CN100364538C

CN100364538C - Application of fugillin in treating immunity entiritis and related immunity disease

Info

Publication number: CN100364538C
Application number: CNB2005100386328A
Authority: CN
Inventors: 徐强; 谭仁祥; 吴雪丰
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2005-03-31
Filing date: 2005-03-31
Publication date: 2008-01-30
Anticipated expiration: 2025-03-31
Also published as: CN1686131A

Abstract

The present invention belongs to the technical field of biologic pharmacy. The present invention aims to research the application of fumigaclavine C used as an immunosuppressive agent to enteritis. The Fumigaclavine C of the present invention can be applied to intestinal tract diseases including a Crohn's disease and ulcerative colitis and other autoimmune diseases participated by various T cells, such as delayed type hypersensitivity, sclerosis, insulin-dependent diabetes, autoimmunity myocarditis, a Hashimoto's disease, a Grave's disease, myasthenia gravis, etc.

Description

The application of fumigaclavine third in treatment immunity enteritis and correlation immunity disease

One, technical field

The invention belongs to biological pharmacy technical field.

Two, background technology

Fumigaclavine third (Fumigaclavine C) since finding in 1977, correlational study to this chemical compound is considerably less, report aspect the pharmacology is rare especially, only have one piece of article show this chemical compound in the autoallergic model, have good efficacy (Zhao et al.J Pharm Pharmcol 2004,56:775-84).On the other hand, the enteritis of autoimmunity is frequently-occurring disease and refractory disease clinically, comprises Crohn disease and ulcerative colitis.Immunity enteritis is just morbidity during often from teenager, it is reported that sickness rate is up to annual 4～10/100000 people.Lifelong difficult, the later stage can develop into various gastroenteropathys and comprise intestinal cancer, is accompanied by higher mortality rate.Though can control disease process clinically at present, still lack effective healing means, and used medicine also have intensive side effect.

Three, summary of the invention

The objective of the invention is to study fumigaclavine third as the application of immunosuppressant in immunity enteritis and correlation immunity disease.

Technical scheme of the present invention:

1. the extraction separation of fumigaclavine third and evaluation thereof

(application number: 200310112694.X Tan Ren is auspicious, Xu Qiang, Zhao Ying, Liu Junyan, Wang Jun to see patent.Title: " application of fumigaclavine third in the anti inflammatory immunity depressant ".Date of application: on December 19th, 2003).

2. the pharmacological testing of fumigaclavine third

1) mice enteritis induces

Get the 6-8 BALB/c female mice in age in week, fasting 20 hours.Then the injection hose of 3.5F is inserted rectum from the mice anus, the degree of depth is about 4 centimetres.Injection hose links to each other with 1 milliliter of syringe, injects 100 microlitre TNBS (2,4 by this equipment in the mice colonic lumen, the 6-trinitro-benzene-sulfonic acid, 2,4,6-trinitrobenzene sulfonic acid) solution (TNBS is with 50% dissolve with ethanol solution, and concentration is 5mg/ml).The normal group mice gives 100 microlitres, 50% alcoholic solution in contrast.Extract injection hose after administration finishes and make mice continue to keep vertical position 30 seconds, so that medicinal liquid spreads all over whole colon.

2) scoring of the observation of mice inflammatory symptom and colon's pathological change

Observe and write down body weight and the survival condition of mice every day.The injection TNBS after the 3rd to the 8th to the mice administration.Every day lumbar injection fumigaclavine third (5,10,20mg/kg) or ciclosporin A (CsA, 10mg/kg), the phosphate buffer (PBS) of matched group lumbar injection respective volume.All mices are all injected execution in back 9 days at TNBS, take out sacral lymph nodes respectively, colonic patches and colon.Colon is fixed with 10% formalin solution, and paraffin embedding is done the crown section of 4-5 micron thickness, and dyes with hematoxylin-eosin.According to bibliographical information histology's variation of colon is marked.Scoring reaches as high as 8 fens, and this moment, comprehensive diffusible pathological changes took place colon.

3) sacral lymph nodes and the lymphocytic preparation of colonic patches

Get the TNBS sensitized mice, aseptic separation sacral lymph nodes and colonic patches.Sacral lymphnodes is placed 5 milliliters of D-Hank ' s solution, roll gently with aseptic nook closing member, and blow and beat repeatedly, make cell enter solution and be dispersed into individual cells with pipet.Filter, centrifugal, with RPMI-1640 solution washing twice, suspendible is counted stand-by.In addition colonic patches was digested 20 minutes with collagenase IV (0.5mg/ml in RPMI-1640), add RPMI-1640 solution piping and druming becoming individual cells then, filter, centrifugal, washing twice is counted stand-by.

4) sacral lymph nodes, the detection of cytokines mRNA level is adjusted into every milliliter 5 * 10 with the cell that obtains in colonic patches and the colon ⁶Individual 24 orifice plates that place, at 37 ℃, 5%CO ₂Environment in cultivate.After 24 hours, the taking-up cell carries out centrifugal and extracts total RNA with the TriPure separation agent.In addition, variant group mice is taken out colon respectively, extracts total RNA with TriPure homogenate.Determine RNA concentration with the ultraviolet absorption value at 260nm place at last.Thereafter, be cDNA with the RNA reverse transcription, reaction system is as follows: the total RNA of 2 μ g, 2000 pmol Oligo dT, 1.0mM dNT P, 200 U M-MLV reverse transcriptases, 5 * transcribe buffer.PCR primer and amplification condition see Table 1.The PCR product develops the color with 2.0% agarose gel electrophoresis and with ethidium bromide.With the software of Labworks 4.0 to electrophoretic band brightness semi-quantitative analysis.

5) detection of interleukin II (IL-2) and interferon gamma (IFN-γ)

Take out as the cell conditioned medium of preceding cultivation after 24 hours, in-70 ℃ of preservations.Detect the protein level of IL-2 and IFN-γ in the cells and supernatant then with ELISA.Detection is limited to 10pg/ml, and standard curve range is 0 to 1000pg/ml.

6) gelatinase analysis of spectrum

Get the supernatant of cell culture, method by gelatin degraded detects metal matrix protease-9 (MMP-9) wherein. other get respectively organize mice colon with containing the 100U/ml penicillin, D-Hank ' the s solution flushing of 100 μ g/ml streptomycins, place then RPMI 1640 solution of 1ml serum-free cultivate 24 hours (37 ℃, 5%CO ₂).Get supernatant equally and measure the MMP-9 activity.Get sample in 20 μ l supernatants and 10 μ l sample buffer (62.5mM Tris-HCl contains 10% glycerol, 0.00125% bromjophenol blue and the 12%SDS) mixing.Electrophoresis uses the SDS-PAGE glue that contains 5% polyacrylamide and 2mg/ml gelatin.Electrophoresis is peeled off this SDS-PAGE glue after 1 hour, with rinsing buffer (50mM Tris-HCl containing 2.5%Triton X-100,5mM CaCl ₂, 1 μ M ZnCl ₂, 0.05% NaN ₃) washed twice, each 30 minutes.And then with incubationbuffer (50mM Tri s-HCl containing 5mM CaCl ₂, 1 μ M ZnCl ₂, 0.05%NaN ₃) bathed 36 hours 37 ℃ of temperature.Again with 0.1% Coomassie brilliant blue R250 dyeing 30 minutes, decolour with 10% glacial acetic acid and 10% aqueous isopropanol then.Characterize the activity of MMP-9 with the white bright band on the blue background.

The PCR parameter of each cytokine of table 1

Primer sequence	Loop parameter	Period	Product (bp)
Primer sequence	Loop parameter	Period	Product (bp)	S5’-GCCCATCCTCTGTGACTC-3’ IL-1β AS5’-GCTCTGCTTGTGAGGTGC-3’ S5’-TGCTCCTTGTCAACAGCG-3’ IL-2 AS5’-TCATCATCGAATTGGCACTC-3’ S5’-GGG ACC AAA CCA GCA CAT-3’ IL-12αAS5’-AAG GCG TGA AGC AGG ATG-3’ S5’-CTTCTTCAGCAACAGCAAGGCGAAAA-3’ IFN-γAS5’-CCCCCAGATACAACCCCGCAATCA-3’ S5’-CATCTTCTCAAAATTCGAGTGACAA-3’ TNF-αAS5’-TGGGAGTAGACAAGGTACAACCC-3’ S5’-CAG CCAACTATGACCAGGAT-3’ MMP-9AS5’-TGC CGT CTATGT CGT CTTTAT-3’ S5’-ACATCTGCTGGAAGGTGGAC-3’ β-actinAS5’-GGTACCACCATGTACCCAGG-3’	94℃ 30 sec， 58℃ 1.5min， 72℃ 1min 94℃ 30sec， 58℃ 1.5min， 72℃ 1min 94℃ 30sec， 55℃ 1.5min， 72℃ 1min 94℃ 30sec， 60℃ 1.5min， 72℃ 1min 94℃ 30 sec， 58℃ 1.5min， 72℃ 1min 94℃ 30sec， 58℃ 1.5min， 72℃ 1min 94℃ 30sec， 60℃ 30sec， 72℃ 30sec	35 35 35 35 35 35 26 or 28	437 391 379 456 175 366 163

S: upstream sequence, AS: downstream sequence

3. the pharmacological tests of fumigaclavine third and analysis

1) after fumigaclavine third lumbar injection was induced influencing the administration of TNBS colonic and inducing enteritis of enteritis to TNBS, mice can produce serious inflammatory symptom, as diarrhoea, weight loss etc., is accompanied by than higher mortality rate.Give behind the TNBS from the 3rd day to the 8th day each treated animal respectively every day lumbar injection fumigaclavine third (5,10,20mg/kg) or CsA (10mg/kg).Compare with matched group, fumigaclavine third is with 10 with can make the body weight gain of mice return to normal level during the dosed administration of 20mg/kg, survival rate that simultaneously can also dose dependent ground raising mice.(as Fig. 1).

2) fumigaclavine third lumbar injection suppresses the enteritis partial pathology damage of mice inflammation and the administration of inflammatory cell infiltration TNBS colonic and induces after the enteritis and to put to death animal on 9th, takes out colon.Perusal is found the intestinal mucosa edema and hemorrhagic ulcer is arranged.The colon wall of major injury can be owing to edema thickens.The feces that not have shaping in the control animals intestinal, and different therewith be that solid manure (as Fig. 2 A) is then arranged in the mouse intestinal behind the drug treatment.The tissue slice inspection finds that the colon of control animals finds the intestinal crypt distortion, the cup cell loss, and monocyte infiltration also has serious mucosa injury; And fumigaclavine third (20mg/kg) and CsA (10mg/kg) treatment back all are significantly improved to partial inflammatory reaction of animal inflammation and mucosal ulcer.(as Fig. 2 B, C).Scoring finds that (5,10, tissue 20mg/kg) is respectively 6.1,3.6 and 2.1 to the fumigaclavine third treatment group, and the mark of matched group is then up to 6.9 (as Fig. 2 D) according to the inflammation standard.

3) fumigaclavine third suppresses the expression of the lymphocytic inflammatory factor of sacral lymph node and the activity of generation and MMP-9

Separate the sacral lymph node cell of control animals and cultivated the external fumigaclavine third that gives 24 hours at 37 ℃.After finishing, cultivation, extracts total RNA to detect the expression of cytokine and MMP-9 with cell centrifugation.Found that fumigaclavine third can suppress to participate in the expression of many cytokines of enteritis (IL-1 β, IL-2, IL-12 α, IFN-γ and TNF-α) and MMP-9 (as Fig. 3 A, B).In addition, the supernatant of collecting cell cultivation detects production of cytokines.The result shows that fumigaclavine third can suppress the generation of IL-2 and IFN-γ (as Fig. 3 E, F).Fumigaclavine third can also suppress another important inflammatory factor MMP-9 (as Fig. 3 C, D) simultaneously.

4) fumigaclavine third suppresses the expression of the lymphocytic inflammatory factor of colonic patch and the activity of generation and MMP-9

Separate the colonic patch cell of control animals and cultivated the external fumigaclavine third that gives 24 hours at 37 ℃.After finishing, cultivation, extracts total RNA to detect the expression of cytokine and MMP-9 with cell centrifugation.The result is similar to the lymphocytic experimental result of sacral lymph node.Shown in Fig. 4 A and B, this medicine can dose dependent ground suppresses the expression of multiple inflammatory factor, comprises IL-1 β, IL-2, IL-12 α, IFN-γ, TNF-α and MMP-9.In addition, it also can protein level suppress IL-2 and IFN-γ (as Fig. 4 E, F).Simultaneously, enzyme spectrum analysis has found that also third pair of active inhibitory action of MMP-9 of fumigaclavine is (as Fig. 4 C, D).

5) third pair of enteritis mice of intravenous injection fumigaclavine colon inflammatory factor is expressed and the active inhibitory action of MMP-9

Separate the colon and the homogenate of each treated animal and extract total RNA.Can be on the mRNA level after fumigaclavine third or the CsA administration significantly the expression of the inflammation-inhibiting factor (as Fig. 5 A, B).And, detect the supernatant of colon's separation back In vitro culture and find that the MMP-9 activity of fumigaclavine third and CsA group also has been subjected to obvious inhibition (as Fig. 5 C, D).

The present invention is that fumigaclavine third (Fumigalavine C) has the good curing effect to the immunity enteritis that is difficult to cure, and can be applied to comprise the intestinal tract disease of Crohn disease and ulcerative colitis than the beneficial effect of prior art.In view of the remarkable inhibitory action of this medicine to various inflammatory factors especially Th1 cytokine, this medicine also can be used as immunosuppressant and is used for the autoimmune disease that other multiple Th1 cell participates in, as delayed hypersensitivity, multiple sclerosis, the diabetes of insulin-dependent, autoimmune myocarditis, Hashimoto's disease, Grave ' s disease, myasthenia gravis etc.

Four, description of drawings

Fig. 1. fumigaclavine third and CsA induce the improvement effect of enteritis to mice TNBS.Fumigaclavine third (5,10,20mg/kg) or CsA (10mg/kg) intraperitoneal administration every day.Every day, body weight change (A) and the death condition (B) of mice respectively organized in record.The result represents with mean ± s.e.m. ^#P＜0.05, ^##P＜0.01, the vs normal mouse; ^*P＜0.05, ^*P＜0.01, the vs model mice (Dunnett ' s test).

Fig. 2. fumigaclavine third and CsA are to the improvement effect of colon perusal of mice inflammation and microscopic examination performance.Fumigaclavine third (5,10,20mg/kg) or CsA (10mg/kg) intraperitoneal administration every day.Taking-up colon on the 9th is observed after the TNBS sensitization.A. the colon outward appearance of enteritis mice.B, the histology of C. enteritis mice colon changes.D. the microcosmic of enteritis mice colon scoring.The result represents with mean ± s.e.m. ^#P＜0.05, ^##P＜0.01, the vs normal mouse; * P＜0.05, * * P＜0.01, the vs model mice (Dunnett ' s test).A, normal group; B, model group; C, d, e, fumigaclavine the third 5,10,20mg/kg group; F, CsA 10mg/kg group.Tissue slice is done HE dyeing.Amplification is B * 10in, C * 100.

Fig. 3. the lymphocytic inflammatory factor expression of third pair of enteritis mice of fumigaclavine sacral lymph node, generation and the active inhibitory action of MMP-9.Detect IL-1 β, IL-2, IL-12 α, IFN-γ and TNF-α with the method for RT-PCR) and the expression (A) of MMP-9.Reuse Lab Work 4.0 softwares are compared with β-actin gene expression and are carried out sxemiquantitative (B).MMP-9 in the cells and supernatant is active to be detected (C) and carries out sxemiquantitative (D) with Lab Work 4.0 softwares with the enzyme spectrum analysis method.IL-2 in the cells and supernatant (E) and IFN-γ (F) level detect with the ELISA method respectively. ^*P＜0.05, ^*P＜0.01, the vs normal mouse (Dunnet ' s test).

Fig. 4. the lymphocytic inflammatory factor expression of third pair of enteritis mice of fumigaclavine colonic patch, generation and the active inhibitory action of MMP-9.Detect IL-1 β, IL-2, IL-12c, IFN-γ and TNF-α with the method for RT-PCR) and the expression (A) of MMP-9.Reuse Lab Work 4.0 softwares are compared with β-actin gene expression and are carried out sxemiquantitative (B).MMP-9 in the cells and supernatant is active to be detected (C) and carries out sxemiquantitative (D) with LabWork 4.0 softwares with the enzyme spectrum analysis method.IL-2 in the cells and supernatant (E) and IFN-γ (F) level detect with the ELISA method respectively. ^*P＜0.05, ^*P＜0.01, the vs normal mouse (Dunnet ' s test).

Fig. 5. fumigaclavine third and CsA express and the active inhibitory action of MMP-9 the inflammatory factor of enteritis mice colon.Fumigaclavine third (5,10,20mg/kg) or CsA (10mg/kg) intraperitoneal administration every day.

Total RNA is extracted in the colon's taking-up and the homogenate of each group.Detect IL-1 β, IL-2, IL-12 α, IFN-γ and TNF-α with the method for RT-PCR) and the expression (A) of MMP-9.Reuse Lab Work 4.0 softwares are compared with β-actin gene expression and are carried out sxemiquantitative (B).Other gets intestinal tissue and carries out In vitro culture, and the MMP-9 in the supernatant is active to be detected (C) and carry out sxemiquantitative (D) with Lab Work 4.0 softwares with the enzyme spectrum analysis method.

Five, the specific embodiment

Fumigaclavine third lumbar injection can significantly improve the inductive mouse immune enteritis of TNBS.Adopt the BALB/c female mice, at first inject TNBS, the 3rd day thereafter to the 8th day to the mice administration: every day, the lumbar injection fumigaclavine third (5,10,20mg/kg) or ciclosporin A (CsA, 10mg/kg), the phosphate buffer (PBS) of matched group lumbar injection respective volume.The result shows: the administration of TNBS colonic is induced after the enteritis, and mice can produce serious inflammatory symptom, as diarrhoea, weight loss etc., is accompanied by than higher mortality rate.Compare with matched group, fumigaclavine third is with 10 with can make mice recover the survival rate (accompanying drawing 1) that body weight gain improves mice during the dosed administration of 20mg/kg.Simultaneously to partial inflammatory reaction of colon and mucosal ulcer all be significantly improved (accompanying drawing 2 B, C).

The participation of many inflammatory cytokine is arranged in the generation evolution of enteritis.This research finds that further fumigaclavine third can suppress to participate in many cytokines (IL-1 β, IL-2, IL-12 α, IFN-γ and TNF-α) of enteritis and expression (accompanying drawing 3A, the B of MMP-9; Fig. 4 A, B; Fig. 5 A, B) and IL-2 and the proteic generation of IFN-γ (as Fig. 3 E, F; Fig. 4 E, F; Fig. 5 E, F).Fumigaclavine third can also suppress the function of another important inflammatory factor MMP-9 (as Fig. 3 C, D simultaneously; Fig. 4 C, D; Fig. 5 C, D).

Above results suggest, fumigaclavine third (Fumigaclavine C) can be applied to comprise the intestinal tract disease of Crohn disease and ulcerative colitis and the autoimmune disease that other multiple Th1 cell participates in, as delayed hypersensitivity, multiple sclerosis, the diabetes of insulin-dependent, autoimmune myocarditis, Hashimoto's disease, Grave ' s disease, myasthenia gravis etc.

Claims

1. the application of fumigaclavine third in the autoimmune disease medicine that preparation treatment T cell participates in, the autoimmune disease that the T cell participates in is a Crohn disease, ulcerative colitis, rheumatoid arthritis, lupus erythematosus, transplant rejection, delayed hypersensitivity, multiple sclerosis, the diabetes of insulin-dependent, autoimmune myocarditis, Hashimoto's disease, Grave ' s disease, myasthenia gravis.