CN115671085A - Application of trans-anethole in preparation of medicine for treating liver ischemia reperfusion injury - Google Patents
Application of trans-anethole in preparation of medicine for treating liver ischemia reperfusion injury Download PDFInfo
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Abstract
The invention relates to application of trans-anethole in preparing a medicine for treating liver ischemia-reperfusion injury, and belongs to new application of a known monomer. Trans-anethole pretreatment can improve liver dysfunction of mouse liver ischemia reperfusion injury, reduce the generation of inflammatory cytokines and infiltration of immunocytes, and simultaneously reduce hepatocyte apoptosis, and the mechanism for treating mouse liver ischemia reperfusion injury is to improve the ratio of metabolite epoxy eicosatrienoic acid to dihydroxyeicosatrienoic acid by regulating and controlling key soluble epoxide hydrolase in arachidonic acid CYP metabolic pathway, inhibit the activation of NLRP3 inflammasome, and reduce inflammatory reaction. Due to the lack of effective drugs for treating liver ischemia reperfusion injury clinically, trans-anethole is expected to become a novel effective potential therapeutic drug with small side effect.
Description
Technical Field
The invention belongs to new application of known monomers, and particularly relates to new application of trans-anethole in preparation of a medicine for treating liver ischemia-reperfusion injury.
Background
Ischemic Reperfusion Injury (IRI) of the liver is a major cause of various postoperative complications in liver surgeries such as liver transplantation and hepatectomy.
Liver ischemia reperfusion injury can cause various adverse reactions such as early graft dysfunction, graft rejection, graft failure and the like after liver transplantation. Liver ischemia reperfusion injury is divided into two typical stages of ischemia and reperfusion, and involves complex physiopathological processes and various mechanisms, including: inflammatory responses, oxidative stress, metabolic dysfunction, apoptosis, autophagy, and the like. Therefore, how to alleviate the ischemia-reperfusion injury of the liver is still an important problem to be clinically urgently solved for improving the quality of a liver transplantation donor and improving the prognosis of the liver transplantation.
The traditional Chinese medicine has the characteristics of multiple target points, less side effects and the like, and in nature, many Chinese herbal medicines contain high-level trans-anethole and trans-anethole [ 1-methoxy-4- (1-propenyl) benzene; trans-anethole, TA belongs to aromatic organic compounds, is colorless and slightly volatile liquid, and has unique smell.
The applicant researches and discovers that the trans-anethole pretreatment can improve the liver ischemia reperfusion injury.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provides application of trans-anethole in preparing a medicament for treating liver ischemia reperfusion injury.
The invention is realized by the following technical scheme: the trans-anethole is used in preparing medicine for treating liver ischemia re-perfusion injury, and is prepared with trans-anethole as the only active component.
The liver ischemia reperfusion injury comprises liver function injury, inflammatory factor production and inflammatory cell infiltration, hepatocyte apoptosis, arachidonic acid CYP metabolic pathway key soluble epoxide hydrolase expression up-regulation, and NLRP3 inflammasome activation.
The medicine is an oral medicine and can be directly orally taken.
The medicament also includes a solvent.
The trans-anethole traditional Chinese medicine monomer has the structure as follows:
C 10 H 12 o, english name Trans-anethole, molecular weight 148.2.
The medicine monomer intervention and animal model construction:
the experiment was divided into 7 groups, which were a normal control group, a normal control + drug solvent group, a model group + drug solvent group, and three drug pretreatment (TA) groups of different concentrations.
Adopting a mouse gastric perfusion administration mode, respectively administering trans-anethole with the concentration of 10mg/kg, 20mg/kg and 40mg/kg to a mouse for continuous gastric perfusion for seven days for pretreatment, constructing a mouse liver 70% ischemia reperfusion injury model on the eighth day, performing ischemia for 1 hour and reperfusion for 6 hours, and immediately taking the mouse liver after the model is successfully constructed and storing peripheral blood for subsequent research. The normal control group and the model group are treated by solvent intragastric administration with the same dosage; the normal control group was identical to the model group and the administration group except that the ischemia and reperfusion periods were not performed.
The research content is as follows:
(1) The improvement effect of the trans-anethole pretreatment on the liver function of the mouse with ischemia-reperfusion injury:
the content of ALT and AST in peripheral blood serum of mice of a normal control group, a model group and different administration concentrations is detected by adopting a full-automatic biochemical analyzer, and the liver tissue damage change of each group of mice is detected by a pathological method. The optimal administration concentration is determined to be 20mg/kg according to liver function index and pathological result, and the optimal administration concentration is determined by the following TA group.
(2) Effect of trans-anethole pretreatment on arachidonic acid regulation of mouse liver ischemia reperfusion injury:
extracting proteins in liver tissues of mice of a normal control group, a model group and a TA group, detecting the transcription level of soluble epoxide esterase (Ephx 2) of key enzyme of arachidonic acid metabolic pathway of the liver tissues by using a qRT-PCR method, detecting the expression of the soluble epoxide esterase (sEH) by using a Western Blot method, and detecting the contents of arachidonic acid metabolites, namely epoxy eicosatrienoic acid (EETs) and docosahexenoic acid (DHETs) in the liver tissues by using an ELISA method.
(3) Anti-apoptotic effects of trans-anethole pretreatment during ischemia reperfusion injury of mouse liver:
detecting the apoptosis condition of the liver cells by adopting a TUNEL immunofluorescence method; extracting proteins in liver tissues of mice of a normal control group, a model group and a TA group, and detecting related apoptosis proteins and related anti-apoptosis proteins in the liver tissues by using a Western Blot method.
(4) The trans-anethole pretreatment inhibits the production of mouse liver ischemia reperfusion injury inflammatory factors and the activation of NLRP3 inflammatory bodies:
the infiltration of inflammatory cells in liver tissues of mice of a normal control group, a model group and a TA group is detected by an immunofluorescence method, the liver tissues of the mice are extracted, and the expression level of inflammatory factor protein and NLRP3 inflammatory corpuscle related protein in the liver tissues is detected by Western Blot.
The beneficial effects of the invention are as follows: the trans-anethole pretreatment can improve the liver dysfunction of the mouse liver ischemia reperfusion injury; can reduce the generation of inflammatory factors and the infiltration of inflammatory cells of the ischemia-reperfusion injury of the liver of a mouse; can reduce the apoptosis of mouse liver ischemia reperfusion injury liver cells; the expression of soluble epoxide hydrolase which is key to the arachidonic acid CYP metabolic pathway of the ischemia reperfusion injury of the liver of a mouse can be regulated and controlled; can inhibit the activation of mouse liver ischemia reperfusion injury NLRP3 inflammasome.
According to the invention, through different methods, the mechanism of relieving the mouse liver ischemia-reperfusion injury by adopting the trans-anethole pretreatment is deeply explored, a theoretical basis is provided for treating the liver ischemia-reperfusion injury, the effect of treating the liver ischemia-reperfusion injury by adopting the trans-anethole pretreatment is proved to be effective, and a new idea is provided for developing a medicament for treating the liver ischemia-reperfusion injury.
Drawings
FIG. 1: effect of trans-anethole pretreatment on liver function of mouse ischemia reperfusion injury, a: peripheral blood serum ALT content; b: peripheral blood serum AST content;
FIG. 2: pathology images of the pathology change study of the mouse liver ischemia reperfusion injury by trans-anethole pretreatment;
FIG. 3: effect of trans-anethole pretreatment on mouse liver ischemia reperfusion injury pathology score;
FIG. 4: influence of trans-anethole pretreatment on liver tissue Ephx2 gene transcription level in mouse liver ischemia reperfusion injury;
FIG. 5: an electrophoresis chart of the influence of trans-anethole pretreatment on the expression of sEH protein in liver tissues in the ischemia-reperfusion injury of the liver of a mouse;
FIG. 6: influence of trans-anethole pretreatment on the expression level of sEH protein in liver tissue in mouse liver ischemia reperfusion injury;
FIG. 7: the effect of trans-anethole pretreatment on the liver tissue arachidonic acid metabolite EET/DHET in the ischemia reperfusion injury of the liver of a mouse;
FIG. 8: staining pattern of mouse liver ischemia reperfusion injury cell apoptosis by trans-anethole pretreatment;
FIG. 9: an electrophoresis chart of the influence of trans-anethole pretreatment on the expression of the mouse liver ischemia reperfusion injury apoptosis related protein;
FIG. 10: influence of trans-anethole pretreatment on apoptosis-related protein expression of mouse liver ischemia reperfusion injury;
FIG. 11: the influence of trans-anethole pretreatment on liver tissue immune cell infiltration in mice liver ischemia reperfusion injury;
FIG. 12: an electrophoresis picture of the influence of trans-anethole pretreatment on the expression of mouse liver ischemia reperfusion injury NF kB-NLRP 3 channel protein;
FIG. 13 is a schematic view of: the effect of trans-anethole pretreatment on the expression of NF kappa B-NLRP3 pathway protein of mouse liver ischemia-reperfusion injury.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the accompanying drawings and preferred embodiments.
The experimental biochemical reagent and the experimental animal related to the invention can be purchased conventionally, and the equipment required by the experiment is from a laboratory.
Experimental materials:
trans-anethol was purchased from Solebao corporation (CAS: 4180-23-8), prepared in three different concentrations of trans-anethol, i.e., a low concentration (10 mg/kg), a medium concentration (20 mg/kg) and a high concentration (40 mg/kg), using sodium carboxymethylcellulose as a solvent, and subjected to a gavage treatment for 7 consecutive days.
Mouse breed C57BL/6J, male, age 6-8 weeks, weight 18 + -2 g, purchased from Beijing Wintolite Corp, and fed normally.
Constructing a mouse liver ischemia reperfusion injury model:
a) Anesthesia: anaesthetizing the mouse by using a respiratory anaesthesia machine and applying an isoflurane inhalation anaesthesia mode, wherein the concentration of the isoflurane is 1.5 percent, and the flow rate of oxygen is 0.6-1L/min;
b) Skin preparation: after anesthesia is finished, skin preparation and disinfection are carried out, the hairs on the abdomen of the mouse are removed by using an animal shaver, the mouse is fixed on an operating table in a supine position, and iodophor solution is disinfected for three times;
c) Opening the abdomen: performing an open abdomen along the midline of the abdomen, wherein the open abdomen is about 2-2.5cm long;
d) Exposing the hepatic portal: soaking a cotton swab and sterile gauze in sterile physiological saline, respectively placing two pieces of moist gauze on the upper edge and the lower edge of an abdominal opening, gently pushing the intestinal system of the mouse out of the abdominal cavity by using the moist cotton swab to the gauze placed on the lower part in advance, covering the gauze with the moist gauze, keeping the moist in the whole operation process, fully exposing portal veins by using the moist cotton swab, spreading liver lobes and left liver lobes in the liver of the mouse towards a diaphragm by using another moist cotton swab, separating connective tissues by using microsurgical forceps, and fully exposing a first liver lobe;
e) Clamping and closing the blood vessel: separating out right lobe branch, clamping a first portal vein, hepatic artery and bile duct above the right lobe branch by using a vascular clamp, immediately changing the color of the middle lobe and the left lobe (accounting for about 70 percent of the whole liver volume) from reddish brown to pale immediately after clamping, and keeping the color of the right lobe ruddy;
f) And (3) maintaining an ischemic state: in the ischemia process, the mouse is placed in a constant temperature box, gauze is kept moist, and vital signs of the mouse are observed;
g) And (3) ending ischemia: after the ischemia time is over, the vascular clamp is taken out gently for reperfusion, and the color of the liver is recovered after reperfusion; the abdomen is simply and continuously sutured, 200 mu L of normal saline is injected under the skin of the mouse after closing the abdomen, and the mouse is placed in a constant temperature box;
h) Material taking: animals were sacrificed at the end of the established reperfusion time and sampled for storage.
Grouping experiments:
a) Normal control (Sham) group (n =10, laparotomy only);
b) Veh (solvent) pre-treatment + Sham group (n =10,7 days given mice with solvent gavage at the same dose as TA);
c) IRI model group (n =10, establish liver IRI model);
d) Veh pretreatment + IRI model group (n =10,7 days given mice with solvent gavage at the same dose as TA);
e) Low concentration TA pretreatment + IRI group (n =10,7 days given mice with 10mg/kgTA gavage);
f) Medium concentration TA pretreatment + IRI group (n =10,7 days given mice 20mg/kgTA gavage);
g) High concentration TA pretreatment + IRI group (n =10,7 days given mice with 40mg/kgTA gavage).
Serological examination of the effects of TA pretreatment on liver function of mice with ischemia-reperfusion injury:
fresh blood was collected from mice via the inferior vena cava. Serum was extracted by centrifugation at 3000rpm for 10min at 4 ℃. Serum alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) levels were measured using a fully automated biochemical analyzer.
As shown in FIG. 1, the levels of AST and ALT in the IRI group were elevated as compared with the Sham group, and the levels of AST and ALT in the serum after ischemia-reperfusion injury in mice were reduced by trans-anethole pretreatment at three concentrations, i.e., low concentration (10 mg/kg), medium concentration (20 mg/kg) and high concentration (40 mg/kg), as compared with the IRI group (p < 0.05). The serological result shows that the Sham group and the Sham + Veh group have no statistical difference, and the IRI group and the IRI + Veh group have no statistical difference, so that the influence of the drug solvent on the experimental result is proved.
Pathology HE staining assessed the effect of trans-anethole pretreatment on mouse liver ischemia reperfusion injury:
fixing liver tissues of each group of mice by 10% formalin, placing tissue blocks in melted paraffin for complete paraffin soaking after gradient dehydration, transparency and other processes, embedding, cooling and solidifying to obtain the section. The paraffin sample is cut into 5 mu m thick, spread on a glass slide after being treated with distilled water of 37 ℃, and dried in a thermostat for use in subsequent experiments. Paraffin sections were dehydrated, cleared, and stained with Hematoxylin (H) to bluish purple for nuclei and intracellular ribosome, and Eosin (E) to red or light red for cytoplasm. The stained sections were stored using gum slides, observed using a microscope, and images were collected.
Liver HE staining scores were graded according to Suzuki criteria, with specific scoring criteria as shown in table 1.
TABLE 1Suzuki Scoring Standard
As shown in fig. 2 and 3, liver tissues of mice in the IRI group showed different degrees of swelling/necrosis, infiltration of inflammatory cells, etc., compared with Sham group; compared with the IRI group, the mice with different concentrations of TA pretreatment group have reduced liver tissue damage degree, which shows that TA pretreatment has protective effect on the liver IRI of the mice. The pathology scores were statistically different (p < 0.05).
According to serological ALT and AST levels and pathological results, TA pretreatment concentration of 20mg/kg is selected as follow-up mechanism research.
qRT-PCR detection of Gene transcript levels of soluble epoxide esterase, a key enzyme in the arachidonic acid metabolic pathway in liver tissue (Ephx 2):
RNA is extracted from liver tissues of each group of mice according to the results, and the transcriptomics results show that the action mechanism of TA is related to the arachidonic acid metabolic pathway and the expression of soluble epoxide hydrolase (sEH, ephx 2) is down regulated.
Extraction of liver RNA: total RNA was extracted by Trizol method. Adding liver tissue, grinding the liver tissue into tissue homogenate by cryogrinding, adding 1mL Trizol and 200 μ L chloroform, respectively, standing for 20min after inversion, centrifuging at 12000rpm for 15min, and sucking supernatant into a centrifugal tube without enzyme. Adding 500 mu L of isopropanol, adding 1mL of 75% ethanol for extracting RNA, and detecting the concentration of the RNA by using a multifunctional microplate reader.
The sequence of the designed PCR primer is shown in table 2, the specific operation steps are the experimental steps described by the Hamr gene Hairpin-itTMreal-Time PCR Kit, and the result is shown in figure 4, compared with the Sham group, the expression of the Ephx2 gene in the IRI group is obviously increased (p is less than 0.05), and the expression of the Ephx2 gene in the TA group can be obviously reduced (p is less than 0.05).
TABLE 2Real-time qRTPCR primer sequences
Western Blot detection of the expression of sEH protein in liver tissue of mice subjected to ischemia-reperfusion injury by using trans-anethole pretreatment:
protein extraction: the liver tissue to be detected is taken out from a refrigerator at the temperature of minus 80 ℃ and placed on dry ice, a liver tissue sample of about 30mg is cut, 800 mu L of RIPA lysis buffer solution containing 1% protease inhibitor mixed solution is added, the tissue is repeatedly ground by a tissue grinder, after centrifugation is carried out for 20min at 13000rpm, supernatant is sucked, 10 mu L of lysis solution is taken, protein quantification is carried out by a BCA kit, and the concentration of the sample is adjusted by using 1 Xloading buffer.
Electrophoresis: the polyacrylamide gel was prepared from 4% concentrated gel and 12% separation gel, and the sample loading was 20 μ g per well. After electrophoresis at 80V for 15-20min, regulating the voltage to 120V, and continuing electrophoresis for 35-45min.
Film transferring: after electrophoresis, the separation gel was fixed by a transfer membrane holder, placed in a transfer membrane solution, and subjected to membrane transfer for 2 hours under an ice bath condition at a constant current of 280 mA.
Sealing the milk: after the membrane transfer is finished, cutting and marking are carried out according to the target protein, and the target protein is placed in a sealing solution and placed on a shaking table to be slowly shaken and sealed for 1.5h under the condition of room temperature.
Primary anti-incubation: after blocking, the membrane was washed with TBST several times, primary antibody was added and incubated overnight at 4 ℃.
And (3) secondary antibody incubation: after the primary antibody incubation is finished, washing the membrane for multiple times by TBST, adding a secondary antibody, and incubating for 1-2h at room temperature.
Exposure and color development: after the secondary antibody incubation is finished, washing the membrane for multiple times by TBST, uniformly dripping ECL developing solution on the strip, and putting the strip into an exposure machine for exposure.
Results as shown in figures 5 and 6, sEH expression was significantly increased in IRI group (p < 0.05) and significantly decreased in TA group (p < 0.05) compared to Sham group.
The ELISA method is used for detecting the influence of the trans-anethole pretreatment on the arachidonic acid metabolite of the liver tissue in the ischemia reperfusion injury of the liver of a mouse:
the liver tissue preserved at-80 deg.C was taken out, about 50mg was weighed, the mouse liver tissue was cut and ground sufficiently, 200. Mu.L PBS was added thereto and sufficiently mixed, and the mixture was centrifuged at 3000rps for 10min to obtain the supernatant. The enzyme-linked immunosorbent assay (ELISA) quantitative kit (Tianjin Anori biological technology, inc. China) is used for detecting the contents of metabolites, namely, the dihydroxy eicosatrienoic acids (DHETs) and the epoxy eicosatrienoic acids (EETs).
According to the standard steps of ELISA kit operation, after operations such as sample adding, diluting, antibody incubation and the like, an enzyme-linked immunosorbent assay (ELISA) instrument is used for reading the absorbance value of each hole under the wavelength of 450 nm.
As shown in FIG. 7, the EETs/DHETs ratio in IRI group was significantly decreased (p < 0.05), while the EETs/DHETs ratio in TA group was significantly increased (p < 0.001) compared to Sham group.
Verifying the influence of trans-anethole pretreatment on hepatocyte apoptosis in the mouse liver ischemia reperfusion injury process:
TUNEL staining to detect apoptotic cell status in liver tissue: the apoptosis of the liver tissue is detected by using a one-step TUNEL apoptosis detection kit (KeygENBIOTECH, jiangsun, china).
Paraffin wax sections of liver tissues of each group of mice are dewaxed by a conventional method, 100 mu L of proteinase K working solution is dripped, reaction is carried out for 30min at 37 ℃, 100 mu L of DNase I reaction solution is dripped, and a positive plate is prepared. TdT enzyme reaction solution (50. Mu.L) and streptavidin-fluorescein labeling solution (50. Mu.L) were added in the dark. Finally, the cell nucleus is re-stained with DAPI staining solution, and an image is collected by a fluorescence microscope.
As a result, as shown in fig. 8, the IRI group apoptotic cells were significantly increased, and the TA group apoptotic cells were significantly decreased, compared to the Sham group.
Western Blot detection of proteins associated with liver tissue apoptosis:
western Blot experimental procedures were as described previously. As a result, as shown in FIGS. 9 and 10, the expression of the TA group-related apoptosis-related proteins (Caspase-3, cleared-Caspase-3, bax) was decreased (p < 0.05) and the expression of the anti-apoptosis-related proteins (Bcl-2, bcl-xl) was increased (p < 0.05) as compared to the IRI group.
Detecting the influence of trans-anethole pretreatment on liver tissue immune cell infiltration in the liver ischemia reperfusion injury of the mice:
paraffin sections of mouse liver tissue were stained using immunofluorescence:
paraffin section of mouse liver is first hydrated/dewaxed and antigen repair is carried out with 0.01M TRIS-EDTA repair solution with pH9.0. Blocked with 5% blank goat serum and incubated with CD11b antibody overnight at 4 ℃. Adding DAPI working solution to stain cell nucleuses, adding an anti-fluorescence attenuation blocking tablet to block, sealing by using a cover glass, and observing and collecting images under a fluorescence microscope.
The results are shown in FIG. 11, where the expression of CD11b positive cells was significantly increased in the IRI group (p < 0.05), while the expression of CD11b positive cells was significantly decreased in the TA group (p < 0.05) compared to the Sham group.
Verifying the influence of trans-anethole pretreatment on the expression of mouse liver ischemia reperfusion injury NF kB/NLRP 3 pathway proteins:
western Blot experimental procedures were as described previously. The results are shown in fig. 12 and fig. 13, the TA group NF κ B signaling pathway proteins (p 65, p-p65, TNF- α, IL-6), and their regulated NLRP3l inflammasome-associated proteins (NLRP 3, IL-1 β) expression were significantly reduced (p < 0.05) compared to the IRI group.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (9)
1. The application of trans-anethole in preparing the medicine for treating liver ischemia reperfusion injury is characterized in that: trans-anethole is used as the only active component to prepare the medicine for treating liver ischemia reperfusion injury.
2. Use according to claim 1, characterized in that: the liver ischemia reperfusion injury is liver dysfunction.
3. Use according to claim 1, characterized in that: the hepatic ischemia reperfusion injury is the production of inflammatory factors and infiltration of inflammatory cells.
4. Use according to claim 1, characterized in that: the hepatic ischemia reperfusion injury is apoptosis of liver cells.
5. Use according to claim 1, characterized in that: the hepatic ischemia reperfusion injury is up-regulated expression of soluble epoxide hydrolase which is key to arachidonic acid CYP metabolic pathway.
6. Use according to claim 1, characterized in that: the hepatic ischemia reperfusion injury is activation of NLRP3 inflammasome.
7. Use according to claim 1, characterized in that: the medicament is an oral medicament.
8. Use according to claim 7, characterized in that: the medicament also includes a solvent.
9. Use according to any one of claims 1 to 8, characterized in that: the trans-anethole structure is as follows:
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| WO2021244010A1 (en) * | 2020-06-02 | 2021-12-09 | 西安交通大学医学院第一附属医院 | Use of heat shock factor 2 binding protein in liver ischemia reperfusion injuries and drug-induced liver injuries |
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| WO2021244010A1 (en) * | 2020-06-02 | 2021-12-09 | 西安交通大学医学院第一附属医院 | Use of heat shock factor 2 binding protein in liver ischemia reperfusion injuries and drug-induced liver injuries |
Non-Patent Citations (5)
| Title |
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| CAIYUN YU等: "Trans-anethole ameliorates LPS-induced inflammation via suppression of TLR4/NF-κB pathway in IEC-6 cells", INTERNATIONAL IMMUNOPHARMACOLOGY, vol. 108 * |
| HONG-IK CHO等: "Protective Mechanism of Anethole on Hepatic Ischemia/Reperfusion Injury in Mice", JOURNAL OF NATURAL PRODUCTS, vol. 76, pages 1717 * |
| KRITIKA PANDIT等: "Amelioration of oxidative stress by trans-Anethole via modulating phase I and phase II enzymes against hepatic damage induced by CCl4 in male Wistar rats", ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH, vol. 29, pages 6317 - 6333, XP037666405, DOI: 10.1007/s11356-021-16070-z * |
| YICHUN TONG等: "Trans-anethole ameliorates lipopolysaccharide-induced acute liver inflammation in broilers via inhibiting NF-kB signaling pathway", POULTRY SCIENCE, vol. 101, no. 8, pages 1 - 9 * |
| 李杨等: "NF-κB与肝脏缺血再灌注损伤的研究进展", 中国现代普通外科进展, vol. 20, no. 04, pages 2 * |
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