CN103611153A - Use of neuregulin 1 - Google Patents
Use of neuregulin 1 Download PDFInfo
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- CN103611153A CN103611153A CN201310695683.2A CN201310695683A CN103611153A CN 103611153 A CN103611153 A CN 103611153A CN 201310695683 A CN201310695683 A CN 201310695683A CN 103611153 A CN103611153 A CN 103611153A
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Abstract
The invention discloses use of neuregulin 1, belongs to the technical field of a biomedicine, and particularly relates to application of the neuregulin 1 inpreparation of a drug for treating lead encephalopathy. The characteristics of the lead encephalopathy are high expression of inflammatory cells and inflammatory factors and wide neuronal cell death. Neuron loss and nerve inflammatory reaction play a crucial role in the lead encephalopathy process. The neuregulin 1 has a significant nerve inflammatory reaction and a role of protecting cell apoptosis in the lead encephalopathy, can inhibit inflammatory activation, reduces the quantities of microglial cells and astrocytes, reduces the expression level of inflammatory cytokines COX-2, and improves the learning and memory defects caused by lead poisoning. In addition, the quantity of ErbB4 positive cells can be increased by the neuregulin 1; the inhibition apoptosis activity of anti-apoptosis protein Bc1-2 is improved; expression of pro-apoptotic protein Bax is reduced, so that apoptosis is inhibited, and neuregulin 1 has significant neuroprotective effects on the lead encephalopathy.
Description
Technical field
The present invention relates to the purposes of neuregulin 1, be specifically related to the application of neuregulin 1 aspect preparation treatment lead encephalopathy medicine, belong to field of biomedicine technology.
Background technology
Plumbous (Lead, Pb) is one of common heavy metal environmental contaminants, become the significant problem of harm humans health.The plumbous damage that can cause multisystem of long-term low dose of contact, nervous system is more obvious.Plumbous expose the inflammatory cell activation such as the astrocyte that makes in mouse brain and microglia, proinflammatory cytokine secretion inflammatory factor after activation is as COX-2, iNOS, IL-6 etc., these inflammatory factors can cell death inducing, thereby causes ability of learning and memory to reduce.Intracerebral ventricle injection lead causes that ability of learning and memory in mice reduces.
Apoptosis and lead encephalopathy in close relations.Jiang Junjun etc. study discovery, and plumbous have promotion apoptotic effect to the mice astrocyte of cultivating, and Bax/Bcl-2 ratio raises.In addition, the apoptosis inhibit of high expressed Bax antagonism Bcl-2 is active.After the lead of 30mg/kg exposes, Bax expresses and raises.Infer that thus lead acetate has very strong neural neurotoxicity, thereby it is even dead to aggravate neuronic apoptosis.Appoint the discoveries such as Zhenhua, mice intracerebroventricular lead acetate can obviously reduce the learning and memory function of mice, increases the autonomic activities number of times of mice.Lead poisoning can directly cause apoptosis, and apoptosis has played important function in neuron loss.Bcl-2 family protein is apoptotic modulin.Neuron loss plays a part very important in saturnine development, explores a kind of new anti-apoptosis method and seems extremely important.
Neuregulin 1 (Neuregulin1, NRG1) is a kind of trophic factors and epidermal growth factor-like domain family, comprises a domain that surpasses 15 transmembrane proteins.NRG1 and its receptor ErbB4 play a significant role in processes such as central nervous system's neurodevelopment, neuronal migration, aixs cylinder stretching, extensions.NRG1 gene has been proved to be can protect Cortical Neurons, dopaminergic neuron and motor neuron etc.NRG1 reduces β
1-42or the lactic acid dehydrogenase of the APP of C-end induction discharges and minimizing TUNEL positive cell number.NRG1 can also protect dopaminergic neuron at parkinson mouse model, and increases the neural generation of veutro Hippocampus and antidepressant effect.NRG1 also can suppress cerebral ischemia reperfusion injury, the apoptosis of the oligodendrocyte progenitor cells of protection Anoxia induction.But can it play a role and have not yet to see report in lead encephalopathy.
Summary of the invention
The purposes that the object of this invention is to provide a kind of neuregulin 1.
In order to realize above object, the technical solution adopted in the present invention is:
The purposes of neuregulin 1, concrete, the application of neuregulin 1 aspect preparation treatment lead encephalopathy medicine.
More specifically, the application aspect the medicine of neuregulin 1 microglia and astrocyte quantity in preparation reduces brain; The application of neuregulin 1 aspect the medicine of preparation reduction inflammatory cytokine COX-2 expression; The application of neuregulin 1 aspect the medicine of preparation increase ErbB4 positive cell quantity; The application of neuregulin 1 aspect the medicine of preparation raising anti-apoptotic proteins Bcl-2 apoptosis inhibit activity; The application of neuregulin 1 aspect the medicine of preparation reduction pro apoptotic protein Bax expression; Neuregulin 1 improves the application aspect the medicine of learning and m emaory obstacle in preparation.
The purposes of neuregulin 1, major embodiment is to get the neuregulin 1 of effective dose and conventional excipients to be prepared into pharmaceutical dosage form as filler (sugar alcohols is as lactose, starch etc.), lubricant (as magnesium stearate, calcium stearate etc.), fluidizer (as aerosil etc.), stabilizing agent (as antioxidant etc.), plasticizer, disintegrating agent etc., as tablet, capsule, injection etc.
Beneficial effect of the present invention:
Accompanying drawing explanation
Fig. 1 is NRG1 β 1 processed group and the interior COX-2 testing result comparison diagram of control group mice brain in the embodiment of the present invention 1;
Fig. 2 is NRG1 β 1 processed group and control group mice Morris water maze Learning and Memory testing result comparison diagram in embodiment 1.
The specific embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
The application of neuregulin 1 aspect preparation treatment lead encephalopathy medicine.
One, materials and methods
1, animal grouping
C57BL/6 is purchased from Zhengzhou University's Experimental Animal Center, male, 6 monthly ages.Lead acetate animal model modeling: adopt classical lead encephalopathy modeling method: lumbar injection lead acetate solution 30mg/kg body weight, the next day once, inject altogether 7 times.After lead acetate injection 7 times, animal is divided into two groups at random: NRG1 β 1 processed group and matched group.Each treated animal is freely looked for food, is drunk water.
2, reagent preparation
People NRG1 β 1(R & D company).NRG1 β 1 or normal saline are treated 14 days (50 nanograms/kg/day, is dissolved in 0.9%NaCl, is called vehicle treated group) by lumbar injection.
3, tissue preparation, SABC
Animal deep anaesthesia, pours into 0.9% normal saline, then 4% ice-cold paraformaldehyde (pH7.4).After getting brain, fix 20% and 30% sucrose dehydration.In the crown section of freezing microtome, thickness 20 μ m.
Immunofluorescence dyeing, the anti-ErbB4 of rabbit is hatched (1:300 dilution, cell signal company, the U.S.), the anti-GFAP(1:800 dilution of mice, Sigma company), the anti-CD11b(1:1000 dilution of mice, Sigma company), the anti-Bcl-2 antibody of mice (1:500 dilution, Sigma company), (the 1:1000 dilution of the anti-Bax antibody of mice, Sigma company), 4 ℃ are spent the night, then the two anti-incubated at room 1 hour of the anti-rabbit igg antibody of the donkey of Alexa Fluor488 labelling and the anti-mouse IgG antibody labelling of Alexa594 donkey.Carry out micro-image observation and cell counting.Every mice is chosen each 3 of hippocampal slices as the object of observation, and frontal cortex is chosen to 3 visuals field.Analytical system is carried out quantitative assay to positive cell number.
4, COX-2ELISA detects
COX-2ELISA test kit is purchased from Shanghai Ya Ji company, the standard substance and the testing sample that add variable concentrations gradient, the 37 ℃ of 60min of detection antibody that add subsequently horseradish peroxidase (HRP) labelling, abandon supernatant, rinse, add substrate A and B, 37 ℃ of 10min, add reaction terminating liquid, 450nm wavelength is measured the OD value in each hole.According to standard curve, calculate the COX-2 concentration of sample to be tested.
5, Morris water maze performance testing
Morris water maze tank is comprised of the round pool of diameter 120cm, a high 55cm, and inwall is painted black, depth of water 41cm, 24 ± 2 ℃ of water temperatures, on pool wall, by 4 equidistant points, pond is divided into 4 quadrants, escapes platform (diameter 6cm) quadrant that is placed in one, the water surface exceeds platform 1cm.Peripheral pool object of reference remains unchanged as space with reference to clue carrying out position in several days times of water maze test.Mouse movement track is connected in the camera acquisition of display system by top, labyrinth.
Morris water maze laboratory program comprises: (1) constant-bearing navigation experiment (place navigation task): for measuring the ability of mice to water maze learning and memory.Experiment lasts 5d, 2 periods of upper and lower noon of every natural gift, per period training 4 times.During training, since a quadrant, by counterclockwise, mice is put into water from all quadrants edge mid-points surface of position to pool wall, observe and be recorded in the time (escape latency, escape latency) of finding platform in 60s.If mice fails to find platform in 60s, with rod, drawn upper mounting plate, and allowed its rest 10s, be designated as 60s incubation period.(2) space exploration experiment (spatial probe test): find after platform the ability to platform space position memory for measuring mice association.Remove platform, then an optional place of entry is put into water by mice towards pool wall, records animal in 60s and passes through target platform (original platform position) number of times.
6, statistical analysis
All data are represented as mean ± standard deviation ，Yong SPSS statistical software and carry out analyzing and processing, between group, relatively with t, check (between two groups, comparing).The measurement result of constant-bearing navigation test escape latency is used the variance analysis of completely randomized design and the multifactor analysis of variance of repeated measurement data; Space exploration result of the test is used the variance analysis of completely randomized design.P<0.05 is that difference has statistical significance.
Two, result
(1) impact of NRG1 on microglia quantity
CD11b is the label of microglia, and immunohistochemistry count detection shows, compares with matched group, and in NRG1 processed group mouse brain, CD11b positive cell number significantly reduces, and difference has statistical significance (in Table 1).
The variation of table 1CD11b positive cell counting (
unit: individual/visual field)
Note: n=5, with matched group comparison, * P<0.05, * * P<0.01
(2) impact of NRG1 on astrocyte quantity
GFAP is the label of astrocyte cell, and immunohistochemistry count detection shows, compares with matched group, and in NRG1 processed group mouse brain, GFAP positive cell number obviously reduces, and difference has statistical significance (in Table 2).Result shows, the effect that NRG1 has significant inhibition inflammatory cell to activate.
The variation of table 2GFAP positive cell counting (
unit: individual/visual field)
Note: n=5, with matched group comparison, * P<0.05, * * P<0.01
(3) impact of NRG1 on inflammatory cytokine COX-2
ELISA testing result shows, compares with matched group, and in NRG1 processed group mouse brain, inflammatory cytokine COX-2 expresses and reduces, and difference has statistical significance (seeing Fig. 1, n=5, with matched group comparison, * * P<0.01).Result shows that NRG1 has the effect of significant inhibition inflammatory factor.
(4) impact of NRG1 on ErbB4 signal intensity
The variation of table 3ErbB4 positive cell counting (
unit: individual/visual field)
Note: n=5, with matched group comparison, * P<0.05
(5) impact of NRG1 on anti-apoptotic proteins Bcl-2
For determining the effect of NRG1 β 1 anti-apoptotic, need to assess apoptosis index, comprise and detect the expression of Bcl-2 and the expression of Bax.Immunohistochemistry count detection shows, compares with matched group, and Bcl-2 positive cell number showed increased in NRG1 processed group mouse brain, difference has statistical significance (in Table 4).
The variation of table 4Bcl-2 positive cell counting (
unit: individual/visual field)
Note: n=5, with matched group comparison, * P<0.05, * * P<0.01
(6) impact of NRG1 on pro apoptotic protein Bax
Immunohistochemistry count detection shows, compares with matched group, and in NRG1 processed group mouse brain, Bax positive cell number obviously reduces, and difference has statistical significance (in Table 5).
The variation of table 5Bax positive cell counting (
unit: individual/visual field)
Note: n=5, with matched group comparison, * * P<0.01
(7) impact of NRG1 on mice behavior
After last Drug therapy, to NRG1 β 1 processed group and control group mice row Morris water maze test, detect the variation of mice space learning memory ability.Result demonstration, the search behavior of NRG1 β 1 processed group animal has certain tendentiousness and purposiveness.NRG1 β 1 treatment can obviously reduce the incubation period of mice with lead poisoning, compares with matched group, and difference has statistical significance (seeing Fig. 2 A).In space exploration test, analyze the number of times discovery that in 60s, mice is passed through target area, the number of times of NRG1 β 1 processed group is than matched group showed increased, and difference has remarkable statistical significance (P<0.01) (to see Fig. 2 B, Veh, n=13; NRG1 β 1, n=14; With matched group comparison, * P<0.05, * * P<0.01, * * * P<0.001).
Three, conclusion
Lead poisoning can activate microglia and the astrocyte in brain, excites inflammatory reaction.The postmortem discovery of Lead Poisoning Patients, has microglia and astrogliosis at tissues such as Hippocampus and cortexes, and the expression of inflammatory factor is also increased.Microglia produces a large amount of proinflammatories, neurotoxic substances and excititoxic molecule, causes Neuron Apoptosis even dead.Except microglia, astrocyte is activation in a large number also.Microglia and astrocyte cause that the release of inflammatory factor COX-2, iNOS etc. and complement system are as the activation of C4, C1q α.These materials make again microglia and Activation of Astrocytes and growth, thereby evoke local cascade inflammatory reaction, increase the weight of the death of neuronal cell.Therefore, suppress the generation that inflammatory reaction can slow down lead encephalopathy.NRG1 β 1 can reduce the quantity of Hippocampus and prefrontal cortex astrocyte and microglia, and the protein expression of inflammatory factor COX-2 also obviously reduces, and suppresses the activation of microglia and astrocyte; Morris water maze shows, the ability of learning and memory of mice with lead poisoning 1 group of NRG1 β be improved significantly.Result shows that NRG1 β 1 has the effect that suppresses microglia and Activation of Astrocytes, can improve the ability of learning and memory obstacle that lead poisoning causes.
Meanwhile, in lead encephalopathy mice, NRG1 can raise the relative level of ErbB4, and the apoptosis inhibit that increases anti-apoptotic proteins Bcl-2 is active, thereby reduces the expression inhibited apoptosis generation of pro apoptotic protein Bax, and neurocyte is had to protective effect.
The purposes of neuregulin 1 in the present embodiment, comprises the following steps: the neuregulin 1 of getting effective dose dissolves in normal saline and is prepared into injection, and intramuscular injection medication, is used for the treatment of the toxic encephalopthy being caused by heavy metal.
Claims (8)
1. the purposes of neuregulin 1, is characterized in that: the application of neuregulin 1 aspect preparation treatment heavy metal poisoning encephalopathy (HIE) medicine.
2. the purposes of neuregulin 1, is characterized in that: the application aspect the medicine of neuregulin 1 microglia quantity in preparation reduces brain.
3. the purposes of neuregulin 1, is characterized in that: the application aspect the medicine of neuregulin 1 astrocyte quantity in preparation reduces brain.
4. the purposes of neuregulin 1, is characterized in that: the application of neuregulin 1 aspect the medicine of preparation reduction inflammatory cytokine COX-2 expression.
5. the purposes of neuregulin 1, is characterized in that: the application of neuregulin 1 aspect the medicine of preparation increase ErbB4 positive cell quantity.
6. the purposes of neuregulin 1, is characterized in that: the application of neuregulin 1 aspect the medicine of preparation raising anti-apoptotic proteins Bcl-2 apoptosis inhibit activity.
7. the purposes of neuregulin 1, is characterized in that: the application of neuregulin 1 aspect the medicine of preparation reduction pro apoptotic protein Bax expression.
8. the purposes of neuregulin 1, is characterized in that: neuregulin 1 improves the application aspect the medicine of learning and m emaory obstacle in preparation.
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| WO2020062407A1 (en) * | 2018-09-27 | 2020-04-02 | 广州中医药大学(广州中医药研究院) | Use of neuregulin 1 |
Citations (2)
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| CN1352391A (en) * | 2000-12-27 | 2002-06-05 | 周明东 | Novel anti-tumor medicine and its screening method and use |
| CN102139095A (en) * | 2010-01-29 | 2011-08-03 | 上海泽生科技开发有限公司 | Method for applying neuregulin to prevent, treat or delay cardiac ischemia reperfusion injuries, and composition for preventing, treating or delaying cardiac ischemia reperfusion injuries |
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Patent Citations (2)
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| CN1352391A (en) * | 2000-12-27 | 2002-06-05 | 周明东 | Novel anti-tumor medicine and its screening method and use |
| CN102139095A (en) * | 2010-01-29 | 2011-08-03 | 上海泽生科技开发有限公司 | Method for applying neuregulin to prevent, treat or delay cardiac ischemia reperfusion injuries, and composition for preventing, treating or delaying cardiac ischemia reperfusion injuries |
Non-Patent Citations (1)
| Title |
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| 李琴: "基因重组NRG-1β对缺血性脑损伤的神经保护作用评价", 《中国博士学位论文全文数据库 医药卫生科技辑》, 15 November 2009 (2009-11-15), pages 070 - 30 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020062407A1 (en) * | 2018-09-27 | 2020-04-02 | 广州中医药大学(广州中医药研究院) | Use of neuregulin 1 |
| CN110946991A (en) * | 2018-09-27 | 2020-04-03 | 广州中医药大学(广州中医药研究院) | Application of neuregulin1 |
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Application publication date: 20140305 |