Application of oridonin in preparation of anti-neuritis medicine

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of oridonin in preparation of anti-neuritis medicines. The oridonin disclosed by the invention can be used for inhibiting neuroinflammation and inhibiting programmed death of neuronal cells, so that neurons are protected. Chemical structural formula of oridonin is as follows: # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to the application of oridonin in the preparation of anti-neuritis drugs. Background Art

[0002] Neuroinflammation generally refers to an inflammatory response in the central nervous system caused by various pathological damages such as infection, trauma, ischemia, and toxins. The hallmark of this process is the production of pro-inflammatory cytokines, including IL-1β, IL-6, TNFα, CCL5, CXCL10, CCL3, and CCL4, etc. The innate immune cells involved in this process are mainly microglia and astrocytes.

[0003] Currently, drugs for treating neuroinflammation include anti-inflammatory drugs such as mecobalamin tablets, oryzanol tablets, doxycycline hydrochloride tablets, and amoxicillin capsules. However, the long-term use of these drugs causes adverse reactions in the gastrointestinal tract: loss of appetite, nausea, vomiting, diarrhea, etc., and also easily causes fatigue, rash, etc.

[0004] Traditional Chinese medicine is a type of drug with high safety and few side effects traditionally. Therefore, it is of great significance to explore the new functions of existing traditional Chinese medicine in the preparation of drugs for treating neuritis. Summary of the Invention

[0005] To explore the new functions of existing traditional Chinese medicine in the preparation of drugs for treating neuritis, the present invention provides the application of oridonin in the preparation of anti-neuritis drugs. The oridonin of the present invention can inhibit neuroinflammation and inhibit the programmed death of neuron cells, thereby protecting neurons.

[0006] The present invention provides the application of oridonin in the preparation of anti-neuritis drugs, and the chemical structural formula of the oridonin is as follows:

[0007]

[0008] The oridonin provided by the present invention can inhibit neuroinflammation and inhibit the programmed death of neuron cells, thereby protecting neurons, and can be used to prepare anti-neuritis drugs.

[0009] Further, the anti-neuritis drug uses oridonin as the only effective active ingredient.

[0010] Further, the drug also includes pharmaceutically acceptable carriers, excipients, diluents, and adjuvants.

[0011] Further, the carrier, excipient, diluent, and auxiliary materials include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, arabic gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil.

[0012] Further, the drug is a solution of oridonin with a concentration of 8 mM to 12 mM.

[0013] Further, the drug is a solution of oridonin with a concentration of 10 mM.

[0014] Further, the oridonin is used for preparing a therapeutic drug for Parkinson's disease caused by neuritis.

[0015] Further, the neuritis is induced by LPS.

[0016] Further, the oridonin is used for preparing an inhibitor of programmed neuronal cell death.

[0017] Further, the oridonin is used for preparing an inhibitor of the expression of inflammatory factors TNFα, IL-6, IL-1β, CCL5, CXCL10, CCL3, and CCL4 mRNA.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The present invention verifies that oridonin can inhibit neuroinflammation and inhibit programmed neuronal cell death, thereby protecting neurons. It can be used for preparing anti-neuritis drugs.

[0020] Neuroinflammation and neuronal death play key roles in the pathogenesis of Parkinson's disease. Oridonin exhibits its neuroprotective effect by reducing neuroinflammation and neuronal death, which helps to alleviate muscle tremors, stiffness, bradykinesia, balance disorders, and cognitive decline caused by Parkinson's disease. Therefore, it can be regarded as a potential new treatment option. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1To detect the secretion of inflammatory factors in BV2 cells after LZ stimulation and Pon administration by ELISA, expressed as the expression rate; and to detect the expression of mRNA of related inflammatory factors and inflammatory mediators in BV2 cells after LZ stimulation and administration by RT-qPCR;

[0023] In the figure, A shows the expression rate of TNFα detected by ELISA;

[0024] B shows the expression rate of IL-6 detected by ELISA;

[0025] C shows the expression of TNFα mRNA detected by RT-qPCR;

[0026] D shows the expression of IL-6 mRNA detected by RT-qPCR;

[0027] E shows the expression of IL-1β mRNA detected by RT-qPCR;

[0028] F shows the expression of CXCL10 mRNA detected by RT-qPCR;

[0029] G shows the expression of CCL5 mRNA detected by RT-qPCR;

[0030] H shows the expression of CCL4 mRNA detected by RT-qPCR;

[0031] I shows the expression of CCL3 mRNA detected by RT-qPCR.

[0032] Figure 2 To detect the mRNA expression of inflammatory factors and related inflammatory mediators in the tissue near the pinhole in the hippocampus of the mouse brain by RT-qPCR;

[0033] In the figure, A shows the expression of TNFα mRNA detected by RT-qPCR;

[0034] B shows the expression of IL-6 mRNA detected by RT-qPCR;

[0035] C shows the expression of IL-1β mRNA detected by RT-qPCR;

[0036] D shows the expression of CCL4 mRNA detected by RT-qPCR;

[0037] E shows the expression of CCL5 mRNA detected by RT-qPCR;

[0038] F shows the expression of CCL3 mRNA detected by RT-qPCR;

[0039] G shows the expression of CXCL10 mRNA detected by RT-qPCR;

[0040] H is the expression of JAK1 mRNA detected by RT-qPCR;

[0041] I is the expression of STAT3 mRNA detected by RT-qPCR;

[0042] Figure 3 It is the result of the JAK-STAT signaling pathway and the expression of inflammatory proteins in the tissue near the pinhole in the hippocampus of the mouse brain after stereotactic injection of LPS into the hippocampus of C57BL / 6J mice detected by Western blot.

[0043] Figure 4 It is the effect of Pon on the HT22 cell line of hippocampal neurons in mice with programmed necrosis;

[0044] In the figure, A is the effect of Pon on the detection result of lactate dehydrogenase;

[0045] B is the effect of Pon on the ATP level;

[0046] Figure 5 It is the result detected by flow cytometry after TNFα and Z-VAD were used to induce programmed necrosis in the HT22 cell line of mouse hippocampal neurons. The Q2 region represents the percentage of dead cells, and Pon can significantly reduce the cell necrosis caused by TZ;

[0047] In the figure, A is the result detected by flow cytometry. The Q1 region represents the Hoechst - PI + cell population, the Q2 region represents the Hoechst + PI + cell population, the Q3 region represents the Hoechst + PI - cell population, and the Q4 region represents the Hoechst - PI - cell population;

[0048] B is the proportion of the Q2 + cell population in different experimental groups of cells;

[0049] Figure 6 It is the result of shooting and statistics by a high-content live cell imaging system after Hoechst and PI staining at different time gradients of modeling the HT22 cell line with TZ;

[0050] In the figure, A is the staining result at 0, 2, 4, 6, 8, 10, and 12 h after modeling with TZ in different experimental groups;

[0051] B is the Hoechst counted by the high-content live cell imaging system+ PI - Percentage of cells

[0052] C is the content of lactate dehydrogenase in the cell culture supernatant used for staining.

[0053] Figure 7 For modeling the HT22 cell line with TZ, the changes in protein expression in different experimental groups detected by Western blotting.

[0054] Figure 8 Results of the protein expression of proteins mediating programmed necrosis in the tissue near the pinhole in the hippocampus of the mouse brain after stereotaxic injection of LPS into the hippocampus of C57BL / 6J mice detected by Western blotting. Detailed implementation manners

[0055] The following is a detailed description of the specific implementation manners of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0056] Example 1: Application of oridonin in the preparation of anti-neuritis drugs.

[0057] I. Test methods

[0058] The reagent consumables and instrument information used in the tests of the present invention are shown in Table 1 and Table 2 respectively.

[0059] Table 1 Reagent consumables

[0060]

[0061]

[0062]

[0063] Table 2 Instruments and models

[0064]

[0065] 1. Inhibitory effect of oridonin (Pon) on the inflammatory response of microglia caused by LPS and Z-VAD (LZ).

[0066] (1) Isolation and extraction of oridonin

[0067] Oridonin was purchased from Hangzhou TargetMol Biotechnology Co., Ltd., and the product number is T2S1975.

[0068] (2) Co-stimulate microglial (BV2) cells with LPS and Z-VAD (LZ)

[0069] The mouse microglial cell line BV2 cell strain used in this experiment was purchased from the ATCC Cell Resource Center, and the product number is CBP60922.

[0070] ① Resuscitation of BV2 cells:

[0071] After taking out the cryopreserved BV2 cells from the liquid nitrogen tank, quickly place them in a 37 °C water bath for thawing. After thawing, transfer the cell suspension to a 3 mL DMEM complete medium, centrifuge at 1000 rmp for 3 min, discard the supernatant, add 1 mL of fresh DMEM complete medium, gently pipette and mix well, transfer the cell suspension to a 10 cm cell culture dish containing 6 mL of DMEM complete medium, use the cross-shaking method to evenly mix the cells, and then place them in a 37 °C, 5% CO2 cell culture incubator.

[0072] ② Culturing and subculturing of BV2 cells:

[0073] The condition for cell subculture is that the confluence of BV2 cells reaches about 90%-95%. When the subculture condition is met, take out the cell culture dish from the incubator, discard the cell culture medium, add PBS to wash the cells, discard the PBS, then add 0.125% trypsin, digest in the incubator for 90 s, add 2 times the volume of DMEM complete medium to terminate digestion, pipette the cells down, centrifuge the cell suspension at 1000 rpm for 3 min, discard the supernatant, add fresh DMEM complete medium, and subculture at a ratio of 1:2 - 1:3.

[0074] ③ Cryopreservation of BV2 cells:

[0075] The condition for cell cryopreservation is that its confluence reaches about 90%-95%. The cell digestion and centrifugation steps are the same as those for cell subculture. After centrifugation, discard the supernatant and add 900 μL of DMEM complete medium, 100 μL of DMSO for cryopreserving the cells. After adding DMSO, quickly pipette and mix well, then transfer to a cell cryopreservation tube, place the cell cryopreservation tube in a cell cryopreservation box and put it in an -80 °C refrigerator for gradient cooling, and transfer to the liquid nitrogen tank for storage after 24 h.

[0076] ④ Cell grouping:

[0077] The cells were divided into three groups, namely the control group, the LPS+Z-VAD group (LZ group), and the LPS+Z-VAD+Pon group (LZ+Pon group);

[0078] Control group: Every 100 μL system (DMEM complete medium + 1‰ DMSO);

[0079] LPS + Z-VAD group (LZ group): Add LPS to every 100 μL system (DMEM complete medium + 1‰ DMSO) to make its final concentration 1 μg / mL, add Z-VAD with a final concentration of 20 μM, and treat for 18 h;

[0080] LPS + Z-VAD + Pon group (LZ + Pon group): Add LPS to every 100 μL system (DMEM complete medium + 1‰ DMSO) to make its final concentration 1 μg / mL, add Z-VAD with a final concentration of 20 μM, add Pon with a final concentration of 10 μM, and treat for 18 h.

[0081] The Pon added in the above system is the Pon stock solution with a concentration of 10 mM, which is obtained by dissolving oridonin (Pon) in DMSO solvent to a concentration of 10 mM.

[0082] (3) Detect inflammatory factors TNFα and IL-6 by enzyme-linked immunosorbent assay (ELISA)

[0083] In a 96-well plate, seed 1×10 4 BV2 cells in each well. After 12 h, the dosing groups are dosed with a final concentration of 10 μM. After 2 h, stimulate with LPS with a final concentration of 1 μg / mL and Z-VAD with a final concentration of 20 μM. After 18 h, collect the cell supernatant and detect IL-6 and TNFα by ELISA; One day before the detection, prepare the coated antibody working solution according to the ratio of coated antibody: coated diluent (1:250), add 100 μL of the coated antibody working solution to each well, and incubate overnight at 4°C; Wash the coated wells 3 times with the washing solution; Add 200 μL of the assay diluent to each well and block at room temperature for 1 h; After washing 3 times with the washing solution, add 100 μL of the standard product or cell culture supernatant (the TNFα detection supernatant is diluted 25×, and the IL-6 detection supernatant is diluted 50×), and incubate at room temperature for 2 h; After washing 5 times with the washing solution, for IL-6 detection, prepare the enzyme-labeled antibody working solution according to the ratio of antibody: detection diluent (1:500), enzyme: detection diluent (1:250), and for TNFα detection, prepare the enzyme-labeled antibody working solution according to the ratio of antibody: detection diluent (1:250), enzyme: detection diluent (1:250). Add 100 μL of the enzyme-labeled antibody working solution to each well and incubate at room temperature for 1 h; After washing 7 times with the washing solution, add 100 μL of the substrate solution and incubate at room temperature in the dark for 30 min; Add 50 μL of the termination solution to terminate the reaction, and detect the absorbance of each well at 450 nm and 570 nm in the ABS mode of the microplate reader. The expression rate in data processing = absorbance value of each group / absorbance value of the LZ group.

[0084] 2. Molecular-level study on the inhibitory effect of oridonin on microglial inflammatory response.

[0085] (1) Detection of mRNA expression of inflammation-related factors IL-6, TNFα and related inflammatory mediators by RT-qPCR.

[0086] ① The resuscitation, cryopreservation, subculture and culture of BV2 cells, as well as the grouping, were the same as above.

[0087] ② RNA extraction: Seed 1×10 6 BV2 cells in a 3-cm culture dish. After 12 h, administer Pon at 10 μM. After 1 h, stimulate with 1 μg / mL LPS + 20 μM Z-VAD. After 4 h, discard the cell culture supernatant, wash once with PBS, then add RNAisolater Total RNA Extraction Reagent and let it stand on ice for 5 min to lyse the cells, then pipette and collect; add chloroform at 1 / 5 volume to the lysate, vortex for 15 s to form an emulsion, and let it stand at 4℃ for 5 min; centrifuge at 12000g at 4℃ for 15 min; take out the centrifuge tube, aspirate the upper aqueous phase into a new centrifuge tube, add an equal volume of pre-cooled isopropanol, invert and mix well, and let it stand at 4℃ for 20 min; centrifuge at 12000g at 4℃ for 10 min, and usually a white precipitate can be seen; aspirate and discard the supernatant, add 1 ml of 75% ethanol, flick the bottom of the tube to suspend the precipitate, and invert several times, then let it stand at room temperature for 3 - 5 min; centrifuge at 12000g at 4℃ for 5 min, discard the supernatant; dry in the open air at room temperature in a clean environment for 2 - 5 min; add 25 μL of enzyme-free water to dissolve, and detect the RNA concentration by Nanodrop.

[0088] ③ Reverse transcribe RNA into cDNA, and detect the mRNA expression of inflammatory factors IL-6, TNFα and related inflammatory mediators by RT-qPCR.

[0089] 3. Inhibitory effect of oridonin on neuroinflammation in mice.

[0090] (1) Use stereotaxic injection of LPS into the hippocampal region of the brain to establish a neuroinflammation model in mice, and study the anti-neuroinflammatory activity of oridonin in vivo. The specific steps are as follows:

[0091] Twenty-four male C57BL / 6J mice (8 weeks old, 20 - 22 g) were purchased from Beijing Sparf Bioscience Co., Ltd. After the C57BL / 6J mice entered the laboratory animal room, they were raised under the conditions of sufficient food and water supply, a temperature of 22 ± 2 °C, a humidity of 60 ± 5%, and a 12 h light / 12 h dark cycle. All animal operations involved in this experiment were approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine. The mice were randomly divided into 4 groups: control group, LPS group, LPS + Pon (5 mg / kg) group, and LPS + Pon (10 mg / kg) group. After continuous intraperitoneal injection for 7 days, stereotaxic injection of LPS into the brain was performed. Prepare 1% sodium pentobarbital, at a dose of 0.2 mL / 10 g, and inject the mice according to their body weight. If there is no response when pinching the mouse's tail hard, it means the mouse has been deeply anesthetized and the next step can be carried out. Use a small animal hair clipper to shave the hair on the mouse's head. After shaving, hair removal cream can be used to further remove the hair. Fix the mouse with prepared skin on the operating table: First, insert the ear rods into the two ear holes of the mouse, adjust the position of the ear rods so that the heights and the depths of insertion of the two ear rods are the same. Hold the mouse's tail and rotate it up and down to ensure that the ear rods have fixed the mouse's head. Then, fix the mouse's front teeth on the bite bar, and adjust the positions of the bite bar and the ear rods so that the mouse's head is on the same plane. Wipe the mouse's head with alcohol for disinfection, use a sterile surgical scissors to cut open the skin on the mouse's head, and then use forceps to bluntly separate the mouse's head tissues to expose the anterior fontanelle, posterior fontanelle, and skull of the mouse. Then, use a sterile cotton swab dipped in physiological saline to gently wipe the skull to make the anterior fontanelle and posterior fontanelle clearer. Open the stereotaxic apparatus, first position the syringe at the anterior fontanelle and zero it. Move the syringe to the left and right respectively. If the difference in the Z-axis readings of the syringe on the left and right is less than or equal to 0.03 mm, it can be considered that the left and right are leveled. Then move the syringe to the posterior fontanelle. If the difference in the Z-axis reading of the syringe is less than or equal to 0.03 mm, it can be considered that the front and back are leveled. Position the syringe at the dentate gyrus area of the mouse hippocampus (AP: -1.7 mm, ML: -1.2 mm, DV: -2.0 mm), mark it with a marker pen, and then use a cranial drill to make a small hole at the marked position for inserting the needle. The insertion depth of the needle is 2 mm. Inject 5 μg / 2 μL of LPS into each mouse at a rate of 0.2 μL / min. After the injection is completed, keep the needle in place for 5 min, and then seal the small hole with bone wax and suture the wound. The blank control was injected with physiological saline under the same conditions.

[0092] 4. Oridonin inhibits neuroinflammation in vivo.

[0093] The expression levels of inflammatory factors in the mouse brain were detected.

[0094] (1) Preparation of protein samples

[0095] First, add RIPA lysis buffer (containing 1% protease inhibitor and phosphatase inhibitor) to the tissue according to the ratio of 10 mg of tissue corresponding to 600 μL of RIPA lysis buffer. Use a tissue grinder at 40 Hz for 2 min, three times for grinding and crushing. After crushing, add 5×SDS-PAGE protein loading buffer according to the ratio of tissue lysate:5×SDS-PAGE protein loading buffer of 4:1, and then place it in a 99°C metal bath and boil for 40 min.

[0096] (2) Western blot

[0097] Gel preparation: First, wipe and clean the experimental tools such as glass plates and combs with a sponge, then spray a large amount of alcohol and dry. Add 4 mL of lower layer solution, 4 mL of lower layer buffer, and 160 μL of coagulant accelerator for pouring the gel to complete the preparation of the lower layer gel. Add 2 mL of lower layer solution, 2 mL of lower layer buffer, and 20 μL of coagulant accelerator, pour the gel and then insert the comb.

[0098] Protein electrophoresis: After clamping the glass plate in the card slot, pour electrophoresis buffer into the two-layer glass plate, pull out the comb and then perform sample loading. After sample loading, supplement the electrophoresis buffer in the electrophoresis tank, set the voltage of the electrophoresis instrument to 150 v for 70 min to complete electrophoresis.

[0099] Protein transfer: Pre-cool the transfer buffer at 4°C in advance, activate the PVDF membrane by putting it into methanol. Pry open the two glass plates from the gap, cut off the upper layer gel, wash the gel with running water, pour the transfer buffer into a porcelain dish, soak the transfer sandwich in the transfer buffer, assemble it in the order of sponge, filter paper, gel, PVDF membrane, filter paper, sponge, roll out the bubbles with a centrifuge tube, clamp the sandwich and put it into the card slot. After filling the slot with transfer buffer, perform transfer at 300 mA for 2.5 h. The whole transfer process is carried out in an environment of 4°C.

[0100] Blocking: Wash the transferred PVDF membrane once with 0.1% TBST, and then block it at room temperature in the blocking solution for 30 min.

[0101] Incubate with primary antibody: Put the strip into the primary antibody and incubate overnight at 4°C.

[0102] Wash the membrane after primary antibody incubation: Wash it four times with 0.1% TBST, each time placing it on a shaker with the maximum rotation speed for 5 min.

[0103] Incubate with secondary antibody: Incubate at room temperature for 2 h.

[0104] Wash the membrane after secondary antibody incubation: Wash it five times with 0.1% TBST, each time placing it on a shaker with the maximum rotation speed for 5 min.

[0105] Development: Prepare ECL luminescent liquid A: ECL luminescent liquid B in a ratio of 1:1. Pay attention to avoid light. Use filter paper to absorb the liquid on the membrane as much as possible, place it on the developing plate, evenly add the luminescent working solution, and develop it through a developer.

[0106] Table 3 Protein electrophoresis buffer

[0107] Glycine 1.44％ 14.4g SDS powder 0.1％ 1g Tris base 0.3％ 3.03g <![CDATA[ddH2O]]> - Volume made up to 1 L

[0108] Table 4 Protein transfer buffer

[0109] Glycine 0.9％ 9g Tris base 0.193％ 1.93g <![CDATA[ddH2O]]> - Volume made up to 1 L

[0110] Table 5 Membrane washing buffer

[0111] Tris base 0.12％ 2.425g NaCl 0.4％ 4g <![CDATA[ddH2O]]> - Volume made up to 1 L Concentrated hydrochloric acid pH adjusted to 7.6

[0112] (4) Genome extraction

[0113] Animal tissue was directly added into RNA isolater Total RNA Extraction Reagent at a ratio of 10 mg to 500 μL, and ground using a tissue grinder at 40 Hz for 2 min, 3 times. After the crushing was completed, the tissue was placed on ice for 30 min for lysis, and the subsequent operation was the same as that of cells.

[0114] (5) cDNA synthesis

[0115] Reverse transcribe the extracted RNA, first remove the genomic DNA: calculate the amount of RNA required (1000ng) and add 2μl of 5×gDNA wiper Mix, make up the volume to 10μL with dd H2O, and mix it gently with a pipette. 42℃2min. Prepare the first-strand cDNA synthesis reaction solution according to Table 6. After the preparation is completed, mix it gently with a pipette. Add it to the solution prepared in the first step. Perform the first-strand cDNA synthesis reaction: 50℃15min, 85℃5s, 4℃∞. After the reaction is completed, use Nanodiop to detect the concentration.

[0116] Table 6 cDNA synthesis reaction solution

[0117] The mixture from the previous step 10 μL 10X RT Mix 2 μL HiScript III Enzyme Mix 2 μL Oligo(dT)20VN 1 μL Random hexamers 1 μL <![CDATA[RNase-free ddH2O]]> 4 μL

[0118] (6) Real-time fluorescence quantitative PCR

[0119] The reaction solution was prepared according to Table 7, the reaction was carried out according to the procedure in Table 8, and real-time monitoring was performed using QuantStudio7. The primer sequences refer to Table 9.

[0120] Table 7 qPCR reaction solution

[0121]

[0122] Table 8 qPCR reaction procedure

[0123]

[0124] Table 9 qPCR primers

[0125]

[0126] II. Test results

[0127] 1. Inhibitory effect of oridonin on microglial inflammatory response caused by LZ

[0128] The results are as Figure 1 shown. Compared with the control group, LZ significantly increased the expression of TNFα and IL-6 in BV2 cells, and the oridonin treatment group significantly inhibited the expression of TNFα and IL-6 induced by LZ ( Figure 1 a of Figure 1 and b of

[0129] ). To further verify whether oridonin can inhibit the inflammatory response of microglia, the expression levels of TNFα, IL-6, IL-1β and related chemokines CCL5, CXCL10, CCL3, CCL4 mRNA in BV2 cells after LZ stimulation were detected in this example.

[0130] The results are as Figure 1 shown. Compared with the control group, LZ significantly increased the expression of TNFα, IL-6, IL-1β and chemokines CCL-5, CXCL10, CCL3, CCL4 mRNA in BV2 cells, and the oridonin treatment group significantly inhibited the increase of TNFα, IL-6, IL-1β, CCL5, CXCL10, CCL3, CCL4 mRNA induced by LZ. These results indicate that oridonin can inhibit the microglial inflammatory response caused by LZ.

[0131] 2. Study on the signaling pathway of oridonin inhibiting microglial inflammatory response

[0132] The results are as Figure 2 shown. Compared with the control group, the expression of pRIPK1, NLRP3, pJAK1 and pSTAT3 was significantly increased after LZ stimulation, while the oridonin treatment group significantly inhibited the expression of pRIPK1, NLRP3, pJAK1 and pSTAT3 after LZ stimulation.

[0133] The results indicate that oridonin inhibits microglial activation by inhibiting the RIPK1-JAK1-STAT3 signaling pathway, thereby reducing the expression of related inflammatory factors such as TNFα.

[0134] 3. Inhibitory effect of oridonin on neuroinflammation in mice.

[0135] In this example, the expression levels of inflammatory factors TNFα, IL-6, IL-1β and related chemokines CCL5, CXCL10, CCL3, CCL4 mRNA in the brains of mice after stereotaxic injection of LPS were detected.

[0136] The results are as Figure 3 shown. Compared with the control group, the expression levels of inflammatory-related factors TNFα, IL-6, IL-1β and chemokines CCL-5, CXCL10, CCL3, CCL4 and JAK1, STAT3 mRNA in the model group were significantly increased. The oridonin treatment group significantly inhibited the increase in the expression of inflammatory-related factors TNFα, IL-6, IL-1β and chemokines CCL-5, CXCL10, CCL3, CCL4 and JAK1, STAT3 mRNA after LPS stimulation. The results indicate that oridonin also has a good inhibitory effect on neuroinflammation in vivo.

[0137] 4. Related signaling pathways of oridonin inhibiting neuroinflammation in vivo.

[0138] By detecting the expression levels of proteins related to the JAK-STAT pathway, an inflammatory-related pathway in the brains of mice. The results are as Figure 3 shown. Compared with the control group, after intracerebral injection of LPS, it would cause an increase in the expression levels of inflammatory-related proteins NLRP3, JAK1, JAK2, pSTAT3, pSTAT1, TNFα, IL-1β. The oridonin treatment group significantly inhibited the increase in the expression levels of inflammatory-related proteins NLRP3, JAK1, JAK2, pSTAT3, pSTAT1, TNFα, IL-1β. The results show that oridonin can also inhibit neuroinflammation in vivo, and it may act by affecting the JAK-STAT pathway.

[0139] Example 2: Application of oridonin in the preparation of drugs against neuronal necrosis.

[0140] I. Test materials

[0141] Cells: Mouse hippocampal neuron cell line HT22 cells, purchased from Auspicious Biotechnology (Shanghai) Co., Ltd. The cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum in a 37°C incubator with 5% CO2 concentration. Cells in the logarithmic growth phase were selected for various tests.

[0142] Animals: Male C57BL / 6J mice (8 weeks old, 20 - 22 g) were purchased from Beijing SPF Biotechnology Co., Ltd. After the C57BL / 6J mice entered the laboratory animal room, they were raised under the conditions of sufficient food and water supply, a temperature of 22 ± 2 °C, a humidity of 60 ± 5%, and a 12 h light / 12 h dark cycle. All animal operations involved in this experiment were approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine.

[0143] II. Test Methods

[0144] 1. LDH Detection

[0145] In a 96-well plate, 8×10 3 HT22 cells were seeded in each well. After 18 h, the Pon administration group was given Pon at a concentration of 2.5 μM. After 1 h, the cells were stimulated with TNF (10 ng / mL) + Z-VAD (20 μM). After 8 h, the cell supernatant was collected; according to the required amount of INT solution (1X), an appropriate amount of INT solution (10X) was diluted to 1X with INT diluent; the LDH detection working solution was prepared according to the ratio of 10 μl of INT solution (1X), 10 μl of lactic acid, and 10 μl of enzyme (per well). 30 μL of the LDH detection working solution and 60 μL of the cell culture supernatant were added to each well. After reacting for 30 min in the dark on a shaker, the absorbance at 490 nm and 888 nm was measured using the ABS mode of an enzyme-linked immunosorbent assay reader. The detection system is shown in Table 10.

[0146] Table 10 LDH Detection System

[0147]

[0148]

[0149] 2. CTG Detection

[0150] The cell treatment conditions were the same as those for LDH detection. The administration concentration was 0.675 μM. 10 μL of CTG detection reagent was added to 100 μL of the cell culture system, and the cells were lysed by shaking on a shaker for 15 min. 75 μL of the supernatant was transferred to a white opaque enzyme-linked immunosorbent assay plate, and colorimetry was performed using the chemiluminescence mode of an enzyme-linked immunosorbent assay reader.

[0151] 3. PI and Hoechst Staining

[0152] The cell treatment conditions were the same as those for LDH detection. The cell culture supernatant was discarded, and the cells were washed once with PBS. The PI stock solution was diluted to a working concentration of 2 μg / mL with F12, and the Hoechst working concentration was 10 μg / mL. The cells were incubated at 37 °C for 10 minutes. Imaging was performed using a high-content live cell imager by selecting the DAPI and CY3 channels (the relevant parameters are shown in Table 11).

[0153] Table 11 Selection of Exposure Time for High-Content Live Cell Imager

[0154] Channel Exposure time (ms) DAPI 30 FITC 140 Cy3 70 TL25 25

[0155] 4. Detection by Western-blot

[0156] (1) Preparation of protein samples: For cell samples, plate 25×10 4 cells in a 3-cm cell culture dish HT22. After 18 h, administer Pon, then give the stimulus 1 h later, and collect the samples 4 h later. Dilute 5×SDS-PAGE protein loading buffer to 1× with RIPA containing protease inhibitor and phosphatase inhibitor, add 200 μL of 1× SDS-PAGE protein loading buffer to each dish, scrape the samples with a cell scraper and collect them into an EP tube, and immediately place them in a 99°C metal bath for 40 min; for tissue samples, add RIPA containing protease inhibitor and phosphatase inhibitor at a ratio of 10 mg:600 μL, lyse the tissue with a tissue grinder, add 5×SDS-PAGE protein loading buffer after lysis is completed, and place it in a 99°C metal bath for 40 min;

[0157] (2) Gel preparation: First, wipe and clean the experimental tools such as glass plates and combs with a sponge, then spray a large amount of alcohol and blow dry; add 4 mL of lower layer solution, 4 mL of lower layer buffer, and 160 μL of coagulant accelerator, and perform gel injection to complete the preparation of the lower layer gel; add 2 mL of lower layer solution, 2 mL of lower layer buffer, and 20 μL of coagulant accelerator, perform gel injection, and then insert the comb;

[0158] (3) Protein electrophoresis: After clamping the glass plate in the card slot, pour electrophoresis buffer into the two-layer glass plate, pull out the comb to perform sample loading. After sample loading is completed, supplement the electrophoresis buffer in the electrophoresis tank, set the voltage of the electrophoresis instrument to 150 v for 70 min to complete electrophoresis;

[0159] (4) Protein transfer: Pre-cool the transfer buffer at 4°C in advance, activate the PVDF membrane by placing it in methanol. Pry open the two glass plates from the gap, cut off the upper layer of the gel, rinse the gel with running water, pour the transfer buffer into a porcelain dish, soak the transfer sandwich in the transfer buffer, assemble it in the order of sponge, filter paper, gel, PVDF membrane, filter paper, sponge, roll out the bubbles with a centrifuge tube, clamp the sandwich and place it in the card slot. After filling the slot with transfer buffer, perform transfer at 300 mA for 2 h. The entire transfer process is carried out in an environment at 4°C;

[0160] (5) Blocking: Wash the transferred PVDF membrane once with 0.05% TBST, and then block it at room temperature in the blocking solution for 30 min;

[0161] Apply primary antibody: Incubate the strips in primary antibody at 4°C overnight.

[0162] Wash the membrane after primary antibody: wash four times with 1 / 1000 TBST, each time on a shaker at the maximum speed for 5 minutes;

[0163] Apply secondary antibody: incubate at room temperature for 2 hours;

[0164] Wash the membrane after secondary antibody: first rinse once with 0.5% TBST, then wash five times with 0.1% TBST, each time on a shaker with the speed set to the maximum for 5 minutes, add 2.5ml to the large box and 1.5ml to the small box;

[0165] Development: ECL luminescent liquid A: ECL luminescent liquid B = 1:1, pay attention to avoid light, absorb the liquid on the membrane as much as possible, place it on the developing plate, evenly add luminescent working solution, and develop it through a developer.

[0166] Table 12 10× Protein Electrophoresis Buffer Master Solution Formula (1L)

[0167] Reagent Dosage Glycine 144g SDS powder 10g Tris base 30.3g <![CDATA[ddH2O]]> Volume made up to 1 L

[0168] Table 13 10× Protein Transfer Buffer Master Solution Formula (1L)

[0169] Reagent Dosage Glycine 90g Tris base 19.3g <![CDATA[ddH2O]]> Volume made up to 1 L

[0170] Table 14 20×TBS stock solution formula (1L)

[0171] Reagent Dosage NaCl 80g Tris base 48.5g <![CDATA[ddH2O]]> Volume made up to 1 L Concentrated hydrochloric acid PH adjusted to 7.6

[0172] 5. Flow cytometry

[0173] 24-well plate with 1×10 5 HT22 cells were cultured in the Pon-treated group. After 18 hours, the Pon-treated group was treated with Pon at a concentration of 5 μM. One hour later, the cells were stimulated with TNF (10 ng / mL) + Z-VAD (20 μM), all diluted with DMEM / F12. After 8 hours of stimulation, Hoechst with a final concentration of 10 μg / mL was added to each well. After 5 minutes at 37°C, the cells were blown down with a pipette and collected into an EP tube. The cells in the negative control wells were digested with trypsin and washed with PBS. The cells to be put on the machine were filtered into the EP tube with a 70 μm cell filter. Before putting the machine on, the liquid circuit was rinsed with PBS until the cell count was zero. The voltage was adjusted with negative control and single positive cells. After the adjustment, the samples were tested. Before the samples were tested on the machine, PI with a final concentration of 4 μg / mL was added for staining. After the test, the software FlowJo was used for data processing and analysis.

[0174] Table 15 Adjustment voltage parameters

[0175] Channel Voltage (V) FSC 230 SSC 400 PE 320 Pacific Blue 270

[0176] 6. Stereotaxic injection of LPS into mice:

[0177] Twenty-four 8-week-old male C57BL / 6J mice were randomly divided into 4 groups: control group, LPS group, LPS + Pon (5 mg / mL) group, and LPS + Pon (10 mg / mL) group. Then, after intraperitoneal injection of Pon for 7 days, stereotaxic injection into the brain was prepared. After anesthetizing the mice with 1% sodium pentobarbital at a dose of 0.2 mL / 10 g, the position of the hippocampal region in the mouse brain (AP: -1.7 mm; ML: -1.2 mm; DV: -2.0 mm) was determined, marked with a marker pen, disinfected with 75% alcohol, and then, through a stereotaxic injection instrument and a microinjector, 2 mm was inserted along the Z-axis to inject normal saline or 2.5 μg / μL LPS solution at a rate of 0.2 μL / min. After the injection was completed, the syringe remained at the injection site for 10 min to allow the drug solution to fully diffuse, and then the needle was slowly and steadily withdrawn. After sealing the wound with bone wax, it was disinfected and sutured. The mice were sacrificed and samples were taken 1 day later.

[0178] 7. Statistical analysis

[0179] All measurement data in this invention were expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparison among multiple groups of samples by the software Graphpad. P < 0.05 indicated that there was a statistically significant difference between the two groups; **** indicated P < 0.0001.

[0180] III. Test results

[0181] 1. Effects of different concentrations of oridonin on necrosis of mouse hippocampal neurons (HT22) induced by TNF + Z-VAD

[0182] Through the detection of LDH and ATP, the results were as Figure 4 shown. At concentrations of 2.5 μM and 0.675 μM respectively, oridonin (Pon) could significantly reduce the necrosis of mouse hippocampal neurons (HT22) induced by TNF + Z-VAD. The IC 50 detected by LDH was 1.317 μM.

[0183] 2. Analysis of the effect of oridonin on mouse hippocampal neuron cells by flow cytometry

[0184] The results were as Figure 5As shown, through PI (propidium iodide) and Hoechst staining and flow cytometry counting, when the concentration of ponciridin (Pon) is 5 μM, it can significantly reduce the necrosis of HT22 cells induced by TNF + Z-VAD, and the proportion of the Q2 region (Hoechst + PI + cell population, dead cells) is significantly reduced.

[0185] The above results indicate that Pon can reduce the number of necrotic HT22 cells, Figure 6 showing the cell death at different time gradients and counting the changes in the number of live cells (Hoechst + PI - ). Pon has a mitigating effect at different time gradients; and the LDH in the culture supernatant of stained cells was detected, and Pon also has an inhibitory effect.

[0186] 3. Detection of the expression of necrosis regulatory proteins Phos-RIP1, Phos-RIP3, and Phos-MLKL by Western-blot

[0187] The results are as Figure 7 shown, and Pon can down-regulate the expression of necrosis regulatory proteins Phos-RIP1, Phos-RIP3, and Phos-MLKL.

[0188] 4. Detection of the expression of necrosis regulatory proteins Phos-RIP1, Phos-RIP3, and Phos-MLKL in the hippocampus after LPS injection into the hippocampal region by Western-blot

[0189] The results are as Figure 8 shown, and Pon can down-regulate the expression of necrosis regulatory proteins Phos-RIP1, Phos-RIP3, and Phos-MLKL in the hippocampus after LPS injection into the hippocampal region.

[0190] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concepts.

[0191] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and deformations.

Claims

1. Use of oridonin in the preparation of an anti-neuritis drug, characterized in that, The chemical structural formula of oridonin is as follows:

2. Use of oridonin according to claim 1 in the preparation of a drug for treating neuritis, characterized in that, The anti-neuritis drug uses oridonin as the only effective active ingredient.

3. Use of oridonin according to claim 1 in the preparation of a drug for treating neuritis, characterized in that, The drug further comprises a pharmaceutically acceptable carrier, excipient, diluent and adjuvant.

4. Use of oridonin according to claim 3 in the preparation of a drug for treating neuritis, characterized in that, The carrier, excipient, diluent and adjuvant include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil.

5. Use of oridonin according to claim 1 in the preparation of a drug for treating neuritis, characterized in that, The drug is an oridonin solution with a concentration of 8 mM to 12 mM.

6. Use of oridonin according to claim 5 in the preparation of a drug for treating neuritis, characterized in that, The drug is an oridonin solution with a concentration of 10 mM.

7. Use of oridonin according to claim 1 in the preparation of a drug for treating neuritis, characterized in that, The oridonin is used for preparing a therapeutic drug for Parkinson's disease caused by neuritis.

8. Use of oridonin according to claim 1 in the preparation of a drug for treating neuritis, characterized in that, The neuritis is induced by LPS.

9. Use of oridonin according to claim 1 in the preparation of an anti-neuritis drug, characterized in that, The oridonin is used for preparing an inhibitor of programmed neuronal cell death.

10. Use of oridonin according to claim 1 in the preparation of a drug for treating neuritis, characterized in that, The oridonin is used for preparing an inhibitor of the expression of inflammatory factors TNFα, IL-6, IL-1β, CCL5, CXCL10, CCL3, CCL4 mRNA.

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