CN106074500A - The application in preparation inflammatory enteropathy medicine of the dehydrogenation lovastatin - Google Patents

Abstract

The invention discloses the application in preparation inflammatory enteropathy medicine of the dehydrogenation lovastatin, the present invention, by setting up chmice acute inflammatory bowel disease model, observing dehydrogenation lovastatin to tests such as the therapeutical effect of inflammatory bowel, discloses preventive and therapeutic effect and the mechanism of action of the mice inflammatory bowel that dextran sulfate sodium is induced by dehydrogenation lovastatin first.Dehydrogenation lovastatin is by suppression NF κ B signal path, hence it is evident that suppression proinflammatory factor TNF α, IL 6, the level of IL 17, improves the level of anti-inflammatory factors IL 10, and plays the protective effect of the mice inflammatory bowel to 4%DSS induction.

Description

The application in preparation inflammatory enteropathy medicine of the dehydrogenation lovastatin

Technical field

The present invention relates to drug world, specifically dehydrogenation lovastatin application in preparation inflammatory enteropathy medicine.

Background technology

Statins is Hydroxymethylglutaryl list acyl coenzyme A (3-hydroxy-3-methyl-glutaryl CoA, HMG- CoA) the special competitive inhibitor of reductase, can reduce the biosynthesis of cholesterol, has significant Bloodlipid-lowering, for blood fat reducing Choice drug in medicine.And reduce the heart by improving the effects such as endothelial function, antiplatelet aggregation, suppression atherosclerosis The sickness rate of angiopathy and case fatality rate.Research in recent years finds that statins still has antiinflammatory and immunoregulation effect, is being permitted Many rheumatism, atrophic diseases and autoimmune disease play certain curative effect.

Dehydrogenation lovastatin (dehydrolovastatin, DLVT) is the newcomer of the Statins family in recent years obtained, and is The dehydrogenation derivant of lovastatin, has obtained national inventing patent mandate.The structural formula of two medicines is as follows.Our early stage pair The acute toxicity testing research of DLVT shows, it is 2000mg kg that its mouse stomach is administered maximum tolerated dose^-1, be equivalent to Lip river and cut down him Being grown up 128 times of one day maximal dose clinically in spit of fland, shows that its toxicity is relatively low.Early-stage Study shows DLVT except having and LVT Also nonspecific inflammation and adjuvant-induced arthritis there is effect outside the same effect for reducing fat.

Lovastatin stereochemical formula is as shown in (1):

Dehydrogenation lovastatin stereochemical formula is as shown in (2):

Inflammatory bowel (inflammatory bowel disease, IBD) be a kind of cause of disease and pathogenesis the most not yet The clearest and the most definite non-specific diseases associated with inflammation, including ulcerative colitis (ulcerative colitis, UC) and Crohn disease (Crohn ' s disease, CD), it is that the intestinal persistence that a kind of inherited genetic factors, environmental factors and immune factor participate in jointly is scorching Disease.In recent years, worldwide, the M & M of IBD rises year by year.This disease pathological change is complicated, clinical Symptom is various, if being eased the most in time and curing, causes the probability for canceration very big.Therefore, arranged by World Health Organization (WHO) For one of modern refractory disease.Conventional medicine has minosalicylates, drugs, glucocorticoid, immunosuppressant and one A little Chinese medicines, but there is no preferable medicine so far, exploitation treating both the principal and secondary aspects of a disease, safely and effectively medicine have important real value.

The IBD cause of disease illustrates the most completely, mainly invades and the chronic nonspecific inflammatory diseases of intestinal mucosa, in a continuous manner Gradually progress may be relevant with the many factors such as dysimmunity, alteration of intestinal flora, heredity and the mental status.It is now recognized that intestinal sticks Film immune dysfunction, pro-inflammatory cytokine secretion increases, and causing inflammatory cascade iodine is its main pathogenesis.

Summary of the invention

It is an object of the invention to provide the application in preparation inflammatory enteropathy medicine of the dehydrogenation lovastatin, to solve The problem proposed in above-mentioned background technology.

For achieving the above object, the present invention provides following technical scheme:

The application in preparation inflammatory enteropathy medicine of the dehydrogenation lovastatin.

As the further scheme of the present invention: the stereochemical formula of dehydrogenation lovastatin is as follows:

As the further scheme of the present invention: dehydrogenation lovastatin is effective for 4%DSS inducing mouse inflammatory bowel Dosage is 33.6mg kg^-1·d^-1。

As the further scheme of the present invention: in described inflammatory enteropathy, proinflammatory factor includes TNF-α, IL-6 and IL- 17, anti-inflammatory factors is IL-10.

Compared with prior art, the invention has the beneficial effects as follows:

The present invention is by setting up chmice acute inflammatory bowel disease model, observing dehydrogenation lovastatin to inflammatory enteropathy Therapeutical effect etc. are tested, and inquire into the preventive and therapeutic effect of the mouse corrosion disease disease property enteropathy that dextran sulfate sodium is induced by dehydrogenation lovastatin And mechanism of action, disclose dehydrogenation lovastatin first to IL-6, IL-10, IL-17, TNF-α and the impact of NF-κ B.Dehydrogenation Lip river is cut down Statin, by suppression NF-κ B signal path, shows obvious inhibitory action to the proinflammatory factor such as TNF-α, IL-6, IL-17, and The level of anti-inflammatory factors IL-10 can be improved and play the effect of inflammatory enteropathy, dehydrogenation lovastatin treatment inflammatory bowel Disease has treating both the principal and secondary aspects of a disease, safely and effectively effect.

Accompanying drawing explanation

Fig. 1 model group and the change of naive mice body weight；

Fig. 2 4%DSS inducing mouse inflammatory bowel activity index；

The impact of the mouse disease activity index that 4%DSS is induced by Fig. 3 DLVT and LVT.

Mouse Colon Histopathology Effect (HE × 100) figure that 4%DSS is induced by Fig. 4 DLVT and LVT, (a) is normal Comparison, (b) model comparison, (c) SASP, (d) DLVT, (e) LVT；

Impact (SP × 200) figure that the mouse Colon tissue NF-κ B that 4%DSS is induced by Fig. 5 DLVT and LVT expresses, (a) Blank group, (b) model comparison, (c) SASP, (d) DLVT, (e) LVT.

Detailed description of the invention

Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, Obviously, described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based in the present invention Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, all Belong to the scope of protection of the invention.

Embodiment 1

One, the foundation of experimental inflammatory enteropathy animal model

1 material and method

1.1 laboratory animals:

Cleaning grade kunming mice (male), body weight 22-25 gram, Medical University Of Chongqing's Experimental Animal Center provide.

1.2 main agents and medicine:

Dextran sulfate sodium salt (Dextran sulfate sodium salt, DSS, MW:36000-50000) (U.S. MP Biomedicals)

Stool blood test kit (Bioengineering Research Institute is built up in Nanjing)

1.3 key instruments:

Electronic balance

A2000S type electronic analytical balance (Germany Sartorius)

Sigma-3k refrigerated centrifuge (U.S. Sigma)

-80 DEG C of ultra cold storage freezers (German)

UV-225 ultraviolet-visible spectrophotometer (Japanese)

BH-2 type optical microscope (Japan Olympus)

The preparation of 1.4 solution:

The sodium carboxymethyl cellulose of 0.5%:

The preparation of 4%DSS aqueous solution: weigh 40gDSS and be dissolved in distilled water, and constant volume is to 1000ml.

The preparation of neutral formalin (10% neutral formalin) solution of 4%: formaldehyde (40%) 100ml, anhydrous phosphoric acid hydrogen Disodium 6.5g, sodium dihydrogen phosphate 4.0g, distilled water 900ml.

2 methods

The foundation of 2.1 mice inflammatory bowel animal models:

Mice random packet, adaptability is fed 7 days, within the 8th day, is freely drunk 4%DSS aqueous solution, continuous 7d.In the meantime, Every day, timing weighed Mouse Weight, carries out occult blood test and observes feces character.Colon samples: the 8th day, at cervical dislocation Dead mice, cuts off abdominal cavity, cleans out colon, cuts 1cm at distance anus and cuts colon 2cm (rectum is to ascending colon), measures long Degree.Ice-cold normal saline is rinsed well repeatedly, with cleaning filter paper suck dry moisture.Then colon sample is cut to two sections: rectum end Section (about 1cm) is inserted 4 DEG C of 4% formalin and is fixed 24h, and routine paraffin wax embeds, section, slice thick about 6 μm, and HE dyes.Remaining Numbering of part, weighs, and wraps with aluminium foil, is immediately placed in quick-freezing in liquid nitrogen (-196 DEG C), proceeds to cryogenic refrigerator (-80 DEG C) and deposits.

2.2 observation index:

2.2.1 clinicing symptom observation scoring/disease activity index, standards of grading (table 1).

Table 1DAI standards of grading

2.2.2 Morphologic observation

Light Microscopic observation respectively organizes specimen section, and often 15 visuals field (× 100, × 200) of group carry out histological damage score, ask Its meansigma methods, standards of grading are shown in Table 2.

Table 2 histological damage score standard

Colonic mucosa feature under light microscopic	Scoring
		Normally	0
Crypts body of gland loses 1/3	1
		Crypts body of gland loses 2/3	2
Crypts body of gland is all lost, and epithelium exists, with inflammatory cell infiltration.	3
		Mucosal epithelium is rotten to the corn, destroys, with obvious inflammatory cell infiltration.	4

2.2.3 the mensuration of colon's myeloperoxidase (MPO) (MPO) activity

Taking colon's block rinsed clean in ice-cold normal saline, clean filter paper blots, and after correct amount, uses eye immediately Section's scissors shred, and are placed in the glass homogenizer of ice bath, are homogenized after adding 0.86% normal saline, and volume ratio by weight is 1:9 Add homogenate medium and be prepared as the tissue homogenate of 10%.Reference reagent cassette method measures the activity of MPO in colon's homogenate.

3 statistical procedures

Experimental data SPSS12.0 statistical software processes, and each group data is with ((x ± s) represents, uses single factor test variance to divide Analysis and the on rank test of nonparametric Wilcox, the difference between each group of data, P ＜ 0.05 has significance for difference.

4 results

4.1 Mouse Weight changes

Fig. 1 model group and the change of naive mice body weight are shown in the change of model group and naive mice body weight.Can be seen that Blank group Mouse Weight increase is relatively more steady, and model group mice is from the beginning of the after modeling the 3rd day, and Mouse Weight is the most not Increase the most on a declining curve.

The clinical symptoms of 4.24%DSS inducing mouse inflammatory bowel

Mice clinical symptoms is observed and is found: the activity of Normal group mice, hair color, defecation, growth are the most normal.Model group Mice, after drinking 4%DSS solution 2d, there is the positive of occulting blood in some animals defecation, and the nearly all stool in mice of 4-5d is occulted blood sun Property, 6-7d is strong positive, even hemafecia；Reducing with mice activity, body weight substantially alleviates simultaneously；Feces is soft, or diarrhoea is sent out Raw, similar to the performance of mankind's IBD clinical symptoms, show the success of 4%DSS inducing mouse inflammatory bowel disease model.As in figure 2 it is shown, 4%DSS model group mice, after 3d DAI start occur continue significantly to increase.Model mice is the most thin and weak, and perianal hair color dissipates Disorderly, there is the vestige that blood sample is defecated.

The mice inflammatory bowel pathomorphology change of 4.34%DSS induction

As shown in accompanying drawing 3 (b), light Microscopic observation DSS model group mouse Colon epithelium mucosa injury is obvious: mucosal epithelium is rotten Rotten, glandular crypts structural deterioration；Goblet cell is lost, with a large amount of inflammatory cell such as neutrophilic granulocyte, lymphocyte, plasma cell Infiltration.Normal group mucosal epithelium is complete, and crypts structure, goblet cell are normal, sees shown in Fig. 3 (a).

MPO activity in the mouse Colon tissue of 4.44%DSS induction

The MPO of model group is significantly raised, and there were significant differences compared with normal group, is shown in Table the inflammatory bowel of 3:4%DSS induction The activity of sick mice MPO.

The activity of the inflammatory bowel mice MPO of table 34%DSS induction

Group	n	MPO(U/g)
			normal	10	0.92±0.22
model	10	2.19±0.42<sup>**</sup>

^**P ＜ 0.01 is to compare with normal group.

5 discuss

According to existing document, DSS can induce inflammatory bowel.One is acute phase inflammatory bowel disease models, and method is 5%DSS aqueous solution is freely drunk to mice, continuous 7 days, the most also has document report 3-5%DSS inducing colitis； We are preliminary experiment when, once attempt being improved this modeling mode: use to mice 5%DSS solution gavage (0.8ml/ Only, bid × 4d), but laboratory animal does not occur that any clinical symptoms, prompting occur that this situation has two kinds of possibilities: one is The amount that DSS takes in is inadequate, and two is this modeling administering mode, it is impossible to maintain certain concentration in enteric cavity, produces epithelium group The toxic action knitted.So, we are based on the observation to preliminary experiment, and the report of pertinent literature, it is believed that use gavage this Mode gives mice DSS solution induction enteritis and is unlikely that, even if because increasing the concentration of DSS solution, taking 0.8ml/ Only, administering mode three times a day, the liquor capacity daily intaked also only has 2.4ml, and normal mouse is typically drunk water 3-6ml/ My god.Therefore, we report according to situation and the relevant document of preliminary experiment, allow mice freely drink 4%DSS solution, continuous 7d Modeling, inducing experimental enteritis；By observing mice clinical symptoms, intestinal mucosa injury and inflammatory cell infiltration situation, with disease Sick activity index (disease activity index, DAI), colon's histological scores and MPO Activity Assessment 4%DSS The mice IBD model of induction is the most successful.MPO is primarily present a kind of enzyme in neutrophilic granulocyte azurophilic granule, and it is lived Property reflect infiltration number and the activity of neutrophilic granulocyte, be acknowledged as evaluating an index of the tissue inflammation order of severity.

Preliminary experiment freely drinks 5%DSS solution by mice, and the 3rd day clinical symptoms starts to occur, such as loose stool, hemafecia, and the 4th It beginning has dead mouse successively, illustrates that 5%DSS concentration is the highest.Early-stage Study 3.5%DSS solution efficacy is inconspicuous.

Experimental result shows, model group mice drinks 4%DSS solution 3d, begins with feces and the positive of occulting blood occurs；4-5d, Mice occur mobility significantly reduce, chaeta in disorder；Defecate shape soft, thick；Losing weight, fecal occult blood positive degree strengthens； 6-7d, it is seen that naked eyes hemafecia, has diarrhoea to occur, and mice is movable, take food and body weight significantly reduces.Light Microscopic observation colon pathology is cut Sheet shows, colonic mucosa epithelial erosion, and almost all crypts is destructurized, and goblet cell is lost, and part focus surface is the fewest Permitted epithelium residual.Inflammation mainly involves mucosa and Submucosa, and minority is up to placenta percreta；Model group mouse Colon tissue MPO lives Compared with normal mice significantly increases property, and the inflammatory cell of infiltration is based on neutrophilic granulocyte, with mononuclear phagocyte, and a small amount of lymph Cell, mononuclear cell.At the position that some pathological changes are lighter, it is seen that part crypts destroys, deformation, arrangement disorder, goblet cell Reduce.The activity of model group MPO substantially increases.

More than pointing out us is reliable by 4%DSS inducing mouse colitis, and model is successful.

Two, the preventive and therapeutic effect research of dehydrogenation lovastatin inflammatory bowel experimental to dextran sulfate sodium inducing mouse

1 experiment material

1.1 laboratory animals:

Male mouse of kunming, body weight 22-25 gram, Medical University Of Chongqing's Experimental Animal Center provide.

1.2 main agents and medicine:

Dextran sulfate sodium (MP company of the U.S.)

Sulfasalazine enteric coatel tablets (Fuda Pharmaceutical Co., Ltd., Shanghai's authentication code: traditional Chinese medicines quasi-word H31020840)

Stool blood test kit (Bioengineering Research Institute is built up in Nanjing)

Myeloperoxidase (MPO) (MPO) measures test kit (Bioengineering Research Institute is built up in Nanjing)

Dehydrogenation lovastatin (dehydrolovastatin, DLVT), is provided by Chongqing Daxin Pharmaceutical Co., Ltd, Outward appearance is creamy white puffy corase meal, is slightly soluble in water.Lot number: 201401001.Lovastatin (lovastatin, LVT), by The new greatly Pharmaceutical share in Chongqing provides.Lot number: 201401001.

IL-6, IL-10, IL-17, TNF-α ELISA kit R&D company

1.3 key instruments:

Electronic balance

A2000S type electronic analytical balance

XiangYiH165-W centrifuge

-80 DEG C of ultra cold storage freezers (German)

Rayto RT-6500 enzyme micro-plate reader

BH-2 type optical microscope (Japan Olympus)

2 methods

2.1 laboratory animal packets:

By 50 kunming mices, male, it is randomly divided into 5 groups, i.e. Normal group (normal), inflammatory bowel model group (model), positive drug (SASP, 250mg kg^-1·d^-1), dehydrogenation lovastatin (DLVT, 33.6mg kg^-1·d^-1) and Lip river Cut down statin (LVT, 33.6mg kg^-1·d^-1), often organize each 10.Each drug treating group mouse stomach relative medicine, normal group and Model group gives the sodium carboxymethyl cellulose of equal-volume 0.5%, every day 1 time, successive administration 10d, and the 3rd day starts, except the most right Beyond group, remaining is respectively organized and freely drinks 4%DSS aqueous solution, and every day is changed once, until experiment terminates, is shown in Table 4 animal packets And dosage regimen.

The packet of table 4 animal and dosage regimen

The evaluation of 2.2 mice inflammatory bowel

Mice clinical symptoms is observed and is found: the activity of Normal group mice, hair color, defecation, growth are the most normal.Model group Mice, after drinking 4%DSS solution 2d, there is the positive of occulting blood in some animals defecation, and the nearly all stool in mice of 4-5d is occulted blood sun Property, 6-7d is strong positive, even hemafecia；Reducing with mice activity, body weight substantially alleviates simultaneously；Feces is soft, or diarrhoea is sent out Raw, similar to the performance of mankind's IBD clinical symptoms, show the success of 4%DSS inducing mouse IBD model.4%DSS model group mice, After 3d DAI start occur continue significantly to increase.Model mice is the most thin and weak, and perianal hair color is at random, has the trace that blood sample is defecated Mark.

The detection of cytokine in 2.3 mice serums

It is administered the 10th day, extracts eyeball and take blood.Centrifuging and taking serum, according to ELISA, (double antibody two step sandwich enzyme-linked immunosorbent is inhaled Attached method) operation of test kit description, respectively IL-6, IL-10, IL-17, TNF-α level in detection mice serum.

Concrete operations are as follows:

(1) prepare: standard substance dilution and preparation biotin antibody working solution and enzyme conjugates working solution.By concentrated cleaning solution Diluting (1:20) with distilled water, standard dilutions 1.0ml is to (concentration is 10ng/ml) in lyophilizing standard substance in addition, then is made into 1000,500,250,125,62.5,31.25,15.625,7.8,0pg/ml concentration.

(2) take out freezing serum to thaw, 12000 × g, 3min.Take out from IL-6, IL-10, IL-17, TNF-α test kit Required lath, in addition to blank well, adds specimen and variable concentrations standard substance (100 μ l/ hole) in respective aperture respectively, seals with shrouding Live reacting hole, hatch 90min for 37 DEG C.

(3) washing plate: get rid of liquid in most hole, every hole adds cleaning mixture 350 μ l, stands 30sec, pats dry in thickness repeatedly absorbent paper, Wash plate 5 times.

(4) in addition to blank well, add biotinylated antibody working solution (100 μ l/ hole), seal reacting hole with shrouding glue, 37 DEG C hatch 60min.

(5) plate is washed 4 times.

(6) in addition to blank well, add enzyme and combine working solution (100 μ l/ hole), seal reacting hole with shrouding glue, hatch for 37 DEG C 30min。

(7) plate is washed 4 times.

(8) adding developer 100 μ l/ hole, lucifuge 37 DEG C hatches 10-20min.

(9) add stop buffer 100 μ l/ hole, at 450nm wavelength, after mixing, measure OD value.

2.4 mouse Colon histopathology morphological observations and mouse Colon tissue MPO Activity determination

Colon's block rinsed clean in ice-cold normal saline, clean filter paper blots, and after correct amount, uses ophthalmology immediately Scissors shred, and are placed in the glass homogenizer of ice bath, are homogenized after adding 0.86% normal saline, and volume ratio by weight is that 1:9 adds Homogenate medium is prepared as the tissue homogenate of 10%.Reference reagent cassette method measures the activity of MPO in colon's homogenate.

2.5 mouse Colon tissue pathology checkings

The formalin of colon 10% is fixed, the specimen that specimens paraffin embedding slices makes, and haematoxylin & eosin (HE) contaminates Color, tissues observed Pathologic changes under light microscopic.

2.6NF-κ B immunohistochemical staining

Paraffin specimen is made colon cross section serial section, is attached on the microscope slide that poly ammonia is acid-treated, inserts 60 DEG C of oven overnight.After sample collects, use Use immunohistochemistrySP SP dyeing.Under concrete operation step:

(1) paraffin section routine dewaxes to water: about 50 DEG C dimethylbenzene I 30min, dimethylbenzene II 30min, 100%, 95%, 85%, 70% ethanol, each 5min of distilled water, then in distilled water, soak 5min.

(2) 3%H₂O₂Incubated at room 10min.(to eliminate the activity of endogenous peroxydase, 3%H₂O₂Join temporarily System.)

(3) distillation washing 2min × 3, PBS soaks 5min.

(4) antigen retrieval: use hot repairing method.Section is placed in and fills antigen retrieval buffers (0.01M citrate buffer PH6.0) in glass container, it is placed on electric furnace heating, controls solution temperature and maintain between 92 DEG C-98 DEG C, continue 10- 15min.After stopping heating, naturally cooling to room temperature (about needing 20-30min, will not cut into slices and take out from buffer), PBS washes 5min×3。

(5) dropping normal mouse closes serum working solution (reagent 1), incubated at room 20min, inclines, does not wash.

(6) dripping an anti-NF-κ B (1:100 dilution) respectively, 4 DEG C overnight.It is negative right that negative control group adds PBS work According to.

(7) PBS rinses 5min × 3.

(8) the dropping anti-working solution of biotinylation two: biotin labeling goat anti-mouse igg, hatches 20min for 37 DEG C.

(9) PBS rinses 5min × 3.

(10) dropping Radix Cochleariae officinalis enzyme labelling streptomycin avidin working solution (reagent 3), hatches 20min for 37 DEG C.

(11) PBS rinses 5min × 3.

(12) (usage: take 1ml distilled water, in test kit, A, B, C reagent respectively adds 1 to DAB chromogenic reagent, drips after being mixed Add to section.Developer should keep in Dark Place, and instant joins), wash 3min from the beginning.

(13) haematoxylin redyes 30 seconds (depending on the time of redying is according to staining conditions), washes 3min from the beginning.

(14) immersing the differentiation liquid 3-4 second, tap water fully rinses, 60 DEG C of stove-dryings.

(15) neutral gum mounting.

The semi-quantitative analysis that 2.7 immunohistochemical staining section NF-kB proteins are expressed

The expression of each layer of basis of microscopic observation colon photographic recording.Positive criteria: NF-κ B is with kytoplasm or born of the same parents Core part is dyed to pale brown (brown) color for the positive；Negative control: using PBS to replace one to resist, remaining step is identical. Use the GD-8 type pathological images Multimedia analysis system section random acquisition to each group of SABC, often organize at least 3 with On animal tissue sections, choose 10 high power fields (200 × 10), measure area integral optical density value (Area integral Optical density, AIOD) show the staining power being detected index, i.e. expression.

3 statistical procedures

Experimental data SPSS12.0 processes in statistical software, and each group data represents with (x ± s), uses single factor test variance Analyzing, the difference between each group of data, P ＜ 0.05 has significance for difference.

4 results

4.1DLVT and the LVT change to the Mouse Weight that 4%DSS induces

Model group mice is from drinking 4%DSS the 3rd day, and compared with naive mice, body weight is decreased obviously；Remaining is respectively given The downward trend of medicine group Mouse Weight is all not as good as model group.

4.2DLVT and the LVT impact on the mice clinical symptoms that 4%DSS induces

Mice clinical symptoms is observed and is found: Normal group mice behavior enlivens, and growth is normal.Model group mice, at drink After 4%DSS solution 2d, there is the positive of occulting blood in some animals feces, and the nearly all stool in mice of 4-5d is occulted blood the positive, 6-7d in Strong positive, even hemafecia；Reducing with mice activity, body weight substantially alleviates simultaneously；Feces is soft, or diarrhoea occurs, with the mankind The performance of IBD clinical symptoms is similar, shows the success of 4%DSS inducing mouse IBD model.As it is shown on figure 3, DSS model group mice, 3d Rear DAI starts appearance and continues significantly to increase；SASP checks DAI and increases, DLVT with LVT has and similar be obviously improved DSS induction The effect of IBD mice clinical symptoms.

The morphologic impact of mouse Colon histopathology that 4%DSS is induced by 4.3DLVT and LVT

Light Microscopic observation DSS model group mouse Colon epithelium mucosa injury is obvious, and mucosal epithelium is rotten to the corn, glandular crypts structure Destroy；Goblet cell is lost, and with massive inflammatory cells infiltrated such as neutrophilic granulocyte, lymphocyte, plasma cells, sees Fig. 4 (b). SASP, DLVT and LVT group Injured colonic mucosa substantially alleviates, and epithelial structure is more complete, and glandular crypts arrangement is relatively neat, and inflammatory is thin Born of the same parents infiltrate minimizing, see Fig. 4 (c), Fig. 4 (d), Fig. 4 (e) respectively.Light Microscopic observation respectively organizes specimen section, often 15 visuals field of group (× 100, × 200) according to " table 2 histological damage score standard ", carry out histological damage score, ask its meansigma methods, appraisal result It is shown in Table 5:DLVT and the LVT impact on 4%DSS inducing mouse colon pathomorphology.

Table 5DLVT and the LVT impact on 4%DSS inducing mouse colon pathomorphology

^**P ＜ 0.01 is to compare with normal group；^##P ＜ 0.01 is to compare with model group.

The cytokine such as IL-6, IL-10, IL-17, TNF-α in the mice serum that 4%DSS is induced by 4.4DLVT and LVT Impact

As shown in table 6, model group mice serum IL-6, IL-17, TNF-α content are all remarkably higher than Normal group (p ＜ 0.01), remaining each administration group mice serum IL-6, IL-17, TNF-α content are all substantially less than model group.Model group mice serum The content of IL-10 is substantially less than Normal group, and the content of remaining each administration group IL-10 is significantly higher than model group.

In the mice serum that 4%DSS is induced by table 6DLVT and LVT the content of IL-6, IL-17, TNF-α impact ( N=10)

^**P ＜ 0.01 is to compare with Normal group；^##P ＜ 0.01 is to compare with model group.

DLVT and LVT shows obvious inhibitory action to the proinflammatory factor such as TNF-α, IL-6, IL-17, and can improve The level of anti-inflammatory factors IL-10.

The activity of MPO in the mouse Colon tissue that 4%DSS is induced by 4.5DLVT and LVT

Mouse Colon tissue MPO Activity determination the results are shown in Table 7.Visible DSS inducing mouse colon's epithelium and lamina propria Middle neutrophil infiltration significantly increases, MPO activity 2.19 ± 0.42U/g, and compared with normal matched group 0.92 ± 0.22U/g substantially increases High.SASP substantially suppresses the infiltration of neutrophilic granulocyte and MPO to increase (P < 0.01).DLVT (33.6mg/kg) and LVT (33.6mg/ Kg) the most substantially suppression MPO activity (P < 0.01).

The impact of the mouse Colon tissue MPO activity that 4%DSS is induced by table 7DLVT and LVT

* P < 0.01 and normal group compares；^##P < 0.01 and model group compares.

4.6NF-κ B immunohistochemical staining

Mouse Colon histogenic immunity histochemical staining shows, in Normal group colon, NF-κ B is almost without expression, sees Fig. 5 (a), and DSS guidance model group mouse Colon tissue is expressed the positive cell number showed increased of NF-κ B, see Fig. 5 (b), in Brown color.In SASP group, DLVT group, LVT group mouse Colon tissue, NF-κ B positive cell not only quantity is compared substantially with model group Reduce, see Fig. 5 (c), Fig. 5 (d), Fig. 5 (e) respectively.

The semi-quantitative analysis that 4.7 immunohistochemical staining section NF-kB proteins are expressed

The expression of each layer of basis of microscopic observation colon photographic recording.Use GD-8 type pathological images multimedia The analysis system section random acquisition to each group of SABC, often organizes the animal tissue sections of at least more than 3, chooses 10 High power field (200 × 10), measures area integral optical density value (Area integral optical density, AIOD) and shows Show the staining power of detected index, i.e. expression.Analysis result is shown in Table 8DLVT and SASP to 4%DSS inducing mouse colon group Knit the impact that NF-κ B expresses.

The impact that 4%DSS inducing mouse colon NF-κ B is expressed by table 8DLVT and LVT

Group	NF-κB(AIOD)
		nromal	0.000±0.000
model	0.282±0.061<sup>**</sup>
		SASP(250mg/kg)	0.181±0.026<sup>##</sup>
DLVT(33.6mg/kg)	0.187±0.028<sup>#</sup>
		LVT(33.6mg/kg)	0.185±0.022<sup>##</sup>

^**P ＜ 0.01 is to compare with normal group；^##P ＜ 0.01 with^#P ＜ 0.05 is to compare with model group.

In sum, the acute colitis mice DAI of the DSS induction of 4% dramatically increases, colon occurs the most scorching In disease damage and tissue, MPO vigor dramatically increases, proinflammatory factor TNF-α in serum, the concentration of IL-6, IL-17 dramatically increase and The concentration of anti-inflammatory factors IL-10 significantly reduces, and in colon, NF-κ B expresses and also dramatically increases.Through dehydrogenation lovastatin and Lip river The DAI that can significantly alleviate DSS inflammatory bowel mice after cutting down statin treatment, the Histological injury improving colon, reduction colon group Knit middle MPO activity, the proinflammatory factor such as TNF-α, IL-6, IL-17 shown obvious inhibitory action, and can improve antiinflammatory because of The level of sub-IL-10, also NF-κ B signal path play the effect of inflammatory enteropathy.

Three, discuss

In recent years basis and clinical research find, statins suppression isoprenoid (isoprenoid) intermediate is such as Isoprenoid method Buddhist nun's ester (farnesylpyrophosphate, FPP) and isoprenoid four Isoprenoid (geranylgeranylpyrophosphate, GGPP) synthesizes, and then the albumen that suppression cellular isoprenoids relies on, and sends out Wave the multiple effects such as anti-Cardiovascular Remodeling, antitumor, prevention dementia, antiinflammatory and immunomodulating, thus to suffering from metabolic disease Have rheumatism, rheumatoid arthritis and inflammatory bowel concurrently and there is good clinical efficacy.

There is cytokine to participate in the whole pathological process of inflammatory bowel, they constitute a cytokine net Network, can regulate and control mutually it and produce, and wherein TNF-α, IL-6, IL-17 etc. are proinflammatory factor, and IL-10 is then anti-inflammatory factors. NF-κ B is the transcription regulatory factor that a class has multidirectional activity, participates in expression and the regulation and control of several genes, anti-in inflammation and immunity Should, the aspect such as cell proliferation, apoptosis and infection plays an important role.NF-κ B/I κ B complex can by cytokine (TNF-α, IL-1 β), oxidant, the many factors such as virus antigen activate, NF-κ B is displaced into karyon, starts related gene and transcribes.

The specific name of the english abbreviation related in the present invention is as shown in table 9.

Table 9

It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie In the case of the spirit or essential attributes of the present invention, it is possible to realize the present invention in other specific forms.Therefore, no matter From the point of view of which point, all should regard embodiment as exemplary, and be nonrestrictive, the scope of the present invention is by appended power Profit requires rather than described above limits, it is intended that all by fall in the implication of equivalency and scope of claim Change is included in the present invention.

Although moreover, it will be appreciated that this specification is been described by according to embodiment, but the most each embodiment only wraps Containing an independent technical scheme, this narrating mode of description is only that for clarity sake those skilled in the art should Description can also be formed those skilled in the art through appropriately combined as an entirety, the technical scheme in each embodiment May be appreciated other embodiments.

Claims (4)

1. dehydrogenation lovastatin application in preparation inflammatory enteropathy medicine.

The dehydrogenation lovastatin the most according to claim 1 application in preparation inflammatory enteropathy medicine, its feature exists In, the stereochemical formula of dehydrogenation lovastatin is as follows:

The dehydrogenation lovastatin the most according to claim 1 application in preparation inflammatory enteropathy medicine, its feature exists In, dehydrogenation lovastatin is 33.6mg kg for the effective dose of 4%DSS inducing mouse inflammatory bowel^-1·d^-1。

The dehydrogenation lovastatin the most according to claim 1 application in preparation inflammatory enteropathy medicine, its feature exists In, in described inflammatory enteropathy, proinflammatory factor includes TNF-α, IL-6 and IL-17, and anti-inflammatory factors is IL-10.