CN106474146B - Lentinan is preparing the purposes in suppressing the impaired medicine of beta Cell of islet - Google Patents
Lentinan is preparing the purposes in suppressing the impaired medicine of beta Cell of islet Download PDFInfo
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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
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (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)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses lentinan to prepare the purposes in suppressing the impaired medicine of beta Cell of islet.Lentinan is being prepared by anti-oxidation protection beta Cell of islet from the purposes in the medicine of damage.The present invention is by the model being damaged with STZ induced rat insulinoma cells INS 1, the effect and possible molecular mechanism that the beta Cell of islet INS 1 that observation lentinan is induced by anti-oxidant mitigation STZ is damaged.As a result show, for the cellular damages of INS 1 of STZ inductions, lentinan can improve apoptosis and the intracellular ROS accumulations of the cells of INS 1, and the expression and NO releases, dose dependent for reducing iNOS mitigate the activation of JNK and p38 MAPK signal paths caused by STZ.In addition, lentinan can also improve the insulin secretion obstacle of STZ inductions by blocking NF κ B paths and strengthening the expression of Pdx 1.
Description
Technical field
The invention belongs to field of medicaments, it is related to the purposes of lentinan, and in particular to lentinan suppresses pancreas islet β preparing
Purposes in the medicine of cell damage.
Background technology
Diabetes (Diabetes mellitus, DM) are the metabolic diseases using chronic blood glucose rise as principal character.With
Socio-economic development and the raising of medical level, diabetes morbidity increases increasingly, and its morbidity and mortality has been located in non-
The 3rd of disease is infected, and developing country is particularly evident.Learned from the 12nd diabetes branch of Chinese Medical Association, in
The big or middle city of state and small towns crowd's diabetes prevalence of more than 20 years old are more than 10%, and prediabetes is up to 15%.To the greatest extent
Pipe type 1 diabetes patient only accounts for the 5%-10% of diabetic's sum, but type 1 diabetes are sent out in child or adolescent's period more
Disease, and easily induced Diabetic DKA, diabetic keratopathy hyperosmotic state acute complicationses, and each systemic blood vessels of whole body, nerve
It is last that death is caused with kidney failure more Deng chronic complicating diseases, bring heavy burden and pressure to family, society, country.
Type 1 diabetes (Type 1Diabetes mellitus, T1DM) are a kind of chronic, multifactor LADA diseases
Disease, in pathogenic process, beta Cell of islet can be destroyed constantly, ultimately result in hypoinsulinism, show as hyperglycaemia
Disease.The factors such as heredity, environment and autoimmunity are all easily caused T1DM generation.Wherein, environmental factor is probably that main T1D is caused
Cause of disease element, but its related mechanism is not yet clear and definite so far.Mankind T1DM pathogenic processes and the mould of metabolic characteristics are preferably simulated in manufacture
Type, Mechanism Study and glycometabolism correlative study tool to diabetes are of great significance.Streptozotocin
(Streptozotocin, STZ) is the medicine of a kind of classical manufacture rat and diabetes mice model, and it can destroy pancreas islet β
Cell, make hypoinsulinism.At present using quite varied.
So far, T1D standard regimens are external source insulin injections, control blood sugar level.Although the therapeutic modality energy
Glycometabolism is effectively improved, improves life in patients, but often induces hypoglycemia and various complicated complication, such as big blood
Pipe patch, PVR and kidney damage etc..So long-term external source insulin injection, can not obtain good intervention and
Therapeutic effect.Therefore, find a kind of beta Cell of islet that effectively suppresses to be damaged, the new method for reaching treatment T1D is most important.
Lentinan (Lentinan, LNT) is a kind of macromolecular polysaccharide polymer extracted from mushroom fruiting body.Research
Show, there is lentinan regulation immunologic function, anti-platelet aggregation, antitumor, protect liver, antiviral and stimulation interferon to be formed
Etc. function, it has also become the focus of research.But the research for lentinan at present is still concentrated mainly on antitumor and raising body
Be immunized etc., the impaired research of effectively suppression beta Cell of islet anti-oxidant to lentinan are rarely reported.
The content of the invention
Therefore, an object of the present invention is a kind of new application for providing lentinan, specifically the mushroom
Purposes in the polysaccharide medicine impaired as beta Cell of islet is suppressed.
Another object of the present invention is to inquire into the possibility molecular mechanism that environmental factor causes T1DM to occur, there is provided Yi Zhongtong
Cross novel drugs of the anti-oxidation protection beta Cell of islet from damage.
The purpose of the present invention can be achieved through the following technical solutions：
Lentinan is preparing the purposes in suppressing the impaired medicine of beta Cell of islet.
Lentinan is being prepared by anti-oxidation protection beta Cell of islet from the purposes in the medicine of damage.
It is a kind of to suppress the impaired pharmaceutical composition of beta Cell of islet, contain lentinan as active ingredient, also contain pharmacy
The auxiliary material of upper permission.
A kind of medicine or health products by anti-oxidation protection beta Cell of islet from damage, contain lentinan.
The beneficial effects of the present invention are：The invention provides a kind of lentinan to suppress the impaired medicine of beta Cell of islet
Purposes in thing.The present invention observes lentinan by the model being damaged with STZ induced rat insulinoma cells INS-1
Pass through the beta Cell of islet INS-1 of the anti-oxidant mitigation STZ inductions effects damaged and possible molecular mechanism.As a result show, for
The INS-1 cellular damages of STZ inductions, lentinan can improve the apoptosis and intracellular reactive oxygen species generation (reactive of INS-1 cells
Oxygen species, ROS) accumulation, iNOS (nitric oxide synthase type) expression and nitric oxide (NO) release are reduced,
(mitogen original activated protein swashs JNK (c-Jun N terminal kinases) and p38MAPK caused by dose dependent mitigation STZ
Enzyme, mitogen-activatedproteinkinases) signal path activation.In addition, lentinan can also pass through blocking
NF- κ B paths and the insulin secretion obstacle for strengthening Pdx-1 (the homologous frame factor -1 of pancreas duodenum) expression improvement STZ inductions.
It can be obtained further by following detailed description of the invention and institute's accompanying drawings on the advantages and spirit of the present invention
Solution.
Brief description of the drawings
It is respectively the schematic diagram of influences of the STZ and LNT to INS-1 cell viabilities shown in Figure 1A and Figure 1B.
Fig. 2A and Fig. 2 B show the schematic diagram of the influence for the INS-1 Apoptosis that LNT is induced STZ.
Fig. 3 A and Fig. 3 B show the schematic diagram of the influence for the INS-1 Apoptosis that LNT is induced STZ.
Fig. 4 A and Fig. 4 B show the INS-1 intracellulars ROS accumulations that LNT induced STZ and NO synthesizes showing for increased influence
It is intended to.
Fig. 5 A and Fig. 5 B show the schematic diagram for the influence that LNT induces STZ INS-1 cells iNOS expression to rise.
Fig. 6 A- Fig. 6 D show the influence that LNT induces INS-1 cell JNK and p38MAPK signal path to activate STZ
Schematic diagram.
Fig. 7 A- Fig. 7 C show schematic diagrames of the LNT to the influence of INS-1 insulin synthesis obstacle caused by STZ.
Fig. 8 A- Fig. 8 D show the influence of NF- κ B and Pdx-1 protein expressions in the INS-1 cells that LNT is handled STZ
Schematic diagram.
Embodiment
The present invention provides a kind of lentinan and is preparing the purposes in suppressing the impaired medicine of beta Cell of islet.
Wherein, the lentinan that the present invention uses, purity can commercially buy, can also make by oneself, preferably up to 98%
Ground, the lentinan are commercially available in Jiangsu Yong Jian Pharmaceutical Technology Co., Ltd.The molecular formula of lentinan is
(C42H70O35) n, molecular weight 1152.999, structural formula are：
To better illustrate the object, technical solutions and advantages of the present invention, the present invention is made by the following examples into one
The elaboration of step.
The influence for the INS-1 Apoptosis that the lentinan of embodiment 1 is induced STZ
Experimental cell：Rat insulin oncocyte INS-1 be incubated at containing 10% hyclone (fetal calf serum,
Abbreviation FBS), 11.1mM glucose, 50 μM of beta -mercaptoethanols, 100U/ml penicillin, 100 μ g/ml streptomysins, 10mM HEPES
In the RPMI-1640 nutrient solutions of (4- hydroxyethyl piperazineethanesulfonic acids), 1mM Sodium Pyruvates.Cell uses the culture dish of different size
Or culture plate is incubated at 5%CO2, in 37 DEG C of constant humidity incubator.
Experimental drug：Lentinan is purchased from Jiangsu Yong Jian Pharmaceutical Technology Co., Ltd.
Experimental procedure：
1) STZ and LNT solution is configured.STZ is dissolved in citrate buffer solution.The preparation of citrate buffer solution：Citric acid
(molecular weight (FW)：210.14) 2.1g is added in distilled water 100mL and is made into A liquid；Sodium citrate (FW：294.10) 2.94g additions
B liquid is made into distilled water 100mL.A, B liquid are mixed (1 by the used time by a certain percentage:1.32), PH meters measure pH value, adjusts PH
4.2-4.5 it is STZ citrate buffer solution.LNT is dissolved in sterile PBS.The STZ and LNT equal matching while using of drug solution,
Lucifuge operates.
2) INS-1 cells are handled.After INS-1 plating cells when its adherent growth density is 70% or so, serum is removed, is used
Containing 0.1%BSA (bovine serum albumin(BSA)), 11.1mM glucose, 50 μM of beta -mercaptoethanols, 100U/ml penicillin, 100 μ g/ml chains
Mycin, 10mM HEPES, STZ or/and LNT the processing cell of the RPMI-1640 nutrient solutions of 1mM Sodium Pyruvates and various concentrations,
Coherent detection is carried out after 24h.The experimental group being jointly processed by through STZ and LNT is incubated with the LNT of concentration containing alignment processing nutrient solution in advance
30min is educated, then dosing stimulates 24h again.All cell experiments for carrying out agent-feeding treatment are respectively provided with solvent control group, molten to exclude
Agent itself influences for the possibility of cell state and experimental result, and ensures that the ultimate density of solvent is not more than 0.1%.
3) MTT detects cell viability.Concrete operation step is as follows：Cell is inoculated in 96 orifice plates, is completed through drug-treated
Afterwards, per hole, (MTT is dissolved in sterile PBS (phosphate buffered saline solution, phosphate buffer saline) to addition MTT 10 μ l
In, concentration is 0.5% (m/v)；96 orifice plates are placed in 37 DEG C of incubators and react 4h；Exhausted and cultivated with syringe after the completion of reaction
Liquid, DMSO (dimethyl sulfoxide (DMSO)) 100 μ l dissolving purple crystal first a ceremonial jade-ladle, used in libations are added per hole.10min is reacted in 37 DEG C of incubators；With enzyme mark
Instrument reads absorbance at 570nm wavelength, and vigor of the cell after drug-treated and the absorbance are proportional.
4) the double dye method detection Apoptosis of Annexin V-PI.About 2 × 105Individual/ml cell is inoculated in 60mm culture
In ware, 37 DEG C of overnight incubations are so that its is adherent, and handling cell 24h, PBS with medicine (STZ or/and LNT) washes cell 2 times, 1000g
(acceleration of gravity) centrifuges 5min, abandons supernatant, and cell is resuspended in the Annexin V-FITC combinations liquid for adding 400 μ l, adds 5 μ l's
Annexin V-FITC, are gently mixed, and after 4-8 DEG C of lucifuge is incubated 15min, PI (propidium iodide) lucifuge for adding 10 μ l is incubated
Flow cytomery apoptosis rate is used in 5min, 1h.
5) TUNEL fluoroscopic examinations Apoptosis.Abandon old nutrient solution, PBS washings cell 1-2 times.Consolidated with 4% paraformaldehyde
Determine cell, 25min is fixed under the conditions of 4 DEG C；PBS washes cell 2-3 times, and 1 is pressed with PBS:100 dilution proportion Proteinase K, 100 μ
L/ holes, washed 1-3 times with PBS after being incubated at room temperature 5min；Mark：1 is pressed with deionized water:5 dilution proportion 5 ×
Equilibration Buffer, 100 μ l/ holes, it is incubated at room temperature 10-30min；Thawed on ice while marking and being incubated
BrightGreen Labeling Mix, prepare TdT (terminal enzyme (DNA), terminal deoxynucleotidyl
Transferase) incubation buffer；TdT incubation buffer incubated cells are added, 50-100 μ l/ holes, 37 DEG C of lucifuges are incubated in wet box
Educate 60min；Surplus liquid is discarded, PBS is washed 1-3 times；Cell dyeing 5min, PBS are washed 1-3 times with Hochest；By cover glass
It is placed on slide, fluorescence microscope is just being put with Zeiss and is being taken pictures.
Statistical analysis：All data of this experiment represent that group difference analysis is adopted using means standard deviation (± s)
Examined with t, with P<0.05 is used as significant difference normative reference.
Experimental result：
1) what as shown in Figure 1A, Figure 1A was shown is：MTT experiment is carried out after the STZ processing INS-1 cells 24h of various concentrations,
The cell viability of detection；As can be seen that when STZ working concentrations are 0.5mM, there is vigor decline, i.e. 0.5mM in INS-1 cells
STZ can cause appropriate INS-1 cellular damages, therefore we will carry out subsequent experimental using 0.5mM STZ.Referring back to figure
What 1B, Figure 1B were shown is：MTT experiment, the cell viability of detection are carried out after the LNT processing INS-1 cells 24h of various concentrations；Can
To find out, cell viability only can not be influenceed with LNT processing INS-1 cells, this prompting LNT does not have obvious cell to INS-1
Toxicity.(*P<0.05, * * P<0.01, * * * P<0.001)
2) importantly, such as Fig. 2A-Fig. 3 B, when LNT concentration reaches 200 μ g/ml and 400 μ g/ml, can substantially mitigate
The INS-1 Apoptosis of 0.5mM STZ inductions.Wherein Fig. 2A represent be：LNT pre-processes INS-1 cell 30min, then uses
The 0.5mM STZ and LNT of various concentrations is jointly processed by cell 24h, and blank control group LNT and STZ are not added with, Annexin V-
Flow cytometer detection each group Apoptosis situation after the double dyes of PI；Fig. 2 B are then the statistical conditions to apoptosis rate in Fig. 2A.Fig. 3 A tables
What is shown is：INS-1 cellular processes are with described in Fig. 2A, and TUNEL decoration methods detect Apoptosis situation, using laser co-focusing
Micro- sem observation cell (400 ×), wherein blue-fluorescence are Hoechst, and green fluorescence is TUNEL positive；Fig. 3 B are then to figure
The statistics of TUNEL positive cell percentages in 3A.
From above experimental result can be seen that the INS-1 Apoptosis that lentinan of the present invention induces for STZ
To certain protective effect.(**P<0.01, #P<0.05)
INS-1 intracellulars ROS accumulations, NO synthesis increase and the iNOS (induction types one that the lentinan of embodiment 2 is induced STZ
Nitric oxide synthase) express the influence risen
In order to explore the effect of LNT possibility, we have studied LNT in inducing INS-1 cellular damage processes in STZ to oxidation
Stress signal path correlation molecule influence.
1) intracellular reactive oxygen species generation detects
Cell presses 1-2 × 104Individual/hole is inoculated in 96 orifice plates of the black wall of white background, and culture is to after growing stabilization, at medicine
After managing 24h, operating procedure is as follows：Culture supernatant is discarded, operation needs soft with PBS cell twice；100 μ l are added per hole
Working solution, 1-2h is incubated in incubator；Reaction working solution is discarded, again PBS cell, fluorescence microplate reader is in wavelength Ex/
Reading at Em=520/605nm.As a result see Fig. 4 A- Fig. 4 B, Fig. 4 A represent be：LNT pre-processes INS-1 cell 30min, then uses
The 0.5mM STZ and LNT of various concentrations is jointly processed by cell 24h, and blank control group LNT and STZ are not added with, intracellular reactive oxygen species generation
Detection kit (red fluorescence) detection INS-1 intracellulars ROS is horizontal, is read with fluorescence microplate reader in wavelength Ex/Em=520/605
Number, takes bottom reading mode；Fig. 4 B represent be：INS-1 cellular processes are horizontal with Fig. 4 A, Griess methods detection NO.
As seen from the figure, compared with control group, STZ processing adds INS-1 intracellulars ROS levels.Meanwhile LNT energy dose dependents
Alleviate ROS increases (Fig. 4 A) caused by STZ.(*P<0.05, #P<0.05)
2) the horizontal detections of NO
INS-1 cells 2 × 104Individual/hole is inoculated in 48 orifice plates, and after agent-feeding treatment 24h, operating procedure is as follows：Collect training
Nutrient solution, 1500g (acceleration of gravity) take supernatant after being centrifuged off cell fragment；Each sample is operated with Griess methods and makes standard
Curve；ELIASA reading at wavelength 540nm；NO contents in each foster supernatant of processing tissue culture are calculated according to standard curve.As a result
See Fig. 4 B, the horizontal up-regulations of NO of STZ inductions can also be suppressed by LNT.
3) Western blot detect iNOS expression
With the INS-1 total protein of cell after the kit extraction drug-treated 24 hours of extraction total protein, with Western
Western blotting method, detect the expression of iNOS albumen.With GAPDH (glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3-
Phosphate dehydrogenase) expression to ensure the protein of identical applied sample amount.As a result Fig. 5 A and Fig. 5 B are seen,
Fig. 5 A represent be：For INS-1 cellular processes with described in Fig. 4 A, Western Blot detect each treatment group INS-1 cells
INOS protein expression situations；Fig. 5 B represent be：The change of other groups of iNOS protein expressions is analyzed on the basis of the independent treatment groups of STZ
Change multiple.Compared with blank control group, 0.5mM STZ clearly enhances the expression of INS-1 cell iNOS albumen, 200 μ g/ml
It can effectively suppress iNOS expression increase caused by STZ with 400 μ g/ml LNT.(*P<0.05, #P<0.05)
This part of test results illustrates that lentinan of the present invention can be by reducing ROS accumulations, NO releases and iNOS tables
Stress up to INS-1 cellular oxidations caused by improvement STZ.
The lentinan of embodiment 3 induces STZ the influence of INS-1 cell JNK and p38MAPK signal path activation
According to STZ, LNT in embodiment 2 on INS-1 cellular oxidations stress correlation molecule influence result, we are following
It has detected the Activation of each treatment group JNK, p38MAPK signal path.
Western blot detects JNK and p38MAPK phosphorylation, and specific steps are shown in the Western described in embodiment 2
Blot methods.As a result see Fig. 6 A-6D, Fig. 6 A represent be：LNT pre-processes INS-1 cell 30min, then with 0.5mM STZ and
The LNT of various concentrations is jointly processed by cell 24h, and blank control group LNT and STZ are not added with, Western Blot detection phosphorylations
JNK protein expression situations；Fig. 6 B are then the levels of other groups of phosphorylation JNK albumen of statistical analysis on the basis of the independent treatment groups of STZ；
Fig. 6 C represent be：LNT pre-processes INS-1 cell 30min, then is jointly processed by carefully with 0.5mM STZ and the LNT of various concentrations
Born of the same parents 24h, blank control group LNT and STZ are not added with, phosphorylated p38 protein expression situation；Fig. 6 D are then that the independent treatment groups of STZ are
Benchmark survey analyzes the level of other groups of phosphorylated p38 albumen.In INS-1 cells, STZ processing causes JNK and p38MAPK
Phosphorylation level increase, and LNT processing can dose dependent reductions STZ induce JNK (Fig. 6 A and 6B) and p38MAPK
The phosphorylation of (Fig. 6 C and 6D).(**P<0.01, #P<0.05, ##P<0.01)
This partial results illustrates, lentinan of the present invention by suppressing the activation of JNK and p38MAPK signal paths,
Improve the INS-1 Apoptosis and oxidative stress status of STZ inductions.
Influence of the lentinan of embodiment 4 to INS-1 insulin synthesis obstacle caused by STZ
In order to further study the INS-1 insulin synthesis function effects that LNT is handled STZ, we are (real using RT-PCR
When fluorescent quantitation, Real-time PCR) method detection Insulin 1 and Insulin 2 mRNA (messenger RNA) water
It is flat, and determine each group cell insulin synthesis situation.
1) real-time fluorescence quantitative RT-PCR
The GAPDH of INS-1 cells, insulin 1 and insulin 2 mRNA level in-site SYBR GREEN dye methods,
The type real-time PCRs of Roche Light 480 II carry out relative quantification detection, and data are analyzed with 2- Δ Δ Ct methods.As a result such as
Shown in Fig. 7 A- Fig. 7 C, what Fig. 7 A were represented is：LNT pre-processes INS-1 cell 30min, then with 0.5mM STZ and various concentrations
LNT is jointly processed by cell 24h, and blank control group LNT and STZ are not added with, and Real-time PCR detect Insulin 1 mRNA；
Fig. 7 B represent be：LNT pre-processes INS-1 cell 30min, then is jointly processed by carefully with 0.5mM STZ and the LNT of various concentrations
Born of the same parents 24h, blank control group LNT and STZ are not added with, and Real-time PCR detect Insulin 2 mRNA.Fig. 7 C represent be：
For INS-1 cellular processes with described in Fig. 7 A, sour ethanol extracted overnight INS-1 intracellular insulin is each with measured by radioimmunoassay
Experimental group intracellular insulin content, and corrected with albumen.STZ can suppress turning for INS-1 cells Insulin 1 and Insulin 2
Record is horizontal.Compared with the independent treatment groups of STZ, when LNT concentration reaches 200 μ g/ml, Insulin 1 (Fig. 7 A) and Insulin 2
The mRNA level in-site of (Fig. 7 B) is obviously improved.(*P<0.05, #P<0.05)
2) insulin inside cells extracting experiment
INS-1 cells are inoculated in 48 orifice plates, and after agent-feeding treatment 24h, operating procedure is as follows：Cell abandons culture supernatant, PBS
Rinsing once, adds 200 μ l sugar-frees and the KRB buffer solutions (Krebs- containing each treatment group corresponding concentration medicine (STZ and LNT)
Ringer bicarbonate HEPES buffer, HEPES containing 10mmol/l, 0.5mmol/l NaH2PO4, 3.6mmol/l
KCl, 135mmol/l NaCl, 2mmol/l NaHCO3, 1.5mmol/l CaCl2, 0.5mmol/l MgSO4, 0.1%BSA.pH
=7.4) 1h, is cultivated；Cell per well adds 200 μ l acid ethanolic extraction liquid (acid-ethanol solution：74%ethanol,
1.4%HCl), after being incubated 12h or so under the conditions of 4 DEG C, supernatant is taken in 2ml EP pipes.Sample can be frozen in -80 DEG C of ultralow temperature ices
It is to be measured in case；Put with insulin and exempt from detection kit, put the method for exempting from (radio immuno assay, RIA) detection insulin level.
As a result as seen in figure 7 c, LNT can improve INS-1 cells insulin synthetic quantity caused by STZ and decline.(***P<0.001, #P<
0.05)
Result above shows that lentinan of the present invention can improve INS-1 cells insulin synthesis caused by STZ and hinder
Hinder.
The influence that the lentinan of embodiment 5 is expressed NF- κ B caused by STZ and Pdx-1
Improve the mechanism of STZ induction INS-1 cell function damages to further explore LNT, we use Diagnosis of Sghistosomiasis
Mark method (Western Blot) have detected the expression of core NF- κ B and intracellular Pdx-1 albumen.
Western blot detects the expression of core NF- κ B and intracellular Pdx-1 albumen, and specific steps are shown in described in embodiment 2
Western blot methods.As a result see Fig. 8 A- Fig. 8 D, Fig. 8 A represent be：LNT pre-processes INS-1 cell 30min, then uses
The 0.5mM STZ and LNT of various concentrations is jointly processed by cell 24h, and blank control group LNT and STZ are not added with, Western
Blot detection NF- κ B p65's enters core amount；Fig. 8 B are then the situations for carrying out statistical analysis to Fig. 8 A using Histone as internal reference.Figure
8C represent be：INS-1 cellular processes carry out Western Blot experiments, detect PDX-1's with Fig. 8 A, extraction total protein
Expression；Fig. 8 D are then to carry out statistical analysis situation to Fig. 8 C using GAPDH as internal reference.
STZ individually handles the expression for enhancing INS-1 nucleus NF- κ B.Compared with the independent treatment groups of STZ, LNT's is common
Processing can reduce the expression (Fig. 8 A and 8B) of core NF- kB proteins；Also, compared with control group, STZ can lower Pdx-1 albumen
Level, Pdx-1 protein level declines (Fig. 8 C and 8D) caused by the LNT reversible STZ of processing.(***P<0.01, #P<
0.05)
This partial results illustrates that LNT can strengthen Pdx-1 protein expressions, improve STZ and cause by suppressing core NF- kB activations
β cell dysfunctions.
To sum up, it is more to observe mushroom by the model being damaged with STZ induced rat insulinoma cells INS-1 by the present invention
Sugar passes through the beta Cell of islet INS-1 of the anti-oxidant mitigation STZ inductions effects damaged and possible molecular mechanism.As a result show, it is right
In the INS-1 cellular damages of STZ inductions, lentinan can improve apoptosis and the intracellular reactive oxygen species generation accumulation of INS-1 cells, reduce
INOS expression and nitric oxide (NO) release, dose dependent mitigate the activation of JNK and p38MAPK paths caused by STZ.
In addition, lentinan can also be improved by blocking NF- κ B paths and strengthening Pdx-1 (the homologous frame factor -1 of pancreas duodenum) expression
The insulin secretion obstacle of STZ inductions.
By the above detailed description of preferred embodiments, it is intended to more clearly describe the feature of the present invention with spiritual,
And not protection scope of the present invention is any limitation as with above-mentioned disclosed preferred embodiment.On the contrary, its purpose
It is intended to cover various changes and has being arranged in the scope of the claims of the invention to be applied of equality.Cause
This, the apllied scope of the claims of the present invention should make most broad explanation according to above-mentioned explanation, to cause it
Cover all possible change and the arrangement of tool equality.
Claims (2)
1. lentinan is preparing the purposes in suppressing the impaired medicine of beta Cell of islet, it is characterised in that prepared by lentinan
Purposes in the medicine damaged by the beta Cell of islet INS-1 of anti-oxidant mitigation STZ inductions.
2. purposes as claimed in claim 1, it is characterised in that lentinan improves the INS-1 insulin of STZ inductions preparing
Purposes in the medicine of dyssynthesis.
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| 1型糖尿病患者淋巴细胞对胰岛β细胞的毒性作用及药物干预研究;梅焕平等;《山东免疫学会、山东微生物学会医学微生物学专业委员会、山东省医学会微生物学和免疫学专业委员会、山东省医药生物技术学会2001年学术年会论文汇编》;20011201;第83页"结论"项下 * |
| 菌类中药多糖降血糖效应机制研究进展;代荣等;《中国中药杂志》;20150115;第40卷(第2期);第174-179页 * |
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