CN103143021A - Application of PPAR alpha-UGT (peroxisome proliferator activated receptor alpha-uridine diphosphate glucuronosyl transferase) pathway inhibitor in treating inflammatory bowel diseases - Google Patents

Abstract

The invention provides an application of a PPAR alpha-UGT (peroxisome proliferator activated receptor alpha-uridine diphosphate glucuronosyl transferase) pathway inhibitor in treating inflammatory bowel diseases, belongs to the field of new medicine research and development, and in particular relates to discovery of the treatment effect of the PPAR alphas-UGT pathway inhibitor, which is used for relieving hepatic and gall disease complications of inflammatory bowel diseases by controlling the steady equilibrium of bile acid, and reducing the risk that inflammatory bowel diseases are transformed to colon cancer. The application proves that the PPAR alpha-UGT pathway can be remarkably activated in a DSS colonitis mouse due to the detection to mice in intestine PPAR alpha and downstream typical target genes of a normal group and DSS (dextran sodium sulfate) induced colonitis mice by a qRT-PCR (quantitative real-time polymerase chain reaction) technology; and the application discovers and proves that an FXR-FGF15 (farnesoid X receptor-fibroblast growth factor15) negative feedback pathway of bile acid is remarkably inhibited in the colonitis mouse and bile acid synthesis is remarkably increased due to the qRT-PCR technology and an immunohistochemical technology. Therefore, activation of the PPAR alpha-UGT pathway is an initial factor of homeostasis imbalance of bile acid in inflammatory bowel diseases, and the PPAR alpha-UGT pathway inhibitor is expected to become a new target for researching and developing medicines for treating vicious transformation of inflammatory bowel diseases and complications thereof.

Description

The application of PPAR α-UGT pathway inhibitor in the treatment inflammatory bowel

Technical field

The present invention relates to the new drug development field, particular content is PPAR α-UGT pathway inhibitor by controlling the homeostasis of bile acid, and then alleviates the liver and gall diseases complication of inflammatory bowel and reduce it and transform the application of risk to colon cancer.

Background technology

UGT is the phase II metabolic enzyme that is positioned on endoplasmic reticulum, and it can strong aglycone and glycosyl donor glucuronic acid (UDPGA) combination of catalysis lipotropy, thereby increases the polarity of lipophilic substrate.Most compound (endogenous material such as bile acid, bilirubin, steroid hormone, alkamineses; Exogenous material such as composition of food, medicine, environmental toxin, carcinogen etc.) the Glucuronidated product be the metabolism inactivation reaction, and water miscible increase is conducive to it from urine and bile discharge.Existing bibliographical information, the expression of UGT and function are subjected to the regulation and control of nuclear receptors PPAR's α.Therefore, PPAR α-UGT path is considered to the detoxification pathways of classics of body.

Inflammatory bowel (IBD) comprises that Crohn disease (CD) and ulcerative colitis (UC) are a kind of and heredity, environment, Ia chronic intestinal tract disease.The IBD patient of 10%-30% is disorderly with liver and gall diseases and bile acid homeostasis, but pathogenesis is not clear.Epidemiological study shows the occurred frequently closely related of IBD and colorectal cancer (CRC), and the risk that patients of ulcerative colitis is suffered from colorectal cancer is up to 10 times of normal population, and this risk increases gradually along with course of disease time and the intensity of enteritis.Most IBD patients show effect repeatedly, protracted course of disease, and wherein the considerable part patient is because developing complications operative treatments such as needing liver transplantation.The IBD course of disease time is long and be difficult to radical cure, and most patient IBD need take the medicine of steroidal anti-inflammatory medicine and immunosuppressant class throughout one's life.But due to selectivity and the specific restriction of said medicine, inevitably can damage patient's immune protection capability in the treatment disease, cause patient's anti-infection ability to descend, the danger of malignant change increases.Therefore, be badly in need of verifying clinically the pathogenesis of IBD, find new treatment target spot, research and development are medicine more safely and effectively.

Bile acid is important endogenous signaling molecule in body, the multiple physiological reactions such as mediation lipid absorption, cholesterol decomposition, energy metabolism and inflammatory reaction.But the damage of the bile acid long term exposure trigger cell of high concentration and tumor generate, therefore in body, the bile acid concentration needs monitor closely.In gallbladder silt patient's urine, the glucuronide conjugate of bile acid can raise 4 to 50 times and not wait, and the bile acid that exists with the glucuronide conjugate form in urine accounts for 35% of TOTAL BILE ACID TBA.It is reported, UGT is the important metabolisable form of bile acid, is also unique deactivation mode.

Suffer from based on patient IBD the clinical fact that liver and gall diseases reaches the colon cancer sickness rate that significantly increases more, the short cancer activity of conjugated bile acid and the theoretical basis of being eliminated by the UGT metabolism, we think: the unbalance of PPAR α-UGT-bile acid biosynthesis regulated and control network is the initiating agent that non-controllability enteritis vicious transformation and serious liver and gall complication occur, and PPAR α-UGT path also is expected to become the medicament research and development novel targets for the treatment of IBD and complication thereof.

Summary of the invention

The medicament research and development novel targets that the objective of the invention is to study the imbalance mechanism of bile acid under the inflammatory bowel state and find to prevent the inflammatory bowel vicious transformation.At first we induce the chronic colitis mouse model by sodium dextran sulfate (DSS), and can the research ulcerative colitis cause the change of PPAR α-UGT path.According to the qRT-PCR experimental result, the mrna expression of the PPAR α at the small intestinal place of the chronic colitis mice that DSS induces itself does not change, but significant rise has all occured for the classical target gene Acox-1 in its downstream and L-Fabp.Consistent is that the expression of the Main Subtype of the small intestinal UGT of place and active rise trend is highly significant also therewith.Found further that by qRT-PCR technology and immunohistochemistry technique the degenerative FXR-FGF15 path of mediation bile acid has been subject to significant inhibition in the enteritis mice, and the mrna expression of bile acid biosynthesis rate-limiting enzyme Cyp7a1 and protein content there is significant rise trend.The increase of bile acid biosynthesis has been brought out in this explanation PPAR α-activation of UGT path, and might cause toxicity bile acid accumulating and the further deterioration of disease in vivo.Therefore, the inhibitor of PPAR-UGT path can be by recovering the bile acid homeostasis and then preventing the deterioration of inflammatory bowel and the generation of liver and gall complication.

Description of drawings

Fig. 1: the mrna expression (a) of PPAR α in normal mouse and DSS enteritis mice; The mrna expression of A cox1 (b); The mrna expression of L-Fabp (c)

Fig. 2: the mrna expression (a) of each hypotype of UGT and functional activity (b) in normal mouse and DSS enteritis mice

Fig. 3: the mrna expression (a) of FXR and FGF15 and immunohistochemical analysis (b) in normal mouse and DSS enteritis mice

Fig. 4: the mrna expression (a) of bile acid biosynthesis rate-limiting enzyme and protein content (b) in normal mouse and DSS enteritis mice

The specific embodiment

Copying of the chronic colitis disease mouse model that embodiment 1.DSS induces

Experiment material: the male C57BL/6 mice of SPF level 20g (8 age in week) is provided by Military Medical Science Institute's Experimental Animal Center;

Dextran sulfate (DSS, 36000-50000) is available from MP company.

Experimental technique:

1. experimental mouse needs to adapt at least 7 days in standardization Animal House environment, gives the standard animal feed, drinks autoclaving water, and in time changes bedding and padding (2-3 time weekly).

2. configure 2.5%DSS solution: use autoclaving water according to w/v configuration 2.5% preparation DSS solution, the DSS solution for preparing can be deposited a week in 4 ℃ of refrigerators.

3. after experiment beginning, calculate by the amount of drinking water 5ml of every mice every day, pour the DSS solution of 2 daily drink amounts at every turn in the mice water bottle, if any residue, should discard after 2 days, again pour the DSS solution of 2 fresh daily drink amounts into.First cycle that gives of DSS solution is 7 days.

4. morning on the 8th, replace 2.5%DSS solution to give model mouse with autoclaving water and freely drink, the cycle of drinking is 14 days.

5. front 14 days of the experiment beginning, to answer diet every day, the amount of drinking water of record cast mice, and weigh in, observation has or not hemafecia to produce.

6. above whole process is a circulation, then repeats 2 above-mentioned circulations, and modeling finishes, and the whole modeling cycle was 9 weeks.Matched group is drunk autoclaving water, and all experimental mouse give standard feed.

Embodiment 2.qRT-PCR gene expression detects

Experiment material: Trizol (Takara, Japan), reverse transcription reagent (Takara, Japan), SYBR Green I PCR test kit (Takara, Japan), Bio-Rad CFX series fluorescent quantitation instrument (Biosystems, Bedford, MA).

Experimental technique: Trizol (Takara, Japan) extracts total RNA, measures RNA concentration and purity.Adopt reverse transcription reagent (Takara, Japan) to carry out reverse transcription.And with the quantitative PCR instrument: Thermal Cycler Dice ^TMTest on Real Time System (Takara, Japan).Primer sequence is as shown in table 1.Adopt SYBR Green I PCR test kit (Takara, Japan) to carry out expression conditions research.The employing three-step approach is carried out, and reaction system is 20 μ L, and annealing temperature is 60 ℃, and each sample carries out three times to be repeated, and date processing adopts Δ Δ C ^tMethod.

Table 1 qRT-PCR primer sequence

Embodiment 3.Western Blot detects

Experiment material: Bio-Rad electrophresis apparatus; Bio-Rad gel imaging instrument.FXR, FGF15, Cyp7a1 antibody and corresponding two resists available from Santa Cruz Biotechnology (Santa Cruz, CA).GAPDH antibody is available from SunShine Biotechnology (Nanjing, China).Cell pyrolysis liquid: 10mM Tris-HCl (pH7.5), 1mM EGTA, 1mM MgCl2,1mM β-ME, 1%glycerine, protease inhibitor (containing 1mM DTT and 2mM PMSF).

Experimental technique:

1. get that appropriate (80～100mg) flesh tissue samples or the correct tissue sample of preserving add the total protein extraction agent (or nucleoprotein extraction agent) that 1ml contains protease inhibitor, extracting total protein (or nucleoprotein) after homogenate.

2. quantification of protein: press the operation of BCA quantification of protein test kit operating instruction, working sample concentration.

3. prepare before the protein sample loading: sample is diluted to same concentrations with lysis buffer, add respectively 4 * sample-loading buffer (sample volume: 4 * sample-loading buffer=3: 1), mixing, and boiling in 95-100 ℃.Cooled on ice after 5min, loading (applied sample amount is every duct 50-100 μ g albumen).

4. electrophoresis: the preparation running gel, carry out SDS-PAGE.Concentrated glue constant voltage 75V, approximately 60min; Separation gel constant voltage 115V, approximately 90min.

5. shift (half dry type transfer):

1. electrophoresis after finishing cuts adhesive tape to suitable size, with transferring film buffer balance.

2. pvdf membrane activation: cut out in advance the pvdf membrane onesize with adhesive tape, soaked 1-2 minute in methanol, then soaked 1-2 minute in ultra-pure water, immersed at last in the transferring film buffer balance 5 minutes.

3. transferring film: membrane-transferring device is put well by the order of carbon anode plate, filter paper, film, gel, filter paper, negative electrode carbon plate from bottom to up successively, removes bubble.Switch on power, (0.2 ampere, the glue of 1cm thickness shifted 1.5 hours in constant current; 1.5cm 0.3 ampere, the glue of thickness, transferase 12 hour).After shifting end, deenergization takes out film, gets film bar to be measured and does immunoblotting.

6. immunoreation:

1. 37 ℃ of 1hr are steadily shaken in 5% milk (TBS/T preparation) sealing; Or spend the night in 4 ℃ of placements.

2. abandon confining liquid.

3. (dilute with 5% milk (TBS/T dilution) by suitable dilution ratio, 4 ℃ of placements are spent the night to add primary antibodie; Or steadily shake 37 ℃ of 1hr-2hr.Abandon primary antibodie, wash film with 1%TBS/T, 5min * 4 time.

4. add two anti-(diluting with 5%BSA by suitable dilution ratio) of horseradish peroxidase, steadily shake 37 ℃ of 1hr

5. abandon two anti-ly, wash film with 1%TBS/T, 5min * 4 time.

6. add nitrite ion, exposure.

The separation of embodiment 4. subcellular structure S9 systems and UGT are active to be detected

Experiment material: UDP-glucuronic acid (UDPGA), single lactone, alamethicin, estradiol, estradiol 3-O-glucuronide, chenodeoxycholic acid, 4-methyl umbelliferone (4-MU), 4-methyl umbelliferone O-glucuronide, mycophenolic acid (MPA), and naloxone is available from Sigma-Aldrich (St.Louis, MO).MPA O-glucuronide and naloxone 3-β-D-Glucose aldehydic acid glycosides is available from Toronto Research Chemicals Inc (Ontario, Canada).

Test method:

1.S9 preparation: treating excess syndrome is tested the respective organization of animal, inserts in the PBS buffer of pre-cooling, makes 20% tissue homogenate.The centrifugal rear removal of 1000g lower floor cell debris, supernatant is sub-packed in 1.5ml Eppendorf pipe, 9000g after centrifugal 20 minutes its supernatant be S9.The S9 that obtains is with BCA kit measurement protein content, inserts-80 degree Refrigerator stores after packing standby.

2.UGT active detection the: adopt the probe substrate temperature method of incubating that the activity of UGT is detected.200 μ l temperature are incubated reaction system should comprise 50mM Tris-HCl buffer (pH7.4), 10mM MgCl2,25 μ g/ml alamethicin, UDPGA, S9, and for the selective probe substrate (comprising estradiol, chenodeoxycholic acid, 4-methyl umbelliferone, mycophenolic acid and naloxone) of different UGT hypotypes.The concentration of UDPGA should guarantee to reach the platform concentration of UGT reactivity.S9 and alamethicin need pre-reaction 30min under 4 degree conditions, then carry out the detection of UGT activity.Temperature adds 600ul acetonitrile precipitation albumen, cessation reaction after incubating the reaction end.After high speed centrifugation (15000r/min, 10min) 2 times, get supernatant 200ul and be placed in the glass sample injection bottle and treat sample introduction.The reaction density of probe substrate should be lower than the Km value of corresponding metabolic enzyme, and the reaction final concentration of estradiol, chenodeoxycholic acid, 4-methyl umbelliferone, mycophenolic acid and naloxone is respectively 20,50 in this experiment, 100,200 and 20 μ M.Concrete temperature is incubated reaction condition and is seen table 2 for details.

The active temperature that detects of table 2 UGT is incubated condition

The immunohistochemical analysis of embodiment 5.FXR-FGF15

Experiment material: FXR and FGF15 antibody and corresponding two resist available from Santa Cruz Biotechnology (Santa Cruz, CA)

Test method:

The tissue to be measured that 1, will be fixed in 10% formalin carries out paraffin embedding and section.

2,3% hydrogen peroxide at room temperature was hatched 5-10 minute, to eliminate the activity of endogenous peroxydase.

3, distilled water flushing, PBS soaks 5 minutes/2 times.

4,5-10% normal goats serum (PBS dilution) sealing, incubated at room 10 minutes, the serum deprivation that inclines is not washed.Drip the primary antibodie working solution, hatch for 37 ℃ and spent the night in 1-2 hour or 4 ℃.

5, PBS rinses, 5 minutes/3 times.

6, drip the anti-working solution of appropriate biotin labeling two, hatched 10-30 minute for 37 ℃.

7, PBS rinses, 5 minutes/3 times.

8, drip appropriate Radix Cochleariae officinalis enzyme or the strepto-avidin working solution of alkali phosphatase enzyme mark, hatched 10-30 minute for 37 ℃.

9, PBS rinses, 5 minutes/3 times.

10, chromogenic reagent 3-15 minute (DAB or NBT/BCIP)

11, tap water fully rinses, redye, and dehydration, transparent, mounting.

12, take pictures and read sheet.

Claims (2)

1.PPAR the application of α-UGT pathway inhibitor in the treatment inflammatory bowel

2.PPAR the application of α-UGT pathway inhibitor in the treatment inflammatory bowel, it is characterized in that: the inhibition of PPAR α-UGT path, can pass through the negative feedback process of the endocrine approach of adjusting bile acid FXR-FGF15, recover the homeostasis of bile acid under the inflammatory bowel state, and then alleviate the liver and gall diseases complication of inflammatory bowel and reduce the risk that it transforms to colon cancer.

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