CN109507337A - A kind of new method based on blood urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism - Google Patents
A kind of new method based on blood urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism Download PDFInfo
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Abstract
The present invention provides a kind of methods based on blood urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism, which is characterized in that shown in specific step is as follows: the selection test group and control group of S1,1:1；S2, sample to be tested is periodically collected；Sample described in S3, processing S2 carries out UPLC-Q-TOF/MS separation analysis；S4, metabolic profiling analysis is carried out to the UPLC-Q-TOF/MS spectrum data that S3 is obtained, obtains data set；S5, PLS-DA model is constructed according to the data set that S4 is obtained；S6, S-PLOT load diagram and VIP score value according to the model of S5 component, filter out the metabolic markers that can distinguish blank group and Gandhi's capsule administration group；S7, enrichment analysis is carried out to the metabolic markers that S6 is screened using IPA analysis method, each metabolic markers is ranked up according to bioaccumulation efficiency.The pharmacological mechanism of Gandhi's capsule for treating diabetic nephropathy can be predicted by this method.
Description
Technical field
It is the present invention relates to metabolism group field, in particular to a kind of to be controlled based on blood/urine metabolite prediction Gandhi's capsule
The method for treating diabetic nephropathy mechanism.
Background technique
Metabolism group is an important branch of systems biology research field, by pathophysiologic factor or gene
Biochemical change and metabolic profiling analysis caused by modification provide massive information for the explaination of complex mechanism, this is multiple with Chinese medicine
The feature that square preparation ingredient is more, pharmacological mechanism is many and diverse is perfectly in harmony, therefore can be used for compound Chinese medicinal preparation pharmaceutical research.Mesh
Before, metabolism group research method is widely used to the research of the pathomechanism and mechanism of drug action of all kinds of metabolic diseases.
Nuclear magnetic resonance (NMR), gas chromatography combined with mass spectrometry (GC-MS) and liquid chromatography mass combination (LC-MS) technology are three kinds common
In the modern analytical technique of metabonomic analysis.
The incidence of Diabetic Nephropathy patients microangiopathies is higher, and blood pressure is the independent hazard factor of kidney function damage,
If hypertensive patient suffers from diabetes simultaneously, diabetic nephropathy incidence will be significantly improved, and clinical manifestation is duration albumen
Urine, patient will enter the kidney failure stage, and the death rate can dramatically increase.Currently, in the market for the Chinese and Western of diabetic nephropathy treatment
Medicine is considerably less.Preparation Gandhi capsule passes through the clinical verification of many years in institute, has the function of significantly reducing albuminuria, long in institute
Treatment of the phase for microangiopathies such as diabetic nephropathies.Pre-clinical assay statistics indicate that, Gandhi's capsule can substantially reduce
The Urinary Microalbumin Excretion for 24 hours of Diabetic Nephropathy patients delays the progress of diabetic nephropathy, protection and improvement renal function,
To improve the quality of life of patient, and patient has clear improvement after taking 2 courses for the treatment of (4 months) of Gandhi's capsule.However, sweet
The molecular mechanism of ground capsule for treating diabetic nephropathy is still not very clear.So a kind of can specify the mechanism of drug action
Method is for study.
Summary of the invention
The present invention aims to overcome the above drawbacks, provides a kind of machine that can specify Gandhi's capsule for treating Diabetic Nephropathy patients
System.
It is provided by the invention a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism side
Method, which is characterized in that shown in specific step is as follows:
The selection test group and control group of S1,1:1；
The test group refers to the sufferer for taking Gandhi's capsule；
The control group refers to the sufferer for not taking Gandhi's capsule.
S2, sample to be tested is periodically collected；
Two groups of sufferer is all made of the collection that same detection cycle carries out sample.
S3, processing S2 above-mentioned sample carries out UPLC-Q-TOF/MS separation analysis；
S4, metabolic profiling analysis is carried out to the UPLC-Q-TOF/MS spectrum data that S3 is obtained, obtains data set；
The metabolic profiling analysis refers to that the relevant default metabolin of similar to structure in specific metabolic process or property uses
Targetedly analytical technology is quantitative determined, such as: a certain class formation is similar, the relevant compound of property.
S5, the data set building OPLS-DA model obtained according to S4 are (using using analysis soft sim CA-P11.0
Modes such as (Umetrics AB, Umea, Sweden)) building Partial Least Squares discriminant analysis (OPLS-DA) model；
S6, according to S-PLOT load diagram in the model of S5 building and VIP score value is calculated, while obtains corresponding P value, with
It is condition that VIP value, which is greater than 1.0 and P value less than 0.05, filters out the metabolism mark that can distinguish blank group and Gandhi's capsule administration group
Will object.Analysis soft sim CA-P11.0 (Umetrics AB, Umea, Sweden) Lai Shixian can be used in the step.
S7, enrichment analysis is carried out to the metabolic markers that S6 is screened using IPA analysis method, by each metabolic markers
It is ranked up according to bioaccumulation efficiency.
Further, of the present invention a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness, also have a characteristic that i.e., above-mentioned metabolic profiling analysis specific steps are as follows: use peak filtering technique, only
Retain the strongest several ion fragments of abundance in identical retention time, deletes other segments.
Or above-mentioned metabolic profiling analysis specific steps are as follows:
S4-1. gamma correction and normalization are carried out to data, retains those RSD in QC sample and is less than X% (X% can
With customized, such as variable 30%)；
The data also can be used the data analysis module built in software Masshunter that instrument is special before carrying out S4-1
MzData format is converted to the LC-MS data of format；
S4-2., the LC-MS data of each sample are standardized as to the sum of all peak areas of each sample, obtain one
New data set.The step can be realized by importing data to 7.0 software of MATLAB (The MathWorks, Inc.) etc..
Further, of the present invention a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness also has a characteristic that the multivariate statistics performance parameter of i.e. above-mentioned OPLS-DA model is R2X=
0.599, R2Y=0.921, Q2=0.745.
Further, of the present invention a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness, also have a characteristic that i.e., above-mentioned sample to be tested be blood sample, urine sample.Also according to the difference of test object
It can be other samples.
Further, of the present invention a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness, also have a characteristic that i.e., the method for above-mentioned processing S2 sample are as follows: by the mixed liquor of methanol and acetonitrile
It is added in sample, is centrifugated after mixing, it is to be measured takes out supernatant.
Further, of the present invention a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness, also have a characteristic that i.e., the volume ratio of above-mentioned methanol and blood sample or urine sample be 4-8:1.
Further, of the present invention a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness, also have a characteristic that i.e., above-mentioned UPLC-Q-TOF/MS separation analysis parameter it is as follows:
The composition of the mobile phase of chromatography are as follows: B:0.1% formic acid acetonitrile, gradient elution are provided that 0-2min:3%B, 2-
11min:3%-15%B, 11-16min:15%-30%B, 16-18min:30%-95%B, 18-19.5min:95%B,
19.5-20min:95%-3%B；4min is balanced later；400 μ L/min of flow velocity, 4 μ L of sample volume；；
Flight mass spectrum uses the source ESI, and parameter is set as positive ion mode；
Capillary voltage: 3500V；Dry gas flow velocity: 11L/min；Spray pressure: 45psig；Dry temperature degree: 350
℃；Fragmentation voltage: 120V；Mass range: m/z 50-1000；Collision energy is set as 10- when MS/MS second mass analysis
30eV。
Further, of the present invention a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness also has a characteristic that i.e., with Student's T-test statistical analysis (such as: using SPSS software
Etc. being calculated) method, judge whether difference metabolin has statistical significance.
Further, of the present invention a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness, also have a characteristic that i.e., above-mentioned metabolite be mainly lipid metabolite and amino acids metabolism
Object.
Further, of the present invention a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness, also have a characteristic that i.e., can be applied to research other drugs therapy mechanism.
Action and effect of the invention:
Diabetic nephropathy is a kind of typical metabolic disease, occurrence and development and energetic supersession, glycometabolism, amino acid
Metabolism and lipid metabolism are in close relations.Therefore, the metabonomic technology based on UPLC-Q-TOF/MS is applied to Gandhi's capsule
The serum of clinical treatment patient and the metabolic profiling analysis of urine specimen are to disclose Gandhi's capsule for treating diabetic nephropathy drug effect machine
The effective means of system.Meanwhile serum sample and urine specimen are acquired, the metabonomic analysis integrated will be more comprehensive
Characterization Gandhi's capsule regulates and controls track and the efficiency of metabolic pathway in human body.
In our current research, we are using UPLC-Q-TOF/MS technology analysis Gandhi's capsule administration group and control group patient's blood
The metabolic profile of cleer and peaceful urine changes, and then uses Multielement statistical analysis method Partial Least Squares discriminant analysis (OPLS-DA)
The variation occurred come metabolic markers after screening Gandhi's capsule for treating 6 months.Clinical generation of this research as Gandhi's capsule for the first time
Xie Zuxue research, it has been found that the influence of Gandhi's capsule metaboilic level, and do in mechanism of action of the metabolic pathway level to it
Preliminary elaboration is gone out, to predict that the pharmacological mechanism of Gandhi's capsule for treating diabetic nephropathy provides new method.
Detailed description of the invention
Fig. 1, typical patients serum UPLC-Q-TOF/MS total ion chromatogram；
Wherein: (A) is that blank group (B) is that 6 months groups are administered in Gandhi's capsule.
Fig. 2, typical patient urine UPLC-Q-TOF/MS total ion chromatogram；
Wherein: (A) is that blank group (B) is that 6 months groups are administered in Gandhi's capsule.
6 months Fig. 3, the blank group (light color) based on UHPLC-Q-TOF/MS data and Gandhi's capsule administration groups (aterrimus)
The OPLS-DA shot chart (A) and S-plot of serum sample scheme (B).
6 months Fig. 4, the blank group (light color) based on UHPLC-Q-TOF/MS data and Gandhi's capsule administration groups (aterrimus)
Urine specimen OPLS-DA shot chart (A) and S-plot figure (B).
The metabolic pathway IPA of Fig. 5, serum and urinary biomarkers object analyze result figure.
Fig. 6, Gandhi's capsule intervene the metabolic pathway analysis chart of diabetic nephropathy.
Specific embodiment
The present embodiment carries out with the following method:
(1) data acquire
The present embodiment is randomly divided into test group and control group using Diabetic Nephropathy patients as study subject, and every group
30 people.
Wherein test group: Diabetic Nephropathy patients add Gandhi's capsule, 3 times a day, 3 tablets each time, are used continuously 6 months.
Control group: Diabetic Nephropathy patients add dregs of a decoction capsule, 3 times a day, 3 tablets each time, are used continuously 6 months.
The patient of acquisition in every 2 months urinates for 24 hours and blood sample.Blood and urine sample use after sample pre-treatments
Acquity UPLC BEH C18 column (2.1mm × 100mm, 1.7 μm, Waters) chromatographic column progress chromatography.Stream
The composition of dynamic phase are as follows: B:0.1% formic acid acetonitrile, gradient elution are provided that 0-2min:3%B, 2-11min:3%-15%B,
11-16min:15%-30%B, 16-18min:30%-95%B, 18-19.5min:95%B, 19.5-20min:95%-3%；
4min is balanced later；400 μ L/min of flow velocity, 4 μ L of sample volume；.Data acquisition uses Agilent 6530Accurate-Mass
Q-TOFMS series connection quadrupole time-of-flight mass spec-trometry instrument, using the source ESI, parameter is set as positive ion mode.Capillary voltage:
3500V；Dry gas flow velocity: 11L/min；Spray pressure: 45psig；Dry temperature degree: 350 DEG C；Fragmentation voltage: 120V；Matter
Measure range: m/z 50-1000.Collision energy is set as 10-30eV when MS/MS second mass analysis.
(2) data acquisition and processing (DAP)
It will be with LC-MS data conversion at NetCDF and mzData format by MassHunter software.It is soft in MATLAB7.0
Using the peak filtering technique inside seminar in part, i.e., only retains the strongest ion fragment of abundance in identical retention time, delete
Except other segments.Next, further being handled with XCMS software data.XCMS software major function is non-to data progress
Linearity correction and normalization.Parameter setting is as follows: full width half max degree (FWHM)=10, bandwidth (bw)=10 and signal-to-noise ratio
(snthresh)=5, other parameters are default value.80% rule generally acknowledged using metabolism group, retains those in QC sample
These data are imported 7.0 software of MATLAB, correction are normalized according to total peak area by variable of the middle RSD less than 30%.
Finally, the Data Integration after the completion of processing is obtained a new data set, and carry out multi-variate statistical analysis.
Using method as above, by the serum and urine sample of 6 months patients after selected blank group and Gandhi's capsule administration
This progress metabolic profiling analysis.The serum UPLC-Q-TOF/MS map of 6 months groups of typical blank group and administration as shown in Figure 1,
Urine UPLC-Q-TOF/MS map is as shown in Figure 2.Data set peak capacity with higher after screening, wherein sera data collection
Containing 579 peaks, the data set of urine then contains 998 peaks.
(3) metabolism group data are analyzed
Partial least squares discriminant analysis (Partial least-squares discriminant analysis, OPLS-
DA) it is a kind of multivariate statistical method for having supervision, is analyzed using 13.0 version of SIMCA-P software, is missed to reduce unit
Difference, data can first pass through log transformation.By analyzing the Loading figure and shot chart of OPLS-DA model, in conjunction with importance parameter
It is condition less than 0.05 that VIP value, which is greater than 1.0 and P value,, filter out the strongest potential metabolic markers of separating capacity between two groups.
Evaluation model quality mainly uses following three parameter (R2X,R2Y and Q2Y), pass through the leave-one-out of default
Process calculates.R2X and R2Y is for evaluating the goodness of fit, Q2Y is for assessment models predictability.
It can be by the characteristic ionic of two groups of differentiations, with UPLC-Q-TOF/MS using as above in the present embodiment in order to obtain
Method, construct OPLS-DA model according to the collected data.As shown in figure 3, multivariate statistics performance parameter is R2X=
0.599, R2Y=0.921, Q2=0.745, show that two models all have highly effective predictive ability.Blank group and Gandhi's glue
The classification specificity that the OPLS-DA model of 6 months group data sets building is administered in capsule has also reached 100%, and sensibility reaches
100%.
According to the S-plot load diagram of OPLS-DA model (Fig. 3 and Fig. 4 shown in) and the score value of VIP, filtering out being capable of area
Divide the metabolic markers of blank group and Gandhi's capsule administration group.It is looked for respectively in the data of UPLC-Q-TOF/MS serum and urine
To 18 and 20 metabolism biological markers, wherein the potential metabolism biological marker in serum is mainly the rouge such as long chain fatty acids
Matter metabolin, also includes purine, creatinine, the potential metabolism biological marker in urine be mainly amino acid, organic acid, purine and
Sphingol etc..
(4) it statisticallys analyze
Difference between different disposal group is compared with Student ' s T-test statistical analysis technique using SPSS software
Whether different metabolin has statistical significance.When P value is less than 0.05, then it is assumed that difference has conspicuousness.
Using statistical method as above, the relative ion intensity of metabolin is imported into SPSS software and carries out t check analysis, knot
Fruit shows that the P value of all differences metabolin is respectively less than 0.05, it was demonstrated that identifies the accurate of difference metabolism biological marker with this method
Property.
(5) metabolic pathway enrichment analysis (IPA)
IPA (Ingenuity Pathway Analysis) is that a kind of Large Scale Biology path analysis based on algorithm is soft
Part.On the one hand it may search for the relevant information of gene, albumen, drug etc., and construct its interactive network model；It is another
Aspect can also analyze the experimental data from genome, micro-RNA, SNP, chip, metabolism group, protein groups etc..This reality
It applies example and enrichment analysis is carried out to the metabolism biological marker that early period, OPLS-DA model discrimination obtained using IPA analysis method, such as scheme
Shown in 5.Wherein the size of circle and shade are the main indicators for judging the access accumulation rate, mainly by the confidence level of access
(- LogP) and disturbance degree (Impact) composition, as shown in the table, the highest access of bioaccumulation efficiency is purine metabolism approach, is come
Second is glycerophosphatide metabolic pathway, and other there are also all kinds of amino acid metabolism approach.It can be seen that purine, glycerophosphatide and
Phenylalanine, arginine, alanine, aspartic acid, proline and glutamine metabolism are that Gandhi's capsule plays treatment diabetes
Nephrosis drug action regulates and controls the critical path of organism metabolism level.
(6) metabolic pathway interaction diagram constructs
The present embodiment is in the metabolism biological marker difference between research Gandhi's capsule administration group and control group, it was found that
38 potential metabolism biological markers.The variation of these markers is in close relations with corresponding metabolic pathway, includes lipid generation
It thanks, sphingolipid metabolism, purine metabolism, phenylalanine, arginine, alanine, aspartic acid, proline and glutamine metabolism etc..
In order to excavate the relationship between these significant metabolins, by searching for KEGG PATHWAY database, metabolic pathway phase is constructed
Interaction figure is as shown in Figure 6.The visualization changed by metabolism biological marker, can preferably conclude, summarize Gandhi's capsule
Administration group changes in the access of metaboilic level.
The effect and effect of the present embodiment:
The present embodiment is constructed sweet by combining UPLC-Q-TOF/MS data and Multielement statistical analysis method (OPLS-DA)
The metabolism group method of ground capsule for treating diabetic nephropathy Mechanism Study.It is studied by the method, it has been found that 39 kinds of serum
And (or) the metabolic markers of urine, it includes lipid generation that changing for these markers is in close relations with corresponding metabolic pathway
It thanks, sphingolipid metabolism, purine metabolism, phenylalanine, arginine, alanine, aspartic acid, proline and glutamine metabolism etc..
The chronic inflammation that diabetes cause can lead to injury of kidney through a variety of ways, cause body apparent oxidation occur and answer
Swash, and generate a large amount of ROS, ROS, which can damage vascular wall, leads to vascular inflammation.In addition, CRP also can directly inducing endothelial cell be generated
The expression of plasma plasminogen activator inhibitor -1 (Pal-1) mRNA and Pal-1 albumen, and increase its activity, it aggravates
Kidney damage.By synthesis adhesion molecule and monocyte chemoattractant protein-1, promote leucocyte synthesis releasing superoxide and egg
White hydrolase, causes renal tissue to damage, and clinically shows as albuminuria and disorders of lipid metabolism.
Phospholipid metabolic disorder is the key factor of the morbidity of diabetic nephropathy, and phosphatide is the primary structure composition of biomembrane
Part, it contains, and there are many fatty acid compositions, such as phosphatidyl glycerol (PG), phosphatide acid imide (PE), phosphatidylinositols (PI), phosphatide
Acyl inositol (PS), phosphatidyl choline (PC), sphingomyelin (sphingom-yelin, SM) and lysophosphatidyl choline (LPC).
Other than this structure function, PC participates in the process that neutral fat and cholesterol deposition are emulsified in blood vessel, can improve intelligence, and activation is thin
Born of the same parents.In addition, their metabolism is closely related with many diseases, such as Alzheimer's disease, obesity and cancer.Due to this
A little important biological functions, PC have obtained more and more concerns in many fields.A large number of studies show that disorders of lipid metabolism with
Type-2 diabetes mellitus and diabetic nephropathy are directly related.In some diseases model, PC molecule has become type-2 diabetes mellitus and glycosuria
Sick nephrosis has the biomarker of important adjusting or adjusting expressional function.
The present embodiment finds Gandhi's capsule administration group compared with the control group, phosphatide in serum by the method for metabolism group
The horizontal of metabolite significantly reduces, be related to LysoPC (18:2 (9Z, 12Z)), PC (0:0/18:0), LysoPE (20:0/0:
0) long-chains saturation or the unsaturated phosphatide constituents such as.Changed according to the concentration of OPLS-DA tendency chart and potential source biomolecule marker, table
It is bright that abnormal phospholipid metabolism has occurred in Diabetic Nephropathy patients.Mechanism includes rope than alcohol approach, oxidative stress, albumen
Three approach of kinase c (PKC) are activated under the glucose high concentration environment of diabetes.And the activation (PLA2) of phosphatide and PKC
Activation it is related.Phosphatidase is enzyme important in human body, can be catalyzed the decomposition of phosphatide, generates free fatty acid.Therefore, it activates
PLA2 will accelerate the decomposition of phosphatide.PC the and LPC metaboilic level obtained in our experiment improves result and this mechanism
It is consistent.With the development of diabetic nephropathy, PLA2 is activated, so the concentration of PC reduces.PC is under phosphatase catalytic
A fatty acid chain is lost, when PC is decomposed, the concentration of PC is increased.Abnormal phosphatization inositol (PI) metabolism is under one kind
Drop trend, this phenomenon may be related with the activation of sorbierite approach (SP).Under the intervention of Gandhi's capsule, LysoPC (18:2
(9Z,12Z))、PC(0:0/18:0)、LysoPE(20:0/0:0)、PI(22:0/20:4(5Z,8Z,11Z,14Z))、PC(18:2
(2E, 4E)/0:0) etc. phosphatide constituents relative amount significantly reduce, this prompts us, Gandhi's capsule can pass through intervene phosphatide
Metabolism, inhibits the decomposition and metabolism of phosphatide, plays adjustment effect to the development of diabetic nephropathy.Many studies have shown that LysoPC contains
The change of amount and the physiological change of Apoptosis are related, including mitochondria dysfunction and oxidative stress.However, specific mechanism
It is not clear, it is still necessary to the further relationship between research Gandhi's capsule and LysoPC variation.
In addition, the present embodiment the results show that compared with the control group, uric acid in Gandhi's capsule administration group, hypoxanthine and
The horizontal of the metabolins such as purine significantly reduces.Uric acid is to be generated by purine metabolism, therefore our result of study shows that Huang is fast
The activation of purine oxidizing ferment and the formation of microalbuminuria are closely related.In Diabetic Nephropathy patients, purine metabolism is close with albuminuria
Cut phase is closed, this shows during disease, and purine metabolism gets muddled phenomenon.Uric acid, hippuric acid, hypoxanthine be all from
The product generated in purine metabolism, two kinds of crucial purine catabolisms (xanthine and hypoxanthine) are by xanthine oxidase
(XO) and active oxygen acts on generation.This result shows that, uric acid, hypoxanthine in the patient urine after Gandhi's capsule for treating
It is significantly reduced with the horizontal of metabolins such as purine, illustrates that the activation of xanthine oxidase is significantly inhibited, purine metabolic disturbance
It also tends to gentle.
Amino acid metabolism is also that IPA analyzes the key metabolism biological marker being important to note that, is related to phenylalanine, color ammonia
The metabolic pathways such as acid, aspartic acid, leucine and proline.Show to include valine, leucine, different bright ammonia in research recently
Branched-chain amino acid (BCAA) and the aromatic amino acids such as tyrosine and phenylalanine including acid be considered as diabetes serum and
The strong predictive factor and biomarker of urine specimen.Equally, serum leucine content or urine metabolite BCAA and aromatic series
Conspicuousness variation also has occurred in amino acid in Diabetic Nephropathy patients.Therefore, the change of amino acid metabolite is largely
It is had a significant impact to kidney filtration state is destroyed, the generation of this and diabetic nephropathy renal tissue lesion has important relation.
Obtaining Gandhi's capsule as a result, mainly influences Diabetic Nephropathy patients amino acid metabolism, lipid-metabolism and purine metabolism
Be consistent as a result, albuminuria can be improved with Gandhi's capsule in clinical practice, also meet with previous research.
To sum up, the present embodiment is using the technique study Gandhi's capsule administration group of UPLC-Q-TOF/MS metabolism group and right
Change according to the metabolic profile of group patients serum and urine, and uses the discriminant analysis of Multielement statistical analysis method Partial Least Squares
(OPLS-DA) variation that patient's serum and urine metabolism marker occur after 6 months of Gandhi's capsule for treating has been screened.As a result table
Bright, UPLC-MS sera data collection contains 579 peaks, and the data set of urine then contains 998 peaks.The statistic property parameter of data
For R2X=0.599, R2Y=0.921, Q2=0.745, what these parameters showed the two models all and have height is effectively predicted energy
Power.We screen potential source biomolecule marker in the data of UPLC-Q-TOF/MS serum and urine, wherein potential in serum
Metabolism biological marker is mainly the lipid-metabolisms object such as long chain fatty acids, also comprising purine, creatinine etc., the potential metabolism in urine
Biomarker is mainly amino acid, organic acid, purine and sphingol etc..Using IPA analysis method to Key Metabolic access into
Row enrichment.The highest access of bioaccumulation efficiency is purine metabolism approach, and coming second is phospholipid metabolism approach, and other there are also all kinds of
Amino acid metabolism approach.It can be seen that purine, glycerophosphatide and phenylalanine, arginine, alanine, aspartic acid, proline
And glutamine metabolism, it is that Gandhi's capsule plays treatment diabetic nephropathy drug action, the horizontal crucial way of regulation organism metabolism
Diameter.
The present embodiment changed using the metabolic markers taken after Gandhi's capsule in blood urine, success prediction its improving
Effect in terms of purine metabolism, phospholipid metabolism, amino acid metabolism.And the result of this and Gandhi's capsule clinical research is consistent,
Reduce microdose urine protein for 24 hours.The present embodiment also for predict other compound Chinese medicinal preparations pharmacological mechanism provide it is feasible
Method.
Claims (10)
1. a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism method, which is characterized in that
Shown in specific step is as follows:
The selection test group and control group of S1,1:1；
S2, sample to be tested is periodically collected；
Sample described in S3, processing S2 carries out UPLC-Q-TOF/MS separation analysis；
S4, metabolic profiling analysis is carried out to the UPLC-Q-TOF/MS spectrum data that S3 is obtained, obtains data set；
S5, OPLS-DA model is constructed according to the data set that S4 is obtained；
S6, S-PLOT load diagram and VIP score value according to the model of S5 component, while corresponding P value is obtained, it is greater than with VIP value
1.0 and P value is condition less than 0.05, filters out the metabolic markers that can distinguish blank group and Gandhi's capsule administration group；
S7, enrichment analysis is carried out to the metabolic markers that S6 is screened using IPA analysis method, by each metabolic markers according to
Bioaccumulation efficiency is ranked up.
2. as described in claim 1 a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism
Method, it is characterised in that:
The metabolic profiling analysis refers to using peak filtering technique, if only retaining the strongest dry ion of abundance in identical retention time
Segment deletes other segments.
3. as described in claim 1 a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism
Method, it is characterised in that:
The step of metabolic profiling analysis, is as follows:
S4-1. gamma correction and normalization are carried out to data, retains the variable that those RSD in QC sample are less than X%；
S4-2., the LC-MS data of each sample are standardized as to the sum of all peak areas of each sample, obtain one it is new
Data set.
4. as described in claim 1 a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism
Method, it is characterised in that:
The multivariate statistics performance parameter of the OPLS-DA model is R2X=0.599, R2Y=0.921, Q2=0.745.
5. as described in claim 1 a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism
Method, it is characterised in that:
The method of the processing S2 sample are as follows: the mixed liquor of methanol and acetonitrile is added in sample, after mixing centrifugation point
From taking-up supernatant is to be measured.
6. as claimed in claim 5 a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism
Method, it is characterised in that:
The volume ratio of the mixed liquor and sample of the methanol and acetonitrile is 4-8:1.
7. as described in claim 1 a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism
Method, it is characterised in that:
The parameter of the UPLC-Q-TOF/MS separation analysis is as follows:
The composition of the mobile phase of chromatography are as follows: B:0.1% formic acid acetonitrile, gradient elution are provided that 0-2min:3%B, 2-
11min:3%-15%B, 11-16min:15%-30%B, 16-18min:30%-95%B, 18-19.5min:95%B,
19.5-20min:95%-3%；4min is balanced later；400 μ L/min of flow velocity, 4 μ L of sample volume；
Flight mass spectrum uses the source ESI, and parameter is set as positive ion mode；
Capillary voltage: 3500V；Dry gas flow velocity: 11L/min；Spray pressure: 45psig；Dry temperature degree: 350 DEG C；It is broken
Split voltage: 120V；Mass range: m/z 50-1000；Collision energy is set as 10-30eV when MS/MS second mass analysis.
8. as claimed in claim 1 a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness, it is characterised in that:
With Student's T-test statistical analysis technique, judge whether difference metabolin has statistical significance.
9. as described in claim 1 a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetic nephropathy mechanism
Method, it is characterised in that:
The metabolite is mainly lipid metabolite and amino acids metabolin.
10. as claimed in claim 1 a kind of based on blood/urine metabolite prediction Gandhi's capsule for treating diabetogenous nephrosis
The method of the interpretation of the cause, onset and process of an illness, it is characterised in that:
The therapy mechanism of detection other drugs can be applied to.
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