CN116650491A - Application of CA in preparation of neuroinflammation inhibitor - Google Patents

Abstract

The invention relates to the field of biological medicine, and mainly relates to application of a chaperone-mediated autophagy (Chaperon-Mediated Autophagy, CMA) agonist CA77.1 (CA) in neuroinflammation. The invention mainly evaluates the effect of small molecule compound CA in Lipopolysaccharide (LPS) induced in vivo and in vitro neuroinflammation models. The medicament can inhibit the expression of inflammatory factors inducible NO synthase (iNOS), cycloxygenase-2 (COX-2), nitric Oxide (NO) and inteleukin 6 (IL-6) in an LPS-induced microglial cell line BV2 and primary microglial cells. In addition, CA can inhibit microglial activation and protect dopaminergic neurons in LPS-stimulated in vivo neuroinflammatory animal models.

Description

Application of CA in preparation of neuroinflammation inhibitor

Technical Field

The invention relates to the field of biological medicine, and mainly relates to an application of CA in neuroinflammation.

Background

CA77.1, the alias CA, is a chaperone mediated autophagy agonist, modified to optimize the retinoic acid receptor antagonist AR7. Recent researches show that CA can penetrate through the blood brain barrier, has no organ toxicity and has oral activity, which suggests that CA has good clinical application prospect. CA has also been shown to be effective in protecting neurons, slowing down the progression of pathology in Alzheimer's disease model animals, improving learning and locomotor ability in model animals. However, there is no relevant report on the role of CA in LPS-induced microglial inflammatory responses.

Disclosure of Invention

The invention provides a new application of CA as a neuroinflammation inhibitor, and the invention discloses the inhibition effect of CA on microglial inflammation effect for the first time, and clarifies the protection effect of CA on dopaminergic neurons in vivo and in vitro.

The invention adopts the following technical scheme:

use of CA as a neuroinflammation inhibitor.

Use of CA as a dopaminergic neuron protective agent.

Use of CA in the preparation of a neuroinflammation inhibitor.

Use of CA for the preparation of a dopaminergic neuron protective agent.

Use of CA in preparing medicine for treating Parkinson disease is provided.

The invention also discloses a neuroinflammation inhibition drug or a parkinsonism treatment drug, which comprises CA and a drug carrier.

The invention discloses a preparation method of the neuroinflammation inhibition drug or the parkinsonism treatment drug, which comprises the step of mixing a drug active ingredient CA with a drug carrier to obtain the neuroinflammation inhibition drug or the parkinsonism treatment drug.

In the above technical scheme, the drug carrier is a conventional drug carrier, such as buffer solution, physiological saline, excipient, etc., and the specific carrier selection and the mixing method of the active drug CA and the drug carrier are conventional techniques.

Further, the neuroinflammation is microglial neuroinflammation.

In the above technical solution, the microglial cell is microglial cell line BV2 or primary microglial cell.

Further, the neuroinflammation is LPS-induced microglial neuroinflammation.

Use of CA in the preparation of an inhibitor of microglial overactivation.

Further, the inflammatory mediators include iNOS, COX-2, NO, and IL-6.

The invention has the advantages that:

the invention discloses a new effect of CA in treating neuroinflammation for the first time, and the CA can be found in a neuroinflammation model of in vitro cells to down regulate the levels of inflammatory factors iNOS, mRNA and protein of COX-2 and the release amount of inflammatory mediators NO and IL-6 of microglial cell line BV2 induced by LPS. In LPS-treated primary microglia, CA is also able to reduce the protein levels of iNOS and COX-2 and the release of the inflammatory mediator NO. The method comprises the steps of administering CA or an equal volume of control solvent to a mouse in an intraperitoneal injection mode, and injecting LPS or PBS into the bilateral midbrain substantia nigra region of the mouse by using a brain stereotactic injection mode, wherein the result shows that compared with the mouse treated by LPS, the pretreatment of CA can obviously reduce the marker IBA1 of microglial activation in the midbrain region of the mouse and increase the number of TH positive dopaminergic neurons, and prompts inhibition of LPS-induced neuroinflammation and protection of the dopaminergic neurons by CA.

Drawings

FIG. 1 shows the results of CA inhibiting the expression of inflammatory protein in BV2 of LPS-treated microglial cell line.

FIG. 2 shows the results of CA inhibiting LPS-treated primary microglial inflammatory protein expression.

FIG. 3 shows the amount of inflammatory factor NO released from the LPS-treated microglial line BV2 inhibited by CA.

FIG. 4 shows the amount of CA inhibiting the release of the primary microglial inflammatory factor NO by LPS treatment.

FIG. 5 shows the release of inflammatory factor IL-6 from the CA-inhibited LPS-treated microglial line BV 2.

FIG. 6 shows the inhibition of LPS-treated microglial cell line BV2 inflammatory factor by CAiNOSIs measured as a result of the amount of mRNA expression of (1).

FIG. 7 shows the inhibition of LPS-treated microglial cell line BV2 inflammatory factor by CACOX-2Is measured as a result of the amount of mRNA expression of (1).

FIG. 8 is a measurement of the inhibition of the dopaminergic neuron cell marker TH in the brain substantia nigra region of mice induced by LPS stimulation by CA at the somatic level.

Fig. 9 is a measurement of the situation of the brain black area microglial marker IBA1 in mice induced by stimulation with CA inhibited LPS at the body level.

Figure 10 is a measurement of the improvement of motor performance impairment in mice caused by LPS stimulation by CA at the body level.

Detailed Description

The instruments and reagents used in the invention are all commercially available, and specific experimental operations and tests are conventional techniques. Wherein DMEM and DMEM/F12 media are available from Gibco corporation of America; fetal Bovine Serum (FBS) was purchased from Gibco company of the united states; dimethyl sulfoxide (DMSO) was purchased from Shanghai, a worker; penicillin, streptomycin was purchased from Shanghai, in; lipopolysaccharide (LPS) was purchased from SIGMA company of America; iNOS antibodies were purchased from us Cell Signal Technology company; COX-2 antibodies were purchased from Abcam, USA; anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from Thermo company, usa; DYY-6C electrophoresis apparatus was purchased from Liuyi Instrument works in Beijing; CO ₂ Cell incubator was purchased from Thermo company of united states; microglial cells BV2 were purchased from American Type Culture Collection (ATCC); reverse transcription kit and real-time fluorescent quantitative PCR instrument were purchased from ABI company, usa; c57BL/6J mice were purchased from Shanghai Laike; stereotactic syringes were purchased from rawald life technologies limited. CA in the present invention is purchased from Selleck corporation, U.S. and has the following chemical formula:

statistical differences between groups were analyzed using GraphPad Prism 6.0. The difference between groups is calculated by single factor analysis of variance (One-way ANOVA)PValue: *P<0.05，**P<0.01，***P <0.001 andP<0.0001。

examples

1. Cell experiment: BV2 cells or primary microglia were divided into different treatments: (1) dmso+pbs group; (2) ca+pbs group; (3) dmso+lps group; (4) CA+LPS group. DMSO and PBS are solvents for CA, LPS, respectively. In the CA+LPS group, cells 12 and h were pretreated with CA and 16 h were then treated with LPS, and related experiments were performed.

2. Culture of microglial cell line BV 2: before passage, the state and density of the cells are observed under a microscope, and the passage condition is reached when the cells grow to about 90% of the culture dish. PBS buffer, 0.05% pancreatin and cell culture medium (DMEM medium containing 10% inactivated serum and 1% diabody) were pre-heated in a water bath for 30 min. Old medium was aspirated off, 1 mL PBS was added, the dish was shaken, and the remaining old medium was washed off. 0.05% pancreatin of 1 mL was added, left to stand for 2 min, and the digestion was stopped by adding fresh medium of 1 mL until the cells were significantly shed. From the edge to the center of the dish, the cells were all detached by gentle pipetting with a pipette and transferred to a centrifuge tube of 5 mL. 1000 Centrifugal 5min at rpm. The supernatant was aspirated, and 1 mL fresh medium was added and gently swirled. Inoculating into a middle dish according to a certain proportion, shaking, standing at 37deg.C and 5% CO ₂ Is cultured in a cell culture incubator.

3. Culture of primary microglial cells: and (3) putting the newly born mice for 2-3 days into 75% alcohol until the mice are inflated, so as to achieve the anesthetic effect. Then put it into PBS buffer solution to wash out the residual alcohol. The whole brain was carefully removed and placed in a dish containing pre-chilled PBS. The meninges and vessels were carefully stripped under a microscope and placed in a new dish containing pre-chilled PBS. The tissue was minced with an ophthalmic scissors, carefully transferred to a large dish containing 0.25% pancreatin, and digested in a 37 ℃ cell incubator for 15 min with shaking every 5 min. After the tissue has been digested, excess pancreatin is removed by aspiration. Washed 3 times with serum-free DMEM, transferred to a 50 mL centrifuge tube, and added with 0.3 mg/mL deoxyribonuclease I (DNase I) to digest DNA in the tissue. The blown tissue mass was filtered through a 40 μm cell filter screen, and the filtrate was collected and centrifuged at 1000 rpm for 5 min. The supernatant was aspirated, the pellet resuspended in pre-warmed primary microglial medium and inoculated into polylysine coated flasks.

4. Western immunoblotting

(1) Preparation of cellular protein samples: the old medium was aspirated, washed once with PBS buffer, 200. Mu.L of 0.05% pancreatin was added, and digested in an incubator at 37℃for 2 min. After macroscopic shedding of cells, 200. Mu.L of fresh medium was added and digested. Gently blow from the edge of the well to the center of the well until all cells fall off. Cells were transferred to labeled 1.5 mL centrifuge tubes and centrifuged at 1500 rpm for 5 min. The supernatant was aspirated, 100. Mu.L of cell lysate was added, and the mixture was blown down. Then the mixture was again inserted into ice, and the mixture was allowed to stand and crack for 10 minutes. Ultrasonic was performed 200W, 5 times with an ultrasonic cell disruptor. Adding 2 Xloading Buffer (Sample Buffer) with the same volume as the cell lysate, mixing, and decocting in boiling water for 10 min to obtain a cell protein Sample.

(2) SDS-PAGE gel electrophoresis: and (3) preparing polyacrylamide separating gel with proper concentration according to the molecular weight of the protein to be detected, pouring the polyacrylamide separating gel into a clamped glass plate, and pressing the liquid level with isopropanol and eliminating bubbles. After the separating glue solidifies 1 h, the upper concentrated glue is arranged and poured into a glass plate, and a comb is inserted. After half an hour, the comb was pulled off, the gel was placed in a vertical electrophoresis tank and poured into the electrophoresis buffer. Protein samples of about 10 μg were added to the wells, and protein markers were added to both sides of the samples as a scale. 120 V electrophoresis 1 h, the front edge of the buffer solution to be loaded runs to the bottom of the gel, and the electrophoresis is finished.

(3) Transferring: pre-chilled 1×tb buffer was prepared in advance, poured into a spin-film dish, and placed into a spin-film clamp, taking care of the front and back of the spin-film clamp. Spreading a layer of sponge, two layers of filter paper, and discharging bubbles by using a scraper. PVDF membranes that have been activated with methanol are placed on filter paper. The gel plate was removed and the thin glass plate carefully pried open and the upper glue was cut off. The lower glue layer was washed with double distilled water and then placed on a PVDF membrane. Two layers of filter paper, a layer of sponge, are covered on the gel, and each step uses a scraper to discharge bubbles. Placing the materials into a film transferring groove after clamping, pouring the materials into a TB, placing the materials into an ice box, and installing an instrument to distinguish the positive electrode from the negative electrode. The film transfer tank is placed in a basin with an ice-water mixture, cooled, and transferred to a film 2 h by 240 mA.

(4) Blocking and antibody incubation: skim milk powder was weighed and placed in 1 XTBST to prepare a 5% skim milk blocking solution. And after the membrane transfer is finished, taking out the PVDF membrane and putting the PVDF membrane into the sealing liquid. Put on a shaker and shake 1 h at room temperature. Parafilm was tiled on the plate and primary antibodies were diluted with 1 XTBE in the proportions indicated, and sequentially dropped onto the seal. Taking out the cut film, and dipping the film in clean toilet paper to dry the water. The membranes were facing down, directly contacted with antibody and incubated overnight at 4 ℃. The incubated membrane was washed with 1 XTBE, 1 XTBE was changed every 10 min, and washed 3 times. After the washing is finished, the secondary antibody is incubated. The dilution ratio of the secondary antibodies is 1:10000, and the membranes are put into the corresponding secondary antibodies according to the species of the primary antibodies, and the membranes are incubated by shaking at room temperature for 2 h.

(5) Developing and imaging: after the secondary antibody incubation was completed, the incubated membranes were washed with 1 XTBE, changing TBST every 10 min, and washing 2 times. Finally, the mixture is changed to 1 XTBS and washed for 10 min. According to the specification, a certain amount of the developer is prepared in a ratio of 1:1. Dipping the film into toilet paper, and soaking in developing solution for 1 min. The Clinx ChemiCapture software was opened and the film was placed in a chemiluminescent imager for development imaging.

FIG. 1 shows the results of inhibiting the expression of inflammatory protein of LPS-treated microglial cell line BV2 by CA, wherein one band shows the expression level of inflammatory protein iNOS, two bands show the expression level of inflammatory protein COX-2, and three bands show the expression level of internal reference protein beta-actin. Group 1 shows the expression levels of three proteins in normal microglial line BV2, group 2 shows the expression levels of three proteins in microglial line BV2 treated with 12. Mu.M CA alone, group 3 shows the expression levels of three proteins in microglial line BV2 treated with LPS alone (1. Mu.g/mL), and group 4 shows the expression levels of three proteins in microglial line BV2 stimulated with LPS (1. Mu.g/mL) and pretreated with 12. Mu.M CA.

FIG. 2 shows the results of inhibiting the expression of inflammatory protein in LPS-treated primary microglial cells by CA, wherein one band shows the expression level of inflammatory protein iNOS, two bands show the expression level of inflammatory protein COX-2, and three bands show the expression level of internal reference protein beta-actin. Group 1 shows the expression levels of three proteins in normal primary microglia, group 2 shows the expression levels of three proteins in primary microglia treated with 20. Mu.M CA alone, group 3 shows the expression levels of three proteins in primary microglia treated with LPS alone (1. Mu.g/mL), and group 4 shows the expression levels of three proteins in primary microglia stimulated with LPS (1. Mu.g/mL) and pretreated with 20. Mu.M CA.

5. NO detection experiment: after the cell treatment is finished, collecting cell supernatant, centrifuging to remove insoluble precipitate, transferring the supernatant into a new centrifuge tube, and uniformly mixing. Add 50. Mu.L of supernatant to 96 well plates, then add 50. Mu. L Griess Reagent I, and mix by patting. Then 50 mu L Griess Reagent II was added and the mixture was stirred and mixed. OD absorbance values of 540 nm were measured in a microplate reader. And (3) making a standard curve according to the concentration and the reading of the standard substance, and calculating the content of inflammatory factors in the sample according to the standard curve.

FIG. 3 is a graph showing the amount of inflammatory factor NO released from the LPS-treated microglial line BV2 by CA, wherein column 1 shows the amount of inflammatory factor NO released from the normal microglial line BV2, column 2 shows the amount of inflammatory factor NO released from the microglial line BV2 treated with 12. Mu.M CA alone, and column 3 shows the amount of inflammatory factor NO released from the microglial line BV2 treated with LPS alone, and column 4 shows the amount of inflammatory factor NO released from the microglial line BV2 stimulated with LPS and pretreated with 12. Mu.M CA.

FIG. 4 shows the amount of inflammatory factor NO released from primary microglia treated with CA to inhibit LPS, wherein column 1 shows the amount of inflammatory factor NO released from normal primary microglia, column 2 shows the amount of inflammatory factor NO released from primary microglia treated with 20. Mu.M CA alone, and column 3 shows the amount of inflammatory factor NO released from primary microglia treated with LPS alone, and column 4 shows the amount of inflammatory factor NO released from primary microglia stimulated with LPS and pretreated with 20. Mu.M CA.

6. Enzyme-linked immunosorbent assay: after the cell treatment was completed, the supernatant was retained and centrifuged at 5000 rpm for 5 min. The supernatant was pipetted into a clean 1.5 mL centrifuge tube. 100 mu L of sample or standard substance is sucked and added into an ELISA plate coated with the antibody, and the ELISA plate is sealed by a sealing plate film and then placed into a 37 ℃ incubator for incubation for 90 min. After incubation was completed, the liquid was pipetted off and the paper was drained off without washing. 100. Mu.L of diluted biotin antibody was added. Sealing with a sealing plate film, and incubating in a 37 ℃ incubator for 60 min. The liquid was removed and washed 3 times with diluted wash solution at 1 min intervals. 100 mu L of avidin-peroxidase complex working solution is added, and the mixture is sealed by a sealing plate film and then placed in a 37 ℃ incubator for incubation for 30 min in a dark place. The liquid was removed and washed with diluted wash solution 5 times at 2 min intervals. Adding 90 mu L of color development liquid, sealing by a sealing plate film, and then incubating for 15 min at 37 ℃ in a dark place. The color change was observed, and 100. Mu.L of stop solution was added until the first four wells of the standard had a clear color difference and the last three wells had a no clear color difference. The absorbance value at 450 nm was measured in a microplate reader. And (3) making a standard curve according to the concentration of the standard substance and the corresponding absorbance value, and calculating the content of inflammatory factors in the sample according to the standard curve.

FIG. 5 shows the amount of inflammatory factor IL-6 released from the LPS-treated microglial line BV2 by CA, wherein bar 1 shows the amount of inflammatory factor IL-6 released from the normal microglial line BV2, bar 2 shows the amount of inflammatory factor IL-6 released from the microglial line BV2 treated with 12. Mu.M CA alone, bar 3 shows the amount of inflammatory factor IL-6 released from the microglial line BV2 treated with LPS alone, and bar 4 shows the amount of inflammatory factor IL-6 released from the microglial line BV2 stimulated with LPS and pretreated with 12. Mu.M CA.

7. Extraction of RNA: after the cell treatment is completed, the old medium is aspirated. The PBS was washed once and discarded. mu.L of Trizol reagent was added and the mixture was shaken on a shaker for 5min to allow the cells to fall off. All cell suspensions were transferred to 1.5 mL centrifuge tubes by gentle pipetting. 100. Mu.L of chloroform was added thereto, and the mixture was turned upside down several tens of times to mix the chloroform and the cell suspension thoroughly, and the mixture was allowed to stand at room temperature for 10 minutes. The sample was placed in a centrifuge at 4℃and centrifuged at 12000 rpm for 15 min. After centrifugation, it was observed that the liquid surface was divided into three layers, the upper layer was RNA, the middle layer was DNA, and the bottom layer was protein. The 150. Mu.L of the uppermost RNA was carefully aspirated and added to a new 1.5 mL centrifuge tube. Then adding equal volume of isopropanol, mixing, standing in a refrigerator at-20deg.C for 30 min. The sample was placed in a centrifuge and centrifuged at 12000 rpm for 15 min. After centrifugation, a white precipitate was observed at the bottom of the centrifuge tube. 500. Mu.L of pre-chilled 75% ethanol was added and the precipitate washed. The sample was further placed in a centrifuge and centrifuged at 12000 rpm for 15 min. The supernatant was slowly aspirated. The centrifuge tube was left to stand in a fume hood and air dried for 20 min. Add 20. Mu.L of DEPC water and determine RNA concentration using NanoDrop, record and save the concentration.

8. Reverse transcription PCR and qRT-PCR

(1) Reverse transcribing the RNA into cDNA in the reaction system of

The reaction procedure was as follows

(2) The qRT-PCR reaction system is

A program was set on ABI7500 and eight connecting tubes were placed. The reaction procedure was as follows

(3) The primer sequences used are

FIG. 6 shows the inhibition of LPS-treated microglial cell line BV2 inflammatory factor by CAiNOSWherein column 1 represents inflammatory factors in the normal microglial cell line BV2iNOSColumn 2 shows inflammatory factors in microglial line BV2 treated with 12. Mu.M CA aloneiNOSColumn 3 shows inflammatory factors in microglial cell line BV2 treated with LPS aloneiNOSColumn 4 shows inflammatory factors in microglial line BV2 stimulated with LPS and pretreated with 12. Mu.M CAiNOSRelative expression amount of mRNA of (2).

FIG. 7 shows the inhibition of LPS-treated microglial cell line BV2 inflammatory factor by CACOX-2Wherein column 1 represents inflammatory factors in the normal microglial cell line BV2COX-2Column 2 shows inflammatory factors in microglial line BV2 treated with 12. Mu.M CA aloneCOX-2Column 3 shows inflammatory factors in microglial cell line BV2 treated with LPS aloneCOX-2Column 4 shows inflammatory factors in microglial line BV2 stimulated with LPS and pretreated with 12. Mu.M CACOX-2Relative expression amount of mRNA of (2).

9. C57BL/6J mice of 6-8 weeks of age were randomly divided into 4 groups: (1) solvent+PBS group; (2) ca+pbs group; (3) solvent+LPS group; (4) CA+LPS group. PBS and Solvent are solvents for LPS and CA, respectively. In the form of intraperitoneal injection, 8 mg/kg of CA or Solvent were pre-treated for 7 days. Next, 2. Mu.g of LPS was injected into the bilateral midbrain substantia nigra region of the mice in a stereotactic manner to construct a model of Parkinson's Disease (PD) neuroinflammation. After the mice wake up, CA or Solvent treatment is continued for 7 days, the mice are sacrificed, and relevant brain tissues are taken out for subsequent experimental treatment and analysis.

10. After the end of the administration, the mice were anesthetized with 0.3% sodium pentobarbital, followed by perfusion with 4% paraformaldehyde; after the perfusion is finished, the whole brain of the mouse is immediately taken off by forceps, soaked in 4% paraformaldehyde and preserved at 4 ℃. Dehydrating brain tissue with 30% sucrose solution, preserving at 4deg.C, and dehydrating for three days; embedding whole brain with embedding medium, slicing with frozen slicer to slice thickness of 20 μm, and collecting in antifreeze solution; brain pieces were washed with PBS, and wells were incubated with 500. Mu.L of rinse solution (0.4% Triton-X100,1% FBS in PBS) for 10 min at room temperature; the brain slice was washed 3 times with PBS, and 500. Mu.L of blocking solution (PBS containing 0.4% Triton-X100,5% FBS) was added to each well and incubated for 60 min at room temperature; diluting primary antibody (TH, 1:1000; IBA1, 1:1000) with rinsing solution, adding 300 μl of primary antibody dilution per well, and incubating overnight at 4deg.C; the primary antibody was blotted and washed three times with PBS; diluting a secondary antibody (Alexa Fluor 594 donkey anti-Mouse IgG (H+L) red fluorescent secondary antibody, 1:300) with a rinsing solution, and incubating 2H with a tinfoil light-shielding table at room temperature; sucking the secondary antibody, and washing with PBS for three times; the nuclear dye DAPI (1:10000) was diluted with PBS, 300. Mu.L DAPI was added to each well, and after incubation for 10 min, washed twice with PBS; then adding an anti-quenching agent sealing piece; the sections were placed on an Olympus IX71 inverted fluorescence microscope, and the stained areas of TH and IBA1 in the midbrain substantia nigra region were observed and photographed.

Fig. 8 is a measurement of the change in the dopaminergic neuron cell marker TH in mice caused by the stimulation with CA at the body level, wherein, group 1 represents the intensity of the dopaminergic neuron cell marker TH in the midbrain substantia nigra region in normal control mice, group 2 represents the intensity of the dopaminergic neuron cell marker TH in the midbrain substantia nigra region in mice treated with CA alone, group 3 represents the intensity of the dopaminergic neuron cell marker TH in mice stimulated with LPS alone, and group 4 represents the intensity of the dopaminergic neuron cell marker TH in the midbrain substantia nigra region in mice stimulated with LPS and pre-administered with CA.

Fig. 9 is a graph showing the activation of the midbrain substantia nigra region microglial marker IBA1 in mice induced by stimulation with CA at the body level, wherein group 1 represents the intensity of the midbrain substantia nigra region microglial marker IBA1 in normal control mice, group 2 represents the intensity of the midbrain substantia nigra region microglial marker IBA1 in mice treated with CA alone, group 3 represents the intensity of the midbrain substantia nigra region microglial marker IBA1 in mice stimulated with LPS alone, and group 4 represents the intensity of the midbrain substantia nigra region microglial marker IBA1 in mice stimulated with LPS and pre-administered with CA.

11. Animal behavioural ability test. Rotating rod experiment: when training the mice, put them on a static rotating rod to move freely 5min, familiarize with the environment. The rotating rod is arranged to rotate at a constant speed of 4 r/min until the mouse can move on the rotating rod 90 s without falling and the direction is correct, and training is continued for 3 days. When testing mice, the rotating rod is set to rotate at an acceleration of 8 r/min from 4 r/min to 40 r/min within 5min ² . The mice were placed on the rotating bars, the running state of the mice was observed after the start, and the time of the mice on the bars was recorded.

Figure 10 is a measurement of the improvement of motor performance impairment in mice caused by LPS stimulation by CA at the body level. Wherein, group 1 represents the on-stick time of normal control mice, group 2 represents the on-stick time of mice treated with CA alone, group 3 represents the on-stick time of mice stimulated with LPS alone, and group 4 represents the on-stick time of mice stimulated with LPS and pre-dosed with CA.

Inflammatory factors released by microglial overactivation are important causes of neuronal death, a pathological feature that can be detected early in many neurodegenerative diseases. Intervention in microglial activation, inhibition of neuroinflammation has become an effective strategy for the treatment of neurodegenerative diseases. Therefore, drug development targeting anti-neuroinflammation is critical for the treatment of neurodegenerative diseases.

Claims (10)

1. Use of CA as a neuroinflammation inhibitor or use of CA in the preparation of a neuroinflammation inhibitor.
2. 2. The use according to claim 1, wherein the neuroinflammation is microglial neuroinflammation.
3. 3. The use according to claim 2, wherein the microglial cell is microglial cell line BV2 or primary microglial cell.
4. 4. The use according to claim 2, wherein the neuroinflammation is LPS-induced microglial neuroinflammation.
5. 5. A neuroinflammation-inhibiting drug or a parkinson's disease-treating drug, comprising CA and a drug carrier.
6. 6. The neuroinflammation suppressing drug or parkinson's disease therapeutic drug according to claim 5, wherein said neuroinflammation is microglial neuroinflammation.
7. Use of ca in the manufacture of a medicament for the treatment of parkinson's disease.
8. Use of ca for the preparation of an inhibitor of microglial overactivation.
9. Use of ca for the preparation of an inhibitor of inflammatory mediators.
10. Use of ca as or in the preparation of a dopaminergic neuron protective agent.