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Application of erodium stephanianum willd element as autophagy-lysosome signal channel blocker

A technology of signaling pathways, Cranedin, which is applied in the field of medicine, can solve the problems of no patent application and no articles showing the blocking effect of autophagy-lysosome signaling pathways, and achieve the effect of inhibiting degradation pathways

Pending Publication Date: 2022-03-04
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no article showing the blocking effect of 6-OA on the autophagy-lysosome signaling pathway, and no patent application yet

Method used

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  • Application of erodium stephanianum willd element as autophagy-lysosome signal channel blocker
  • Application of erodium stephanianum willd element as autophagy-lysosome signal channel blocker
  • Application of erodium stephanianum willd element as autophagy-lysosome signal channel blocker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Detection of changes in intracellular autophagy proteins by Western blot experiments after 6-OA treatment

[0022] 1. Experimental method

[0023] ① Treat HCT116 and HT29 cells with different concentrations of 6-OA (0 μM, 1.6 μM, 3.2 μM, 6.4 μM) for 24 hours. HCT116 and HT29 cells were treated with 6-OA (3.2 μM) for 0 hours, 6 hours, 12 hours, and 24 hours.

[0024] ② Lyse the cells: wash three times with pre-cooled PBS (0.01M, pH=7.4), add 100 μL of RIPA cell lysate (Beiyuntian, P0013B), lyse on ice for 30 minutes, gently invert and mix once every 10 minutes; Centrifuge at 4°C and 13200rpm for 30min, and take the supernatant for protein concentration determination;

[0025] ③Sample preparation: Add 30 μg of protein to 20 μL of 1× protein loading buffer (P0015), mix well, boil in a boiling water bath for 10 minutes to obtain samples;

[0026] ④ Gel preparation: prepare 10% separating gel (5mL): 1.5mM Tris-HCl (pH8.8) 1.3mL, ddH2O 1.9mL, 30% acrylamide 1.7m...

experiment example 2

[0035] Experimental example 2, laser confocal experiment

[0036] 1. Experimental method

[0037] Construction of pLVX-Puro-GFP-LC3 plasmid: using homologous recombination method, using C112-ClonExpress IIOne Step Cloning Kit kit (Novizyme, Cat#C112-01) to construct, as follows:

[0038] Combined with the ClonExpress II recombination reaction system (Novizan Biotechnology Co., Ltd.) to design amplification-specific primers, high-fidelity PCR polymerase was used to amplify the target gene LC3 and GFP sequences. Cut the empty PLVX-puro with the fast-cutting enzyme ECOR I (TaKaRa), and connect the LC3 and GFP sequences of the target gene with the digested PLVX-puro through the ligase ExnaseⅡ. The connection conditions are as follows: 37°C for 0.5h, then Immediately cool in an ice bath for 5 minutes, then gently mix the ligation product with 100 μL of Escherichia coli DH5α competent cells (OD value 0.5), let stand on ice for 30 minutes; incubate in an ice-water bath for 90 second...

experiment example 3

[0048] Experimental example 3, observation of lysosome morphology under electron microscope

[0049] 1. Experimental method

[0050] ① Cell collection: HCT116 cells treated with 6-OA were digested with trypsin, collected into a 1.5mL conical centrifuge tube, and centrifuged at 1800r / min for 10min.

[0051] ②Fixation and cleaning: Remove the supernatant after centrifugation, add 3% glutaraldehyde immediately, and put it in a 4°C refrigerator for fixed storage (at least 2 hours). Wash with 0.1mol / L phosphate buffered saline twice, each time for 10min, then fix with 1% osmic acid in a refrigerator at 4°C for 1h, then wash twice with 0.1mol / L phosphate buffered saline, each time 10 minutes each time. After osmium fixation, the cells were wrapped in lens tissue and placed in penicillin vials for subsequent processing.

[0052] ③Dehydration and soaking: using gradient dehydration method, 30%, 50%, 70%, 90% ethanol, 90% ethanol: 90% acetone = 1:1 mixture, 90% acetone, 100% acetone...

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Abstract

The invention discloses an application of erodium brevifolium willd as an autophagy-lysosome signal channel blocking agent. The research shows that the erodium brachyphyllum element can inhibit an autophagy-lysosome pathway and inhibit autophagy. The erodium stephanianum willd element is extracted from the traditional Chinese herbal medicine centipeda minima, the centipeda minima is always used for treating respiratory system diseases over the years, the safety is high, the development and utilization prospects are good, and the price is low. The invention provides a new application of the erodium brachyphyllum willd element, the erodium brachyphyllum willd element is used as an autophagy-lysosome signal channel blocking agent, the autophagosomes of HCT116 cells and HT29 cells treated by the erodium brachyphyllum willd element are increased, and the autophagy-lysosome degradation pathway is blocked.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to the application of geranidin as an autophagy-lysosome signal pathway blocker. Background technique [0002] Goose is not herbivorous (Centipeda minima (L.) A.Br.et Aschers.) is a plant of the genus Asteraceae, also known as Centipeda minima, and chicken intestine grass. The fact that geese do not eat grass was first recorded in the "Eat Habit of Materia Medica" in the Southern Tang Dynasty, and it is also recorded in the famous Chinese pharmacopoeia "Compendium of Materia Medica", "The goose does not eat grass can clear the nose, sharpen the seven orifices, spit out wind and phlegm, and stop the nose from falling." . Goose does not eat grass and grows in damp places at an altitude of 300 to 1900 meters. It is mainly produced in Zhejiang, Hubei, Jiangsu, Guangdong and other places in my country. Abroad, it is mainly distributed in the plains of India and Ceylon. [...

Claims

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Application Information

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IPC IPC(8): A61K31/365A61P35/00
CPCA61K31/365A61P35/00
Inventor 汪洋何庆瑜余楠楠
Owner JINAN UNIVERSITY
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