CN113750258A - Novel method for treating hepatic fibrosis diseases by pirfenidone - Google Patents
Novel method for treating hepatic fibrosis diseases by pirfenidone Download PDFInfo
- Publication number
- CN113750258A CN113750258A CN202010521441.1A CN202010521441A CN113750258A CN 113750258 A CN113750258 A CN 113750258A CN 202010521441 A CN202010521441 A CN 202010521441A CN 113750258 A CN113750258 A CN 113750258A
- Authority
- CN
- China
- Prior art keywords
- pirfenidone
- mouse
- hepatic fibrosis
- fibrosis
- mice
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- ISWRGOKTTBVCFA-UHFFFAOYSA-N pirfenidone Chemical compound C1=C(C)C=CC(=O)N1C1=CC=CC=C1 ISWRGOKTTBVCFA-UHFFFAOYSA-N 0.000 title claims abstract description 43
- 229960003073 pirfenidone Drugs 0.000 title claims abstract description 43
- 206010019668 Hepatic fibrosis Diseases 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title description 7
- 208000019425 cirrhosis of liver Diseases 0.000 claims abstract description 18
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims abstract description 18
- 230000000694 effects Effects 0.000 claims abstract description 10
- 230000014509 gene expression Effects 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 238000010172 mouse model Methods 0.000 claims abstract description 6
- 230000007246 mechanism Effects 0.000 claims abstract description 4
- 210000005229 liver cell Anatomy 0.000 claims abstract description 3
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 10
- 229920002674 hyaluronan Polymers 0.000 claims description 10
- 229960003160 hyaluronic acid Drugs 0.000 claims description 10
- 102000008186 Collagen Human genes 0.000 claims description 8
- 108010035532 Collagen Proteins 0.000 claims description 8
- 229920001436 collagen Polymers 0.000 claims description 8
- 238000002474 experimental method Methods 0.000 claims description 7
- 210000002966 serum Anatomy 0.000 claims description 7
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 6
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 6
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 claims description 5
- 108010082126 Alanine transaminase Proteins 0.000 claims description 5
- 238000012795 verification Methods 0.000 claims description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims description 2
- 108090000340 Transaminases Proteins 0.000 claims description 2
- 230000009471 action Effects 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 230000011382 collagen catabolic process Effects 0.000 claims description 2
- 210000002744 extracellular matrix Anatomy 0.000 claims description 2
- 239000000835 fiber Substances 0.000 claims description 2
- 210000004024 hepatic stellate cell Anatomy 0.000 claims description 2
- 210000003494 hepatocyte Anatomy 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 102000014898 transaminase activity proteins Human genes 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 2
- 238000010171 animal model Methods 0.000 claims 1
- 230000000903 blocking effect Effects 0.000 claims 1
- 230000020411 cell activation Effects 0.000 claims 1
- 230000028709 inflammatory response Effects 0.000 claims 1
- 239000002547 new drug Substances 0.000 claims 1
- 230000001737 promoting effect Effects 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 abstract description 15
- 239000007928 intraperitoneal injection Substances 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 239000000243 solution Substances 0.000 abstract description 4
- 238000003304 gavage Methods 0.000 abstract description 3
- 230000001575 pathological effect Effects 0.000 abstract description 2
- 235000012424 soybean oil Nutrition 0.000 abstract description 2
- 239000003549 soybean oil Substances 0.000 abstract description 2
- 108091092878 Microsatellite Proteins 0.000 abstract 1
- 230000001225 therapeutic effect Effects 0.000 abstract 1
- 210000005228 liver tissue Anatomy 0.000 description 6
- 206010016654 Fibrosis Diseases 0.000 description 4
- 230000007882 cirrhosis Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 3
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- SNBCLPGEMZEWLU-QXFUBDJGSA-N 2-chloro-n-[[(2r,3s,5r)-3-hydroxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methyl]acetamide Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CNC(=O)CCl)[C@@H](O)C1 SNBCLPGEMZEWLU-QXFUBDJGSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108010009583 Transforming Growth Factors Proteins 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000002300 anti-fibrosis Effects 0.000 description 1
- 230000003510 anti-fibrotic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- 230000005182 global health Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002206 pro-fibrotic effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 208000005069 pulmonary fibrosis Diseases 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- -1 small molecule compound Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Environmental Sciences (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Toxicology (AREA)
- Diabetes (AREA)
- Endocrinology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Rheumatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Unlike other previous studies, the present study discusses the therapeutic mechanism of Pirfenidone (PFD) on carbon tetrachloride-induced liver fibrosis in mice. A CCL4 liver fibrosis pathological model group is prepared by taking 40 healthy male SPF-grade ICR mice of 8 weeks as experimental objects, performing intraperitoneal injection on the mice by adopting a CCL4 soybean oil solution of 0.4 ml/10 percent, performing intragastric gavage on the mice by using low-dose and high-dose pirfenidone, analyzing the expression condition of alpha-SMA (short tandem repeat) related genes of liver cells of the mice, and discussing the influence mechanism of the pirfenidone on the liver fibrosis. At present, a lot of reports are made abroad, and the influence of the reports on the hepatic fibrosis is still lack of enough research. The research discusses the treatment effect of pirfenidone on hepatic fibrosis by using a hepatic fibrosis mouse model caused by CCL4, and provides a new direction for PFD treatment of early intervention of hepatic fibrosis.
Description
Technical Field
The field of the treatment of hepatic fibrosis related diseases by Pirfenidone (PFD), in particular to the verification of the feasibility of the treatment of the hepatic fibrosis diseases induced by carbon tetrachloride by pirfenidone.
Background
Research has shown that cirrhosis has become a serious endocrine-metabolic disease threatening human health. Cirrhosis is a 14 th adult lethal disease worldwide, has a very high risk of developing liver cancer, and is a major global health problem. Liver fibrosis is the common pathological basis and inevitable stage of cirrhosis and even liver failure. Prevention and early intervention on hepatic fibrosis are the best measures for stabilizing the disease condition and preventing the hepatic fibrosis from developing into cirrhosis and liver cancer. To successfully reverse liver fibrosis, excessive collagen deposition, excessive scar degradation and recovery of damaged liver tissue to normal are degraded. Pirfenidone has been shown to have some effect in anti-fibrosis.
Pirfenidone (PFD), a novel oral small molecule compound, chemically named 5-methyl-1-phenyl-2- (1 hydro) -pyridone, has anti-fibrotic, anti-inflammatory and anti-oxidative effects. In vitro and animal experiments, PFD is able to inhibit pro-fibrotic and pro-inflammatory cytokines, including transforming growth factor and tumor necrosis factor, acting to inhibit fibroblast proliferation and collagen deposition. Clinical tests show that the traditional Chinese medicine composition plays a certain role in inhibiting fibrosis of lung, heart, kidney and the like.
Pirfenidone (PFD) has the effect of remarkably reducing the hepatic fibrosis of carbon tetrachloride-induced hepatic fibrosis mice. The inhibition of the alpha-SMA related gene in liver is determined, and then the influence of hepatic fibrosis indexes HA (hyaluronic acid), LN (laminin) and IV-C (IV type collagen) is detected by an enzyme-linked reaction method, so that a theoretical basis is provided for the treatment of hepatic fibrosis.
At present, the effect and the action mechanism of pirfenidone are proved at home and abroad that pirfenidone can inhibit HSC activation through targeting, so that the expression level of alpha-SMA gene is obviously reduced, HSC expression is inhibited, ECM generation is reduced, and the effect of resisting hepatic fibrosis is achieved. Healthy male SPF-grade ICR mice are divided into a liver fibrosis model group (CCL4 group), a pirfenidone low dose group (PFD-L group), a pirfenidone high dose group (PFD-H group) and a normal control group, the stomach is irrigated with 120mg/kg and 240mg/kg Pirfenidone (PFD), and the improvement effect of pirfenidone on liver fibrosis is verified by detecting ALT (glutamic pyruvic transaminase), AST (glutamic oxalacetic transaminase), ALP (alkaline phosphatase) and liver fibrosis indexes HA (hyaluronic acid), LN (laminin) and IV-C (IV collagen) in serum. Pirfenidone remarkably reduces the content of HA, LN and IV-C in serum, reduces the expression level of genes encoding proinflammatory cytokines, protects liver cells, relieves inflammatory reaction, promotes collagen degradation to reduce the synthesis of extracellular matrix, reduces the expression of alpha-SMA genes, blocks the activation of hepatic stellate cells and inhibits the increase of collagen fibers, so that PFD treatment can provide a theoretical basis for the treatment of hepatic fibrosis.
Disclosure of Invention
In order to explore the problem of treatment of hepatic fibrosis related diseases by pirfenidone, the invention constructs a hepatic fibrosis mouse model in advance and further detects various indexes of the mouse. Histology level, comparing and observing the tissue characteristics of a common mouse control group (NC), a liver fibrosis model group (CCL4 group), a pirfenidone low dose group (PFD-L group) and a pirfenidone high dose group (PFD-H group); at the cellular level, pirfenidone improves hepatocyte sensitivity; performing biochemical detection on related indexes in serum of each group of mice at a molecular level, monitoring the content of each corresponding index in liver in an important way, and measuring the content of each group of mice; gene level, detecting the expression condition of alpha-SMA related genes in liver, and comprehensively detecting the influence of pirfenidone on hepatic fibrosis.
The invention researches an experiment on the treatment effect of pirfenidone by constructing a hepatic fibrosis mouse model. Provides a novel method for verification experiments of pirfenidone in hepatic fibrosis treatment, promotes the development of pirfenidone development and clinical treatment to a certain extent, and has good popularization and feasibility.
Detailed Description
1. Preparation and grouping processing of hepatic fibrosis mouse model and histological examination
The male SPF grade ICR mice (purchased from Shanghai laboratory animal center of Chinese academy of sciences) are 40 healthy at 8 weeks old, and the weight of the mice is 25-30 g. Adaptive feeding for one week. Mice were randomly divided into 4 groups: liver fibrosis model group (CCL4 group) (n ═ 10): intraperitoneal injection of 0.4 ml/10% carbon tetrachloride (CCL4) soybean oil solution for 1 time in 2 days for 42 days (6 weeks); pirfenidone low dose group (PFD-L group) (n ═ 10): gavage was given simultaneously with 120mg/kg PFD solution for 28 days (4 weeks) 1 time, except for intraperitoneal injection of CCL 4; pirfenidone high dose group (PFD-H group) (n ═ 10): gavage was given concurrently with 240mg/kg PFD solution for 28d (4 weeks) 1 day, except for intraperitoneal injection of CCL 4; normal control group (n ═ 10), normal feeding. Each group of mice was housed under standard laboratory conditions and had free access to food and water. Each group of mice was sacrificed 42 days after model creation, serum was retained, and liver tissues were rapidly collected and stored at-80 ℃ for real-time fluorescent quantitative PCR detection. Fixing liver tissues by 4% paraformaldehyde, performing gradient dehydration, performing transparency in xylene, soaking in wax, performing paraffin embedding treatment, preparing into 3 μm slices, staining with hematoxylin-eosin, observing with ECTIPSE50I microscopic image processing system, collecting pictures, and comparing morphological changes of liver tissues.
2. Detecting related indexes by using a full-automatic biochemical analyzer:
(1) indices of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and alkaline phosphatase (ALP) in each group in serum.
3. Liver fibrosis related indexes HA (hyaluronic acid), LN (laminin) and IV-C (IV collagen) content determination:
the mice are taken blood and then are stood for 1h at room temperature, 3000 r.min < -1 >, centrifuged for 15min, and serum is separated. Detecting hepatic fibrosis indexes HA, LN and IV-C by using a radioimmunoassay method, and strictly operating according to the instruction of the kit.
℃15s。
4. Determination of liver tissue alpha-SMA Gene expression:
real-time fluorescent quantitative PCR method is used. The total RNA of the liver tissue of the mouse is extracted by a Trizol one-step method and is reversely transcribed to synthesize cDNA, and the operation is carried out according to the instruction of a kit. Real-time quantitative amplification is carried out on a real-time fluorescence quantitative PCR instrument, beta-actin is used as an internal reference, and an alpha-SMA primer is synthesized by a biological engineering (Shanghai) corporation. alpha-SMA upstream primer: 5'-AGC GGG CAT CCA CGA AAC-3', respectively; a downstream primer: 5'-TGA TCT TCA TGG TGC TGG GTG-3' are provided. Beta-actin upstream primer: 5'-AAC AGT CCG CCT AGA AGC AC-3', respectively; a downstream primer: 5'-CAT TGA CAT CCG TAA AGA CC-3' are provided. The Real-time PCR reaction system is 20 ul, wherein the cDNA is 1 ul, SYBR Green10 ul, the upstream primer is 0.5 ul, the downstream primer is 0.5 ul, the ddH2O8 ul, the amplification program is pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 10s, annealing at 55 ℃ for 20s, extension at 72 ℃ for 30s, and the reaction is carried out for 45 cycles. Extension at 72 ℃ for 5 min. Data analysis was performed using SDS software, and results were analyzed by comparing Ct values (cycle threshold).
Description of the drawings: FIG. 1 is a circuit diagram of a hepatic fibrosis mouse model constructed in advance and used for monitoring various corresponding indexes in the liver.
Claims (4)
1. A novel mouse experiment mode is to use carbon tetrachloride to induce mouse hepatic fibrosis animal model to treat pirfenidone.
2. The novel mouse experimental method of claim 1, wherein in the experiment for confirming the effect and action mechanism of pirfenidone on liver fibrosis, a liver fibrosis mouse model is constructed in advance, and further, various indexes of the mouse are detected. Measuring the expression of the mouse hepatocyte alpha-SMA related gene; the influence of pirfenidone on hepatic fibrosis is comprehensively detected by ALT (glutamic-pyruvic transaminase), AST (glutamic-oxalacetic transaminase), ALP (alkaline phosphatase), HA (hyaluronic acid), LN (laminin) and IV-C (collagen IV) in serum, and the pirfenidone HAs obvious effects on protecting liver cells, relieving inflammatory response, promoting collagen degradation to reduce synthesis of extracellular matrix, reducing alpha-SMA gene expression, blocking hepatic stellate cell activation and inhibiting collagen fiber increase.
3. The novel mouse assay of claim 1, wherein the assay is performed on a mouse at a histological level and a molecular level.
4. The novel mouse experimental mode of claim 1 provides a novel and simple method for verification experiment of the curative effect of pirfenidone, which suggests that PFD can become a new drug for early prevention and treatment of liver fibrosis, and has good popularization and feasibility.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010521441.1A CN113750258A (en) | 2020-06-02 | 2020-06-02 | Novel method for treating hepatic fibrosis diseases by pirfenidone |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010521441.1A CN113750258A (en) | 2020-06-02 | 2020-06-02 | Novel method for treating hepatic fibrosis diseases by pirfenidone |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113750258A true CN113750258A (en) | 2021-12-07 |
Family
ID=78785377
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010521441.1A Withdrawn CN113750258A (en) | 2020-06-02 | 2020-06-02 | Novel method for treating hepatic fibrosis diseases by pirfenidone |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113750258A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103550242A (en) * | 2013-11-22 | 2014-02-05 | 四川国康药业有限公司 | Pharmaceutical composition for treating hepatic fibrosis and preparation method thereof |
-
2020
- 2020-06-02 CN CN202010521441.1A patent/CN113750258A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103550242A (en) * | 2013-11-22 | 2014-02-05 | 四川国康药业有限公司 | Pharmaceutical composition for treating hepatic fibrosis and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 肖敏 等: "吡非尼酮对四氯化碳诱导的小鼠肝纤维化的影响", 《中国应用生理学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11135160B2 (en) | Use of chlorogenic acid in preparing pharmaceuticals for treatment of LAG-3-mediated disease | |
| CA3112695C (en) | Composition for treating fibrotic diseases, comprising benzhydryl thioacetamide compound as active ingredient | |
| CN113616644B (en) | Application of RNA helicase DHX33 inhibitor in preparation of drugs for treating leukemia | |
| JP2023033425A (en) | Novel spiro and cyclic bis-benzylidine proteasome inhibitor for treatment of cancer, diabetes and neurological disorders | |
| US20220008420A1 (en) | Calmodulin inhibitors, chk2 inhibitors and rsk inhibitors for the treatment of ribosomal disorders and ribosomapathies | |
| KR102277739B1 (en) | A composition for treating fibrosis diseases comprising benzhydrylthio acetamide compounds as an active ingredient | |
| US10744114B2 (en) | Use of eupatilin as pharmaceutical composition for prevention and treatment of fibrosis using EMT inhibitory activity | |
| CN113750258A (en) | Novel method for treating hepatic fibrosis diseases by pirfenidone | |
| CN106390123B (en) | MiR-29 and its inhibitor are preparing the application in anti-organ-graft refection's drug | |
| Zhao et al. | Huaier extract attenuates acute kidney injury to chronic kidney disease transition by inhibiting endoplasmic reticulum stress and apoptosis via miR‐1271 upregulation | |
| CN111170980B (en) | Calycosin derivative and synthesis method and application thereof | |
| CN112522263A (en) | siRNA for treating hepatic fibrosis and delivery preparation thereof | |
| CN109953989B (en) | Pharmaceutical use of 2- (4-piperidylstyrene) -1,3, 3-trimethyl-3H-indolium iodide | |
| Allison | A single-cell, 2D atlas of the normal human kidney using imaging mass cytometry | |
| CN103417988A (en) | Application of CDK2 gene to preparation of leukemia induced differentiation therapeutic drug | |
| CN117224558B (en) | Application of RNA editing enzyme inhibitor in preparation of medicines for preventing and/or treating hepatic fibrosis | |
| CN114984220B (en) | Application of Mas receptor inhibitor in preparing medicine for preventing and treating acute liver failure | |
| CN109276567B (en) | Application of 7-hydroxycoumarin in preparation of medicine for treating acute kidney injury | |
| CN111973743B (en) | Application of targeted drug of RNA binding protein ZCCHC4 | |
| CN116751311A (en) | Polypeptide based on RIP3 methylation modification and application thereof in cell programmed necrosis related diseases | |
| CN108191875B (en) | Compound and application thereof in preparation of medicine for treating immune-related diseases | |
| CN117942346A (en) | Application of drospirenone in preparing medicine for preventing and treating fibrosis diseases | |
| Allison | ER calcium stabilizers in NS | |
| CN115990255A (en) | Application of METTL3 in preparation of medicine for preventing and treating radioactive lung injury | |
| WO2019199911A1 (en) | Photoswitchable dibenzothienylmethyl triphenylphosphonium derivatives and methods of treating cancer therewith |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20211207 |
|
| WW01 | Invention patent application withdrawn after publication |