CN110327360A - Application and its experimental method of the baicalin in preparation treatment silicosis drug - Google Patents
Application and its experimental method of the baicalin in preparation treatment silicosis drug Download PDFInfo
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Abstract
The invention discloses application and its experimental method of the baicalin in preparation treatment silicosis drug, belong to silicosis treatment technology field；Baicalin treats the application in silicosis drug in preparation, contains baicalin component in the silicosis drug；The experimental method that baicalin is applied in preparation treatment silicosis drug, specifically includes the following steps: experimental animal selection and grouping, the foundation of Silicotic Rats pulmonary fibrosis model, the collection of BALF supernatant, pathologic state colony inspection, lung fibrosis evaluation, measure IL-1, IL-6, the expression of TNF-α inflammatory factor, TLR4, NF- κ B measurement, baicalin is in the application in preparation treatment silicosis drug in the present invention, baicalin can effectively inhibit the speed of silicotic fibrosis, and it can actively find the novel targets of lung fibrosis therapeutic effect, scutelloside is formed with antagonistic effect to silicosis pulmonary fibrosis, and then it can effectively apply in the application of silicosis therapeutic agent.
Description
Technical field
The answering in preparation treatment silicosis drug the present invention relates to silicosis therapeutic agent technical field more particularly to baicalin
With and its experimental method.
Background technique
Silicosis is fine with lung tissue Chronic persistent inflammation and progressive caused by referring to after Long Term Contact silica dust
Dimension turns to the systemic disease of main pathological characteristic, and basic pathology change procedure is pulmonary alveolitis, Granuloma in lung, pulmonary fibrosis；
Currently, silicosis is that China's disease incidence is higher, endanger one of occupational disease of most serious, clinic there is no special effect medicine therapeutic, mainly with
It is main means that symptomatic treatment, which alleviates the state of an illness, the main reason is that recognizing deficiency to pneumoconiosis mechanism, makes to develop and develop medicine
Object is restricted.
Pathogenesis of Silicosis is sufficiently complex, so far not yet clarification completely, and pulmonary alveolar macrophage mediated intrinsic in recent years
Immunologic mechanism becomes the hot spot of current Pathogenesis of Silicosis research；Imbedibility SiO2 grit form is very similar to pathogen correlation point
Subpattern or dangerous associated molecular pattern, silicosis pathogenic process early stage, AMs can identify SiO2 by pattern recognition receptors
The AMs of grain, activation generates phagocytosis；During swallowing SiO2, activated by intracellular signaling pathways such as ROS, ATP
NLRP3 inflammatory body to mediate the cell coke of pulmonary alveolar macrophage to die by activating NLRP3 inflammatory body intracellular, and then discharges
The proinflammatory factors such as IL-1 β, IL-6, prostaglandin induce alveolar inflammation.
Summary of the invention
The purpose of the present invention is to solve the problems of the prior art, and the baicalin proposed treats silicosis medicine in preparation
Application and its experimental method in object.
To achieve the goals above, present invention employs following technical solutions:
Baicalin treats the application in silicosis drug in preparation, contains baicalin component in the silicosis drug.
The experimental method that baicalin is applied in preparation treatment silicosis drug, specifically includes following experimental procedure: dynamic in vivo
Object experiment:
1., using disposable intratracheal perfusion tested material SiO2 is filled into Wister male rat, establish silicosis lung
Fibrosis animal model；
2., set up be added baicalin treatment group a, be added baicalin treatment group b, be added baicalin treatment group c, the Chinese
Root of fangji A prime positive controls and normal negative control group；
3., from whole, tissue, cell factor and molecular level observation scutelloside to the SiO2 Pulmonary Fibrosis in Rats induced
Antagonistic effect and mechanism of action.
Preferably, in step A interior animal experiment specifically includes the following steps:
S1, experimental animal selection and grouping: selection Wistar male rat 200, according to step 2. in the model set up
200 rats are randomly divided into 5 groups, every group 40 by group；
S2, Silicotic rat preparation: with ether by rat anesthesia, direct oral cavity exposure tracheostomy takes lmlSiO2 dust
Suspension is slowly injected into tracheae, is injected in hanfangchin A positive controls and normal negative control group using same method
1ml sterile saline；
The collection of S3, BALF supernatant: after rat is put to death, 2.5ml, 37 DEG C of sterile salines are slowly injected into lung in body
Interior, interval was drawn back, is carried out repeatedly after 30 seconds, until being collected into BAL fluid about 14-16ml, will fill
Washing lotion is centrifuged 10min with 4 DEG C, 1500rpm, extracts supernatant and saves backup under -80 DEG C of environment；
S4, pathologic state colony inspection: taking left lung, fix through formalin, paraffin embedding, and slice does HE dyeing, in light
The variation of microscopic observation pathologic；
S5, lung fibrosis evaluation；
S6, the expression for measuring IL-1, IL-6, TNF-α inflammatory factor: measurement each group lung tissue of rats, BALF and serum IL-
1, the expression of IL-6, TNF-α inflammatory factor；
S7, TLR4, NF- κ B measurement: measurement lung tissue, BALF and serum T LR4, NF- kB protein expression, RtPCR method
Detect TLR4, NF- κ BmRNA expression.
Preferably, interior animal experiment further includes administration in step A:
The daily oral gastric infusion for the treatment of group a, treatment group b, the animal in treatment group c is primary, Silicotic model group and just
Normal negative control group gives the physiological saline of isometric(al).
Preferably, baicalin dosage is 40-60mg/kg in step ②Zhong treatment group a, baicalin dosage is in treatment group b
Baicalin dosage is 10-15mg/kg in 20-30mg/kg, treatment group c.
Preferably, in step s5 lung fibrosis evaluation the following steps are included:
Lung tissue Masson dyeing, observes lung fibrosis lesion degree；
Lung tissue hydroxyproline content is measured, judges collagen aggregate level.
Preferably, each group lung tissue of rats, BALF and serum IL -1, IL-6, TNF-α inflammatory factor are measured in step s6
The measuring method of expression is ELISA method.
Preferably, lung tissue, BALF and serum T LR4, NF- kB protein expression, the inspection of RtPCR method are measured in step s7
Survey TLR4, the measuring method of NF- κ BmRNA expression is Western Blot method.
Compared with prior art, the present invention provides baicalin treats application and its experiment side in silicosis drug in preparation
Method, have it is following the utility model has the advantages that
1, the experimental method of the treatment silicosis drug in vivo during zoopery, selects Wistar male rat 200
Only, 5 groups, and respectively Silicotic model group, Normal group, hanfangchin A positive controls, scutelloside are randomly divided into
Large dosage, middle dosage and low dose for the treatment of group, every group 40；After grouping, with ether by rat anesthesia, direct oral cavity exposure tracheae is opened
Mouthful, take lmlSiO2 dust suspension to be slowly injected into tracheae, control group injects 1ml sterile saline using same method；Place
After the completion of reason, primary to each oral gastric infusion for the treatment of group animal daily, Silicotic model group and normal negative control group are given
The physiological saline of volume；After rats death, 2.5ml37 DEG C of sterile saline is slowly injected into intrapulmonary in body, is spaced 30 seconds
Afterwards, drawn back, carried out repeatedly, until be collected into BAL fluid about 15ml, by irrigating solution with 4 DEG C,
1500rpm is centrifuged 10min, extracts -80 DEG C of supernatant and saves backup；After treatment, left lung is taken, is fixed through formalin, paraffin
Embedding, slice, does HE dyeing, changes in light microscopic observation pathologic, and lung tissue is observed in observation lung tissue Masson dyeing
Fibrosis lesion degree；Lung tissue hydroxyproline content is measured, judges collagen aggregate level；It is big using ELISA method measurement each group
Mouse lung tissue, BALF and serum IL -1, IL-6, TNF-α inflammatory factor expression, using Western Blot method measure lung
Tissue, BALF and serum T LR4, NF- kB protein expression, RtPCR method detect TLR4, NF- κ BmRNA expression, measurement
Data are recorded after the completion, and different time points each group mouse pulmonary fibrosis changes situation after observation scutelloside administration；Scutelloside administration
Afterwards different time points each group lung tissue of rats, BALF and serum IL -1, IL-6, TNF-α inflammatory factor expression；Scutelloside
Different time points each group lung tissue of rats, BALF and serum T LR4, NF- kB protein expression after administration, the detection of RtPCR method
TLR4, NF- κ BmRNA expression.
2, the experimental method of the treatment silicosis drug in vitro during cell culture experiments, chooses I phase, II phase, III phase
Silicosis male patient each 6, bronchoalveolar lavage is carried out to the silicotic of selection: firstly, in the lung section for wanting lavation through work
It examines hole and 2% lidocaine 1.5ml is injected by a thin silicone tube；Section or sub- segmental bronchus opening are closely wedged into Bronchofiberscope top
Place, then 37 DEG C of sterile salines, each 35ml, total amount 175ml are rapidly injected by silicone tube through biopsy hole；It uses immediately
BALF is recycled in 80mmHg vacuum suction；Withdrawal liquid is used double-layer sterile filtered through gauze remove mucus immediately, and records total amount；?
After lavation, irrigating solution is collected, after filtering using 4 layers of sterile gauze, filtrate is taken to be centrifuged 15min through 1500r/min, PBS washing adds
Enter 10%FBS-DMEM culture solution, macrophage is routinely counted after mixing, cell survival rate is calculated after Trypan Blue, with containing
10%FBS-DMEM culture solution diluting cells are the cell suspension of 1 × 106/ml, are placed in culture bottle, adherent to cell, are led to
Microscopic observation form, conventional H E dyeing, acid-fast stain and Wright's staining is crossed to identify it；Macrophage-conditioned media is taken, is added
Enter to be free of the culture solution of serum, macrophage is divided into 2 groups: the suspension of the dust containing SiO2, final concentration of 50 μ is added in experimental group
g/ml；The culture medium without serum is only added in control group；Two groups, at 37 DEG C, are cultivated under the conditions of 5%CO2, and in culture
Culture supernatant in different time periods, detection supernatant inflammatory factor IL-1, IL-6, TNF-α and cell TLR4, NF- κ are drawn respectively
The expression of B albumen and mRNA selects the culture supernatant of the highest time point of expression, and -80 DEG C save backup, and silicious dust macrophage is thin
Born of the same parents culture supernatant night work is that culture substrate is added in fibroblast, establishes silicious dust co-culture model；Cell is divided into dye dirt
Group, TLR4/NF- kB inhibitor group inhibitor group, scutelloside group；Culture medium containing 10%FBS-DMEM is added, in 37 DEG C, 5%
It is cultivated under conditions of CO2；Every group sets 15 parallel holes, and each group is cultivated for 24 hours, after 48h, 72h, culture solution is sucked out, through filter membrane respectively
Filtering, -80 DEG C of preservations, it is spare to collect cell；Group of cells culture for 24 hours, after 48h, 72h, collect cell, surveyed using mtt assay
Determine cell activation situation, cell generation cycle is measured using flow cytometer, Annexin V/PI double-staining measurement cell withers
It dies；Use immunocytochemical method measurement group of cells I, the expression of type III collagen；The supernatant of -80 DEG C of preservations is taken, is used
ELISA method measures the horizontal measurement of IL-1, IL-6 in supernatant, TNF-α 1；Group of cells culture for 24 hours, after 48h, 72h, collect
Cell detects protein expression level using Western Blot method；Group of cells culture for 24 hours, after 48h, 72h, collect cell,
MRNA expression is detected using Real time PCR method.
Changed by different time points each group mouse pulmonary fibrosis after observing scutelloside administration in above-mentioned interior animal experiment
Become situation, observe different time points each group lung tissue of rats, BALF and serum IL -1, IL-6, TNF-α inflammatory after scutelloside administration
Different time points each group lung tissue of rats, BALF and serum T LR4, NF- κ B after expression, the observation scutelloside of the factor are administered
Protein expression level, RtPCR method detect TLR4, NF- κ BmRNA expression；
By in above-mentioned cell culture experiments in vitro observe the culture of each group macrophage for 24 hours, tri- groups of differences of 48h, 72h when
Between section supernatant IL-1, IL-6, TNF-α inflammatory factor expression, cell TLR4, NF- kB protein mRNA expression, observation
Each group Fibroblast cell-culture for 24 hours, tri- groups of 48h, 72h activation, proliferation and apoptosis situations in different time periods, observation each composition fibre
Tie up cell culture for 24 hours, tri- groups of 48h, 72h supernatant hydroxyproline contents in different time periods, observation each group fibroblast training
Support for 24 hours, after tri- groups of different time sections of 48h, 72h the case where I, type III collagen expression, observation each group Fibroblast cell-culture for 24 hours,
The expression of tri- groups of 48h, 72h supernatant IL-1, IL-6, TNF-α inflammatory factor in different time periods, cell TLR4, NF- kB protein
And mRNA expression.
Detailed description of the invention
Fig. 1 is the Technology Roadmap of interior animal experiment in the present invention.
Specific embodiment
Below in conjunction in the embodiment of the present invention, technical solution in the embodiment of the present invention is clearly and completely retouched
It states, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.
Embodiment 1:
Baicalin treats the application in silicosis drug in preparation, contains baicalin component in the silicosis drug.
The experimental method that baicalin is applied in preparation treatment silicosis drug, specifically includes following experimental procedure:
A, interior animal experiment:
Tested material SiO2 is filled into Wister male rat using disposable intratracheal perfusion, establishes silicosis lung fiber
Change animal model；
If the treatment group of three groups of various doses, hanfangchin A positive controls and normal negative control group；
From whole, tissue, cell factor and molecular level observation scutelloside to the short of money of the SiO2 Pulmonary Fibrosis in Rats induced
Antiatherosclerotic effect and mechanism of action；
B, cell culture experiments in vitro:
Silicious dust co-culture model is established, following model group is broadly divided into:
Model group；
TLR4/NF- kB inhibitor group；
Scutelloside intervention group；
The influence that scutelloside generates fibroblast activation, proliferation, apoptosis and fibrosis is observed, measurement each group macrophage is thin
The table of born of the same parents and Fibroblast cell-culture supernatant inflammatory factor IL-1, IL-6, TNF-α and cell TLR4, NF- kB protein and mRNA
It reaches.
Interior animal experiment in step A specifically includes the following steps:
S1, experimental animal selection and grouping: selection Wistar male rat 200 is randomly divided into 5 groups, respectively silicosis
Model group, Normal group, hanfangchin A positive controls, scutelloside large dosage, middle dosage and low dose for the treatment of group, every group
40；
S2, Silicotic rat preparation: with ether by rat anesthesia, direct oral cavity exposure tracheostomy takes lmlSiO2 dust
Suspension is slowly injected into tracheae, and control group injects 1ml sterile saline using same method；
The collection of S3, BALF supernatant: after rat is put to death, 2.5ml37 DEG C of sterile saline is slowly injected into lung in body
Interior, interval was drawn back, is carried out repeatedly after 30 seconds, until being collected into BAL fluid about 14-16ml, will fill
Washing lotion is centrifuged 10min with 4 DEG C, 1500rpm, extracts -80 DEG C of supernatant and saves backup；
S4, pathologic state colony inspection: after rat is put to death, taking left lung, fix through formalin, paraffin embedding, and slice is done
HE dyeing changes in light microscopic observation pathologic；
S5, lung fibrosis evaluation；
The expression of S6, IL-1, IL-6, TNF-α inflammatory factor: measurement each group lung tissue of rats, BALF and serum IL -1,
The expression of IL-6, TNF-α inflammatory factor；
S7, TLR4, NF- κ B measurement: measurement lung tissue, BALF and serum T LR4, NF- kB protein expression, RtPCR method
Detect TLR4, NF- κ BmRNA expression.
Cell culture experiments in vitro in step B specifically includes the following steps:
The selection of P1, silicotic: choosing I phase, II phase, III phase silicotic each 6, be male, and the age is close, and
Carry out questionnaire survey；
P2, lavation bronchovesicular；
P3, the separation of macrophage, culture and identification: after silicotic carries out bronchoalveolar lavage, lavation is collected
Liquid, then after 4 layers of sterile gauze filter, filtrate is taken to be centrifuged 15min through 1500r/min, 10%FBS-DMEM is added in PBS washing
Culture solution routinely counts macrophage after mixing, cell survival rate is calculated after Trypan Blue, is trained with containing 10%FBS-DMEM
Nutrient solution diluting cells be 1 × 106/ml cell suspension, be placed in culture bottle, it is adherent to cell, by microscopic observation form,
Conventional H E dyeing, acid-fast stain and Wright's staining identify it；
The foundation of P4, silicious dust co-culture model: taking macrophage-conditioned media, the culture solution for being free of serum is added, by macrophage
Cell is divided into 2 groups:
The suspension of the dust containing SiO2, final concentration of 50 μ g/ml is added in experimental group；
The culture medium without serum is only added in control group；
Two groups, at 37 DEG C, are cultivated under the conditions of 5%CO2, and for 24 hours, centre is drawn respectively in culture in different time periods for culture
Clearly, the expression of supernatant inflammatory factor IL-1, IL-6, TNF-α and cell TLR4, NF- kB protein and mRNA, selection expression are detected
The culture supernatant of horizontal highest time point, -80 DEG C save backup, and silicious dust macrophage culture supernatant night work is added for culture substrate
It adds in fibroblast, establishes silicious dust co-culture model；
P5, fibroblast grouping: cell is divided into dye dirt group, TLR4/NF- kB inhibitor group inhibitor group, scutelloside
Group；Culture medium containing 10%FBS-DMEM is added, is cultivated under conditions of 37 DEG C, 5%CO2；Every group sets 15 parallel holes, each group
It cultivates respectively for 24 hours, after 48h, 72h, culture solution is sucked out, through membrane filtration, it is spare to collect cell for -80 DEG C of preservations；
P6, cell activation, proliferation and apoptosis detection: group of cells culture for 24 hours, after 48h, 72h, collect cell, using MTT
Method measures cell activation situation, measures cell generation cycle using flow cytometer, Annexin V/PI double-staining measures carefully
Born of the same parents' apoptosis；
The expression of P7, I, type III collagen: group of cells culture for 24 hours, after 48h, 72h, collect cell, measure group of cells
I, the expression of type III collagen；
The horizontal measurement of P8, supernatant IL-1, IL-6, TNF-α 1: the supernatant of -80 DEG C of preservations in p6 is taken, is surveyed with ELISA method
It is fixed；
The expression of P9, cell TLR4/NF- kB protein: group of cells culture for 24 hours, after 48h, 72h, collect cell, adopt
Protein expression level is detected with Western Blot method；
P10, cell TLR4/NF- κ BmRNA expression: group of cells culture for 24 hours, after 48h, 72h, collect cell, inspection
Survey mRNA expression.
Interior animal experiment further includes administration in step A:
The daily oral gastric infusion of each treatment group animal is primary, and Silicotic model group and normal negative control group, which are given, etc. holds
Long-pending physiological saline.
In step s1 baicalin large dosage, middle dosage, the specific value of low dose be followed successively by 50mg/kg, 25mg/kg,
12.5mg/kg。
In step s5 lung fibrosis evaluation the following steps are included:
Lung tissue Masson dyeing, observes lung fibrosis lesion degree；
Lung tissue hydroxyproline content is measured, judges collagen aggregate level.
Measured in step s6 each group lung tissue of rats, BALF and serum IL -1, IL-6, TNF-α inflammatory factor expression water
Flat measuring method is ELISA method.
Measurement lung tissue, BALF and serum T LR4, NF- kB protein expression in step s7, RtPCR method detection TLR4,
The measuring method of NF- κ BmRNA expression is Western Blot method.
Lavation bronchovesicular specifically includes following steps in step p2；
Preoperative Method: conventional to be done before biopsy brush inspection after Bronchofiberscope air flue inspection with preparing before Fibrobronchoscopy
BAL, local anesthetic are 2% lidocaine；
The selection of lavation position: lavation position is determined according to high kilovoltage rabat；
BAL procedure step: firstly, injecting 2% lidocaine 1- by a thin silicone tube through biopsy hole in the lung section for wanting lavation
2ml；Section or sub- segmental bronchus opening are closely wedged into Bronchofiberscope top, then is rapidly injected 37 DEG C by silicone tube through biopsy hole
Sterile saline, each 25-50ml, total amount 100-250ml；BALF is recycled with 50-100mmHg vacuum suction immediately；It will return
Receive liquid uses double-layer sterile filtered through gauze to remove mucus immediately, and records total amount.
In step p7 measure group of cells I, type III collagen expression measuring method be immunocytochemical method.
The method that mRNA expression is detected in step p10 uses Real time PCR method.
In vivo during zoopery, Wistar male rat 200 are selected, is randomly divided into 5 groups, and be respectively
Silicotic model group, Normal group, hanfangchin A positive controls, scutelloside large dosage, middle dosage and low dose for the treatment of group,
Every group 40；After grouping, with ether by rat anesthesia, direct oral cavity exposure tracheostomy takes lmlSiO2 dust suspension slowly to infuse
Enter tracheae, control group injects 1ml sterile saline using same method；After the completion of processing, each treatment group animal is given daily
Oral gastric infusion is primary, and Silicotic model group and normal negative control group give the physiological saline of isometric(al)；After rats death,
2.5ml37 DEG C of sterile saline is slowly injected into intrapulmonary in body, interval was drawn back, is carried out repeatedly after 30 seconds, until
It is collected into BAL fluid about 15ml, irrigating solution is centrifuged 10min with 4 DEG C, 1500rpm, extracts -80 DEG C of supernatant guarantors
It deposits spare；After treatment, left lung is taken, is fixed through formalin, paraffin embedding, slice does HE dyeing, in light microscopic observation lung group
Pathological change is knitted, lung fibrosis lesion degree is observed in observation lung tissue Masson dyeing；Measurement lung tissue hydroxyproline contains
Amount, judges collagen aggregate level；It is scorching using ELISA method measurement each group lung tissue of rats, BALF and serum IL -1, IL-6, TNF-α
The expression of sex factor expresses water using Western Blot method measurement lung tissue, BALF and serum T LR4, NF- kB protein
Flat, RtPCR method detects TLR4, NF- κ BmRNA expression, and data are recorded after the completion of measurement, different after observation scutelloside administration
Time point each group mouse pulmonary fibrosis changes situation；Different time points each group lung tissue of rats, BALF and blood after scutelloside administration
The expression of clear IL-1, IL-6, TNF-α inflammatory factor；Different time points each group lung tissue of rats, BALF after scutelloside administration
With serum T LR4, NF- kB protein expression, RtPCR method detects TLR4, NF- κ BmRNA expression.
In vitro during cell culture experiments, I phase, II phase, each 6 of III phase silicosis male patient are chosen, to selection
Silicotic carries out bronchoalveolar lavage: firstly, injecting 2% benefit by a thin silicone tube through biopsy hole in the lung section for wanting lavation
Cacaine 1.5ml；Section or sub- segmental bronchus opening are closely wedged into Bronchofiberscope top, then is quickly infused through biopsy hole by silicone tube
Enter 37 DEG C of sterile salines, each 35ml, total amount 175ml；BALF is recycled with 80mmHg vacuum suction immediately；By withdrawal liquid
Mucus is removed with double-layer sterile filtered through gauze immediately, and records total amount；After lavation, irrigating solution is collected, 4 layers of sterile gauze are used
After filtering, filtrate is taken to be centrifuged 15min through 1500r/min, PBS washing is added 10%FBS-DMEM culture solution, routinely counts after mixing
Number macrophages, calculate cell survival rate after Trypan Blue, with the diluting cells of culture solution containing 10%FBS-DMEM for 1 ×
The cell suspension of 106/ml, is placed in culture bottle, adherent to cell, passes through microscopic observation form, conventional H E dyeing, antiacid dye
Color and Wright's staining identify it；Macrophage-conditioned media is taken, the culture solution for being free of serum is added, macrophage is divided into
2 groups: the suspension of the dust containing SiO2, final concentration of 50 μ g/ml is added in experimental group；The culture without serum is only added in control group
Base；Two groups, at 37 DEG C, are cultivated under the conditions of 5%CO2, and draw culture supernatant in different time periods respectively in culture, are detected
The expression of supernatant inflammatory factor IL-1, IL-6, TNF-α and cell TLR4, NF- kB protein and mRNA select expression most
The culture supernatant of high time point, -80 DEG C save backup, and are that culture substrate is added to by silicious dust macrophage culture supernatant night work
In fibrocyte, silicious dust co-culture model is established；Cell is divided into dye dirt group, TLR4/NF- kB inhibitor group inhibitor group, Huang
A kind of reed mentioned in ancient books glycosides group；Culture medium containing 10%FBS-DMEM is added, is cultivated under conditions of 37 DEG C, 5%CO2；Every group sets 15 parallel holes,
Each group is cultivated for 24 hours, after 48h, 72h, culture solution is sucked out respectively, and through membrane filtration, it is spare to collect cell for -80 DEG C of preservations；In each group
Cell culture for 24 hours, after 48h, 72h, collect cell, is measured cell activation situation using mtt assay, is measured using flow cytometer thin
Born of the same parents proliferating cycle, Annexin V/PI double-staining measure Apoptosis；Using immunocytochemical method measurement group of cells I,
The expression of type III collagen；The supernatant for taking -80 DEG C of preservations, with IL-1, IL-6, TNF-α 1 in ELISA method measurement supernatant
Level measurement；Group of cells culture for 24 hours, after 48h, 72h, collect cell, using Western Blot method detect protein expression
It is horizontal；Group of cells culture for 24 hours, after 48h, 72h, collect cell, using Real time PCR method detection mRNA express water
It is flat.
Changed by different time points each group mouse pulmonary fibrosis after observing scutelloside administration in above-mentioned interior animal experiment
Become situation, observe different time points each group lung tissue of rats, BALF and serum IL -1, IL-6, TNF-α inflammatory after scutelloside administration
Different time points each group lung tissue of rats, BALF and serum T LR4, NF- κ B after expression, the observation scutelloside of the factor are administered
Protein expression level, RtPCR method detect TLR4, NF- κ BmRNA expression；
By in above-mentioned cell culture experiments in vitro observe the culture of each group macrophage for 24 hours, tri- groups of differences of 48h, 72h when
Between section supernatant IL-1, IL-6, TNF-α inflammatory factor expression, cell TLR4, NF- kB protein mRNA expression, observation
Each group Fibroblast cell-culture for 24 hours, tri- groups of 48h, 72h activation, proliferation and apoptosis situations in different time periods, observation each composition fibre
Tie up cell culture for 24 hours, tri- groups of 48h, 72h supernatant hydroxyproline contents in different time periods, observation each group fibroblast training
Support for 24 hours, after tri- groups of different time sections of 48h, 72h the case where I, type III collagen expression, observation each group Fibroblast cell-culture for 24 hours,
The expression of tri- groups of 48h, 72h supernatant IL-1, IL-6, TNF-α inflammatory factor in different time periods, cell TLR4, NF- kB protein
And mRNA expression.
Toll-like receptor is a kind of transmembrane protein of newfound I type, is a kind of important PRR.TLR4 is TLRs family
Important member in race is mainly expressed in the cell surfaces such as bronchial epithelial cell, human embryonic kidney cells, pulmonary alveolar macrophage,
TLR4 mediate signal path be activated after, can specifically with corresponding pathogen-associated molecular pattern ining conjunction with, and then activation under
Signaling pathway protein such as nuclear factor NF- κ B, Activating protein-1 etc. is swum, TNF-α, IL-1 β, IFN-γ, monocyte chemoattractant egg are eventually led to
The synthesis and secretion of the inflammatory factors such as white 1 or chemotactic protein, thus the generation of inducing lung fibrosis；Zoopery also confirms that hair
Existing, silicious dust can raise mouse lung tissue TLR4mRNA and the expression with NF- κ B p65mRNA, prompt SiO2 that can pass through activation
TLR4/NF- κ B p65 signal path starts pulmonary fibrosis.
Scutelloside is able to suppress TLR4/Myd88/NF- κ B signal pathway activated, by scutelloside intervene mouse silicotic fibrosis and
By the signal transduction mechanism of the TLR4/Myd88/NF- κ B fibroblast activation mediated, scutelloside being capable of that modulates fibrosis for regulation
And novel targets are explored, new thinking and new therapeutic agent are provided for clinical treatment silicosis.
Comparative example:
The experimental method that baicalin is applied in preparation treatment silicosis drug, specific experiment operating procedure and 1 base of embodiment
This is identical, the difference is that being added without baicalin in experimentation.
Experimental test is carried out to the experimental method of above-described embodiment 1, test result is as follows table 1:
Table 1
It can be obtained by experimental result and table 1, for baicalin in the application in preparation treatment silicosis drug, baicalin can
The effective speed for inhibiting silicotic fibrosis, and the novel targets of lung fibrosis therapeutic effect can be actively found, scutelloside is to silicon
Lung pulmonary fibrosis is formed with antagonistic effect, and then can effectively apply in the application of silicosis therapeutic agent.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (8)
1. application of the baicalin in preparation treatment silicosis drug, which is characterized in that contain baicalin in the silicosis drug
Component.
2. the experimental method that baicalin is applied in preparation treatment silicosis drug: it is characterized in that, specifically including following experiment step
It is rapid: interior animal experiment:
1., using disposable intratracheal perfusion tested material SiO2 is filled into Wister male rat, establish silicosis lung fiber
Change animal model；
2., set up be added baicalin treatment group a, be added baicalin treatment group b, be added baicalin treatment group c, Stephania tetrandra
A prime positive controls and normal negative control group；
3., from whole, tissue, cell factor and molecular level observation scutelloside to the antagonism of the SiO2 Pulmonary Fibrosis in Rats induced
Effect and mechanism of action.
3. the experimental method that baicalin according to claim 2 is applied in preparation treatment silicosis drug, which is characterized in that
Interior animal experiment in step A specifically includes the following steps:
S1, experimental animal selection and grouping: selection Wistar male rat 200, according to step 2. in the model group set up will
200 rats are randomly divided into 5 groups, every group 40；
The foundation of S2, Silicotic Rats pulmonary fibrosis model: with ether by rat anesthesia, direct oral cavity exposure tracheostomy is taken
LmlSiO2 dust suspension is slowly injected into tracheae, in hanfangchin A positive controls and normal negative control group using same
Method inject 1ml sterile saline；
The collection of S3, BALF supernatant: after rat is put to death, being slowly injected into intrapulmonary in body for 2.5ml, 37 DEG C of sterile salines,
Interval was drawn back, is carried out repeatedly after 30 seconds, until BAL fluid about 14-16ml is collected into, by irrigating solution
It is centrifuged 10min with 4 DEG C, 1500rpm, supernatant is extracted and is saved backup under -80 DEG C of environment；
S4, pathologic state colony inspection: taking left lung, fix through formalin, paraffin embedding, and slice does HE dyeing, under light microscopic
Observe pathologic variation；
S5, lung fibrosis evaluation；
S6, the expression for measuring IL-1, IL-6, TNF-α inflammatory factor: measurement each group lung tissue of rats, BALF and serum IL -1,
The expression of IL-6, TNF-α inflammatory factor；
S7, TLR4, NF- κ B measurement: measurement lung tissue, BALF and serum T LR4, NF- kB protein expression, the detection of RtPCR method
TLR4, NF- κ BmRNA expression.
4. the experimental method that baicalin according to claim 2 is applied in preparation treatment silicosis drug, which is characterized in that
Interior animal experiment further includes administration in step A:
The daily oral gastric infusion for the treatment of group a, treatment group b, the animal in treatment group c is primary, Silicotic model group and normal yin
Property control group gives the physiological saline of isometric(al).
5. the experimental method that baicalin according to claim 2 is applied in preparation treatment silicosis drug, which is characterized in that
Baicalin dosage is 40-60mg/kg in step ②Zhong treatment group a, baicalin dosage is 20-30mg/kg, treats in treatment group b
Baicalin dosage is 10-15mg/kg in group c.
6. the experimental method that baicalin according to claim 3 is applied in preparation treatment silicosis drug, which is characterized in that
In step s5 lung fibrosis evaluation the following steps are included:
Lung tissue Masson dyeing, observes lung fibrosis lesion degree；
Lung tissue hydroxyproline content is measured, judges collagen aggregate level.
7. the experimental method that baicalin according to claim 3 is applied in preparation treatment silicosis drug, which is characterized in that
Measured in step s6 each group lung tissue of rats, BALF and serum IL -1, IL-6, TNF-α inflammatory factor expression measurement
Method is ELISA method.
8. the experimental method that baicalin according to claim 3 is applied in preparation treatment silicosis drug, which is characterized in that
Lung tissue, BALF and serum T LR4, NF- kB protein expression are measured in step s7, RtPCR method detects TLR4, NF- κ BmRNA
The measuring method of expression is Western Blot method.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113662952A (en) * | 2021-08-25 | 2021-11-19 | 天津中医药大学 | Compound dry powder inhalant and application thereof |
-
2019
- 2019-07-12 CN CN201910628192.3A patent/CN110327360A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| TAO LIU等: "Baicalin Alleviates Silica-Induced Lung Inflammation and Fibrosis by Inhibiting the Th17 Response in C57BL/6 Mice", 《JOURNAL OF NATURAL PRODUCTS》 * |
| 李春红等: "实验性矽肺模型大鼠外周血CD4+CD25+Foxp3+调节性T细胞的变化及意义", 《环境与职业医学》 * |
| 郭敬文等: "矽肺肺纤维化治疗药物的研究进展", 《中国工业医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113662952A (en) * | 2021-08-25 | 2021-11-19 | 天津中医药大学 | Compound dry powder inhalant and application thereof |
| CN113662952B (en) * | 2021-08-25 | 2022-11-22 | 天津中医药大学 | Compound dry powder inhalant for treating idiopathic pulmonary fibrosis and application thereof |
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