25-羟基胆固醇 z-bo 2020-12-11 363

　　Introduction: Cholesterol plays an important role in the development and function of neurons. In particular, it involves the biophysical properties of membranes and the biogenesis of synapses. As part of its functional pathway, cholesterol is metabolized into different natural oxygen-containing sterols, including 24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. 24-Hydroxycholesterol (24-HC) regulates the expression of cholesterol homeostasis-related enzymes in the brain. It has recently been found to play an important role in midbrain dopaminergic neurogenesis. On the other hand, 25-hydroxycholesterol (25-HC) is a stronger inhibitor of sterol synthesis. It is produced by cholesterol 25 hydroxylase (CH25 H), which is produced by cells in response to changes in cholesterol levels, and can also be produced during infection. It is an amplifying agent of inflammatory response and has various effects on lipid metabolism. 25-HC regulates the activity of sterol regulatory element binding proteins (SREBPs) and liver X receptor (LXRS) signaling pathways by reducing cholesterol synthesis and increasing cholesterol efflux and clearance. Increased production of oxidized cholesterol is associated with several diseases. For example, in Alzheimer's disease patients, the level of 24-HC has been shown to be increased. In fact, the role of 24-HC in Alzheimer's disease pathogenesis and neuronal death has been fully demonstrated. High levels of 25-HC are also found in neurological diseases such as amyotrophic lateral sclerosis (ALS), neuromyelitis optica, and x-linked adrenal leukodystrophy. 25-HC amplification products were observed during virus infection. However, the relationship between neuronal damage/loss in neurological diseases and/or viral infections and 25-HC is unclear. Interestingly, some reports show that 25-HC has toxic effects on different cell lines such as PC12 cells, mouse CATH, neural cell lines and oligodendrocyte 158 N cell lines. These findings have yet to be verified in vivo. The zebrafish is a powerful model organism for neurodevelopment and toxicity studies. They provide a simple, fast, economic and reliable model for neurodevelopment and toxicology research. The purpose of this study is to use a zebrafish model to investigate the effect of 25-HC on the survival of living neurons. We found that 25-HC affects early neurodevelopment and movement, and increases brain and spinal cord cell death.

　　Zebrafish: According to standard procedures, the wild type (AB/TL strain), isl1-GFP and huc-GFP transgenic zebrafish are cultivated and reared. Add 0.003% phenylthiourea (PTU) to the zebrafish hatching water 24 hours after fertilization to block pigment formation. Treatment and analysis of 25-hydroxy cholesterol: 4 days after fertilization, it was exposed to 25-hydroxy cholesterol (25-HC) at concentrations of 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, 50 μM, 60 μM, 80 μM, and 100 μM for four days. Prepare 25 hydroxycholesterol at 1 μMg/ml and store at -20°C. In each experiment, embryos were collected from three mating pools and treated as n=1. 20 embryos (n=20) and 10 replicates (n=10) were used as batches, with different concentrations of 25-hydroxyl Cholesterol processes embryos and quantifies their survival rate, hatch rate, gross morphological and behavioral changes. For imaging analysis, each treatment group consisted of 7 fish, and the experiment was repeated 3-4 times (n=3-4). Observe the embryo survival rate and hatch rate. The morphology of brain neurons of huc:GFP and isl1:GFP at 2dpf were analyzed. Simply put, 2dpf zebrafish were fixed overnight in 4% paraformaldehyde at 4°C, and then the difference was analyzed using a Zeiss LSM780 confocal microscope.

　　Motion activity measurement: implant 20hpf embryos into low melting point agarose, record their movement in the chorion for 20 minutes with a camera, and keep their moisture during this period. Then use the danioscope software to quantify the percentage of time the embryo is active. The behavioral exposure response was also monitored at 2 dpf. Simply put, use a pair of blunt tweezers to gently touch the tails of zebrafish larvae and observe how they respond to touch. Use EyoVisual XT 12 software (NOLDUS) for analysis to quantify distance swimming.

　　 motor axon visualization: immunohistochemical analysis of 2dpf zebrafish to observe the axon projection of motor neurons. Fix the embryos with 2dpf in the dent (20% dimethyl sulfoxide and 80% methanol) overnight at 4°C. After fixation, the embryos were rehydrated with successively decreasing methanol concentrations (75%, 50%, and 25%) for 30 min. Then rinse with PBST (0.1% Tween) several times (1 h). On this basis, embryos were incubated in block solution (2% BSA and 10% NGS PBST) for 1 hour, and the primary antibody (anti-acetylated tubulin, 1:500) was added, and incubated overnight at 4°C. The next day, the embryos were rinsed with PBST (0.1% Tween) and blocked again for 1 h (PBST containing 2% BSA and 10% NGS). Then add the secondary antibody (Alexa Furor 488, 1:1000), and incubate overnight at 4°C. The next day, the embryos were rinsed again with PBST (1 h) and placed on glass slides with 80% glycerol. A Zeiss LSM780 confocal microscope was used to take Z-Stack images.

　　 mauthner cells, muscle and α-Bungarotoxin staining: 2dpf zebrafish were observed by immunohistochemical methods to observe mauthner cells, muscle morphology and post-synaptic receptors at neuromuscular junctions in the back of the zebrafish. The embryos (2dpf) were fixed overnight in 4% paraformaldehyde (PFA) at 4°C, and washed twice with PBST (0.1% Tween) after fixation for 10 minutes each. After this, the embryos were incubated in collagenase (1 mg/ml in PBS) for 30 min. Then rinse the collagenase several times with PBST (0.5% Triton-X). Then the embryos were incubated in a blocking solution (2% NGS, 1% BSA, 1% DMSO, 1% Triton) for 1 h. Finally, the embryos were incubated with 10ug/ml sulfohdamine-conjugated α-Bungarotoxin (molecular probe ) Incubate in PBST for 30 min. Then rinse the embryos with PBST (0.5% Triton-X) several times, and then image. Phalloidin staining method was used to visualize the muscles. The embryos were fixed as described above, then infiltrated in 2% Triton X-100/PBS for 1.5 h, and cultured overnight in 1:20 Alexa-Fluor 488 conjugated phalloidin on a shaker at 4 C. The embryos are then rinsed and imaged.

　　 mauthner cells are stained, and the embryos are washed with PBST (0.5% Triton-x) and dH2O-triton-X (0.5%) after fixation. Then incubate with acetone (100%) at -20°C for 10 min. Subsequently, the embryos were washed with PBST (0.5% Triton-X) and PBS-DT (1% BSA, 1% DMSO, 0.5% Triton-X), and further blocked in 5% NGS PBS-DT for 1 h. The primary antibody (anti-3A10, 1:200) was added to the blocking solution and incubated overnight at 4°C. The next day, rinse the embryos with PBS-DT, then add the secondary antibody (alexa fluor 488, 1:1000), and incubate overnight at 4°C as before. Observe and image the embryo with a confocal microscope.

　　 TUNEL staining: fix 2dpf fish in 4% PFA, use 25, 50, 75% MeOH in PBST (0.1% Tween) for continuous dehydration and rehydration, rinse with PBST several times. The embryos were digested with proteinase k (10μg/ml) for 20 min, washed with PBST, and fixed with 4% PFA for 20 min. Followed by 2 quick washes and 3 long washes of 20 minutes. , Then rinse again with PBS and incubate in the TUNEL reaction mixture at 37°C for 1 hour. Observe under the microscope.

　　 acridine orange staining: 48hpf embryos in E3 medium are placed in separate wells of a 6-well plate, and incubated with acridine orange 1x for 30 min at room temperature. Acridine orange raw material is prepared in 1 mg/ml (100X) MILIQ water and stored at -20 degrees. Then the embryos were washed three times for 10 min with E3 medium. Then treated with tricaine, the dead cells were observed under a microscope. 24 hpf and 2 dpf zebrafish were also incubated with 25-HC (10μM and 40μM) overnight to evaluate the effect of 25-HC on the later stages of the main development process. After 25-HC treatment, the larvae were evaluated for cell death and neuronal defects. Results: 25-Hydroxycholesterol (25-HC) affects development and is fatal in long-term high-dose exposure: Zebrafish embryos were treated with 9 concentrations of 25-HC 2, 5, 10, 20, 40, 50, 60, 80, 100μM (2-4 cell period), for 4 days, analyze its survival rate, hatch rate and general morphology every day. The effects of 25-HC exposure on the survival rate, hatchability and morphology of zebrafish embryonic larvae are shown in Figure 1. Compared with the control group, the 2-40μM25-HC treatment group had no significant changes in the embryo mortality rate of 24-96hpf. However, at high doses of 25-HC (50-100μM), the survival rate of the 80μM and 100μM treatment groups was significantly reduced, resulting in 100% embryo death at 2 dpf. Embryos exposed to 25-HC will cause obvious morphological defects, such as curved spine and mild to extreme pericardial edema, but at 0-40μM, the entire body size does not change significantly. The total morphological defect rate of 40μM25-HC group and above tissues increased. When the concentration exceeds 40 μM, the embryos die within 2-3 days after fertilization, which is considered impossible for early development studies. Exposure to 25-HC at a concentration of 40 μM had no significant effect on the hatchability. In order to further study the effect of 25-HC on the early nervous system of zebrafish. In follow-up studies, representative low and high doses (10μM and 40μM) were selected, at which embryos survived and had relatively consistent developmental effects .

　　The effect of 26-HC on the early movement of zebrafish larvae: In order to evaluate the effect of chronic exposure to 25-HC on the nervous system, we first evaluated the movement behavior of zebrafish embryos and larvae. Interestingly, the early spontaneous winding of embryos showed a downward trend, which was significantly lower at 40 μM. A similar abnormal locomotor behavior was also observed in 2 dpf, where the larvae exposed to a concentration of 40 μM hardly moved in the tactile-induced swimming response of larvae exposed to 10 and 40 μM 25-HC. At 5dpf, we observed that the locomotor activity of juveniles treated with 10μM 25-HC was significantly reduced, while the locomotor activity of fish treated with 40μM 25-HC was not. In addition to motor deficits, 40μM 25-HC treated zebrafish larval motor neuron axons were abnormally shortened. In addition, 40μM 25-HC treatment destroyed neuromuscular connections and muscle morphology. We observed an abnormal aggregation of postsynaptic receptors in the middle of each somite. Compared with the control group, larvae exposed to low concentrations of 25-HC had no significant differences in motor axon morphology, number of postsynaptic receptors at neuromuscular junctions, and muscle morphology. We did not observe any major changes in the morphology of mauthner cells. These data indicate that high concentrations of 25-HC may have a serious impact on the early development of the motor system, mainly at the level of motor axons.

　　25-HC changes the development of the zebrafish central nervous system and leads to cell death: We next tried to further evaluate the effect of elevated 25-HC levels on the central nervous system. For this, we used a transgenic line expressing GFP [huc:GFP]. The overall structure of the 2dpf larval brain shows that the volume of the head is reduced, and the number of neurons in the brain is reduced, which is more obvious at 40μM than at 10μM. Next, we used the [isl1:GFP] transgenic line to detect the brain motor neurons of zebrafish larvae exposed to 25-HC. Compared with the control group, zebrafish injected with 40μM 25-HC significantly reduced the location and number of trigeminal nerve (NV) and facial nerve (NVi) branch motor neurons at 2dpf. In addition, in these fishes, vagus nerve (nx) motor neurons show abnormal mid-lateral positioning and intercellular spacing. Although zebrafish exposed to 10μM 25-HC showed abnormal localization of facial (NVii) gill motor neurons, these defects were not serious compared to zebrafish exposed to higher concentrations of 25-HC. We also use an acetylated tubulin that marks mature axons to analyze brain axons. 25-HC treatment resulted in an overall reduction in the number of brain axons. At high doses, the staining of acetylated tubulin in the axons of trigeminal ganglion cells almost disappeared. Compared with untreated larvae, 25-HC exposure resulted in a significant increase in apoptotic cells in the brain and spinal cord in a dose-dependent manner.

　　 Late exposure to 25-HC has no effect on the neurodevelopment of zebrafish: Whether the defect caused by 25-HC depends on the developmental stage of administration. Treat 24 hpf and 2dpf zebrafish embryos with 25-HC, and analyze whether there are abnormalities at 2dpf and 3dpf, respectively. Interestingly, when many developmental processes have already begun or occurred, if 25-HC is injected after 24hpf or 2dpf, no significant death or changes in motor axon characteristics are observed.

　　 Conclusion: Our research shows that 25-HC has an adverse effect on the neuromuscular development, survival and behavior of zebrafish. This study provides new information about the toxic effects of oxidized cholesterol 25-HC on nerve and muscle development. However, additional experiments are needed to fully characterize the mechanism by which 25-HC causes these defects. For example, binding partners for 25-HC, such as LXRS, can also be genetically or pharmacologically tested on zebrafish to assess the potential of 25-HC to alleviate effects. Before evaluating whether inhibiting the production of 25-HC may play a neuroprotective effect under certain pathological conditions, we need to further verify our findings on other model systems (such as mice).