CN103356611A

CN103356611A - Application of berberine in preparation of weight-loss drug

Info

Publication number: CN103356611A
Application number: CN2013102544710A
Authority: CN
Inventors: 宁光; 张晓燕; 杨颖�
Original assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Current assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Priority date: 2013-06-24
Filing date: 2013-06-24
Publication date: 2013-10-23

Abstract

The invention belongs to the field of pharmaceutical preparation, and particularly provides an application of berberine in preparation of a medicine used for inhibiting autophagy of adipocyte and a medicine used for treating obesity. According to the application, C3H10T1/2 cell is cultured and induced to be differentiated into mature adipocyte, then the test for the impact of berberine to the mature C3H10T1/2 adipocyte autophagy marker protein LC3 and an autophagy substrate P62, and the test for detecting the C3H10T1/2 flat autophagy flux variation through chloroquine serving as a lysosomal inhibitor are performed, and the variation of the quantity of autophagosome can be observed through a transmission electron microscope; the results of various tests show that the berberine can effectively inhibit the autophagy of C3H10T1/2 adipocyte and provides theoretical basis for clinically reasonably applying the berberine to the treatment of obese patients; in addition, the berberine also can be applied to the preparation of the medicine for inhibiting the autophagy of adipocyte.

Description

The application of berberine in the preparation slimming medicine

Technical field

The invention belongs to medical preparation field, the particularly application of berberine in preparation inhibition adipose cell autophagy medicine and preparation obesity treating medicine.

Background technology

Along with the raising of economic level, the change of people's dietary structure, momental minimizing, the prevalence of the disease of causeing fat rises rapidly, and at present overweight the or population of being obese in the whole world approximately reaches 1,000,000,000 according to estimates.Obesity is as one of key component of metabolism syndrome, and is closely related with various diseases such as type 2 diabetes mellitus, dyslipidemia, hypertension, coronary heart disease, the serious harm mankind's health.

Recent study is found, autophagy is playing an important role aspect the lipid metabolism of liver and fatty tissue, autophagy is one and engulfs self cytoplasm protein or organelle and make its coated vesicle that enters, and merge to form the autophagy lysosome with lysosome, the degrade process of its content that wraps up, the metabolism that realizes by this cell itself need and the renewal of some organelle.So the autophagy phenomenon provides new thinking for the treatment of obesity.Under the normal growth condition, nutrient substance is abundant, autophagocytosis remains on a foundation level, be mainly used in house keeper's purpose, it for cell provide a kind of routine-the garbage-cleaning service, such as the degraded of long-lived protein and damaged cell device, such continuous renewal is particularly important to the cell of static termination differentiation; Under the condition of hunger, autophagy is further induced, and for cells survival provides extra inside nutrition, autophagy can be by energy stress or other stress-induceds widely, great majority are the synthetic re-using aminoacid for important albumen, and perhaps the generation for ATP provides catabolic substrate.Although autophagy can not be removed all metabolism rubbish, but autophagy can prevent the generation of disease, the defective of autophagy and human numerous disease are closely related, important energy metabolism organ and endocrine organ such as hepatopathy, nerve retrograde affection, cancer and other metabolism syndrome fat, studies show that in adipose cell, autophagy can degrade mitochondrion and kytoplasm are regulated Adipocyte Differentiation.

Berberine is a kind of isoquinoline alkaloid, is the main component of Chinese Drug Rhizomes of Coptis, and its chemical structural formula is as follows:

Former studies has confirmed that berberine has a lot of pharmacological actions and comprises antibiotic, antitumor, expression that can inflammation-inhibiting factor TNF-α, IL-6 etc. especially has obvious anti-inflammatory effect at the individuality of obesity, and nearest research finds that berberine can blood sugar lowering, improves Insulin Resistance.

C3H10T1/2 cell line be in 1973 first from 14 to 17 days C3H mouse embryonic cell separate, these cells show the fibroblast form in incubation, be similar to mescenchymal stem cell in function, existing document confirms that Gdf6 and Bmp4 all can induce C3H10T1/2 to break up to adipose cell lines, show the feature of adipose cell.

Whether the autophagy approach participates in berberine to the regulating action of lipid metabolism, not relevant research.The present invention is by the impact of berberine on C3H10T1/2 adipose cell autophagy, and autophagy is to the regulation and control of lipid metabolism, for the treatment of obesity provides new drug target and Therapeutic Method in the monitoring mature fat cell.

Summary of the invention

The purpose of this invention is to provide berberine and suppress C3H10T1/2 adipose cell autophagy aspect definite experimental data is provided, for the treatment that clinically can be more rational berberine be applied to the obese patient provides theoretical foundation.

The present invention relates to the application of berberine in the preparation obesity treating medicine.

The present invention relates to the application of berberine in preparation inhibition adipose cell autophagy medicine.

Described adipose cell is the C3H10T1/2 adipose cell.

Autophagy (autophagy) is the distinctive biosis of eukaryotic cell, and then autophagosome parcel self impaired or aging organelle and the macromole of cell by forming duplicature be combined with lysosome, realizes the degraded to content.

The light chain 3(LC3 of microtubule-associated protein 1) is the homologous protein of yeast Atg8 in mammalian cell, during cell autophagy, Atg-8 can excise one section aminoacid of its C end, after the latter and PHOSPHATIDYL ETHANOLAMINE (PE) combination, forms LC3-II (LC3-PE) on the cell membrane thereby be attached to.The LC3-II is unique a kind of reliably protein labeling relevant with autophagy.In yeast, the content of Atg8 increased by ten times at least when autophagy was induced.At mammalian cell, when autophagy is induced, the not too large variation of the total content of LC3, but the LC3-I changes increase to the LC3-II.P62/SOSTM1 is coupled to LC3, and the substrate as autophagy is degraded late, so expression and the autophagy activity of whole p62 level are negative correlation in the cell.Therefore, the method for available Western trace detects the conversion of LC3 in the cell and the expression of p62 changes, and a situation arises to react autophagy.

The present invention now cultivates the C3H10T1/2 cell and induce the differentiation and maturation adipose cell, then by berberine on the C3H10T1/2 adipose cell autophagy marker protein LC3 of maturation and autophagy substrate P62 affect test, lysosome inhibitor chloroquine detects the variations of flux test of C3H10T1/2 fat autophagy and transmission electron microscope observing autophagosome number change situation, detect berberine and can suppress C3H10T1/2 adipose cell autophagy phenomenon, for the treatment that clinically can be more rational berberine be applied to the obese patient provides theoretical foundation.By detection of the present invention, all testing results show: berberine can suppress C3H10T1/2 adipose cell autophagy, so berberine has good curative effect in the medicine for the treatment of of obesity.

The present invention has the following advantages:

1, berberine can suppress C3H10T1/2 adipose cell autophagy and be dose dependent, berberine suppresses the autophagy of C3H10T1/2 adipose cell and is time dependence, and autophagosome quantity obviously reduces after processing from the electron microscopic observation to the berberine, so berberine is remarkable to the inhibition of C3H10T1/2 adipose cell autophagy, can be used on preparation and suppress in the adipose cell autophagy medicine.2, experimental data and theoretical foundation are provided for the more rational treatment that berberine is used for the obese patient clinically.

Description of drawings

Fig. 1 is berberine on the comparison diagram that affects of the different action times of C3H10T1/2 adipose cell autophagy marker protein LC3 II/I.

Fig. 2 is berberine on the comparison diagram that affects of the different action times of C3H10T1/2 adipose cell autophagy substrate p62.

Fig. 3 is that the berberine of variable concentrations is on the comparison diagram that affects of C3H10T1/2 adipose cell autophagy marker protein LC3 II/I.

Fig. 4 is that the berberine of variable concentrations is on the comparison diagram that affects of C3H10T1/2 adipose cell autophagy substrate p62.

Fig. 5 adds respectively chloroquine (CQ) in the C3H10T1/2 adipose cell, rapamycin+chloroquine (RAPA+CQ), and behind berberine+chloroquine (BBR+CQ) effect 12h, the comparison diagram of autophagy marker protein LC3 II/I.

Fig. 6 is the transmission electron microscope picture of three groups of autophagosomes, compares with matched group (CON group), and positive control rapamycin group (RAPA group) autophagosome quantity showed increased, and berberine group (BBR group) autophagosome quantity significantly reduces.

The specific embodiment

Below in conjunction with embodiment, the invention will be further described:

The berberine of using in the experiment (Berberine) is available from Shanghai City Drug Supervisory testing institute; Mesoblastic pluripotent stem cell strain C3H10T1/2 cell is available from U.S. ATCC (American Type Culture Collection).

1, cell culture and induce differentiation

(1), the cultivation of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain with go down to posterity:

The C3H10T1/2 cell routine is incubated in the 10%FBS/DMEM culture fluid, puts 5%CO ₂, 37 ℃ of incubators hatch, after cell reaches the 80%-90% fusion, in super-clean bench with pipet with the culture fluid sucking-off, add 0.25% pancreatin 5mL digestion, microscopically sees that cellular contraction is rounded, add normal 10%FBS/DMEM culture fluid and stop the trypsinization reaction, repeatedly blow and beat cell residual on bottle wall with pipet, make cell detachment culture bottle wall, suck and be equipped with in the centrifuge tube of culture fluid, the centrifugal 3min of 1000rpm, sedimentation cell, centrifugal after, supernatant discarded, add again normal culture fluid, with pipet piping and druming, cell is scatter.Go down to posterity in the 1:4 ratio and to cultivate or be inoculated into 6 orifice plates or 12 orifice plates, put 5%CO ₂, 37 ℃ of incubators cultivate, and change culture fluid 1 time in per 2 days.

(2), the strain of C3H10T1/2 mesoderm pluripotency embryonic stem cell induce differentiation:

Cell on culture plate covers with the normal culture fluid 10%FBS/DMEM of rear adding induced liquid A(, contain recombinant human insulin 5ug/ml, 3-isobutyl-1-methylxanthine 0.5mmol/L, dexamethasone 1umol/L, indomethacin 125nmol/L, triiodothyronine 1nmol/L, rosiglitazone 1umol/L), remove induced liquid A after 2 days and change the normal culture fluid 10%FBS/DMEM of induced liquid B(into, contain recombinant human insulin 5ug/ml, triiodothyronine 1nmol/L, rosiglitazone 1umol/L), remove induced liquid B after 2 days and change normal culture fluid 10%FBS/DMEM into, change every other day later on culture fluid one time.Treating that 90% above cell is rounded and a large amount of fat occur drips, and represents that namely the C3H10T1/2 cell induced successfully, becomes ripe adipose cell.The adipose cell of inducing is hatched 12h with 0.2%BSA/DMEM, can carry out subsequent experimental.

2, test process and test result (* among the figure represents P＜0.05, and * * represents P＜0.01):

(1), berberine is on the impact of C3H10T1/2 adipose cell autophagy marker protein LC3 and autophagy substrate P62

After C3H10T1/2 is induced to differentiate into mature fat cell, add

berberine effect

3h, 6h and 12h, Western Blot result shows: after the berberine effect, the ratio of autophagy marker protein LC3 II/I reduces along with the prolongation of berberine action time, specifically as shown in Figure 1; P62 albumen is along with the prolongation of berberine action time and increase, specifically as shown in Figure 2.Illustrate that berberine can suppress C3H10T1/2 adipose cell autophagy and presentative time dependency.

After C3H10T1/2 is induced to differentiate into mature fat cell, the berberine that adds variable concentrations, be respectively 0.2umol/L, 1umol/L, 5umol/L, effect 12h, Western Blot result shows: autophagy marker protein LC3 II/I reduces along with increasing of berberine concentration, specifically as described in Figure 3; Autophagy substrate p62 increases along with increasing of berberine concentration, specifically as shown in Figure 4.Illustrate that berberine can suppress C3H10T1/2 adipose cell autophagy and be dose dependent.

(2), lysosome inhibitor chloroquine detects C3H10T1/2 fat autophagy variations of flux

Chloroquine is a kind of lysosome inhibitor, can suppress the lysosomal degraded of autophagy, thereby increases the level of LC3-II in the cell, and this is a kind of method that detects the autophagy flux, namely by suppressing lysosomal degraded, detects the generation of autophagy in the certain hour.Experiment adopts chloroquine to process the C3H10T1/2 adipose cell, detects C3H10T1/2 fat autophagy variations of flux.

Ripe C3H10T1/2 adipose cell adds respectively chloroquine (CQ), rapamycin+chloroquine (RAPA+CQ), behind berberine+chloroquine (BBR+CQ) effect 12h, Western Blot result is as shown in Figure 5: compare with matched group (CON group), CQ group autophagy marker protein LC3 II/I significantly raises; Compare with the CQ group, RAPA+CQ organizes as positive control, and autophagy marker protein LC3 II/I significantly raises, and BBR+CQ group autophagy marker protein LC3 II/I significantly reduces, and illustrates that berberine can suppress C3H10T1/2 adipose cell autophagy.

(3), transmission electron microscope observing autophagosome number change

The transmission electron microscope observing result is as shown in Figure 6: autophagosome is the little body structure of blister not of uniform size that duplicature or multilayer film hold, and the inside is wrapped in still indigested kytoplasm and organelle, such as mitochondrion, endoplasmic reticulum etc.Compare with matched group (CON group), positive control rapamycin group (RAPA group) autophagosome quantity showed increased, and berberine group (BBR group) autophagosome quantity significantly reduces.

In sum, by berberine the affect test, lysosome inhibitor chloroquine of C3H10T1/2 adipose cell autophagy marker protein LC3 and autophagy substrate P62 detected the variations of flux test of C3H10T1/2 fat autophagy and transmission electron microscope observing autophagosome number change situation, show that all berberine can suppress C3H10T1/2 adipose cell autophagy.

Claims

1. the application of berberine in the preparation obesity treating medicine.

2. the application of berberine in preparation inhibition adipose cell autophagy medicine.

3. the application in the claim 2, described adipose cell are the C3H10T1/2 adipose cell.