CN106619619A - Application of compound HUBIN-1 in preparation of drug for preventing and/or treating liver inflammatory diseases - Google Patents
Application of compound HUBIN-1 in preparation of drug for preventing and/or treating liver inflammatory diseases Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
Abstract
The invention relates to the field of drug research and development and particularly discloses an application of a compound HUBIN-1 in preparation of a drug for preventing and/or treating liver inflammatory diseases. Extensive and deep research discovers the hepatocyte protection function and the anti-inflammatory treatment function of the compound HUBIN-1 for the first time, the compound HUBIN-1 is applicable to various types of liver inflammations, and the application field of the compound HUBIN-1 is broadened. The novel drug for preventing and/or treating various liver inflammatory diseases such as acute hepatitis, chronic hepatitis and severe hepatitis (hepatic failure) which include viral hepatitis, drug-induced hepatitis, nonalcoholic fatty liver disease, alcoholic hepatitis, autoimmune hepatitis and the like is provided and has remarkable social benefits.
Description
Technical field
The present invention relates to Field of Drug Discovery, compound HUBIN-1 is specifically disclosed to prepare inflammation disease pre-
Purposes in anti-and/or medicine.
Background technology
Inflammation disease is a kind of common disease at present clinically, is classified from etiology angle, can be divided into disease
Virus hepatitis, drug induced hepatitis, fat hepatitis, alcoholic hepatitis and autoimmune hepatitiss etc..China belongs to hepatitis B
The high popular country of malicious (HBV) infection, the people of national existing hepatitis B patient about 30,000,000.According to onset emergency and course of disease length,
Acute hepatitises, chronic hepatitiss, hepatitis graviies (liver failure) etc. can be divided into.Arrange due to lacking appropriate and effective intervention and treatment
Apply, part of Acute hepatitis and the chronic hepatitis patient state of an illness can be developed rapidly and become hepatitis graviies.Clinical report shows heavy
The case fatality rate of hepatitis is very high, and case fatality rate is up to 50%～70%.Hepatitis viruss (being mainly hepatitis B viruss in China) are felt
Dye, chemical poisonous substance or medicine, ischemia, ethanol, radiation sexual stimuluses, many factors such as inherited metabolic obstacle and autoimmune disease
Slow or acute hepatitises can be caused, hepatitis graviies (liver failure) are even resulted in.Hepatitis graviies (liver failure) PD is rapid, fierce
Danger, with hepatocyte extensive necrosis, causes the functions such as liver synthesis, removing toxic substances, excretion and bioconversion that serious hindrance or mistake occur
It is compensatory, show as with one group of clinical syndrome of disturbances of blood coagulation and jaundice, hepatic encephalopathy, ascites etc..
At present, for hepatitis graviies (liver failure) still lack effective treatment meanss and medicine, its case fatality rate is caused always
It is high.Clinically it is typically only capable to take the composite treatments such as corresponding metabolism and nutritional support.Although liver transplantation can be used for one
Acute hepatic failure patient caused by a little concrete reasons, but because the problem of internal organs source and medical expense, it is impossible in clinic
Upper large-scale use.And biological artificial liver support system regulating liver-QI, stem cell transplantation are in treatment acute severe hepatitises (liver failure)
Effect be still in clinical research early stage.Therefore new medicine is urgently researched and developed, new treatment meanss and method are opened up,
To lift the treatment survival rate of hepatitis graviies (liver failure), and the degree of inflammation of various acute and chronic hepatitis can be improved.
The content of the invention
It is an object of the invention to overcome the defect of prior art, there is provided compound HUBIN-1 is preparing inflammation
Purposes in disease prevention and/or medicine.
In order to realize the purpose of the present invention and related purpose, the present invention is adopted the following technical scheme that：
A first aspect of the present invention, there is provided compound HUBIN-1 is preparing inflammation disease prevention and/or treatment
Purposes in medicine, the structural formula of the compound HUBIN-1 is as shown in formula I：
The molecular formula of the compound HUBIN-1 is C13H11CIFN3O2.The molecular weight of the compound HUBIN-1 is
295.0524。
The compound HUBIN-1 is prior art, can be prepared by existing method, and these are in art technology
In the knowledge category that personnel understand that.
Preferably, the medicine possesses following active any one or multinomial：(1) ALT levels can be reduced；(2) energy
Enough reduce AST levels；(3) hepatocellular degeneration and degree of necrosis can be mitigated；(4) hepatocellular apoptosis are suppressed；(5) inflammation is reduced thin
Born of the same parents infiltrate；(6) hepatic macrophages (Kupffer Cell) activation and inflammatory factor secretion are suppressed；(7) suppress hepatocyte esterified and thin
Born of the same parents' Lipotoxicity；(8) apoptosis of hepatic stellate cell is promoted.
Preferably, the medicine possesses following active any one or multinomial：(1) anti-liver injury；(2) anti-inflammatory cell
Infiltration；(3) anti-hepatocyte is esterified；(4) anti-hepatic fibrosis.
Preferably, the inflammation disease includes but is not limited to acute hepatitises, chronic hepatitiss, hepatitis graviies, fat
Liver, hepatic fibrosis and liver cirrhosis.
Preferably, the inflammation disease includes but is not limited to viral hepatitis, drug induced hepatitis, fatty liver
Various inflammation diseases such as inflammation, alcoholic hepatitis, autoimmune hepatitiss, including acute hepatitises, chronic hepatitiss and acute
Hepatitis graviies, subacute severe hepatitises, slow extra urgaent dispatch hepatitis graviies.
Preferably, the compound HUBIN-1 is used as one of effective ingredient or sole active ingredient.
Second aspect present invention discloses a kind of inflammation disease prevention and/or medicine, containing safe and effective
The compound HUBIN-1 of amount, and pharmaceutically acceptable carrier.
Preferably, the compound HUBIN-1 is used as one of effective ingredient or sole active ingredient.
Pharmaceutically acceptable carrier is various pharmaceutically conventional adjuvants and/or excipient, including but not limited to sugar
Class (such as Lactose, dextrose and saccharose), starch (such as corn starch and potato starch), cellulose and its derivates are (such as carboxymethyl
Sodium cellulosate, ethyl cellulose and methylcellulose), tragacanth gum powder, Fructus Hordei Germinatus, gelatin, Talcum, kollag is (such as Hard Fat
Acid and magnesium stearate), calcium sulfate, vegetable oil, such as Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, Oleum sesami, olive oil, Semen Maydis oil and cupu oil are polynary
Alcohol (such as Propylene Glycol, glycerol, Sorbitol, mannitol and Polyethylene Glycol), alginic acid, emulsifying agent is (such as Tween, Polyethylene oxide
Oleum Ricini), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, tablet agent, stabilizer, antioxidant, preservative, nothing
Pyrogen water, isotonic saline solution and phosphate buffer etc.；The carrier can as needed improve stability, activity and the biology of formula
Effectiveness etc..
When medicine of the present invention is used, the compound HUBIN-1 is used as sole active ingredient, or the compound
HUBIN-1 can be mixed different as one of effective ingredient from one or more pharmaceutically acceptable carrier or excipient
The pharmaceutical dosage form of route of administration.
The medicine of the present invention can make any conventional dosage form according to the universal method on pharmaceuticss.
The dosage form of the medicine can be tablet, capsule, powder, granule, syrup, solution, oral liquid, fine wine
Agent, tincture, aerosol, powder spray, injection, injectable sterile powder, or suppository.Above-mentioned preparation type can be according to medicament
The related definition learned in (sixth version, People's Health Publisher, Cui Fude) understands that the preparation of above-mentioned preparation can be according to pharmaceuticss
The method of the related preparations in (sixth version, People's Health Publisher, Cui Fude) is prepared.
A third aspect of the present invention, there is provided a kind of method of prevention and/or treatment inflammation disease, including step
Suddenly：By the medicament administration of the compound HUBIN-1 containing safe and effective amount in effective object.
Preferably, the inflammation disease is acute hepatitises, chronic hepatitiss and hepatitis graviies (liver failure), including disease
Various inflammation diseases such as virus hepatitis, drug induced hepatitis, fat hepatitis, alcoholic hepatitis and autoimmune hepatitiss
Disease.
Using dosage can be 1.25～10mg/kg, can be selected according to the concrete condition using object.These are in doctor
In the ken for knowing and grasping.
Methods described can be internal.Can also be external, for example it is only used for scientific research.
A fourth aspect of the present invention, there is provided the new application of compound HUBIN-1, the structure of the compound HUBIN-1
Formula is as shown in formula I：
Formula I,
Including any one of following purposes or multinomial：(1) ALT levels can be reduced；(2) AST levels can be reduced；(3) energy
Enough mitigate hepatocellular degeneration and degree of necrosis；(4) hepatocellular apoptosis are suppressed；(5) inflammatory cell infiltration is reduced；(6) liver is suppressed
Macrophage (Kupffer Cell) is activated and inflammatory factor secretion；(7) suppress hepatocyte esterified and cytolipin toxicity；(8) promote
The apoptosis of hepatic stellate cell.Compared with prior art, beneficial effects of the present invention are：
The present invention through extensively in-depth study, be found that first compound HUBIN-1 hepatocytoprotection and
Anti-inflammatory treatment is acted on, and suitable for polytype inflammation, has opened up the application of compound HUBIN-1.The present invention
For acute hepatitises, chronic hepatitiss and hepatitis graviies (liver failure), including viral hepatitis, drug induced hepatitis, fat hepatitis, wine
Various inflammation diseases such as essence hepatitis and autoimmune hepatitiss provide new prevention and/or medicine, have
Obvious social benefit.
Description of the drawings
Fig. 1：Protective effect of the compound HUBIN-1 different dosings dosage to mouse hepatitis model；Wherein, HUBIN-1-
0.625 group, 1.25 groups, 2.5 groups, 5 groups, 10 groups, respectively represent LPS/D-GalN modelings before carry out within 30 minutes 0.625mg/kg,
The treatment group of the compound HUBIN-1 intraperitoneal administrations of 1.25mg/kg, 2.5mg/kg, 5mg/kg and 10mg/kg dosage；Chemical combination
Thing HUBIN-1 treatment groups hepatic pathology is obviously improved, 2.5mg/kg, 5mg/kg and 10mg/kg treatment group best results, hepatic tissue
It is several without hepatic necrosis and cell infiltration.
Fig. 2：Therapeutical effect of the compound HUBIN-1 different dosing times to mouse hepatitis model；Compound HUBIN-1 in
Administration in 30 minutes and 1 hour 30 minutes before LPS/D-GalN modelings, after modeling can significantly alleviate hepatic necrosis and inflammatory cell
Infiltration, improves hepatic pathology.
Fig. 3：Compound HUBIN-1 and comparitive studies of the Nec-1 to mice hepatitis；30 minutes before LPS/D-GalN modelings
Carry out Isodose (5mg/kg) compound HUBIN-1 or Nec-1 (Nec-1 1., 2., 3. respectively be purchased from Sigma-
Aldrich, Merck Millipore, BioVision) intraperitoneal administration, compound HUBIN-1 more can significantly change than Nec-1
Kind hepatic pathology.
Fig. 4：Hepatocellular apoptosis and the impact of necrosis of the compound HUBIN-1 to TNF-α/ActD inductions；Model group is represented
With the hepatocellular apoptosis/Necrosis Model of TNF-α/ActD inductions, HUBIN-1-25 μM, -50 μM, -100 μM represents respectively with 25 μ
M, 50 μM, 100 μM of compound HUBIN-1 pretreatment 1 hour, then add TNF-α/ActD to stimulate；TNF-α/ActD stimulates 6 hours
Post analysis early apoptosis of cells ratio, compound HUBIN-1 pretreatment can significantly lower the early apoptosis ratio of L02 cells
(4A)；TNF-α/ActD stimulates 24 hours post analysis necrosis ratios, compound HUBIN-1 pretreatment significantly to lower L02 cells
Downright bad ratio.
Fig. 5：The Western Blot of LPS/D-GalN model mices hepatic tissue Caspase-3 spliced bodies and RIP1 albumen point
Analysis；1 is Normal group, and 2-4 is LPS/D-GalN model group, and 5-7 is 2.5mg/kg dosage HUBIN-1 treatment groups, and 8-10 is
5mg/kg dosage HUBIN-1 treatment groups, 11 is simple compound HUBIN-1 injection groups；2.5mg/kg and 5mg/kg dosage
Caspase-3 spliced bodies and RIP1 protein expression levels are substantially less than model group in HUBIN-1 treatment groups, point out compound
The protective effect of the mice hepatitis that HUBIN-1 is induced LPS/D-GalN is by suppressing hepatocellular apoptosis and necrosis.
Fig. 6：The impact of the hepatocyte Lipotoxicity that compound HUBIN-1 is induced Palmic acid；Model group is represented with highly concentrated
Degree Palmic acid (PA) inducing hepatocyte Lipotoxicity model, HUBIN-1-25 μM, -50 μM, -100 μM represent respectively with 25 μM, 50
μM, 100 μM of compound HUBIN-1 pretreatment 1 hour, then add PA to stimulate；Compound HUBIN-1 pretreatment can significantly reduce PA
Caused cytotoxicity, and in dose dependent.
Fig. 7：The impact of the hepatic stellate cells LX2 apoptosis that compound HUBIN-1 is induced STS；Model group is represented with STS
The LX2 model of cell apoptosis of induction, HUBIN-1-25 μM, -50 μM, -100 μM represents respectively with 25 μM, 50 μM, 100 μM of change
Compound HUBIN-1 pretreatment 1 hour, then add STS inductions.Compound HUBIN-1 pretreatment can promote the HSC apoptosis that STS is induced,
And in dose dependent.
Specific embodiment
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific concrete in order to describe
Embodiment, rather than in order to limit the scope of the invention.
When embodiment provides numerical range, it should be appreciated that except non-invention is otherwise noted, two ends of each numerical range
Any one numerical value can select between point and two end points.Unless otherwise defined, the present invention used in all technologies and
The same meaning that scientific terminology is generally understood that with those skilled in the art of the present technique.Except the concrete grammar used in embodiment, equipment,
Outside material, according to those skilled in the art to the grasp of prior art and the record of the present invention, can also use and this
Any method of the similar or equivalent prior art of method, equipment described in inventive embodiments, material, equipment and material come real
The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental technique, detection method, preparation method using this technology lead
The conventional molecular biology in domain, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniquess of association area.The perfect explanation in existing document of these technologies, specifically can be found in Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The preparation of the compound HUBIN-1 solution of embodiment 1
Compound HUBIN-1, using the storing liquid of the compound HUBIN-1 being configured to after DMSO hydrotropies, before injection with
PBS dilutions are made into the solvent of compound HUBIN-1.
Protective effect-the dose-effect relationship of the mice hepatitis that the compound HUBIN-1 of embodiment 2 is induced LPS/D-GalN is ground
Study carefully
Animal model is new medicine, the whether effective research tool of new treatment meanss of checking, at present in the world
Using simulation hepatitis graviies animal model have Drug hepatitis graviies model (APAP model, LPS/D-GalN
Model, thioacetamide model, carbon tetrachloride model etc.), Acute Hepatic ischemia model, partially hepatectomized model etc..Wherein LPS/
D-GalN models, because of its favorable repeatability, the outer toxicity of liver is not obvious, and hepatic injury shows the advantages such as close clinic, be it is a kind of more
Preferable hepatitis graviies animal model.
Six week old male C57BL/6 mices (being purchased from Nanjing biological medicine academy) are randomly divided into Normal group, chemical combination
Thing HUBIN-1 groups, model group (LPS/D-GalN groups), treatment group (0.625mg/kg HUBIN-1+LPS/D-GalN groups,
1.25mg/kg HUBIN-1+LPS/D-GalN groups, 2.5mg/kg HUBIN-1+LPS/D-GalN groups, 5mg/kg HUBIN-1+
LPS/D-GalN groups, 10mg/kg HUBIN-1+LPS/D-GalN groups).Treatment group press respectively 0.625mg/kg, 1.25mg/kg,
The dosage of 2.5mg/kg, 5mg/kg and 10mg/kg carries out the lumbar injection of the solvent of compound HUBIN-1, after 30 minutes
LPS/D-GalN (10 μ g/kg LPS+400mg/kg D-GalN) lumbar injection modeling；Model group is replaced with isopyknic solvent
The solvent of compound HUBIN-1；Compound HUBIN-1 groups replace LPS/D-GalN with isopyknic PBS；Normal group does not do
Process.LPS/D-GalN modelings are plucked eyeball method for 6 hours and take blood, and take liver organization fixation and frozen, biochemical and pathological examination
Assessment each group mouse liver injury degree.
Experimental result：
LPS/D-GalN model group ALT, AST level is significantly raised (table 1) compared with Normal group, hepatic tissue pathology section
Also show that hepatic necrosis and cell infiltration are obvious；The treatment group of each dosage is compared with model group, and its ALT, AST level is notable
Reduce, especially (no difference of science of statistics between this 3 dosage) is become apparent from 2.5mg/kg, 5mg/kg and 10mg/kg group reduction, this three
Group murine liver tissue pathological section also shows that hepatocyte is several without necrosis, and cell infiltration is also reduced (Fig. 1)；Simple compound
The liver function and pathology of HUBIN-1 injection groups (10mg/kg dosage) is similar with Normal group.
Result above shows that compound HUBIN-1 is with 2.5mg/kg, 5mg/kg and 10mg/kg dosed administration to LPS/D-
The mice hepatitis of GalN inductions is respectively provided with protective effect.Preferred dose is 2.5mg/kg, 5mg/kg.
Table 1
| ALT(U/L) | AST(U/L) | Statistical analysiss | |
| Normal control | 26.3±1.4 | 86±1.4 | a,c |
| Individual compounds HUBIN-1 | 25.3±0.7 | 87.1±3.9 | a,c |
| LPS/GalN model group | 1631.3±258.4 | 932.5±88.9 | b,c |
| Compound HUBIN-1-0.625 treatment groups | 639.5±114.7 | 522.4±61.8 | a,b,c |
| Compound HUBIN-1-1.25 treatment groups | 228.7±45.2 | 225.3±40.1 | a,b,c |
| Compound HUBIN-1-2.5 treatment groups | 79.4±3.9 | 134.4±5.7 | a,b |
| Compound HUBIN-1-5 treatment groups | 93.6±7.1 | 151.8±8.5 | a,b |
| Compound HUBIN-1-10 treatment groups | 111.3±12.3 | 161.6±13.2 | a,b |
Note：Numerical value is MEAN ± SE；A-have significant difference compared with model group；B-have statistics with normal control ratio
Difference；C-have significant difference compared with the dosage treatment group of compound -2.5.
Therapeutical effect-the time-effect relationship of the mice hepatitis that the compound HUBIN-1 of embodiment 3 is induced LPS/D-GalN grinds
Study carefully
Six week old male C57BL/6 mices (being purchased from Nanjing biological medicine academy) are randomly divided into Normal group, model
Group (LPS/D-GalN groups), treatment group's (2.5mg/kg HUBIN-1+LPS/D-GalN groups).Treatment group lumbar injection LPS/D-
GalN modelings (10 μ g/kg LPS+400mg/kg D-GalN), before the LPS/D-GalN modelings after 30 minutes and modeling
Give within 30 minutes, 1 hour, 2 hours compound HUBIN-1 2.5mg/kg dosage lumbar injections；Model group is with isopyknic solvent
Replace compound HUBIN-1；Normal group is not processed.LPS/D-GalN modelings are plucked eyeball method for 6 hours and take blood, and take liver
Tissue is fixed and frozen, biochemical and pathological examination assessment each group murine liver tissue degree of injury.
Experimental result：
LPS/D-GalN model group ALT, AST level is significantly raised compared with Normal group, and hepatic tissue pathology section also shows
Show that hepatic necrosis and cell infiltration are obvious；Give compound HUBIN-1 (2.5mg/ within 30 minutes before LPS/D-GalN modelings
Kg) lumbar injection, can mitigate the hepatic injury degree of LPS/D-GalN inductions.Compared with model group, compound HUBIN-1 intervention groups
Mice serum ALT and AST level is remarkably decreased (table 2).Pathological section also shows that hepatocyte is several without necrosis, and cell infiltration also subtracts
Few (Fig. 2)；Administration in 30 minutes and 1 hour after LPS/D-GalN modelings can also alleviate inflammation, reduce Serum ALT, AST water
It is flat, but its therapeutic effect is not as good as administration before modeling；And 2 hours after modeling are administered again, hepatic tissue pathology and the biochemistry for the treatment of group refer to
Mark is compared with model group without significant difference.
Result above shows that the mice hepatitis that compound HUBIN-1 is induced LPS/D-GalN not only has prevention ＆ protection
Effect, early stage administration also has therapeutical effect.
Table 2
| ALT(U/L) | AST(U/L) | Statistical analysiss | |
| Normal control | 26.3±1.4 | 86±1.4 | a,c |
| LPS/GalN model group | 1631.3±258.4 | 932.5±88.9 | b,c |
| 30 minutes administration groups before modeling | 79.4±3.9 | 134.4±5.7 | a,b |
| 30 minutes administration groups after modeling | 448.3±48.8 | 416.7±34.3 | a,b,c |
| 1 hour administration group after modeling | 718.3±121.9 | 528.3±72.3 | a,b,c |
| 2 hours administration groups after modeling | 1461.3±212.7 | 819.4±130.2 | b,c |
Note：Numerical value is MEAN ± SE；A-have significant difference compared with model group；B-have statistics with normal control ratio
Difference；C-had significant difference compared with administration group with 30 minutes before modeling.
The comparitive study of the mice hepatitis that the compound HUBIN-1 of embodiment 4 and Nec-1 is induced in LPS/D-GalN
Nec-1 is bought respectively from following company：
Table 3
Six week old male C57BL/6 mices (being purchased from Nanjing biological medicine academy) are randomly divided into Normal group, model
Group (LPS/D-GalN groups), compound HUBIN-1 treatment groups (5mg/kg+LPS/D-GalN groups), Nec-1 treatment groups (Nec-1
1., 2., 5mg/kg dosage is 3. adopted).5mg/kg dosage lumbar injection compound HUBIN-1 or Nec-1 press respectively in treatment group
1., 2., 3., LPS/D-GalN (10 μ g/kg LPS+400mg/kg D-GalN) lumbar injection modeling after 30 minutes；Model group with
Isopyknic solvent replaces compound HUBIN-1；Normal group is not processed.LPS/D-GalN modelings pluck eyeball method in 6 hours
Blood is taken, and takes liver organization and fixed and frozen, biochemical and pathological examination assessment each group mouse liver injury degree.
Experimental result：
LPS/D-GalN model group ALT, AST level is significantly raised compared with Normal group, and hepatic tissue pathology section also shows
Show that hepatic necrosis and cell infiltration are obvious；Compound HUBIN-1 treatment groups and Nec-1 treatment groups compared with model group, its ALT,
AST levels, and hepatic necrosis degree and cell infiltration significantly reduce；It is especially optimal with the curative effect of compound HUBIN-1,
Its compared with Nec-1 1., 2., 3. treatment group, Serum ALT, AST levels reduce becoming apparent from (and Nec-1 1., 2., 3. nothing between this three groups
Significant difference), murine liver tissue pathological section also shows that hepatocyte is several without necrosis, and cell infiltration is also less (Fig. 3).
Result above shows, the mice hepatitis that compound HUBIN-1 is induced LPS/D-GalN under the conditions of Isodose
Protective effect is better than commercialization Nec-1.
Table 4
| ALT(U/L) | AST(U/L) | Statistical analysiss | |
| Normal control | 26.3±1.4 | 86±1.4 | a,c |
| LPS/GalN model group | 1631.3±258.4 | 932.5±88.9 | b,c |
| Compound HUBIN-1 treatment groups | 93.6±7.1 | 151.8±8.5 | a,b |
| Nec-1 1. treatment groups | 765.3±134.4 | 585.7±69.8 | a,b,c |
| Nec-1 2. treatment groups | 692.3±111.3 | 515.3±72.8 | a,b,c |
| Nec-1 3. treatment groups | 559.5±104.7 | 492.4±61.8 | a,b,c |
Note：Numerical value is MEAN ± SE；A-have significant difference compared with model group；B-have statistics with normal control ratio
Difference；C-have significant difference compared with compound HUBIN-1 treatment groups.
The compound HUBIN-1 of embodiment 5 suppresses the macrophage inflammatory factor secretion of LPS inductions
Kupffer Cell (hepatic macrophages) plays an important role in all kinds of inflammation diseases develop, clinical
With the phenomenon for finding to there is activation of Kupffer cells and inflammatory factor diacrisises during hepatitis in basic research.Often adopt in vitro
Stimulated with LPS, inducing macrophage activation and inflammatory factor secrete to model, and the cell model can be used for the body of Antihepatitis medicament
Outer screening and curative effect evaluation.
We set up macrophage activation model using mouse primary hepatic macrophages (Kupffer Cell), and LPS is purchased from
Sigma companies.The primary Kupffer Cell of separating mouse, is divided into matched group, and LPS model group (500ng/ml), compound HUBIN-1 does
Pre- group (respectively with 25 μM, 50 μM, 100 μM of compound HUBIN-1 pretreatment 1 hour, then adding LPS to stimulate).LPS stimulations 24 are little
Shi Hou, collects cells and supernatant, ELISA detection inflammatory factor TNF-αs, the level of HMGB1.
Experimental result：
LPS stimulates can promote Kupffer Cell TNF secretion-α, HMGB1, and compound HUBIN-1 pretreatment Kupffer Cell energy
The secretion level of these inflammatory factors is significantly reduced, and in dose dependent (table 5).
Result above shows that compound HUBIN-1 can suppress the macrophage activation of LPS inductions and inflammatory factor secretion,
With anti-inflammatory efficacy.
Table 5
Note：Numerical value is MEAN ± SD；A-have significant difference compared with model group；B-have statistics poor with contrast ratio
It is different；C-have significant difference compared with HUBIN-1-25 μM of group of compound；D-have compared with HUBIN-1-100 μM of group of compound
Significant difference.
The compound HUBIN-1 of embodiment 6 suppresses the hepatocellular apoptosis of TNF-α/ActD inductions and necrosis
All there is hepatocellular apoptosis and necrosis in the hepatitis caused by the factors such as viral infection, ethanol, high fat and medicine irritation
Phenomenon.External stimulation frequently with TNF-α/ActD comes inducing hepatocyte apoptosis, necrosis, sets up the cell model of hepatocyte injury,
The cell model can be used for the in-vitro screening and curative effect evaluation of hepatosis treating medicine.
We set up hepatocyte injury model, TNF-α and ActD using people's liver epithelial cell system L02 cells (being purchased from ATCC)
PeproTech and MCE companies are purchased from respectively.L02 cell attachments overnight after, be divided into matched group, TNF-α/ActD model group
(+0.2 μM of ActD of 10ng/ml TNF-αs), compound HUBIN-1 intervention groups are (respectively with 25,50,100 μM of compound
HUBIN-1 pretreatment 1 hour, then add TNF-α/ActD to stimulate).TNF-α/ActD collects cell after stimulating 6 hours and 24 hours,
The detection of hepatocellular apoptosis and necrosis is carried out using the apoptosis/necrosis detection kit of BD.
Experimental result：
TNF-α/ActD stimulates the early apoptosis that can induce L02 cells for 6 hours, and compound HUBIN-1 pretreatment can be significantly
The early apoptosis ratio of L02 cells is lowered, and in dose dependent (Fig. 4 A)；TNF-α/ActD can be lured substantially after stimulating 24 hours
The necrosis of L02 cells is led, compound HUBIN-1 pretreatment can significantly lower the downright bad ratio of L02 cells, and in dose dependent
(Fig. 4 B).
Result above shows that hepatocellular apoptosis and necrosis of the compound HUBIN-1 to TNF-α/ActD inductions has obvious
Mitigation, have hepatocytoprotection.
The compound HUBIN-1 of embodiment 7 suppresses the apoptosis of LPS/D-GalN model mice hepatic tissues and necrosis
Six week old male C57BL/6 mices (being purchased from Nanjing biological medicine academy) are randomly divided into Normal group, chemical combination
Thing HUBIN-1 groups, model group (LPS/D-GalN groups), treatment group (2.5mg/kg HUBIN-1+LPS/D-GalN groups, 5mg/kg
HUBIN-1+LPS/D-GalN groups).Treatment group is carried out respectively the solvent of compound HUBIN-1 by 2.5mg/kg or 5mg/kg dosage
Lumbar injection, LPS/D-GalN (10 μ g/kg LPS+400mg/kg D-GalN) lumbar injection modeling after 30 minutes；Model group
With the solvent that isopyknic solvent replaces compound HUBIN-1；Compound HUBIN-1 groups replace LPS/D- with isopyknic PBS
GalN；Normal group is not processed.After LPS/D-GalN modelings 6 hours, execution mice takes its liver organization to be carried out
(antibody is purchased from for Caspase-3 (its spliced body prompting apoptosis) and the Western Blot detections of RIP1 (prompting necrosis) molecule
CST companies).
Experimental result：
The expression of Caspase-3 spliced bodies and RIP1 albumen is dramatically increased (Fig. 5) in LPS/D-GalN model group；
The expression of above-mentioned molecule is significantly lowered in the HUBIN-1 treatment groups of 2.5mg/kg and 5mg/kg dosage；Simple compound
The expression of the above-mentioned molecule of HUBIN-1 injection groups is also substantially less than model group.
In conjunction with the embodiments 2, the embodiment result shows, the compound HUBIN-1 of preferred dose is induced LPS/D-GalN
The protective effect of mice hepatitis be by suppressing hepatocellular apoptosis and necrosis.
The compound HUBIN-1 of embodiment 8 suppresses the hepatocyte of fatty acid-induced esterified and cytolipin toxicity
Fatty liver refers to the excessive pathological changes of athero in the hepatocyte caused due to a variety of causes.Fatty liver disease is serious
Compatriots' health is threatened, the common cause of disguised liver cirrhosis has been acknowledged as.It is external frequently with fatty acid (e.g., Oleic acid (OA) and
Palmic acid (PA)) revulsion sets up the esterified model of hepatocyte, and the cell model can be used for the in-vitro screening of fatty liver medicine
And curative effect evaluation.
We set up the esterified model of hepatocyte, Oleic acid and Petiolus Trachycarpi using people's liver epithelial cell system L02 cells (being purchased from ATCC)
Acid is purchased from Sigma companies.L02 cell attachments overnight after, be divided into matched group, OA/PA model group (OA:PA presses 2:1 mixed preparing
Into the fatty acid-induced culture medium of final concentration 0.5mM), compound HUBIN-1 intervention groups are (respectively with 50 μM, 100 μM of compound
HUBIN-1 pretreatment 1 hour, then carry out OA/PA inducing culture).OA/PA carries out the esterified detection of hepatocyte after inducing 48 hours.
Or stimulating L02 cells to set up hepatocyte Lipotoxicity model using 1mM Palmic acids, analysis of compounds HUBIN-1 is (respectively with 25 μ
M, 50 μM, 100 μM of compound HUBIN-1 pretreatment 1 hour, then carry out PA stimulations) to cytolipin toxicity caused by PA
Intervention effect, PA analyzes cytotoxicity after stimulating 24 hours using MTS methods.
Experimental result：
OA/PA can induce L02 cells and obvious steatosis, compound HUBIN-1 pretreatment occurs after processing 48 hours
The steatosis degree of L02 cells can be substantially reduced；High concentration PA can cause obvious cytolipin toxicity, compound HUBIN-
1 pretreatment can significantly reduce cytotoxicity caused by PA, and in dose dependent (Fig. 6).
Result above shows that compound HUBIN-1 can suppress the hepatocyte of fatty acid-induced esterified and cytolipin toxicity,
There is potential fatty liver curative effect.
The compound HUBIN-1 of embodiment 9 promotes the apoptosis of hepatic stellate cell
Hepatic stellate cell (HSC) is the main effects cell during hepatic fibrosis/liver cirrhosis, when liver is subject to inflammation etc.
During damage, HSC is activated, the HSC of activation on the one hand by hypertrophy and extracellular matrix secretion participate in hepatic fibrosis formation and
The reconstruction of liver inner structure, on the other hand makes the intrinsic pressure rising of sinus hepaticus by cellular contraction.Various fibrosis factors using HSC as
Final target cell.Human liver microsome proteins system LX2 can be used for the in-vitro screening and curative effect of anti-fibrosis medicine Jing after induced activation to be commented
Estimate.
Staurosporine (STS) can be used for the induced activation of HSC, and the overactivity of the inducible HSC of high dose STS is further sent out
Raw apoptosis.We set up HSC activation-apoptotic cell using STS (being purchased from MCE) induction LX2 cells (being purchased from ATCC) activation
Model.It is divided into matched group, STS model group (50nM), compound HUBIN-1 intervention groups are (respectively with 25 μM, 50 μM, 100 μM of change
Compound HUBIN-1 pretreatment 1 hour, then add STS to stimulate).STS collects cell after stimulating 24 hours, is detected using the apoptosis of BD
Test kit carries out fluidic cell apoptosis detection.
Experimental result：
STS stimulates the late apoptic that can induce LX2 cells for 24 hours, compound HUBIN-1 pretreatment further to improve
The late apoptic ratio of LX2 cells, and in dose dependent (Fig. 7).
Result above shows that compound HUBIN-1 can promote the HSC apoptosis that STS is induced, and treats with potential fibrosis
Effect.
Embodiment above is, in order to illustrate embodiment disclosed by the invention, can not to be interpreted as the limit to the present invention
System.Additionally, method, the change of compositionss in various modifications listed herein and invention, without departing from the scope of the present invention
With for those skilled in the art be obvious on the premise of spirit.Although having combined the various concrete of the present invention
Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.
In fact, various modifications obvious for those skilled in the art as above all should include obtaining invention
Within the scope of the invention.
Claims (10)
1. compound HUBIN-1 is preparing inflammation disease prevention and/or the purposes in medicine, the compound
The structural formula of HUBIN-1 is as shown in formula I：
2. purposes according to claim 1, it is characterised in that the medicine possesses following active any one or many
：
(1) ALT levels can be reduced；(2) AST levels can be reduced；(3) hepatocellular degeneration and degree of necrosis can be mitigated；(4)
Suppress hepatocellular apoptosis；(5) inflammatory cell infiltration is reduced；(6) hepatic macrophages/activation of Kupffer cells and inflammatory factor are suppressed
Secretion；(7) suppress hepatocyte esterified and cytolipin toxicity；(8) apoptosis of hepatic stellate cell is promoted.
3. purposes according to claim 1, it is characterised in that the medicine possesses following active any one or many
：
(1) anti-liver injury；(2) anti-inflammatory cellular infiltration；(3) anti-hepatocyte is esterified；(4) anti-hepatic fibrosis.
4. purposes according to claim 1, it is characterised in that the inflammation disease includes acute hepatitises, chronic
Hepatitis, hepatitis graviies, liver failure, fatty liver, hepatic fibrosis and liver cirrhosis.
5. purposes according to claim 1, it is characterised in that the inflammation disease includes viral hepatitis, medicine
Physical property hepatitis, fat hepatitis, alcoholic hepatitis, autoimmune hepatitiss.
6. a kind of inflammation disease prevention and/or medicine, the compound HUBIN-1 containing safe and effective amount, and
Pharmaceutically acceptable carrier.
7. medicine according to claim 6, it is characterised in that the compound HUBIN-1 as one of effective ingredient or
Sole active ingredient.
8. a kind of method of prevention and/or treatment inflammation disease, including step：By the compound containing safe and effective amount
The medicament administration of HUBIN-1 is in effective object.
9. method according to claim 8, it is characterised in that the inflammation disease includes acute hepatitises, chronic
Hepatitis, hepatitis graviies, liver failure, fatty liver, hepatic fibrosis and liver cirrhosis.
10. the new application of compound HUBIN-1, the structural formula of the compound HUBIN-1 is as shown in formula I：
Including any one of following purposes or multinomial：(1) ALT levels can be reduced；(2) AST levels can be reduced；(3) energy
Enough mitigate hepatocellular degeneration and degree of necrosis；(4) hepatocellular apoptosis are suppressed；(5) inflammatory cell infiltration is reduced；(6) liver is suppressed
Macrophage/activation of Kupffer cells and inflammatory factor are secreted；(7) suppress hepatocyte esterified and cytolipin toxicity；(8) liver is promoted
The apoptosis of sternzellen.
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| WO2020020119A1 (en) * | 2018-07-25 | 2020-01-30 | 浙江大学 | Rip1 inhibitor and use thereof in medicine |
| CN112263576A (en) * | 2020-09-30 | 2021-01-26 | 浙江大学 | Application of compound ZJU-37 in preparation of drugs for preventing and/or treating liver diseases |
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| WO2014152182A1 (en) * | 2013-03-15 | 2014-09-25 | President And Fellows Of Harvard College | Deuterated heterocyclic inhibitors of necroptosis |
| WO2016094846A1 (en) * | 2014-12-11 | 2016-06-16 | President And Fellows Of Harvard College | Inhibitors of cellular necrosis and related methods |
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| CN101674826A (en) * | 2005-12-20 | 2010-03-17 | 哈佛大学校长及研究员协会 | Chemical compound, screening and Therapeutic Method |
| WO2014152182A1 (en) * | 2013-03-15 | 2014-09-25 | President And Fellows Of Harvard College | Deuterated heterocyclic inhibitors of necroptosis |
| WO2016094846A1 (en) * | 2014-12-11 | 2016-06-16 | President And Fellows Of Harvard College | Inhibitors of cellular necrosis and related methods |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020020119A1 (en) * | 2018-07-25 | 2020-01-30 | 浙江大学 | Rip1 inhibitor and use thereof in medicine |
| CN110759895A (en) * | 2018-07-25 | 2020-02-07 | 浙江大学 | Novel RIP1/RIP3 dual-target inhibitor and application thereof in treatment of multi-target disease with one drug |
| CN110759895B (en) * | 2018-07-25 | 2020-10-27 | 浙江大学 | Novel RIP1/RIP3 dual-target inhibitor and application thereof in treatment of multi-target disease with one drug |
| CN112351979A (en) * | 2018-07-25 | 2021-02-09 | 浙江大学 | RIP1 inhibitor and application thereof in medicine |
| CN112351979B (en) * | 2018-07-25 | 2021-07-06 | 浙江大学 | RIP1 inhibitor and application thereof in medicine |
| CN112263576A (en) * | 2020-09-30 | 2021-01-26 | 浙江大学 | Application of compound ZJU-37 in preparation of drugs for preventing and/or treating liver diseases |
| WO2022068075A1 (en) * | 2020-09-30 | 2022-04-07 | 浙江大学 | Application of compound zju-37 in preparation of drug for preventing and/or treating liver diseases |
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