CN110025767A - A kind of osteocalcin is preparing the application in Alzheimer disease drugs - Google Patents
A kind of osteocalcin is preparing the application in Alzheimer disease drugs Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
Abstract
The present invention relates to a kind of osteocalcin to prepare the application in Alzheimer disease drugs, present invention discover that OCN can improve the cognition dysfunction and starch fiber sample plaque deposition, the activation that can inhibit spongiocyte, controllable AKT/mTOR access of AD mouse, the drug for preparation prevention and/or treatment Alzheimer disease provides new possibility direction and molecular target.
Description
Technical field
The invention belongs to treatment of alzheimer field, in particular to a kind of osteocalcin is preparing Alzheimer disease medicine
Application in object.
Background technique
Body is in aging course usually with different degrees of bone density decline and neurodegeneration.Alzheimer
Sick (Alzheimer ' s disease, AD) is the most common neurodegenerative disease, while being also that dull-witted principal pathogenetic is former
Cause, the cause of disease are still not clear, and show as various exceptions such as memory, thinking, behavior and mood, seriously affect the life matter of patient
Amount.Epidemiological investigation show AD patient's osteoporosis risk with higher, and patients with osteoporosis there is also
Higher AD risk prompts the two to exist and suffers from altogether.The common feature of primary osteoporosis and AD are no region, kind
Race, socio-economic status boundary, increase with the age and incidence increases, seriously affect the quality of life of patient, prevention and treatment is appointed
Business is arduous.At present for AD drug therapy can only part patients in remission, it is very limited to the effect of disease itself.If energy
Certain inner link or common signal access are found from both neurodegenerative diseases, it is new by being provided for the treatment of the two
Direction.
In recent years, bone is constantly being mined as the function of endocrine organ.Osteocalcin (osteocalcin, OCN) conduct
One of the major hormone of osteoblast secretion, is no longer merely considered as a classical bon e formation marker, even more whole body
The important Auto-regulator of energetic supersession can enhance insulin secretion, improve insulin resistance, improve sugar tolerance and plasma lipid profile, adjust
Brown adipose tissue differentiation is controlled, Male reproduction function and development are influenced.Clinical research from cross section and that part is longitudinal
Also indicate that OCN level and blood glucose level, insulin resistance and fat mass exist negatively correlated in circulation, with normal individual, 1 type or 2
The non-fat content of the patients such as patients with type Ⅰ DM, gestational diabetes mellitus, metabolic syndrome, polycystic ovary syndrome is positively correlated, can also
Predict diabetes risk.
But the current research in relation to bone Central nervous system regulation more lacks, and majority is not bone specific secretion
Substance.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of osteocalcin to prepare answering in Alzheimer disease drugs
With there is no the correlative studys that osteocalcin is used to prepare prevention and/or treatment Alzheimer disease drugs before this.
The present invention provides a kind of osteocalcin to prepare the application in Alzheimer disease drugs.
Further, the drug is using osteocalcin as active constituent, be equipped with pharmaceutically acceptable auxiliary material or it is complementary at
Divide and is prepared into preparation use.
Further, the active form of the osteocalcin is non-carboxylation osteocalcin ucOCN.
Further, the preparation be selected from injection, subdermal implants, tablet, pulvis, granule, capsule, oral solution,
One of sustained release agent.
ImmunohistochemistryResults Results of the present invention show: either intraperitoneal injection or Central injection ucOCN substantially reduced can form sediment
For powder fiber-like patch in the deposition of hippocampus of mice and cortex, the protecting effect of Central injection ucOCN is especially significant, can reduce close
72% fiber-like plaque deposition.OCN cannot be only used for improving the cognitive disorder symptom of AD mouse, and the pathology that can also treat AD changes
Become, the related drugs for disclosing OCN can become a kind of effective means for the treatment of early onset AD.
Beneficial effect
Present invention discover that OCN can improve the cognition dysfunction of AD mouse and starch fiber sample plaque deposition, can inhibit glue
The activation of cell plastid, controllable AKT/mTOR access, for preparation prevention and/or treatment Alzheimer disease drug provide it is new
Possible direction and molecular target.
Detailed description of the invention
Fig. 1 is that osteocalcin is injected intraperitoneally to improve the schematic diagram of AD mouse anxiety corelation behaviour；Wherein, A is the entrance of AD mouse
The time of open field；B is the number that AD mouse enters open field；C is the time of Elevated plus-maze open arms；D is elevated plus
The number of labyrinth open arms；E is the time that AD mouse enters camera-lucida；F is the number that AD mouse enters camera-lucida.
Fig. 2 is the schematic diagram that the Spatial memory impairment that osteocalcin improves AD mouse is injected intraperitoneally；Wherein, A is
AD mouse finds the time of underwater platform in the water maze training stage, and B is the walk time of the quadrant where original platform, and C is to wear
Cross the number of original platform.
Fig. 3 is the schematic diagram that the amyloid plaque deposits that osteocalcin mitigates AD hippocampus of mice and cortex are injected intraperitoneally；Wherein,
A is WT mouse, and B is WT+10ug/kg OCN mouse, and C is AD mouse, and D is the AD mouse that 1ug/kg ucOCN is injected intraperitoneally, and E is
The AD mouse of 10ug/kg ucOCN is injected intraperitoneally, F is that immunohistochemistry A β dyes quantitative analysis results, and G is Western Blot inspection
Survey result.
Fig. 4 is the schematic diagram that intracerebroventricular injection osteocalcin improves AD mouse anxiety corelation behaviour；Wherein, A be AD mouse into
Enter the time of open field；B is the number that AD mouse enters open field；C is the time of Elevated plus-maze open arms；D is overhead ten
The number of word labyrinth open arms；E is the time that AD mouse enters camera-lucida；F is the number that AD mouse enters camera-lucida.
Fig. 5 is the schematic diagram for the Spatial memory impairment that intracerebroventricular injection osteocalcin improves AD mouse；Wherein, A
The time of underwater platform is found in the water maze training stage for AD mouse, B is the walk time of the quadrant where original platform, and C is
Across the number of original platform.
Fig. 6 is the schematic diagram for the amyloid plaque deposits that intracerebroventricular injection osteocalcin mitigates AD hippocampus of mice and cortex；Its
In, A is WT mouse, and B is AD mouse, and C is the AD mouse of intracerebroventricular injection 20ng/h ucOCN, and D is that immunohistochemistry A β dyeing is fixed
Amount analysis is as a result, E is Western Blot testing result.
Fig. 7 A is the schematic diagram that various concentration aging A β 1-42 intervenes PC12 cell；
Fig. 7 B is to give ucOCN to be incubated for the schematic diagram that cell activity caused by improving A β 1-42 is damaged.
Fig. 8 A-D is the schematic diagram for the AKT/mTOR access that osteocalcin regulates and controls AD mouse and PC12 cell.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
The present embodiment selects 7 monthly age APP/PS1 male mices and brood negative control hero mouse as experimental animal, purchase
From model animal research institute, Nanjing University.
Embodiment 1
(1) OCN intervenes
The mode of intraperitoneal injection ucOCN is taken to intervene APP/PS1 mouse first.It is 4 weeks a length of when entire intervention, it is daily fixed
Time point intraperitoneal injection ucOCN is primary, and dosage is respectively 1ug/kg and 10ug/kg.Therefore, pass through the mice group of intraperitoneal injection
It is respectively as follows: WT, WT+10ug/kg OCN, AD, AD+1ug/kg OCN and AD+10ug/kg OCN, it is every group 15, totally 75 small
Mouse.The configuration method of ucOCN is that 1mg ucOCN powder is dissolved in the sterile N.S. of 1ml, averagely dispenses and is stored in -80 DEG C
Refrigerator is spare, and when use can be diluted to corresponding concentration with sterile N.S..
It participates in promoting insulin secretion, improvement insulin resistance, improvement sugar tolerance and blood lipid etc. one in periphery in view of ucOCN
The adjusting of serial physiological function, in order to which clear ucOCN is directly to enter brain in maincenter performance protective effect or by improving periphery
Function of organization and participate in the adjusting to AD pathophysiological process indirectly, while in such a way that telocoele directly gives ucOCN into
Row is intervened.Telocoele ucOCN means of intervention is microdialysis press pump continuous injection 2 weeks, and the rate of release of microdialysis press pump is
0.5ul/h, releasing dosage 20ng/h.Therefore, intracerebroventricular injection animal packet is tri- groups of WT, AD, AD+20ng/h, every group 8
Only, totally 24 mouse.
Two ways successively carries out open field, Elevated plus-maze and light and shade shuttle experiment detection respectively after intervening small
Mouse anxiety-like behavior, determined with Morris water mouse Spatial memory ability；It draws materials after the completion of behaviouristics detection, paraffin section is immune
Groupization contaminates hippocampus and cortical amyloid threadiness patch, and Western Blot detects A β GAP-associated protein GAP and AKT/mTOR pathway protein table
It reaches.In vitro for 24 hours using aging A β 1-42 damage PC12 cell, and 2h is intervened using various concentration ucOCN in advance, observes ucOCN
Protective effect and the expression of AKT/mTOR pathway protein to cell.
(2) experimental result
1. use first the mode of intraperitoneal injection observe ucOCN intervene whether can improve the anxiety corelation behaviour of AD mouse with
And ability of learning and memory obstacle.
Anxiety corelation behaviour is shuttled by open field, Elevated plus-maze and light and shade, and 3 experiments are mutual to confirm reflection.It is opening
Put field experiment 5min in, relative to WT mouse, AD mouse time locating for open field central area have decreasing trend (F (4,
65)=1.880, P=0.125, post hoc:WT vs AD, P=0.078, Figure 1A), and 28 days AD of ucOCN are injected intraperitoneally
Mouse has increase trend, especially 1ug/kg ucOCN intervention group in the open field central area time, but is not up to statistics
Difference (P > 0.05, Figure 1A)；And the number that AD mouse enters central area significantly reduces (F (4,65)=6.463, P compared with WT mouse
< 0.001, post hoc:WT vs AD, P=0.004, Figure 1B), 1ug/kg and 10ug/kg ucOCN intervene so that AD mouse into
The number for entering central area obviously increases (P=0.006and 0.011respectivelly, Figure 1B), especially 1ug/kg
UcOCN intervenes.
In the 5min of elevated plus-maze test, although that there are statistics is poor for total variances of each group mouse in the open arms time
Different (F (4,65)=2.888, P=0.029, Fig. 1 C), but individually compare simultaneously no difference of science of statistics between two groups；Enter open arms
Number for, AD mouse is reduced trend (F (4,65)=4.507, P=0.003, post hoc:WT vs compared with WT mouse
AD, P=0.641, Fig. 1 D), ucOCN intervenes so that the number that AD mouse enters open arms obviously increases (P=0.002and
0.032respectivelly, Fig. 1 D), especially 1ug/kg ucOCN intervenes.In light and shade shuttles and tests (5min), AD mouse
The time locating for camera-lucida is significantly shorter than WT mouse (F (4,32)=4.160, P=0.008, post hoc:WT vs AD, P=
0.033, Fig. 1 E), and 1ug/kg (P=0.001) and 10ug/kg (P=0.006) ucOCN intervenes so that AD mouse is in camera-lucida
The locating time obviously increases (Fig. 1 E)；Each group does not find notable difference (F (4,32)=0.741, P in terms of entering camera-lucida number
=0.571, Fig. 1 F).In above-mentioned 3 experiments, WT+OCN equal no significant difference (P > 0.05) compared with WT group.
2. passing through the Spatial memory ability of Morris determined with Morris water each group mouse.In 1-7 days training stages, AD
Group mouse finds average time of underwater platform and is longer than other four groups of mouse respectively, wherein compared with WT group mouse the 1st, 3,
4, have within 6 and 7 days statistical difference (P < 0.05, Fig. 2A), had system at the 3rd, 4,5,6 and 7 day compared with WT+OCN group mouse
Meter learns difference (P < 0.05, Fig. 2A), reaches statistical difference (P=at the 7th day compared with 1ug/kg ucOCN intervention group mouse
0.009, Fig. 2A), reach statistical difference (P=0.008, figure on day 4 compared with 10ug/kg ucOCN intervention group mouse
2A).On the day of probe test, after removing platform, relative to WT and WT+OCN group mouse, AD mouse quadrant where original platform
The time of travelling significantly reduces (F (4,55)=2.789, P=0.035, posthoc:WT vs AD, P=0.019；post
Hoc:WT+OCN vs AD, P=0.013, Fig. 2 B), and 10ug/kg ucOCN intervention can obviously increase AD group mouse in original platform
The time (P=0.014, Fig. 2 B) of place quadrant travelling；In addition, AD mouse passes through the number of original platform position compared with WT mouse
It is decreased obviously (F (4,55)=1.390, P=0.249, post hoc:WTvs AD, P=0.024, Fig. 2 C), ucOCN intervention group
There are increased trend, but no difference of science of statistics (P > 0.05, Fig. 2 C).
3. using the Pathologic changes of immunohistochemistry and Western Blot detection hippocampus of mice and cortical amyloid sample lesion.
As shown in figure 3, two groups of mouse of the WT and WT+OCN in August age are without amyloid plaque deposits (Fig. 3 A, B), and AD hippocampus of mice and
There is obviously amyloid plaques (Fig. 3 C), 1ug/kg (F (2,12)=11.18, P=0.003, posthoc:AD in cortex
Vs AD+1ug/kg ucOCN, P=0.004, Fig. 3 D) and 10ug/kg (P=0.007, Fig. 3 E) ucOCN intervene 28 days can be bright
The aobvious amyloid plaque deposits area for reducing AD mouse；Quantitative result is shown in Fig. 3 F.There is also consistent trend, WT by Western Blot
It is expressed without obvious source of people APP and aβ protein with two groups of hippocampus of mice of WT+OCN and cortex, and AD group hippocampus and cortex APP and A β
Albumen great expression, ucOCN intervene the latter two protein expressions and are substantially reduced；OCN receptor GPR158, p-CREB, LC3, BDNF and
The protein expressions such as Bcl-2 have no significant change (Fig. 3 G).
4. observing it in the direct effect of maincenter by mini-osmotic pump telocoele continuous injection ucOCN 2 weeks.
In open field experiment, time of the AD mouse in open field central area be substantially reduced compared with WT mouse (F (2,21)=
5.111, P=0.016, post hoc:WT vs AD, P=0.024, Fig. 4 A) but enter central area number and WT mouse
Without significant difference (F (2,21)=3.747, P=0.041, post hoc:WT vs AD, P=0.870, Fig. 4 B), 20ng/
HucOCN, which intervenes 2 weeks, can obviously increase time (P=0.037, Fig. 4 A) and number (P that AD mouse enters open field central area
=0.044, Fig. 4 B)；In elevated plus-maze test, AD mouse enter open arms time (F (2,21)=12.78, P <
0.001, post hoc:WT vs AD, P=0.550, Fig. 4 C) and number (F (2,21)=0.477, P=0.627, post
Hoc:WT vs AD, P=0.847, Fig. 4 D) equal and WT mouse is without significant difference, and ucOCN intervention can obviously increase AD mouse and exist
The locating time (P=0.003, Fig. 4 C) of open arms；In light and shade shuttles and tests, three groups of mouse enter the time (F (2,21) of camera-lucida
=1.074, P=0.360, Fig. 4 E) and number (F (2,21)=0.296, P=0.747, Fig. 4 F) without significant difference.
In the water maze training stage, WT mouse and 20ng/h ucOCN intervention AD mouse were found underwater flat since the 2nd day
The average time of platform is significantly shorter than AD mouse, AD mouse and WT mouse reached at the 2nd, 4,5 and 7 day statistical difference (P <
0.05, Fig. 5 A), and reach statistical difference (P < 0.05, Fig. 5 A) within the 5th and 7 day in training with AD+20ng/h ucOCN mouse.
On the day of probe test, after removing platform, the walk time (F (2,21)=6.084, P of AD mouse quadrant where original platform
=0.008, post hoc:WT vs AD, P=0.006, Fig. 5 B) and the number (F (2,21)=5.311, P=that passes through original platform
0.014, post hoc:WT vs AD, P=0.010, Fig. 5 C) it is considerably less than WT mouse, though and 20ng/h OCN intervenes mouse
Have the tendency that improving above-mentioned phenotype, but and not up to statistical difference (P=0.063and 0.140respectively, Fig. 5 B,
C)。
5. intracerebroventricular injection ucOCN can reduce the amyloid plaque deposits lesion of APP/PS1 hippocampus of mice.
Intervene unanimously with intraperitoneal injection ucOCN, immunohistochemistry A β is dyed it has also been found that WT hippocampus of mice and cortex are without amyloid
There are apparent amyloid plaque deposits (Fig. 6 B) for plaque deposition (Fig. 6 A), AD hippocampus of mice and cortex, and 20ng/hucOCN is dry
(t=11.06, df=4, P < 0.001, Fig. 6 C, D) is obviously reduced in pre- mouse amyloid plaques area；Western Blot is also shown
Showing analog result, AD hippocampus of mice and cortex, there are obvious APP and aβ protein to express, and 20ng/h ucOCN mouse APP and A β
Protein expression is remarkably decreased (Fig. 6 E).
6. ucOCN can reduce damage of the aging A β 1-42 to PC12 cell activity.
It is provided with various concentration gradient first, gropes the optimum concentration that aging A β 1-42 intervenes PC12 cell.The results show that
1uM, 5uM, 10uM, 25uM intervene activity (F (4,10)=62.81, P < 0.001, the figure that PC12 cell can be effectively suppressed for 24 hours
7A), especially 10uM A β 1-42 can inhibit about 45% cell activity (P < 0.001), therefore be selected as the A β of subsequent cell experiment
1-42 intervenes concentration.2h gives ucOCN incubation in advance before A β 1-42 intervention can obviously improve the work of cell caused by A β 1-42
Property damage (F (5,12)=54.78, P < 0.001, Fig. 7 B), either 1ng/ml (P=0.0287), 10ng/ml (P=
0.0155) or 100ng/ml (P < 0.001), and there are dose-dependent effects.
The variation of the access after 7. vitro detection ucOCN intervenes in vivo.The result shows that 10uM A β 1-42 in vitro
Incubation can obviously reduce AKT (F (5,12)=12.682, P < 0.001, post hoc:Ctr vs A β 1-42, P=0.033) for 24 hours
With the phosphorylation level of mTOR (F (5,12)=2.072, P=0.140, post hoc:Ctr vs A β 1-42, P=0.018),
And the OCN of various concentration shifts to an earlier date 2h and intervenes the expression (P=0.001, Fig. 8 B, D) that can obviously increase p-AKT, 100ng/ml
UcOCN shifts to an earlier date 2h and intervenes the expression that can obviously increase p-mTOR (P=0.035, Fig. 8 B, D).Telocoele is taken to infuse in vivo experiment
Penetrate that OCN intervenes the hippocampus of mouse and cortex carries out Western Blot, it has been found that analog result (Fig. 8 A, C), as the result is shown phase
For WT mouse, AD hippocampus of mice (F (2,6)=30.13, P=0.001, post hoc:WT vs AD, P=0.001) and skin
Under the AKT phosphorylation level of matter (F (2,6)=20.739, P=0.002, post hoc:WT vs AD, P=0.002) is obvious
Drop, 20ng/h OCN persistently intervene can obviously increase within 2 weeks AKT phosphorylation level (P=0.027and P=0.011,
respectively)；And AD hippocampus of mice and the mTOR phosphorylation level of cortex have downward trend, 20ng/h OCN persistently intervenes
Have the tendency that within 2 weeks increasing AKT phosphorylation level, but is to reach statistical difference (P > 0.05).
The present embodiment is passed through using external model in the AD body of APP/PS1 mouse and aging A β 1-42 damage PC12 cell
Intraperitoneal injection and two kinds of means of intervention of intracerebroventricular injection, illustrate therapeutic effect of the OCN for AD for the first time.It was found that OCN can improve AD
The cognition dysfunction and starch fiber sample plaque deposition of mouse, the activation that can inhibit spongiocyte, controllable AKT/mTOR are logical
Road.The present embodiment extreme enrichment theory of the bone as endocrine organ, the regulating and controlling effect for bone Central nervous system provide
Foundation provides new possibility direction and molecular target to prevent and treat Alzheimer disease.
Claims (4)
1. a kind of osteocalcin is preparing the application in Alzheimer disease drugs.
2. application according to claim 1, it is characterised in that: the drug is equipped with pharmacy using osteocalcin as active constituent
Upper acceptable auxiliary material or complementary ingredient are prepared into preparation use.
3. application according to claim 2, it is characterised in that: the active form of the osteocalcin is non-carboxylation osteocalcin
ucOCN。
4. application according to claim 2, it is characterised in that: the preparation be selected from injection, subdermal implants, tablet,
One of pulvis, granule, capsule, oral solution, sustained release agent.
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