CN102727505A - Application of salidroside in preventing and treating amyotrophy diseases - Google Patents
Application of salidroside in preventing and treating amyotrophy diseases Download PDFInfo
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Abstract
The invention relates to an application of salidroside in preventing and treating amyotrophy diseases. Experiments prove that salidroside can inhibit the expression of amyotrophy peculiar factor Atrogin-1, myotube atrophy and reduction of skeletal muscle fiber cross section area during the amyotrophy generating process, and has the functions of increasing the content of slow-twitch fiber protein in skeletal muscle and inhibiting fast-twitch fiber protein expression, and thus the amyotropy is prevented and resisted.
Description
Technical field:
The present invention relates to the new purposes of chemical compound rhodioside, be specifically related to rhodioside and prevent and treat the application in the amyotrophic medicine in preparation.
Background technology:
Rhodioside (formula I) is the chemical compound of known chemical structure, English name Salidroside, molecular formula C14H20O7, CAS accession number 10338-51-9.
Research both at home and abroad shows that rhodioside plays an important role in resisting fatigue, defying age, immunomodulating, removing free radical, hypermnesis, improvement sleep.Clinical basic is also pointed out, the formation that rhodioside can obviously reverse kidney injury that pulmonary hypertension, diabetes or hypoxia cause, the weakening of vascular smooth muscle cell proliferation differentiation potential etc. and disturb hepatic fibrosis.But the not effect of show Herba hylotelephii erythrosticti glycosides in skeletal muscle and the report of Mechanism Study up to now.
Skeletal muscle can be divided into slow muscle and fast muscle according to the metabolic type characteristic of itself, is made up of slow switch fibers albumen and fast muscle fiber albumen respectively.Slow muscle is called red muscle again, is main with aerobic metabolism, and contraction is difficult for producing fatigue slowly, lastingly, mainly is to keep posture and carry out slow and persistent work; Fast muscle is also claimed white muscle, is main with the anerobic glycolysis metabolism, shrinks soon, and explosive force is strong, but occurs tired easily.Variation fast in the skeletal muscle, the slow switch fibers protein content has important regulation to amyotrophic generation, development, i.e. the increase of slow switch fibers protein content can improve body movement endurance, and having of muscular atrophy diseases prevented and antagonism.And normal increase with the fast muscle fiber protein content in muscular atrophy diseases.Thus, the proteic increase of slow switch fibers or with the proteic minimizing of fast muscle fiber can prevent and treat skeletal muscle atrophy.
Under the physiological status, the synthetic and degraded of skeletal muscle albumen and speed parapeptone thereof is in dynamic equilibrium.But clinical muscular dystrophy property disease (like Du Shi muscular dystrophy, lateral sclerosis of spinal cord etc.), muscular atrophy of disuse (in long-term bed, wound braking, the space travel weightlessness amyotrophy etc.), go/denervated muscle atrophy, the amyotrophy in senile muscular atrophy, malignant disease (like diseases such as cancer of late stage cachexia, heart failure, renal failure, diabetes, chronic obstructive pulmonary disease, AIDS and prolonged application glucocorticoids) late period etc.; The skeletal muscle protein metabolic imbalance; Characteristic changes to show as that skeletal muscle weight obviously alleviates, the selectivity of the reducing of muscle fiber cross-sectional area, muscle fiber types GAP-associated protein GAP (being slow switch fibers albumen and fast muscle fiber albumen) is lost etc.; Cause muscular strength decline, the dyskinesia, fatiguability and metabolism disorder etc., have a strong impact on routine work and life.But the present clinical good Therapeutic Method that still do not have.
In the amyotrophy process, the dynamic equilibrium of protein synthesis and degraded is damaged, and the protein degradation increase is basic reason (the Russell AP that causes skeletal muscle atrophy; Clinical and Experimental Pharmacology and Physiology; 37,378 – 384,2010; Glass DJ, Curr Opin Clin Nutr Metab Care13:225 – 229,2010).Generally acknowledged at present; Comprise nearly all type amyotrophy such as serious disease concurrency amyotrophy such as skeletal muscle dystrophic disease, muscular atrophy of disuse, denervation property amyotrophy and cancer of late stage cachexia and AIDS; Its final common change is that the special atrophy factors A trogin-1 of skeletal muscle expresses increase, and the skeletal muscle PD causes that greater than synthetic the skeletal muscle fiber cross-sectional area reduces and the muscle fiber types GAP-associated protein GAP changes; Cause amyotrophic generation (Bodine SC; Et al.Science, 294:1704-1708,2001; Foletta VC, et al.Pflugers Arch-Eur J Physiol, 461:325 – 335,2011).Therefore, through expression, increase muscle fiber cross-sectional area and the regulating muscle fiber type GAP-associated protein GAP content that suppresses Atrogin-1, can prevent and treat the incidence and development of skeletal muscle atrophy disease, raising work and viability.
Summary of the invention:
The objective of the invention is to develop the new purposes of rhodioside.Specific purposes are the minimizings that can suppress expression, myotube diameter and the skeletal muscle fiber cross-sectional area of amyotrophy specific factor Atrogin-1 in the skeletal muscle through experiment proof rhodioside; And find that rhodioside has the proteic content of slow switch fibers in the skeletal muscle of increasing, suppress the effect of fast muscle fiber protein expression, thereby play prevention and resist amyotrophic effect.Finally prepare the medicine of prevention and treatment skeletal muscle atrophy property disease with rhodioside.
The present invention with following experiment confirm the new function of rhodioside:
1, rhodioside is to the influence of amyotrophy specific factor Atrogin-1 expression
Myotube (myotubes) is the multinucleated syncytia greater than three nuclears that skeletal myoblast (myoblasts) forms in vitro differentiation, fusion; Have myofibrillar structure and functional characteristic; Be widely used in the experiment in vitro research of skeletal muscle, be particularly useful in the pharmacological experiment observation analysis medicine the direct action effect of skeletal muscle atrophy.
This experiment forms the multinuclear myotube through inducing sarcoplast C2C12 vitro differentiation, and adopts internationally recognized external myotube atrophy model (Sandri M et al, Cell, 30; 117 (3): 399-412,2004) carry out myotube atrophy experiment, analyzed the influence that rhodioside is expressed amyotrophy specific factor Atrogin-1 mRNA with real-time quantitative real time PCR method.Experimental result shows, outside inductor, in the myotube atrophy process, compares with matched group, and the expression of rhodioside processed group amyotrophy specific factor Atrogin-1mRNA obviously suppresses (P < 0.01).The proof rhodioside can suppress amyotrophy specific factor Atrogin-1mRNA expresses, and has the effect that prevention and treatment amyotrophy take place.
See experiment one for details.
2, rhodioside is to the influence of myotube atrophy
It is the positive evidence of external myotube generation atrophy that the myotube diameter reduces.This experiment utilizes above-described myotube atrophy model, through the method for cellular immunofluorescence, has analyzed rhodioside to amyotrophic direct effect.Experimental result shows, in external myotube atrophy, compares with matched group, and the diameter of rhodioside processed group myotube obviously increases (P < 0.01).The proof rhodioside has direct inhibitory action to amyotrophy.
See experiment two for details.
3, rhodioside is to amyotrophic influence
For further the inhibition of checking rhodioside is amyotrophic in bulk effect, we use rhodioside (50mg/ kg/day) solution and equal-volume PBS thereof, and irritate stomach every day and handle the C57 mice, continuous 14 days, preparation rhodioside processed group and control group mice thereof.And adopt internationally recognized tail suspention method (Gregory R.Adams, et al.J Appl Physiol95:2185 – 2201,2003), the tail suspention induced amyotrophy to form in 14 days, was prepared in body skeletal muscle atrophy model.
Reducing of muscle fiber cross-sectional area is the positive evidence that takes place at the body skeletal muscle atrophy.In this experiment, utilize tail suspention to induce skeletal muscle atrophy after, adopt immunohistochemical method, analyzed the variation of skeletal muscle fiber cross-sectional area.Experimental result shows, compares with matched group, and rhodioside processed group mouse muscle fiber cross-sectional area significantly increases (P < 0.05).Proof is on integral level, and rhodioside has also been brought into play and suppressed amyotrophic action effect.
See experiment three for details.
4, rhodioside is to the influence of muscle fiber types correlative protein expression
Variation fast in the skeletal muscle, the slow switch fibers protein content has important regulation to amyotrophic generation, development, i.e. the increase of slow switch fibers protein content can improve body movement endurance, and having of muscular atrophy diseases prevented and antagonism.And normal increase with the fast muscle fiber protein content in muscular atrophy diseases.Thus, the proteic increase of slow switch fibers or with the proteic minimizing of fast muscle fiber can prevent and treat skeletal muscle atrophy.
In this experiment, use rhodioside (50mg/ kg/day) solution and equal-volume PBS thereof, irritate stomach every day and handle the C57 mice, continuous 14 days, preparation rhodioside processed group and control group mice thereof.Adopt Western Blot method, analyzed that rhodioside is fast to mice, the influence of slow switch fibers protein content.Experimental result shows, compares with matched group, and the expression of the significant albumen Troponin of rhodioside processed group mice slow switch fibers I-SS significantly increases (P < 0.01), and the significant albumen Troponin of fast muscle fiber I-FS has reduction trend.The proof rhodioside has prevention and resists amyotrophic effect.
See experiment four for details.
More than the used test product rhodioside that receives of experiment is bought from Chengdu general think of bio tech ltd, purity (detecting with the HPLC method): 99%.
The invention has the beneficial effects as follows:
Find and confirmed the new function of rhodioside first for prevention and treatment disease of muscular dystrophy.
The present invention has the amyotrophy of reduction specific factor through experiment proof rhodioside and expresses; Myotube diameter and muscle fiber cross-sectional area reduces in the inhibition amyotrophy generating process; Increase slow switch fibers protein content and the effect that suppresses the fast muscle fiber protein expression in the skeletal muscle.
And suppress the expression of amyotrophy specific factor and increase the proteic content of slow switch fibers in myotube diameter or muscle fiber cross-sectional area and the muscle, be the prerequisite that prevents and improve the amyotrophy state.Therefore, the present invention has proved the medicine that utilizes rhodioside to can be used for preparing prevention and treatment disease of muscular dystrophy as material medicine.
Four, description of drawings
Fig. 1 is the expression of Atrogin-1 after real-time quantitative real time PCR detection matched group and the atrophy of rhodioside processed group myotube.
Fig. 2 is the form after cellular immunofluorescence shows matched group and the atrophy of rhodioside processed group myotube.
Fig. 3 is a diameter variation after Image-Pro Plus6.0 software statistics matched group and the atrophy of rhodioside group processed group myotube.
The myofibrillar cross-sectional area of gastrocnemius after Fig. 4 histogenic immunity fluorescence demonstration matched group and the rhodioside group mice amyotrophy.Scale is 50 microns.
The variation of gastrocnemius muscle fiber cross-sectional area after Fig. 5 Image-Pro Plus6.0 software statistics matched group and the rhodioside group mice amyotrophy.
Fig. 6 is that Western blot detects matched group and the interior expression fast, slow switch fibers albumen (Troponin I-FS, Troponin I-SS) of rhodioside group mice skeletal changes.Troponin I-FS and Troponin I-SS are that inhibitory subunit of troponin is present in the hypotype in fast muscle fiber and the slow switch fibers, can reflect the proteic content of fast muscle fiber albumen and slow switch fibers respectively; Beta Actin is confidential reference items, can reflect the unanimity of total protein applied sample amount.
Fig. 7 is the proteic relative expression quantity of slow switch fibers in Image-Pro Plus6.0 software statistics matched group and the rhodioside group mice skeletal.
Fig. 8 is the proteic relative expression quantity of fast muscle fiber in Image-Pro Plus6.0 software statistics matched group and the rhodioside group mice skeletal.
The specific embodiment
Laboratory animal and material source used in below testing are following:
Laboratory animal: male C57 mice is available from Beijing dimension tonneau China animal center, production licence number: SCXK (capital) 2012-0001; The laboratory animal certification of fitness number: 0261086.
C2C12 cell line is purchased in the Chinese Academy of Medical Sciences.
Myotube is the multinucleated syncytia that the external one-tenth flesh differentiation of the mononuclear cell C2C12 of this Experiment Preparation back forms.
Receive the reagent thing: rhodioside, buy from Chengdu general think of bio tech ltd, HPLC:99%.
Other material source:
The organization embedding agent is available from Thermo company;
Rabbit source laminin antibody is available from Abcam company;
Goat-anti rabbit FITC fluorescent labeling two is anti-available from Invitrogen company;
Sheep anti mouse FITC fluorescent labeling two is anti-available from Invitrogen company;
Trizol, reverse transcription are available from Invitrogen company;
SYBR is available from ABI company;
Rabbit source Troponin I-SS antibody is available from Aviva company;
Rabbit source Troponin I-FS antibody is available from Aviva company;
Rabbit source beta actin antibody is available from Santa Cruz company;
Mus source myosin antibody is bought from Beijing Zhong Shan company
The DMEM culture medium is available from U.S. GIBCO company;
Hyclone (FBS), horse serum (HS), trypsin are available from HyClone company;
Glutamine is available from Sigma company;
Penicillin and streptomycin are available from Sigma company;
Cell culture fluid: growth-promoting media is DMEM, adds 20%FBS, 1% glutamine, and 1% penicillin and streptomycin are two anti-; Differentiation liquid is DMEM, adds 2%HS, 1% glutamine, and 1% penicillin and streptomycin are two anti-;
Key instrument equipment: Bio-Rad electrophresis apparatus, Bio-Rad electrophoresis tank and wet electric turn trough, ABI Step-One Plus real time PCR appearance, Sanyo's freezing microtome, Leica laser confocal microscope, the Format CO of changeing
2Incubator, the Great Wall superclean bench; The Olympus inverted microscope.
Experiment one: the influence that rhodioside is expressed amyotrophy specific factor Atrogin-1
1. experiment purpose:
Matched group and rhodioside processed group myotube are adopted in this experiment, through external evoked myotube atrophy, with the method for real-time quantitative realtime PCR, analyze the influence of rhodioside to amyotrophy specific factor Atrogin-1 expression in the myotube atrophy.
2. experimental technique:
2.1 sarcoplast recovery, cultivation
A, from liquid nitrogen container, take out fast frozen sarcoplast C2C12, frozen pipe is put in 37 ℃ of water-baths fast, jog makes its quick dissolving.
B, change in the 15mL primary sterilization centrifuge tube, add the 5mL cell growth medium, 1000rpm, centrifugal 5min removes cryoprotective agent.
Supernatant is removed in C, suction, adds the 5mL cell growth medium, uses the piping and druming pipe to blow and beat gently and is cell suspension, is inoculated in the culture bottle of 75mm.
D, 5%CO
2Cultivate under 37 ℃ of conditions of incubator.
2.2 sarcoplast experiment
A, when recovery cell stand density reaches 80%, discard old culture medium, with PBS with cell washing 2 times.
B, adding 2mL0.25% trypsin-EDTA observe cell and will separate, when presenting circle, add the 3mL cell growth medium and stop digestion under inverted microscope.
C, blow and beat culture bottle gently, make cell detachment complete with suction pipe.Sucking-off cell suspension is put into the 15mL centrifuge tube, with the rotating speed of 1000rpm, and centrifugal 5min.
D, sop up supernatant, add cell growth medium 5mL, and blow and beat cell suspension, become individual cells until the cell homodisperse with the apicule head straw that draws.
E, according to conventional method, carry out cell counting with blood-counter system, be concentration with cell dilution: 104/ML cell suspension.
F, according to every 60mm culture dish inoculating cell suspension 1mL, and add the 2mL growth-promoting media, each 60mm culture dish is cultivated under the 3mL growth-promoting media.
G, cell were grown about 24 hours, arrived about 80% stand density.
H, culture dish is grouped into: each 4 ware of matched group and rhodioside processed group mark.
2.3 myotube preparation
Growth-promoting media in A, removal matched group and the rhodioside processed group ware, be replaced by:
A. matched group: the differentiation liquid of 3ml/ ware;
B. rhodioside processed group: final concentration is the 3ml/ ware differentiation liquid of 30ug/ml rhodioside.
B, induce sarcoplast differentiation, changed culture fluid in per 24 hours.
C, differentiation are after 72 hours, and visible sarcoplast forms the multinuclear myotube greater than three nuclears.
2.4 external myotube atrophy experiment
Use the myotube that above experiment is differentiated to form, adopt internationally recognized external amyotrophy model experiment method (Sandri M et al, Cell, 30; 117 (3): 399-412,2004), inhale and remove the culture fluid in matched group and the rhodioside group ware, add 3mL PBS and induced the myotube atrophy 6 hours, choose 4 wares for every group, be used for real time pcr analysis.
2.5RNA extraction, detect with quantitative
A, cracking: inhale and to remove the PBS in matched group and the rhodioside group, every ware adds 1ml Trizol and blows and beats the cracking myotube repeatedly, places 30min during the EP that is drawn into 1.5ml manages.
B, respectively be separated: the ratio in 0.2ml chloroform/1ml Trizol adds chloroform, the tight violent mixing of lid 15 seconds, and room temperature was placed 2-3 minute.4 ℃, centrifugal 15 minutes of 12000g.Draw the supernatant water afterwards and place another new EP pipe.
C, precipitated rna: in the ratio of 0.5ml isopropyl alcohol/1ml Trizol, add isopropyl alcohol to the aqueous phase of drawing, put upside down mixing, room temperature was placed 15 minutes.4 ℃, 12000g obtained the RNA deposition in centrifugal 10 minutes.
D, rinsing RNA: supernatant discarded, in the ratio of 1ml75% ethanol/1ml Trizol, add 75% washing with alcohol RNA deposition twice, 4 ℃ then, 7500g centrifugation 5 minutes, supernatant discarded.
E, heavily molten RNA: dry RNA deposition is 5-10 minute in the air at room temperature, and with an amount of DEPC water dissolution RNA deposition, 55 ℃-60 ℃ water-soluble 10 minutes.
F, RNA purity detecting and quantitative: get 1 μ l RNA solution and add mixing in the 99 μ l DEPC water, measure ratio and the concentration of A260/A280 with ultraviolet spectrophotometer.
2.6 reverse transcription reaction
25 μ L reaction system operating procedures:
After b gently got rid of, 70 ℃ of 10min were put-20 ℃ of 3～5min, gently get rid of.
C adds in a pipe: 5 * Buffer, 5 μ L
dNTP 10μL
RNasin 1μL
M-MLV 1μL
d42℃90min,95℃10min
The synthetic cDNA of e preserves subsequent use in-20C.
2.7 design of primers
Utilize DNAMAN software design primer according to the ncbi database sequence, select suitable primer to synthesize (seeing table 1) according to annealing temperature and sequence-specific then.
Table 1 genes of interest Atrogin1 and confidential reference items GAPDH primer sequence
2.8real time PCR detects the expression of Atrogin1mRNA
50 μ L reaction system operating procedures:
PCR reaction condition: 94 ℃ of degeneration 5 minutes.94 ℃ 40 seconds, 62 ℃ 15 seconds, 72 ℃ 20 seconds, 40 circulations.72 ℃ were extended 10 minutes.
3. data analysing method
Data are represented with mean+SD, t check between data analysis employing group.P≤0.01 expression has notable statistics difference.
4. experimental result
As shown in Figure 1.It is thus clear that the expression of amyotrophy specific factor Atrogin-1mRNA is merely 39% of matched group after the atrophy of rhodioside group processed group myotube, compare with matched group, the expression of Atrogin-1mRNA received obvious inhibition (
*P<0.01).
5. experiment conclusion
Rhodioside can significantly suppress the expression of amyotrophy specific factor Atrogin-1mRNA, antagonist atrophy.
Experiment two: rhodioside is to the influence of myotube atrophy
1. experiment purpose:
Matched group and rhodioside processed group myotube are adopted in this experiment, through external evoked myotube atrophy, use the cellular immunofluorescence chemical method, analyze the influence of rhodioside to external myotube atrophy.
2. experimental technique:
2.1 sarcoplast recovery, cultivation
A, from liquid nitrogen container, take out fast frozen sarcoplast C2C12, frozen pipe is put in 37 ℃ of water-baths fast, jog makes its quick dissolving.
B, change in the 15mL primary sterilization centrifuge tube, add the 5mL cell growth medium, 1000rpm, centrifugal 5min removes cryoprotective agent.
Supernatant is removed in C, suction, adds the 5mL cell growth medium, uses the piping and druming pipe to blow and beat gently and is cell suspension, is inoculated in the culture bottle of 75mm.
D, 5%CO
2Cultivate under 37 ℃ of conditions of incubator.
2.2 sarcoplast experiment
A, when recovery cell stand density reaches 80%, discard old culture medium, with PBS with cell washing 2 times.
B, adding 2mL0.25% trypsin-EDTA observe cell and will separate, when presenting circle, add the 3mL cell growth medium and stop digestion under inverted microscope.
C, blow and beat culture bottle gently, make cell detachment complete with suction pipe.Sucking-off cell suspension is put into the 15mL centrifuge tube, with the rotating speed of 1000rpm, and centrifugal 5min.
D, sop up supernatant, add cell growth medium 5mL, and blow and beat cell suspension, become individual cells until the cell homodisperse with the apicule head straw that draws.
E, according to conventional method, carry out cell counting with blood-counter system, be concentration with cell dilution: 104/ML cell suspension.
F, according to every 60mm culture dish inoculating cell suspension 1mL, and add the 2mL growth-promoting media, each 60mm culture dish is cultivated under the 3mL growth-promoting media.
G, cell were grown about 24 hours, arrived about 80% stand density.
H, culture dish is grouped into: each 4 ware of matched group and rhodioside processed group mark.
2.3 myotube preparation
Growth-promoting media in A, removal matched group and the rhodioside processed group ware, be replaced by:
A. matched group: the differentiation liquid of 3ml/ ware;
B. rhodioside processed group: final concentration is the 3ml/ ware differentiation liquid of 30ug/ml rhodioside.
B, induce sarcoplast differentiation, changed culture fluid in per 24 hours.
C, differentiation are after 72 hours, and visible sarcoplast forms the multinuclear myotube greater than three nuclears.
2.4 external myotube atrophy experiment
Use the myotube that above experiment is differentiated to form, adopt internationally recognized external amyotrophy model experiment method (Sandri M et al, Cell, 30; 117 (3): 399-412,2004), inhale and remove the culture fluid in matched group and the rhodioside group ware, add 3mLPBS and induced the myotube atrophy 6 hours, choose 4 wares for every group, be used for the analysis of cellular immunofluorescence chemical staining.
2.5 the cellular immunofluorescence chemical staining is analyzed
A, the PBS in matched group and the rhodioside group myotube culture dish inhaled go.
B, adding 4% paraformaldehyde 2ml, room temperature is placed 30min.
C, inhale nor-aldehyde, add the 0.3%Triton X-100 room temperature treatment 30 minutes of PBS dilution.
0.3%Triton X-100 is removed in D, suction, the sheep blood serum room temperature sealing of adding 5% 1 hour.
E, discard confining liquid, add anti-myosin one anti-(1:100 dilution), 4 ℃ are spent the night.
F, 0.1%PBST wash 3 times, each 10 minutes.
G, adding FITC-labelling IgG two anti-(1:1000 dilutions), room temperature 30 minutes.
H, PBST wash 3 times, each 10 minutes.
I, DAPI mounting, laser confocal microscope are observed down and are taken pictures.
2.6 Application of I mage-Pro Plus6.0 software carries out quantitative analysis to image.
3. data analysis
Data are represented with mean+SD, t check between data analysis employing group.P≤0.01 expression has notable statistics difference.
4. experimental result
As shown in Figure 2.It is thus clear that the matched group myotube is after hungry 6 hours, the myotube diameter obviously diminishes; And rhodioside processed group myotube obviously increases than matched group myotube diameter; Show after the analysis of Application of I mage-Pro Plus6.0 software statistics, rhodioside processed group myotube diameter be 2.15 times of matched group (
*P<0.01) (see figure 3).Explain that rhodioside has suppressed the myotube atrophy.
5. experiment conclusion
Rhodioside can be through increasing the myotube diameter in the myotube atrophy, antagonist shrink tube.
Experiment three: rhodioside is to the influence of muscle fiber cross-sectional area
1. experiment purpose:
This experiment adopts the tail suspention to induce skeletal muscle atrophy, with histogenic immunity fluorescence chemical method, analyzes the influence that rhodioside reduces muscle fiber cross-sectional area in the mice amyotrophy.
2. experimental technique:
2.1 rhodioside is handled and the untreated mice Preparation of model
The rhodioside powder is diluted to 10mg/ml concentration with PBS.24 male C57 are divided into two groups according to the close principle of body weight; Mice was suspended in midair preceding 14 days in tail; The rhodioside processed group is irritated stomach, continuous 14 days according to per kilogram of body weight 50mg every day (50mg/kg/day) rhodioside dosage, untreated control group with processed group equal-volume solvent PBS synchronous every day.Irritating the stomach time unification is 8-9 point in the morning.
2.2 tail suspention mice amyotrophy Preparation of model
With rhodioside processed group and untreated control group C57 mice; According to internationally recognized mouse tail suspention method (Gregory R.Adams, et al.J Appl Physiol95:2185 – 2201,2003); Make mouse hind leg just liftoff, the angle of inclination on health and ground is approximately 30 °.Mice can be in mouse cage a low level free movable, ingest and drink water.The all single cage of animal is raised, and room temperature remains on 23 ± 2 ℃, keeps illumination in 12 hours every day.The tail suspention induced amyotrophy to form in 14 days, and after the end, cervical region dislocation sudden death mice is got mouse hind leg dorsal part gastrocnemius.
2.3 tissue freezing section
A, pre-cooling: the small beaker that will fill isopentane is placed in the liquid nitrogen, pre-cooling 10 minutes.
B, embedding: make holder with platinum paper, drip and go up embedding medium, the flesh tissue that takes out is put into embedding medium, placed isopentane 30 seconds-1 minute, treat that embedding medium becomes milky.
C, section: on rotary microtome, cut into slices.
D, fixing: will cut into slices to place in the acetone and fix 5 minutes, and place-20 ℃ of preservations afterwards.
2.4 histogenic immunity fluorescence chemical staining analysis
A, section is placed among the PBST, washed 5 minutes.
B, the effect of 0.3%Triton X-100 room temperature 30 minutes.
C, the sealing of 5% sheep blood serum room temperature 1 hour.
D, the anti-Laminin one of adding anti-(1:500 dilution), 4 ℃ are spent the night.
E, PBST wash 3 times, each 10 minutes.
F, adding FITC-labelling IgG two anti-(1:1000 dilutions), room temperature 30 minutes.
G, PBST wash 3 times, each 10 minutes.
H, DAPI mounting, laser confocal microscope are observed down and are taken pictures.
2.5 Application of I mage-Pro Plus6.0 software is analyzed image.
3. statistical analysis
Data are represented with mean+SD, t check between data analysis employing group.0.01＜p≤0.05 expression has significant difference.
4. experimental result
The control group mice tail suspended in midair after 14 days, the myofibrillar cross-sectional area average out to 1415 μ m of gastrocnemius
2, and feeding rhodioside group mouse tail was suspended in midair after 14 days, the myofibrillar cross-sectional area average out to 1669 μ m of gastrocnemius
2, with respect to matched group, after the atrophy of rhodioside group mice myofibrillar cross-sectional area obviously increase (
*P<0.05) (see Fig. 4, Fig. 5).
5. experiment conclusion
Myofibrillar cross-sectional area during rhodioside can form through increase mice amyotrophy, the antagonist atrophy.
Experiment four: rhodioside is to the influence of mouse muscle fiber type correlative protein expression
1. experiment purpose:
This experiment utilizes the method for Western blot through extracting matched group and rhodioside group mice gastrocnemius albumen, detects rhodioside and handles the influence of back to slow switch fibers albumen in the mice gastrocnemius and fast muscle fiber protein expression.
2. experimental technique
2.1 the extraction of histone
A, the preparation of histone lysate:
The constituent and the content of table 2 histone lysate
| Reagent | Concentration | Volume |
| Tris | 1M | 1ml |
| EDTA | 0.5M | 0.4ml |
| NaCl | 5M | 3ml |
| Briji96 | 10% | 8.75ml |
| NP40 | 10% | 1.25ml |
Above composition adds ddH2O to 100ml, adds protease inhibitor PMSF, Leupeptin, Aprotinin during use to final concentration 100 μ g/ml, 2 μ g/ml and 2 μ g/ml.
B, get 50-100mg muscular tissue and join 1mL and organize in the lysate and (added protease inhibitor), with homogenizer in fully homogenate on ice.
C, cracking on ice 30 minutes.
D, 4 ℃, centrifugal 20 minutes of 12000g gets supernatant, puts-80 ℃ of preservations.
E, the BSA of 0.2mg/ml is diluted to series of standards solution (0,25,50,100,200 μ g/ml); get each 20 μ l of testing sample of standard solution and suitably dilution; add in the protein concentration analytical reagent of 5 times of dilutions, behind the vibration mixing, photometry density value under the 595nm wavelength.OD value with standard protein concentration is made standard curve, calculates the testing sample protein concentration.
2.2SDS-PAGE electrophoresis
A, encapsulating: prepare 12% separation gel and 5% spacer gel respectively, record separation gel and spacer gel;
B, sample preparation: add 5 * gel loading buffer (final concentration 1 *), boiling water degeneration 3-5min at sample;
C, last appearance: cathepsin 18 0 μ g/ hole, molecular weight of albumen marker10 μ l/ hole;
D, electrophoresis: constant voltage electrophoresis (spacer gel 90V, separation gel 180V), electrophoresis to gel bottom;
E, take off gel, the electricity of putting into pre-cooling changes buffer balance 5min.
2.3 commentaries on classics film
A, cutting thick filter paper and the NC film identical with the gel size, balance 5min in the electricity commentaries on classics buffer of pre-cooling.
B, on the anode plate of half dry type electroporation, place 4 bed thickness filter paper, NC film, gel and 4 bed thickness filter paper successively, drive the bubble of each interlayer away, press negative plates.
C, carry out electricity with the constant current of 1.25mA/cm2 and change.
After D, electricity change end, film is placed the TBS rinsing 3 times, each 5min.
2.4 antigen antibody reaction
A, sealing: transfer film is put into hybridization bag, add an amount of confining liquid, room temperature jog 2h.
B, an anti-reaction: incline and fall confining liquid, add one anti-(Troponin I-SS or Troponin I-FS) with the confining liquid dilution according to the ratio of 0.1ml/cm2, eliminate bubble, seal, 4 degree jogs spend the night.
C, rinsing: take out transfer film, wash 3 times, each 10min with TBS-T.
D, two anti-reactions: transfer film is put into hybridization bag, add the two anti-of confining liquid dilution, catch up with except that sealing behind the bubble, at room temperature vibration 2h. according to the ratio of 0.1ml/cm2
E, rinsing: take out transfer film, wash 3 times, each 10min with TBS-T.
2.5ECL colour developing
A, respectively get equal-volume ECL reagent A liquid, B liquid and mix (quoting film 0.125ml ECL mixed liquor according to every square centimeter calculates).
B, transfer film is contacted filter paper gently blot liquid, have proteinic one to face up and be placed on the preservative film.
C, the ECL mixed liquor is added on the transfer film, reaction 1min mentions transfer film, blots liquid with filter paper.
D, cover transfer film, notice that the surface is smooth with preservative film.
E, the transfer film that preservative film covers is fixed in the magazine, has proteic one to face up with adhesive tape.Carry out the X-ray exposure in the darkroom, the punching observed result.
2.6 Application of I mage-Pro Plus6.0 software is analyzed gradation of image.
3. data analysis
Data are represented with mean+SD, t check between data analysis employing group.P≤0.01 expression has notable statistics difference.
4. experimental result
Rhodioside was irritated stomach after 14 days, in the gastrocnemius expression ratio matched group of slow switch fibers albumen (Troponin I-SS) increased by 6.2 times (
*P<0.01) (see Fig. 6, Fig. 7), and the expression ratio matched group of fast muscle fiber albumen (Troponin I-FS) has reduced 8%, (sees Fig. 6, Fig. 8).
5. experiment conclusion
Rhodioside can significantly increase the proteic content of slow switch fibers in the skeletal muscle, and has the trend that suppresses the fast muscle fiber protein expression, can bring into play prevention and treat amyotrophic effect.
Claims (7)
1. rhodioside prevents and treats the application in the amyotrophic medicine in preparation.
2. the described application of claim 1, said amyotrophy are that skeletal muscle myotube diameter reduces.
3. the described application of claim 1, said amyotrophy are that the skeletal muscle fiber cross-sectional area reduces.
4. the described application of claim 1, said amyotrophy are the increases that amyotrophy specific factor (Atrogin-1) is expressed.
5. the described application of claim 1, said amyotrophy are that the slow switch fibers protein content reduces.
6. the described application of claim 1, said amyotrophy are that the fast muscle fiber protein content increases.
One kind the prevention and treat amyotrophic medicine, its active component is a rhodioside.
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| CN107624069A (en) * | 2015-02-03 | 2018-01-23 | 纳图瑞克斯有限公司 | For improving the composition and method of muscle metabolism |
| CN113694043A (en) * | 2021-08-17 | 2021-11-26 | 西安电子科技大学 | Salidroside patch for treating muscular atrophy |
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| CN107624069A (en) * | 2015-02-03 | 2018-01-23 | 纳图瑞克斯有限公司 | For improving the composition and method of muscle metabolism |
| CN113694043A (en) * | 2021-08-17 | 2021-11-26 | 西安电子科技大学 | Salidroside patch for treating muscular atrophy |
| CN113694043B (en) * | 2021-08-17 | 2023-12-22 | 西安电子科技大学 | Rhodiola rosea glycoside patch for treating muscular atrophy |
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