CN115487302A - Application of Toll-like receptor 3 in treating clonorchis sinensis liver fibrosis - Google Patents
Application of Toll-like receptor 3 in treating clonorchis sinensis liver fibrosis Download PDFInfo
- Publication number
- CN115487302A CN115487302A CN202211399559.7A CN202211399559A CN115487302A CN 115487302 A CN115487302 A CN 115487302A CN 202211399559 A CN202211399559 A CN 202211399559A CN 115487302 A CN115487302 A CN 115487302A
- Authority
- CN
- China
- Prior art keywords
- clonorchis sinensis
- liver
- mice
- tlr3
- infection
- 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.)
- Pending
Links
- 241001327965 Clonorchis sinensis Species 0.000 title claims abstract description 87
- 102000008230 Toll-like receptor 3 Human genes 0.000 title claims abstract description 9
- 108010060885 Toll-like receptor 3 Proteins 0.000 title claims abstract description 9
- 208000019425 cirrhosis of liver Diseases 0.000 title abstract description 21
- 206010019668 Hepatic fibrosis Diseases 0.000 claims abstract description 35
- 206010009344 Clonorchiasis Diseases 0.000 claims abstract description 17
- 102000002689 Toll-like receptor Human genes 0.000 claims description 11
- 108020000411 Toll-like receptor Proteins 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 5
- 230000015788 innate immune response Effects 0.000 claims description 2
- 230000001404 mediated effect Effects 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims 1
- 229940079593 drug Drugs 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 abstract description 107
- 101000831496 Homo sapiens Toll-like receptor 3 Proteins 0.000 abstract description 81
- 102100024324 Toll-like receptor 3 Human genes 0.000 abstract description 81
- 210000004185 liver Anatomy 0.000 abstract description 76
- 210000000013 bile duct Anatomy 0.000 abstract description 29
- 230000003902 lesion Effects 0.000 abstract description 21
- 239000000556 agonist Substances 0.000 abstract description 16
- 210000000651 myofibroblast Anatomy 0.000 abstract description 15
- 241000238631 Hexapoda Species 0.000 abstract description 14
- 230000004913 activation Effects 0.000 abstract description 14
- 206010061218 Inflammation Diseases 0.000 abstract description 7
- 102000004889 Interleukin-6 Human genes 0.000 abstract description 7
- 108090001005 Interleukin-6 Proteins 0.000 abstract description 7
- 230000004054 inflammatory process Effects 0.000 abstract description 7
- 208000027418 Wounds and injury Diseases 0.000 abstract description 5
- 230000006378 damage Effects 0.000 abstract description 5
- 208000014674 injury Diseases 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 4
- 230000026731 phosphorylation Effects 0.000 abstract description 4
- 238000006366 phosphorylation reaction Methods 0.000 abstract description 4
- 208000006454 hepatitis Diseases 0.000 abstract description 3
- 208000018191 liver inflammation Diseases 0.000 abstract description 3
- 229960005486 vaccine Drugs 0.000 abstract description 3
- 239000002547 new drug Substances 0.000 abstract description 2
- 230000028327 secretion Effects 0.000 abstract description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 34
- 208000015181 infectious disease Diseases 0.000 description 30
- 102000008186 Collagen Human genes 0.000 description 19
- 108010035532 Collagen Proteins 0.000 description 19
- 229920001436 collagen Polymers 0.000 description 19
- 230000001965 increasing effect Effects 0.000 description 15
- 230000008021 deposition Effects 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 9
- 210000002919 epithelial cell Anatomy 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 230000002950 deficient Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 210000005228 liver tissue Anatomy 0.000 description 7
- 230000036285 pathological change Effects 0.000 description 7
- 231100000915 pathological change Toxicity 0.000 description 7
- 229940115272 polyinosinic:polycytidylic acid Drugs 0.000 description 7
- 230000037396 body weight Effects 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 238000010172 mouse model Methods 0.000 description 6
- 244000045947 parasite Species 0.000 description 6
- 241001327942 Clonorchis Species 0.000 description 5
- 201000000077 Cysticercosis Diseases 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000004043 dyeing Methods 0.000 description 5
- 239000000835 fiber Substances 0.000 description 5
- 230000001338 necrotic effect Effects 0.000 description 5
- 208000004441 taeniasis Diseases 0.000 description 5
- 241000018650 Pinus massoniana Species 0.000 description 4
- 210000000683 abdominal cavity Anatomy 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000003203 everyday effect Effects 0.000 description 4
- 206010020718 hyperplasia Diseases 0.000 description 4
- 238000003364 immunohistochemistry Methods 0.000 description 4
- 210000004969 inflammatory cell Anatomy 0.000 description 4
- 230000028709 inflammatory response Effects 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 206010016654 Fibrosis Diseases 0.000 description 3
- 102000004388 Interleukin-4 Human genes 0.000 description 3
- 108090000978 Interleukin-4 Proteins 0.000 description 3
- 235000011610 Pinus tabuliformis Nutrition 0.000 description 3
- 230000002354 daily effect Effects 0.000 description 3
- 230000004761 fibrosis Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 206010019842 Hepatomegaly Diseases 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000011888 autopsy Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 244000079386 endoparasite Species 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 206010004593 Bile duct cancer Diseases 0.000 description 1
- 206010008635 Cholestasis Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010019695 Hepatic neoplasm Diseases 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 235000011609 Pinus massoniana Nutrition 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 208000026900 bile duct neoplasm Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000006990 cholangiocarcinoma Diseases 0.000 description 1
- 231100000359 cholestasis Toxicity 0.000 description 1
- 230000007870 cholestasis Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 230000003176 fibrotic effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000003304 gavage Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000006951 hyperphosphorylation Effects 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 108091005434 innate immune receptors Proteins 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000024241 parasitism Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
- 206010048282 zoonosis Diseases 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/10—Anthelmintics
Abstract
The invention discloses application of Toll-like receptor 3 in treating clonorchis sinensis hepatic fibrosis, and relieving clonorchis sinensis hepatic fibrosis by using a TLR3 agonist. Experiments prove that the TLR3 high expression regulates the phosphorylation of the ERK/P38 signal channel of a host, and inhibits the secretion of IL-6, thereby reducing the liver inflammation injury caused by clonorchis sinensis infection and reducing the hepatic fibrosis; TTLR3 agonist treatment further proves that TLR3 high expression blocks clonorchis sinensis infection to cause death of mice, reduces the amount of liver charged insects, and lightens liver lesion, inflammation and bile duct lesion caused by clonorchis sinensis infection; reduce the activation quantity of liver myofibroblasts caused by the clonorchis sinensis and relieve the liver fibrosis. TLR3 has better application prospect as a target spot of developing new drugs and vaccines for treating and preventing clonorchis sinensis hepatic fibrosis in the future.
Description
Technical Field
The invention relates to a new medical application of an innate immune receptor Toll-like receptor 3 (TLR 3 for short), in particular discloses an application of Toll-like receptor 3 in treating clonorchis sinensis liver fibrosis, and specifically relates to an application of Toll-like receptor 3 and an agonist thereof in treating clonorchis sinensis liver fibrosis, belonging to the technical field of biomedical pharmacy.
Background
Clonorchis sinensis is prepared from clonorchis sinensis (Clonorchis sinensis)Clonorchis sinensis) An important food-borne zoonosis caused by parasitism in liver bile ducts of human and animals is closely related to liver fibrosis, liver cirrhosis and bile duct cancer. At present, no commercial vaccine for effectively preventing and controlling clonorchiasis sinensis exists, and no specific medicine for treating or reversing the hepatic fibrosis caused by clonorchiasis sinensis exists. Therefore, the pathogenic mechanism of clonorchis sinensis induced hepatic fibrosis is deeply known, and the development of the medicine for relieving clonorchis sinensis hepatic fibrosis is very important for the prevention and treatment of clonorchis sinensis diseases.
Disclosure of Invention
The invention aims to provide application of Toll-like receptor 3 in treating clonorchis sinensis hepatic fibrosis, novel application of TLR 3-mediated host innate immunity in resisting clonorchis sinensis infection, and verification of the effect of TLR3 agonist Poly (I: C) on treating clonorchis sinensis hepatic fibrosis by using TLR 3.
In order to achieve the purpose, the invention adopts the following technical scheme:
establishing a mouse model of hepatic fibrosis caused by clonorchis sinensis infection, detecting the survival rate, the weight and the number of parasites in the liver of the mouse, collecting the liver of the mouse 7 days, 15 days and 35 days after infection, detecting indexes such as liver lesion, bile duct injury, hepatic fibrosis change and the like of the infected mouse, and exploring the new effect of TLR3 in treating the clonorchis sinensis hepatic fibrosis. Based on the effect of TLR3 in clonorchis sinensis pathogenesis, the agonist Poly (I: C) of TLR3 is used for treating clonorchis sinensis infected WT mice, the indexes are detected, and the effect of TLR3 on treating clonorchis sinensis hepatic fibrosis is verified and discussed.
The TLR3 regulated immune response of the invention has therapeutic effect on hepatic fibrosis caused by clonorchis sinensis, and the steps are as follows:
1. establishment of clonorchis sinensis induced liver fibrosis mouse model
Wild Type (WT) C57BL/6 mice were divided into 6 groups of 10 mice, and given 0, 50, 100, 200, 400, 800 cysticercosis/mouse, respectively. Weighing and recording the weight and the death rate of the mice on the current day of insect attack and every day after the insect attack, respectively euthanizing the mice 7d, 15d and 35 d after the insect attack, and taking livers to carry out statistics on the amount of the live insects in the livers so as to determine the quantity of cysticercosis infection of the mice hepatic fibrosis caused by the clonorchis sinensis. As a result, after the C57BL/6 mice are infected with clonorchis sinensis by 200 cysticercus/mouse, the recovery rate of the intrahepatic adults of the mice averagely ranges from 13% to 15%, the infection rate is 100%, and the death rate of the mice averagely ranges from 40%. Obvious hepatic fibrosis symptoms appear 35 days after infection, and the liver fibrosis symptom can be used as the infection amount established by a mouse model for subsequently testing hepatic fibrosis caused by the clonorchis sinensis.
2. Detection of liver fibrosis condition of TLR 3-deficient mouse infected with clonorchis sinensis
Establishment of liver fibrosis WT and TLR3 deletion (TLR 3) caused by clonorchis sinensis -/- ) Mouse model, PBS treated mice as negative control. The body weight and mortality of the mice were weighed and recorded daily after infection and the mice were euthanized 7d, 15d, 35 d after infestation, respectively, livers were isolated and statistics for the amount of intrahepatic parasites were performed. Observation of clonorchis sinensis-infected WT and TLR3 by ELISA, western blot, pathological section, immunohistochemistry and Masson staining -/- Researches on condition of liver lesion and hepatic fibrosis of mice on TLR3 serving as a therapeutic target for treating clonorchis sinensis liver fibersThe possibility. Separated mouse bile duct epithelial cells are stimulated by the clonorchis sinensis exocrine products, and the mechanism of TLR3 for treating clonorchis sinensis hepatic fibrosis is explored. As a result, TLR3 inhibits the expression of IL-6 and reduces inflammatory response by regulating the phosphorylation of ERK/P38 protein. Clonorchis sinensis-infected TLR3 compared to WT-infected mice -/- The body weight of the mice is greatly reduced, the death rate is increased from 40 percent to 50 percent on average, and the recovery rate of the liver endoparasites is increased from 15 percent to 20 percent at most. TLR3 -/- After mice are infected with clonorchis sinensis, liver tissue is seriously injured, and intrahepatic inflammation is increased. The activation of the myofibroblasts around the bile duct is continuously increased, and the collagen deposition is more obvious; at infection 35 d, the area of the tract is completely encapsulated by the deposited collagen, and the collagen has already extended to the parenchyma of the liver, creating extensive fibrosis. TLR3 can be used as a target protein for treating clonorchis sinensis hepatic fibrosis.
3. Detection of liver fibrosis treatment condition of clonorchis sinensis infected WT mice by TLR3 agonist treatment
Establishing a mouse hepatic fibrosis model caused by clonorchis sinensis, injecting a TLR3 agonist Poly (I: C) solution into the abdominal cavity of a mouse according to the dose of 100 microgram/mouse 8 h before infecting cysticercus, and respectively administering equal dose of Poly (I: C) to the 10 th d and the 20 th d of the clonorchis sinensis infected mouse again for treatment by the abdominal cavity injection. And (4) counting the death rate, weight change and the quantity of the liver lotus insects of the mice. And the conditions of liver lesion, inflammatory reaction, myofibroblast activation and hepatic fibrosis of mice infected by the clonorchis sinensis are observed by ELISA, pathological section, immunohistochemistry and Masson staining method. The result shows that compared with untreated mice, the expression level of TLR3 in the liver of the mice treated by the TLR3 agonist is obviously increased, the mice are prevented from dying due to the infection of the clonorchis sinensis, the amount of the liver-loaded insects is reduced, the weight of the infected mice is greatly increased, and liver lesion, inflammation and bile duct lesion caused by the infection of the clonorchis sinensis are reduced; reduce the activation quantity of liver myofibroblasts caused by the clonorchis sinensis and relieve the liver fibrosis. These results indicate that the TLR3 agonist stimulates TLR3 high expression in treatment, helps an organism to reduce inflammatory reaction and injury of liver induced by clonorchis sinensis infection, greatly relieves clonorchis sinensis hepatic fibrosis, and improves survival rate of mice.
The invention has the positive effects that:
discloses the application of Toll-like receptor 3 in treating clonorchis sinensis liver fibrosis, and relieving clonorchis sinensis liver fibrosis by using TLR3 agonist. Experiments prove that the TLR3 high expression regulates the phosphorylation of the ERK/P38 signal channel of a host, and inhibits the secretion of IL-6, thereby reducing the liver inflammation injury caused by the clonorchis sinensis infection and reducing the hepatic fibrosis; TTLR3 agonist treatment further proves that TLR3 high expression blocks clonorchis sinensis infection to cause death of mice, reduces the amount of liver charged insects, and lightens liver lesion, inflammation and bile duct lesion caused by clonorchis sinensis infection; reduce the activation quantity of liver myofibroblasts caused by the clonorchis sinensis and relieve the liver fibrosis. TLR3 has better application prospect as a target spot of developing new drugs and vaccines for treating and preventing clonorchis sinensis hepatic fibrosis in the future.
Drawings
FIG. 1 is a screening result of the number of mouse metacercaria infection in the mouse liver fibrosis model established in the present invention;
FIG. 2 shows the collagen deposition in mouse model liver caused by liver fibrosis due to Clonorchis sinensis infection;
FIG. 3 shows the weight, mortality, intra-hepatic TLR3 expression and the amount of loaded insects of WT mice and TLR3 deficient mice infected with Clonorchis sinensis;
FIG. 4 shows the liver pathological changes and inflammatory injuries of WT mice and TLR3 deficient mice infected with clonorchis sinensis;
FIG. 5 shows the activation of fibroblast and collagen deposition in liver muscle of mice after infection of Clonorchis sinensis by WT mice and TLR3 deficient mice in the invention;
FIG. 6 is a diagram of the mechanism of TLR3 activation in the present invention that regulates the immune response of the host;
FIG. 7 shows the improvement of the body weight, mortality and the amount of liver-born insects of mice infected with clonorchis sinensis by the treatment with the TLR3 agonist of the invention;
FIG. 8 shows the improvement of liver pathological changes and inflammatory responses of mice infected with clonorchis sinensis by treatment with the TLR3 agonist of the invention;
FIG. 9 shows the improvement of clonorchis sinensis infected mouse liver myofibroblast activation and collagen deposition by TLR3 agonist treatment according to the invention.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting in any way. The following examples are provided to further illustrate the essence of the present invention, but the present invention is not limited thereto.
Example 1
Establishment of mouse model of hepatic fibrosis caused by clonorchis sinensis and mechanism of TLR3 regulating hepatic fibrosis caused by clonorchis sinensis
1. Screening of the amount of cysticercosis
Wild Type (WT) C57BL/6 mice were divided into 8 groups of 10 mice each, given 0, 50, 100, 200, 400, 800 metacercaria/mouse, respectively. Weighing and recording the weight and the death rate of the mice every day after the attack of the insects, respectively euthanizing the mice 7d, 15d and 35 d after the attack of the insects, and taking livers to carry out statistics on the amount of the insects in the livers (the results are shown in figure 1);
2. establishment of WT mouse fibrosis model
After the number of attack insects was determined, 200 cysticercosis/mouse were gavaged to inoculate clonorchis sinensis cysticercosis, and 100 μ L sterile PBS gavaged mouse was used as a blank control. Collecting feces of mice every day after infection, euthanizing the mice 7d, 15d and 35 d after infection, separating livers for total RNA extraction and masson pine staining, and showing the hepatic fibrosis condition of the mice as shown in figure 2;
3. fluorescent quantitative PCR (RT-PCR) for detecting TLR3 expression
Total RNA from cells/liver tissues was isolated using Trizol reagent, and cDNA was synthesized using a one-step gDNA reverse kit. All primers used in RT-PCR were synthesized by Jilin province Cumei Biotechnology, inc.; preparing an RT-PCR system by using a fluorescent dye FastStart Universal SYBR Green Master (ROX) strictly according to the use instruction; the RT-PCR procedure is divided into three steps: 1 min at 95 ℃; then repeating 30 cycles at 95 ℃ for 45 s,60 ℃ for 45 s, and 72 ℃ for 45 s; then, the temperature is 72 ℃ for 10 min; the primers are as follows:
TLR3-F CGCAGTTCAGCAAGCTATTG ;
TLR3-R TCTTCGCAAACAGAGTGCAT ;
β-actin-F TGCTGTCCCTGTATGCCTCT;
β-actin-R GGTCTTTACGGATGTCAACG;
analysis of mRNA expression levels β -actin was used as an internal reference gene. Melting curves were automatically analyzed by collecting fluorescence signals, using 22 -ΔΔCt Calculating the relative expression quantity of mRNA by a formula, wherein delta Ct represents Ct (sample) -Ct (control); each sample was tested in 3 replicates and the data used for the final analysis was from 3 independent experiments; TLR3 expression is shown in figure 3A;
4. mechanism for regulating hepatic fibrosis caused by clonorchis sinensis by TLR3
Separating the clonorchis sinensis extracellular vesicles, stimulating biliary epithelial cells, collecting total cell protein, and detecting the phosphorylation level of immunoregulation related pathway protein by Western blot. Collecting culture supernatant, and detecting the expression condition of the cell factor. It was found that host cell TLR3 recognizes dsRNA in the extracellular vesicle of clonorchis sinensis, thereby activating high expression of TLR3 and reducing expression of IL-6 and TNF-alpha by reducing the hyperphosphorylation of EKR and P38 pathways (FIG. 3).
Test example 1
The following experiments show that the effect evaluation of the invention for clonorchis sinensis hepatic fibrosis
1. Weight, death rate and change of quantity of intrahepatic dutchmanspidae after TLR3 deficient mice are infected with clonorchis sinensis
Establishing a mouse liver fiber model caused by the clonorchis sinensis according to the method, recording the weight and the death rate of the mouse every day, and counting the number of parasites in the liver of the infected mouse 35 days after infection. The body weight of mice infected with clonorchis sinensis decreased significantly and began to rise slowly 20 d after infection. TLR3 infected by clonorchis sinensis -/- The weight loss was more pronounced in mice (fig. 4A). Mortality monitoring of infected mice showed that clonorchis sinensis-infected TLR3 -/- The mortality rate of the mice is 50 percent and is obviously higher than 40 percent of the clonorchis sinensis infected WT mice (figure)4B) .1. The Statistical analysis is carried out on liver endoparasites of infected mice, and the result shows that the TLR3 infected by clonorchis sinensis -/- The number of parasites in the liver of mice (35) was significantly higher than that of WT mice (27). In addition we have found TLR3 -/- The number of adults in the liver of mice (20) was significantly increased compared to WT mice (13) (fig. 4C) ((C))p<0.001)。
2. Effect of TLR3 deletion on liver injury and inflammatory response of mice infected with clonorchis sinensis
We performed on clonorchis sinensis-infected WT mice and TLR3 -/- The mice were examined for liver autopsy lesions, pathological sections of liver tissue and morphology of biliary epithelial cells, and PBS-treated mice were used as negative controls. Compared with wild mice, the TLR3 deficient mice have aggravated liver lesions, obvious protuberant connective tissue of cord-shaped bile ducts can be seen, hepatomegaly is achieved, and liver nodules are more obvious. PBS control mice had no lesions in liver (fig. 5A);
the liver tissue of the mice treated with PBS was intact and well-defined. In contrast, WT and TLR3 were infected with Clonorchis sinensis at 7d, 15d and 35 d -/- The mouse liver has necrotic foci, and a large number of inflammatory cells accumulate around the necrotic foci and around the portal vein. TLR3 infected with clonorchis sinensis compared to WT mice -/- The liver inflammation in mice was more severe, with a large number of necrotic foci at 7d infection, and significant myofibroblast accumulation around the bile ducts with clogged worms were observed at 15d and 35 d infections (fig. 5B). The pathological changes of the bile ducts of the mice after the clonorchis sinensis infection are observed, and the result shows that the epithelial cells of the bile ducts of the mice in the PBS control group are orderly arranged, the walls of the bile ducts have clear hierarchical structures, and hyperplasia and deformation are not generated. The WT infected mice had bile duct dilatation, bile duct epithelial cell hyperplasia, and lumen deformation, which failed to maintain normal morphology. TLR3 compared to WT infected mice -/- The proliferation and expansion of the bile duct of the mouse are more serious, and the small hyperplastic bile duct appears, which indicates that the bile duct lesion caused by clonorchis sinensis is further aggravated after TLR3 is deleted (figure 5C); liver HAI score, TLR3 -/- The mouse score was significantly higher than WT mice (fig. 5D) ((r))p<0.05, p<0.05, p<0.05);
The regulation and control effect of TLR3 on inflammatory mediators of clonorchis sinensis infected host liver is detected by an ELISA method. The results showed that clonorchis sinensis was infected with TLR3 compared to WT mice -/- IL-6 in mouse liver (FIG. 5E) (see)p<0.001, p<0.001, p<0.001 TNF-. Alpha. (FIG. 5F) ((B))p<0.001, p<0.001, p<0.001 IL-4 (FIG. 5G) ((B))p<0.05, p<0.01, p<0.05 ) is significantly increased. However, expression of IFN- γ was significantly reduced (fig. 5H) ((ii))p<0.001, p<0.001, p<0.001)。
3. Influence of TLR3 deletion on hepatic myofibroblast activation and hepatic fibrosis caused by clonorchis sinensis
Observation of clonorchis sinensis infected WT and TLR3 by Masson staining -/- Liver fibrosis of mice, PBS treated mice as negative control. The control mice had no collagen deposition in the liver. In sharp contrast, clonorchis sinensis-infected WT and TLR3 -/- Collagen fibers continue to accumulate around the bile ducts of the mice. TLR3 infected with clonorchis sinensis compared to WT mice -/- Collagen deposition around bile ducts is more obvious when the mice are infected for 7 d; when the infection lasts for 15d, the area of collagen around the bile duct is increased sharply, and obvious fibrous bridging is formed; at infection 35 d, the area of the tract is completely encapsulated by the deposited collagen, and the collagen has extended to the parenchyma of the liver, creating extensive fibrosis (fig. 6A). The result of quantitative analysis of mouse hepatic fibrosis by using Image J shows that TLR3 deletion causes rapid deterioration of clonorchis sinensis hepatic fibrosis and the area of deposited collagen fiber is significantly increased (p<0.001, p<0.001, p<0.001). These results suggest that TLR3 can reduce liver fibrosis caused by clonorchis sinensis (fig. 6B);
immunohistochemistry was performed on α -SMA protein in the liver to determine the number and location of myofibroblasts in the liver after infection with clonorchis sinensis. The results showed that clonorchis sinensis infection significantly activated myofibroblast activation in the liver of the host, with activated myofibroblasts mainly distributed around the bile duct (fig. 6C,D)(p<0.001, p<0.001, p<0.001). The expression level of α -SMA protein was significantly increased in the liver of infected 7d, 15d and 35 d, TLR 3-deficient mice compared to WT mice, suggesting that TLR3 deletion enhances myofibroblast activation (fig. 6C). Image J quantitative analysis of alpha-SMA positive area in mouse liver showed that alpha-SMA positive area in mouse liver increased after 15D and 35D post infection, tlr3 deletion compared to WT mice (fig. 6D) ((p>0.05, p<0.01, p<0.001)。
4. Implementation of TLR3 agonist therapeutic regimens
Establishing a mouse liver fiber model caused by clonorchis sinensis according to the method, injecting 100 microgram of Poly (I: C) solution into the abdominal cavity of the mouse 8 h before infecting cysticercus, and respectively injecting 100 microgram of Poly (I: C) into the abdominal cavity at the 10 th d and the 20 th d after infecting the WT mouse by the clonorchis sinensis.
5. Influence of TLR3 high expression on body weight, liver parasite number and survival rate of infected mice
From the day of the gavage, mice were weighed and recorded at the same time daily, and mice were recorded daily for mortality. TLR3 agonist Poly (I: C) treatment of mice significantly increased TLR3 expression in mouse-infected liver (fig. 6A). Mice with high TLR3 expression gained significantly higher body weight than untreated mice (fig. 7A). The death rate of clonorchis sinensis infected WT mice in the acute phase was 40% on average, and the TLR3 high expression mice were all alive, completely blocking the acute-phase death caused by clonorchis sinensis (FIG. 7B). Mice were euthanized at 7d, 15d, 35 d of infection, and livers were removed, and mice were observed and recorded for liver lesions. High expression of TLR3 can significantly reduce the number of parasites in liver of mice infected with clonorchis sinensis from 31 to 8 (FIG. 7C) ((C))p<0.001 And the number of adults in the mice of the group with high expression of TLR3 is obviously reduced (figure 7C) ((C))p<0.001)。
6. Influence of TLR3 high expression treatment on liver lesion and liver tissue injury of infected mice
The liver pathological changes and the tissue damage conditions of the infected mice are observed through the combination of the autopsy and the pathological section. Compared with untreated mice, the liver lesion degree of the mice with high expression of TLR3 is obviously reduced, no obvious liver lesion is seen in infected 7d, no obvious hepatomegaly or cholestasis phenomenon is seen, a slight bleeding point appears when 15d is infected, and a slight fibrous lesion appears when 35 d is infected (FIG. 8A).
(fig. 8B) pathological changes were significantly alleviated in TLR 3-highly expressed mice compared to untreated mice (see fig. 8B)p<0.05, p<0.01, p<0.001). The inflammatory reaction of the liver of the mouse with the high TLR3 expression level is obviously reduced, no inflammatory pathological change is seen in the liver at 7d after infection, and no inflammatory cell aggregation and necrotic foci appear: (p<0.05 ); at 15d infection, only a small inflammatory cell aggregation occurs: (p<0.01 No necrotic foci and no obvious bile duct dilatation are seen; after 35 d infection, pathological changes in the liver increased and inflammatory cell numbers increased, and lesions and inflammatory responses remained mild compared to WT mice (fig. 8B) ((B))p<0.001)。
The results of immunohistochemical observation of the change of the bile duct of the mice infected with clonorchis sinensis show that the epithelial cells of the bile duct are enlarged, the lumen is obviously expanded, the epithelial cells of the bile duct are proliferated and denatured after the WT mice are infected with clonorchis sinensis, and clonorchis sinensis at different stages of development exists in the bile duct (figure 8C). The hepatobiliary lesions of the mice in the TLR3 high expression group at different infection times are obviously improved compared with those in the untreated group, bile duct lesions hardly occur after 7d and 15d infection, the bile duct morphology is regular, the structure is clear, and slight epithelial cell hyperplasia occurs only after 35 d infection (figure 8C). Liver HAI score, TLR 3-high expressing group mice scored significantly lower than WT mice (fig. 8D) ((b))p<0.05, p<0.01, p<0.001);
The liver was taken, placed in a 50 mL centrifuge tube, cut into small pieces with scissors, 3 mL sterile PBS was added, and the liver tissue was thoroughly ground and disrupted using a tissue homogenizer. Supernatants were collected and expression of IL-4, IL-6, IFN-. Gamma., TNF-. Alpha.and TGF-. Beta.1 was detected using an ELISA detection kit. ELISA detection results show that TLR3 high expression obviously reduces IL-6 in mouse liverp<0.001, p<0.001, p<0.001)、TNF-α(p<0.001, p<0.001, p<0.001)、IL-4(p<0.01, p<0.01, p<0.05 And promoting expression of IFN-gamma: (p<0.001, p<0.001, p<0.001 (FIGS. 8E-H).
7. Observation of TLR3 high expression on activation of liver myofibroblasts and collagen deposition of infected mice
Mouse liver was taken, formalin fixed, paraffin embedded liver tissue was used for α -SMA immunohistochemical analysis. Protein expression was observed by light microscopy and the protein expression level was digitally analyzed by Image-Pro Plus software. We tested liver activated fibroblast marker protein α -SMA by immunohistochemistry. The results showed that clonorchis sinensis infection caused massive activation of liver myofibroblasts of WT mice. High expression of TLR3 significantly reduced activation of liver myofibroblasts by clonorchis sinensis (fig. 9A). Statistical analysis showed that 35 d of the area of α -SMA positive cells in the liver of TLR 3-highly expressed mice was reduced by 25.73% when infected with clonorchis sinensis 35 d compared to the untreated group (FIG. 9B) ((p>0.05, p<0.01, p<0.001)。
Collagen deposition in the liver of infected mice was detected by masson staining. The dyeing preorder treatment of the Chinese pines is the same as immunohistochemical dyeing, gradient alcohol hydration is carried out after xylene dewaxing, chinese pines dyeing sections are manufactured strictly according to the specification of a Solambio common Chinese pines dyeing kit, and the degree of hepatic fibrosis of the mice is observed by an optical microscope. The masson dyeing result shows that compared with untreated mice, the collagen deposition condition of liver fibers of the TLR3 high-expression mice is obviously improved. After 7D and 15D of infection, the liver of TLR 3-high expressing mice had almost no collagen deposition (fig. 9C, D) ((r))p<0.05, p<0.001 ); infection 35D, fibrotic deposition of collagen around bile ducts, but significantly decreased compared to treatment group (fig. 9C, D) ((D))p<0.001). These results indicate that high expression of TLR3 helps the body reduce collagen deposition in liver fibrosis induced by clonorchis sinensis infection.
Claims (3)
1. The application of the TLR 3-mediated host innate immunity in preparing the drug for resisting the clonorchis sinensis infection.
2. The application of the Toll-like receptor 3 in preparing the medicine for treating clonorchis sinensis hepatic fibrosis.
3. A medicament according to claim 1 or 2 in any pharmaceutical dosage form referred to in pharmacopeia.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211399559.7A CN115487302A (en) | 2022-11-09 | 2022-11-09 | Application of Toll-like receptor 3 in treating clonorchis sinensis liver fibrosis |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211399559.7A CN115487302A (en) | 2022-11-09 | 2022-11-09 | Application of Toll-like receptor 3 in treating clonorchis sinensis liver fibrosis |
Publications (1)
Publication Number | Publication Date |
---|---|
CN115487302A true CN115487302A (en) | 2022-12-20 |
Family
ID=85116353
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211399559.7A Pending CN115487302A (en) | 2022-11-09 | 2022-11-09 | Application of Toll-like receptor 3 in treating clonorchis sinensis liver fibrosis |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115487302A (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100310600A1 (en) * | 2008-02-15 | 2010-12-09 | Carter William A | Selective agonist of toll-like receptor 3 |
CN102258527A (en) * | 2010-05-28 | 2011-11-30 | 中国医学科学院药物研究所 | Application of stimulating agent CRX-675 of Toll-like receiver in resisting pulmonary fibrosis |
-
2022
- 2022-11-09 CN CN202211399559.7A patent/CN115487302A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100310600A1 (en) * | 2008-02-15 | 2010-12-09 | Carter William A | Selective agonist of toll-like receptor 3 |
CN102258527A (en) * | 2010-05-28 | 2011-11-30 | 中国医学科学院药物研究所 | Application of stimulating agent CRX-675 of Toll-like receiver in resisting pulmonary fibrosis |
Non-Patent Citations (1)
Title |
---|
王玉茹: "TLR2、TLR3 在华支睾吸虫致肝纤维化中的作用机制", 万方学位论文, pages 85 - 119 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2010143065A1 (en) | Compositions and methods for increasing lifespan and health span | |
CN108686211A (en) | A kind of drug and therapy for treating liver fibrosis | |
US10220019B2 (en) | Method for treating pulmonary fibrosis comprising application of dimethylamino micheliolide | |
CN114191423B (en) | Application of small-molecule diterpene compound or salt thereof in preparation of medicine for preventing and treating acute lung injury | |
CN110734972B (en) | Application of miR-181c-3p as kidney fibrosis marker | |
CN115487302A (en) | Application of Toll-like receptor 3 in treating clonorchis sinensis liver fibrosis | |
CN107157980B (en) | Application of oridonin in preparation of anti-myocardial remodeling drugs | |
CN113521285A (en) | Application of intervention BOK in preparation of medicine for treating new coronary pneumonia | |
KR102056118B1 (en) | A pharmaceutical composition for alleviating, treating or preventing allergic rhinitis | |
KR20180028890A (en) | Pharmaceutical Composition for Treating Macular Degeneration Containing mTOR Inhibitor | |
CN113181376A (en) | Application of Htra2 gene expression inhibitor in preventing acquired sensorineural deafness | |
CN107951918B (en) | Application of honeysuckle petroleum ether part extract in preparation of medicine for treating pulmonary fibrosis | |
CN113194926A (en) | Pharmaceutical composition comprising bio-implant containing mesenchymal stem cells for preventing or treating liver disease | |
CN110917351A (en) | Use of MBD2 inhibitors for the prevention and treatment of fibrotic diseases | |
CN109674774B (en) | Application of propane-1-alcohol compound in preparation of medicine for treating cerebral hemorrhage | |
CN114306594A (en) | Application of desulzumab ozogamicin in preparation of medicine for treating knee osteoarthritis | |
CN114832000B (en) | Application of LPE16:0 in preparation of medicines for resisting respiratory syncytial virus infection | |
CN114917346B (en) | Medicine and pharmaceutical composition for treating ischemic heart disease | |
CN111481509B (en) | siRNA (small interfering ribonucleic acid) nanoparticle for relieving neuroimmune response and treating cerebral apoplexy | |
CN112294961B (en) | ACP5 inhibitors and their use in the prevention and treatment of fibrotic diseases | |
TWI779941B (en) | Extraction method of clinacanthus nutans, and use of it's extract for treating osteoporosis, neurodegenerative diseases, skeletal muscle atrophy and improving wound healing | |
CN113304249B (en) | Application of thymosin beta 4 in preparation of medicine for treating pulmonary fibrosis complicated with lung cancer | |
CN114306306B (en) | Application of 2-bromopalmitic acid in preparation of medicine for treating diseases related to spermatogenic dysfunction | |
CN102247349A (en) | Purpose of salvianolic acid A on prevention and / or treatment of liver pathology caused by diabetes | |
CN116036280A (en) | Application of MYO9B inhibitor in preparing medicines for preventing and/or treating individual fibrosis diseases |
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 | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20221220 |