CN110251549A - Application of the barren wort total chromocor extract in the drug or health care product of preparation prevention and treatment Hashimoto thyroiditis - Google Patents

Application of the barren wort total chromocor extract in the drug or health care product of preparation prevention and treatment Hashimoto thyroiditis Download PDF

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Publication number
CN110251549A
CN110251549A CN201910548638.1A CN201910548638A CN110251549A CN 110251549 A CN110251549 A CN 110251549A CN 201910548638 A CN201910548638 A CN 201910548638A CN 110251549 A CN110251549 A CN 110251549A
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group
barren wort
total chromocor
wort total
rat
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CN110251549B (en
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李清林
葛明华
郑传铭
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Zhejiang Cancer Hospital
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Zhejiang Cancer Hospital
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    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
    • A61K36/296—Epimedium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

Abstract

The present invention relates to a kind of Chinese medicine new application, in particular to application of a kind of barren wort total chromocor extract in the drug or health care product of preparation prevention and treatment Hashimoto thyroiditis belongs to technical field of Chinese medicine.A kind of application of barren wort total chromocor extract in the drug or health care product of preparation prevention and treatment Hashimoto thyroiditis.It is proved through results of animal, barren wort total chromocor extract of the present invention has the function of Hashimoto thyroiditis rat significantly to treat Hashimoto thyroiditis；The safety of barren wort total chromocor extract is good, and curative effect is good, can effectively treat Hashimoto thyroiditis.The barren wort total chromocor extract is that a kind of improvement clinical symptoms effect is good, and toxic side effect is small, the Chinese medical extract with good potential applicability in clinical practice.

Description

Drug or health care of the barren wort total chromocor extract in preparation prevention and treatment Hashimoto thyroiditis Application in product
Technical field
The present invention relates to a kind of Chinese medicine new application, in particular to a kind of barren wort total chromocor extract prevents and treats bridge sheet in preparation The drug of thyroiditis or the application in health care product, belong to technical field of Chinese medicine.
Background technique
Hashimoto thyroiditis (hashimoto thyroiditis) is also known as chronic lymphocytic thyroiditis, is a kind of Using itself parathyroid tissue as the chronic auto-immune disease of antigen.Reason Kyushu University Hashimoto is according to tissue It learns feature to report first, therefore is named as Hashimoto thyroiditis again, for the most common thyroid gland inflammation in clinic.It is more common in middle aged female Property, sending out the age well is 40~50 years old.Also there are many cases in children.
Currently, this disease of western medical treatment mainly replaces the treatment such as cure, immunotherapy and operation using thyroxine Means alleviate thyroid gland local symptom, but to the constitutional symptom unsatisfactory curative effect of patient.Wherein, most routine treatment is hormone replacement Therapy, and hormone does not have immunoregulation effect, cannot reduce Thyroid autoantibodies titre, and patient needs lifelong medication. Secondly, patient needs continuous physical examination, the dosage of long-term steroid is adjusted according to the index of thyroid function, adverse reaction is more, stops Medicine is easily repeatedly.Therefore, seeking the effective drug of new Hashimoto thyroiditis has apparent society and economic value.
Summary of the invention
The purpose of the present invention is to provide a kind of barren wort total chromocor extracts in the medicine for preparing prevention and treatment Hashimoto thyroiditis Application in object or health care product.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of application of barren wort total chromocor extract in the drug or health care product of preparation prevention and treatment Hashimoto thyroiditis.Medicine Reason achieves satisfied effect studies have shown that inventor's barren wort total chromocor extract prevention and treatment Hashimoto thyroiditis, Have found the new application of barren wort total chromocor extract.
Barren wort total chromocor extract of the present invention be according to the conventional method of this field from Herba Epimedii extraction purification. The extraction processing technology can be used Chinese medicine extracting flavonoids method known in the art and be prepared, such as but unlimited In cold soaking, diacolation, ultrasonic wave, Soxhlet extraction, circumfluence distillation, supercritical extract etc., and it is combined such as cold soaking-diacolation, Cold soaking-ultrasonic wave, cold soaking-Soxhlet extraction, the extracting methods such as cold soaking-circumfluence distillation.The barren wort total chromocor extract Mainly according to known Chinese medicine flavones purification process, such as, but not limited to column chromatography, solvent extraction, Solid Phase Extraction, ion is handed over for purifying The methods of change.
Preferably, the drug or health care product of the prevention and treatment Hashimoto thyroiditis are with barren wort total chromocor extract for work Property ingredient, in addition the preparation that pharmaceutically acceptable auxiliary material is prepared.Barren wort total chromocor extract of the present invention can be with Directly or various oral medicinal herb conventional doses are made by the conventional method of this field with pharmaceutically acceptable pharmaceutical carrier to be used for It prevents and treats in Hashimoto thyroiditis.
The pharmaceutical preparation form can be any pharmaceutical dosage form, these dosage forms include: tablet, sugar coated tablet, Film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral solution, mouth containing agent, granule, electuary, ball Agent, powder, paste, sublimed preparation, suspension, pulvis, solution, injection, suppository, ointment, emplastrum, creme, spray, drop Agent, patch；It is preferred that peroral dosage form, such as: capsule, tablet, oral solution, granule, pill, powder, sublimed preparation, paste.It is described Peroral dosage form can contain common excipient, such as adhesive, filler, diluent, tablet agent, lubricant, disintegrating agent, Toner, flavoring agent and wetting agent when necessary can be coated tablet.Suitable filler includes cellulose, mannitol, cream It is sugared with other similar fillers；Suitable disintegrating agent includes starch, polyvinylpyrrolidone and starch derivatives, such as hydroxyl Amylcose acetate sodium；Suitable lubricant includes, such as magnesium stearate.Suitable pharmaceutically acceptable wetting agent includes dodecyl Sodium sulphate etc..
It is proved through results of animal, barren wort total chromocor extract of the present invention has Hashimoto thyroiditis rat obvious Treatment Hashimoto thyroiditis effect；The safety of barren wort total chromocor extract is good, and curative effect is good, can effectively treat this first of bridge Shape adenositis.The barren wort total chromocor extract is that a kind of improvement clinical symptoms effect is good, and toxic side effect is small, has good clinic The Chinese medical extract of application prospect.
Detailed description of the invention
Fig. 1 is HE dyeing observation Hashimoto thyroiditis rat model thyroid pathology photo (HE dyeing, 200 times)；
Fig. 2 is HE dyeing observation Hashimoto thyroiditis rat model thyroid pathology photo (HE dyeing, 400 times)；
Fig. 3 is content of the barren wort total chromocor extract to FT3, FT4, TOP-Ab, ATGA, T3 and T4 in HT rat blood serum Influence curve (x ± s, n=6)；
Fig. 4 is barren wort total chromocor extract to IFN-γ in HT rat blood serum, IL-17, IL-6, IL-10, IL-23 and The influence curve (x ± s, n=6) of the content of TGF-β；
Fig. 5 is each group rat peripheral blood Treg cell streaming scatter plot, note: A blank control group；B model group；C Herba Epimedii Extractive of general flavone low dose group；D barren wort total chromocor extract middle dose group；E barren wort total chromocor extract high dose group； F levothyroxine sodium tablet positive drug group；
Fig. 6 is each group rat peripheral blood Th17 cell streaming scatter plot, note: A blank control group；B model group；C Herba Epimedii Extractive of general flavone low dose group；D barren wort total chromocor extract middle dose group；E barren wort total chromocor extract high dose group； F levothyroxine sodium tablet positive drug group；
Fig. 7 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue FoxP3 expression photo (n=6, × 200 Times)；
Fig. 8 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue FoxP3 expression photo (n=6, × 400 Times)；
Fig. 9 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue ROR γ t expression photo (n=6, × 200 Times)；
Figure 10 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue ROR γ t expression photo (n=6, × 400 Times)；
Figure 11 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue IL-17 expression photo (n=6, × 200 Times)；
Figure 12 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue IL-17 expression photo (n=6, × 400 Times)；
Figure 13 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue IL-23 expression photo (n=6, × 200 Times)；
Figure 14 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue IL-23 expression photo (n=6, × 400 Times)；
Figure 15 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue IL-6 expression photo (n=6, × 200 Times)；
Figure 16 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue IL-6 expression photo (n=6, × 400 Times)；
Figure 17 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue IL-10 expression photo (n=6, × 200 Times)；
Figure 18 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue IL-10 expression photo (n=6, × 400 Times)；
Figure 19 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue IFN-γ expression photo (n=6, × 200 Times)；
Figure 20 be epimedium flavone to Hashimoto thyroiditis rat thyroid tissue IFN-γ expression photo (n=6, × 400 Times)；
Figure 21 is rat thyroid related gene relative expression quantity statistic curve
Figure 22 is rat thyroid correlative protein expression figure
Figure 23 is rat thyroid GAP-associated protein GAP relative expression's situation statistic curve
Figure 24 is rat thyroid primary cell photo (100 ×)；
Figure 25 is Contained Serum to rat thyroid cell activity photo, note: A blank control group；B model group；C Herba Epimedii Flavones low dose group；D epimedium flavone middle dose group；E epimedium flavone high dose group；F levothyroxine sodium tablet positive drug Group；
Fig. 3, Fig. 4, Figure 21, Figure 23, note: compared with normal group,▲P<0.05,▲▲P<0.01；Compared with model group:★P < 0.05,★★P < 0.01.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be further explained in detail.It should be appreciated that this hair Bright implementation is not limited by the following examples, and the accommodation in any form made to the present invention and/or changed will all be fallen Enter the scope of the present invention.
In the present invention, if not refering in particular to, all parts, percentage are unit of weight, used equipment and raw material etc. It is commercially available or commonly used in the art.Method in following embodiments is unless otherwise instructed the normal of this field Rule method.The main agents source that following embodiment is used is as follows:
The barren wort total chromocor extract of the present invention of embodiment 1 acts on the curative effect of Hashimoto thyroiditis rat
The preparation of barren wort total chromocor extract:
Herba Epimedii Preparata: taking Herba Epimedii crude drug, removes impurity, wins blade, sprays clear water, slightly moistens, chopping, dry.It takes It refines suet oil and heats fusing, Herba Epimedii silk is added, be fried with slow fire the aobvious uniform glossy gloss in surface, when being in yellowish, take out, put It is cool.Herba Epimedii every 100kg refining suet oil 20kg.
The preparation of extractive of general flavone: taking Herba Epimedii Preparata appropriate, and respectively plus 70% ethyl alcohol, 10 times of amounts extract 2 times, every time 2h.Medical fluid is concentrated into 1mL medical fluid crude drug containing 1g, the technique using HPD300 macroporous resin purification general icariin passes through investigation It determines are as follows: medical fluid is diluted with water into every 1mL medical fluid crude drug containing 0.1g, passes through HPD300 large pore resin absorption column with 4BV/h flow velocity (resin column diameter height compares for 1:11), first 5 times of column volume washings, after with 7 times of 50% ethanol elutions of column volume, elution flow rate 4BV/ H collects eluent, ethyl alcohol is recovered under reduced pressure, and filters, and filtrate adds 3% gelatin solution to precipitating is not generated, ethyl alcohol is added to reach alcohol content 80%, it stands overnight, filters, ethyl alcohol is recovered under reduced pressure, barren wort total chromocor extract is made, and (general icariin content is about 80%).
1 experiment purpose
1.HE dyeing observation Hashimoto thyroiditis rat model thyroid pathology changes；
FT3, FT4, TOP-Ab, ATGA, T3 in 2.ELISA detection rat blood serum, IFN-γ, IL-17, IL-6, IL-10, The level of IL-23 and TGF-β and T4；
3. barren wort total chromocor extract is to Treg, Th17 impact cell in HT rat peripheral blood；
4. FoxP3 in immunohistochemical observation Hashimoto thyroiditis rat model thyroid gland, ROR γ t, IL-17, IL-23, The expression of IL-6, IL-10 and IFN-γ；
5.qRT-PCR detect rat thyroid FoxP3, ROR γ t, TGF-β 1, IL-17, IL-23, IL-6, IL-10 and IFN-γ gene relative expression quantity；
6.WB detects rat thyroid FoxP3, ROR γ t, TGF-β 1, IL-17, IL-23, IL-6, IL-10 and IFN-γ Protein expression situation；
7. dextran sulfate sodium (DSS) causes rat thyroid epithelial cell damage model foundation；
8. drug-treated and cell Proliferation detection；
9. rat thyroid epithelial cell proliferation and toxicity detection；
10.Annexin V-FITC/PI method detects influence of the Contained Serum to Apoptosis.
2 experimental methods
The preparation of 2.1 animal models and grouping administration
SPF grades of female wistar rats, weight 150-180g.By Shanghai, western Poole-Bi Kai experimental animal Co., Ltd is mentioned For animal productiong licensing SCXK (Shanghai) 2013-0016.
Rearing conditions: constant temperature, 22 ± 2 DEG C of temperature, humidity 50%-60%, the every 12 hours light and shades alternating of illumination changes wind number 15-20 times/hour.It is raised by Zhejiang University of Traditional Chinese Medicine's animal experiment study center.It tests in receptacle credit number SYXK (Zhejiang) 2013-0184。
Take 60 rats, adaptability culture one week, handled according to following grouping: mouse is divided into 6 groups, and every group 10, Totally 60:
A blank control group
B model group
C barren wort total chromocor extract high dose group (240mg/kg)
D barren wort total chromocor extract middle dose group (120mg/kg)
E barren wort total chromocor extract low dose group (60mg/kg)
F positive drug group
Dosage
A group gives 0.4ml physiological saline, once a day, continuous 14 days.
B group gives 0.4ml physiological saline, once a day, continuous 14 days.
C group gives the barren wort total chromocor extract solution of 240mg/kg, once a day, continuous 14 days.
D group gives the barren wort total chromocor extract solution of 120mg/kg, once a day, continuous 14 days.(dosage: 400mg/kg)
E group gives the barren wort total chromocor extract solution of 60mg/kg, once a day, continuous 14 days.
F group is given levothyroxine sodium tablet and is treated, and 10 μ g/ times, 1 time/d continuous 14 days.
The preparation of 2.2 nude mice models
1) initial immunity: rat adaptable fed 1 week, the thyroglobulin PTG antigen physiological saline that will be prepared It is mixed with complete Freund's adjuvant adjuvant by 1:1 after dissolution, final concentration of every milliliter of antigen containing adjuvant 0.25mgPTg is buffered with PBS Liquid is dissolved into the solution that PTg content is 1mg/mL, mixes in equal volume with complete Freund's adjuvant CFA fully emulsified, initial dose is 100 μ g/ only, are subcutaneously injected in rat back.Rats in normal control group is without any processing.
2) booster immunization: the PBS solution of above-mentioned PTg and IFA are mixed in equal volume it is fully emulsified, dosage be 100 μ g/ only, In the 2nd week after initial immunity, booster immunization was primary weekly, made multiple spot subcutaneous injection in rat toe, back etc. respectively Terminated to the 6th week.
2.3 HE dyeing observation transplanting lung carcinoma cell nude mouse tumor changes
Detection time point: model dissects rat after preparing 6 weeks, takes parathyroid tissue to be put into 10% neutral formalin and fixes, takes Material embedded section.
1) slice is conventional is dewaxed with dimethylbenzene, through ethyl alcohol at different levels to distilling washing: dimethylbenzene (I) 5min → dimethylbenzene (II) 5min
2) ethyl alcohol of ethyl alcohol 1min → 80% ethyl alcohol of lmin → 75% lmin of dehydrated alcohol 2min → 95% → distillation washing 2min
3) haematoxylin dyeing 5min, tap water rinse 5min
4) acidic alcohol breaks up 30s
5) tap water impregnates 15min
6) Yihong liquid 2min is set, tap water rinses
7) conventional dehydration, transparent, mounting: 95% ethyl alcohol (I) ethyl alcohol of 1min → 95% (II) 3min → dehydrated alcohol (I) The transparent transparent 5min of 5min → dimethylbenzene (the II) → resinene sealing of 5min → dehydrated alcohol (II) 5min → dimethylbenzene (I)
8) Image Acquisition and analysis: being taken pictures (200 times and 400 times of microscopic observations) by microscope, collection analysis sample phase Close position.
2.4 ELISA detect rat blood serum FT3, FT4, TOP-Ab, ATGA, T3, T4, IFN-γ, IL-17, IL-6, IL- 10, IL-23 and TGF-β level
Detection time point: after administration 2 weeks, empty stomach fasting 12h (can't help water), heart extracting blood, after standing 30min, 3000r/min is centrifuged 10min, and it is spare to prepare serum.
(1) prepare: taking out kit from refrigerator, room temperature rewarming balances 20min.
(2) match liquid: 20 times of concentrated cleaning solutions being diluted to former cleaning solution again with distilled water.
(3) add standard items and sample to be tested: taking sufficient amount of enzyme mark coating plate, be fixed on frame, mark is respectively set Quasi- sample wells, sample to be tested hole and blank control wells, record each hole site, and 50 μ L of standard items is added in standard sample wells；To test sample 10 μ L of sample to be tested, then plus 40 μ L of Sample dilution (i.e. 5 times of Sample Dilution) are first added in this hole；Blank control wells are not added.
(4) in addition to blank well, the detection of horseradish peroxidase (HRP) label is added in every hole in standard sample wells and sample aperture 100 μ L of antibody.
(5) it incubates: sealing reacting hole with envelope version film, 37 DEG C of water-baths or insulating box incubate 60min.
(6) board-washing: discarding liquid, pat dry on blotting paper, and cleaning solution is filled it up in every hole, stands 1min, gets rid of cleaning solution, absorbs water It is patted dry on paper, so repeats board-washing 5 times (board-washing can also be operated with board-washing machine by specification).
(7) develop the color: 50 μ L of color developing agent A liquid is first added in every hole, adds 50 μ L of color developing agent B liquid, and plate vortex mixer mixes 30s (or gently shaken with hand and mix 30s), 37 DEG C are protected from light colour developing 15min.
(8) it terminates: taking out ELISA Plate, every hole adds 50 μ L of terminate liquid, terminates reaction.
(9) it measures: being returned to zero with blank well, after termination in 15min, with the light absorption value (OD in each hole of 450nm wavelength measurement Value).
2.5 barren wort total chromocor extracts are to Treg impact cell in HT rat peripheral blood
After administration 3 days, taking in every 100 μ L injecting tubes of each group rat peripheral blood, every pipe injects 25 μ L heparin sodium aquas, Then it is separately added into CD4-FITC (5 μ g/mL), CD25-ApC (2 μ g/ml) each 10 μ l in each pipe, is put into 4 DEG C of refrigerators and is incubated for 20min takes out each pipe and each 2ml of erythrocyte cracked liquid is added, is sufficiently mixed (5min at 37 DEG C), is then centrifuged for (1500r/min) 5min abandons supernatant, and Hanks is washed 2 times, and 20 μ LPE-Foxp3 are added, and 4 DEG C of incubation 50min are protected from light.Perm Buffer(200μ L) cleaning twice, is then resuspended with 400 μ L Hanks buffers.(the upper machine final concentration 5 of machine final volume on 400 μ L is taken in each pipe × 106 cells) it the streaming pipe that mark of cell injection is resuspended is resuspended in Hanks liquid, 100 μ L resuspension cell is taken in each pipe It injects in the streaming pipe marked, up flow type detects the variation of Treg quantity in each group rat peripheral blood.
Influence of the 2.6 barren wort total chromocor extracts to Th17 cell in HT rat peripheral blood
After administration 3 days, take fresh 100 μ L of heparin anti-coagulating, be added 860 μ L, IRPMI 1640 culture medium mix, then plus people 10 μ L IPMA working solutions, 10 μ L Ionomycin working solutions, 20 μ LBFA working solutions, PMA, Ionomycin, BFA are dense in system Degree is respectively 50ng/mL, 750ng/mL, 10 μ L/mL.At 37 DEG C, 5%CO2 incubator moderate stimulation culture 3h；It will be thin after culture Born of the same parents' suspension mixes gently, and is added in 1 streaming test tube, centrifugation；Supernatant is abandoned, oscillation mixes, and adds 2 μ LFITC-Anti rat- of people CD4 is protected from light and is incubated for 15min；Add 100 μ L fixers, avoid light place 15min；1 × PBS of 2mL is added to wash, centrifugation；Abandon supernatant, vibration Mixing is swung, adds 100 μ L rupture of membranes agent, avoids vibrating, mix gently, be protected from light 5min；2 μ LPE-Anti rat-IL- are added in pipe 17, it mixes gently, is protected from light and is incubated for 15min；Add 1 × PBS of 2mL washed once, be centrifuged, abandons supernatant, flow cytometer detection.
FoxP3 in 2.7 immunohistochemical observation Hashimoto thyroiditis rat model thyroid glands, ROR γ t, IL-17, IL-23, The expression of IL-6, IL-10 and IFN-γ.
Paraffin section through 60 DEG C baking piece 1 hour, through dimethylbenzene dewaxing (dimethylbenzene I 10min, dimethylbenzene II 10min), ladder Spending alcohol, (100%3min, 100%3min, 95%3min, 90%3min, 85%3min, 75%3min, distilled water 3min steam Distilled water 3min, PBS 3min).
Antigen retrieval: the citron acid solution that slice is placed in 0.1mol/L and PH=6.0 is repaired in liquid, with 100 fire of micro-wave oven It boils to pico-, then is maintained 7 minutes with 50 firepower within power three minutes, stop after heating natural cooling 20-30 minutes.It is rinsed with PBS 5min × 3/ time.
Immuning hybridization: 3%H2O210 minutes are incubated for eliminate endogenous peroxidase activity.PBS is rinsed, and 5 minutes × 3 It is secondary.It is added dropwise confining liquid (5%BSA), room temperature 30 minutes in wet box.Confining liquid is wiped with filter paper, is not washed.Suitable concentration is added dropwise FoxP3, ROR γ t, IL-17, IL-23, IL-6, IL-10 and IFN-γ primary antibody set in wet box 4 DEG C of overnight incubations, then with PBS 5 Minute × wash away primary antibody 3 times, the PBS outside sample is wiped with filter paper.Biotinylation secondary antibody working solution is added dropwise, room temperature, which is set in wet box, incubates It educates 20 minutes, washes away secondary antibody with PBS 5 minutes × 3 times, the PBS outside sample is wiped with filter paper.Horseradish enzyme is added dropwise and marks strepto- ovum White element working solution, room temperature are set in wet box and are incubated for 20 minutes, washed away with PBS 5 minutes × 3 times, the PBS outside sample is wiped with filter paper. DAB chromogenic reagent, tap water sufficiently rinse.Haematoxylin is carried out again to redye, and is dehydrated, transparent, neutral gum mounting.Slice is 10 Thyroid gland is randomly selected under × 20 times and 10 × 40 power microscopes does not repeat 6 visuals field.
2.8 qRT-PCR detect rat thyroid FoxP3, ROR γ t, TGF-β 1, IL-17, IL-23, IL-6, IL-10 and IFN-γ gene relative expression quantity
QRT-PCR the primer sequence information is as follows:
RNA is extracted
1) sample process: by parathyroid tissue (every 200mg adds 1ml) and Thyroid follicular epithelial cell (every 1 × 107A cell Add 1ml) it is put into the homogenate tube of Trizol, sample after cracking is placed at room temperature for 5-10min, so that nucleoprotein divides completely with nucleic acid From.
2) it is placed in super-clean bench, incubates 5min, 12000rpm, be centrifuged 10min.
3) it sucts clearly in new 1.5mL centrifuge tube, the chloroform of 200 μ L is added, shakes up, is stored at room temperature 2min, 4 DEG C, 12000rpm is centrifuged 10min.
4) supernatant is drawn in new 1.5mL centrifuge tube, and the isopropanol of 600 μ L is added, is uniformly mixed, is stored at room temperature 15min 4 DEG C, 12000rpm, is centrifuged 15min, abandons supernatant.
5) dehydrated alcohol (750 μ L dehydrated alcohols and 250 μ L DEPC water) that 1mL75% is added, which rinses, to be precipitated, and 4 DEG C, 12000rpm is centrifuged 5min, abandons supernatant.
6) 1mL dehydrated alcohol is added, rinsing precipitating 4 DEG C, 12000rpm, is centrifuged 5min, abandons supernatant, drying at room temperature 10min。
7) the DEPC water that 40 μ L are added dissolves RNA, is placed in -80 DEG C of refrigerators and saves backup.
Reverse transcription reaction
It prepares following reaction system and carries out reverse transcription reaction.Reaction condition: 42 DEG C, 15min；85 DEG C, 5min.
QPCR reaction
1) it prepares following reaction system and carries out real-time fluorescence quantitative PCR reaction.It is with vortex oscillator that solution in pipe is thorough It is uniformly mixed,
Of short duration low-speed centrifugal.Reaction condition: 95 DEG C, 10min denaturation；95 DEG C, 15s；60 DEG C, 60s；40 circulations.
2) point sample: the liquid mixed in step (1) is added in orifice plate, and each gene guarantee 3 of each sample is multiple Hole.
3) PCR reacts: Real time PCR instrument uses bio-red real-time fluorescence quantitative PCR instrument, and PCR program has optimized. Excellent 96 orifice plate plate will have been put in step (2) is placed in progress PCR reaction in Realtime PCR instrument.
4) reaction condition: 95 DEG C, 10min denaturation；95 DEG C, 15s；60 DEG C, 60s；40 circulations.
2.9 WB detect rat thyroid FoxP3, ROR γ t, TGF-β 1, IL-17, IL-23, IL-6, IL-10 and IFN- γ protein expression situation
Protein sample preparation
1) by parathyroid tissue (every 200mg adds 1ml) and Thyroid follicular epithelial cell (every 1 × 107 cell adds 1ml) RIPA Lysate (containing PMSF and protease inhibitors), is ground using manual homogenizer.It is placed in and cracks 30min on ice；
2) 12000rpm is centrifuged 5min, takes supernatant.
The total protein concentration measurement (BCA method) organized after drug-treated
1) BCA determination of protein concentration kit Solarbio Cat pc0020
2) 96 drafting board Costow 3599
3) BCA reagent: Cu reagent: 50:1 volume ratio prepares 8ml, is configured to BCA working solution；
4) 15 μ L of standard items is diluted to 150 μ L, compound concentration 0.5mg/ml with PBS；
5) sample is diluted to 2 times and 8 times, adds 20 μ L to 96 orifice plates；
6) plus 200 μ L BCA working solutions, 37 DEG C 20 minutes are measured with microplate reader A562nm wavelength.
SDS-PAGE electrophoresis and transferring film
1) clean 1.5mm glass plate is taken, by specification installs on gum-making rack.
2) 10% separation gel and 5% concentration glue are prepared
It is mixed well after each substance is added, fills separation gel, then sealed with aqueous.
It is mixed after each substance is added, fills concentration glue.
3) it prepares the SDS-PAGE concentration glue of 5% concentration and injects the upper end of separation gel, careful insertion and glass plate The adaptable sample comb of thickness avoids generating bubble.
4) after spacer gel polymerization, sample comb is carefully taken out, the electrophoretic buffer of 1 × Tris-Gly is then added.
5) it draws appropriate amount of sample supernatant to be added in sample well, the albumen Marker of pre-dyed is added in the hole by sample, not In the hole for adding Sample supernatants, 1 × SDS sample-loading buffer is added and keeps glue surface balance.
6) power supply is opened, voltage starts setting up as 80V, and after protein sample enters separation gel, 120V is can be improved in voltage. Referring to the position of pre-dyed Marker, when purpose band enters gel optimal separation area (about the 2/3 of gel), stop electrophoresis.
7) 4 DEG C of transferring film liquid are pre-chilled in advance.
8) transfer box is opened on pallet, spreading close to the inner face of cathode side has Kong Wei with what transferring film buffer soaked Pad, puts three layers of filter paper for being soaked with transferring film buffer thereon, pays attention to emptying bubble.
9) glass plate is carefully pried open, glue is placed in the pallet containing transferring film liquid, the separation gel containing purpose band is cut Under, it is placed on filter paper with the immersion of transferring film liquid.
10) pvdf membrane soaked through methanol and transferring film liquid is spread on gel, cannot have bubble between glue and film, film, The size of filter paper and gel, it is roughly the same.
11) filter paper for putting three layers of dipped transferring film liquid again on pvdf membrane pays attention to emptying bubble.
12) second block of foam-rubber cushion is put, makes entirely to transfer interlayer and sequentially forms " fiber mat-filter paper-gel-NC film- Filter paper-fiber mat " level closes transfer folder, is put into transfer groove, transferring film liquid is filled in slot.
13) power supply, current stabilization 200mA, 120min are opened.
14) it after transferring film, takes out pvdf membrane and performs label, wash film 10min × 3 time with TBST.
Immunoblotting
Closing and antigen-antibody reaction
1) it closes: pvdf membrane being put into and is incubated in box, confining liquid of the addition containing 5% skimmed milk power, shaking table oscillation 1.5~ 2h；
2) after closing, film 10min × 3 time are washed with TBST；
3) film is put into the nti-IL17A of diluent A containing primary antibody (1:1000)；Anti-IL6(1:50)；Anti-IL23(1: 2000)；Anti-INFγ(1:1000)；Anti-RORγt(1:2000)；Anti-Foxp3(1:2000)；GAPDH(1:1000) Incubation box in, 4 DEG C of shaking table oscillation incubations are stayed overnight；
4) it takes out within second day, shaken at room temperature 30min inhales and abandons primary antibody, and TBST washes 10min × 3 time；
5) secondary antibody, 1~2h of room temperature shaker oscillating reactions are diluted with 5% skimmed milk power confining liquid；
6) secondary antibody after reaction, recycles secondary antibody.Then film 5~10min × 3 time are washed with TBST.
X light reaching the film, development, fixing are used in darkroom
1) by two kinds of reagents of A and B volume mixtures such as on preservative film；After 1min, by memebrane protein down with this mixed liquor It comes into full contact with；After 1min, film is moved on another preservative film, most raffinate is removed, wraps, is put into X- mating plate folder.
2) in darkroom, by 1 × developer solution and fixing solution respectively to entering in vinyl disc；X- mating plate is taken out under red light, is used Hand papercutter cuts out appropriately sized (long and wide than film is both needed to big 1cm)；
3) X- mating plate folder is opened, X- mating plate is placed on film, once it puts, it is just immovable, X- mating plate folder is shut, is opened Beginning timing；According to the strong and weak appropriate adjustment time for exposure of signal, generally 1min or 5min also may be selected different time and repeatedly press Piece, to reach optimum efficiency；
4) after the completion of exposing, X- mating plate folder is opened, X- mating plate is taken out, immerses in developer solution develop rapidly, it is to appear obvious After band, development is terminated at once.Developing time is generally 1~2min (20~25 DEG C), and (being lower than 16 DEG C) when temperature is too low need to fit When extension developing time；
5) after developing, X- mating plate is immersed in fixing solution at once, fixing time is generally 5~10min, saturating with film Until bright；After washing away remaining fixing solution with tap water, dry at room temperature.
It should be noted that taking one jiao of film as far as possible when development and fixing need to move film, finger nail not scratch film, Otherwise result can be had an impact.
2.10 IL-23 secrete influence (6 groups * 3 * 2 fingers of IL-17 and IFN-γ to HT peripheral blood mononuclear cells of rats Marked for * 2 time points)
Administration front and back, takes rat peripheral blood, after separating peripheral blood mononuclear cells, is respectively added in two Tissue Culture Dish 1mL cell suspension (cell concentration is about 2 × 10 6/mL), wherein 15 μ L IL-23 (50ng/mL) of addition is mixed well, Another 1 not adduction understand label.37 DEG C, 5%CO2Sterile culture case culture 72 hours.Culture supernatant is collected, adduction is not added The supernatant of IL-23 respectively respectively loaded in 2 sterile EP tubes, -20 DEG C freeze it is spare.Culture supernatant is detected using ELISA kit The IL-17 of liquid, IFN-γ content.
The preparation of 2.11 rat thyroid epithelial cell suspensions
1. taking the thyrocytes tissue of normal SD rats, Hank ' the s liquid with 37 DEG C containing penicillin and streptomycin is washed repeatedly immediately Brush 5 times cleans the substance of blood and other attachments.
2. being put into tissue in the culture dish of the PBS solution containing penicillin and streptomycin, net adipose connective tissue is picked with eye scissors, Isolate thyrocytes tissue.
3. thyrocytes tissue is put into another sterile petri dish, again with containing after dual anti-PBS flushing 3 times, move Enter sterile containing in dual anti-solution bottle, thyrocytes tissue is cut into the fragment of 1-2mm size, sample during whole operation It is soaked in PBS.
4. rear tissue block will be shredded to filter through sterile 70 μm of cell sieves, and with PBS repeated flushing, it is mixed with removing as far as possible Blood cell and small fat cell.
5. collecting fine grained chippings tissue to be put into a sterile petri dish, 0.2% clostridiopetidase A II is added, the cunning shredded is completely covered Membrane tissue is placed in 37 degree of cell incubators and is incubated for 2.5h, and 0.25% trypsase is added, and incubator is incubated for 0.5h.
6. the culture medium containing 10%FBS, which is added, terminates digestion, postdigestive small tissue blocks are filtered through 70um cell sieve, instead It is multiplexed PBS and elutes small tissue blocks.The cell for being attached to filter and ware bottom is rinsed with PBS, filtered fluid moves into 10ml sterile centrifugation tube. 1000rpm centrifugation, abandons supernatant, takes precipitating.(centrifugation 3 times can be resuspended with PBS in centre, as far as possible reduction pancreas enzyme concentration)
7. being resuspended with 1640 containing 10%FBS, counts and plant in culture bottle.Progress culture medium changes liquid within 3-4 days.
8. making to purify using limiting dilution Colony Culture method.
9. cell is divided into 5 groups: be respectively Normal group, model control group, barren wort total chromocor extract low dosage, Barren wort total chromocor extract middle dosage, barren wort total chromocor extract high dose group.With trypan blue liquid living cell counting.
2.12 dextran sulfate sodiums (DSS) cause rat thyroid epithelial cell damage model foundation
After rat thyroid epithelial cell culture to logarithmic growth phase, full dose changes liquid, is separately added into 1 μ g/ml, 2 μ g/ml, 5 μ g/ml, 10 μ g/ml, 15 five kinds of various concentrations of μ g/ml dextran sulfate sodium (DSS), administration time act on 12-72h (12, 24,36,72h).Full dose continues culture for 24 hours after changing liquid.Using CCK-8 colorimetric determination epithelial cell growth situation, to determine Portugal The optium concentration and Best administration time of glycan sodium sulphate (DSS) liquid damage.Cell viability=(dosing cell OD- blank group OD/ Control cell-blank group OD) * 100%.
2.13 rat thyroid epithelial cell proliferations and toxicity detection
It is thin to normal rat thyrocytes using CCK-8 colorimetric determination barren wort total chromocor extract Contained Serum The influence of born of the same parents' proliferation, using LDH detection kit detection barren wort total chromocor extract Contained Serum to rat thyroid epithelium The influence of cytotoxicity.
2.14 drug-treateds and cell Proliferation detection
The rat thyroid epithelial cell of logarithmic growth phase,
The barren wort total chromocor extract Contained Serum of first group of addition optium concentration sum is incubated for the best use time altogether, so The DSS liquid that optium concentration is added afterwards is incubated for Best Times altogether；
Second group of normal fetal calf serum of addition 10%, then be added optium concentration DSS liquid be incubated for altogether Best Times (according to Experimental result determine)；
Third group cell is normally cultivated, and is not processed.Cell proliferative conditions are detected using CCK-8.
2.15 Annexin V-FITC/PI methods detect influence of the Contained Serum to Apoptosis
(Normal group, model control group, barren wort total chromocor extract low dose group, barren wort total chromocor mention each group Taking object middle dose group, barren wort total chromocor extract high dose group and levoid group) cell adds Contained Serum culture After 48h, suspension cell is collected by centrifugation, centrifuge speed 2000rpm is centrifuged 5min, and abandoning culture medium, (attached cell is digested with pancreatin Time unsuitable too long, about lmin, to prevent causing false positive).Washing cell with cold PBS, (2000rpm, centrifugation 5min are collected twice Cell), experiment uses the bis- transfection reagent boxes of Annexin-V FITC/PI.Cell suspending liquid (cell concentration is about 1 × 10 6/ ML 5 μ LAnnexin-V FITC dyeing liquors are added in), mixes gently after 10 μ L PI dyeing liquors and is incubated under the conditions of being protected from light in 4 DEG C 30min uses flow cytomery Apoptosis in l h.
3 results and analysis
3.1 HE dyeing observation parathyroid tissue changes
From Fig. 1,2 it is found that normal group: normal rat Thyroid Structure is complete, and thyroid follicular cells (TEC) arrangement is whole Together, folliculus is rounded or oval, uniform in size consistent, and follicular cavity includes colloid and enriches, and dyes pale red, has no lymphocyte And plasmocyte infiltrating；Model group: Mouse thyroid morphosis, compared with normal group, rat thyroid follicular structure destroys tight Weight, part folliculus atrophy or disappearance, atrophy folliculus form is imperfect, and intracavitary colloid is rare, dyes pale red, sees around folliculus aobvious Lymphocytic infiltration is write, sees plasmocyte infiltrating in folliculus；Barren wort total chromocor extract high dose group: rat thyroid structure is big Part is more complete, and folliculus form primitive rule, size is more consistent, accidental lymphocytic infiltration between TEC, has no that slurry is thin in folliculus Born of the same parents, gum level are less slightly；Barren wort total chromocor extract middle dose group: rat thyroid structure is more complete, and folliculus form is basic Rule, it is not of uniform size, there are part lymphocyte and thick liquid cell in folliculus and between TEC, the person of being destroyed is few, and gum level is less； Barren wort total chromocor extract low dose group: rat thyroid structure is destroyed, and thyroid follicle is not of uniform size, and colloid contains Amount is few, and karyon dyeing is deep, has lymphocyte and plasmocyte infiltrating in folliculus and between TEC；Positive drug group: rat thyroid structure is complete Whole, folliculus form primitive rule is not of uniform size, accidental lymphocytic infiltration between TEC, has no thick liquid cell in folliculus, colloid contains It measures less slightly, has no lymphocyte and plasmocyte infiltrating.
3.2 ELISA detect rat blood serum FT3, FT4, TOP-Ab, ATGA, T3, T4, IFN-γ, IL-17, IL-6, IL- 10, IL-23 and the horizontal of TGF-β become
Shadow of the 1 barren wort total chromocor extract of table to the content of FT4, ATGA, T3, T4, FT3 and FT4 in HT rat blood serum It rings (x ± s, n=6)
Note: compared with normal group,▲P<0.05,▲▲P<0.01；Compared with model group:★P < 0.05,★★P < 0.01
By table 1 and Fig. 3 it is found that after modeling, model group is compared to normal group, and ATGA, TPO-Ab, T3 content are obvious in serum Rise, index T4, FT3, FT4 content is decreased obviously, and is had significant difference (P < 0.05 or P < 0.01).Each administration group is being administered After two weeks, index ATGA, TPO-Ab, T3 has different degrees of decline compared to model group, wherein barren wort total chromocor extract High dose group and the decline of levothyroxine sodium tablet treatment group are obvious, have significant difference (P < 0.05 or P < 0.01).Index T4, FT3, FT4 upon administration, each administration group compared to model group rise, wherein barren wort total chromocor extract high dose group and Levothyroxine sodium tablet treatment group rising degree is maximum, has significant difference (P < 0.05).
After epimedium flavone and levothyroxine sodium tablet two weeks in rat blood serum under ATGA, TPO-Ab, T3 content Drop degree height is followed successively by barren wort total chromocor extract low, middle and high dose groups, levothyroxine sodium tablet treatment group.Rat T4, FT3, FT4 content rising degree height are followed successively by barren wort total chromocor extract low, middle and high dose groups, left-handed first in serum Shape parathyrine Na Pian treatment group.
2 barren wort total chromocor extract of table is to IFN-γ in HT rat blood serum, IL-17, IL-6, IL-10, IL-23 and The influence (x ± s, n=6) of the content of TGF-β
Note: compared with normal group,▲P<0.05,▲▲P<0.01；Compared with model group:★P < 0.05,★★P < 0.01
By table 2 and Fig. 4 it is found that compared with normal group, IFN-γ in serum in model group, IL-17, IL-6, IL-23 and The content of TGF-β obviously rises, and index IL-10 content is decreased obviously, and has significant difference (P < 0.05).Each administration group is being given After two weeks, index IFN-γ, IL-17, IL-6, IL-23 and TGF-β have different degrees of decline compared to model group to medicine, wherein The decline of barren wort total chromocor extract high dose group is obvious, has significant difference (P < 0.01).Index IL-10 upon administration, Each administration group rises compared to model group, wherein barren wort total chromocor extract high dose group and levothyroxine sodium tablet treatment Group rising degree is maximum, has significant difference (P < 0.05).
3.3 barren wort total chromocor extracts are to Treg, Th17 impact cell in HT rat peripheral blood
Table 3 and Fig. 5 are it is found that model group is decreased obviously compared with the Treg cell proportion of blank control group, and there are statistical significances (P<0.01).The Treg cell proportion of each dosage group of barren wort total chromocor extract and levothyroxine sodium tablet positive drug group is bright It is aobvious to increase, it is statistically significant compared with model group (P < 0.01 or P < 0.05), as barren wort total chromocor extract is administered Dosage increases, and Treg cell proportion rises.
By table 3 and Fig. 6 it is found that model group is significantly raised (P < 0.01) compared with the Th17 cell proportion of blank control group.Excessive sheep Corresponding Th17 cell ratio in each dosage group of leaves of pulse plants extractive of general flavone and levothyroxine sodium tablet positive drug group rat peripheral blood Example significantly reduces (P < 0.01 or P < 0.05) compared with model group, as barren wort total chromocor extract dosage increases, Th17 cell proportion declines therewith.
3 each group rat peripheral blood Treg and Th17 cell proportion of table
Note: compared with blank control group,▲▲P < 0.01；Compared with model group, * P < 0.05, * * P < 0.01
FoxP3 in 3.4 immunohistochemical observation Hashimoto thyroiditis rat model thyroid glands, ROR γ t, IL-17, IL-23, The expression of IL-6, IL-10 and IFN-γ
The ImmunohistochemistryResults Results as shown in table 4-10 and Fig. 7-20 show each group rat thyroid tissue have FoxP3, ROR γ t, IL-17, IL-23, IL-6, IL-10 and IFN-γ positive expression.Compared to normal group, model group rats ROR γ t, IL-17, IL-23, IL-6 and IFN-γ positive expression are significantly raised (P < 0.01).Upon administration, each to be administered relative to model group Group rat ROR γ t, IL-17, IL-23, IL-6 and IFN-γ positive expression, positive staining area and accumulation OD value are bright Aobvious to reduce, reduction degree is followed successively by levothyroxine sodium tablet positive drug group, epimedium flavone high dose group, excessive sheep from high to low Leaves of pulse plants flavones middle dose group, epimedium flavone low dose group.
FoxP3 and IL-10 index, compared to normal group, model group positive expression is substantially reduced (P < 0.01), after administration, Each administration group expression is significantly raised.
Influence that 4 epimedium flavone of table expresses Hashimoto thyroiditis rat thyroid tissue FoxP3 (N=6)
Note: compared with Normal group,▲: P < 0.05,▲▲: P < 0.01；Compared with model control group,★: P < 0.05,★★: P <0.01。
Influence that 5 epimedium flavone of table expresses Hashimoto thyroiditis rat thyroid tissue ROR γ t (N=6)
Note: compared with Normal group,▲: P < 0.05,▲▲: P < 0.01；Compared with model control group,★: P < 0.05,★★: P <0.01。
Influence that 6 epimedium flavone of table expresses Hashimoto thyroiditis rat thyroid tissue IL-17 (N=6)
Note: compared with Normal group,▲: P < 0.05,▲▲: P < 0.01；Compared with model control group,★: P < 0.05,★★: P <0.01。
Influence that 7 epimedium flavone of table expresses Hashimoto thyroiditis rat thyroid tissue IL-23 (N=6)
Note: compared with Normal group,▲: P < 0.05,▲▲: P < 0.01；Compared with model control group,★: P < 0.05,★★: P <0.01。
Influence that 8 epimedium flavone of table expresses Hashimoto thyroiditis rat thyroid tissue IL-6 (N=6)
Note: compared with Normal group,▲: P < 0.05,▲▲: P < 0.01；Compared with model control group,★: P < 0.05,★★: P <0.01。
Influence that 9 epimedium flavone of table expresses Hashimoto thyroiditis rat thyroid tissue IL-10 (N=6)
Note: compared with Normal group,▲: P < 0.05,▲▲: P < 0.01；Compared with model control group,★: P < 0.05,★★: P <0.01。
Influence that 10 epimedium flavone of table expresses Hashimoto thyroiditis rat thyroid tissue IFN-γ (N=6)
Note: compared with Normal group,▲: P < 0.05,▲▲: P < 0.01；Compared with model control group,★: P < 0.05,★★: P <0.01。
3.6 qRT-PCR detect rat thyroid FoxP3, ROR γ t, TGF-β 1, IL-17, IL-23, IL-6, IL-10 and IFN-γ gene relative expression quantity
It can be seen that compared with the control group from Figure 21 and table 11, gene ROR γ t, IL-17, IL-23, IL-6 in model group It is significantly increased with the expression quantity of IFN-γ and IL10, the expression quantity of TGF-β and Foxp3 significantly reduce (P < 0.01)；With model group Compare, barren wort total chromocor extract low dose group, barren wort total chromocor extract middle dose group, barren wort total chromocor extract The expression quantity of high dose group and positive drug group ROR γ t, IL-17, IL-23, IL-6 and IFN-γ gene gradually decrease, and IL10, The expression quantity of TGF-β and Foxp3 gene gradually rises (P < 0.01 or P < 0.05), and dose dependent is presented.
11 rat thyroid related gene relative expression quantity statistical form of table
Note:▲▲Indicate p < 0.01 compared with Normal group；★Indicate p < 0.05 compared with model group；★★Expression and model Group compares p < 0.01
3.7 WB detect rat thyroid FoxP3, ROR γ t, TGF-β 1, IL-17, IL-23, IL-6, IL-10 and IFN- γ protein expression situation
It can be seen that compared with the control group from Figure 22, Figure 23 and table 12, ROR γ t, IL-17, IL-23, IL- in model group 6 and the expressing quantity of IFN-γ significantly increase and IL10, the expressing quantity of TGF-β and Foxp3 significantly reduce (P < 0.01)； Compared with model group, barren wort total chromocor extract low dose group, barren wort total chromocor extract middle dose group, Herba Epimedii is always yellow The expressing quantity of ketone extract high dose group and positive drug group ROR γ t, IL-17, IL-23, IL-6 and IFN-γ gradually drop It is low, and the expressing quantity of IL10, TGF-β and Foxp3 gradually rise (P < 0.01 or P < 0.05), and dose dependent is presented.
12 rat thyroid GAP-associated protein GAP relative expression quantity statistical form of table
Note:▲▲Indicate p < 0.01 compared with Normal group；★Indicate p < 0.05 compared with model group；★★Expression and model Group compares p < 0.01
3.8 IL-23 secrete the influence of IL-17 and IFN-γ to HT peripheral blood mononuclear cells of rats
By table 13,14 it is found that before non-administration, model group and each administration group without IL-23 stimulation are compared with blank control group IL-17 and IFN-γ expression obviously rise, and there are statistical significance (P < 0.01)；After IL-23 is stimulated, model group IL-17 and IFN-γ expression with each administration group compared with blank control group obviously rise, there are statistical significance (P < 0.01).After IL-23 is stimulated, each group IL-17 and IFN-γ expression are above the same group of cell without IL-23 stimulation.
After administration, for the model group without IL-23 stimulation compared with blank control group, IL-17 and IFN-γ expression are equal Obvious to rise, there are statistical significance (P < 0.01), for each administration group compared with model group, IL-17 and IFN-γ expression are equal Be substantially reduced, the expression of levoid group is minimum, IL-17 and IFN-γ expression with Contained Serum administration concentration It increases and gradually decreases, it is statistically significant (P < 0.01 or P < 0.05).After IL-23 is stimulated, each administration group and model Group compares, and IL-17 and IFN-γ expression are substantially reduced, levoid positive drug group expression it is minimum, IL-17 and IFN-γ expression is gradually decreased as the administration concentration of Contained Serum increases, statistically significant (P < 0.01 or P < 0.05)。
Cytokines in Peripheral Blood Mononuclear IL-17 and IFN-γ express water after IL-23 is stimulated before the administration of 13 each group rat of table Flat influence (Pg/ml, n=3)
Note: compared with blank control group,▲▲P < 0.01；Compared with model group, * P < 0.05, * * P < 0.01
Cytokines in Peripheral Blood Mononuclear IL-17 and IFN-γ express water after IL-23 is stimulated after the administration of 14 each group rat of table Flat influence (Pg/ml, n=3)
Note: compared with blank control group,▲P < 0.05,▲▲P < 0.01；Compared with model group, * P < 0.05, * * P < 0.01
The preparation of 3.9 rat thyroid epithelium primary cells
Rat thyroid tissue purifies after digesting obtains the irregular attached cell of paving stone shape, such as Figure 24.Cell warp Cell survival rate is obtained after crossing Trypan Blue as (97.43 ± 5.62) %.
3.10 dextran sulfate sodiums cause rat thyroid epithelial cell damage model foundation
With blank control group ratio, rat thyroid epithelial cell after various concentration DSS and different role temporal induction, OD value reduces after each administration group cell.As shown in Table 15, DSS unites when concentration is greater than 15 μ g/ml and effect for 24 hours or more Meter learns difference (p < 0.05, p < 0.01), wherein for the DSS of 20 μ g/ml when acting on for 24 hours, difference is most significant (p < 0.01).
15 various concentration DSS of table act on rat thyroid cell different time OD value (N=4)
Note: compared with blank control group,▲P < 0.05,▲▲P < 0.01
3.11 rat thyroid epithelial cell proliferations and toxicity detection
After the barren wort total chromocor extract Contained Serum group of various concentration is used for cell, compared with blank control group, respectively The cell OD value of administration group slightly reduces, but no difference of science of statistics (P > 0.05).With blank control group ratio, the LDH of group of cells Activity increases with the increase of administration concentration, and wherein barren wort total chromocor extract high dose group dramatically increases, and has statistics Difference (P < 0.05).No difference of science of statistics (P > 0.05) is compared between barren wort total chromocor extract administration group.
16 various concentration Contained Serum of table to rat thyroid cell proliferative and toxic effect (N=4)
Note: compared with blank control group,▲P < 0.05,▲▲P < 0.01 and barren wort total chromocor extract low dose group ratio Compared with * P < 0.05, * * P < 0.01.
3.13 drug-treateds and cell Proliferation detection
With blank control group ratio, model group cell OD value conspicuousness is reduced, and there are statistical difference (P < 0.01)；With model Group ratio, barren wort total chromocor extract group cell OD value conspicuousness rise, and have statistical difference (P < 0.05).
17 Contained Serum of table act on rat thyroid cell activity influence (N=4)
Note: compared with blank control group,▲P < 0.05,▲▲P < 0.01；Compared with model group, * P < 0.05, * * P < 0.01
Influence of the 3.14 medicine serum to Apoptosis
By table -18 and Figure 25 it is found that model group obviously rises compared with the apoptosis rate of blank control group, there are statistics meanings Adopted (P < 0.01).The apoptosis rate ratio of each dosage group of epimedium flavone and levothyroxine sodium tablet positive drug group obviously drops It is low, it is statistically significant compared with model group (P < 0.01 or P < 0.05), as epimedium flavone dosage increases, cell Apoptosis rate reduces.
18 medicine serum of table to Apoptosis influence (%,N=3)
Note: compared with blank control group,▲P < 0.05,▲▲P < 0.01；Compared with model group, * P < 0.05, * * P < 0.01
To sum up, barren wort total chromocor extract has apparent treatment Hashimoto thyroiditis to Hashimoto thyroiditis rat Effect.Barren wort total chromocor extract of the present invention proves that safety is good, and curative effect is good through results of animal, can effectively treat Hashimoto thyroiditis, the barren wort total chromocor extract are that a kind of improvement clinical symptoms effect is good, and toxic side effect is small, are had good Potential applicability in clinical practice Chinese medical extract.
2 barren wort total chromocor extract tablet of embodiment or other health care products
The ingredient of barren wort total chromocor extract tablet is
Ingredient Every dosage
Barren wort total chromocor extract 100mg
Microcrystalline cellulose 37.5mg
Superfine silica gel powder 10.5mg
Stearic acid 17.5mg
Lactose 10.5mg
Magnesium stearate 1.05mg
Microcrystalline cellulose, micro mist silicon is added in barren wort total chromocor extract (being made by 1 method of embodiment) by recipe quantity Glue, stearic acid, lactose mix, and magnesium stearate is added after particle is made, and mix, and tabletting is to get (every contains barren wort total chromocor 80mg).Adult recommends oral 2-4 pieces/times of dosage, 2-3 times daily.
3 barren wort total chromocor extract capsule of embodiment or other health care products
The ingredient of barren wort total chromocor extract capsule is
Ingredient Every dosage
Barren wort total chromocor extract 100mg
Microcrystalline cellulose 37.45mg
Superfine silica gel powder 10.5mg
Stearic acid 17.5mg
Lactose 10.5mg
Microcrystalline cellulose, micro mist silicon is added in barren wort total chromocor extract (being made by 1 method of embodiment) by recipe quantity Glue, stearic acid, lactose mix, and after particle is made, are filled into No. 0 hard capsule case, and polishing obtains that (every capsule is total containing Herba Epimedii Flavones 80mg).Adult recommends oral 2-4 tablets/time of dosage, 2-3 times daily.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form Limitation, there are also other variations and modifications on the premise of not exceeding the technical scheme recorded in the claims.
Sequence table
<110>Zhejiang Provincial People's Hospital
<120>application of the barren wort total chromocor in the drug or health care product of preparation prevention and treatment Hashimoto thyroiditis
<130> W-HLY19-031
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Rat IL6-F)
<400> 1
ttccagccag ttgccttctt 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Rat IL6-R)
<400> 2
ctggtctgtt gtgggtggta 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Rat IL17-F)
<400> 3
gtgaaggcag cggtactcat 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Rat IL17-R)
<400> 4
gggtgaagtg gaacggttga 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Rat IL23-F)
<400> 5
caaaggatcc gccaaggtct 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Rat IL23-R)
<400> 6
tggaacggag aagagaacgc 20
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Rat INF- γ-F)
<400> 7
ggcaaaagga cggtaacacg 20
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Rat INF- γ-R)
<400> 8
tctgtgggtt gttcacctcg 20
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Rat IL10-F)
<400> 9
aagggttact tgggttgcca 20
<210> 10
<211> 21
<212> DNA
<213>artificial sequence (Rat IL10-R)
<400> 10
tggccttgta gacacctttg t 21
<210> 11
<211> 20
<212> DNA
<213>artificial sequence (Rat ROR γ t-F)
<400> 11
cctcctgcca ccttgagtat 20
<210> 12
<211> 20
<212> DNA
<213>artificial sequence (Rat ROR γ t-R)
<400> 12
tctgagccct gttctggttc 20
<210> 13
<211> 20
<212> DNA
<213>artificial sequence (Rat TGF-β-F)
<400> 13
ctgctgaccc ccactgatac 20
<210> 14
<211> 20
<212> DNA
<213>artificial sequence (Rat TGF-β-R)
<400> 14
agccctgtat tccgtctcct 20
<210> 15
<211> 20
<212> DNA
<213>artificial sequence (Rat Foxp3-F)
<400> 15
gcccacacct cctcttcttc 20
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Rat Foxp3-R)
<400> 16
ctgtcctgga gaagtgcctg 20
<210> 17
<211> 22
<212> DNA
<213>artificial sequence (Rat β-actin-F)
<400> 17
cagggtgtga tggtgggtat gg 22
<210> 18
<211> 22
<212> DNA
<213>artificial sequence (Rat β-actin-R)
<400> 18
agttggtgac aatgccgtgt tc 22

Claims (4)

1. a kind of application of barren wort total chromocor in the drug or health care product of preparation prevention and treatment Hashimoto thyroiditis.
2. application according to claim 1, it is characterised in that: the drug or health care product of the prevention and treatment Hashimoto thyroiditis It is using barren wort total chromocor as active constituent, in addition the preparation that pharmaceutically acceptable auxiliary material is prepared.
3. application according to claim 1, it is characterised in that the preparation method of the barren wort total chromocor is:
(1) Herba Epimedii Preparata: taking Herba Epimedii crude drug, removes impurity, wins blade, sprays clear water, slightly moistens, chopping, dry；Take refining Suet oil heats fusing, and Herba Epimedii silk is added, and is fried with slow fire the aobvious uniform glossy gloss in surface, when being in yellowish, takes out, put It is cool；Herba Epimedii every 100kg refining suet oil 20kg；
(2) preparation of extractive of general flavone: taking Herba Epimedii Preparata appropriate, and respectively plus 70% ethyl alcohol, 10 times of amounts extract 2 times, every time 2h；Medical fluid is concentrated into 1mL medical fluid crude drug containing 1g, the technique using HPD300 macroporous resin purification general icariin passes through investigation It determining are as follows: medical fluid is diluted with water into every 1mL medical fluid crude drug containing 0.1g, passes through HPD300 large pore resin absorption column with 4BV/h flow velocity, First 5 times of column volume washings, after with 7 times of 50% ethanol elutions of column volume, elution flow rate 4BV/h collects eluent, is recovered under reduced pressure Ethyl alcohol, filtration, filtrate add 3% gelatin solution to precipitating is not generated, add ethyl alcohol to make alcohol content up to 80%, stand overnight, and filter, subtract Receipts ethyl alcohol is pushed back, barren wort total chromocor extract is made.
4. application according to claim 1, it is characterised in that: in the drug or health care product, barren wort total chromocor contains Amount is 10% or more.
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