CN102526023A - Application of EGCG (epigallocatechin gallate) in preparing medicine for treating type-II diabetes - Google Patents
Application of EGCG (epigallocatechin gallate) in preparing medicine for treating type-II diabetes Download PDFInfo
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Abstract
The invention discloses application of EGCG (epigallocatechin gallate) in preparing a medicine for treating type-II diabetes, relates to the field of biology and medicine, and aims to provide an application of EGCG in treating the type-II diabetes. The major technical characteristics are as follows: EGCG can be used for effectively preventing and treating the type-II diabetes and the associated mitochondrial dysfunction in myocardial cell, autophagy increase and oxidative stress up-regulation; and EGCG prompts that the inhibition of EGCG on insulin resistance is possibly realized by inhibiting the regulation of F0x01 on autophagy, and prompts that F0x01 is possibly a target for treating diabetes.
Description
Technical field
The present invention relates to biology and pharmaceutical field, the particularly application of EGCG in preparation treatment type ii diabetes medicine.
Background technology
In recent years, the sickness rate of diabetes on full boundary scope have to go to the toilet to increase severely and add.Relevant supposition shows that by 2025, the quantity of whole world diabetes adult patient will increase to 300,000,000 by 1.35 hundred million of nineteen ninety-five.India, China and the U.S. are the highest countries of onset diabetes rate in the world.At present, about 90% diabetics must have and living habit and fat closely-related type ii diabetes, and its principal character is an insulin resistant.A lot of researchs show that all insulin resistant and mitochondrial dysfunction are closely related, think that mitochondrial dysfunction is to cause the part factor of insulin resistant at least.
Green tea is considered to have significant sanatory effect as a kind of beverage that can generally consume.It is believed that green tea can prevent and treat a lot of diseases, and to form the custom and the longevity of drinking green tea also be closely-related.In recent years, people are more and more to the research of green tea, find that it mainly is to be rich in polyphenol content because of it to cause that green tea can be urged health, and particularly flavonol and flavonol account for the weight of stem tea 30% greatly.Catechin is the principal mode of flavonol, and it is by (-)-epigallocatechin-3-gallate (EGCG), EGC, and ECG and EC form, and wherein most important composition is EGCG.
EGCG accounts for the 9%-13% of green tea gross weight, is main activity of green tea and water-soluble components, has extremely strong antioxidant activity.The chemical molecular structural formula of EGCG is:
Molecular formula is C
22H
18O
11, molecular weight is 458.4 gram/moles.Discover that EGCG can reduce cancer, the neurodegenerative diseases that oxidative stress causes and the sickness rate of diabetes.Because the obesity that diabetes are relevant becomes one of maximum threat of health of people, and EGCG can effectively reduce body weight and fat content.Rat is carried out the EGCG of peritoneal injection 70-92 milligram/kg body weight, can in 2-7 days, reduce the body weight of 20%-30%; Feed the SD male rat for a long time with EGCG, can reduce the subcutaneous fat of 40%-70% and the stomach fat of 20%-35%.Therefore, the mechanism of research EGCG regulation and control body weight and diabetes is very important.
In vivo, the effect that contains the hyperglycemia medicine of EGCG is that the incident by a series of complicacies constitutes.2004, in the volunteer of a group young healthy of Japan, carried out the acute experiment of green tea for the glucose dosis tolerata, the result shows that oral glucose is taken 1.5 grams again after 20 minutes green tea extract can significantly reduce the sugared content in the blood.Discoveries such as Hase, the green tea catechins of taking for 12 weeks continuously can reduce glucose and insulin level, and this shows that taking green tea for a long time can improve insulin sensitivity.Experiment shows that in the SD rat blood serum that EGCG feeds, protein, the content of fatty acid and glycerol all do not have to change, but sugared content significantly reduces.In the mice that high fat is raised, also found similar variation, low-level blood sugar content can reduce oxidative stress and osmotic pressure, thereby slows down the complication of diabetes.
Summary of the invention
The object of the present invention is to provide the new purposes of EGCG in the treatment diabetes, EGCG can effectively recover the heart mitochondrial function, and suppressing autophagy is increased by the two-phase expression of enzymes that the oxidative stress rise causes with minimizing; In the myocardial cell H9c2 that handles with high sugar, EGCG can effectively reduce ROS and produce, and suppresses the autophagy of FoxO1 regulation and control, recovers mitochondrial function, reduces the insulin resistant of high sugar to being caused.
Technical scheme of the present invention is achieved in that
The application of EGCG in preparation treatment type ii diabetes medicine, the imbalance of the heart mitochondrial function that EGCG suppresses to be caused by autophagy.
EGCG also is used to suppress the autophagy rise of high sugared inductive myocardial cell H9c2.
EGCG also is used to suppress the activation of the oxidative stress of high sugared inductive myocardial cell H9c2.
EGCG also is used to recover the mitochondrial function disorder of high sugared inductive myocardial cell H9c2.
EGCG also is used to reduce the insulin resistant of high sugared inductive myocardial cell H9c2.Major advantage of the present invention is:
(1) develops the new purposes of EGCG aspect the treatment type ii diabetes first.
(2) disclosed the important function of the autophagy that FoxO1 regulated and control in type ii diabetes.
(3) set forth the restitution of significant variations and the EGCG of organelle mitochondrion important in the myocardial cell in type ii diabetes to it.
Description of drawings
Fig. 1 is the column sketch map that EGCG recovers GK rat heart mitochondrial function.Wherein, Figure 1A is the protein immunoblot sketch map of mitochondrion complex and VDAC1 albumen variation in the GK rat heart of EGCG nursing; Figure 1B is mitochondrial respiratory chain complex I in the GK rat heart of EGCG nursing, the column sketch map of III and a-ketoglutaric acid dehydrogenase activity.Data from 6 groups of rats, represent with the form of meansigma methods ± standard error; + P＜0.05, ++ P＜0.01, +++P＜0.001vs.Wistar matched group; * P＜0.05, * * P＜0.01, * * * P＜0.001vs.GK matched group.
Fig. 2 is that EGCG reduces the protein immunoblot sketch map that the two-phase enzyme protein expression raises in the GK rat heart.+ P＜0.05, +++P＜0.001vs.Wistar matched group; * P＜0.05, * * P＜0.01vs.GK matched group.
Fig. 3 is the protein immunoblot sketch map of EGCG to mitochondrion generation in the GK rat heart and the influence of mitochondrion dynamics correlative protein expression.Wherein, Fig. 3 A is that mitochondrion generates the protein immunoblot sketch map that correlative protein expression reduces in the EGCG GK rat heart of feeding; Fig. 3 B is the protein immunoblot sketch map of mitochondrion dynamics correlative protein expression in the EGCG GK rat heart of feeding.Data from 6 groups of rats, represent with the form of meansigma methods ± standard error; + P＜0.05, ++ P＜0.01, +++P＜0.001vs.Wistar matched group; * P＜0.05, * * P＜0.01, * * * P＜0.001vs.GK matched group.
Fig. 4 is the protein immunoblot sketch map that EGCG suppresses autophagy correlative protein expression in the GK rat heart.Wherein, Fig. 4 A, Fig. 4 B are that the autophagy marker molecule is expressed the protein immunoblot sketch map that reduces in the EGCG GK rat heart of feeding; Fig. 4 C is the protein immunoblot sketch map that regulation and control autophagy upper reaches protein expression reduces in the EGCG GK rat heart of feeding.Data from 6 groups of rats, represent with the form of meansigma methods ± standard error; + P＜0.05, ++ P＜0.01, +++P＜0.001vs.Wistar matched group; * P＜0.05, * * P＜0.01, * * * P＜0.001vs.GK matched group.
Fig. 5 is that EGCG can have among the efficient recovery myocardial cell H9c2 and induces the insulin resistant that is produced by high sugar, and autophagy raises, the column sketch map that mitochondrion loss and oxidative stress increase.Wherein Fig. 5 A is that EGCG can effectively stop the protein immunoblot sketch map that insulin resistant GAP-associated protein GAP phosphorylation reduces in the high sugared inductive H9c2 cell; Fig. 5 B, Fig. 5 C are that EGCG can effectively stop the protein immunoblot sketch map that the autophagy correlative protein expression raises in the high sugared inductive H9c2 cell; Fig. 5 D is that EGCG can effectively stop minimizing of mitochondria number correlative protein expression and two-phase enzyme correlative protein expression increase protein immunoblot sketch map in the high sugared inductive H9c2 cell; Fig. 5 E is that EGCG can effectively stop the column sketch map that the ROS generation increases in the high sugared inductive H9c2 cell; Fig. 5 F, Fig. 5 G are that EGCG can reduce the laser co-focusing sketch map that autophagic vacuole forms in the high sugared inductive H9c2 cell; Fig. 5 H is that the EGCG processing can reduce the active immunofluorescence dyeing sketch map of autophagic vacuole in the high sugared inductive H9c2 cell.Figure A-E, the H9c2 cell is with 20 μ mol/L EGCG pretreatment 24 hours, subsequently with the variation of 25mmol/L glucose detection of active oxygen generation in 24 hours; Handled 10 minutes with the 100nmol/L insulin at last, collect albumen and be used for immunoblotting assay.Data from 5 independent repeated experiments, represent with the form of meansigma methods ± standard error.+ P＜0.05, ++ P＜0.01, +++P＜0.001vs. normal control group, * P＜0.05, the high sugared processed group of * * P＜0.01vs..Figure F-G, H9c2 cell after 12 hours, earlier with 20 μ mol/L EGCG pretreatment 24 hours, used the 25mmol/L glucose 24 hours with the EGFP-LC3 transfection subsequently, and laser confocal microscope detects the formation of autophagic vacuole.Figure H, the H9c2 cell was used the 25mmol/L glucose 24 hours subsequently with 20 μ mol/L EGCG pretreatment 24 hours, identified the variation of autophagic vacuole then through the painted method of MDC.
Fig. 6 is that myocardial cell H9c2 regulates and control the protein immunoblot sketch map of autophagy through FoxO1 under high sugar effect.Wherein, Fig. 6 A, Fig. 6 B can suppress the protein immunoblot sketch map of autophagy rise in the sugared inductive H9c2 cell of height after disturbing for FoxO1 RNA; Fig. 6 C, Fig. 6 D are that FoxO1 crosses the expression back increases the H9c2 cell autophagy through the mode of non-transcribed regulation and control protein immunoblot sketch map.Data from 3 independent repeated experiments, represent with the form of meansigma methods ± standard error.* P＜0.05, * * P＜0.01, * * * P＜0.001vs. matched group.
The specific embodiment
Below in conjunction with the accompanying drawing and the specific embodiment the present invention is done detailed elaboration.
I, materials and methods
1, material
EGCG ((-)-epigallocatechin-3-gallate), glucose (Glucose), insulin (Insulin); Ubiquinone 1 (CoQ1), ubiquinone in the last of the ten Heavenly stems (decylubiquinone), cytochrome C (Cytochrome C); 2,6-Dichlorobenzenone-indophenol (DCIP), 2-(P-iodophenyl)-3 (P-nitrobenzophenone)-5-phenyl tetrazolium chloride (2-(p-iodophenyl)-3 (p-nitrophenyl)-5-phenyl tetrazolium chloride; INT), coenzyme A (CoASH), diphosphothiamine (thiamine pyrophosphate); Monodansylcadaverine (MDC) and to Tubulin; GAPDH one anti-from Sigma aldrich company (Sigma, St Louis, MO).The L-DMEM culture medium, penicillin (penicillin), streptomycin (streptomycin); 2,7-dichloro-dihydro fluorescein(e) diacetate (2 ', 7 '-dichlorodihydrofluorescein diacetate; H2DCFDA), trypsin trypsin) and to OxPhos Complexes I, II; III and IV one anti-from Invitrogen company (Carlsbad, USA); To NQO1, HO-1, SOD2, VDAC1, PGC1, mtTFA, DRP1, Mfn1, Mfn2, OPA1 and Ac-FoxO1 one anti-from Santa Cruz company (Heidelberg, Germany); To FoxO3a, FoxO1, p-FoxO1 (Thr24), Atg5; Atg7, Beclin1, LC3B, p-Akt (Ser 473); Akt, p-AMPK α (Thr172), p-GSK3 β, AMPK α; P-mTOR (Ser2448), mTOR one anti-from Cell Signaling Technology company (Beverly, MA, USA); Horseradish peroxidase to Mus/rabbit/goat two anti-available from Jackson's immune Research laboratory (Jackson ImmunoResearch Laboratories, Inc); Hyclone available from PAA Laboratories GmbH (Linz, Austria); Cell pyrolysis liquid is available from the green skies, Haimen, Jiangsu biotech company.Other reagent are available from local supply and marketing merchant.
2, experimental technique
Animal feeding
The non-diabetic Wistar rat of big all around male diabetes GK rat and corresponding size is available from SLAC Laboratory Animal Co.Ltd (Shanghai; China). according to NIH animal feeding principle raising rat; All mouse are divided into three groups, Wistar matched group, the GK rat group that GK matched group and EGCG feed; The feeding volume of EGCG is 100mg/kg/day, and matched group gives the salt of respective volume.Raise after three months, collect heart tissue in order to experiment.
Cell culture
Rat myocardial cell H9c2 is available from U.S. ATCC company; Be grown in and contain 10% hyclone; In the L-DMEM culture medium of 100 units per ml penicillins and 100 mcg/ml streptomycins; And be placed in 5% CO2 gas incubator of constant temperature (37 ℃) constant humidity, changing liquid once in 2 days, all cells was in 10 generations.
Protein immunoblot is analyzed
The H9c2 cell of collecting heart tissue (100mg) or handling adds cell pyrolysis liquid in hatching 30 minutes on ice.17, centrifugal 15 minutes of 000g collects supernatant, protein quantification, and supernatant is stored in-20 ℃.Getting about 20 μ g albumen separates through sds polyacrylamide gel electrophoresis and goes on the NC film; The NC film is with 5% defatted milk powder (being dissolved in TBST) sealing 1 hour, then with an anti-incubated at room 1 hour, and TBST rinsing 3 times 15 minutes; Two anti-incubated at room are 1 hour then; TBST rinsing 3 times 15 minutes is at last through chemical luminous substrate HRP reaction, the variation of usability ray film recording light brightness.
Mitochondrial respiratory chain complex enzyme I, the detection of III and a-ketoglutaric acid dehydrogenase activity
The mitochondrion extracting.With A liquid (120mM NaCl, 20mM HEPES, 2mM MgCl2,1mM EGTA, the and 5g/l bovine serum albumin of the rat heart tissue that shreds with pre-cooling; PH 7.4) resuspended, after leaving standstill 5 minutes on ice with homogenizer with its homogenate, with tissue homogenate centrifugal 10 minutes in 600g.The supernatant that collection obtains centrifugal 10 minutes again in 17000g.Mitochondrion deposition with A liquid resuspended after, in 7000g centrifugal 10 minutes, precipitate with B liquid (300mM sucrose, 2mM HEPES, 0.1mM EGTA; PH7.4) resuspended, and in 3500g centrifugal 10 minutes, the mitochondrion that obtains deposition is carried out protein quantification with B liquid after resuspended.Above all operations all carries out at 4 ℃.Resulting mitochondrion is stored in-80 ℃ and surveys until the enzyme biopsy.
NADH-CoQ oxidoreductase (complex I)
Reaction system is: 50mmol/L Tris-HCl pH8.1,0.05mmol/L DCIP, 0.35% bovine serum albumin (BSA), 1 μ mol/L antimycin A (antimycin A), 0.2mmol/L sodium azide (NaN
3), 0.05mmol/L ubiquinone 1 (coenzyme Q1), through 200 μ mol/L also the NADH of ortho states (NADH) start reaction, under 30 ℃ of conditions, read the variation 2 minutes of 600nm place light absorption value then.
CoQ-cytochrome c reductase (complex III)
Reaction system is: 50mmol/L Tris-HCl pH 7.8; 0.2mmol/L sodium azide (NaN3); 0.05% tween 20 (Tween-20), 0.01% bovine serum albumin (BSA), 0.05mmol/L cytochrome C (Cytochrome C); Start reaction through 0.05mmol/Ldecylubiquinol, under 30 ℃ of conditions, read the variation 2 minutes of 550nm place light absorption value then.
A-ketoglutaric acid dehydrogenase complex (a-KGDH)
Reaction system is: 35mmol/L kaliumphosphate buffer (potassium phosphate buffer pH7.25), 2mmol/L sodium azide (NaN
3), 0.5mmol/L ethylenediaminetetraacetic acid (EDTA), 2.5 μ mol/L rotenone (rotenone), 5mmol/L magnesium chloride (MgCl
2), 0.5mmol/L NADH (NAD
+); 0.2mmol/L diphosphothiamine (thiamine pyrophosphate); 2mmol/L a-ketoglutaric acid (a-Ketoglutarate) starts reaction through 0.04mmol/L coenzyme A (CoASH), under 30 ℃ of conditions, reads the variation 2 minutes of 340nm place light absorption value then.
The mensuration of oxidative stress in the cell
By the ratio in 600,000/ holes, with myocardial cell H9c2 kind in 6 well culture plates; Leave standstill the cell attachment state that restore normal growth of treating; Use 20 μ mol/L EGCG pretreatment 24 hours, used the 25mmol/L glucose subsequently 24 hours, abandon culture medium; Clean one time with PBS, add the H in the L-DMEM culture medium that is dissolved in serum-free then
2DCFDA was hatched 0.5 hour in 37 ℃ of incubators, then removed H2DCFDA solution, cleaned one time with PBS, added cell pyrolysis liquid (10mmol/L Tris; 150mmol/L NaCl, 0.1mmol/L EDTA, 0.5%Triton X-100, pH 7.5); Centrifugal (15000g, 10min, 4 ℃) obtain supernatant and detect (exciting light 485nm with fluorescence analyser; Emission light 538 nm), use BCA kit measurement protein concentration simultaneously, finally represent the situation of change of active oxygen with fluorescence OD value/protein content.
The observation of autophagic vacuole (autophagosome and autophagy lysosome)
The H9c2 cell is with the EGFP-LC3 transfection after 12 hours; With 20 μ mol/L EGCG pretreatment 24 hours, use the 25mmol/L glucose subsequently 24 hours earlier, abandon culture medium; After fixing 15 minutes, detect the formation of autophagic vacuole with paraformaldehyde in laser confocal microscope.
The H9c2 cell was with 20 μ mol/L EGCG pretreatment 24 hours; Use the 25mmol/L glucose after 24 hours subsequently, abandon culture medium, add the L-DMEM culture medium of serum-free; With MDC dyeing 30 minutes; Then remove MDC solution, clean one time, under fluorescence microscope, observe the variation and the Taking Pictures recording of blue and white fluorescence then with PBS.
FoxO1 siRNA disturbs and crosses and express
Ratio according to 150000/ hole; With the H9c2 cell inoculation in 6 orifice plates; Operated according to the description of liposome 2000 in second day, and changed the siRNA (sequence is 5 '-UGAAUAGCAAGGUGUCUGCTT-3 ') of FoxO1 over to the H9c2 cell, after 24 hours; Handled again 24 hours with the 25mmol/L glucose, collect albumen and be used for immunoblotting assay.
Description according to liposome 2000 is operated; Expression vector pCDNA3-FoxO1 (the 3A)-FLAG of FoxO1 wild type expression vector pCDNA3-FoxO1-FLAG and phosphorylation site sudden change is changed among the myocardial cell H9c2; After 48 hours, collect albumen and be used for immunoblotting assay.
Statistical analysis
Meansigma methods ± standard error that experimental data draws from least 3 independent repeated experiments is represented.Fisher ' s LSD method in the one way analysis of variance method (ANOVA) of use SPSS statistical software is carried out result's significance of difference analysis.The significance of difference is explained by following :+P＜0.05, ++ P＜0.01, +++P＜0.001; * P＜0.05, * * P＜0.01, * * * P＜0.001.
Embodiment one
EGCG is to the therapeutic effect of mitochondrion dysfunction in the GK rat heart
Respectively from normal Wistar rats; Extract protein and mitochondrion in the GK rat heart that diabetes GK rat and EGCG feed; Come the expression of detection line plastochondria GAP-associated protein GAP then with the method for protein immunoblot; The result sees Figure 1A, and the expression of mitochondrial respiratory chain complex and VDAC1 all descends to some extent in the GK rat heart, and EGCG has good restitution to it; Come the enzymatic activity of detection line plastochondria relevant enzyme with the method for enzyme activity determination, the result sees Figure 1B, EGCG for increase GK rat heart intersome respiratory chain complex enzyme (I, III) and the enzymatic activity of a-ketoglutaric acid dehydrogenase complex enzyme significant effect is arranged.
Embodiment two
The recovery effects that EGCG raises two-phase expression of enzymes in the GK rat heart.
The expression of two-phase enzyme is the important indicator of antioxidant system in the body, and its rising also can be reacted intravital oxidative stress from the side to be increased.From normal Wistar rats, extract protein in the GK rat heart that diabetes GK rat and EGCG feed respectively, detect the expression of two-phase enzyme with the method for protein immunoblot.As can be seen from Figure 2, (MnSOD) expression has significant rise with respect to the Wistar rat to the two-phase enzyme in the diabetes GK rat heart for NQO1, HO1, and the raising of EGCG can effectively reduce the expression of two-phase enzyme.
Embodiment three
EGCG is to the influence of mitochondrion generation and dynamics correlative protein expression amount in the GK rat heart.
We find that mitochondrion in the GK rat heart has the phenomenon of dysfunction and decreased number, infer that these phenomenons possibly generate with mitochondrion and the variation of dynamics is relevant.Can find out that from Fig. 3 A the expression that GK rat heart mitochondrion generates GAP-associated protein GAP increases to some extent, the raising of EGCG has restitution to it; Can find out that from Fig. 3 B GK rat heart mitochondrion merges GAP-associated protein GAP and do not change, reduce to some extent that EGCG then can recover its expression to normal rat Wistar but divide GAP-associated protein GAP Drp1.
Embodiment four
The inhibition effect that EGCG raises autophagy correlative protein expression amount in the GK rat heart.
According to The above results, we infer that the handicapped phenomenon of mitochondrion is because the autophagy increase is caused in the GK rat heart, reach the effect of recovering mitochondrial function thereby EGCG has certain inhibitory action to autophagy.We detect the expression of autophagy GAP-associated protein GAP with the method for protein immunoblot, and the result sees Fig. 4 A, B, and EGCG raises (LC3B, the increase of Atg family protein expression) for the autophagy in the GK rat heart has the obvious suppression effect.And can find out that from Fig. 4 C in two paths of control autophagy under the effect of insulin, mTOR does not have significant change, and the expression rising meeting of FoxOs family protein amount in the GK rat heart suppressed by EGCG.
Embodiment five
EGCG can suppress among the myocardial cell H9c2 by the sugared inductive insulin resistant of height, and autophagy increases, and oxidative stress raises and mitochondria number reduces.
The H9c2 cell was used the 25mmol/L glucose 24 hours subsequently with 20 μ mol/L EGCG pretreatment 24 hours, handled 10 minutes with the insulin of 100nmol/L at last, and protein immunoblot hybridization detects the expression of GAP-associated protein GAP.Shown in Fig. 5 A; P-AKT and p-GSK3 β are two marker molecules of insulin resistant; P-AKT and p-GSK3 β have tangible decline in the high sugared processed group; Explain that the 25mmol/L glucose can induce the H9c2 cell to produce insulin resistant, 20 μ mol/L EGCG pretreatment then can effectively prevent the generation of insulin resistant; Can find out that from Fig. 5 B EGCG induces autophagy GAP-associated protein GAP FoxO1 for high sugar, FoxO3a, Atg7, the expression of Atg12-Atg5 and LC3B raises all has the obvious suppression effect; The ratio of LC3-II/LC3-I is being represented the intensity of autophagy, and the autophagy that Fig. 5 C explanation EGCG can effectively suppress to be caused by high sugar in the H9c2 cell raises; Mitochondrial function is disorderly closely related with insulin resistant, can find out that from Fig. 5 D high sugar is induced and can be caused mitochondrion composition Complex III in the H9c2 cell, the minimizing of Complex IV and VDAC1, and EGCG then can stop the generation of this phenomenon; Active oxygen mainly causes owing to electronics seepage in the mitochondrion oxidative phosphorylation process, Fig. 5 D, and the active oxygen that E explanation EGCG can suppress to be caused by high sugar produces and corresponding two-phase enzyme raises; When autophagy took place, intracellular LC3 can gather together, and formed autophagic vacuole; After 12 hours,, used the 25mmol/L glucose subsequently 24 hours with EGFP-LC3 transfection H9c2 cell earlier with 20 μ mol/L EGCG pretreatment 24 hours; Laser confocal microscope detects the formation of autophagic vacuole; As scheming F, G, the LC3 speckle in the EGCG processed group cell obviously are less than high sugar and induce group; MDC usually is used to the unobstructed detection of autophagy approach.Can see that from Fig. 5 H the H9c2 cell is after 20 μ mol/L EGCG handle 24 hours, fluorescence intensity dies down, and means that autophagic vacuole lacks the sour environment of normal function.
Embodiment six
The autophagy that FoxO1 regulated and control plays an important role in the sugared inductive myocardial cell H9c2 of height.
We find that the rise of myocardial cell autophagy is relevant with FoxO1 in the animal experiment; We used the 25mmol/L glucose 24 hours subsequently with FoxO1 siRNA transfection H9c2 cell, and the result finds autophagy downward modulation in the interferential H9c2 cell of FoxO1; Isochrone plastochondria composition Complex IV and VDAC1 rise to some extent; Explain that FoxO1 induces the autophagy of H9c2 cell to play an important role in raising at high sugar, like Fig. 6 A, B; Expression vector pCDNA3-FoxO1 (the 3A)-FLAG transfection myocardial cell H9c2 of FoxO1 wild type expression vector pCDNA3-FoxO1-FLAG and phosphorylation site sudden change; The result shows that the FoxO1 of phosphorylation site sudden change crosses the increase that expression can not cause autophagy; Explain that FoxO1 crosses the mode of regulating and control through non-transcribed the expression back and increases the H9c2 cell autophagy; Like Fig. 6 C, D.
Visible by The above results; The mitochondria dysfunction that EGCG can effectively prevent and treat type ii diabetes and followed; Autophagy increases and oxidative stress raises, and prompting FoxO1 possibly play important effect as the treatment target spot of an EGCG in the regulation and control autophagy.This time finding has extraordinary suggesting effect for the mechanism of action of the effective EGCG of understanding on diabetes mellitus prevention.
Claims (5)
1.EGCG the application in preparation treatment type ii diabetes medicine is characterized in that, the imbalance of the heart mitochondrial function that EGCG suppresses to be caused by autophagy.
2.EGCG the application in preparation treatment type ii diabetes medicine is characterized in that EGCG is used to suppress the autophagy rise of high sugared inductive myocardial cell H9c2.
3.EGCG the application in preparation treatment type ii diabetes medicine is characterized in that, EGCG is used to suppress the activation of the oxidative stress of high sugared inductive myocardial cell H9c2.
4.EGCG the application in preparation treatment type ii diabetes medicine is characterized in that EGCG is used to recover the mitochondrial function disorder of high sugared inductive myocardial cell H9c2.
5.EGCG the application in preparation treatment type ii diabetes medicine is characterized in that EGCG is used to reduce the insulin resistant of high sugared inductive myocardial cell H9c2.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104042605A (en) * | 2014-01-20 | 2014-09-17 | 西安交通大学 | Use of epigallocatechin gallate (EGCG) in preparation of food and drug for preventing and treating myocardial energy metabolism disorders |
| CN109211817A (en) * | 2018-09-06 | 2019-01-15 | 苏州科铭生物技术有限公司 | A kind of CoA contents assay kit and its method based on micromethod |
| CN109758475A (en) * | 2017-11-03 | 2019-05-17 | 绿恩生化科技股份有限公司 | The purposes that medical composition is damaged in drug in preparation for treating pancreas |
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2012
- 2012-01-13 CN CN2012100099605A patent/CN102526023A/en active Pending
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| 杨晓等: "EGCG对实验性糖尿病大鼠肾脏的保护作用", 《中国药理学通报》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104042605A (en) * | 2014-01-20 | 2014-09-17 | 西安交通大学 | Use of epigallocatechin gallate (EGCG) in preparation of food and drug for preventing and treating myocardial energy metabolism disorders |
| CN109758475A (en) * | 2017-11-03 | 2019-05-17 | 绿恩生化科技股份有限公司 | The purposes that medical composition is damaged in drug in preparation for treating pancreas |
| CN109211817A (en) * | 2018-09-06 | 2019-01-15 | 苏州科铭生物技术有限公司 | A kind of CoA contents assay kit and its method based on micromethod |
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Application publication date: 20120704 |