CN105738630A - Application of serine-threonine Raf kinase inhibitor protein in preparation of medicine for treating immune hepatic fibrosis - Google Patents

Abstract

The invention discloses research on the action of serine-threonine Raf kinase inhibitor protein in medicine for treating immune hepatic fibrosis.The action mechanism of the RKIP in the immune hepatic fibrosis forming process is discussed for the first time, animal experiments and in-vitro cell experiments show that in the immune hepatic fibrosis forming process, activation of an Raf-1/MEK/ERK1,2 signal transduction pathway is closely related to protein expression decreasing and phosphorylation increasing of the RKIP, and the RKIP has the important regulating and controlling effect in the hepatic fibrosis generating mechanism.Therefore, the fact that the Raf-1/MEK/ERK1,2 signal transduction pathway is inhibited through RKIP overexpression is an important target of new medicine for treating the immune hepatic fibrosis.The serine-threonine Raf kinase inhibitor protein can be applied to preparation of the medicine for treating the immune hepatic fibrosis.

Description

Serine/threonine kinase inhibition albumen is in treatment fibrosis medicine Application

Technical field

The present invention relates to serine/threonine kinase inhibition albumen (Raf kinase inhibitory protein, RKIP) Purposes, specifically RKIP as drug target be marked on preparation treatment fibrosis medicine in application.

Background technology

Hepatic fibrosis is the pathological change being caused chronic hepatopathy to have by Different types of etiopathogenises, shows as liver inner cell characteristically Excessively synthesized and the deposition of epimatrix (extracellu larmatrix, ECM).Multinomial research is had to confirm, hepatic stellate cell (hepatic stellate cells, HSCs) is the key cellular constituents in process of hepatic fibrosis, is the master of ECM in liver Want cell derived.It is now recognized that suppression HSCs activation is one of prevention the main path reversing hepatic fibrosis with propagation.

Recent study finds, Raf-1/MEK/ERK1, and 2 signal paths take part in the regulation and control of HSCs activation and propagation Journey, close with the generation development relationship of hepatic fibrosis, the formation of hepatic fibrosis can be affected from many aspects.

Serine/threonine (Ser/Thr) kinase inhibition albumen (is called for short: Raf kinase inhibition albumen, English name: Raf kinase Inhibitory protein, RKIP) it is phosphotidylethanolabinding binding protein (phosphatidylethanolamine Binding protein, PEBP) important a member in family.This albumen is a kind of and Raf-1 interacts and suppresses Raf-1/ MEK/ERK1, the albumen of 2 signal paths, RKIP is also that discovery and Raf-1 play suppression in be combineding with each other the most in vivo The protein molecular of effect, indicates that it is likely to be of important biological function.RKIP is exempting from as the inhibitive factor of signal path Effect in epidemic disease hepatic fibrosis is still known few.

Summary of the invention

It is an object of the invention to research and develop serine/threonine kinase inhibition albumen RKIP and be marked on system as drug target Application in standby treatment fibrosis medicine.

The present invention is experimentation based on inventor and completes.

(1) Raf kinase inhibition albumen Expression in immunologic liver injury induced rat process of hepatic fibrosis Situation

By SD male rat with aseptic porcine blood serum induced synthesis fibrosis animal model, observational study RKIP, p- The distribution in hepatic fibrosis different times hepatic tissue such as RKIP and express change, and Raf-1/MEK/ERK1, the work of 2 paths The effect of change situation.Result shows, porcine blood serum is successfully, reproduced out Rat Liver Fibrosis Model, and this model is adjoint in forming process The change of liver function mark, hepatocyte chronic inflammation cellular infiltration, liver rope arrangement disorder, portal area and central vein knot occur Forming tissue thickening, hepatic tissue collagen fiber showed increased, the content of serological index HA, LN, COL-I, PC III raises simultaneously.

Use SABC detection hepatic tissue RKIP, ERK and phosphorylated protein express, RT-PCR check RKIP, ERK, The expression of Col-I and Col-III mRNA, Western blot detection RKIP, ERK etc. and the expression of phosphorylated protein, result Showing, in process of hepatic fibrosis, RKIP expression declines, and phosphorylation RKIP level raises simultaneously.Phosphorylation simultaneously ERK, Raf-1, MEK-1 express substantially to be increased, and shows to there is Raf-1/MEK/ERK1 in hepatic fibrosis generating process, and 2 signals turn The activation of guiding path.

(2) Raf kinase inhibition albumen activation HSCs in expression and phosphorylation level

Cell results shows, the rat HSCs's of IL-1 β stimulation group, Locostatin+ IL-1 β group and Locostatin group ERK1 mrna expression amount significantly raises.The most aobvious through the expressing quantity of RKIP, ERK, p-ERK of the HSCs of IL-1 β effect 48h Write and raise, be remarkably decreased through the RKIP expressing quantity of the HSCs of Locostatin effect 48h, and the albumen of p-ERK, α-SMA Expression significantly raises, and result above is pointed out, and RKIP expression after Locostatin suppresses declines, Raf-1/MEK/ ERK1, 2 signal transduction pathways are activated and promote the activation of HSCs, propagation.

In a word, RKIP effect in treatment immunologic liver injury, hepatic fibrosis is studied by the present invention first, real Testing and show, in process of hepatic fibrosis, RKIP expression declines and phosphorylation RKIP level raises and Raf-1/MEK/ ERK1, the activation of 2 signal transduction pathways is closely related, raises RKIP level and hepatic fibrosis can be suppressed to develop, thus be medicine The important target of thing treatment hepatic fibrosis.Serine/threonine kinase inhibition albumen can be applicable to preparation treatment fibrosis Medicine in.

Detailed description of the invention

Experimental program:

(1), RKIP Expression situation in immunologic liver injury induced rat process of hepatic fibrosis

(1) experimental animal model preparation, be grouped and process

SD male rat 88, body weight is (140 ± 20) g, is divided into Normal group 28, model group 60.Normal group Intraperitoneal injection of saline 0.5mL/, twice a week.Model group with aseptic porcine blood serum lumbar injection 0.5mL/ only, weekly 2 Secondary, totally 18 weeks, replicate Rat Liver Fibrosis Model.Model group rats is randomly selected respectively at 8wk, 11wk, 14wk, 18wk 15, normal rats 7, pull out after eyeball takes blood and put to death rat, and aseptic condition cuts skin and enters abdominal cavity, leaves and takes lobus sinister immediately Hepatic tissue is in-80 DEG C of preservations, then cuts partial liver lobus sinister and fix with 4% paraformaldehyde, for future use.Hepatic tissue is taken from liver The same area of lobus sinister.

(2) Testing index

1. automatic clinical chemistry analyzer detection Serum ALT, AST.

2. ELISA kit detection serum I Collagen Type VI (COL-I), III procollagen type (PC III), hyaluronic acid (HA), layer Fibronectin (LN).

3. ELISA kit detection hepatic tissue III procollagen type (PC III), laminin，LN (LN).

4. HE dyeing: paraffin-embedded hepatic tissue is with the thickness serial section of 5 μm, and experimental procedure contaminates routinely Color.

5. Masson trichrome stain: hepatic tissue is with 5 μm thickness serial section, and experimental procedure dyes routinely.

6. Immunohistochemical detection: the expression of RKIP, p-RKIP, p-ERK1/2, ERK1/2.

7. RT-PCR measures RKIP mRNA, COL-I mRNA, COL-III mRNA, ERK1 mRNA, ERK2 mRNA Expression.

8. Western blot method detection RKIP, p-RKIP, p-ERK1/2, ERK1/2, COL-I, COL-III, α-SMA, The expression of MEK, p-MEK.

(3) experimental result:

1. the change of rat vital sign, liver index

Normal group and the model group rats mental status are all good, hair smoothing, and activity and diet amount of drinking water are normal, and body weight increases Long no difference of science of statistics.Starting at the end of experiment from experiment, Normal group and model group rats are all without dead.When dissected can Seeing that model group rats liver occurs a certain degree of enlargement, hypertrophy, compared with Normal group, model group rats liver index increases Greatly (p ＜ 0.05).The results are shown in Table 1-1.

The change (± s) of table 1-1 fibrosis rat liver index

Note: compare with Normal group, * P < 0.05, * * P < 0.01

2. the pathological change of fibrosis rat liver tissue

HE coloration result shows, normal rats lobules of liver clear in structure and complete, centered by central vein, to surrounding in putting Penetrating shape arrangement, hepatocyte has no degeneration or necrosis.Chronic inflammation cellular infiltration seen from model group hepatocyte, liver rope arrangement disorder, with Modeling time lengthening portal area and central vein connective tissue are thickening, form thick fibre gap, separate liver parenchyma in lobule Or lobule, form the pseudolobuli differed in size.

Masson coloration result shows, has a small amount of blue collagen expression around rats in normal control group hepatic tissue blood vessel.Mould Type control rats hepatic tissue collagen fiber showed increased, blue collagen is positioned at around portal area and blood vessel more and stretches out, Connecting and form thicker fibrous septum, hepatocyte is in enlargement in various degree, and kytoplasm loosens.

3. the changes of contents of fibrosis rat blood serum HA, LN, COL-I, PC III

Result shows, the content of model group rats serum HA, LN, COL-I, PC III is all remarkably higher than normal rats (p ＜ 0.05 Or p ＜ 0.01).The results are shown in Table 1-2.

The changes of contents (± s) of table 1-2 fibrosis rat blood serum HA, LN, COL-I, PC III

Note: compare with Normal group, * P < 0.05, * * P < 0.01

4. on liver tissues of rats with hepatic fibrosis LN, PC III impact of content

Result shows, model group rats hepatic tissue PC III content is higher than normal rats, and along with the prolongation of modeling time, content is protected Hold in level relatively smoothly；And the content of hepatic tissue LN was significantly higher than normal group at the 8th, 11 weeks, the results are shown in Table 1-3.

The changes of contents (± s) of table 1-3 rat with liver fibrosis LN, PC III

Note: compare with Normal group, * P < 0.05, * * P < 0.01

5. the phosphorylation level of immunohistochemical method detection RKIP and ERK

Immunohistochemical method testing result shows, in normal rats hepatic tissue, RKIP expression is higher, model group rats In liver, RKIP positive cell significantly reduces；P-RKIP is relatively low at the expression of normal rat hepatic tissue, with modeling time lengthening, In model group rats liver, p-RKIP expresses and strengthens；Normal rat hepatic tissue less expression ERK and p-ERK, and model group rats In hepatic tissue, the expression of ERK and p-ERK higher (p ＜ 0.05 or p ＜ 0.01), is distributed mainly on around portal area and vein, And along with the prolongation of modeling time, the expression of ERK and p-ERK is increasing trend, and statistical result is as shown in table 1-4.

The changes of contents of table 1-4 rat with liver fibrosis RKIP, p-RKIP, ERK1/2, p-ERK1/2 (± s)

Note: compare with Normal group, * P < 0.05, * * P < 0.01

6. fibrosis rat tissue RKIP mRNA, COL-I mRNA, COL-III mRNA, ERK1 mRNA, ERK2 The expression change of mRNA

RT-PCR testing result shows, in model group rats hepatic tissue, RKIP mrna expression fall occurs with the prolongation of modeling time Low trend, the expression of ERK1 mRNA and ERK2 mRNA is in rising trend with modeling time lengthening, COL-I mRNA, COL-III The expression of mRNA is significantly higher than normal group, and result is as shown in tables 1 to 5.

Table 1-5 immunity liver tissues of rats RKIPmRNA, ERK1mRNA, ERK2mRNA, COL-I mRNA, COL-III The Expression (± s, n=8) of mRNA

Note: compare with Normal group, * P < 0.05, * * P < 0.01

7. Western blot method detection RKIP and Raf-1/MEK/ERK1,2 signal path associated protein and phosphorylation water thereof Flat

Western blot method testing result shows, RKIP expression in normal rats hepatic tissue is higher, and model group is big In rat liver, RKIP expression reduces；ERK expression in normal liver tissue is less, and in model group, ERK expresses and increases；p- ERK expression in normal rats hepatic tissue is less, and with the prolongation of modeling time, in model group, the expression of p-ERK first rises Downward trend occurs after height；With the prolongation of modeling time, the expression of Raf is on a declining curve, and p-Raf is in rising trend； The expression in normal group of MEK is higher, downward trend occurs with the prolongation of modeling time, and the expression of p-MEK is then presented The trend of liter；COL-I, COL-III, the expression of α-SMA rise (p ＜ 0.05 or p ＜ 0.01) with the prolongation of modeling time.Knot Fruit is as shown in table 1-6 ~ 1-8.

The Expression (± s, n=8) of table 1-6 fibrosis rat RKIP, ERK, Raf, MEK albumen

Note: compare with Normal group, * P < 0.05, * * P < 0.01

Table 1-7 fibrosis rat COL-I, COL-III, the Expression (± s, n=8) of α-SMA albumen

Note: compare with Normal group, * P < 0.05, * * P < 0.01

The Expression (± s, n=8) of table 1-8 fibrosis rat p-RKIP, p-ERK, p-Raf, p-MEK albumen

Note: compare with Normal group, * P < 0.05, * * P < 0.01

(2), RKIP activation HSCs in expression and phosphorylation level

(1) experimental technique and Testing index

1. extract separation rat primary hepatic stellate cell successfully cultivate and pass on, and do following relevant identification experiment:

Platform expects blue dyeing, observation of cell survival rate；The autofluorescence of application fluorescence microscope HSCs；Activating cell is identified: Hepatic stellate cell and the cultivation hepatic stellate cell α-SMA of more than 8 days that immunocytochemical stain detection has just separated dye.

The impact of the rat HSCs propagation that 2. IL-1 β is stimulated by MTT method detection Locostatin

3. RKIP between the hepatic stellate cell of the specific inhibitor Locostatin effect that RT-PCR measures use RKIP MRNA, ERK1 mRNA, COL-I mRNA, COL-III mrna expression amount.

4. Western blot experiment detection IL-1 β is with the hepatic stellate cell of time excessive activation, and uses simultaneously RKIP, ERK, p-ERK, COL-I, COL-III and α expression of-SMA albumen between the hepatic stellate cell of Locostatin effect

(2) experimental result:

1. the impact that rat HSCs is bred by MTT method detection IL-1 β and Locostatin

MTT method test experience result shows, compares with normal group, and the cell proliferation rate using IL-1 β stimulation group is 80.2%, The cell proliferation rate of Locostatin+IL-1 β group is 25.1%, and the cell proliferation rate of Locostatin group is 7.7%.

2. the Locostatin impact on cultured rat hepatic stellate cells ERK signal path related gene expression

RT-PCR method testing result shows, compares with normal group, use IL-1 β stimulation group, Locostatin+IL-1 β group and The expression no difference of science of statistics of the RKIP mRNA of the rat HSCs of Locostatin group, and ERK1 mRNA, COL-I MRNA, COL-III mrna expression amount significantly raise (p ＜ 0.05 or p ＜ 0.01).Result is as shown in table 2-1.

Table 2-1. cultured rat hepatic stellate cells RKIPmRNA, ERK1mRNA, COL-1mRNA, COL-III impact of mrna expression (2^-△△CtMethod) (± s)

Note: compare with blank group, * P < 0.05, * * P < 0.01

3. the Locostatin impact on cultured rat hepatic stellate cells signal path correlative protein expression

Western blot testing result shows, compares with normal group, through RKIP, ERK, p-of the HSCs of IL-1 β effect 48h ERK and COL-I, the expressing quantity of COL-III significantly raise, through Locostatin effect 48h HSCs RKIP, COL-I, COL-III expressing quantity be remarkably decreased, and the expressing quantity of p-ERK, α-SMA significantly raises, through Locostatin The expressing quantity of the RKIP acting on the HSCs of 47 h after effect 1h through IL-1 β is remarkably decreased, and the expressing quantity of p-ERK shows Write and raise (p ＜ 0.05 or p ＜ 0.01).Result is as shown in table 2-2,2-3.

The expression (± s) of table 2-2. cultured rat hepatic stellate cells RKIP, ERK, p-ERK albumen

Note: compare with blank group, * P < 0.05, * * P < 0.01

Table 2-3. cultured rat hepatic stellate cells COL-I, COL-III, the expression (± s) of α-SMA albumen

Note: compare with blank group, * P < 0.05, * * P < 0.01.

Claims (2)

1. serine/threonine kinase inhibition albumen application in treatment fibrosis.

2. serine/threonine kinase inhibition albumen is marked on answering in preparation treatment fibrosis medicine as drug target With.