufficiently expose the gap between the ankle and the tibiofibula, and after local sterilization, 20 μ L of 3.5% MSU needle crystal suspension was injected, and the blank control group was injected with the same amount of physiological saline. After MSU injection, the posterior ankle joint on the side of the model is observed, and if obvious swelling exists, the success of the model is shown. 30min after molding, injecting 20 μ L10 μ M corresponding drug into ankle joint of mouse with sinoaconitine group, hypaconitine group, benzoylhypaconine group, and benzoylmesaconine group.

(1) After the model is built, the toe swelling volume of the tested joints 3h,6h,9h,12h,24h and 48h is measured, and the swelling rate is calculated.

(2) And (3) killing the mice by conventional cervical dislocation after the detection of relevant indexes of the mice is finished, extracting joint effusion, injecting 0.5ml of normal saline into a joint cavity by using an injector, mixing the synovial fluid with the normal saline by properly bending and rotating the hind limbs of the mice, puncturing and recovering lavage fluid from the outer side of the trifurcations, centrifuging at 3000r/min for 10min, collecting supernatant, and detecting the expression of inflammation related factors such as IL-1 beta, IL-18, NLRP3 and the like in the ankle joint fluid of each group of mice by referring to an ELISA kit.

2.4 DSS (direct sequence-induced plasticity) induction establishment of ulcerative colitis model

48C 57BL/6 male mice were randomized into 6 groups: blank group, model group, mesaconine group, hypaconitine group, benzoylhypaconine group, benzoylmesaconine group, each group contains 8 individuals. The molding method comprises the following steps: the ulcerative colitis model was prepared by drinking 3% DSS solution (distilled water preparation, ready-to-use preparation) freely in each of the remaining groups except the blank group with distilled water, replacing the fresh DSS solution every day in time, and drinking continuously for 7 d. The appearance of hematochezia in the mice indicates that the molding is successful. On the same day of molding, the neoaconitine group, the hypaconitine group, the benzoylhypaconine group and the benzoylmesaconine group were separately administered with 10 μ M of the corresponding drugs by gavage, the administration volume was 1mL per 100g of the mouse body weight, and the equal volume of 0.5% sodium carboxymethylcellulose solution was administered to the blank group and the model group for 7 days.

(1) The change of the body weight of the mice is recorded every day, the fecal characters (soft stool, pasty stool or watery stool) of the mice are observed, and the fecal occult blood condition is detected by a benzidine method. Finishing DAI scoring according to the change of the feces and body weight of the mice, wherein the score is 0: the weight is not reduced, the stool character is normal, no naked eye bloody stool exists, and the stool is negative in occult blood; 1 minute: the body mass is reduced by 1 to 5 percent, the stool is loose, and the stool is positive in occult blood; and 2, dividing: the body mass is reduced by 5 to 10 percent, the stool is soft and has positive occult blood; and 3, dividing: the body mass is reduced by 10 to 15 percent, and the stool is positive in occult blood; and 4, dividing: the body mass decreased by >15%, watery stool, and macroscopic bloody stool.

(2) At the end of the experiment, each group of mice was sacrificed, colon tissue was removed, and the colon length was measured.

(3) After the experiment is finished, anesthetizing the mouse, collecting blood in the eye socket, centrifuging for 100min at 3500r/min, collecting supernatant, marking according to component package, and placing in a refrigerator at-80 ℃ for later use. 20mg of colon tissue is taken and put into a tissue homogenizer, 9 times of PBS is added to the tissue homogenizer for grinding on ice, then the mixture is centrifuged for 20min at 12000r/min in a 4 ℃ centrifuge, and the supernatant is taken and placed at minus 80 ℃ for standby after being subpackaged and marked according to the group of mice. IL-1. Beta. IL-6 and NLRP3 levels in serum and colon tissue homogenate supernatants were assayed according to ELISA kit instructions.

2.5 LPS (lipopolysaccharide) induced establishment of mouse sepsis model

48C 57BL/6 male mice were randomized into 6 groups: blank group, model group, mesaconine group, hypaconitine group, benzoylhypaconine group, benzoylmesaconine group, each group contains 8 individuals. The molding method comprises the following steps: each of the groups was subjected to a single intraperitoneal injection of 200. Mu. LLPS (10 mg/kg) except for the blank group, which was injected with the same volume of 0.9% saline. At the same time, 100 μ L of the corresponding group of drugs with the concentration of 10 μ M was given to the treatment group by intraperitoneal injection. The level of proinflammatory factors (IL-1 beta and IL-6) in the serum of the mice is obviously increased, which indicates that the modeling is successful.

(1) 20h after LPS injection, anesthetizing the mouse, collecting blood from the orbit, centrifuging at 3500r/min for 100min, collecting supernatant, marking according to component, and placing in a refrigerator at-80 ℃ for later use. After blood collection, animals are sacrificed, 0.5mL PBS is injected into the left lung bronchus of each mouse, after 3 times of repeated suction, BALF is collected, and the operation is repeated for 3 times, so that the recovery rate of each sample is ensured to exceed 80%. Centrifuging at 12000r/min for 20min at 4 deg.C. Dividing the supernatant into small parts (0.1 mL/part), storing at-80 deg.C, collecting supernatant, labeling according to mouse group, and standing at-80 deg.C for use. IL-1. Beta., IL-6 and NLRP3 levels in BALF were determined according to ELISA kit instructions.

(2) Total RNA (1. Mu.g) extracted from lung tissue by TRIzol reagent was reverse-transcribed into cDNA using cDNA reverse transcription kit. qRT-PCR was performed using mRNA level of GAPDH as an internal control to detect the expression of mRNA of IL-1. Beta., IL-6, NLRP3 genes. IL-1 β upstream primer: 5 'GGCAACTGTTCCTGAACTCAAC-3', downstream primer: 5 'CTGCGAGATTTGAAGCTGGATG-3'; IL-6 upstream primer: 5 'CTGCAAGACTTCCATCCAGT-3', downstream primer: 5 'GTTGGGAGTGGTATCCTCTGTG-3'; NLPR3 upstream primer: 5 'TTCTGGTATAGGGTCTGGAGCA-3', a downstream primer: 5 'CTGAGGATATATGATGGGAAGGGC-3'; GAPDH upstream primer: 5 'AATGGATTTGGACGCATTGGTT-3', a downstream primer: 5 'TTTGCACTGTACGTGTTGAT-3'. The reaction conditions were set for qRT-PCR as follows: pre-denaturation at 95 ℃ for 2min; amplification cycles of 95 ℃ 30s,60 ℃ 1min,72 ℃ 1min for a total of 42 cycles; extension at 72 ℃ for 5min. 3 wells are prepared for each sample, GAPDH is used as an internal reference primer, and the expression level of the related gene mRNA is calculated according to a calculation formula of a relative quantitative method by using the Ct value of the collected fluorescence signal.

2.6 high fat diet induced ApoE ^-/- Mouse establishment of atherosclerosis model

8 WT mice of 8 weeks of age were used as a blank group, and 40 ApoE mice of the same week of age ^-/- The mice are randomly divided into a blank group, a model group, a mesaconine group, an hypaconitine group, a benzoylhypaconine group and a benzoylmesaconine group, and each group contains 8 mice. The molding method comprises the following steps: the blank group was routinely fed with normal diet, and the remainder were given High Fat Diet (HFD). Common feed (wheat flour 20%, raw bean cake 10%, corn flour 20%, standard flour 36%, table salt 4%, eggshell powder 5%, egg 5%), high fat feed (common feed 82.3%, refined lard 10%,Sugar 5%, cholesterol 2%, bile salt 0.5%, propylthiouracil 0.2%). After a mouse atherosclerosis model is successfully established after high-fat diet for 12 weeks, 100 mu L of corresponding medicine with the concentration of 10 mu M is respectively injected into the abdominal cavity of each administration group every day, and the administration is continuously carried out for 6 weeks.

(1) On the 6 th week of administration, mice received an insulin resistance test (ITT), fasted for 6h, and were intraperitoneally injected with insulin at a quality of 0.75U/kg. Blood glucose was measured before (0 min) and 15, 30, 60 and 120min after insulin injection, respectively.

(2) After administration for 6 weeks, mice are fasted for 12h, the eyeballs are picked up and blood is taken, centrifugation is carried out at 3500r/min for 15min, and serum is separated and stored for standby under the condition of 80 ℃ below zero. Serum Total Cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and Low density lipoprotein cholesterol (LDL-C) levels were measured by a fully automatic enzyme scale.

(3) And (3) detecting the expression of IL-1 beta and NLRP3 inflammatory factors in the serum of each group of mice by referring to an ELISA kit.

2.7 Method for establishing senile dementia rat model by inducing rats through D-galactose + beta-amyloid

48 adult SD rats were taken and randomized into 6 groups: blank group, model group, mesaconine group, hypaconitine group, benzoylhypaconine group, benzoylmesaconine group, and 8 groups. Adopts intraperitoneal injection of D-galactose and bilateral hippocampal CA1 area to inject aggregated Abeta at one time _25～35 A molding method, preparing a rat model with senile dementia. The specific molding method comprises the following steps: in each group except the blank group, 150mg/kg of D-galactose was intraperitoneally injected, and 4nmol/L of 0.9% normal saline-dissolved Abeta was bilaterally injected into the hippocampus at 6 weeks and 7 weeks continuously _25～35 . Injecting equal amount of normal saline subcutaneously in the blank group, injecting equal amount of normal saline into hippocampus on both sides for 6 weeks and 7 weeks, using behavior verification model, and injecting 100 μ L of corresponding drug with concentration of 10 μ M into abdominal cavity every day for 8 weeks after model building is successful.

(1) Each group of animals was subjected to Morris water maze behavioural test on day 9 of molding. Positioning navigation test: rats were placed in water facing the pool wall from 2 entry points, respectively, and the time (Sca latency) and swimming path for finding the platform within 2min were recorded. If the rat did not find the platform at 2min, the latency was recorded as 120s and the rat was returned to the cage. And (3) space exploration test: after the above experiment was completed, the platform was removed and the rats were launched from any of the same water entry points, the swimming trajectory and time of the rats were recorded, and within 2min the rats crossed the virtual platform and the frequency and residence time in the platform quadrant were measured. The higher the frequency recorded, the longer the residence time in the platform, the stronger the learning and memory ability of the rat, and conversely the worse.

(2) The hippocampal tissue is ground with 0.9% normal saline on ice according to a ratio of 1. IL-1. Beta., IL-6 and NLRP3 levels were measured in the supernatant of hippocampal homogenates according to the ELISA kit instructions.

2.8 statistical data

Data are expressed as Means ± SD. In comparing the mean of the two groups, a two-tailed student's t-test was used to determine significance. In the case of comparing multiple treatment groups or genotypes, significance between the means was calculated using one-way analysis of variance and Tukey test to calculate P-value. * P values <0.05 were considered statistically significant.

3 results

3.1 inhibitory Effect of Aconitum alkaloids on GSDMD in MSU and Nigericin stimulated BMDMs

To test the inhibitory effect of aconitoids on GSDMD, we used primary mouse bone marrow macrophages (BMDMs) to study their effects on Caspase-1 activation, IL-1 β secretion, and GSDMD. As shown in figure 1, in BMDMs initiated by LPS, the influence of aconite alkaloids on Caspase-1 activation, IL-1 beta secretion, NLRP3 and GSDMD when stimulated by MSU/Nigericin is analyzed by ELISA, and the result shows that the aconite alkaloids can obviously inhibit Caspase-1, NLPR3 activation, IL-1 beta secretion and the levels of NLRP3 and GSMDM (P is less than 0.001).

3.2 Effect of Aconitum alkaloids on NLRP3 in MSU-induced gouty arthritis

MSU crystals are important inducers of gouty arthritis and are closely associated with NLRP3 inflammasome activation and mature IL-1 beta secretion. To assess the anti-inflammatory properties of aconite alkaloids in vivo, we established a mouse model of acute gouty arthritis by injecting MSU crystals into the mouse ankle. MSU crystals can activate NLRP3 inflammasome in synovial macrophages and induce ankle inflammation. Our findings indicate that aconite alkaloids are effective in reducing ankle swelling compared to MSU group mice (figure 2A). ELISA results showed that aconite alkaloids decreased IL-1 β and IL-18 release, and decreased level of ankle synovial macrophage NLRP3 (FIGS. 2B-D). The aconite alkaloids can inhibit the generation and activation of NLRP3 inflammasome, reduce the level of inflammatory factors, and more effectively relieve the inflammatory reactions of mouse such as joint pain, swelling, dyskinesia and the like.

3.3 Effect of Aconitum alkaloids on NLRP3 in DSS-induced ulcerative colitis

As shown in table 1, compared with the mice in the blank group, the weight of the mice induced by DSS was significantly reduced after 7 days, while the administration of aconite alkaloid-treated group significantly slowed the weight reduction of the mice induced by DSS, and showed less colitis symptoms. In addition, the DAI score (a composite score representing weight loss, diarrhea and rectal bleeding) was also significantly reduced in aconite alkaloid treated animals. Compared with the blank group, the length of the colon of the animals in the DSS group is obviously reduced, and the length of the colon of each group of aconite alkaloids is reduced less. FIG. 3 shows that DSS induced elevated IL-1 β, IL-6 and NLRP3 levels in mouse serum and colon, while the aconitine group treated with alkaloids all significantly reduced IL-1 β, IL-6 and NLRP3 levels in serum and colon (FIGS. 3A-F). The aconitine alkaloid is proved to have the functions of improving the inflammatory state of the colitis mouse and inhibiting the inflammatory level, and the action mechanism of the aconitine alkaloid is closely related to the regulation and control of NLRP3 inflammasome.

Table 1 effect of aconite alkaloids on DSS-induced colitis mice body weight, colon length, DAI score.

Note: control: blank group, DSS: and (4) model groups. Values are expressed as mean ± SD. Statistical differences were calculated by one-way analysis of variance and Tukey HSD test. Compared with the blank: ^### P<0.001; compared with the model: * P<0.01，***P<0.001。

3.4 Effect of Aconitum alkaloids on LPS-induced sepsis

We examined LPS-induced expression of inflammatory factors and NLRP3 and mRNA expression in sepsis lung tissue. After 20h of intraperitoneal injection of LPS, ELISA results show that compared with a blank group, the expressions of IL-1 beta, IL-6 and NLRP3 in alveolar lavage fluid BALF of mice in the LPS group are obviously increased, while the expressions of IL-1 beta, IL-6 and NLRP3 in BALF are obviously reduced in an aconitine alkaloid treated group (figures 4A-C). In addition, LPS injection significantly increased the mRNA levels of inflammatory genes including IL-1 β, IL-6, NLRP3 in lung tissue of mice, while aconitine alkaloids inhibited LPS-induced elevation of these genes (FIGS. 4D-F). The aconitine alkaloid can inhibit NLRP3 inflammasome and improve inflammatory reaction.

3.5 Effect of Aconitum alkaloids on NLRP3 in high fat diet-induced atherosclerosis

The ITT results show (table 2) that compared with the control group, the blood glucose concentration of the model group is significantly increased (P < 0.05), aconitine alkaloids can reduce blood glucose, HC, BH, and BM have significant blood glucose reducing effects at 15min of insulin injection (P < 0.05), and MC, HC, BH, and BM have significant blood glucose reducing effects at 120min of insulin injection (P < 0.05). After 6 weeks of drug intervention, the model group had significantly increased levels of TC, TG and LDL-C, significantly decreased levels of HDL-C, and the differences were statistically significant (P < 0.05), as compared to the blank group. Compared with the model group, the levels of TC, TG and LDL-C in the aconitine alkaloid group are obviously reduced, the level of HDL-C is obviously increased, and the differences have statistical significance (P <0.05, figures 5A-D). ELISA results showed that the serum IL-1. Beta. And NLRP3 levels of the model group were significantly increased compared to the blank group, and the IL-1. Beta. And NLRP3 levels of the aconitine alkaloid group were significantly decreased compared to the model group (FIGS. 5E and F). The result shows that aconitine alkaloid can inhibit IL-1 beta secretion and inhibit inflammatory reaction of atherosclerosis by regulating NLRP3 inflammasome, and has anti-inflammatory and anti-atherosclerosis effects.

Table 2 aconite alkaloids are resistant to high fat diet induced glucose tolerance (ITT) in atherosclerotic mice.

Note: control: blank set, HFD: and (4) model groups. Values are expressed as mean ± SD. Statistical differences were calculated by one-way anova and Tukey HSD test. Compared with the blank: ^### P<0.001; compared to the model group: * P is<0.05，**P<0.01，***P<0.001。

3.6 Effect of Aconitum alkaloids on NLRP3 in D-galactose + beta-amyloid induced Alzheimer's disease

The results of the Morris water maze experiment show that the blank group has very obvious difference (P is less than 0.01) compared with the model group no matter the escape latency, the times of passing the original platform position or the residence time of the original platform quadrant, and the model group animals have obvious memory impairment and show the symptoms of dementia (figures 6A-C). The time of the aconite alkaloid treatment group in the escape latency period is lower than that of the model group, the times of passing through the original platform position and the time of staying in the original platform quadrant are higher than those of the model group (P < 0.05), and the senile dementia symptom is improved. ELISA results showed that injection of D-galactose + amyloid-beta protein in animals resulted in elevated IL-1 β, IL-6 and NLRP3 levels in hippocampal tissues of brain, whereas IL-1 β, IL-6 and NLRP3 levels in the aconitine-treated group were significantly reduced compared to the model group (FIGS. 6D-F). It is demonstrated that aconite alkaloids can improve spatial learning and cognitive ability of AD rats by inhibiting NLPR3 inflammasome.

Claims

1. Use of aconite alkaloids in preparing medicine for treating GSDMD protein related diseases, wherein the aconite alkaloids are selected from at least one of mesaconine, hypaconitine, benzoylhypaconine and benzoylmesaconine; the disease related to the GSDMD protein is gouty arthritis, ulcerative colitis, sepsis, atherosclerosis and/or senile dementia.

2. The use of an aconitine alkaloid according to claim 1in the preparation of a medicament for the treatment of disorders associated with GSDMD protein, wherein said gouty arthritis is pathogenic induced by crystallization of sodium urate.

3. Use of an aconitine alkaloid according to claim 1 for the preparation of a medicament for the treatment of disorders associated with the protein GSDMD, wherein ulcerative colitis is induced by DSS.

4. Use of an aconitine alkaloid according to claim 1in the manufacture of a medicament for the treatment of disorders associated with the GSDMD protein wherein the sepsis is LPS induced pathogenic.

5. Use of an aconitine alkaloid according to claim 1 for the preparation of a medicament for the treatment of disorders associated with GSDMD protein, wherein said atherosclerosis is induced pathogenic by high fat diet.

6. Use of an aconitine alkaloid according to claim 1 for the preparation of a medicament for the treatment of disorders related to GSDMD protein, wherein said senile dementia is caused by the combination of D-galactose and beta-amyloid.