CN110339188A - - 2 ketone of 4- methyl -5,6- dihydropyran is preparing the application in anti-hepatic fibrosis and medicines resistant to liver cancer - Google Patents
- 2 ketone of 4- methyl -5,6- dihydropyran is preparing the application in anti-hepatic fibrosis and medicines resistant to liver cancer Download PDFInfo
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Abstract
The present invention provides -2 ketone of 4- methyl -5,6- dihydropyran to prepare the application in anti-hepatic fibrosis and medicines resistant to liver cancer, belongs to anti-hepatic fibrosis and liver-cancer medicine technical field.Present invention discover that -2 ketone of 4- methyl -5,6- dihydropyran has good anti-hepatic fibrosis activity and inhibits the activity of liver cancer cells transfer, can be used in preparing anti-hepatic fibrosis medicines and medicines resistant to liver cancer.Embodiment is as the result is shown, 4- methyl -5, -2 ketone of 6- dihydropyran can inhibit the activity of 1 promoter of Type I collagen protein alpha, reduce the expression of the hepatic fibrosis markers such as COL1A1, ACTA2 in hepatic stellate cells LX-2, improve BDL rat liver tissue pathologic structure and degree of hepatic fibrosis, is able to suppress the transfer of hepatocellular carcinoma H22.
Description
Technical field
The present invention relates to anti-hepatic fibrosis and liver-cancer medicine technical field, in particular to 4- methyl -5,6- dihydropyran -2
Ketone is preparing the application in anti-hepatic fibrosis and medicines resistant to liver cancer.
Background technique
Chronic hepatic diseases are a kind of global disease for seriously endangering human health, virus infection (HBV HCV), poison
The many factors such as object (aflatoxins), alcohol, inborn errors of metabolism can cause chronic liver disease.Liver fibrosis be secondary to
Compensation response after hepar damnification caused by various chronic factors and inflammation in process of tissue reparation, the further hair of liver fibrosis
Exhibition leads to cirrhosis, liver failure and liver cancer.The study found that liver fibrosis is a kind of invertibity lesion, if can be at this
One stage gave effective treatment, the treatment to chronic hepatitis, and the prevention of the serious fatal disease such as cirrhosis and liver cancer has
Important meaning.In recent years, the clinical treatment of liver fibrosis obtains some new developments, but only approval has listed again for China at present
Several Chinese patent drugs such as square shell liver softening tablet and FUZHENG HUAYU JIAONANG, alternative anti-hepatic fibrosis medicines are less.
Liver cancer is the very high cancer of whole world morbidity and mortality, and China is the whole world highest state of onset of liver cancer rate
One of family and area.Operation is the most common treatment means of liver cancer, but by tumor size, position and grade malignancy etc. it is a variety of because
The influence of element, and it has generally been developed to middle and advanced stage when liver cancer discovery, the only small number of patients that can be treated using operation,
For the liver cancer patient for the excision that can not perform the operation, therapeutic choice is very limited, poor prognosis.Sorafenib is as a kind of Azaindole kinase
Inhibitor can act on tumour cell and tumor vessel simultaneously, as breakthrough hepatoma-targeting therapeutic agent, suffer to liver cancer
The existence improvement of person is still relatively limited, needs to develop novel liver cancer treatment strategy.
Therefore, in the evolutionary process from liver fibrosis to liver cancer, the medicine that can inhibit liver fibrosis and liver cancer simultaneously is found
Object is possible to have important meaning to the prevention and treatment of liver cancer.
Summary of the invention
In view of this, it is an object of that present invention to provide a kind of -2 ketone of 4- methyl -5,6- dihydropyran to prepare anti-liver fiber
Change and the application in medicines resistant to liver cancer.- 2 ketone of 4- methyl -5,6- dihydropyran is used to prepare anti-hepatic fibrosis medicines by the present invention
And medicines resistant to liver cancer, there is good anti-hepatic fibrosis and resisting liver cancer activity.
In order to achieve the above-mentioned object of the invention, the present invention provides following scheme:
The present invention provides -2 ketone of 4- methyl -5,6- dihydropyran in preparing anti-hepatic fibrosis medicines and medicines resistant to liver cancer
Application.
It is that effective component and pharmaceutically acceptable carrier form the present invention provides -2 ketone of 4- methyl -5,6- dihydropyran
Composition preparing the application in anti-hepatic fibrosis medicines and medicines resistant to liver cancer.
The present invention provides -2 ketone of 4- methyl -5,6- dihydropyran in preparation liver fibrosis and prevention of hcc health care product
Using.
The present invention provides a kind of drug for anti-hepatic fibrosis and anti-liver cancer and anti-, the effective component of the drug includes 4-
The derivative of -2 ketone of methyl -5,6- dihydropyran, pharmaceutically acceptable salt, solvate, metabolite, stereoisomer,
Tautomer, polymorph, prodrug.
The present invention provides -2 ketone of 4- methyl -5,6- dihydropyran to apply in preparing anti-hepatic fibrosis medicines, the present invention
It was found that -2 ketone (CAS:2381-87-5) of 4- methyl -5,6- dihydropyran has good anti-hepatic fibrosis activity and inhibits liver cancer
Transcellular activity can be used in preparing anti-hepatic fibrosis medicines and medicines resistant to liver cancer.Hepatic stellate cells is liver fibrosis
Cytological Basis, the activation of hepatic stellate cells can induce the table of the hepatic fibrosis markers such as COL1A1, ACTA2, TGFB1, MMP2
It reaches, leads to a large amount of collagen deposition of liver.Embodiment is the results show that -2 ketone of 4- methyl -5,6- dihydropyran can inhibit Type I collagen
The activity of 1 promoter of protein alpha reduces the expression of the hepatic fibrosis markers such as COL1A1, ACTA2 in LX-2 cell, it is big to reduce BDL
Mouse serum alt, AST, ALP, TG, LDL are horizontal, improve BDL rat liver tissue pathologic structure and degree of hepatic fibrosis, suppression
The mRNA expression of Col1a1, Acta2, Mmp2, Tgfb1 in rat liver tissue processed.In addition, 4- methyl -5,6- dihydropyran -2
Ketone has the apparent transfer ability for inhibiting liver cancer cells.These the result shows that -2 ketone of 4- methyl -5,6- dihydropyran have it is good
Good anti-hepatic fibrosis activity, being expected to research and development becomes the new drug of anti-hepatic fibrosis and anti-liver cancer and anti-.
Detailed description of the invention
Fig. 1 is 1 gained LX2 cell survival rate histogram of embodiment；
Fig. 2 is that -2 ketone of 4- methyl -5,6- dihydropyran of various concentration in embodiment 2 presses down the activity of COL1A1 promoter
System compares schematic diagram；
Fig. 3 is the expression of results schematic diagram of the mRNA of COL1A1 in embodiment 3；
Fig. 4 is the expression of results schematic diagram of the mRNA of α-SMA in embodiment 3；
Fig. 5 is the detection that -2 ketone of 4- methyl -5,6- dihydropyran influences target gene protein expression level in embodiment 3
Result schematic diagram；
Fig. 6 be in embodiment 4 BDL operation after rat liver/weight ratio figure；
Fig. 7 be in embodiment 4 BDL operation after rat body weight variation diagram；
Fig. 8 is BDL rat liver tissue figure in embodiment 4；
Fig. 9 is BDL rat liver pathological section figure in embodiment 4；
Figure 10 is rat liver bile duct proliferation double blind scoring figure in embodiment 4；
Figure 11 is rat liver tissue necrosis double blind scoring figure in embodiment 4；
Figure 12 is that rat liver tissue is sliced sirius red stains figure in embodiment 4；Wherein, A) it is sham group coloration result
Figure, B) be BDL group coloration result figure, C) be -2 ketone processing group coloration result figure of 4- methyl -5,6- dihydropyran, D) it is that each group contaminates
Color area percentage composition schematic diagram；
Figure 13 is hepatic fibrosis markers mRNA expression schematic diagram in embodiment 4；Wherein, A is the mRNA of COL1A1
Expression schematic diagram, B are the mRNA expression schematic diagram of acta2, and C is the mRNA expression schematic diagram of Mmp2, and D is
The mRNA expression schematic diagram of Tgfb1；
Figure 14 is liver cancer cells scratch experiment result figure in embodiment 5.
Specific embodiment
The present invention provides -2 ketone of 4- methyl -5,6- dihydropyran in preparing anti-hepatic fibrosis medicines and medicines resistant to liver cancer
Application.The present invention is to the 4- methyl -5,6- dihydropyran -2 in preparing anti-hepatic fibrosis medicines and medicines resistant to liver cancer
Application mode does not have special requirement, according to specific needs, using method well known to those skilled in the art by the 4- methyl-
5,6- dihydropyran -2 is prepared into the drug of different dosage forms.In the present invention, -2 ketone of 4- methyl -5,6- dihydropyran
Effective concentration be preferably 3~100 μm of olL-1, more preferably 20~80 μm of olL-1。
The present invention does not have special requirement to the source of -2 ketone of 4- methyl -5,6- dihydropyran, uses commercially available 4-
- 2 ketone of methyl -5,6- dihydropyran is voluntarily prepared.When need to voluntarily prepare 4- methyl -5,6- -2 ketone of dihydropyran, institute
The preparation method for stating -2 ketone of 4- methyl -5,6- dihydropyran preferably includes following steps:
Under the effect of the catalyst, make mevalonolactone that elimination reaction occur, obtain 4- methyl -5,6- dihydropyran -2
Ketone.
In the present invention, the catalyst is preferably a hydration p-methyl benzenesulfonic acid；The mevalonolactone and catalyst
Mass ratio be preferably 20:1；In the present invention, the solvent that the elimination reaction uses is preferably toluene, and the present invention is to described molten
The dosage of agent does not have special requirement, can dissolve the mevalonolactone and catalyst.In the present invention, described
The temperature of elimination reaction is preferably the reflux temperature of reaction solution, and the time is preferably 19h.
In the present invention, further include post-processing to elimination reaction liquid after the elimination reaction, the post-processing include with
Lower step:
The elimination reaction liquid is successively filtered, silica gel column chromatography and elution, obtains pure 4- methyl -5,6- bis-
- 2 ketone of hydrogen pyrans.
In the present invention, the suction filtration preferably decompression filters, and the time of the suction filtration is preferably 1h；The present invention is to described
The mode of silica gel column chromatography does not have special requirement, uses the mode of silica gel column chromatography well known to those skilled in the art.
In the present invention, the mode of the elution is preferably petroleum ether-ethyl acetate gradient elution, the petroleum ether-ethyl acetate
Volume ratio is preferably 10:1~2:1.
In the present invention, the reaction equation of the elimination reaction is shown in formula I:
It is that effective component and pharmaceutically acceptable carrier form the present invention provides -2 ketone of 4- methyl -5,6- dihydropyran
Composition preparing the application in anti-hepatic fibrosis medicines and medicines resistant to liver cancer.The present invention is to the type of the carrier without spy
It is different to require, it may include one or more pharmaceutically acceptable carriers.
In the present invention, the composition can be prepared into a variety of dosage forms, in order to be administered.In the present invention, described group
The dosage form concretely parenteral preparation of object is closed, it is specific such as injection, suppository；The injection can be solution, mix
The dried powder of suspension or injectable (injection water is added before the injection to be used immediately)；The injection remove include effectively at
It exceptionally, further preferably include following carrier or auxiliary agent: physiologically acceptable sterile, aqueous or non-aqueous liquor, dispersing agent, suspension
Agent, emulsion, suitable aqueous or nonaqueous carrier, diluent, solvent or medium, it is specific such as water, ethyl alcohol, polyalcohol (the third two
Alcohol, polyethylene glycol, glycerol etc.), one of vegetable oil (such as olive oil) and injectable organic ester or a variety of, it is described to can be injected with
Machine ester preferably includes ethyl oleate.
In the present invention, concretely oral preparation, the oral preparation have for the anti-hepatic fibrosis and medicines resistant to liver cancer
Body can be solid dosage forms or liquid dosage form；The solid dosage forms is specifically such as tablet, dragee, pill, powder, particle
Agent, capsule or coating agent, the liquid dosage form specifically can be emulsion, solution, suspension, syrup or elixir.
The present invention provides -2 ketone of 4- methyl -5,6- dihydropyran in preparation liver fibrosis and prevention of hcc health care product
Using.When -2 ketone of 4- methyl -5,6- dihydropyran of the present invention is used for liver fibrosis and prevention of hcc health care product, 4- methyl -
Any conventional oral dosage formulations, such as tablet, capsule, powder, particle can be made in -2 ketone of 5,6- dihydropyran；Or injection is made
The non-oral dosage forms such as injection.- 2 ketone of 4- methyl -5,6- dihydropyran of the present invention is used for liver fibrosis and prevention of hcc health care
When product, pharmaceutically acceptable any form is can be selected in auxiliary material.
The present invention provides a kind of drug for anti-hepatic fibrosis and anti-liver cancer and anti-, the effective component of the drug includes 4-
The derivative of -2 ketone of methyl -5,6- dihydropyran, pharmaceutically acceptable salt, solvate, metabolite, stereoisomer,
Tautomer, polymorph, prodrug.
In the present invention, described to need for the dosage of anti-hepatic fibrosis and the drug of anti-liver cancer and anti-according to administration mode, suffer from
Course of disease degree, patient age, whether there is or not the considerations of the combined factors such as medical history to be adjusted.
Below with reference to embodiment to -2 ketone of 4- methyl -5,6- dihydropyran provided by the invention prepare anti-hepatic fibrosis and
Application in medicines resistant to liver cancer is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
- 2 ketone of 1 4- methyl -5,6- dihydropyran of embodiment does not have toxicity to LX2 cell
Using 4- methyl -5,6- dihydropyran -2 ketone (hereinafter referred to as C8) (50,100,150,200,300 μ of various concentration
mol·L-1) the human liver microsome proteins LX2 in logarithmic growth phase is handled for 24 hours, use Sulforhodamine B protein staining (SRB)
The influence of colorimetric determination compound on intracellular proliferation, concrete operations mode are as follows:
(1) plating cells: by the cell inoculation in logarithmic growth phase in 96 well culture plates, in 37 DEG C, 5%CO2Condition
Lower culture is for 24 hours；
(2) it is administered: after cell is adherent, discarding former culture medium, Compound Stock solution is formulated as gradient concentration with culture medium, point
Not She Zhi blank control group, vehicle control group and administration group, each concentration sets 3 multiple holes, and every 200 μ L of pore volume continues to train
It supports for 24 hours；
(3) cell is fixed: being taken out culture plate, is first carefully rinsed with PBS buffer solution one time, every hole is added 10% (m/v's)
Trichloroacetic acid (TCA) 50uL fixes cell, 4 DEG C of placement lh；Then fixer is abandoned, is washed with distilled water 5 times, is done naturally in air
It is dry；
(4) dye: after drying in air, every hole adds SRB solution 100uL, places 10~30min at room temperature.Then it goes
Supernatant is washed 5 times with 1% acetic acid, is air-dried；
(5) it dissolves: being eventually adding 10mM Tris solution (pH10.5) solution in 150 holes μ L/, shake on oscillator plate
Swing 5min；
(6) it measures: measuring OD (optical density) value in enzyme-linked immunosorbent assay instrument, returned to zero with blank control, wavelength used is
515nm；
(7) it calculates: inhibitory rate of cell growth=[(OD control wells-OD dosing holes)/OD control wells] × 100%.
Through detecting, gained LX2 cell survival rate histogram is as shown in Figure 1.As shown in Figure 1, each dense set by experiment
Spend the stage, -2 ketone of 4- methyl -5,6- dihydropyran to LX2 cell without apparent cytotoxicity, in 300 μm of olL-1When pair
The inhibiting rate of LX2 cell Proliferation is 8.7%, shows that -2 ketone of 4- methyl -5,6- dihydropyran does not influence the proliferation of LX2 cell, because
This -2 ketone of 4- methyl -5,6- dihydropyran is substantially without cytotoxicity.
The activity of -2 ketone of 2 4- methyl -5,6- dihydropyran of embodiment inhibition hepatic stellate cells COL1A1 promoter
The most important feature of liver fibrosis is the overexpression of collagen, and especially Type I collagen α 1 (COL1A1), COL1A1 is
A kind of important collagen for leading to liver fibrosis high can express in liver fibrosis process, and this height is shown
On transcriptional level.If being able to suppress the promoter activity, the generation of collagen can suppress, and then inhibit liver fibrosis.Therefore
Influence using -2 ketone of 4- methyl -5,6- dihydropyran to collagen promoter activity judges potential drug anti-hepatic fibrosis as preliminary
The index of effect.
Using the high-throughput anti-hepatic fibrosis screening model based on COL1A1 promoter, reported using single luciferase gene
- 2 ketone of 4- methyl -5,6- dihydropyran (3,20,50 and 100 μm of olL of detection system detection various concentration-1) and positive control
25μmol·L-1Indicate nutgall catechin gallic acid ester (EGCG) to COL1A1 promoter activity in LX-2-COL1A1 cell
Influence, wherein the specific method is as follows for single luciferase reporter gene detection system detection compound:
(1) drug-treated
(1) monoclonal cell strain (LX-2-COL cell) of expression COL1A1 promoter is stablized in building before experiment, and freezes
In liquid nitrogen；It recovers the cell strain, and in DMEM culture medium, 5%CO2It is cultivated in 37 DEG C of cell incubators of constant temperature, in cell
Logarithmic growth phase, when cell confluency degree is to 90%~95%, with 1.5 × 104A/hole spreads cell into 96 orifice plates, continues to train
It supports for 24 hours；
After cell in (2) 96 orifice plates is adherent, former culture medium is discarded, is diluted the liquid storage of untested compound with new culture medium
To required concentration, be added the untested compound of gradient concentration, drug effect for 24 hours after, use Bright-GloTMLuciferase
Assay System (Luciferase Assay System) kit detects promoter activity.
(2) Activity determination
(1)Bright-GloTMAssay Reagent freezes in -40 DEG C of refrigerators, is placed in equilibrium at room temperature with preceding taking-up；
(2) by plasma-free DMEM medium and Bright-GloTMAssay Reagent is mixed in the ratio of 1:1, according to 96
The every 50 μ L of hole of orifice plate prepares working solution；
(3) it inhales and abandons former culture medium, after carefully rinsing 1 time with PBS, inhale and abandon PBS and working solution described in step (2) is added；
(4) orifice plate is placed on oscillator, room temperature shakes 5min, cracks cell thoroughly；
(5) liquid in orifice plate is transferred in lighttight dedicated 96 orifice plate using multi-pore channel pipettor, is put into single function
It can chemiluminescence microplate readerThe Activity determination of COL1A1 promoter is carried out in L.
- 2 ketone of 4- methyl -5,6- dihydropyran of various concentration is to the activity suppression of COL1A1 promoter than as shown in Figure 2.
As shown in Figure 2, -2 ketone of 4- methyl -5,6- dihydropyran of various concentration can significantly inhibit the activity of COL1A1 promoter, 30 μ
mol·L-1With 100 μm of olL-1- 2 ketone of 4- methyl -5,6- dihydropyran to the active inhibiting rate point of COL1A1 promoter
It Wei 54.6% and 59.7%.
- 2 ketone of 3 4- methyl -5,6- dihydropyran of embodiment inhibit hepatic stellate cells in liver fibrosis related genes mRNA and
Protein expression level
The influence of target gene mRNA expression is examined using real-time fluorescence PCR detection method (Real-time PCR)
It surveys, the expression of results difference of the opposite mRNA of COL1A1, α-SMA are as shown in Figure 3, Figure 4；Use immunoblotting (Western
Blot) influence of target gene protein expression level is detected, testing result is as shown in Figure 5.By Fig. 3~5 it is found that 4- first
The LX2 cell that the processing of -2 ketone of base -5,6- dihydropyran is induced by TGF β 1 for 24 hours after, can inhibit liver fibrosis to concentration dependent
The mRNA and protein expression level of marker COL1A1 and α-SMA.
Wherein, the concrete operations mode of real-time fluorescence PCR detection method (Real-time PCR) are as follows:
The Real of Taqman is carried out using the Fast Start Universal Probe Master (ROX) of Roche company
Time PCR reaction using cDNA obtained by reverse transcription as template, and is compareed using GAPDH gene as internal reference, is formulated as follows reactant
It is (20 μ L):
2×FastStart Universal Probe Master
According to sample number and different primers, the above reaction system in addition to cDNA is prepared, is added to Real-time PCR use
In 96 orifice plates, every 17 μ L of hole.3 μ L cDNA templates are added in every hole again to be mixed the above system with 96 orifice plate of ParafilmTM
It uniformly and is centrifuged (1000rpm, 5min), is put into 700FastStartReal-time PCR System, setting response procedures are such as
Under:
Wherein, fluorescence signal value is collected at the end of each circulation, after reaction, each sample provided according to instrument
Target gene ct value and reference gene ct value, using opposite ct value method analysis target gene and reference gene expression quantity, sentence
Influence of the off-drug object to the related gene expression of cell or animal tissue.
The concrete operations mode of immunoblotting (Western blot) are as follows:
(1) pvdf membrane: the pvdf membrane that will be cut in advance is pre-processed, 15S in methyl alcohol is impregnated and is activated, then impregnated
In 1 × transferring film buffer；One jiao can be cut off in the upper left corner or the upper right corner, as label, to distinguish front and back sides and the sequence of film；
(2) it unloads gel: gel is removed from plate, excision concentration glue and bromophenol blue front edge portion are immersed in 1 × transferring film
It is balanced in buffer；
(3) install gel and pvdf membrane: clamping plate white clear one is placed on bottom end, successively puts sponge, filter paper, PVDF
Film, gel, filter paper, sponge, then by clamping plate black side pressure, fasten；During assembling, every layer will avoid gas as far as possible
Bubble, especially between pvdf membrane and gel；
(4) transferring film: electric current, 180mA constant current transferring film 1.5h are adjusted, or adjusts voltage 90V constant pressure, transferring film 1h~1.5h；This
Condition will suitably shorten according to destination protein molecular size appropriate adjustment, molecule small time；
(5) it closes: after transferring film, taking out pvdf membrane, be put into 5% skim milk, room temperature closes 1~2h；
(6) primary antibody is incubated for: being cut out film according to the size of the marked molecular size range of pre-dyed Marker and purpose band to be measured
It opens, it is peaceful how not few；With 1%BSA or primary antibody diluent preparing primary antibody, primary antibody dilution ratio by specification；Be ready to antibody and
After film, film is put into hybridization bag, adds corresponding primary antibody, is incubated overnight in 4 DEG C of shaking tables；
(7) it rinses: taking out film item, three times with 1 × PBS-T rinsing, each 10min；
(8) secondary antibody is incubated for: being got out secondary antibody in advance, is prepared with 1%BSA, antibody concentration by specification is prepared；Film is put into
Secondary antibody is incubated in box, and corresponding secondary antibody is added, in incubation at room temperature 2h；
(9) it rinses: taking out film item from hybridization bag, three times with 1 × PBS-T rinsing, each 10min；
(10) ECL colour developing exposure: fresh enhanced HRP substrate chemiluminescence liquid (ECL) is prepared, by substrate and buffer
It mixes, is uniformly added drop-wise on film item in the ratio of 1:1 (V/V), colour developing is exposed in gel imager；
(11) interpretation of result: save exposure image, with Image J handle, compared with internal reference after, statistics protein expression variation
Situation.
- 2 ketone of 4 4- methyl -5,6- dihydropyran of embodiment improves common bile duct ligation (Bile Duct Ligation, BDL)
The hepatic pathology and liver fibrosis situation of rat
- 2 ketone of 4.1 4- methyl -5,6- dihydropyran does not reduce the weight of rat after BDL operation
By Sprague-Dawley (SD) male rat (weight is in 180~220g) adaptable fed 1~2 day, random point
For sham-operation group (sham), BDL group, -2 ketone processing group of 4- methyl -5,6- dihydropyran, every group 6~8, and meanwhile it is pre- more than every group
2~3 are reserved, is died unexpectedly during surgery to prevent stop object or postoperative because complication shifts to an earlier date death.
BDL operation is carried out to SD rat, method particularly includes:
(1) one evening of operation consent, by animal fasting, normal water；
(2) preoperative to give super-clean bench ultraviolet disinfection 30min, after surgical instrument, sewing needle high pressure sterilization, be immersed in 75% ethyl alcohol
In, toy Anesthesia machine is opened, enough isofluranes are added；
(3) after animal holonarcosis, the hair of abdomen is shaved off, after iodophor disinfection, abdomen is opened in upper abdomen center, with sterile swab stick
Liver edge is raised, duodenum is pulled open, finds choledochus, is separated choledochus with the adipose tissue of surrounding with fine-pointed forceps, in nearly liver
Twice are respectively ligatured at door and at nearly duodenum with suture, cut ductuli hepaticus communis from two ligation sites centre, internal organ are restored former
Vitamin K and penicillin are sprinkled after sample and are sewed up the incision；Sham-operation group only opens abdomen, then sutures；Animal is set to lie on one's side after suture, to
Free diet and drinking-water after awake.
Receive BDL operation the same day be considered as the 0th day, since first day give BDL group intraperitoneal injection of saline, give
- 2 ketone (100mgkg of 4- methyl -5,6- dihydropyran is injected intraperitoneally in -2 ketone processing group of 4- methyl -5,6- dihydropyran-1), simultaneously
It weighs in, is administered 14 days daily, until the 15th day execution animal, weighs in and liver weight, and collect blood, bile, liver, urine
It is analyzed, while sham-operation group (sham group) is set and is compareed.Gained rat liver/weight ratio figure is as shown in fig. 6, big
Mouse changes of weight figure is as shown in Figure 7.
It will be appreciated from fig. 6 that BDL group is increased relative to sham group, liver weight and the ratio conspicuousness of weight, and 4- methyl -5,6-
- 2 ketone processing group of dihydropyran can't reduce the ratio；As shown in Figure 7, during administration, 4- methyl -5,6- dihydropyran -2
Ketone processing group the weight of animals steady growth, remains basically stable with BDL group, show -2 ketone of 4- methyl -5,6- dihydropyran administration after not
The weight of BDL rat can be reduced, there is good internal safety at this dose.
- 2 ketone of 4.2 4- methyl -5,6- dihydropyran can significantly improve Liver Function
To liver every in sham group, BDL group and -2 ketone processing group rat blood serum of 4- methyl -5,6- dihydropyran and urine
Relevant biochemical indicator is detected, and detection uses 7180 type automatic clinical chemistry analyzer of Hitachi and middle raw north control biotechnology stock
The biochemical indicator detection kit of part Co., Ltd, Testing index are as follows:
(1) serum (Serum): alanine aminotransferase (ALT), aspartate amino transferase (AST), alkaline phosphorus
Sour enzyme (ALP), total bile acid (TBA), total bilirubin (TBIL), total cholesterol (CHO), total triglycerides (TG), low density lipoprotein
Protein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C)；If testing result goes beyond the scope, with distilled water dilution 10
Times, it detects again；
(2) urine sample (Urine) and bile (Bile): total bile acid (TBA) and total bilirubin (TBIL).Wherein, urine sample and
Bile dilutes 10 times with distilled water in advance.
It is as shown in table 1 to detect acquired results.
The relevant biochemical indicator of items liver in the serum and urine of 1 BDL rat of table
| Sham (n=7) | BDL (n=8) | C8 100mg·kg-1(n=8) | |
| Serum AST(U·L-1) | 125.71±24.69 | 757±255.49** | 424.63±265.3# |
| Serum ALT(U·L-1) | 27.71±9.48 | 124.11±62.52** | 68.38±39.81# |
| Serum ALP(U·L-1) | 216.71±33.62 | 417.22±70.40** | 271.63±56.30## |
| Serum CHO(mmol·L-1) | 1.77±0.23 | 3.38±0.41** | 2.74±0.83 |
| Serum LDL(mmol·L-1) | 0.41±0.05 | 1.59±0.22** | 1.11±0.49# |
| Serum HDL(mmol·L-1) | 0.93±0.13 | 0.91±0.22** | 1.03±0.32 |
| Serum TG(mmol·L-1) | 0.34±0.14 | 1.11±0.31** | 0.71±0.37# |
| Serumγ-GT(U·L-1) | / | 46.33±16.77** | 34.50±28.76 |
| Serum TBA(μmol·L-1) | 14.19±4.65 | 257.39±43.10** | 164.65±142.25 |
| Serum TBiLi(μmol·L-1) | 1.97±0.22 | 162.70±21.91** | 99.69±82.08 |
| Urine TBA(μmol·L-1) | 6.88±1.20 | 472.5±197.6 | 356.41±325.53 |
| Urine TBiLi(μmol·L-1) | 1.82±0.47 | 83±42.62 | 53.25±51.10 |
| Bile TBA(μmol·L-1) | / | 487.16±131.08 | 671.33±287.60 |
| Bile TBiLi(μmol·L-1) | / | 14.967±3.64 | 9.225±7.90 |
C8 is -2 ketone of 4- methyl -5,6- dihydropyran in table 1,*P < 0.05,**P < 0.01vs.sham group；#P < 0.05,##p<
0.01vs.BDL group.
As shown in Table 1, after BDL operation, the relevant biochemical indicator of items liver all significantly deteriorates in rat blood serum and urine,
And -2 ketone of 4- methyl -5,6- dihydropyran can reduce the enzymes content such as ALT, AST, ALP in BDL rat blood serum not only with conspicuousness,
It is horizontal that TG and LDL in serum can also be improved, should the result shows that, can be significant after the administration of -2 ketone of 4- methyl -5,6- dihydropyran
Improve the postoperative Liver Function of BDL.
- 2 ketone of 4.3 4- methyl -5,6- dihydropyran can improve BDL rat liver tissue lesion
Bile duct ligation will cause more serious influence to animal pattern, after causing cholestasis, will cause various generations
It thanks to disorder, and causes hepar damnification and further fibrosis.Fig. 8 is BDL rat liver tissue figure；As shown in Figure 8, BDL group
Rat liver enlargement, liver surface is coarse and uneven, and liver is hardened, and -2 ketone of 4- methyl -5,6- dihydropyran handle
For group compared with BDL group, liver volume is small, and surface is more smooth, and tissue is relatively soft, therefore -2 ketone of 4- methyl -5,6- dihydropyran exists
Damage of the bile duct ligation to animal's liver can be improved to a certain extent.
Rat liver pathological section figure is as shown in Figure 9.Pathological section is the results show that sham group rat liver tissue liver cell
Regularly arranged, lobuli hepatis is complete, and sinus gap is uniform, and endochylema is abundant, no inflammatory cell infiltration, necrosis and bile duct proliferation situation；BDL
Group liver organization pathologic structure is substantially change, and a large amount of bile duct proliferation occurs, tissue necrosis increased significantly；Methyl -5 4-,
- 2 ketone processing group of 6- dihydropyran can significantly improve bile duct proliferation and tissue necrosis in liver.
Double blind scoring is carried out to all animal's liver tissue H&E pathological sections, wherein bile duct proliferation double blind scoring such as Figure 10
Shown, tissue necrosis double blind scoring is as shown in figure 11.By Figure 10,11 it is found that relative to BDL group, 4- methyl -5,6- dihydro pyrrole
- 2 ketone processing group bile duct proliferation average marks of muttering reduce, and necrosis of liver tissue degree is substantially reduced.
- 2 ketone of 4.4 4- methyl -5,6- dihydropyran inhibits BDL rat liver fibrosis degree
Sirius red stains (Sirius Red) is carried out to liver tissue slices, the A of coloration result such as Figure 12)~C) institute
Show, the D of each group stained area percentage composition such as Figure 12) shown in.As shown in Figure 12, sham group only has a small amount of collagenous fibres dye
Color, the dyeing of BDL group collagenous fibres largely increases, and the dyeing of -2 ketone processing group collagenous fibres of 4- methyl -5,6- dihydropyran is obvious
It reduces；The positive staining area of slice is counted, collagen is fine after the administration of -2 ketone of 4- methyl -5,6- dihydropyran as the result is shown
Dimension area is reduced significantly, and illustrates that -2 ketone of 4- methyl -5,6- dihydropyran is able to suppress the hepatic fibrosis progression of BDL rat.
The expression of -2 ketone of 4.5 4- methyl -5,6- dihydropyran inhibition BDL rat liver fibrosis marker
Rat liver tissue total serum IgE is extracted, detects COL1A1, ACTA2, Mmp2 and Tgfb1 liver fibrosis using RT-PCR
The mRNA level in-site of marker, acquired results are as shown in figure 13.As shown in Figure 13, after BDL operation, ACTA2, COL1A1, Mmp2,
The mRNA expression of the liver fibrosis Research of predicting markers such as Tgfb1 is significant to be increased, after the administration of -2 ketone of 4- methyl -5,6- dihydropyran,
The mRNA level in-site of COL1A1, Mmp2, Tgfb1 significantly reduce (ACTA2, p ≈ 0.1).Should the result shows that, 4- methyl -5,6- dihydro
The expression of BDL rat liver fibrosis marker can be significantly inhibited after the administration of -2 ketone of pyrans.
The transfer ability of -2 ketone of 5 4- methyl -5,6- dihydropyran of embodiment inhibition liver cancer cells
Using -2 ketone of 4- methyl -5,6- dihydropyran (80,160,240 μm of olL of various concentration-1) to raw in logarithm
The case where long-term human liver cancer cell HepG2 handles 48h, and microscopically observation scar closes up under inverted microscope, it is specific to grasp
It is as follows to make mode:
(1) it plating cells: by the cell dissociation of logarithmic growth phase, counts, with 3 × 105The cell number in the hole /mL/ is inoculated in 6
In orifice plate；
(2) scratch: after when cell growth 48 is small, when coverage is close to 100%, with 10 μ L Tip on cell monolayer
Scratch；
(3) it cleans: rinsing cell with PBS, wash away cell fragment；
(4) be administered: cell continues to cultivate in the culture medium containing certain concentration drug；
(5) it observes: at regular intervals, the case where microscopically observation scar closes up under inverted microscope, and in 48
Hour photograph.
It is analyzed through scratch experiment, after being handled HepG2 cell 48 hours after scratch with -2 ketone of 4- methyl -5,6- dihydropyran,
The healing state of cell is observed under the microscope, and gained liver cancer cells scratch experiment result figure is as shown in figure 14.As shown in Figure 14,
Cell is gradually to travel motion at scratch after 48 hrs for HepG2 cell, and scratch narrows, and 4- methyl -5,6- dihydropyran -2
Then scratch is high-visible for ketone processing group cell, and expands with the increase of administration concentration, under highest 240 μM of concentration, cell
Scratch does not narrow substantially.As a result after illustrating the processing of -2 ketone of 4- methyl -5,6- dihydropyran, hepatoma Hep G 2 cells hypomotility,
Thus illustrate that -2 ketone of 4- methyl -5,6- dihydropyran can inhibit the transfer of liver cancer cells.
In summary result is it is found that -2 ketone of 4- methyl -5,6- dihydropyran can effectively inhibit COL1A1 promoter
Activity inhibits to concentration dependent the mRNA and protein expression level of hepatic fibrosis markers COL1A1 and α-SMA, improves big
Liver function, bile duct proliferation and the degree of necrosis of mouse reduce liver fiber level, and are able to suppress the transfer of liver cancer cells, show it
With the anti-liver fibration in good inside and outside and anti-liver cancer cell transferance.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (4)
- - 2 ketone of 1.4- methyl -5,6- dihydropyran is preparing the application in anti-hepatic fibrosis medicines and medicines resistant to liver cancer.
- - 2 ketone of 2.4- methyl -5,6- dihydropyran is prepared by the composition that effective component is formed with pharmaceutically acceptable carrier Application in anti-hepatic fibrosis medicines and medicines resistant to liver cancer.
- Application of -2 ketone of 3.4- methyl -5,6- dihydropyran in preparation liver fibrosis and prevention of hcc health care product.
- 4. a kind of drug for anti-hepatic fibrosis and anti-liver cancer and anti-, which is characterized in that the effective component of the drug includes 4- first The derivative of -2 ketone of base -5,6- dihydropyran, pharmaceutically acceptable salt, solvate, metabolite, stereoisomer, mutually Tautomeric, polymorph, prodrug.
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