CN113730583A - Application of lipid droplets as drug targets of neurodegenerative diseases - Google Patents
Application of lipid droplets as drug targets of neurodegenerative diseases Download PDFInfo
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Abstract
The invention discloses an application of lipid droplets as drug targets of neurodegenerative diseases, firstly applies a lipid droplet accumulation microglia phenotype transformation inhibitor to the preparation of drugs for treating or preventing neurodegenerative diseases related to neuroinflammation, applies a lipid droplet generation inhibitor as a lipid droplet accumulation microglia phenotype transformation inhibitor to the preparation of drugs for treating or preventing neurodegenerative diseases related to neuroinflammation, and firstly uses (i) urolithin B or pharmaceutically acceptable salts thereof or (ii) a long fatty acid acyl coenzyme A synthetase inhibitor as an effective component of the lipid droplet generation inhibitor and the lipid droplet accumulation microglia phenotype transformation inhibitor Quickly and accurately screening out the medicine for treating or preventing the neurodegenerative diseases related to neuroinflammation.
Description
Technical Field
The invention belongs to the technical field of medicine research and molecular biology methodology, and particularly relates to application of lipid droplets as a drug target of neurodegenerative diseases.
Background
Microglia (microglia) is an inherent immune effector cell in the central nervous system, wherein one kind of microglia rich in Lipid droplets has different transcriptomic characteristics with other subtypes of microglia, and is called Lipid Droplet Accumulation Microglia (LDAM), the down-regulated gene part of the microglia rich in Lipid droplets coincides with the two microglia subtypes of DAM and MGnD, but the phenomena that genes such as AXL, CD74, CLEC7A and CYBB are down-regulated in the microglia rich in Lipid droplets and are up-regulated in DAM and MGnD also exist.
Existing studies indicate that microglia are involved in the occurrence of a series of neurodegenerative diseases, and microglial activation and neuroinflammation are main characteristics of neuropathology. Alzheimer's Disease (AD) is a neurodegenerative disease with extraneural amyloid deposits caused by a β aggregation and neurofibrillary tangles caused by tau protein hyperphosphorylation as main pathological features, and its clinical manifestations are characterized by global dementia such as memory impairment, aphasia, disuse, agnosia, impairment of visual spatial skills, executive dysfunction and personality and behavior changes, and the etiology is unknown so far, and it is not clear whether the phenomenon of microglial cell LDAM phenotype activation also exists in the AD pathogenesis.
Currently, research on AD pathogenesis and therapeutic approaches is limited. For many years, drugs such as cholinesterase inhibitors and excitatory aspartate receptor antagonists have been recommended by clinical guidelines as the first treatment modality. However, these drugs also only improve some of the patients' clinical symptoms and cognitive function levels to some extent, and can cause drug intolerance and severe adverse effects (ADRs) with long-term use. Meanwhile, in recent years, research and development of a series of drugs developed by taking A beta and tau as targets are continuously carried out, but finally, clinical trials fail, and the A beta and the tau are not optimal therapeutic targets. Therefore, the method has important significance for exploring the pathogenesis of AD, searching safe and effective drug action targets, developing molecular mechanism research aiming at the targets, and providing theoretical and experimental basis for perfecting and explaining the pathogenesis of AD and accelerating the development and development of new anti-AD drug processes.
Disclosure of Invention
The invention discovers that (i) urolithin B or pharmaceutically acceptable salts thereof or (ii) long fatty acid acyl coenzyme A synthetase inhibitors (triacin C, TRC) can inhibit the generation of lipid droplets in microglia, thereby inhibiting the conversion of the microglia to LDAM phenotype, and also discovers that the memory disorder of AD mice can be obviously improved after the proportion of LDAM is reduced, and the expression level and the neuron vitality of neuron synapse-related protein PSD95 and Syn are improved, thereby determining that the lipid droplets can become targets of neurodegenerative disease drugs, and inhibiting the conversion of the microglia LDAM phenotype by inhibiting the generation of the lipid droplets can become a new means for treating AD.
Based on the above findings, the present invention provides the following technical solutions:
in a first aspect, the present invention provides an application of an inhibitor of phenotypic transformation of lipid droplet accumulation microglia in the preparation of a medicament for treating or preventing a neurodegenerative disease associated with neuroinflammation, wherein the medicament takes the inhibitor of phenotypic transformation of lipid droplet accumulation microglia as an active ingredient, takes microglia as a target for treating the neurodegenerative disease associated with neuroinflammation, and delays, prevents and treats the neurodegenerative disease associated with neuroinflammation by inhibiting the phenotype of the microglia transformed into the lipid droplet accumulation microglia.
In a second aspect, the invention provides an application of a lipid droplet generation inhibitor in preparing a medicament for treating or preventing neurodegenerative diseases related to neuroinflammation, wherein the medicament takes the lipid droplet generation inhibitor as an effective component, and inhibits the microglia from being converted into lipid droplets to accumulate the microglia by inhibiting the generation of lipid droplets in the microglia, so that the neurodegenerative diseases related to neuroinflammation are delayed, prevented and treated.
In a third aspect, the invention provides an application of (i) urolithin B or a pharmaceutically acceptable salt thereof or (ii) a long fatty acid acyl coenzyme a synthetase inhibitor in preparing a medicament for treating or preventing a neurodegenerative disease related to neuroinflammation, wherein the medicament adopts (i) urolithin B or a pharmaceutically acceptable salt thereof or (ii) a long fatty acid acyl coenzyme a synthetase inhibitor as an effective component for inhibiting the generation of lipid droplets in microglia, and inhibits the conversion of the microglia into the lipid droplets to accumulate the microglia by inhibiting the generation of the lipid droplets, so that the neurodegenerative disease related to neuroinflammation is delayed, prevented and treated.
In a fourth aspect, the present invention provides use of (i) urolithin B or a pharmaceutically acceptable salt thereof or (ii) a long fatty acid acyl-CoA synthetase inhibitor for the preparation of a lipid droplet formation inhibitor comprising, as an active ingredient, either (i) urolithin B or a pharmaceutically acceptable salt thereof or (ii) a long fatty acid acyl-CoA synthetase inhibitor against beta-amyloid polypeptide1-42The primary microglia is induced to generate lipid droplets with an inhibiting effect.
In a fifth aspect, the present invention provides use of (i) urolithin B or a pharmaceutically acceptable salt thereof, or (ii) a long fatty acid acyl-coa synthetase inhibitor for the preparation of a lipid droplet accumulation microglia phenotype transformation inhibitor comprising, as an effective ingredient, (i) urolithin B or a pharmaceutically acceptable salt thereof, or (ii) a long fatty acid acyl-coa synthetase inhibitor, for inhibiting the transformation of microglia into lipid droplet accumulation microglia by inhibiting lipid droplet production.
In a sixth aspect, the present invention provides a method for screening a drug that is an inhibitor of lipid droplet generation, an inhibitor of phenotypic transformation of lipid droplet accumulation microglia, or a drug for treating or preventing a neurodegenerative disease associated with neuroinflammation, comprising the steps of:
adding beta amyloid polypeptide 1-42 (Abeta) into a primary microglia in-vitro culture system1-42) Co-culturing as a blank;
adding beta amyloid polypeptide 1-42 and a drug to be detected or a drug combination to be detected into a primary microglia in-vitro culture system for co-culture to serve as an intervention group;
counting the proportion of the lipid droplets accumulating the microglia in each group;
selecting an intervention group effective to reduce the proportion of microglia accumulated by lipid droplets relative to the blank group.
On the basis of the technical scheme, the step of counting the proportion of the lipid droplets accumulating the microglia in each group comprises the following steps: the proportion of lipid droplets accumulating microglia was determined by observing the number of lipid droplets under a confocal laser fluorescence microscope.
Specifically, the neurodegenerative disease related to neuroinflammation is one or more of Parkinson's disease, Alzheimer's disease and multiple sclerosis.
The mode for detecting the proportion of the microglia accumulated by the lipid droplets at least comprises one of the following modes: (i) detecting the number of intracellular lipid droplets by using a laser confocal transmission electron microscope; (ii) detecting microglia related markers accumulated by BODIPY and Perilipin2 lipid droplets by an immunofluorescence method; (iii) detecting the characteristic gene transcription level of the microglia accumulated by the lipid droplets by a qPCR method; (iv) WB measures lipid droplet accumulation microglia characteristic protein number.
The invention has the following advantages and beneficial effects:
(1) the invention takes (i) the urolithin B or the pharmaceutically acceptable salt thereof or (ii) the long fatty acid acyl coenzyme A synthetase inhibitor as the effective component of the lipid drop generation inhibitor and the lipid drop accumulation microglia phenotype transformation inhibitor for the first time.
(2) The lipid droplet generation inhibitor and the lipid droplet accumulation microglia phenotype transformation inhibitor are used as effective components of the medicine for treating or preventing the neurodegenerative diseases related to neuroinflammation.
(3) The drug screening method provided by the invention takes the ratio of the lipid droplets to accumulate the microglia as the index of drug screening for the first time, and the method can simply, quickly and accurately screen out the drug for treating or preventing the neurodegenerative disease related to neuroinflammation.
Drawings
FIG. 1 shows the results of immunofluorescence dual-labeling experiments on brain tissues of WT aged mice 12 months old and APP/PS1/tau tri-transgenic AD mice 12 months old; wherein, fig. 1A shows the expression of the specific marker of LDAM, Perilipin2 (PLIN 2 for short), and fig. 1B shows the expression of the microglia marker IBA 1.
FIG. 2 shows the results of animal behavioral experiments in AD mice; wherein, fig. 2A shows a flow chart of AD mouse model construction, administration, behavioral experiments; FIG. 2B shows the results of the aging score test; FIG. 2C shows the body weight change of the mice in each group; FIG. 2D shows the results of the intelligent nesting experiment; FIG. 2E shows the latency of four groups of mice to find the hidden platform during the Morris water maze training phase; FIG. 2F shows the Morris water maze training phase Abeta1-42+ TRC groups (a) and Abeta1-42The motion trajectory of group (b); figure 2G shows basal levels of rigor in four groups of mice in a conditioned fear memory experiment.
Figure 3 shows the results of immunohistochemical staining of AD mouse brain tissue IBA 1.
Fig. 4 shows the statistical results of fig. 3.
FIG. 5 shows the experimental electrophoretogram of CD68 and IBA1 Western blotting (see FIG. 5A) of AD mouse brain tissue and the optical density analysis result (see FIG. 5B).
FIG. 6 shows the experimental electrophoretogram of Western blotting (see FIG. 6A) and densitometric analysis results (see FIG. 6B) of AD mouse brain tissues TIP47, Perilipin2 and Perilipin 1.
FIG. 7 shows the microglial ultrastructure observed in mouse hippocampal tissue by transmission electron microscopy.
FIGS. 8 and 9 show immunofluorescence dual-labeling results for brain tissue of AD mice; among them, fig. 8 shows the expression of the LDAM-specific marker Perilipin2 in AD cells in vitro, and fig. 9 shows the expression of the microglia marker IBA1 in AD cells in vitro.
FIG. 10 shows the results of in vitro laser confocal fluorescence experiments with BODIPY493/503 (green) in AD cells.
Fig. 11 is the statistical results of fig. 10.
FIG. 12 shows the results of lipid droplet quantification by in vitro AD cells using a flow-through single-label semi-quantitative assay; in which, FIG. 12A shows BODIPY in AD cells in vitro+Number of positive cells, fig. 12B shows the average number of lipid droplets in AD cells in vitro, fig. 12C shows the average particle size of lipid droplets in AD cells in vitro, a represents Ctrl group; b represents a TRC group; c represents Abeta1-42Group (d); d represents Abeta1-42+And (5) TRC group.
FIG. 13 shows the experimental profile of in vitro AD cell BODIPY493/503 flow-type single-label semiquantitative analysis (see FIG. 13A) and its statistical results (see FIG. 13B).
FIG. 14 shows the Western blot electropherograms of TIP47, Perilipin2, and Perilipin1 of AD cells in vitro (see FIG. 14A) and their densitometry analysis results (see FIG. 14B).
FIG. 15 shows UB inhibition of lipid droplet formation by microglia in vitro by laser confocal fluorescence microscopy.
Figure 16 shows results of animal behavioural experiments in AD mice under UB intervention; wherein, fig. 16A is the aging degree scoring experimental result; FIGS. 16B-F show the latency of four groups of mice to find the hidden platform and the motion trajectories of three groups of mice recorded during the 5 consecutive days of the Morris water maze training phase; figure 16B shows escape latency for five groups of mice during Morris water maze training phase; FIG. 16C shows escape latency on day 5 of mouse training in the Morris water maze experiment; FIG. 16D shows the number of times a mouse crossed the platform in the Morris water maze experiment; FIG. 16E shows the residence time of the mouse in the target quadrant in the Morris water maze experiment; fig. 16F shows a graph of the movement trajectories of the groups of mice, wherein a represents Ctrl group; b represents Abeta1-425μm groups; c represents UB150mg/kg group; d represents UB100mg/kg group; e represents UB50mg/kg group; figure 16G shows basal levels of rigidity in three groups of mice during the conditioned fear testing period.
FIG. 17 shows H in hippocampal DG region of 5 groups of mice under UB intervention&E staining (HE), NISS staining (NISS), IBA1 immunohistochemical staining results; wherein, a represents Ctrl group; b represents Abeta 1-425 μm group; c represents UB150mg/kg group; d represents UB100mg/kg group; e represents UB50mg/kg group.
FIG. 18 shows the Western blot electrophoresis of PSD95 and Synapsin I in brain tissue of AD mice under UB intervention (see FIG. 18A) and its densitometric analysis results (see FIG. 18B); wherein, a represents Ctrl group; b represents Abeta 1-425 μm group; c represents UB150mg/kg group; d represents UB100mg/kg group; e represents UB50mg/kg group.
Figure 19 shows transmission electron microscopy images of hippocampal brain tissue of 5 AD mice under UB intervention.
FIGS. 20 and 21 show the results of immunofluorescence dual-labeling experiments in hippocampal regions of brain tissue of AD mice under UB intervention; among them, fig. 20 shows the expression of the LDAM-specific marker Perilipin2, and fig. 21 shows the expression of the microglia marker IBA 1.
FIG. 22 shows the results of lipid droplet quantification of microglia in brain tissue of AD mice by flow single-label semi-quantitative analysis experiments, the main indicators including the average number of lipid droplets (see FIG. 22 a); BODIPY + positive cell number (see fig. 22 b); the average diameter size of the lipid droplets (see fig. 22 c).
FIG. 23 shows Western blot electropherograms of the hippocampal TIP47, Perilipin1, and Perilipin2 of AD mice (see FIG. 23A) and densitometry analysis thereof (see FIG. 23B); wherein, a represents Ctrl group; b represents Abeta 1-425 μm group; c represents UB150mg/kg group; d represents UB100mg/kg group; e represents UB50mg/kg group.
Figure 24 is an in vitro microglia immunofluorescence image under UB intervention.
FIG. 25 shows BODIPY in microglia in vitro under UB intervention obtained by flow single-label semi-quantitative analysis experiment+Number of positive cells (see FIG. 25A) and mean number of lipid droplets (see FIG. 25B).
Fig. 26 shows the experimental profile of flow-through single-label semi-quantitative analysis of primary microglia (see fig. 26A) and its statistical results (see fig. 26B).
FIG. 27 shows the Western blot experimental electropherograms of primary microglia TIP47, PIN1 and Perilipin2 (see FIG. 27A) and their densitometric analysis results (see FIG. 27B); wherein, a represents Ctrl group; b represents Abeta 1-425 μm group; c represents UB10 μm group; d represents UB5 μm group; e represents UB1 μm group.
Fig. 28 shows the primary microglia green fluorescent particle microsphere phagocytosis experiment results.
FIG. 29 shows the result of DCFH-DA assay for the release of reactive oxygen species from microglia.
FIG. 30 shows the results of ELISA assay for the expression of CCL3, CXCL10, IL-4 and TNF-. alpha.in microglia.
FIG. 31 is a flow chart of an experiment for co-culturing primary microglia and Neuron-2a cells.
FIG. 32 shows the death of Neuron-2a cells in coculture system detected by PI fluorescent staining.
FIG. 33 shows the Western blot electropherograms of MAP-2, PSD95 and Synapsin I (see FIG. 33A) and their densitometric analysis results (see FIG. 33B); wherein, a represents Ctrl group; b represents Abeta 1-425 μm group; c represents UB10 μm group; d represents UB5 μm group; e represents UB1 μm group.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
In this example, the conditions of microglial cell activation and LDAM phenotype transformation in brain tissues of 12-month-old WT aged mice and 12-month-old APP/PS1/tau triple-transgenic AD mice are compared, 2 groups of mouse brain tissue samples (12-month-old WT group, 12-month-old APP/PS1/tau group) are selected to perform IBA1 (microglial cell marker) and PLIN2 (perilipin 2, as LDAM specific marker) immunofluorescence double-label experiment, and from the results of the immunofluorescence double-label experiment shown in fig. 1, IBA1 and PLIN2 in hippocampal DG region of 12-month-old APP/PS1/tau group AD mice have obvious co-localization, and the co-localization percentage is obviously higher than that of 12-month-old WT aged mice. Furthermore, it was also observed that the expression levels of IBA1 and Perilipin2 were significantly increased in the hippocampal DG region of 12-month old APP/PS1/tau triple transgenic AD mice compared to 12-month old WT aged mice.
Example 2
Selecting SPF (specific pathogen free) grade 7-8 week-old male C57BL/6J mice, equivalently dividing the mice into 5 groups according to a random number table method, constructing an AD mouse model according to a flow chart shown in figure 2A, and administering the AD mouse model:
Aβ1-42group (2): using Abeta1-42Mice were induced to become AD mice by lateral intracerebroventricular injection (30. mu.M), and double distilled water was administered at 20. mu.g/kg body weight per day, and administration was stopped on day 21.
Ctrl group: double distilled water was administered daily to mice at 20. mu.g/kg body weight, and administration was stopped on day 21.
TRC group: TRC was intraperitoneally injected at a dose of 8mg/kg/d daily on a Ctrl group basis, and the administration was stopped on day 21.
Aβ1-42+ TRC group: using Abeta1-42Mice were induced to become AD mice by lateral intracerebroventricular injection (30. mu.M), and TRC was intraperitoneally injected daily at a dose of 8mg/kg/d, with the administration being stopped on day 21.
As shown in fig. 2A, animal behavioral tests such as an aging degree scoring test, an intelligence nesting test, a Morris water maze test, a conditioned fear test, and the like, including and recording the body weight and food intake of each group of mice, were performed within one week after the administration was stopped.
The results of the aging degree scoring experiments shown in FIG. 2B show that A beta1-42The aging degree score of the group is obviously improved in 7 days, 14 days and 21 days compared with that of Ctrl group, and Abeta (amyloid beta)1-42The aging degree score of the + TRC group is obviously reduced。
As can be seen from the weight change of the mice in each group shown in FIG. 2C, the weight growth rate is sequentially Abeta from low to high1-42Group, Abeta1-42+ TRC, Ctrl.
As can be seen from the intelligence nesting test results shown in FIG. 2D, A β1-42Lowest score of group, Abeta1-42The scoring results of the + TRC group, TRC group and Ctrl group were almost indistinguishable.
As can be seen from the results of the Morris water maze test shown in FIG. 2E, A.beta.was observed during the 5 day duration of the training phase1-42Latency for group finding of hidden platforms is significantly extended compared to Ctrl group, Abeta1-42Incubation period and Abeta for searching hidden platform in + TRC group1-42The group comparison is obviously shortened, and the fact that the incubation period of searching for a hidden platform of an AD mouse can be obviously reduced by injecting TRC into the abdominal cavity is proved.
As can be seen from the movement trace of the mouse in the Morris water maze experiment shown in FIG. 2F, A beta1-42+ TRC group (a) vs. A β1-42The trajectory of group (b) is simple.
In order to further verify the cognitive function levels of four groups of mice, the conditioned fear memory test was performed in this example, and the results are shown in fig. 2G, in the training period of conditioned fear, the basal stiffness levels of four groups of mice are similar without obvious difference, and in the memory detection period of conditioned fear, a β is1-42The basal rigidity level of the group mice is obviously reduced compared with Ctrl group, and Abeta1-42Base rigidity level and Abeta of mice in + TRC group1-42The mice in the group are obviously improved, which indicates that the memory disorder of AD mice can be obviously improved by injecting TRC into the abdominal cavity.
The above results demonstrate that the expression of A.beta.1-42The induction of the constructed AD mouse model was successful, and the administration of TRC intervention could improve memory impairment in AD mice.
Example 3
This example further verifies the presence of LDAM in brain tissue of AD mice based on example 2.
First, the IBA1 (microglia marker) immunohistochemical results and CD68 (microglia) shown in fig. 5 are shown by fig. 3 and 4Activation marker) as can be seen from the results of the immunoblot experiments, a β1-42Microglia in hippocampal region of group mice were significantly abnormally activated compared to Ctrl group.
FIG. 6 shows the results of Western blotting experiments at A.beta.1-42Under stimulation, Abeta1-42The LDAM markers Perilipin TIP47, Perilipin2 and Perilipin1 of the group mice were significantly higher than those of Ctrl group.
FIG. 7 shows TEM results showing Abeta1-42There were lipid droplets present in the microglia of the group mice; the immunofluorescence double-label result of the brain tissue of the AD mouse shown in FIG. 8 and FIG. 9 shows that the A beta1-42The expression level of LDAM marker Perilipin Perilipin2 in the group mice was significantly up-regulated compared to Ctrl group.
The above results illustrate A β1-42LDAM was present in brain tissue of AD mice constructed by lateral ventricle injection. More interestingly, activation of microglia in brain tissues of AD mice and expression and generation of intracellular lipid droplets can be effectively reduced by the TRC, and LDAM phenotypic transformation is inhibited, so that cognitive dysfunction of the AD mice is relieved.
Example 4
To further clarify the LDAM phenotype in AD cells in vitro, this example utilized a β at a concentration of 5 μ M1-42BV2 microglia was subjected to in vitro time point stimulation experiments to investigate the rule of lipid droplet generation in microglia in vitro.
In this example, BV2 microglia was characterized by using three methods of confocal laser, flow, WB, etc. as shown in fig. 10 and 11 and BODIPY493/503 flow-type single-label semi-quantitative analysis shown in fig. 12 and 13, it was found that a β at a concentration of 5 μ M was detected1-42When the stimulation time reaches 24 hours, the number of lipid droplets and the lipid content in BV2 microglia are the most, and the Western blotting experimental results shown in FIG. 14 also show that the expression levels of Perilipin TIP47, Perilipin2 and Perilipin1 which are markers of LDAM are the highest at this time; and over time, as A beta1-42When the stimulation reaches 48 hours, the lipid drop quantity and the lipid content in BV2 microglia are reducedDuring the dynamic process of LDAM generation, lipid droplets with larger diameters will continuously fuse into lipids or form small lipid droplets. In summary, A β1-42The LDAM phenotype is also present in AD cells in vitro under stimulation.
Example 5
Examples 3 and 4 demonstrate that LDAM exists in both AD mouse brain tissue and AD cells in vitro, and this example verifies whether UB has a regulatory effect on the generation of lipid droplets in LDAM by intervening AD cells in vitro with different concentrations of UB based on example 4.
TABLE 1
| Group of | Stimulation model making | Intervention |
| Ctrl group | PBS | PBS |
| Aβ1-42Group of | Aβ1-42(5μM) | PBS |
| UB10 μ M group | Aβ1-42(5μM) | UB(10μM) |
| UB5 μ M group | Aβ1-42(5μM) | UB(5μM) |
| UB1 μ M group | Aβ1-42(5μM) | UB(1μM) |
4', 6-diamidino-2-phenylindole (DAPI) and BODIPY green dye with excitation wavelength of 493nm and emission wavelength of 503nm were used to stain cells, and photographs were taken under a confocal laser fluorescence microscope, and FIG. 15 shows that UB concentrations of 1-10 μ M were effective in inhibiting A β from affecting in vitro BV2 microglia1-42The number of lipid droplets generated during induction initially suggested that UB could regulate the phenotypic shift of LDAM.
Example 6
This example demonstrates whether UB can be an active ingredient of an inhibitor of lipid droplet generation, an inhibitor of phenotypic transformation of lipid droplet accumulation microglia or a drug for treating or preventing neurodegenerative diseases associated with neuroinflammation, by the steps of:
(1) SPF-grade 7-8 week-old male C57BL/6J mice are selected and equally divided into 5 groups according to a random number table method, and the grouping conditions are shown in table 2:
TABLE 2
All mice were injected with physiological saline for 2h, and Abeta1-42Group and treatment group mice were given a lateral ventricle injection of a β1-42(10umol/L) Induction of AD model, Ctrl group given equal amount of physiological saline instead of A beta1-42And (4) injecting.
After 21 days of model administration, the change conditions of the cognitive and memory behaviors related to the AD mice are observed, for example, the detection of the corresponding indexes is completed by adopting an aging degree scoring experiment, a water maze experiment, a condition fear-based memory experiment and other experiments, and the result is shown in figure 16. After completion of the above experiments, mice were sacrificed and heart perfusion and material was taken. HE staining method and Niss detection method for observing survival (marking and semi-quantifying) of mouse hippocampal tissue neuron cells, and high content of mouse hippocampal tissue neuron cellsErzhi staining for observing change of neuron dendritic spine in mouse brain tissue, and immunohistochemical detection of IBA1+Microglial activation was observed, and the results are shown in fig. 17; western blot experiments were performed to detect the expression levels of synaptophysin PSD95 and Syn, and the results are shown in FIG. 18.
(2) On the basis of the step (1), detecting the expression change condition of microglia lipid droplets in the brain tissue of the AD mouse by using a transmission electron microscope and a flow type single-standard semi-quantitative analysis experiment, wherein the result is shown in fig. 19 and 22; the expression of the LDAM specific marker, namely the Perilipin2 and the microglia marker, namely the IBA1, in the brain tissue of the AD mouse is determined by detecting the BODIPY493/503 and the Perilipin2 through an immunofluorescence double-label experiment, and the results are shown in figures 20 and 21; the qPCR detects the change of the transcription level of the DAM characteristic gene, and the Western blot experiment verifies that the qPCR regulates and controls LDAM transcriptome characteristic proteins TIP47, Perilipin1 and Perilipin2, and the result is shown in FIG. 23.
(3) Establishing an in vitro cell model of inflammatory stimulation by taking primary microglia as a culture system, wherein the group is shown in table 3:
TABLE 3
| Group of | Stimulation model making | Treatment of |
| Ctrl group | PBS | PBS |
| Aβ1-42Group of | Aβ1-42(5μM) | PBS |
| UB10 μ M group | Aβ1-42(5μM) | UB(10μM) |
| UB5 μ M group | Aβ1-42(5μM) | UB(5μM) |
| UB1 μ M group | Aβ1-42(5μM) | UB(1μM) |
Immunofluorescence assay BODIPY and Perilipin2 determined markers associated with the LDAM phenotype in vitro, the results are shown in fig. 24; the inhibition of UB on microglia lipid droplet expression in the brain tissue of AD mice is verified by a flow type single-label semi-quantitative analysis experiment, and the result is shown in FIG. 25 and FIG. 26; qPCR detects the change of LDAM characteristic gene transcription level, WB verifies the regulation and control of qPCR on LDAM characteristic protein, and the result is shown in FIG. 27.
The fluorescent latex particle phagocytosis experiment detects the capability of primary microglia to phagocytize the green fluorescent particle microspheres, and the result is shown in fig. 28; the DCFH-DA method detects ROS levels in microglia cells, and the results are shown in FIG. 29; the results of the qPCR and ELISA assays for changes in inflammation-related markers such as CCL3, CXCL10, IL-4, and TNF-. alpha.are shown in FIG. 30.
(4) As shown in fig. 31, an in vitro co-culture system of primary microglia and Neuron cells of Neuron-2a was constructed by using primary microglia as a culture system, and the groups are shown in table 4:
TABLE 4
| Group of | Stimulation model making | Treatment of |
| Ctrl group | PBS | PBS |
| Aβ1-42Group of | Aβ1-42(5μmol/L) | PBS |
| UB10 μ M group | Aβ1-42(5μmol/L) | UB(10μM) |
| UB5 μ M group | Aβ1-42(5μmol/L) | UB(5μM) |
| UB1 μ M group | Aβ1-42(5μmol/L) | UB(1μM) |
Adding the supernatant of the microglia cell culture medium into a Neuron culture system of Neuron-2a, wherein PBS or normal saline is used for replacing A beta in Ctrl group1-42The supernatant of the microglia cell culture medium under the stimulation condition is used for detecting the death condition of the neuron cells by using a PI fluorescent probe, and the result is shown in FIG. 32; WB was used to detect the expression of PSD95 and Syn, and the results are shown in FIG. 33.
Claims (10)
1. Use of an inhibitor of phenotypic conversion of lipid droplet accumulation microglia for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disease associated with neuroinflammation, characterized in that: the drug contains a lipid droplet accumulation microglial cell phenotype transformation inhibitor as an active ingredient, wherein the lipid droplet accumulation microglial cell phenotype transformation inhibitor can inhibit a lipid droplet accumulation microglial cell phenotype.
2. Use according to claim 1, characterized in that: the lipid droplet accumulation microglial cell phenotype transformation inhibitor contains a lipid droplet production inhibitor as an active ingredient, wherein the lipid droplet production inhibitor can inhibit the production of lipid droplets in microglia.
3. Use according to claim 2, characterized in that: the lipid droplet generation inhibitor contains (i) urolithin B or a pharmaceutically acceptable salt thereof or (ii) a long fatty acid acyl-CoA synthetase inhibitor as an active ingredient.
4. Use according to any one of claims 1 to 3, characterized in that: the neurodegenerative disease related to neuroinflammation is one or more of Parkinson's disease, Alzheimer's disease and multiple sclerosis.
5. A lipid droplet generation inhibitor characterized by: comprising (i) urolithin B or a pharmaceutically acceptable salt thereof or (ii) a long fatty acid acyl-CoA synthetase inhibitor as an active ingredient.
6. The lipid droplet production inhibitor according to claim 5, wherein: the lipid droplet generation inhibitor can inhibit beta amyloid polypeptide 1-42 from inducing primary microglia to generate lipid droplets.
7. An inhibitor of phenotypic transformation of lipid droplet accumulation microglia, wherein: comprising (i) urolithin B or a pharmaceutically acceptable salt thereof or (ii) a long fatty acid acyl-CoA synthetase inhibitor as an active ingredient.
8. A method for screening a drug which is an inhibitor of lipid droplet generation, an inhibitor of phenotypic transformation of lipid droplet accumulation microglia or a drug for treating or preventing a neurodegenerative disease associated with neuroinflammation, comprising the steps of:
adding beta amyloid polypeptide 1-42 into a primary microglia in-vitro culture system for co-culture to serve as a blank group;
adding beta amyloid polypeptide 1-42 and a drug to be detected or a drug combination to be detected into a primary microglia in-vitro culture system for co-culture to serve as an intervention group;
counting the proportion of the lipid droplets accumulating the microglia in each group;
selecting an intervention group effective to reduce the proportion of microglia accumulated by lipid droplets relative to the blank group.
9. The drug screening method according to claim 8, characterized in that: the step of counting the proportion of the lipid droplets accumulating the microglia in each group comprises the following steps: the proportion of lipid droplets accumulating microglia was determined by observing the number of lipid droplets under a confocal laser fluorescence microscope.
10. The drug screening method according to claim 8, characterized in that: the neurodegenerative disease related to neuroinflammation is one or more of Parkinson's disease, Alzheimer's disease and multiple sclerosis.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115791727A (en) * | 2022-11-26 | 2023-03-14 | 吉林大学 | Application of fat-soluble fluorescent probe in neuronal death detection and detection method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104582730A (en) * | 2013-05-17 | 2015-04-29 | 曾嘉 | Method for improving metabolic function of mitochondria and use thereof |
| WO2020146632A1 (en) * | 2019-01-11 | 2020-07-16 | The Board Of Trustees Of The Leland Stanford Junior University | Methods of treating neurodegenerative disorders and identifying targets therefore |
| CN111995610A (en) * | 2019-05-27 | 2020-11-27 | 首都医科大学 | Application of urolithin compound in medicine for treating glioblastoma |
| CN112023049A (en) * | 2020-09-10 | 2020-12-04 | 四川大学华西医院 | New application of inhibitor of long-chain acyl coenzyme A synthetase 4 and medicine for treating cerebral ischemic stroke |
-
2021
- 2021-08-27 CN CN202110996135.8A patent/CN113730583A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104582730A (en) * | 2013-05-17 | 2015-04-29 | 曾嘉 | Method for improving metabolic function of mitochondria and use thereof |
| WO2020146632A1 (en) * | 2019-01-11 | 2020-07-16 | The Board Of Trustees Of The Leland Stanford Junior University | Methods of treating neurodegenerative disorders and identifying targets therefore |
| CN111995610A (en) * | 2019-05-27 | 2020-11-27 | 首都医科大学 | Application of urolithin compound in medicine for treating glioblastoma |
| CN112023049A (en) * | 2020-09-10 | 2020-12-04 | 四川大学华西医院 | New application of inhibitor of long-chain acyl coenzyme A synthetase 4 and medicine for treating cerebral ischemic stroke |
Non-Patent Citations (4)
| Title |
|---|
| ABDULRAHMAN: "Urolithins Attenuate Multiple Symptoms of Obesity in Rats Fed on a High-Fat Diet", 《糖尿病代谢综合征和肥胖——目标和治疗》 * |
| JIALIN XU: "Urolithins Attenuate LPS-Induced Neuroinflammation in BV2Microglia via MAPK, Akt, and NF- B Signaling Pathways", 《J. AGRIC. FOOD CHEM》 * |
| 孙永馨: "阿尔茨海默病发病与淀粉样β蛋白1～42/α1-抗糜蛋白酶复合物及脂质代谢紊乱的关系", 《中国临床康复》 * |
| 陶永光: "《肿瘤分子生物学与细胞生物学实验手册》", 30 November 2014, 湖南科学技术出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115791727A (en) * | 2022-11-26 | 2023-03-14 | 吉林大学 | Application of fat-soluble fluorescent probe in neuronal death detection and detection method |
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