CN106474424B - Treat application of the psoriasis Chinese medicine in the drug of preparation prevention and treatment type 1 diabetes - Google Patents

Abstract

The present invention relates to application of the treatment psoriasis Chinese medicine in the drug of preparation prevention and treatment type 1 diabetes, wherein the effective component of the prevention and treatment type 1 diabetes drug is made by following raw material medicine: 15 parts of Glabrous Sarcandra Herb, 9 parts of curcuma zedoary, 15 parts of dark plum, 60 parts of smilax, 9 parts of radix paeoniae rubra, 15 parts of Asian puccoon, 6 parts of Radix Glycyrrhizae.The drug of prevention and treatment type 1 diabetes of the present invention can reduce the infiltration of inflammation in mice pancreatic tissue, protects islet cells, prevents and treats the significant effect of type 1 diabetes.

Description

Treat application of the psoriasis Chinese medicine in the drug of preparation prevention and treatment type 1 diabetes

Technical field

The present invention relates to medical preparations, and in particular to contains the pharmaceutical preparation for not determining structure from plant.

Background technique

Type 1 diabetes are that a kind of mediated by T lymphocyte causes pancreas islet to damage itself beta Cell of islet generation immune response Wound, the anxious hair property chronic disease that insulin absolutely lacks, needs insulin life-long therapy.Foreign data shows whole world different regions Type 1 diabetes disease incidence have decades of times difference, highest is Finland white man, and up to 36.0/10 ten thousand man-years, and south east asia is then Lower, the result of report is 2.0/10 ten thousand man-years or so, and the disease disease incidence is up to 0.6/10 ten thousand in 0~14 years old crowd in China.By This apparently, it is harm that although China's type 1 diabetes disease incidence is lower, the numerous of population, which determine patient and not within minority, One of disease of human health.Have the tendency that research data shows that China teenager type 1 diabetes patient shows and rises year by year, Disease incidence also increases with the growth at age, and the disease incidence difference between nationality is up to 12 times, and disease incidence is between different regions The now low north Gao Xianxiang in apparent south.Therefore it finds effective drug treated and improve the disease and has become an extremely urgent times Business.

In type 1 diabetes (i.e. insulin-dependent, IDDM) patient, pancreas often has apparent pathological change, β cell number Amount is only normal 10% hereinafter, mostly scorching with pancreas island.Main pathological change has: 1. pancreas island atrophys: this is type 1 diabetes The most common pathological change, shows as cell shrinkage, narrows in pencil.The islet cell form of atrophy is smaller, and core is fine and close, Endochylema is few.Such pyknotic cells can still secrete glucagon, growth hormone release inhibiting hormone and pancreatic polypeptide hormone, but seldom excreting insulin. 2. pancreas island hyperplasia fertilizer exists: this is another more rare material alterations, is more common in that the course of disease is short or the patient of neopathy.Pancreas island Hyperplasia, in addition it is loose, and diameter is more than 300~400 μm.Cell is larger, and shape rule is clear, and core is larger.After granules stain Observation, island are mainly contained with epidemic β cell, and particle is almost sloughed entirely, also contain α cell.For " honeymooners " state of an illness The performance of alleviation, β cell can almost disappear when aggravation after the several years.3. β cell vacuolar degeneration: being mainly seen in this disease Acute, feature are that cytoplasm is emptying, and particle or other organelles are had no under light microscopic.Mainly due to glycogen daughter nucleus it is calm caused by, β cell permanent damage is not caused, reversible change is belonged to.4. pancreas island is scorching: 50%~70% type 1 diabetes patient, especially It is the children dead shortly after that fall ill, and Chang Kejian has pancreas island and its surrounding to have the infiltration of lymphocyte and monocyte, is Inflammation of pancreatic islet (insulitis).Thus the pathogenesis of autoimmune response system type 1 diabetes is prompted.

Although doctor trained in Western medicine can not also cure type 1 diabetes, great progress is achieved in clinic, treatment means are increasingly various Change, delayed the generation of disease to some extent, mitigate the symptom of disease, improves the quality of life of type 1 diabetes patient.It is close several Year, the treatment of type 1 diabetes achieves many new breakthroughs, such as pancreatic islets transplantation, insulin analog and administration route, gene, The research of immunologic intervention, stem-cell therapy etc. provides new idea and method for the treatment of type 1 diabetes.But pure west Doctor's preparation side effect is larger, and blood glucose fluctuation is obvious, and it is poor that injection of insulin treats patient compliance, so being badly in need of developing for controlling Treat the Chinese medicine compound prescription of type 1 diabetes.

The research about Chinese medicine treatment type 1 diabetes domestic at present is confined to traditional Chinese medicine monomer or active constituent more, and Chinese medicine The research that composition treats type 1 diabetes is seldom.Chen Wei etc. the study found that apply astragalus polyose (Astragalus in early days Polysacharin, APS) the pre- immune defect that can correct NOD mouse Ts cell, restore its cell function, makes immunosupress Effect is reinforced, and Thl type cell/cell factor immune imbalance state is corrected, to prevent or delay NOD diabetes mice to send out Disease.The discovery ginseng extract such as Kobayashi can reduce the IFN-γ content of NOD mouse, increase IL-4 content to prevent glycosuria The occurrence and development of disease.Green onion acrylic component Rhein has the function of obviously treating NOD diabetes mice and its nephrosis, can be with bright The aobvious damage for improving db/db diabetes mice nephrosis, delays renal hypofunction.Research finds mulberry leaf water extract to streptozotocin The therapeutic effect of the type 1 diabetes mouse islets of (Streptozotocin, STZ) induction and health care function to kidney and liver Effect.The results of study such as Kang Min show that black tartary buckwheat powder causes diabetic mice to have certain blood sugar reducing function alloxan.Shi Nianwei etc. Research finds tripterygium glycosides by lowering the Th1 expression in NOD mouse pancreas to prevent the generation of diabetes.Cao Li etc. has found Pueraria lobota Root element can significantly improve the insulin resistance of tissue of experimental diabetic mice.The discoveries such as Wang Liying early stage applies pre- be immunized of tea polysaccharide can To prevent or delay the generation of NOD mouse type 1 diabetes.About the Chinese medicine of prevention and treatment type 1 diabetes, state is known in the patent document of office Successively disclose various Chinese medicine compound prescriptions.Wherein, CN104042928 patent application is disclosed by 40-60 parts of dendrobium nobile, faenum graecum 5-15 The compound of part preparation；CN103816438 patent application is disclosed by pueraria lobata 50g；Balsam pear 50g；Corn stigma 50g；Guava Leaf 25g；Radix Ophiopogonis 20g；Kelp 20g；Fructus lycii 15g；Malt 10g；Radix Rehmanniae 9g；Radix paeoniae rubra 6g；The compound of melbine 0.03g preparation； CN103316101 patent application is disclosed by 6-15 parts of faenum graecum, 6-15 parts of Radix Astragali, 3-8 parts of Herba Epimedii, 3-8 parts of psoralea corylifolia, mountain 3-8 parts of the fruit of medicinal cornel, 1-5 parts of rheum officinale, 3-8 parts of cortex cinnamomi, 2-6 parts of the coptis compounds prepared；CN103005446 patent application disclose by The pure natural composite tablet that corn stigma, Cordyceps militaris synthesize is function of blood sugar reduction food；CN103585431 patent application discloses By 3-9 parts of elecampane, 3-9 parts of folium artemisiae argyi, 6-12 parts of raspberry, 15-30 parts of Japanese polygala, 3-6 parts of root of kirilow rhodiola, 10-15 parts of semen ziziphi spinosae, 6-12 parts of rhizoma anemarrhenae, 9-15 parts of Gorgon fruit, the compound of 6-12 parts of Semen Cuscutae preparations.

The patent application of Publication No. CN101632827A, which discloses, a kind of treats psoriasis Chinese medicine composition, the composition It is formulated by radix paeoniae rubra, Glabrous Sarcandra Herb, curcuma zedoary, dark plum, Asian puccoon, smilax, 7 taste Chinese traditional herbs of Radix Glycyrrhizae, there is promoting blood circulation Ecchymose removing, the antipruritic function of dispelling wind and eliminating dampness.Said medicine is answered by national distinguished veteran doctors of TCM, famous skin disease Zhuan man Huen state dimension professor's clinic It is added and subtracted with the Empirical formula of many decades, is the common medicine that Guangdong Provincial TCM Hospital is used to treat psoriasis, disappears with promoting blood circulation The effect of spot, dispelling wind and eliminating dampness is antipruritic, the psoriasis for being clinically used for treatment blood-deficiency and wind-dry type are curative for effect.

Modern pharmacological studies have shown that for there are the Chinese medicine composition a variety of pharmacological activity, overview to rise, it is published at present Pharmacological action reported in the literature is as follows:

(1) index components such as the Paeoniflorin and glycyrrhizic acid that contain in side have the effects that anti-inflammatory, immunosupress, Glabrous Sarcandra Herb In contain the compounds such as chlorogenic acid, Rosmarinic acid, astilbin, it may have (Feng Limin, Zhao Rui the effects of anti-inflammatory, immunosupress Sesame, Wang Yinjie, Han Ling, when the preferred silver of Lu Chuanjian orthogonal design considers extraction process [J] of Glabrous Sarcandra Herb effective component in clever piece to be worth doing Precious traditional Chinese medical science traditional Chinese medicines, 2011,08:1860-1861.).

(2) the alkannin Human Keratinocytes strain proliferation in Asian puccoon plays the role of inhibition and apoptosis (Wu Xiaoxia, Zhou Wu The inhibition of alkannin Human Keratinocytes cell strain proliferation and effect [J] the China leprosy dermatology magazine of apoptosis are celebrated, 2003,19 (6): 563-565.).

(3) curcuma zedoary can make natural parakeratosis epidermal cell be converted into normal cell, and epithelial cell can be inhibited to have silk point It splits and plays effect (Xu Houqian, Li Yingdong curcuma zedoary research overview [J] Gansu Chinese of Traditional Chinese Medicine for inhibiting epidermal hyperplasia too fast Report, 1995,12 (1): 46.).

(4) all medicines of the activating microcirculation and removing stasis medicinal such as curcuma zedoary, Glabrous Sarcandra Herb, red spoon, which have the pathology of skin disease microcirculation disorder, significantly changes (Huang Yongjing, Wu Yuansheng, Liao Liehui, Wang Lei Acitretin Capsules joint silver consider the clinic of clever piece treatment psoriasis vulgaris to be worth doing for kind effect Observation [J] China skin cypridology magazine, 2007,10:643-644.).

(5) inhibited (Lu Chuanjian, Liu Fengnian the " silver for the IL-8 that Chinese medicine extract generates TNF-α stimulation The Liaoning influence [J] of the clever piece of bits " Chinese medicine extract to tumor necrosis factor-alpha stimulation relief angle protein cell secretion of cytokines IL-8 Journal of Traditional Chinese Medicine, 2009,11:1862-1863.).

The above research prompt, treatment psoriasis Chinese medicine composition have certain anti-inflammatory, immunosuppressive action.Currently, should Side is there is not yet be applied to prevention and treatment type 1 diabetes (insulin-dependent diabetes mellitus, insulin-dependent Diabetes mellitus, abbreviation IDDM) research report.

Summary of the invention

The technical problem to be solved in the present invention is to provide new application of the treatment psoriasis Chinese medicine in pharmacy.

New application of the above-mentioned treatment psoriasis Chinese medicine in pharmacy is specifically:

Treat application of the psoriasis Chinese medicine in the drug of preparation prevention and treatment type 1 diabetes；The medicine of the prevention and treatment type 1 diabetes Object be it is a kind of described in the patent application of Publication No. CN101632827A treat psoriasis Chinese medicine composition, effective component by Following raw material is made:

15 parts of Glabrous Sarcandra Herb, 9 parts of curcuma zedoary, 15 parts of dark plum, 60 parts of smilax, 9 parts of radix paeoniae rubra, 15 parts of Asian puccoon, 6 parts of Radix Glycyrrhizae.

The drug of above-mentioned prevention and treatment type 1 diabetes is tablet, and the preparation method of the tablet comprises the steps of:

(1) it by the bulk pharmaceutical chemicals, directly smashs into coarse powder to pieces, the ethyl alcohol that 10 times of amount volumetric concentrations are 70% is added and extracts two It is secondary, 2 hours every time, combined extract；

(2) it is concentrated under reduced pressure, recycles ethyl alcohol, medicinal extract is made；

(3) medicinal extract is taken, the starch of 3 times of weight is added, is mixed, is pelletized, it is dry, stearic acid is added by the 5% of tablet weight Magnesium is mixed, tabletting.

The present invention is moved NOD (shi/Ltj) female mice splenic lymphocytes using treatment psoriasis traditional Chinese medicine composition for treating It plants in NOD-SCID female mice body and induces the mouse model of type 1 diabetes and converted using clinically adult taking dose And the drug concentration come intervenes type 1 diabetes mouse model 33 days, 35 days after observation mouse blood sugar to modeling, then materials are seen Survey index.

The result shows that treatment psoriasis Chinese medicine composition is remarkably improved islet cells in type 1 diabetes model mouse pancreas Survival rate, delay mouse invasion, reduce the occurring degree of mouse type 1 diabetes, extend the life cycle of type 1 diabetes mouse. Beta Cell of islet is attacked the important immune factor for immune response occur and being initiation type 1 diabetes, the Chinese traditional medicine composition by self T-cell Object for lymphocyte transplanting establish animal mold have effective protection effect, imply can be used for type 1 diabetes prevention and Treatment.According to the Couple herbs of psoriasis, the Chinese medicine composition usage is as follows: adult every consumption per day is equivalent to above-mentioned effective component 2.15g crude drug/kg weight, adds water to cook 2-3 amalgamation liquid, takes in three times, and 150 milliliters every time, 30 days courses for the treatment of.

In order to which the public is better understood when beneficial effects of the present invention, will be furtherd elucidate below by zoopery.

Detailed description of the invention

Fig. 1 is the change of blood sugar trend curve figure of mouse after modeling." Control " indicates normal group, and " Model " indicates mould Type group, " Treantment " indicate Chinese medicine processing group, i.e. treatment group.

Fig. 2 is the disease incidence curve graph of mouse after modeling." Control " indicates normal group, and " Model " indicates model group, " Treantment " indicates Chinese medicine processing group, i.e. treatment group.

Fig. 3 is the average weight curve graph of mouse after modeling." Control " indicates normal group, and " Model " indicates model Group, " Treantment " indicate Chinese medicine processing group, i.e. treatment group.

Fig. 4 is the average amount of drinking water curve graph of mouse after modeling." Control " indicates normal group, and " Model " indicates model Group, " Treantment " indicate Chinese medicine processing group, i.e. treatment group.

Fig. 5 is the average appetite curve graph of mouse after modeling." Control " indicates normal group, and " Model " indicates model Group, " Treantment " indicate Chinese medicine processing group, i.e. treatment group.

Fig. 6 is that the HE of each group mice pancreatic tissue dyes picture." Control " indicates normal group, and " Model " indicates model Group, " Treantment " indicate Chinese medicine processing group, i.e. treatment group." 10X " indicates 100 times of amplification, and " 20X " indicates 200 times of amplification, " 40X " indicates 400 times of amplification.

Fig. 7 is the immunohistochemical staining picture of each group mice pancreatic tissue insulin antibody." Control " is indicated just Normal group, " Model " indicates model group, and " Treantment " indicates Chinese medicine processing group, i.e. treatment group." 10X " indicates amplification 100 Times, " 20X " indicates 200 times of amplification, and " 40X " indicates 400 times of amplification.

Fig. 8 is the immunohistochemical staining picture of each group mice pancreatic tissue CD3 antibody." Control " indicates normal Group, " Model " indicate model group, and " Treantment " indicates Chinese medicine processing group, i.e. treatment group." 10X " indicates 100 times of amplification, " 20X " indicates 200 times of amplification, and " 40X " indicates 400 times of amplification.

Fig. 9 is the result histogram of the Q-PCR of purpose gene." Control " indicates normal group, and " Model " indicates model Group, " Treantment " indicate Chinese medicine processing group, i.e. treatment group.

Specific embodiment

Following experiments observe the blood glucose of different time points by the model using the animal model of simulation people's type 1 diabetes Therapeutic effect of the value verifying treatment psoriasis Chinese medicine composition for type 1 diabetes.Firstly, groping modeling by tentative experiment Method establishes stable type 1 diabetes mouse model, and then by subsequent experiment, further psoriasis Chinese medicine group is treated in verifying Closing object has the function of reducing blood glucose and delays morbidity.

1 treatment psoriasis Chinese medicine composition reduces blood glucose for type 1 diabetes mouse model and delays the effect of morbidity

The foundation of 1.1 type 1 diabetes mouse models

1.1.1 instrument, material and reagent

Superclean bench, computer, tubular type low speed room temperature centrifuge (German Eppendorf company)；Several sets-of disscting instrument Scissors and tweezers, 5mL syringe needle handle, Tissue Culture Dish (35mm × 10mm), 45mL centrifuge tube, 15mL centrifuge tube, strainer carry Object platform, tissue, emgloves, pipe support, waste cylinder, 1mL syringe, ice chest, cell counting board (Countstart), 1mL/ 200 μ L/10 μ L liquid-transfering guns (German Eppendorf company) and pipette tips, electronic liquor-transferring rifle, 10mL pipette etc.；PBS (1 ×) is molten Liquid, erythrocyte cracked liquid (1 ×, ACK Lysis Buffer), 10%FBS/PBS solution, 0.2% trypan blue, ethanol for disinfection.

1.1.2 spontaneous type 1 diabetes NOD (shi/Ltj) female mice screening

At raising NOD (shi/Ltj) female mice ﹥ 4 months, pass through the wet of observation mouse padding, adhesion degree and drink Whether water preliminary judgement mouse falls ill, start detect blood glucose, by blood glucose >=11.1mmol/L mouse be marked and with not Falling ill, mouse is separated, and modeling is spare.

1.1.3 the separation and transplanting of spontaneous type 1 diabetes NOD (shi/Ltj) female mice splenic lymphocytes

1. super-clean bench shines ultraviolet disinfection, ACK solution rewarming is taken out, takes out idiopathy modeling mouse, preparation from animal house Ice chest holds spleen with the 45mL centrifuge tube equipped with a small amount of 10%FBS/PBS solution.

2. modeling mouse cervical dislocation is put to death, 75% ethanol disinfection is sprayed, abdomen is opened from side and takes out spleen, be put into In 10%FBS/PBS solution, until the spleen of all mouse all takes.

3. spleen is placed on strainer, it is placed in the culture dish equipped with a certain amount of 10%FBS/PBS solution, one by one It shreds, grinds, then be placed in ice chest by strainer filtering into 45mL centrifuge tube.

4. being centrifuged with tubular type low speed room temperature centrifuge, 1300 revs/min or 300g, it is centrifuged 7 minutes.

5. pouring out supernatant after centrifugation, the ACK solution of 2mL is first added, is blown and beaten uniformly, is stored at room temperature 30 seconds with pipette tips, Supernatant is shifted into new centrifuge tube, discards white precipitate, then the ACK solution of 14mL, mixing of turning upside down, room are added in new pipe Temperature is stood to 5 points, is shoaled to solution colour.

6. suitable PBS solution, which is then added, stops reaction, mixing of turning upside down, 300g is centrifuged 7 minutes.

7. pouring out supernatant after centrifugation, 2mLPBS piping and druming is added uniformly, then add to the PBS solution of 30mL pre-cooling, up and down It is reverse, same to pelleted by centrifugation.

8. pouring out supernatant after centrifugation, 4mLPBS is added, is blown and beaten uniformly with pipette, takes out 10 μ L cell suspensions, then PBS to 30mL cleaning, same pelleted by centrifugation is added.Meanwhile 10 the PBS solution that 490 μ L are added in μ L cell suspension dilute 50 times, use After pipette tips piping and druming uniformly, 20 μ L cell suspensions are further taken out, the trypan blue that the concentration of 20 μ L is 0.2% is added, is uniformly mixed, takes out 20 μ L count (diluting 400 times) with countstart Counting software, obtain 3.5 × 10⁶A cell, then total number of cells be 3.5 × 10⁶× 50 × 2 × 4=1.4 × 10⁹It is a.

9. pouring out supernatant after the complete heart, 10mLPBS is added, then cell concentration is 1.4 × 10⁸A/mL, because every small Mouse transplants 0.2mL cell suspension, then the cell quantity of every mouse transplanting is 2.8 × 10⁷It is a.

10. the cell suspension of 10mL is transferred in 15mL centrifuge tube, piping and druming uniformly, is placed in ice chest, brings animal house into.

11. drawing cell suspension, the cell suspension of every NOD-SCID mouse injection 0.2mL with the syringe of 1mL.

12. the NOD-SCID mouse for having made mould is grouped, it is numbered with picric acid.

1.1.4 result

It finally counts, we will send out NOD (shi/Ltj) mouse of IDDM (blood glucose >=11.1mmol/L) from 8 Part splenic lymphocytes successful implantation to 20 NOD-SCID Mice Bodies in, every mouse is implanted into splenic lymphocytes amount about 2.8 ×10⁷It is a.Modeling mouse is randomly divided into 2 groups, and model group 10, stomach-filling distilled water is used for curative effect comparision；Treatment group 10 Only, with treatment psoriasis Chinese medicine composition intervention processing.Normal NOD-SCID mouse 8 is only used as Normal group, is used for model Control.Due to operation error in the observation period, leading to stomach-filling model group early period and treatment group respectively has 1 dead mouse.

The preparation of 1.2 experimental drugs

Provide that the selection of mouse dosage is according to equivalent between different genera animal according to " herbal pharmacology experimental methodology " The direct convert formula of dosage calculates, and adult is daily equivalent to 129g crude drug, and the dosage of mouse is 12 times of adult, I.e. every 10g weight takes in 0.258g crude drug amount.Take leaching obtained by the patent application embodiment 1 of Publication No. CN101632827A Cream is diluted with water to and is equivalent to every milliliter of suspension and contains crude drug amount 1.89g, and 4 DEG C save backup.

1.3 stomach-fillings and blood sugar monitoring

Third day starts stomach-filling after modeling, and Normal group is not necessarily to any processing, model group stomach-filling distilled water, treatment group Stomach-filling medical fluid, two groups of stomach-filling capacity are consistent, and mouse weight weighing is carried out before stomach-filling, is made a record, and stomach-filling 33 days is to drawing materials. The random blood sugar that mouse tail vein blood sampling detects all mouse is begun through within the 15th day after modeling, Abbott Laboratories' ANTU blood glucose meter is used And test paper quickly detects.Blood sugar monitoring point is arranged at the 15th day, the 21st day, the 23rd day, the 25th day, the 26th day, the 27th day, always By the 35th day, mouse blood sugar record is carried out.

1.4 experimental result

1.4.1 variation of each group mouse general status before and after experiment

Normal group frequent activity, it is agile, it is swift in response；Model group is apathetic, slow to respond, slow to move, hair The withered tarnish of dried and withered hair, the back of a bow are curled up body；Treatment group's mental status, reaction, hair color activity increased significantly compared with model group.

1.4.2 variation of each group mouse average blood sugar before and after experiment

The 30th day after modeling, model group and treatment group's mouse blood sugar and three comparison among groups of normal group have significant difference (P < 0.01), hints model modeling success.Compared with normal group, treatment group is variant, but the model group significant difference that is far from, explanation Drug therapy is effective.(the results are shown in Table 1 and Fig. 1)

Mouse blood sugar (mmol/L) changes after 1 NOD-SCID mouse of table and modeling

Note: the comparison between totality, P=0.006 < 0.01, statistics are carried out using the variance analysis of single factor test duplicate measurements Significant difference.Mouse later period blood glucose is very high, and blood glucose meter can not detect, and shows " HI ", is indicated when statistical data with " 27.8 ".Due to It falls ill the later period, model group has reached " HI ", indicates zero deflection with " 27.8 ", for convenience of counting, indicates standard error with " 0.01 ".

1.4.3 variation of each group mouse invasion rate after modeling

Using mouse blood sugar >=11.1mmol/L as standard, determine whether the NOD-SCID mouse of modeling falls ill.3 weeks after modeling It is interior, each group mouse invasion rate no significant difference (P > 0.05), but model group (n=9) has obvious incidence trend, has after 4 weeks obvious Difference (P < 0.01) illustrates type 1 diabetes modeling success, and treatment group (n=9) morbidity is slowly, just has morbidity later by 4 weeks Trend has obvious incidence trend (P < 0.01), illustrates that drug therapy is effective when by the 32nd day.(the results are shown in Table 2 and Fig. 2)

NOD-SCID mouse IDDM disease incidence (%) after 2 modeling of table

Note: being compared two-by-two between three groups, since total sample size between two groups is less than 40, using definite in Chi-square Test Probabilistic method is compared, model group, treatment group compared with normal group,^△P < 0.05,^*P < 0.01,^**P < 0.001,^***P < 0.0001。

1.4.4 variation of each group average mice body weight before and after experiment

Test each group early period mouse weight no significant difference, each group mouse weight increases during the experiment, but three it Between without significant difference, it is on a declining curve to test later period model group mouse body weight, has significant difference compared with treatment group, illustrates reality Test the survival condition that medication can be obviously improved mouse.(the results are shown in Table 3 and Fig. 3)

Each group NOD-SCID average mice body weight changes after 3 modeling of table

Note: statistical method of three comparison among groups using the one-way analysis of variance of duplicate measurements, three comparison among groups difference poles Significantly.

1.4.5 each group mouse is averaged variation of the amount of drinking water before and after experiment

Each group early period mouse amount of drinking water no significant difference is tested, normally the fluctuation of group mouse amount of drinking water is little during the experiment, Substantially without what difference, and model group and treatment group are obviously in rising trend, illustrate modeling success.But model group with control Treatment group compares, and ascendant trend is significantly greater than treatment group, with the extremely significant property for the treatment of group's comparing difference, illustrates that experimental drug can obviously change The survival condition of kind mouse reduces average amount of drinking water.(the results are shown in Table 4 and Fig. 4)

After 4 modeling of table each group NOD-SCID mouse be averaged amount of drinking water variation

Note: statistical method of three comparison among groups using the one-way analysis of variance of duplicate measurements, three comparison among groups difference poles Significantly.

1.4.6 each group mouse is averaged variation of the appetite before and after experiment

Each group early period mouse appetite no significant difference is tested, during the experiment normal group and the fluctuation for the treatment of group mouse amount of drinking water Less, front and back is substantially without what difference, but treatment group is averaged appetite significantly lower than normal group, and model group is obviously in that rising becomes Gesture, it is overall to be higher than other two groups, illustrate modeling success.Treatment group's average amount of food is lower, it may be possible to due to stomach-filling experimental drug Reason.For model group compared with treatment group, the extremely significant property of difference illustrates that experimental drug can be obviously improved the survival condition of mouse, Reduce average amount of food.(the results are shown in Table 5 and Fig. 5)

Each group NOD-SCID mouse average amount of food changes after 5 modeling of table

Note: two comparison among groups use the statistical method of two independent samples t tests, and two comparison among groups differences are extremely significant.

2 explore treatment of the treatment psoriasis Chinese medicine composition for type 1 diabetes mouse model from albumen and molecular level Effect

The detection of 2.1 pathology

2.1.1 materials

All groups of mouse are drawn materials on the 35th day after modeling, and cervical dislocation is put to death, and take pancreatic tissue, a point three parts save.One It is partially disposed in 10% neutral formalin and fixes 18 hours, then tissue dewatering, carry out paraffin embedding；A part is placed in frost packet It buries and is put in cooling in liquid nitrogen in liquid (OCT) rapidly, -80 DEG C save backup；A part is placed in RNAlater (RNA StabilizationSolution, RNA stabilizer are purchased from life company) in save, with extract RNA detection tissue sample in base The expression of cause.

2.1.2 Hematoxylin-eosin dyes

The slice of paraffin sample is carried out with paraffin slicing machine, 3 μm of thickness, serial section, the common glass slide of a part fishes out piece It is dyed as HE, a part adherency glass slide fishing piece is used as immunohistochemical staining.Paraffin section is put in room temperature in slide holding frame Overnight, secondary daily HE dyeing-mounting all-in-one machine (ST5020-CV5030, Leica, Nussloch, Germany) is dyed, dye Color program is shown in Table 6, terminates direct mounting machine mounting after dyeing.

6 HE engine dyeing program of table

2.1.3 each group mice pancreatic histopathologic change

Light microscopic observation: amplify 100 times, 200 times, 400 times of observations under light microscopic respectively.First amplify 100 times under light microscopic Observation finds the pancreas islet in pancreatic tissue and takes pictures, then takes pictures under 200 times and 400 times of mirrors.Under low power lens, it is seen that sample It is with a less complete connective tissue envelope outside.Envelope, which protrudes into, essentially forms interlobular septum, glandular substance of prostate is separated into many small Leaf.The section of visible pancreatic duct and blood vessel in interlobular connective tissue.Largely dyeing in leaflet deeper is serous gland It steeps (exocrine portion), visible some dyeing are shallower between acinus, the cell mass that differs in size, this is pancreas islet (endocrine portion). Pancreas islet is the more light cell mass of dyeing, is differed in size, outsourcing thin layer connective tissue, between the acinus of exocrine portion.Pancreas Island inner cell arranges agglomerating or strand, and iuntercellular has capillary abundant.Pancreas in pancreatic tissue is normally organized in HE dyeing picture Island coating is complete, beta Cell of islet marshalling, and nuclear targeting is limpid, uniform coloring, and nuclear membrane is complete；Model group pancreatic tissue Middle beta Cell of islet reduction, particle depigmentation, vacuolar degeneration, necrosis, disappearance, proliferation of fibrous tissue, hyalinization, alpha Cell of islet are bright Aobvious to increase, β compensatory cellular is loose, and inside pancreas islet and there is a large amount of inflammatory cell infiltration on periphery；Pancreas in treatment group's pancreatic tissue There is inflammatory cell infiltration on island close to conduit side and has alpha Cell of islet proliferation, and the beta Cell of islet that the other side retains is more, form Completely, arranged distribution is neat.(see Fig. 6)

2.1.4 result and analysis

It sees on the whole, normal group without inflammation of pancreatic islet；Beta Cell of islet is obviously more than model group in treatment group's pancreatic tissue, scorching The range and degree of disease infiltration are obviously smaller than model group, light.It can be seen that treatment psoriasis Chinese medicine group from pathology picture It is obvious to close object treatment type 1 diabetes model mice effect.(see Fig. 6)

3.1 immunohistochemical staining

3.1.1 insulin antibody detects

3.1.1.1 colouring method

3 μm of pancreatic tissue paraffin section, 37 DEG C overnight, and next day is put into 65 DEG C of baking ovens and bakes piece 2 hours.Then de- with dimethylbenzene Wax twice, by dehydrated alcohol, 95% ethyl alcohol, 70% ethyl alcohol, 50% ethanol gradient rehydration.Liquid reparation is repaired with 1 × citric acid, 3%H₂O₂It eliminates endogenous peroxydase to disappear enzyme, secondary antibody is closed with derived sera, then incubates primary antibody, 4 DEG C of refrigerator overnights, next day Incubate secondary antibody, DAB colour developing redyes, breaks up, return indigo plant, is dehydrated, neutral gum mounting, under the microscope with take pictures.

3.1.1.2 under the microscope and analyzing

Amplify 100 times, 200 times, 400 times of observations under light microscopic respectively.First amplify 100 times of observations under light microscopic, finds pancreas Pancreas islet in glandular tissue is simultaneously taken pictures, and is then taken pictures under 200 times and 400 times of mirrors.Observation is as it can be seen that Normal group insulin is anti- The region of the body positive diffuses entire pancreas islet, and only pancreas islet periphery α cell is not caught；Model group is caught only fragmentary several Beta Cell of islet, the beta Cell of islet of the insulin positive is more in treatment group's pancreas islet.(see Fig. 7)

3.1.2 CD3 antibody test

3.1.2.1 colouring method

3 μm of pancreatic tissue paraffin section, 37 DEG C overnight, and next day is put into 65 DEG C of baking ovens and bakes piece 2 hours.Then de- with dimethylbenzene Wax twice, by dehydrated alcohol, 95% ethyl alcohol, 70% ethyl alcohol, 50% ethanol gradient rehydration.Liquid reparation is repaired with 1 × citric acid, 3%H₂O₂It eliminates endogenous peroxydase to disappear enzyme, secondary antibody is closed with derived sera, then incubates primary antibody, 4 DEG C of refrigerator overnights, next day Incubate secondary antibody, DAB colour developing redyes, breaks up, return indigo plant, is dehydrated, neutral gum mounting, under the microscope with take pictures.

3.1.2.2 under the microscope and analyzing

Amplify 100 times, 200 times, 400 times of observations under light microscopic respectively.First amplify 100 times of observations under light microscopic, finds pancreas Pancreas islet in glandular tissue is simultaneously taken pictures, and is then taken pictures under 200 times and 400 times of mirrors.Observation is as it can be seen that normally organize CD3 negative antibody；Mould There are many T cell that type group catches the CD3 positive, and the T cell of the CD3 positive is less in treatment group's pancreas islet.(see Fig. 8)

4.1 molecular Biological Detection

4.1.1 experimental method

WithMicro Kit extracts pancreatic tissue total serum IgE, then surveys the concentration of RNA sample, adjusts to properly doing The concentration of RNA reverse transcription.The reverse transcription of RNA sample is carried out with the Reverse Transcriptase kit of Promega, and it is fixed finally to carry out real-time fluorescence Measure PCR, detect pancreatic samples in Insulin II, CD3 ε, IL-4, IL-10, IL-1 β, IFN-γ, each gene of IL-17A it is opposite Expression quantity.Gene order is shown in Table 7.

7 gene order of table

4.1.2 result and analysis

Insulin II is the characterizing gene of islet cells in pancreas, the function of pancreas islet in how much reflection tissues of content Situation.Normal group (n=8) mouse islets function is normal, the relative expression quantity of model group (n=9) and treatment group (n=9) gene Significantly lower than normal group, treatment group retains the function of pancreas islet half, and model group almost loses islet function, using single factor test Variance analysis carries out overall comparison, and difference is extremely significant (P < 0.0001), the results are shown in Table 8 and Fig. 9-A.

CD3 ε is the characterizing gene of T lymphocyte, the degree of inflammatory infiltration in how much reflection tissues of content.Normally CD3 ε is not expressed in NOD-SCID mouse islets tissue, the relative expression quantity of model group (n=9) CD3 ε gene is much higher than treatment group (n=9) and normal group (n=8), illustrate that model group inflammatory infiltration is quite serious, overall ratio is carried out using one-way analysis of variance Compared with difference is extremely significant (P < 0.0001), the results are shown in Table 8 and Fig. 9-B.

IL-4 is the characterization factor of Th2 cell, and the relative expression quantity for the treatment of group (n=9) IL-4 gene is much higher than model group (n=9), it is compared using two independent samples t test methods, difference is extremely significant (P < 0.0001), the results are shown in Table 8 and Fig. 9-C.

IL-10 is a kind of important anti-inflammatory factors.The relative expression quantity for the treatment of group (n=9) IL-10 gene is apparently higher than mould Type group (n=9) illustrates that drug therapy can be adjustable pancreatic tissue lesion portion T cell to Th2 cell differentiation, using using two Independent samples t test method is compared, and difference is extremely significant (P < 0.0001), the results are shown in Table 8 and Fig. 9-D.

IL-1 β is that a kind of and antigen acts synergistically, and promotes CD4⁺The inflammatory cytokine of T cell activation.Model group (n= 9) relative expression quantity of IL-1 β gene is higher than treatment group (n=9), is compared using two independent samples t test methods, difference has Statistical significance (P=0.0103 < 0.05), the results are shown in Table 8 and Fig. 9-E.

IFN-γ is the characterization factor of Th1 cell, and the relative expression quantity of model group (n=9) IFN-γ gene is higher than treatment Group (n=9) is compared using two independent samples t test methods, and difference is more significant (P=0.0001 < 0.001), the results are shown in Table 8 And Fig. 9-F.

IL-17A is the characterization factor for developing closely related Th-17 cell in Th with type 1 diabetes, is proinflammatory factor.Mould The relative expression quantity of type group (n=9) IL-17A gene is much higher than treatment group (n=9), illustrates the serious journey of model group inflammatory infiltration Degree, is compared, difference is more significant (P=0.0008 < 0.001), the results are shown in Table 8 and Fig. 9-using two independent samples t test methods G。

Specific gene relative expression quantity in 8 pancreatic tissue of table

Note: insulin II and CD3 ε is using β-actin as reference gene, using normal group as control group；IL-4, IL-10, IL-1 β, IFN-γ, IL-17A are using CD3 ε as reference gene, using model group as control group.Insulin II and tri- groups of CD3 ε Data first carry out homogeneity test of variance, P < 0.05 illustrates variance using totally comparing between three groups of one-way analysis of variance progress It is uneven, property is strengthened using the key of mean value equality and examines (Welch/Brown-Forsythe), P < 0.0001, Multiple range test between group Using Tamhane's T2 method of inspection, normal group and model group, model group and treatment group, treatment group compared with normal group,^***P < 0.001,^**P < 0.01.Two groups of independent samples of model group and treatment group of IL-4, IL-10, IL-1 β, IFN-γ, IL-17A use T is examined, the test of normality and homogeneity test of variance of advanced row data, IL-4, IL-10, IL-1 β, IFN-γ, the number of IL-17A Though meet normality distribution according to having, heterogeneity of variance, so using Wilcoxon method of inspection in non-parametric test method, P value difference For < 0.0001,0.0001,0.0103 < of <, 0.05,0.0001 <, 0.001,0.0008 < 0.001.

3 statistical analysis

All quantitative datas with(SEM) it indicates, sample size uses Graphpad depending on experiment needs The mapping of 5 software of Prism, statistical analysis use 5 software of Graphpad Prism, 20.0 software of SPSS, the number of duplicate measurements According to Repeated Measurements method of analysis of variance is used, Dunnett ' s T3 method of inspection is used when heterogeneity of variance.Between three groups of quantitative data Comparison use one-way analysis of variance method；When variance is neat, Multiple range test uses Tukey HSD method of inspection, heterogeneity of variance between group When, property is strengthened using the key of mean value equality and examines (Welch/Brown-Forsythe), Multiple range test uses Tamhane's between group T2 method of inspection.Comparison between two groups of quantitative data uses two independent samples t tests, does not meet normality distribution and heterogeneity of variance Two groups of data are using Wilcoxon method of inspection in non-parametric test method.Grouped data uses Fisher's Exact method of inspection, and 1 The existence evaluation data of patients with type Ⅰ DM mouse uses Log-rank Log-Rank test method.The comparison of ranked data is using in rank sum test Kruskal Wallis H method of inspection.P < 0.05 thinks that difference is statistically significant.

4 conclusions

Type 1 diabetes animal model plays by treatment psoriasis Chinese medicine composition intervention processing and delays very much the state of an illness greatly With the effect for the treatment of.The Chinese medicine composition can reduce the infiltration of inflammation in mice pancreatic tissue, protect the function of islet cells. In short, it is of today research shows that the Chinese medicine composition can significantly improve the survival rate of islet cells in T1D model mouse pancreas, subtract The infiltration of inflammatory cell such as T cell, delays mouse invasion in few pancreas islet, reduces the disease incidence of mouse T1D, extends the existence of mouse Phase prompts the Chinese medicine composition to islet cells damage tool in the T1D model mice pancreas of simulation people spontaneous type 1 diabetes foundation There is good protective effect, reduce the infiltration of inflammatory cell, implies that the Chinese medicine composition has very in terms of preventing and treating type 1 diabetes Big development and application prospect.

SEQUENCE LISTING

<110>Guangdong Provincial TCM Hospital

<120>application of the treatment psoriasis Chinese medicine in the drug of preparation prevention and treatment type 1 diabetes

<160> 16

<170> PatentIn version 3.3

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Claims (3)

1. application of the psoriasis Chinese medicine in the drug of preparation prevention and treatment type 1 diabetes is treated, wherein the treatment psoriasis Chinese medicine Effective component be made by following raw material medicine:

15 parts of Glabrous Sarcandra Herb, 9 parts of curcuma zedoary, 15 parts of dark plum, 60 parts of smilax, 9 parts of radix paeoniae rubra, 15 parts of Asian puccoon, 6 parts of Radix Glycyrrhizae.

2. application according to claim 1, which is characterized in that the drug of the prevention and treatment type 1 diabetes is tablet.

3. application according to claim 2, which is characterized in that the tablet is made of following methods:

(1) it by the bulk pharmaceutical chemicals, directly smashs into coarse powder to pieces, the ethyl alcohol that 10 times of amount volumetric concentrations are 70% is added and extracts twice, 2 hours every time, combined extract；

(2) it is concentrated under reduced pressure, recycles ethyl alcohol, medicinal extract is made；

(3) medicinal extract is taken, the starch of 3 times of weight is added, is mixed, is pelletized, it is dry, magnesium stearate is added by the 5% of tablet weight, mixes It is even, tabletting.