CN107158016A - The application of timosaponin and its aglycon in prevention and treatment early diabetic nephropathy medicine is prepared - Google Patents
The application of timosaponin and its aglycon in prevention and treatment early diabetic nephropathy medicine is prepared Download PDFInfo
- Publication number
- CN107158016A CN107158016A CN201710432405.6A CN201710432405A CN107158016A CN 107158016 A CN107158016 A CN 107158016A CN 201710432405 A CN201710432405 A CN 201710432405A CN 107158016 A CN107158016 A CN 107158016A
- Authority
- CN
- China
- Prior art keywords
- sarsasapogenin
- rats
- significantly reduce
- rat
- diabetic nephropathy
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to the new pharmacological action of Sarsasapogenin in the wind-weed, the i.e. preventive and therapeutic action to early diabetic nephropathy.Sarsasapogenin can be obviously improved the infringement of renal function caused by diabetes and form, that is, substantially reduce the excretion of Urine proteins, significantly reduce kidney weight index, significantly reduce serum uric acid, significantly reduce cortex renis advanced glycation end products（AGEs）And interleukin-18（IL‑18）Level, substantially reduces kidney basement membrane thickened（Glycogen deposition）And extracellular matrix（Four Collagen Type VIs and fibronectin）Accumulation, significantly reduce renal inflammation corpusculum NLRP3 and activation caspase 1 protein level.Sarsasapogenin can be used for the prevention and treatment of the Diabetic microvascular complications such as early diabetic nephropathy.
Description
Technical field
The present invention relates to the new pharmacological action of Sarsasapogenin in the wind-weed, especially Sarsasapogenin is to early stage glycosuria
The preventive and therapeutic effect of the Diabetic microvascular complications such as sick nephrosis.
Background technology
Diabetic nephropathy (diabetic nephropathy, DN) is most commonly seen one in Diabetic microvascular complication
Kind, it is that patient is lethal, the main cause that disables for diabetes (diabetes mellitus, DM), is also to cause ESRD
(ESRD) main reason.Diabetes number of patients is in cumulative year after year.IDF (IDF) issue " the IDF whole world
Diabetes general view " the 7th edition, ends 2015, world's DM adult patients reach 4.15 hundred million, wherein China is 1.096 hundred million, ranks
First, next to that India and the U.S., respectively 0.692 hundred million and 0.293 hundred million.If diabetic is chronically at hyperglycemia state,
Even if blood sugar level recovers normal, still can occur the related complication of diabetes.The clinical manifestation master of early diabetic nephropathy
Have：The high filtration rate of albuminuria, glomerulus, glomerular basement membrane are slightly thickened, Glomerular mesangium component * is expanded etc..DN generation
With development mainly as caused by the interaction of genetic predisposition and environmental factor (hyperglycaemia)；Environmental factor also includes high blood
The factors such as fat, hypertension, but hyperglycaemia is more important.DN pathogenesis is considerably complicated, but Main Viewpoints are thought at present：For a long time
Oxidative stress, inflammation, Advanced glycation endproducts (AGEs) formation, RAS axles and polyalcohol Pathway Activation caused by hyperglycaemia
Deng playing leading role in DN pathogenic process.Moreover, numerous studies in recent years are confirmed, metabolic memory (metabolic
Memory) it is a unique phenomenon in chronic complicating diseases of diabetes evolution.Metabolic memory highlights such a fact：
Long term hyperglycemia exposure before and the caused long-term damage effect to histoorgans such as angiocarpy of diabetic disease states, even in
Still persistently exist after blood sugar recovery is normal.Metabolic memory research shows, prevents chronic complicating diseases of diabetes from occurring development not only
Only it is strict glycemic control (Metabolic memory appears to be more than just tight
glucose control necessary to prevent diabetic complications).Lasting hyperglycemia state
The formation of the oxidative stress and Advanced glycation endproducts of induction is probably the basis to form high glycometabolism memory.In clinic
On, metabolic memory is presented as that patient is taken after hypoglycemic medicine, although blood sugar level recovers normal, but diabetogenous nephrosis illness not with
Blood glucose reduction and therefore substantially improve, hypoglycemic contacting with diabetic nephropathy and in the absence of certainty.Therefore, diabetic nephropathy
Pathogenesis and study on prevention be still one of important research topic of this century whole world the world of medicine.
The wind-weed (Rhizome Anemarrhena) is the classical Chinese medicine for treating diabete (diabetes in modern times), in treatment
The frequency occurred in the Chinese medicine compound prescription of diabete is higher.Timosaponin (timosaponin) be Anemarrhena asphodeloides Bge chief active into
Point, it is steroid saponin, is also content highest active component, about 6% in the wind-weed；In the wind-weed comprising A-I, A-II, A-III,
30 kinds of structures such as A-IV, B-I, B-II, B-III, C, E1 and I clear and definite monomer saponin, wherein A-III, B-II equal size
It is higher.A-III can the direct desugar base generation Sarsasapogenin of the generation of desugar base A-I, A-III and A-I
(sarsasapogenin, Sar), B-II etc. can be transformed into A-III by glucosides ferment treatment desugar base.Sarsasapogenin is again
Referred to as sarsasapogenin, is topmost sapogenin in the wind-weed, and molecular formula is C27H44O3, relative molecular weight is 416.64Da.This
Outside, also containing double benzene pyrrone constituents in the wind-weed, such as mangiferin, content is 1% or so.Modern pharmacology research shows, knows
There are female saponin(e hypoglycemic, lipid-loweringing, anti-inflammatory, anti-oxidant, a variety of pharmacology of suppression platelet aggregation and antithrombus formation, anticancer etc. to live
Property, available for preventing and treating atherosclerosis, diabetes, aging, cognitive impairment, osteoporosis, headstroke and cancer etc..
The molecular structural formula of Sarsasapogenin (sarsasapogenin)
The molecular structural formula of Timosaponin A-III (timosaponin A-III)
Timosaponin A-III (timosaponin A-III), A-I molecular structural formula
Timosaponin B-II (timosaponin B-II) molecular structural formula
Timosaponin B-III (timosaponin B-III) molecular structural formula
The content of the invention
1. goal of the invention
It is an object of the invention to be found that the new pharmacological action of Sarsasapogenin, i.e. Sarsasapogenin pair in the wind-weed
The preventive and therapeutic effect of early diabetic nephropathy.In fact, the present invention relates to main steroid saponin in the wind-weed and its member to early stage glycosuria
The preventive and therapeutic effect of sick nephrosis and its possible mechanism of action.
2. technical scheme
The application of timosaponin and its aglycon in prevention and treatment early diabetic nephropathy medicine is prepared.
Application of the described timosaponin and its aglycon in prevention and treatment early diabetic nephropathy medicine is prepared,
It is characterized in that described timosaponin structural formula is：
Sarsasapogenin is Sarsasapogenin, and its structural formula is：
Described timosaponin and its aglycon are in prevention and treatment diabetes other microvascular complication medicines are prepared
Using.
The invention discloses topmost sapogenin in the wind-weed --- and Sarsasapogenin is induced Streptozotocin (STZ)
Diabetic nephropathy model rat preventive and therapeutic effect and its with AGEs formation, NLRP3 inflammation corpusculum activation mediation inflammatory factor
The relation of synthesis and release increase etc., MAIN OUTCOME MEASURES includes：Fasting blood-glucose, body weight, kidney weight index, Urine proteins, serum urine
Acid, AGEs, IL-18 level, basilar memebrane glycogen deposition, extracellular matrix (four Collagen Type VI Col-IV and fibronectin FN) aggregation,
NLRP3 and activated form caspase 1 protein expression etc..Pass through the various pharmacological testings and its knot of above-mentioned Sarsasapogenin
Really, its preventive and therapeutic effect to early diabetic nephropathy is illustrated, so as to further understand the essence of the present invention.
Influence of the Sarsasapogenin to DM rat fasting blood-glucoses and body weight.It was found that Sarsasapogenin is low, high by two
Individual dosage has not significant impact behind administration 2,5 and 9 weeks to the blood sugar level after DM rat empty stomaches on daytime 7-8h；Meanwhile, Sa
You also have not significant impact the body weight of DM rat of Sa two dosage of sapogenin to monitoring weekly.High dose is to normal rat limosis
Blood glucose and body weight do not influence.
Influence of the Sarsasapogenin to DM Renal Functions.The results show Sarsasapogenin is low, high two
Dosage (20,60mg/kg) has obvious reduction to act on the urinary protein excretion, kidney weight index and serum uric acid level of DM rats.
And high dose has not significant impact to normal Renal Function.
Influence of the Sarsasapogenin to DM rat kidney base film thickness and extracellular matrix.The results show thayer
Sa sapogenin is low, high two dosage can significantly reduce the deposition of DM Renal Glomeruli In Rats basilar memebrane glycogens, is thickened so as to suppress it；
Meanwhile, Sarsasapogenin is low, high two dosage can significantly reduce the Collagen Type VI of DM rat kidney four and fibronectin levels, from
And suppress extracellular matrix accumulation.High dose has not significant impact to the above index of normal rat kidney.
Influence of the Sarsasapogenin to DM rat kidney AGEs levels.The results show Sarsasapogenin is low, height
Two dosage can significantly reduce AGEs levels in DM renal cortex of rats, show that Sarsasapogenin can substantially suppress under DM states
Kidney AGEs formation.High dose has not significant impact to AGEs levels in normal renal cortex of rats.
Influence of the Sarsasapogenin to DM rat kidney NLRP3 inflammation corpusculums.The results show Sarsasapogenin
Low, high two dosage can significantly reduce the protein expression of NLRP3 in DM renal cortex of rats, hence it is evident that reduction activated form
The levels of caspase 1, and IL-18 level, show that Sarsasapogenin has and significantly suppress the activation of NLRP3 inflammation corpusculum
Effect.
3. beneficial effect
Above pharmacological tests show that the present invention has advantages below：
(1) present invention is to timosaponin and its main aglycon --- Sarsasapogenin be found that new pharmacological action and
Potential novel medical use, has opened up a new application field.
(2) present invention demonstrates that the wind-weed is hypoglycemic as one of traditional Chinese medicine application, although Sarsasapogenin is the wind-weed
One of main component, but it does not have effect of lowering blood sugar, and it is not hypoglycemic main component to illustrate it, but is found first anti-
Control early diabetic nephropathy pharmacological effect good, effect is strong, and to the biochemical indicator and renal function and form of normal rat without
Harmful effect, therefore the present invention imply that good prospect in medicine.According to metabolic memory theory, hypoglycemic drug and treatment glycosuria
Sick nephrosis has no direct loic relation, hypoglycemic to improve salsa saponin(e in diabetic nephropathy illness, the present invention
Member is to find that it has anti diabetes and kidney disease effect first.1-timosaponin A-1-I, A-III, B-II possess identical Chinaroot Greenbier Rhizome saponin(e
Member, metabolism forms Sarsasapogenin in vivo, therefore also plays the role of similar.
(3) present invention demonstrates that Sarsasapogenin can obviously reduce the urinary protein excretion of DM rats, kidney weight index and serum
Uric acid level, illustrates that Sarsasapogenin can substantially mitigate the infringement of DM rat early stage renal functions.
(4) present invention demonstrates that Sarsasapogenin can significantly inhibit DM Renal Glomeruli In Rats basilar memebrane PAS positive stainings, show
It can reduce the deposition of glycogen to suppress basement membrane thickened；Meanwhile, Sarsasapogenin can significantly reduce DM rat kidney four
Collagen Type VI and fibronectin levels, so as to suppress extracellular matrix accumulation.These show that Sarsasapogenin can substantially mitigate
Infringement of the kidney of DM rat early stages in terms of form.
(5) present invention demonstrates that Sarsasapogenin has no significant effect to the fasting blood-glucose and body weight of DM rats, but substantially resistance
Rat DN development is stopped, it is not to play renal protection by controlling blood sugar condition to illustrate Sarsasapogenin.
(6) present invention demonstrates that Sarsasapogenin has AGEs horizontal forces, explanation in substantially reduction DM renal cortex of rats
Sarsasapogenin can substantially suppress the non-enzyme glycoslysis of albumen and the infringement of AGEs/RAGE axles mediation.
(7) present invention demonstrates that Sarsasapogenin have significantly reduce in DM renal cortex of rats NLRP3 protein expression and
The level of the activated forms of key molecule caspase 1 of NLRP3 inflammation corpusculum activation, and the production that NLRP3 inflammation corpusculum is activated
Thing IL-18 level, shows that Sarsasapogenin has the effect for significantly suppressing the activation of NLRP3 inflammation corpusculum.
Brief description of the drawings
Fig. 1 Sarsasapogenins substantially reduce PAS stained positives region in DM renal cortex of rats.Representative PAS colored graphs
Piece, C groups (A), C+Sar-H groups (B), DM groups (C), DM+Sar-L groups (D), DM+Sar-H groups (E), multiplication factor：╳400；Respectively
Column diagram (F) of the group containing analytic statistics, mean ± S.E.M., n=3, * * P<0.01, compared with C groups；##P<0.01, with DM groups
Compare；
Fig. 2 Sarsasapogenins substantially reduce FN and Co-IV levels in DM renal cortex of rats.Representative FN IHC dyeing
Picture (A), multiplication factor：╳ 400, column diagram (B) of each group containing analytic statistics, mean ± S.E.M., n=3, * * P<0.01,
Compared with C groups；##P<0.01, compared with DM groups.Representative Co-IV IHC dyeing pictures (C), multiplication factor：╳ 400, each group
Column diagram (D) containing analytic statistics, mean ± S.E.M., n=3, * * P<0.01, compared with C groups；##P<0.01, compare with DM groups
Compared with；
Fig. 3 Sarsasapogenins substantially reduce the protein expressions of cleaved caspase 1 in DM Renal Glomeruli In Rats.It is representative
Immunofluorescence dyeing picture, red represents the albumen of cleaved caspase 1, and blueness represents nucleus.
Embodiment
Embodiment 1：Influence of the Sarsasapogenin to DM rat early stage renal functions
1 materials and methods
1.1 Experimental Animals Male Sprague-Dawley strain rats, 8-9 week old, 190-220g, by Xuzhou medical university
Experimental Animal Center is provided.
1.2 medicines are purchased from Beijing Medicass Biotechnologies Co., Ltd.'s (purity with reagent Sarsasapogenin>98%),
STZ is purchased from sigma companies of the U.S., and the reagent such as CMC-Na is purchased from Chemical Reagent Co., Ltd., Sinopharm Group, glucose testing kit, urine
Protein quantification kit builds up Bioengineering Research Institute purchased from Nanjing, and testing uric acid kit has purchased from the poly- wound medical sci-tech in Shanghai
Limit company.
1.3 method
1.3.1 model sets up male Sprague-Dawley rat fasting and can't help more than water 12h, disposable by 60mg/kg
STZ (being dissolved in 0.1mol/L sodium citrate buffer solutions, pH 4.4 before use) is injected intraperitoneally to carry out after modeling, modeling five days, socket of the eye is quiet
Arteries and veins clump takes blood about 0.3ml, 4 DEG C, 3000rpm centrifugation 10min, separates serum.Fasting blood-glucose is detected with glucose kit
(FBG) rat of the FBG values more than 250mg/dl (13.9mmol/L) and less than 600mg/dl (33.3mmol/L), is taken as success
Diabetes rat.
1.3.2 it is grouped and the successful diabetes rat of modeling is randomly divided into 3 groups, respectively glycosuria by administration according to blood glucose value
Disease model group (DM), Sarsasapogenin low (DM+Sar-L), high (DM+Sar-H) dosage group, every group of 11 rats.Separately set just
Normal control group (C) and normally increase dosage group (C+Sar-H), every group of 10 rats.Sarsasapogenin is low, high dose group difference
Sarsasapogenin (suspension is made with 1%CMC-Na), daily one are given by 20,60mg/kg gavages (by 10ml/kg volumes)
It is secondary, continuous 9 weeks.C groups and DM groups give same volume 1%CMC-Na.A body weight is monitored weekly, is adjusted administered volume, is supervised every two weeks
Survey a fasting blood-glucose.
1.3.3 when index determining is administered about 9 weeks, each group rat is put into rat metabolism cage, 24h urines, measurement is collected
Volume, and determine its 24h urine protein content.Afterwards, each group rat femoral vein takes blood, separates serum, determines uric acid level, remaining
Cord blood, it is standby.At once put to death, strip both sides kidney, after weighing respectively, a part is fixed with 10% formalin, remaining
It is organized in -80 DEG C of preservations.
1.3.3.1 Urine proteins, which are determined, uses quantity of proteinuria kit, and Coomassie Brilliant Blue (CBB methods) determines egg in urine
White concentration, is carried out in strict accordance with the determination step of specification.24h urine albumen amounts (mg)=urinary protein concentrations (mg/L) × dilution times
Number × 24h volume of urine (ml).
1.3.3.2 kidney weight assessment of indices takes out rat both sides kidney, peels off structural envelope, physiological saline cleaning, filter
Paper is blotted, scales/electronic balance weighing.
1.3.3.3 testing uric acid uses testing uric acid kit, and urinary acidification determines serum uric acid level, in strict accordance with saying
The determination step of bright book is carried out.
All results of 1.4 statistical procedures are represented with mean ± S.E.M., and multigroup is compared and use one-way analysis of variance,
Compare after heterogeneity of variance between two groups and use Dunnett ' s T3 to examine progress statistical analysis, P<0.05 expression difference has statistics
Meaning.
2 results
Influence of 2.1 Sarsasapogenins to DM rat blood sugars and body weight
In whole experiment process, the FBG of DM rats is constantly in high-level state, is significantly higher than normal rat (P<
0.01).DM rats are given after the dosage 2,5 and 9 weeks of Sarsasapogenin two, and FBG is still within high-level state, with non-administration
DM rats are compared, and no difference of science of statistics (is shown in Table 1).The body weight of DM rats is substantially less than the body weight (P of normal rat<0.01), Sa
Body weight of two dosage of your Sa sapogenin to DM rats during being administered does not make significant difference and (is shown in Table 2).In addition, salsa soap
Aglycon is on the blood glucose and body weight of normal rat without influence (being shown in Table 1,2).
Influence of the Sarsasapogenin of table 1 to DM rat fasting blood-glucoses
Note mean ± S.E.M., n=9-11, * * P<0.01, compared with C groups.
Influence of the Sarsasapogenin of table 2 to DM rat body weights
Note：Mean ± S.E.M., n=9-11, * * P<0.01, compared with C groups.
Influence of 2.2 Sarsasapogenins to DM Rat renals weight index, urinary protein excretion and serum uric acid
The kidney weight index of DM rats substantially increases, the statistically significant (P of the comparison with normal rat<0.01).Salsa
Sapogenin is low, high dose can significantly reduce the kidney weight index (P of DM rats<0.01 and P<0.05) 3, be the results are shown in Table.
The urinary protein excretion of DM rats is dramatically increased, the statistically significant (P of the comparison with normal rat<0.01).Thayer
Sa sapogenin is low, high dose can significantly reduce urinary protein excretion (the equal P of DM rats<0.01) 4, be the results are shown in Table.
The serum uric acid level of DM rats substantially increases, the statistically significant (P of the comparison with normal rat<0.01).Sa
Your Sa sapogenin is low, serum uric acid level (the equal P that high dose can significantly reduce DM rats<0.01) 5, be the results are shown in Table.
Influence of the Sarsasapogenin of table 3 to DM Rat renals weight index
Note：Mean ± S.E.M., * * P<0.01, compared with C groups；#P<0.05, ##P<0.01, compared with DM groups.
Influence of the Sarsasapogenin of table 4 to DM rat urinary protein excretions
Note：Mean ± S.E.M., * * P<0.01, compared with C groups；#P<0.05, ##P<0.01, compared with DM groups.
Influence of the Sarsasapogenin of table 5 to DM rat blood serum uric acid
Note：Mean ± S.E.M., * * P<0.01, compared with C groups；##P<0.01, compared with DM groups.
Embodiment 2：Influence of the Sarsasapogenin to DM Renal Glomeruli In Rats base film thickness and Extracellular Matrix Content
1 materials and methods
1.1 experimental animal be the same as Example 1.
1.2 medicines are purchased from Nanjing Sen Beijia bio tech ltd, bush uniformly dyeing with reagent glycogen PAS staining kits
Liquid is purchased from Shanghai bio tech ltd conversant with things of the past, and Fibronectin antibody is purchased from abcam companies of Britain, and Collagen- IV resists
Body is purchased from Bioworld Reagent Companies of the U.S..
1.3 method
1.3.1 PAS is dyed
The cortex renis for taking formalin to fix, carries out routine paraffin wax embedding.PAS staining procedures are as follows：
1) nephridial tissue paraffin section thickness is 4 μm；
2) dewaxing and aquation：Section is dewaxed each 15min in dimethylbenzene I, dimethylbenzene II successively, respectively at 100%,
95%th, distilled water is entered in 80% ethanol after each 5min；
3) liquid unnecessary around tissue is wiped away, 1% periodic acid is added dropwise is capped it in tissue, room temperature, oxidation
6min；
4) flowing water rinses 5min, then embathes 5min with distilled water；
5) wipe liquid away, Schiff reagents are added dropwise in tissue, room temperature lucifuge dyeing 23min；
6) running water rinses 10min, and distilled water embathes 5min；
7) wipe liquid away, haematoxylin is added dropwise in tissue, room temperature dye core 2min, distilled water flushing 2min；
8) 2s is broken up with 1% hydrochloride alcohol；
9) distilled water flushing 2min；
10) conventional be dehydrated in transparent, neutral gum mounting, fume hood is dried.
11) result is observed：
PAS stained positives material (polysaccharide and glycogen) takes on a red color or aubergine, and nucleus is in blueness.PAS decoration methods are to see
Kidney morphosis is examined, the main method of glomerular mesangium, basilar memebrane and renal tubule structure is especially observed.PAS is taken to dye
Section, cuts into slices for every and takes the cortex renis visual field of 5 non-overlapping copies, determine positive stained area area and sight plane under each visual field
The ratio between product, and its average value is taken, for evaluating kidney basement membrane thickened degree.
1.3.2 ImmunohistochemistryMethods Methods detect the level of extracellular matrix fibronectin and collagen- IV
1) nephridial tissue of FFPE is cut into slices：4μm；
2) dewax aquation：Section is dewaxed each 15min in dimethylbenzene I, dimethylbenzene II successively, then respectively at 100%,
95%th, rinsed 3 times with PBS solution after each leaching 5min in 80% ethanol, each 3min；
3) wipe liquid unnecessary around tissue away with chipless paper, 3%H is added dropwise2O2Covered, be incubated at room temperature in tissue
10min；
4) PBS is rinsed 3 times, each 3min；Wipe liquid away, 0.4% pepsin be added dropwise and repairs liquid to organizing to repair,
37 DEG C of incubation 30min.PBS is rinsed 3 times, each 3min；
5) wipe liquid away, 2%BSA confining liquids, room temperature closing 20min is added dropwise.Confining liquid is got rid of, liquid around tissue is wiped
Body；
6) by fibronectin and the antibody of collagen- IV according to 1:400 ratio is diluted with primary antibody dilution,
Antibody covering tissue is added dropwise, is placed in 4 DEG C of refrigerators of wet box and is incubated overnight；
7) PBS is rinsed 3 times, each 3min, wipes liquid away with paper, and reagent 1 is added dropwise in tissue, 37 DEG C of baking ovens of wet box are placed in
It is incubated 20min；
8) PBS develops a film 3 times, and each 3min wipes liquid away.Reagent 2 is added dropwise, 37 DEG C of incubation 25min of wet box are placed in；
9) PBS is rinsed 3 times, each 3min.Wipe liquid away, haematoxylin is added dropwise in tissue, dye core 2min, PBS flushing；
10) 2s, distilled water flushing 2min are broken up with 1% hydrochloride alcohol；
11) it is conventional to be dehydrated transparent, neutral gum mounting.
Immunohistochemical staining result is mainly the expression water of observation fibronectin and collagen- IV in cortex renis
Flat, yellowish-brown is presented in positive findings.SABC is taken to cut into slices, 5 non-overlapping renal interstitials of micro- Microscopic observation each column section
The visual field, using image analysis software Image-pro-plus 6.0, and with integral optical density (IOD) be index to each group immune group
Change dyeing picture and carry out quantitative analysis, be integral optical density (IOD) and picture statistics area (area) with average integral optical density
Ratio quantized result, its size represents the immune response intensity of GAP-associated protein GAP.
1.4 statistical procedures be the same as Examples 1.
2 results
The influence that 2.1 Sarsasapogenins are dyed to PAS in DM renal cortex of rats
PAS stained positives region is dramatically increased in DM renal cortex of rats, with relatively having statistics in normal rat cortex renis
Learn meaning (P<0.01).Sarsasapogenin is low, high dose can significantly reduce PAS stained positives region in DM renal cortex of rats
(equal P<0.01) Fig. 1, is as a result seen.
Influence of 2.2 Sarsasapogenins to Extracellular Matrix Content in DM renal cortex of rats
FN and Co-IV levels are dramatically increased in DM renal cortex of rats, with relatively having statistics in normal rat cortex renis
Meaning (P<0.01).Sarsasapogenin is low, high dose can significantly reduce FN and Co-IV levels (equal P in DM renal cortex of rats<
0.01) Fig. 2 B, D, are as a result seen.
Embodiment 3：Sarsasapogenin to AGEs levels in DM renal cortex of rats and influence
1 materials and methods
1.1 experimental animal be the same as Examples 1.
1.2 medicines are purchased from sigma companies of the U.S. with reagent I Collagenase Type, and other conventional chemical reagent are purchased from Chinese medicines group
(Shanghai) chemical reagent Co., Ltd.
1.3 method
1.3.1 AGEs levels are determined
Fluorescence spectrophotometry detects AGEs levels.By the kidney sample of preservation, added after weighing by 10 times of amounts (w/v)
100mM PBS (pH7.4) buffer solution, fine granularity is cut into eye scissors, then in ultrasound homogenate under low temperature.Homogenate is in 4
10000rpm is centrifuged after 15min at DEG C, and supernatant is used for total protein content and other indexs of correlation are determined.Precipitation is taken, with double
Steam water washing 3 times, chloroform/methanol (1 is added in precipitation:1) 1.0ml, shaken overnight.Add methanol/water (4:1) 0.5ml,
4000rpm centrifuges 5min at 4 DEG C.Precipitation is washed 2 times with methanol 1.0ml, and distilled water is washed 2 times, then with pH7.5,0.02mol/L
Hepes buffer solutions (CaCl containing 0.1mol/L2) wash 2 times.It is deposited in 1.0ml Hepes buffer solutions at 4 DEG C overnight.Centrifugation is gone
Except buffer solution, in the Hepes buffer solutions that particle is suspended in 1.0ml enzymes containing NTx (290U), add toluene and chloroform is each
2.0μl.Using the blank tube containing only Hepes buffer solutions and clostridiopetidase A as standard, digestive juice is centrifuged, left and taken by 37 DEG C of vibration 24h
Clear liquid.The fluorescence intensity (reflection AGEs content) in supernatant is detected with fluorescence spectrophotometry, excitation wavelength is 370nm,
Absorbing wavelength is 440nm.AGEs content is represented with U/mg nephridial tissue albumen.
1.4 statistical procedures be the same as Examples 1.
2 results
Influence of 2.1 Sarsasapogenins to AGEs contents in DM renal cortex of rats
AGEs contents are dramatically increased in DM renal cortex of rats, with the statistically significant (P of comparison in normal rat cortex renis
<0.01).Sarsasapogenin is low, high dose can significantly reduce AGEs contents (the equal P in DM renal cortex of rats<0.01)；High agent
Amount gives normal rat, and AGEs contents do not have significant change, the results are shown in Table 6.
Table 6 knows influence of the Sarsasapogenin to AGEs contents in DM renal cortex of rats
Note：Mean ± S.E.M., * * P<0.01, compared with C groups；##P<0.01, compared with DM groups.
Embodiment 4：Sarsasapogenin is to NLRP3 protein expressions, the caspase1 and IL- that activate in DM renal cortex of rats
18 levels influence
1 materials and methods
1.1 experimental animal be the same as Examples 1.
1.2 medicines and reagent N LRP3 antibody are purchased from Cell Signaling Technology companies of the U.S., and rabbit-anti β-
Actin antibody is purchased from Bioworld companies of the U.S., and the antibody of cleaved caspase 1 is purchased from Santa Cruz companies of the U.S.,
Western blot primary antibodies, secondary antibody dilution, alkaline phosphatase secondary antibody, phenylmethyl sulfonylfluoride (PMSF) and BCA protein
Testing cassete, DAPI dyeing liquors are purchased from the green skies biotech firm in Jiangsu, Na3VO4Chinese medicines group (Shanghai) chemical reagent is purchased from NaF
Co., Ltd, DAB developers are purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, Dylight 594affinipure
Donkey anti-rabbit are purchased from EarthOx companies of the U.S., and rat IL-18ELISA kits are purchased from Wuhan BOSTER companies.
1.3 method
1.3.1 NLRP3 protein expressions are determined
Western blotting methods determine NLRP3 protein expressions.The renal tissue slurries for taking packing to preserve (were once used for
RAGE expression is determined), sample is separated by electrophoresis, transferring film (nitrocellulose filter) washes film, closes, primary antibody reaction, secondary antibody reaction shows
Color, scans or takes pictures, the change of Image J software analysis protein expression levels.Internal reference is used as using β-actin.
1.3.2 the protein expressions of cleaved caspase 1 are determined
Immuno-fluorescence assay NLRP3 protein expressions.
1) histotomy：4μm；
2) histotomy dewaxing aquation：Dimethylbenzene I 15min, dimethylbenzene II 15min, 100% ethanol 5min, 95% second
Alcohol 5min, 80% ethanol 5min；
3) it is placed in distilled water and soaks 15min, PBS develops a film, 5min ╳ 3 times wipes surplus liquid；
4) Microwave method：Slide is put into the citric acid solutions of PH 6.0, closes and covers, big fire 5min, small fire 15min,
Uncap and naturally cool to room temperature.PBS develops a film, 5min ╳ 3 times, gets rid of liquid and wipes dry unnecessary liquid；
5) close：Hyclone:PBS proportionings are 1:200 preparation confining liquids, each μ l of tissue 40, adding to tissue makes it
Cover tissue surface, room temperature closing 40min；
6) get rid of the liquid of histotomy excess surface and wipe dry surrounding liquid, do not wash, cleaved caspase-1 are added dropwise
(1:100) primary antibody dilution is placed in 4 DEG C of overnight incubations in wet box in slice surface；
7) 4 DEG C of taking-up wet box, 37 DEG C of incubation 30min；
8) wash：PBS develops a film, 5min ╳ 3 times；
9) two antiantibodys (1 that fluorescence labeling is added dropwise under the conditions of tissue surrounding liquid, lucifuge are dried:200), each section group
40 μ l are knitted, tissue surface are covered, and 1h is incubated in 37 DEG C of lucifuges；
10) liquid is got rid of, PBS develops a film, DAPI dyeing liquors are added dropwise in 5min ╳ 3 times, dyes 3min, PBS washing 5min ╳ 3
Secondary (lucifuge operation)；
11) anti-fluorescent quenching mounting liquid is added dropwise in tissue, covered, it is to avoid produce bubble, corner uses colourless
Nail polish is fixed (lucifuge operation)；
12) taken pictures under fluorescence microscope.
Final result, the albumen of cleaved caspase 1 is red fluorescence, and nucleus is blue-fluorescence.
1.3.3 IL-18 levels are determined
Enzyme Linked Immunoadsorbent Assay (ELISA) method determines IL-18 levels.The supernatant of AGEs assay samples is taken, is determined
IL-18 levels in kidney.IL-18 levels are determined to be carried out in strict accordance with the operating procedure of respective kit.
1.4 statistical procedures be the same as Examples 1.
2 results
Influence of 2.1 Sarsasapogenins to NLRP3 protein expressions in DM rat kidney
NLRP3 protein expressions are dramatically increased in DM rat kidney, the statistically significant (P of the comparison with normal rat<
0.01).Sarsasapogenin is low, high dose can significantly reduce NLRP3 protein expressions (equal P in DM rat kidney<0.01)；High agent
Amount gives normal rat, and NLRP3 protein expressions do not change, and the results are shown in Table 7.
Influence of the Sarsasapogenin of table 7 to NLRP3 protein expressions in DM rat kidney
Note：mean±S.E.M.**P<0.01, compared with C groups；##P<0.01, compared with DM groups.2.2 Sarsasapogenin
Influence to the protein expressions of cleaved caspase 1 in DM Renal Glomeruli In Rats
Compared with normal rat, the protein expressions of cleaved caspase 1 are dramatically increased in DM Renal Glomeruli In Rats, salsa
Sapogenin is low, high dose can obviously reduce the protein expressions of cleaved caspase 1 in DM Renal Glomeruli In Rats, and high dose is given
The protein expressions of cleaved caspase 1 do not change in normal rat, glomerulus, the results are shown in Table Fig. 3.
Influence of 2.3 Sarsasapogenins to IL-18 levels in DM renal cortex of rats
IL-18 levels are dramatically increased in DM renal cortex of rats, the statistically significant (P of the comparison with normal rat<0.01).
Sarsasapogenin is low, high dose can significantly reduce IL-18 levels (equal P in DM renal cortex of rats<0.01)；High dose is given just
Normal rat, IL-18 levels do not change, and the results are shown in Table 8.
Influence of the Sarsasapogenin of table 8 to IL-18 levels in DM renal cortex of rats
Note：mean±S.E.M.**P<0.01, compared with C groups；##P<0.01, compared with DM groups.
Claims (3)
1. the application of timosaponin and its aglycon in prevention and treatment early diabetic nephropathy medicine is prepared.
2. timosaponin as claimed in claim 1 and its aglycon are in prevention and treatment early diabetic nephropathy medicine is prepared
It is using, it is characterised in that described timosaponin structural formula：
Sarsasapogenin is Sarsasapogenin, and its structural formula is：
3. timosaponin as claimed in claim 1 or 2 and its aglycon are preparing prevention and treatment diabetes other capilaries simultaneously
Send out the application in disease drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710432405.6A CN107158016B (en) | 2017-06-09 | 2017-06-09 | The application of timosaponin and its aglycon in preparation prevention and treatment early diabetic nephropathy drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710432405.6A CN107158016B (en) | 2017-06-09 | 2017-06-09 | The application of timosaponin and its aglycon in preparation prevention and treatment early diabetic nephropathy drug |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107158016A true CN107158016A (en) | 2017-09-15 |
| CN107158016B CN107158016B (en) | 2019-10-11 |
Family
ID=59825035
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710432405.6A Active CN107158016B (en) | 2017-06-09 | 2017-06-09 | The application of timosaponin and its aglycon in preparation prevention and treatment early diabetic nephropathy drug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107158016B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111732624A (en) * | 2020-07-03 | 2020-10-02 | 河南大学 | New steroid saponin compound, preparation method thereof and application thereof in preparing medicament for treating type 2 diabetes |
| CN115141288A (en) * | 2022-07-27 | 2022-10-04 | 浙江省立同德医院(浙江省精神卫生研究院) | Rhizoma anemarrhenae active polysaccharide, rhizoma anemarrhenae crude polysaccharide, and preparation method and application thereof |
| CN117298132A (en) * | 2023-11-30 | 2023-12-29 | 唐宁医药科技(济南)有限公司 | Application of effective ingredient formula of Guizhi cellulitis in preparing medicine for treating diabetic nephropathy |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1513463A (en) * | 2002-12-31 | 2004-07-21 | 中国科学院上海药物研究所 | Application of timosaponin A3 for preparing medicine for treating No.2 type diabetes mellitus |
| CN1814149A (en) * | 2005-11-25 | 2006-08-09 | 中国科学院上海药物研究所 | Medicine composition for treating diabetes or diabetes kidney-disease, and preparing method |
-
2017
- 2017-06-09 CN CN201710432405.6A patent/CN107158016B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1513463A (en) * | 2002-12-31 | 2004-07-21 | 中国科学院上海药物研究所 | Application of timosaponin A3 for preparing medicine for treating No.2 type diabetes mellitus |
| CN1814149A (en) * | 2005-11-25 | 2006-08-09 | 中国科学院上海药物研究所 | Medicine composition for treating diabetes or diabetes kidney-disease, and preparing method |
Non-Patent Citations (1)
| Title |
|---|
| YONG-LIANG YUAN等: "Timosaponin B-Ⅱ ameliorates diabetic nephropathy via TXNIP, mTOR, and NF-κB signaling pathways in alloxan-induced mice", 《DRUG DESIGN, DEVELOPMENT AND THERAPY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111732624A (en) * | 2020-07-03 | 2020-10-02 | 河南大学 | New steroid saponin compound, preparation method thereof and application thereof in preparing medicament for treating type 2 diabetes |
| CN111732624B (en) * | 2020-07-03 | 2022-09-20 | 河南大学 | Steroid saponin compound, preparation method thereof and application thereof in preparing medicament for treating type 2 diabetes |
| CN115141288A (en) * | 2022-07-27 | 2022-10-04 | 浙江省立同德医院(浙江省精神卫生研究院) | Rhizoma anemarrhenae active polysaccharide, rhizoma anemarrhenae crude polysaccharide, and preparation method and application thereof |
| CN115141288B (en) * | 2022-07-27 | 2023-09-08 | 浙江省立同德医院(浙江省精神卫生研究院) | Rhizoma anemarrhenae active polysaccharide, rhizoma anemarrhenae crude polysaccharide, and preparation method and application thereof |
| CN117298132A (en) * | 2023-11-30 | 2023-12-29 | 唐宁医药科技(济南)有限公司 | Application of effective ingredient formula of Guizhi cellulitis in preparing medicine for treating diabetic nephropathy |
| CN117298132B (en) * | 2023-11-30 | 2024-02-23 | 唐宁医药科技(济南)有限公司 | Application of effective ingredient formula of Guizhi cellulitis in preparing medicine for treating diabetic nephropathy |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107158016B (en) | 2019-10-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kusano et al. | Antidiabetic activity of white skinned sweet potato (Ipomoea batatas L.) in obese Zucker fatty rats | |
| Chakrabarti et al. | Antidiabetic activity of Caesalpinia bonducella F. in chronic type 2 diabetic model in Long-Evans rats and evaluation of insulin secretagogue property of its fractions on isolated islets | |
| Muthukumaran et al. | Syringic acid, a novel natural phenolic acid, normalizes hyperglycemia with special reference to glycoprotein components in experimental diabetic rats | |
| Lee et al. | Delayed treatment with lithospermate B attenuates experimental diabetic renal injury | |
| Bavbek et al. | Elevated concentrations of soluble adhesion molecules and large platelets in diabetic patients: are they markers of vascular disease and diabetic nephropathy? | |
| CN107158016B (en) | The application of timosaponin and its aglycon in preparation prevention and treatment early diabetic nephropathy drug | |
| SIDMAN et al. | Histochemical observations on rods and cones in retinas of vertebrates | |
| Qiao et al. | Astragaloside IV protects rat retinal capillary endothelial cells against high glucose-induced oxidative injury | |
| Freund et al. | Loss of cholinergic muscarinic receptors in the frontal cortex of alcohol abusers | |
| Awotunde et al. | Subacute toxicity study of aqueous root extract of Terminalia schimperiana in male Wistar rats | |
| Duan et al. | Protective effects of fufang xueshuantong on diabetic retinopathy in rats | |
| Saravanan et al. | Modulating efficacy of Rebaudioside A, a diterpenoid on antioxidant and circulatory lipids in experimental diabetic rats | |
| Ino-ue et al. | Polyol metabolism of retrograde axonal transport in diabetic rat large optic nerve fiber | |
| van Veen et al. | A developmental study of interphotoreceptor retinoid-binding protein (IRBP) in single and double homozygous rd and rds mutant mouse retinae | |
| US20190351001A1 (en) | Herbal compound extract to moderate diabetes with liver necrosis and fibrosis and use thereof | |
| CN109985251A (en) | Regulation and the intervention effect measuring method of CMKLR1 agonist of the Chemerin to insulin resistance | |
| Gabriel et al. | Anti-diuretic and anti-glycemic properties of Jatropha gossypiifolia L. leave extract on wistar rats | |
| Tilaye et al. | Vernonia amygdalina del (bitter leaf) extract ameliorates isoniazid (INH) induced liver injury in Swiss albino mice | |
| Haensly | Metaplasia of the parietal layer of Bowman's capsule in the human kidney. Incidence in alcoholic liver disease and hypertension | |
| CN108309986A (en) | Applications of the PCN in preparing the drug for treating type 1 diabetes nephrosis associated disease | |
| George et al. | Hypoglycemic properties of some local herbs extracts in streptozotocin-induced diabetic Wistar Albino rats | |
| CN108685925A (en) | A kind of compound AB-38b treats the application of medicine for treating diabetic nephropathy or health-care products in preparation | |
| AU2021105048A4 (en) | Synthesis and assessment of anticataract activity of metal-nsaid complex in hyperinsulinemic cataract | |
| Katiukhin et al. | Seasonal changes in blood rheology in little ground squirrels | |
| Abdel-Dayem et al. | Histological and immunohistochemical study on nitric oxide synthase and effects of angiotensin receptor blockade in early phase of diabetes in rat kidney |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |