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A method of screening a marker of renal toxicity caused by aristolochic acid by utilizing cell metabolic profiling in vitro

An aristolochic acid and cell metabolism technology, which is applied in the fields of analytical chemistry and medicinal chemistry, can solve the problems of unstable analysis sequences of instruments, difficulty in data modeling, and deviation of mass spectrometry response, and achieves less difficulty in clinical translation, high sensitivity, and high The effect of separation

Inactive Publication Date: 2016-06-08
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

Due to the complexity of biological samples, the instability of the instrument itself and the long analysis sequence lead to a certain deviation in the mass spectrometry response, which will bring difficulties to subsequent data modeling

Method used

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  • A method of screening a marker of renal toxicity caused by aristolochic acid by utilizing cell metabolic profiling in vitro
  • A method of screening a marker of renal toxicity caused by aristolochic acid by utilizing cell metabolic profiling in vitro
  • A method of screening a marker of renal toxicity caused by aristolochic acid by utilizing cell metabolic profiling in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Study on the effective dose of aristolochic acid-induced renal cell damage

[0047] Inoculate equal amounts of humanized normal kidney cells HL7702 suspended in K-SFM medium into 96-well plates (104 cells / well), add 200ul of K-SFM cell culture medium to each well, place at 37°C, 5% CO2 constant temperature cell incubator. Overnight, remove the original medium, add 200ul 0.312, 0.625, 1.25, 2.5, 5, 10, 20, 40, and 80 μg / ml of aristolochic acid respectively, and the aristolochic acid aqueous solution uses K-SFM cell culture medium Make a dilution. Three parallel wells were designed for each dose, and a cell control group (control group) without drug addition and only an equal volume of medium was set in parallel, and a blank group was not inoculated with cells but only an equal volume of medium was added. After culturing in a constant temperature cell incubator at 37°C and 5% CO2 for 24 hours, add 20 μL of 5 mg / ml MTT phosphate buffer solution (pH=7.0) to each well, ...

Embodiment 2

[0077] 1. Nephrotoxic dose study, cell sample collection, processing, analysis and metabolic profile analysis are similar to those in Example 1, the main difference parameters are as follows:

[0078] 1) Set up groups: control group, 24h aristolochic acid injury group. There are 12 parallel samples in each group, a total of 24 samples.

[0079] 2) The number of cells in each sample is about 50 mg after centrifugation. The mobile phase A of metabolic profiling was 0.2% formic acid aqueous solution.

[0080] 3) The main parameters of the PLSDA model (model 2) are A (principal component number) = 2, R2X = 0.609, R2Y = 0.985, and Q2 = 0.96.

[0081] The normal group and the injury group were also well separated in model 2. And the ions in Table 1 are included in the candidate markers screened out by Model 2. It shows that the markers screened by this method have the characteristics of stability and reproducibility.

[0082] The predictive ability of the potential markers of m...

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Abstract

The invention discloses a method of screening a marker of acute renal toxicity caused by aristolochic acid by utilizing cell metabolic profiling in vitro. The effective dose causing renal cell damage is determined by adopting a 3-(4,5-dimethylthiahiazo-2)-2,5-diphenytetrazoliumromide (MTT) staining manner. Renal cell metabolites are analyzed by adopting an ultra-high performance liquid chromatography-mass chromatography technique to obtain metabolic profiles. Metabolic profile data of normal groups and medicine damage groups are analyzed by adopting a multivariate statistic method. A potential renal toxicity marker is screened by combining a change rule of metabolites along with medicine acting time. The screened marker is good in predicting performance. The average accuracy of outer verification is 90% or above. The method comprehensively represents variation situations of metabolites of normal renal cells under medicine damages, and can provide technique supports for early diagnosis of renal toxicity caused by medicines or medicine safety evaluation.

Description

technical field [0001] The present invention relates to the fields of analytical chemistry and medicinal chemistry, and also relates to metabolomics, especially metabolomics based on liquid chromatography-mass spectrometry; specifically, it is a method for screening aristolochic acid-induced nephropathy in vitro by using cell metabolic profiles. Methods for Toxicity Markers. Background technique [0002] Aristolochic acid (AA) is an important chemical component present in Aristolochia plants. Traditional Chinese medicines containing aristolochic acid are mainly used to treat arthritis, gout, rheumatism and festering wounds. However, in the past 10 years, there have been more than 100 cases of severe nephrotoxicity caused by taking Fangji (Aristolochia). Studies have shown that aristolochic acid is the main factor that causes nephrotoxicity, and its mechanism is mainly that aristolochic acid forms adducts with DNA in the body and induces mutations in the A:T gene. Gene muta...

Claims

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Application Information

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IPC IPC(8): G01N30/02
Inventor 肖红斌刘晓燕程孟春王莉
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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