CN104001152B - De-arginine complement component 3 f(DRC3f) treating the application in hepatic injury - Google Patents
De-arginine complement component 3 f(DRC3f) treating the application in hepatic injury Download PDFInfo
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Abstract
The invention discloses a kind of de-arginine complement component 3 f(DRC3f) treating the application in hepatic injury.The invention discloses de-arginine complement component 3 f(DRC3f) preparing the application prevented and/or treated in the product of hepatic injury.De-arginine complement component 3 f(DRC3f disclosed by the invention) can pretreatment weaken Con? the hepatic injury of A induction; this polypeptide to the protective effect of ConA induced liver injury may with suppression hepatocellular apoptosis; promote hepatocyte growth; suppressor T cell, NKT cell infiltrate relevant to hepatic tissue; Con? can A gives DRC3f treatment also alleviate Con after stimulating? the degree of A induced liver injury, and improve Survival.In addition, in the Experiment on therapy of fatty liver, the DRC3f of high dose significantly can reduce ALT, AST, CHOL level in serum, also obviously can reduce the content of hepatic steatosis degree and liver free-fat.Therefore, the present invention is that the treatment of clinical hepatopathy provides new possible direction and concrete measure.
Description
Technical field
The present invention relates to a kind of de-arginine complement component 3 f(DRC3f) treating the application in hepatic injury, belong to field of biological pharmacy.
Background technology
Hepatic disease is a huge global public health problem, and it threatens the health of billions of people.More seriously, the sickness rate of many hepatic disease raises gradually.The increase of immigrant's number, travelling and economic globalization all result in the wide-scale distribution of virus frequently.The fat hepatitis that obesity causes also becomes new health problem; The absorption of a large amount of ethanol can cause fatty liver, alcoholic hepatitis, even liver cirrhosis.Current global chronic hepatitis-B infection person has reached 400,000,000 populations, and nearly 2,000 ten thousand populations have exposure evidence, and about 100,017,000 populations infect hepatitis C.The 26S Proteasome Structure and Function of liver is all more complicated, as the vitals of protein synthesis and storage, for body running with regulate the stable of other histoorgans to provide various material, these factors all imply that the treatment of hepatic disease is very complicated, is full of difficulty.
The chmice acute Immune liver injury of ConA induction is similar with the pathology of autoimmune hepatitis to human viral's hepatitis, stimulate through ConA and cause mouse liver cell necrosis and apoptosis, the rising of adjoint Serum ALT and AST level, further, the lymphocytes such as a large amount of T cell, NKT cell and Kuffer cell are had to infiltrate to liver.To have the modeling cycle short for the acute hepatic injury model of ConA induction, repeatable high, with the advantage such as human hepatitis pathogenesis is similar, has been widely used in the research for the treatment of hepatitis method and mechanism.
Along with the change of modern people's living and diet structure, the sickness rate being called as the fatty liver disease (also claiming fatty liver) of modern times " affluenza " significantly improves, and become the second largest frequently-occurring hepatopathy being only second to viral hepatitis, human health in serious threat.Fatty liver disease can be caused by multiple inducement; pathological changes is mainly positioned at lobules of liver; be one of common DHD; with triglyceride in hepatocyte (TG) excessive accumulation and diffusivity hepatic cell fattydegeneration for major pathologic features; can be in progress as hepatic fibrosis, liver cirrhosis, liver failure, therefore to the early diagnosis of this disease and treat extremely important.The treatment of non-alcoholic fatty liver disease is divided into non-drug therapy and Drug therapy, and the former is only applicable to mild fatty hepatopath, comprises physical training, loses weight, keeps on a diet, removes the means such as the cause of disease.Cholesterol in Patients with Fatty Liver blood and triglyceride levels again often with cholesterol in meals and fat intake closely related, the balanced diet of low calorie, low fat, high dietary-fiber can improve blood lipid metabolism, reduce the accumulation of liver fat, and excess fat, toxin etc. in intestinal can be excreted, thus play effect for reducing fat; Keep a diet and effectively can reduce body weight, and can liver function be improved, promote that the liver retraction of enlargement and intrahepatic fat reduce.Effective and reasonable motion, can reduce body fat, lose weight; Promote that fatty tissue is decomposed, reduce blood fat, reduce the deposition of interior fat, thus play lipotropic object.But; middle severe fatty liver patient effectively cannot remove liver fat by non-drug therapy, and chemotherapy is main is treated by medicines such as antioxidant, liver cell protective agent and function of gallbladder promoting hepatoprotective, cytokine inhibitor and CYP inhibitor such as the appetrol such as the Insulin receptor INSR such as metformin and thiazolidinedione agonist (euglycemic agent), orlistat and sibutramine, Statins and Bei Te class blood lipid-lowering medicine, vitamin E, vitamin Cs.But most of fat-reducing medicament is limited to intrahepatic fat eliminating effect, many lipid lowerers can cause hepatocyte injury in various degree, and therefore the function and position of lipid lowerers in fatty liver treatment still has arguement.
DRC3f is complement component 3 f de-arginic derivant under carboxypeptidase-N acts on, be made up of 16 aminoacid, sequence is NH2-Ser-Ser-Lys-Ile-Thr-His-Arg-Ile-His-Trp-Glu-Ser-Ala-Ser-Leu-Leu-COOH, and molecular weight is 1865Da, isoelectric point, IP 8.51.Complement is the important regulatory factor of inflammation and immunne response, has at least 30 kinds of glycoproteins to have the effect regulating liver regeneration, immunne response and other important physiological process in complement system.In complement, C3 content is the highest, and in the process of complement activation, the C3b that C3 activation is formed is decomposed into iC3b and the 17 PEPC 3f of deactivation under Complement receptor Ⅰ or the effect of the H factor, and then generates DRC3f.It is reported, DRC3f can detect in systemic sclerosis patients serum, DRC3f or C3f of synthesis and the serum containing DRC3f all can promote the hypertrophy of the capillary endothelium of Skin Cell and pneumonocyte, the generation of TGF-β 1 can also be promoted in Skin Cell, show that DRC3f is one of serum small molecular somatomedin.
Summary of the invention
The object of this invention is to provide a kind of de-arginine complement component 3 f(DRC3f) treating the application in hepatic injury, de-arginine complement component 3 f(DRC3f) significantly can reduce Serum ALT, AST level in the acute liver damage of ConA induction, suppress hepatic necrosis and/apoptosis, the survival rate of acute hepatic injury mice can be significantly improved and extend life span, suppress T cell, NKT cell in hepatic injury to infiltrate to hepatic tissue, promote hepatocellular propagation; In chronic hepatic injury, significantly can not only reduce Serum ALT, AST, CHOL, TG level, and hepatic cell fattydegeneration and fat content can be suppressed to rise.
The invention provides de-arginine complement component 3 f(DRC3f) preparing the application prevented and/or treated in the product of hepatic injury.
In above-mentioned application, described hepatic injury is acute liver damage or chronic hepatic injury;
Described acute liver damage is specially acute immune hepatic injury;
Described chronic hepatic injury is specially fatty liver.
De-arginine complement component 3 f(DRC3f) prepare suppress hepatic injury time serum in application in the product that rises of glutamate pyruvate transaminase and/or glutamic oxaloacetic transaminase, GOT level also belong to protection scope of the present invention.
De-arginine complement component 3 f(DRC3f) suppress the application in the product of hepatic necrosis and/or apoptosis in hepatic injury also to belong to protection scope of the present invention in preparation.
De-arginine complement component 3 f(DRC3f) promote that the application in the product of hepatocyte growth in hepatic injury also belongs to protection scope of the present invention in preparation;
Or,
De-arginine complement component 3 f(DRC3f) suppress the application in the product that in hepatic injury, T cell and/or NKT cell infiltrate to hepatic tissue also to belong to protection scope of the present invention in preparation.
In above-mentioned arbitrary described application, described hepatic injury is acute liver damage;
Described acute liver damage is specially acute immune hepatic injury.
De-arginine complement component 3 f(DRC3f) prepare suppress hepatic injury time serum in application in the product that rises of glutamate pyruvate transaminase and/or glutamic oxaloacetic transaminase, GOT and/or serum total cholesterol and/or serum triglyceride level also belong to protection scope of the present invention.
In above-mentioned application, described hepatic injury is chronic hepatic injury;
Described chronic hepatic injury is specially fatty liver.
De-arginine complement component 3 f(DRC3f) suppress the application in the product of hepatic cell fattydegeneration also to belong to protection scope of the present invention in preparation;
Or,
De-arginine complement component 3 f(DRC3f) suppress the application in the product of the content of hepatic tissue Free Fat rising also to belong to protection scope of the present invention in preparation.
In above-mentioned arbitrary described application, described de-arginine complement component 3 f(DRC3f) aminoacid sequence as shown in SEQIDNo.1.
De-arginine complement component 3 f(DRC3f provided by the invention) pretreatment can weaken ConA induction hepatic injury; this polypeptide to the protective effect of ConA induced liver injury may with suppression hepatocellular apoptosis; promote hepatocyte growth; suppressor T cell, NKT cell infiltrate relevant to hepatic tissue; ConA gives the degree that DRC3f treatment also can alleviate ConA induced liver injury after stimulating, and improves Survival.In addition, in the Experiment on therapy of fatty liver, the DRC3f of high dose significantly can reduce ALT, AST, CHOL level in serum, also obviously can reduce the content of hepatic steatosis degree and liver free-fat.Therefore, the present invention is that the treatment of clinical hepatopathy provides new possible direction and concrete measure.
Accompanying drawing explanation
Measurement result and the hepatic tissue HE coloration result of the rear 12h mice serum ALT of ConA stimulation and AST level is given in Fig. 1 acute liver damage prevention experiment.
Each hepatocellular apoptosis and cell cycle detection organizing mice in Fig. 2 acute liver damage prevention experiment.
In Fig. 3 acute liver damage prevention experiment, each lymphocyte of Flow Cytometry Assay hepatic tissue (T cell, NK cell, NKT cell) infiltrates ratiometric result.
Survival rate in Fig. 4 acute liver damage prevention experiment after each group injected in mice ConA within 72h.
Measurement result and the hepatic tissue HE coloration result of the rear 12h mice serum ALT of ConA stimulation and AST level is given in Fig. 5 acute liver damage Experiment on therapy.
Survival rate in Fig. 6 acute liver damage Experiment on therapy after each group injected in mice ConA within 72h.
Fig. 7 fatty liver respectively organizes the Serum ALT testing result of rat.
Fig. 8 fatty liver respectively organizes the serum AST testing result of rat.
Fig. 9 fatty liver respectively organizes the serum T BIL testing result of rat.
Figure 10 fatty liver respectively organizes the change of serum C HOL testing result of rat.
Figure 11 fatty liver respectively organizes the serum TG testing result of rat.
The HE coloration result of Figure 12 fatty liver tissue.
The Sudan stains result of Figure 13 fatty liver tissue freezing section.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Experimental data in following embodiment is with mean ± standard deviation
represent, the comparison of many Sets of Measurement Datas adopts variance analysis or Kruskal-WallisH inspection, compares between two and adopt LSD inspection between group.Survival analysis adopts Kaplan-Meier method and Log-rank inspection.All data all adopt SPSS13.0 software to carry out statistical analysis, and P<0.05 is that difference has statistical significance.
Male Balb/c inbred mouse (6-8 week, 18-22g) is purchased from Hebei Medical University's Experimental Animal Center.
De-arginine complement component 3 f(DRC3f) aminoacid sequence as shown in SEQIDNo.1, for NH2-Ser-Ser-Lys-Ile-Thr-His-Arg-Ile-His-Trp-Glu-Ser-Ala-Ser-Leu-Leu-COOH, synthesized by Sangon Biotech (Shanghai) Co., Ltd., be greater than 98.17% through HPLC purity analysis synthesis content.
IV type concanavalin A, Con A (ConA) purchased from American Sigma company.
Dexamethasone injection is purchased from Ou Yi Shi Pharmaceutical Group Pharmaceutical Co.
0.9% normal saline is purchased from Shijiazhuang Siyao Co., Ltd.
Mouse lymphocyte separating medium is purchased from Beijing Suo Laibao Science and Technology Ltd..
Pancreatin is purchased from Beijing Suo Laibao Science and Technology Ltd..
Hyclone purchased from American Hyclone company.
FITC labelling anti-mouse CD3e antibody, PE labelling anti-mouse DX5 antibody purchased from American BD company.
ConA injection:
ConA lyophilized powder 100mg
Physiological saline solution 5ml
Repeatedly blow and beat with rifle head until ConA lyophilized powder dissolves completely, be divided in 1.5mlEP pipe according to every pipe 1ml ,-20 DEG C of preservations.Front physiological saline solution is used to dilute administration.
DRC3f injection:
DRC3f lyophilized powder 5mg
Physiological saline solution 2ml
Repeatedly blow and beat until after DRC3f lyophilized powder dissolves completely with rifle head ,-20 DEG C of preservations.Be divided in 1.5mlEP pipe according to every pipe 500 μ l, use front physiological saline solution to dilute administration.
The preparation of 4% paraformaldehyde solution:
Paraformaldehyde 40g
Na
2HPO
4·12H
2O29g
NaH
2PO
4·2H
2O2.95g
Distilled water 600ml
By said components under 50 DEG C of conditions, fully stir, until dissolve completely, adjust pH=7.4, add distilled water and be settled to 1L.
Male SD rat (180-200g) is purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
SNMC is purchased from Xi'an Lijun pharmaceutical Co., Ltd.
Blood fat reducing channel activating soft capsule is purchased from Shineway Pharmaceutical Group Limited.
Olive oil is purchased from Sangon Biotech (Shanghai) Co., Ltd..
Sodium cholate is purchased from Sangon Biotech (Shanghai) Co., Ltd..
Cholesterol is purchased from Sangon Biotech (Shanghai) Co., Ltd..
Methylthiouracil is purchased from Sangon Biotech (Shanghai) Co., Ltd..
High lipid food is prepared: Adeps Sus domestica 15%, T-CHOL (Tc) 2.8%, methylthiouracil 0.28%, sodium cholate 0.7%, normal diet 81.2%, % representation quality percentage composition.
Normal diet is purchased from Hebei Medical University's Experimental Animal Center.
The preparation of PI dye liquor: normal saline 129.6ml, PI10mg, RNase2mg, volumn concentration 1.0%TritonX-1000.5ml, sodium citrate 200mg, adding distil water are settled to 200ml, adjust pH to 7.2 ~ 7.6, put keep in Dark Place in 4 DEG C of refrigerators for subsequent use.
Normal saline in following embodiment is physiological saline solution.
The acute liver damage that in embodiment 1, ConA induces is acute immune hepatic injury.
Embodiment 1, de-arginine complement component 3 f(DRC3f) treating the application in acute liver damage
One, preventive effect experiment
(1) experiment grouping
56 male Balb/c inbred mouses (6-8 week, 18-22g) are divided into 7 groups, i.e. Normal group, ConA model group, dexamethasone positive controls, DRC3f1, DRC3f2, DRC3f3 and DRC3f4 group at random, often organize 8.
(2) tail vein injection ConA injection (ConA12mg/kg body weight is being carried out to each group of mice (except normal mouse group), volume is 0.25ml) front 1h, give ConA model group mouse tail vein injection 0.25ml normal saline respectively, dexamethasone positive controls mice dexamethasone injection (dexamethasone 500 μ g/kg body weight, volume is 0.25ml), give DRC3f1, DRC3f2, DRC3f3 and DRC3f4 group mice DRC3f injection (DRC3f is respectively 200,400,800,1600 μ g/kg body weight, and volume is 0.25ml) respectively simultaneously.
The mice of Normal group gives tail vein injection 0.25ml normal saline twice, and for getting rid of time effects, the double injection normal saline time is respectively organized identical with above-mentioned other.
After ConA injection, 12h puts to death each group of mice, and blood and the hepatic tissue of collecting each group of mice carry out follow-up index determining.
(3) mensuration of mice serum glutamate pyruvate transaminase (ALT) and glutamic oxaloacetic transaminase, GOT (AST) level
Serum glutamic pyruvic transminase (ALT) and glutamic oxaloacetic transaminase, GOT (AST) level weigh the index of liver function.
Giving ConA stimulate after 12h by mouse anesthesia, intracardiacly get blood, blood be placed in the aseptic EP pipe not adding anticoagulant, and EP pipe is placed in 37 DEG C of calorstat temperature and bathes 30min, then be placed in 4 DEG C of refrigerator 1h; Take out EP pipe, put into refrigerated centrifuge, 4 DEG C, the centrifugal 15min of 3000g; Careful absorption upper serum is put in another aseptic EP pipe, and precipitation discards.After being diluted by the serum physiological saline solution 4 times be separated, alanine aminotransferase (ALT) detection kit and AST (AST) detection kit (all purchased from Beijing Jiuqiang Biotechnology Co., Ltd.) is used to detect glutamate pyruvate transaminase (ALT) and glutamic oxaloacetic transaminase, GOT (AST) level.
Give the measurement result of the rear 12h mice serum ALT of ConA stimulation and AST level as shown in Figure 1A and 1B.
In Figure 1A and 1B,
*represent P<0.05,
*represent P<0.01,
* *represent P<0.001, all represent compared with ConA model group;
+represent P<0.05,
++p represents <0.01,
+++represent P<0.001, all represent compared with dexamethasone positive controls.
Figure 1A shows, stimulate after 12h through ConA, ConA model group mice serum ALT and AST level are significantly higher than Normal group (P<0.001); Compared with ConA model group mice, dexamethasone positive controls mice is giving significantly to reduce serum alt and AST level (P<0.001) under the pretreated condition of dexamethasone; Compared with ConA model group mice, the DRC3f pretreated group mice of various dose (DRC3f200,400,800,1600 μ g/kg body weight groups) also significantly reduce Serum ALT and AST level, there is dose-response relationship, compared with ConA model group, difference all has statistical significance (P<0.05), and wherein, the effect of DRC3f3 group is the most obvious, compared with dexamethasone positive controls effect, difference not statistically significant.
(4) hepatic tissue paraffin embedding and HE dyeing
After giving ConA stimulation, 12h puts to death mice; Open abdominal cavity fast, be separated liver, cut the hepatic tissue of the suitable size of right lobe of liver ad-hoc location, be placed in volumn concentration be 4% paraformaldehyde solution fix 24h; Carry out paraffin embedding after fixing, carry out the serial section of 5 μm respectively, carry out HE dyeing according to following steps afterwards:
Ammonia aqueous solution 30s → tap water 4min → eosin stains 1min → ethanol serial dehydration → the dimethylbenzene of the aqueous hydrochloric acid solution 30s → tap water 3min → volumn concentration 1% of dimethylbenzene dewaxing → gradient alcohol dehydration → tap water 1min → brazilwood extract dyeing 5min → tap water 3min → volumn concentration 1% is transparent → neutral gum mounting.
Give ConA and stimulate rear 12h murine liver tissue paraffin embedding and HE coloration result as shown in Figure 1B.
In Figure 1B, a represents Normal group, and b represents ConA model group, and c represents dexamethasone positive controls, and d, e, f, g represent DRC3f1, DRC3f2, DRC3f3 and DRC3f4 group respectively.What arrow indicated is necrotic area, Scalebar, 50 μm.
Figure 1B shows, the visible large-area hepatic necrosis of ConA model group murine liver tissue; Compared with ConA model group mice, dexamethasone positive controls mice is giving can significantly improve hepatic necrosis situation under the pretreated condition of dexamethasone; Compared with ConA model group mice, give various dose DRC3f pretreated group (DRC3f200,400,800,1600 μ g/kg body weight groups) mice also significantly reduce ConA induction hepatic necrosis, there is no significant difference between each dosage group of DRC3f and dexamethasone positive controls.Illustrate that DRC3f pretreatment significantly can improve the mouse liver injury of ConA induction, improve effect and dexamethasone positive controls is close.
(5) Flow cytometry hepatocellular apoptosis and cell cycle
1, hepatocyte is separated
After giving ConA stimulation, 12h puts to death mice, opens abdominal cavity and breast chamber; Left ventricle inserting needle, with shears at right auricle place clip, carries out intracardiac perfusion with the physiological saline solution of pre-cooling, rinses liver until become canescence; Be separated liver, remove connective tissue and gallbladder, be cut into small pieces hepatic tissue shape, gently rubbed 200 order steel sieves with the hands, and obtained single cell suspension with normal saline flushing with ophthalmic tweezers.Single cell suspension is crossed 300 order nylon wires, through the centrifugal 4min of 1000rpm, remove supernatant.Precipitation 1ml0.5% trypsin 0.5g pancreatin is dissolved in 100mlHanks liquid) resuspended, add hyclone after digestion 2min and stop digestion, with 1000rpm, centrifugal 4min.Remove supernatant, add the resuspended precipitation of 5ml normal saline, precipitate and to be resuspended in after repeatedly washing 2 times in normal saline and to adjust cell concentration to 1 × 10
6~ 1 × 10
7/ ml, obtains the single cell suspension of liver.
2, hepatocyte dyeing and mensuration
Get single cell suspension 1ml prepared by step 1, the centrifugal 4min of 1000rpm, removes supernatant, obtains precipitation.Get the PI dye liquor that 1ml prepares, resuspended precipitation, 4 DEG C of lucifuges place dyeing 30min, and proceed in streaming pipe, flow cytometer measures apoptosis and cell cycle.
Normal group, ConA model group, the percentage of cerebral apoptosis of dexamethasone positive controls and DRC3f each dosage group mice and cell cycle are respectively as shown in Figure 2 A and 2B.
In Fig. 2, * * * represents P<0.001, represents compared with ConA model group.
Fig. 2 A shows, only there is the hepatocellular apoptosis of (5.32 ± 0.58) % in Normal group mice, and the hepatocyte ratio of ConA model group mouse apoptosis is (30.25 ± 1.29) %, compared with Normal group, ConA model group mouse liver cell apoptosis showed increased (P<0.001); Compared with ConA model group, dexamethasone positive controls and DRC3f tetra-dosage groups all significantly can improve the hepatocellular apoptosis situation (being P<0.001) of being induced by ConA.
Fig. 2 B shows, the hepatocyte of Normal group mice almost remains static, and after ConA stimulates, hepatocyte starts division, and G2/M phase cell percentages increases; DRC3f pretreated group mice (DRC3f1, DRC3f2, DRC3f3 and DRC3f4 group) the G2/M phase hepatocyte percentage ratio of various dose increases (P<0.01) further, G1 phase cell percentages declines (P<0.05), significant difference is had, S phase percentage ratio no significant difference compared with ConA model group compared with ConA model group.DRC3f1 group is the equal not statistically significant of each phase cell percentages difference compared with model group; Compared with ConA model group, dexamethasone positive controls G1 phase cell percentages showed increased, S phase and G2/M phase cell percentages all decline (P<0.01).
Fig. 2 shows, DRC3f pretreatment can suppress hepatocellular apoptosis, also can promote that hepatocyte transformed to G2/M division stage, namely promote hepatocyte growth.
3, each lymphocyte of Flow Cytometry Assay hepatic tissue (T cell, NK cell, NKT cell) infiltrates ratio
1) hepatic tissue separation of lymphocytes
Giving ConA stimulate after 12h put to death mice, the process of liver and hepatic tissue is separated with the hepatocyte of step 1, then the shape that is cut into small pieces by hepatic tissue, gently rubs 200 order steel with the hands and sieves, and obtained single cell suspension with normal saline flushing with ophthalmic tweezers.Single cell suspension is crossed 300 order nylon wires, through the centrifugal 4min of 1000rpm, remove supernatant.Precipitation is resuspended in 2ml physiological saline solution, is carefully added on 2ml mouse lymphocyte separating medium liquid level, with 2000rpm, centrifugal 15min.In careful collection pipe, canescence intermediate layer is in new pipe, adds physiological saline solution that about tetraploid is long-pending and blows and beats with 2000rpm after mixing gently, centrifugal 20min.Remove supernatant, precipitate and be resuspended in 300 μ l normal saline after physiological saline solution washes 2 times repeatedly, obtain lymphocyte suspension.
2) lymphocyte antibody is hatched and mensuration
Lymphocyte suspension step 1) obtained moves in streaming pipe, each sample two is managed, FITC labelling anti-mouse CD3e antibody and PE labelling anti-mouse DX5 antibody is added wherein in a pipe, add corresponding isotype control Ab in another pipe and (be specially the IgG1 isotype control Ab of FITC labelling and the IgM isotype control Ab of PE labelling, equal purchased from American BD company), room temperature lucifuge hatches 30min, according to each lymphocytic cell surface with antigen and the interaction of fluorescently-labeled antibody obtain the good lymphocyte to be measured of labelling, proceeded to streaming pipe again, flow cytomery.
Result as shown in Figure 3.
In Fig. 3, * * represents P<0.01, and * * * represents P<0.001, all represents compared with ConA model group; + represent P<0.05, ++ represent P<0.01, +++ represent P<0.001, represent compared with dexamethasone positive controls.
T cell and NKT cells into hepatocytes infiltrate and play crucial effect in the hepatic injury of ConA induction, and Fig. 3 shows, Normal group mouse liver cell only has a small amount of lymphocyte.Compared with Normal group mice, ConA model group mice is after ConA stimulates, and T cell and NKT cell percentage increase.DRC3f2, DRC3f3 group significantly can reduce T cell and NKT cell proportion (compared with ConA model group, P is all less than 0.01).The result of dexamethasone positive controls is similar with the result of DRC3f3 group (compared with Con model group, P<0.001).
(6) survival rate experiment
1, experiment grouping
30 male Balb/c inbred mouses (6-8 week, 18-22g) are divided into 3 groups, i.e. ConA model group, dexamethasone positive controls and DRC3f processed group at random, often organize 10.
2, in ConA injection (the ConA25mg/kg body weight of tail vein injection fatal dose, volume is 0.25ml) front 1h, give ConA model group, dexamethasone positive controls and DRC3f processed group mouse tail vein injection 0.25ml normal saline, dexamethasone injection (dexamethasone 500 μ g/kg body weight respectively, volume is 0.25ml) and DRC3f injection (DRC3f800 μ g/kg body weight, volume is 0.25ml).
Observe the Survival within 72h after each group of injected in mice ConA, once, then calculate the survival rate of each group of mice respectively, result as shown in Figure 4 for every 1h record.
In Fig. 4, * represents P<0.05, and * * represents P<0.01, all represents compared with ConA model group.
Fig. 4 shows, ConA model group mice 72h survival rate is 20%, and median survival time is 11h; And dexamethasone positive controls and DRC3f processed group are carrying out the Survival that all significantly can improve mice under dexamethasone preventive administration and DRC3f preventive administration condition respectively, survival rate be respectively 80% and 70%(be respectively P<0.004 and P<0.01), median survival time is 72h.
Two, therapeutical effect experiment
(1) experiment grouping
32 male Balb/c inbred mouses (6-8 week, 18-22g) are divided into 4 groups, i.e. Normal group, ConA model group, dexamethasone positive controls and DRC3f3 group at random, often organize 8.
(2) ConA model group, dexamethasone positive controls and DRC3f3 group mouse tail vein injection ConA injection (ConA12mg/kg body weight is given, volume is 0.25ml) after 1h, give dexamethasone positive controls mouse tail vein injection dexamethasone injection (dexamethasone 500 μ g/kg body weight respectively, volume is 0.25ml), DRC3f3 group mouse tail vein injection DRC3f injection (DRC3f800 μ g/kg body weight, volume is 0.25ml), ConA model group mouse tail vein injection 0.25ml normal saline.
Normal group: mice gives tail vein injection 0.25ml normal saline twice, for getting rid of time effects, the time of double injection normal saline is identical with the above-mentioned inject time that other are organized.
After ConA stimulates, 12h puts to death each group of mice, and collection blood and hepatic tissue carry out follow-up index determining.
(3) mensuration of mice serum ALT and AST level is with (three) in step one
Give the measurement result of the rear 12h mice serum ALT of ConA stimulation and AST level as shown in Figure 5A.
In Fig. 5 A, * * * represents P<0.001, represents compared with ConA model group; + represent P<0.05, represent compared with dexamethasone positive controls.
Fig. 5 A shows, after ConA stimulates, ConA model group mice serum ALT and AST level are significantly higher than Normal group; Compared with ConA model group mice, DRC3f3 group give mice 800 μ g/kg body weight DRC3f treatment can significantly reduce in Serum ALT and AST level (being P<0.001); Equally, the experimental result of dexamethasone positive controls similar to the experimental result of DRC3f3 group (being P<0.001).
(4) hepatic tissue paraffin embedding and HE dyeing are as (four) in step one
Give ConA and stimulate rear 12h murine liver tissue paraffin embedding and HE coloration result as shown in Figure 5 B.
In Fig. 5 B, a represents Normal group, and b represents ConA model group, and c represents dexamethasone positive controls, and d represents DRC3f3 group.What arrow indicated is necrotic area, Scalebar, 50 μm.
Fig. 5 B shows, the visible large-area hepatic necrosis of ConA model group murine liver tissue; Compared with ConA model group mice, dexamethasone positive controls and DRC3f3 group significantly can reduce the hepatic necrosis of ConA induction.
(5) survival rate experiment
1, experiment grouping
30 male Balb/c inbred mouses are divided into 3 groups at random, i.e. ConA model group, dexamethasone positive controls and DRC3f processed group, often organize 10.
2, in ConA injection (the ConA25mg/kg body weight of tail vein injection fatal dose, volume is 0.25ml) after 30min, give ConA model group, dexamethasone positive controls and DRC3f processed group mouse tail vein injection 0.25ml normal saline, dexamethasone injection (dexamethasone 500 μ g/kg body weight respectively, volume is 0.25ml) and DRC3f injection (DRC3f800 μ g/kg body weight, volume is 0.25ml).
Observe the Survival within 72h after each group of injected in mice ConA, once, then calculate the survival rate of each group of mice respectively, result as shown in Figure 6 for every 1h record.
In Fig. 6, * represents P<0.05, represents compared with ConA model group.
Fig. 6 shows, ConA model group mice 72h survival rate is 20%; Median survival time is 13h; DRC3f processed group mouse survival rate is increased to 40%, and median survival time is 37h, and compared with ConA model group, difference has statistical significance, P=0.029; Dexamethasone positive controls mouse survival rate is increased to 60%, and median survival time is 72h, and compared with ConA model group, difference has statistical significance, P=0.011; DRC3f processed group compared with dexamethasone positive controls, no significant difference, P=0.507.DRC3f and dexamethasone all can improve the survival rate of injection fatal dose ConA mice, extend life span, but all comparatively preventive effect are slightly poor for therapeutic effect.
Embodiment 2, de-arginine complement component 3 f(DRC3f) treating the application in chronic hepatic injury
SNMC, be a kind of common medicine of clinical treatment fatty liver, it can protect liver plasma membrane, is reached the effect of antiinflammatory protection liver plasma membrane by the activity of inhibition of phospholipase A 2; The effect of steroid sample can be played simultaneously, there is the effects such as antiinflammatory, antiallergic action and the immunomodulating of steroid sample.Meanwhile it also has the effect of NK cell activation, the outer T lymphocyte differentiation potentiation of thymus, has inhibition to fibrosis in chronic liver disease and canceration.
Another kind of positive drug blood fat reducing channel activating soft capsule, main component is Turmeric, it has the effect of blood circulation promoting and blood stasis dispelling promoting the circulation of QI to relieve pain, find to confirm for dyslipidemia study on prevention according to Chinese Journal of Cardiology editorial board dyslipidemia Preventing Countermeasures special topic group, its effect for reducing blood fat mechanism with promotion gallbladder to Cholesterol Excretion with to suppress fatty acid to synthesize relevant, may have certain repair to medicamentous liver lesion.
One, the quick foundation of fatty liver model of rats
Male SD rat (180-200g) is cultivated one week at Animal House adaptability after buying, and weighs rat body weight, every 3 days according to 3ml/kg lumbar injection 10%CCl
4(olive oil solution of the carbon tetrachloride of volumn concentration 10%) totally 4 weeks, simultaneously high lipid food feeding 4 weeks.
Two, experiment grouping and processing mode
Normal group: male SD rat is cultivated one week at Animal House adaptability after buying, chow diet raising+physiological saline 10 weeks (weekly 3 times).
Models of Fatty Liver rat step one built is divided into following group at random and accepts following process:
DRC3f low dose group (200 μ g/kg): according to rat body weight tail vein injection DRC3f(200 μ g/kg, dosage 2ml/kg. time), 3 times weekly, treat 6 weeks;
Dosage group (400 μ g/kg) in DRC3f: according to rat body weight tail vein injection DRC3f(400 μ g/kg, dosage 2ml/kg. time), 3 times weekly, treat 6 weeks;
DRC3f high dose group (800 μ g/kg): according to rat body weight tail vein injection DRC3f(800 μ g/kg, dosage 2ml/kg. time), 3 times weekly, treat 6 weeks;
Western medicine group (SNMC): SNMC diluent gavage treatment (SNMC normal saline dilution to 60mg/ml, dosage 2ml/kg. time), secondary on every Wendesdays, treat 6 weeks;
Chinese drug-treated group (blood fat reducing channel activating soft capsule): blood fat reducing channel activating soft capsule diluent gavage treatment (blood fat reducing channel activating soft capsule normal saline dilution to 40mg/ml, dosage 2ml/kg. time), secondary on every Wendesdays, treat 6 weeks;
Model group: give rat normal saline (dosage 2ml/kg. time) treatment 6 weeks at corresponding administration time point respectively by tail vein.
Above-mentioned experiment, the basic, normal, high dosage group of DRC3f and model group often organize 8, and Western medicine group, Chinese drug-treated group often organize 12, and each group administration time is all consistent.
Three, serological index detects
By after Rat Fast 12h after last therapeutic, cut open and kill rat collection blood, separation of serum application CHEMIX-180 automatic clinical chemistry analyzer (purchased from Japanese Sysmex company) detects the situation of change of the index such as liver function (Serum ALT, AST are active, serum total bilirubin TBIL level), serum triglycerides (TG), serum total cholesterol (CHOL).
The Serum ALT testing result of each group of rat as shown in Figure 7.
In Fig. 7, * represents P<0.05, and * * represents P<0.01, all compared with model group.
Fig. 7 shows, DRC3f low dose group, DRC3f high dose group, Western medicine group, Chinese drug-treated group and normal group Serum ALT levels are all remarkable in model group; Dosage group Serum ALT levels and model group no significant difference (P>0.05), 3 DRC3f dosage groups and Western medicine group no significant difference (P>0.05) in DRC3f.
The serum AST testing result of each group of rat as shown in Figure 8.
In Fig. 8, * represents P<0.05, and * * represents P<0.01, all compared with model group; ▲ represent p<0.05, compared with Western medicine group.
Fig. 8 shows, DRC3f low dose group, DRC3f high dose group and normal group serum AST levels are all remarkable in model group (P<0.05); Dosage group, Western medicine group, Chinese drug-treated group serum AST levels and model group no significant difference (P>0.05) in DRC3f, the serum AST levels of DRC3f high dose group and Western medicine group have notable difference (P<0.05).
The serum T BIL testing result of each group of rat as shown in Figure 9.
Fig. 9 shows, dosage group, DRC3f high dose group, Western medicine group, Chinese drug-treated group and normal group serum T BIL level and model group no significant difference (P>0.05) in DRC3f low dose group, DRC3f.The serum T BIL level of 3 DRC3f dosage groups and Western medicine group no significant difference (P>0.05).
The change of serum C HOL testing result of each group of rat as shown in Figure 10.
In Figure 10, * * represents P<0.01, compared with model group.▲ ▲ represent p<0.01, compared with Western medicine group.
Figure 10 shows, dosage group in DRC3f low dose group, DRC3f, DRC3f high dose group change of serum C HOL level are all remarkable in model group (P<0.01); Western medicine group, Chinese drug-treated group and normal group change of serum C HOL level and model group no significant difference (P>0.05).In DRC3f, the change of serum C HOL level of dosage group, DRC3f high dose group and Western medicine group have notable difference (P<0.01).
The serum TG testing result of each group of rat as shown in figure 11.
In Figure 11, * * represents P<0.01, compared with model group.▲ ▲ represent p<0.01, compared with Western medicine group.
Figure 11 shows, DRC3f low dose group, Western medicine group, Chinese drug-treated group, normal group serum TG levels are all remarkable in model group (P<0.01); Dosage group, DRC3f high dose group serum TG levels and model group no significant difference (P>0.05) in DRC3f.In DRC3f, the serum TG levels of dosage group, DRC3f high dose group is higher than Western medicine group, has notable difference (P<0.01).
Four, pathology detect
By after Rat Fast 12h after last therapeutic, cut open and kill rat, each group rat gets a fritter hepatic tissue in the same position of leftlobe of liver, and the paraformaldehyde of 4% is fixed, and conventional dehydration, paraffin embedding, section, carry out HE dyeing.
Each group of rat be organized in light Microscopic observation, the steatosis degree of assessment liver:
0 grade (the fat-free degeneration of hepatocyte);
1 grade (< 25% hepatic cell fattydegeneration);
2 grades (25% ~ 50% hepatic cell fattydegeneration);
3 grades (50% ~ 75% hepatic cell fattydegeneration);
4 grades (> 75% hepatic cell fattydegeneration);
HE coloration result as shown in figure 12.
Figure 12 shows, lobules of liver clear in structure in normal rats hepatic tissue, and hepatocyte, around central vein and portal area ordered arrangement, forms liver rope, steatosis degree 0 grade; Large stretch of fat vacuole is there is, > 75% hepatic cell fattydegeneration, steatosis degree 4 grades in model group rats hepatic tissue; Dosage group, Chinese drug-treated group in DRC3f low dose group, DRC3f, 50% ~ 75% hepatic cell fattydegeneration, steatosis degree 3 grades; Western medicine group, DRc3f high dose group 25% ~ 50% hepatic cell fattydegeneration, steatosis degree 2 grades.
Five, hepatic tissue frozen section and Sudan stains
(1) get a fritter hepatic tissue at the same position of hypomere outside each group of rats'liver lobus sinister, loose hepatic tissue volume is 24 × 24 × 2mm.
(2) supporter is organized in taking-up, and set level and set hepatic tissue, periphery drips embedding medium, and speed is put on freezing stage, freezing.
(3) hepatic tissue blocking will freezed, is clamped in microtome and holds and hold on device, and start key of slightly retreating, turning knob, will organize equating.
(4) mixing up the thickness for cutting, determining according to different tissues, be intensive thinly-sliced of cell in principle, rare can thickly a little the cutting of fiber many cells, generally between 5 ~ 10mm.
(5) mix up anti-roll bending, make frozen section, mix up anti-roll bending exactly, adjustment is to suitable position.During section, the section cut out can be smoothly through passage between the anti-roll bending of cutter in the very first time, entirely lies on the iron plate of mes holder.At this moment just can start anti-roll bending, get a microscope slide, it is affixed upper.
(6) the freezing degree that organizational choice is different should be looked different.The height of freezing degree in household freezer, mainly determines according to different tissues, cannot treat different things as the same, when cutting lipoferous organizing, should be adjusted to about-25 DEG C, cut containing a large amount of fatty time, should-30 DEG C be adjusted to.Sudan stains is carried out after section.
Result as shown in figure 13.
Figure 13 shows, normal group hepatic tissue minute quantity fat, and free fat content is less; There is a large amount of free-fat model group hepatic tissue portal area; In DRC3f low dose group, DRC3f, dosage group still has more free-fat tissue, but reduces to some extent relative to model group; Western medicine group, Chinese drug-treated group, DRC3f high dose group free fat content decline fairly obvious relative to model group.
Above result shows, DRC3f high dose can significantly reduce ALT, AST, CHOL level, and HE dyeing and fatty specific stain result display DRC3f high dose also obviously can reduce the content of hepatic steatosis degree and liver free-fat.
Claims (5)
1. the application in de-arginine complement component 3 f (DRC3f) product that in serum, glutamate pyruvate transaminase and/or glutamic oxaloacetic transaminase, GOT level rise when preparing suppression hepatic injury; The aminoacid sequence of described de-arginine complement component 3 f (DRC3f) is as shown in SEQIDNo.1.
2. the application in de-arginine complement component 3 f (DRC3f) product that in serum, serum total cholesterol and/or serum triglyceride level rise when preparing suppression hepatic injury.
3. application according to claim 2, is characterized in that: described hepatic injury is chronic hepatic injury;
Described chronic hepatic injury is fatty liver.
4. de-arginine complement component 3 f (DRC3f) suppresses the application in the product of hepatic cell fattydegeneration in preparation.
5. de-arginine complement component 3 f (DRC3f) suppresses the application in the product of the content rising of hepatic tissue Free Fat in preparation.
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| DRC3f及eRF3b在肝病发生发展中作用机制的初步研究;李曼;《中国博士学位论文全文数据库》;20111015(第10期);全文 * |
| 乙型病毒性肝炎及相关疾病蛋白质组学及临床诊断的研究;王剑;《中国博士学位论文全文数据库》;20091015(第10期);全文 * |
| 慢性乙型肝炎血清差异性多肽DRC3f的鉴定及作用研究;李曼 等;《卫生研究》;20110531;第40卷(第3期);315-319 * |
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