CN106236812B - A kind of application of barren wort total chromocor on the drug for preparing anti-body aging - Google Patents
A kind of application of barren wort total chromocor on the drug for preparing anti-body aging Download PDFInfo
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Abstract
The present invention provides a kind of application of sheep leaves of pulse plants general flavone on the drug for preparing anti-body aging.Application of the barren wort total chromocor in the application and the adjusting small intestinal stem cell microenvironment of aging in the quantity proliferation and differentiation capability for promoting the small intestinal stem cell of aging, the cell micro-environment is Wnt/ β-catenin signal path and BMP signal path.
Description
Technical field
The present invention provides a kind of application of barren wort total chromocor on the drug for preparing anti-body aging, belongs to medicine pharmacy
Field.
Background technique
With the continuous improvement of human longevity, aging relevant issues are got worse, and correlative study in the recent period prompts body
Aging starts from the aging of enteron aisle.The epithelium layer for being covered in small intestine surface is that self-renewal capacity is strongest in the mammalian body
Institutional framework, the small intestinal stem cell (ISC) in crypts of small intestine bottommost are the sources that small intestinal mucosa epithelial cell is constantly updated.
Have now been found that there are two types of different types of small intestine population of stem cells: one kind can recycle rapidly, between crypt base paneth's cell,
Referred to as crypt base columnar cell (cryp base columnar cells, CBC), for the stem cell of activation, specificity mark
Note object is Lgr5 (leucine-rich-repeat-containingG-protein-coupledreceptor- 5)；And it is another
Kind then remains static, referred to as DNA marker retained cells (DNA-label-retaining cells, LRC), specificity
Marker is Bmi1 (Bmilpolycombringfingeroncogene 1).
Herba Epimedii is China's Chinese traditional herbs, has the effect of kidney-replenishing, strengthening the bones and muscles, barren wort total chromocor
(TotalFlavonesof Epimedium, TFE) is its main pharmacodynamics ingredient, and icariin (Icariin, ICA) is TEF
Main active.Studies have shown that TFE can improve the aging-related degeneration of multiple species experimental models, delay senescence related
The occurrence and development of disease extend the service life.But whether TFE there is adjusting to make the quantity and function of the small intestinal stem cell of aging rats
With being still not clear.This research and inquirement variation of aging rats small intestine stem cell population and Proliferation, Differentiation ability, and further grind
The variation for having studied carefully its microenvironment Wnt and BMP signal path and barren wort total chromocor are to the adjustment effect of above-mentioned change.
Summary of the invention
The present invention provides a kind of application of barren wort total chromocor on the drug for preparing anti-body aging.The specially excessive sheep
Application of the leaves of pulse plants general flavone in the quantity proliferation and differentiation capability for promoting the small intestinal stem cell of aging.Barren wort total chromocor adjusting declines
Application in grown-ups and children's intestinal stem cell microenvironment.Wnt/ β-catenin signal path and BMP signal path is adjusted in Herba Epimedii.
Detailed description of the invention
Fig. 1 is influence (× 100) of the barren wort total chromocor to Intestinal morphology, wherein the a:6 month age group b:24 monthly age
Group c: barren wort total chromocor low dose group (10mg/kg)；D: barren wort total chromocor middle dose group (20mg/kg)；E:
Barren wort total chromocor high dose group (60mg/kg).
Fig. 2 is the influence (× 200) that barren wort total chromocor breaks up small intestine stem cells hyperplasia.Wherein A, B group are as follows: PAS dye
Color and counting；C, D group are as follows: PCNA dyeing and counting.A, in C, a:6 month age group b:24 month age group c: barren wort total chromocor low dosage
Group (10mg/kg)；D: barren wort total chromocor middle dose group (20mg/kg)；E: barren wort total chromocor high dose group (60mg/kg).
Fig. 3 be influence (× 200) of the barren wort total chromocor to small intestine stem cell population, wherein A be Immunofluorescence test not
With the expression of group Lgr5, B is the expression that Western Blot detects different group Bmi1, and C is Western Blot band ash
Degree analyzes the expression of different group Bmi1.A, b, c, d, e are respectively a:6 month age group b:24 month age group c: barren wort total chromocor in A
Low dose group (10mg/kg)；D: barren wort total chromocor middle dose group (20mg/kg)；E: barren wort total chromocor high dose group
(60mg/kg).Compared with age group in June:*P ﹤ 0.05,**P ﹤ 0.01；Compared with 24 months age groups,#P ﹤ 0.05,##P ﹤ 0.01.Fig. 4 is
Influence of the barren wort total chromocor to small intestine Wnt/ β-catenin signal path, wherein A is that Western Blot detects different groups
The expression of other Wnt/ β-catenin signal path GAP-associated protein GAP, B are Western Blot band gray analysis difference group Wnt/
The expression of β-catenin signal path GAP-associated protein GAP.In A, adult:6 month age group；Aging:24 month age group；TFE-10: excessive sheep
Leaves of pulse plants general flavone low dose group (10mg/kg)；TFE-20: barren wort total chromocor middle dose group (20mg/kg)；TFE-60: Herba Epimedii
General flavone high dose group (60mg/kg)；In B, a is Western Blot band gray analysis difference group p-GSK-3 β/GSK-3
β ratio, b are Western Blot band gray analysis difference group Dkk1/actin ratio, and c is Western Blot band ash
Degree analyzes different group β-catenin/actin ratios.
Compared with age group in June:*P ﹤ 0.05,**P ﹤ 0.01；Compared with 24 months age groups,#P ﹤ 0.05,##P ﹤ 0.01.
Fig. 5 is influence of the barren wort total chromocor to small intestine BMP signal, and wherein A is that BMP4 immunohistochemistry detects different groups
Expression, B are the image analysis of BMP4 immunohistochemistry, and C is the expression that Western Blot detects different group BMP2, D Western
Blot band gray analysis difference group BMP2/actin ratio, in A, a, b, c, d, e are respectively a:6 month age group b:24 month age group
C: barren wort total chromocor low dose group (10mg/kg)；D: barren wort total chromocor middle dose group (20mg/kg)；E: Herba Epimedii is always yellow
Ketone high dose group (60mg/kg).Adult:6 month age group；Aging:24 month age group；TFE-10: barren wort total chromocor low dose group
(10mg/kg)；TFE-20: barren wort total chromocor middle dose group (20mg/kg)；TFE-60: barren wort total chromocor high dose group
(60mg/kg).Compared with age group in June:*P ﹤ 0.05,**P ﹤ 0.01；Compared with 24 months age groups,#P ﹤ 0.05,##P ﹤ 0.01.
Specific embodiment
Materials and methods
Experimental drug
Barren wort total chromocor is purchased from Ren Cheng Biotechnology Co., Ltd, carries out ultrasound using 1% sodium carboxymethylcellulose
Hydrotropy prepares the TFE solution of corresponding each dosage.
Experimental animal
SPF grades of male SD rats, are purchased from Medical College of Sanxia University's Experimental Animal Center, production licence number: SCXK (Hubei Province)
2011-0061, sub-cage rearing and are ingested at free water.It is divided into 6 monthly ages adult control group (adult) by its different developmental phases
With 24 monthly age aging model groups (aging), since 18 monthly ages, barren wort total chromocor low, middle and high dose groups give rat respectively
TFE 10mg/kg, 20mg/kg, 60mg/kg stomach-filling to 24 monthly ages, 1 time a day, weekly administration 6 days, continuous 6 months.Experiment examination
Agent
Total protein extraction kit, BCA method protein quantification kit: Beijing Puli's lema gene Technology Co., Ltd.；Primary antibody
β-actin, Lgr5, Dkk1:Santa Cruz company；β: Abcam company of Bmi1, BMP4, β-catenin, GSK3 β, p-GSK3；
PCNA, BMP2: the farsighted Access-was Bioisystech Co., Ltd in Suzhou.Goat-anti rabbit, rabbit-anti mouse, rabbit-anti sheep secondary antibody: the auspicious biotechnology of Wuhan section
Co., Ltd, DAB kit: Wuhan doctor's moral biology.
Key instrument
Western blot Vertial electrophorestic tank: Bio-Rad company, the U.S.；Chemiluminescence imaging system (Bioshine
ChemiQ4800mini): Ou Xiang (Shanghai) scientific instrument Co., Ltd；Ultramicrotome (ULTRACUTR): Leica company (moral
State) booth piece bake piece machine (CS-V1): medical (Hubei Xiaogan) Instrument Ltd. of great achievement；Optics inverted microscope (CX41):
Olympus company (Japan)；Just setting fluorescence microscope (NIKON ECLIPSE CI), Japanese Nikon.
HE dyeing and PAS dyeing
Row heart perfusion after rat anesthesia, separation intestinal tissue take 0.3-0.5mm jejunal tissue at the 20cm of stomach lower end, pass through
4% paraformaldehyde is fixed for 24 hours, rear conventional dehydration, paraffin embedding, slice, HE dyeing and PSA dyeing.Every group randomly selects 5 greatly
Mouse, every rat small intestine take 5 discontinuous slices, choose 5 columnar cell's marshallings and longest intestines in every slice
Villus, HE dye the variation of optics microscopically observation difference group Intestinal morphology, measure villus length, Crypt depth, and count
It calculates villus/crypts value (V/C value).The number of goblet cell between every 100 intestinal epithelial cells of PAS dyeing counting difference group
Amount.Immuning tissue (fluorescence) chemical staining
Small intestine's paraffin mass is sliced (4 μm), anticreep glass slide patch, conventional dewaxing to water.Eurya plant lemon phthalate buffer is high
Pressure repairs 20min, is cooled to room temperature, 3%H2O2Serum room temperature closes 1h after 37 DEG C of incubation 10min, and 4 DEG C of refrigerators of primary antibody are added dropwise and incubate
It educates overnight, biotin labeling secondary antibody is added dropwise in immunohistochemistry, is incubated at room temperature 1h, DAB/H2O2Reaction solution, haematoxylin redyeing, hydrochloric acid
Ethyl alcohol differentiation, conventional to be dehydrated, transparent, dry, mounting.The secondary antibody that Cy3 label is added dropwise in immunofluorescence, which is protected from light, is incubated for 1h, DAPI dye
Color, mounting, microscopy.
Western-blot detection
According to total protein extraction kit specification, small intestine's albumen is extracted, the upper of 5X is added in BCA method protein quantification
Sample buffer, in 95 DEG C of heat denatureds, PAGE gel electrophoresis simultaneously turns pvdf membrane, and 5% skim milk room temperature closes 1h, primary antibody
4 DEG C of overnight incubations, secondary antibody are incubated at room temperature 1h, the development of ECL chemoluminescence method, and GelDoc gel imager acquires image.Statistics
Processing
Experimental data is analyzed using SPSS18.0 software, and IHC and Western blot data are using Image J software point
Analysis, data mean ± standard deviationIndicate, use one-way analysis of variance (one-wayANOVA) between group, with P <
0.05 is statistically significant for difference.
Barren wort total chromocor is on the morphologic influence of naturally-aged rat small intestine
As a result as shown in Figure 1, adult control rats intestinal villi it is elongated at finger-like, marshalling is close, naturally-aged
In model group, villus length obviously shortens, and arrangement is loose and not regular enough, and villus width dramatically increases.It is always yellow to give Herba Epimedii
After ketone 6 months, compared with naturally-aged model group, rat height of naps and arrangement situation all make moderate progress.Utilize Image-Pro
Plus7.0 software measures small intestinal villous height, Crypt depth, calculates V/C value, the experimental results showed that, aging model group and adult
Control group is compared, and small intestinal villous height, Crypt depth V/C value reduce, and after giving barren wort total chromocor, each dosage group villus is long
Degree dramatically increases (P ﹤ 0.01).Crypt depth increased, but only middle dosage is statistically significant (P ﹤ 0.05), and V/C value is only
High dose is statistically significant (P ﹤ 0.05).It the results are shown in Table 1.
Influence of 1 barren wort total chromocor of table to aging rats mucous membrane of small intestine layer morphological change
Note: adult:6 month age group；Aging:24 month age group；TFE-10: barren wort total chromocor low dose group (10mg/kg)；
TFE-20: barren wort total chromocor middle dose group (20mg/kg)；TFE-60: barren wort total chromocor high dose group
(60mg/kg), compared with age group in June:*P ﹤ 0.05,**P ﹤ 0.01；Compared with 24 months age groups,#P ﹤ 0.05,##P ﹤ 0.01.
Barren wort total chromocor is proliferated to naturally-aged rat small intestine and the adjustment effect of differentiation capability
PAS coloration result is as shown in Figure 2 A, and goblet cell is located at small intestine ciliated border, rounded or goblet shape, dyeing
Afterwards in distinct red.Compared with control group of growing up, aging model group goblet cell quantity is remarkably decreased (P < 0.01).Give medicine
The goblet cell quantity of low dosage and high dose significantly rises (P < 0.01) after object, and middle dosage is without significant change.
PCNA coloration result is as shown in Figure 2 C, mainly crypts of small intestine express, aging model group compared with control group of growing up,
PCNA expression quantity significantly reduces (P < 0.01), after giving barren wort total chromocor treatment, middle dose group and high dose group positive cell
Significantly increase (P < 0.01), low dosage is without significant change.
Influence of the barren wort total chromocor to naturally-aged rat small intestine stem cell population
Immunofluorescence results are as shown in Figure 3A, and the marker Lgr5 (green fluorescence) of CBC is mainly expressed in crypt base,
Compared with the control group, Lgr5 expression reduces model group, after giving barren wort total chromocor treatment, is increased.We use simultaneously
The expression of western blot detection Bmi-1.The results show that model group is compared with the control group, expression quantity significantly reduces (P ﹤
0.01) it, is increased after medication, as a result sees Fig. 3 B, C.
Barren wort total chromocor is to naturally-aged rat small intestine stem cell Microenvironment
Adjusting to Wnt/ β-catenin signal
Wnt/ β-catenin signal path is the most important signal for maintaining small intestine stem cells hyperplasia, and small under normal circumstances
Intestinal crypts basal part height expresses Wnt/ β-catenin.This research western blot the results show that compared with control group of growing up,
The expression quantity of aging model group β-catenin significantly reduces (P < 0.05), and Wnt/ β-catenin access obtains inhibitor Dkk1's
Expression quantity then increases and has statistical significance (P < 0.05), meanwhile, the activated state p-Tyr216-GSK-3 β expression quantity of GSK-3 β
Increase, has statistical significance (P < 0.05).After giving barren wort total chromocor treatment, β-catenin, p-Tyr216-GSK-3 β
Expression quantity up-regulation, Dkk1 expression quantity is lowered, and three dosage groups all have statistical significance (P < 0.05).See Fig. 4.
Adjusting to BMP signal
BMP signal path is another important access for adjusting small intestinal stem cell function, is detected by western blot
The expression of BMP2, compared with the control group, expression quantity is substantially reduced aging model group as the result is shown, have significant difference (P <
0.05), give barren wort total chromocor treatment after, compared with aging model group, 3 dosage group BMP2 significantly increase (P <
0.05).Simultaneously Showed by immune group result, BMP4 mainly enteric epithelium express, aging model group compared with the control group, expression quantity
It significantly reduces, gives low dosage and high dose expression quantity up-regulation (P < 0.05) after barren wort total chromocor treatment, middle dosage is without obvious
Change.See Fig. 5.
Claims (1)
1. a kind of application of barren wort total chromocor on the drug for preparing anti-body aging, the specially barren wort total chromocor are promoting
Increased amount of application or the barren wort total chromocor into the small intestinal stem cell of naturally-aged adjust the small intestinal stem cell microenvironment of aging
On application, specially β-catenin, p-Tyr216-GSK-3 β expression quantity up-regulation, Dkk1 expression quantity lower, BMP2 expression quantity
Up-regulation, the up-regulation of BMP4 expression quantity.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1565582A (en) * | 2003-07-02 | 2005-01-19 | 广东医学院 | Chinese medicine formulation with functions of resisting senescence and preventing and treating posteoporosis and cerebral lesion |
| CN1569017A (en) * | 2003-07-24 | 2005-01-26 | 复旦大学附属华山医院 | Total epimeddium flavone tablet and its preparation method |
| CN105641015A (en) * | 2016-01-20 | 2016-06-08 | 三峡大学 | Application of epimedium total flavone in intervention of intestinal tract barrier dysfunction |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1565582A (en) * | 2003-07-02 | 2005-01-19 | 广东医学院 | Chinese medicine formulation with functions of resisting senescence and preventing and treating posteoporosis and cerebral lesion |
| CN1569017A (en) * | 2003-07-24 | 2005-01-26 | 复旦大学附属华山医院 | Total epimeddium flavone tablet and its preparation method |
| CN105641015A (en) * | 2016-01-20 | 2016-06-08 | 三峡大学 | Application of epimedium total flavone in intervention of intestinal tract barrier dysfunction |
Non-Patent Citations (2)
| Title |
|---|
| 淫羊藿黄酮诱导的骨髓间充质干细胞成骨分化中WNT信号通路的研究;张锦芳等;《现代生物医学进展》;20100613;第1006-1008页 * |
| 炎性衰老机制与干预的实验研究;夏世金等;《中国老年杂志》;20091031;第2595-2598页 * |
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