CN106551930B - Application of the tetrahydropalmatine in anti-toxicity of cisplatin medicine is prepared - Google Patents
Application of the tetrahydropalmatine in anti-toxicity of cisplatin medicine is prepared Download PDFInfo
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Abstract
The present invention provides application of the tetrahydropalmatine in anti-toxicity of cisplatin medicine is prepared.The toxicity includes Toxicity of Kidney, ototoxicity and neurotoxicity.Research shows that tetrahydropalmatine is inhibited with MATE1 to OCT2, cytotoxicity caused by energy antagonism cis-platinum, and is preferably verified on the renal cells model of original cuiture.And using () THP as representative, research finds that tetrahydropalmatine has good protective effect to cis-platinum induced mice injury of kidney.Therefore, tetrahydropalmatine shares with cis-platinum, is likely to reduced cis-platinum and is accumulated in kidney, cells,cochlear and DRGs, so as to reach prevention or reduce the expected results of toxicity of cisplatin.The present invention has expanded the new application of tetrahydropalmatine；Due to the high expression OCT2 of kidney, cochlear hair cell and DRGs, and often lack OCT2 expression on tumour cell, cis-platinum has certain passive permeability, therefore, tetrahydropalmatine shares with cis-platinum, while decrease cis-platinum institute is causing toxicity, can't reduce the antitumous effect of cis-platinum.
Description
Technical field
The invention belongs to medicinal usage, is related to application of the tetrahydropalmatine in drug toxicity caused by cis-platinum is reduced, including kidney
Dysentery, ototoxicity and neurotoxicity.
Technical background
Cis-platinum is one of current clinically maximally effective broad-spectrum anti-cancer drug, is widely used in oophoroma, prostate cancer, lung
A variety for the treatment of of solid tumor such as cancer and breast cancer, but its serious toxicity, including renal toxicity, ototoxicity, neurotoxicity etc.
Its clinical practice is limited, it is especially the most serious with renal toxicity and ototoxicity.Clinic is although with hydration therapy or forces profit
The safeguard procedures such as urine, the renal toxicity incidence of cis-platinum is still up to 25 ~ 35%, and occurs immediately initial stage in treatment.Should by oxidation
The mechanism such as sharp, DNA damage and inflammatory reaction, cis-platinum can cause renal tubular cell dead, Renal tissues damage, so that glomerulus is filtered
Rate decline is crossed, kidney excretion substantially reduces, causes it largely to be accumulated in kidney, cause serious kidney failure.Cis-platinum also can
Cochlear hair cell is damaged, causes hearing disability, causes deaf or tinnitus.Therefore, cis-platinum institute is causing toxicity be clinical chemotherapy during
Urgent problem to be solved, the research of its safeguard procedures are also study hotspot in recent years.The conventional anti-inflammatory of clinic, anti-oxidant etc. are arranged
Apply, to protect kidney injury caused by cis-platinum, cells,cochlear damage and peripheral nerve injury, but effect is unsatisfactory.
The generation of cisplatin-induced nephrotoxicity, ototoxicity and neurotoxicity is a complicated multiple-factor participation process, but it is in kidney
Height concentration in Tubular epithelial cell, cochlear hair cell and dorsal root ganglion neurons cell is the key of its toxicity.Cis-platinum
After intravenously administrable, it is distributed with blood circulation to each histoorgan of whole body, wherein concentration highest is nephridial tissue, and especially kidney is small
Pipe epithelial cell.A variety of intake types and outer row's type drug transporters, including basilar memebrane side spy are expressed on renal cells film
The Organic cation transporter 2 of different in nature high expression（organic cation transporter, OCT2）And teleblem side multiple medicine and
Toxin efflux protein (multidrug and toxin extrusion proteins, MATEs), in the kidney of cationic drug
Played an important role in dirty transmembrane transport and kidney disposal.Cis-platinum is absorbed into renal tubular cell through OCT2 by blood, small in kidney
Pipe is accumulated；Cis-platinum is pumped out renal tubular cell by outer row's type transporter MATEs (MATE1 and MATE2-K), reduces it in nephrocyte
Concentration.Because OCT2 is to the huge uptake of cis-platinum, and outer row weaker to it MATEs, cis-platinum is seriously accumulated in kidney, from
And cause Toxicity of Kidney.Equally, OCT2 transporters height is expressed in cochlear hair cell and Dorsal Root Ganglion Neurons, mediates the thin of cis-platinum
Born of the same parents absorb, and cause cis-platinum to cause serious ototoxicity and neurotoxicity in cells accumulation.Clinic is attempted with Imatinib, western miaow
Cis-platinum nephrocyte is absorbed by suppressing OCT2 for the OCT2 such as fourth inhibitor, to reduce the Toxicity of Kidney caused by cis-platinum.It is however, western
Miaow is better than OCT2 for fourth Reverse transcriptase MATEs effects, strengthens cisplatin-induced nephrotoxicity on the contrary.For Cisplatin Ototoxicity or Nervous toxicity
Property, the method for the conventional oxidation and removing free radicals of clinic carrys out antagonism, but it is very small to produce effects.Therefore, find to OCT2 high inhibitions, it is right
MATEs unrestraints or the medicine of weak suppression, to reduce accumulation of the cis-platinum in kidney, cells,cochlear and DRGs, so as to drop
Its low toxicity is to expand the fine approach of cis-platinum clinical practice.
Tetrahydropalmatine (tetrahydropalmatine, THP), including rotundine ((-)-THP), dextrorotation
Tetrahydropalmatine ((+)-THP), and dltetrahydropalmatine ((±)-THP), it is the main effects composition of corydalis tuber (corydalis tuber)
One of, there are a variety of physiologically actives such as analgesia, anti-oxidant.Wherein (-)-THP is usually used in alleviating a variety of pain such as headache, pectoralgia,
And there is the advantages of non-additive, toxic side effect is small.This research is first in stable expression people OCT2, MATE1, coexpression human OCT2
Examined with the mouse renal epithelial cell of MATE1 transgenic cell and original cuiture, cochlear hair cell, Dorsal Root Ganglion Neurons
Tetrahydropalmatine is examined, including (-)-THP, (+)-THP and (±)-THP can be reduced thin to OCT2 and MATE1 inhibitory action
Intracellular cis-platinum gathers, and so as to antagonism cis-platinum, institute is cytotoxicity caused；Further it is studied to cis-platinum small by representative of (-)-THP
Mouse kidney, cochlea and DRGs accumulation and toxicity influence, using illustrate tetrahydropalmatine as cisplatin induced renal toxicity,
The meaning of ototoxicity and neurotoxicity toxicity-reducing medicament, provided for tumor patient and have attenuation, analgesia concurrently, aided in without addicted tumour
Medicine.Tetrahydropalmatine structural formula：
The content of the invention
It is an object of the invention to provide application of the tetrahydropalmatine in anti-toxicity of cisplatin medicine is prepared.The toxicity includes
Toxicity of Kidney, ototoxicity and neurotoxicity.
Research shows that tetrahydropalmatine, including (-)-THP, (+)-THP and (±)-THP have to OCT2 and MATE1
Inhibitory action, cytotoxicity caused by energy antagonism cis-platinum, and obtained preferably on the renal cells model of original cuiture
Checking.And using (-)-THP as representative, research finds that tetrahydropalmatine has protection well to cis-platinum induced mice injury of kidney
Effect.Therefore, tetrahydropalmatine shares with cis-platinum, is likely to reduced cis-platinum and is accumulated in kidney, cells,cochlear and DRGs, from
And reach prevention or reduce the expected results of toxicity of cisplatin.
Usefulness of the present invention is：(1) tetrahydropalmatine is the principle active component of one of " eight Zhe's " corydalis tuber, and
Yuanhu Zhitong Prescription principle active component, (-)-THP are even more clinical conventional antalgesic, have been used for treating pain caused by tumour, and
Without into recessiveness.Therefore the present invention will expand corydalis tuber, Yuanhu Zhitong Prescription and (-)-THP new application；(2) although existing researcher
Wish by suppression of the Cisplatin medicine to OCT2 to reduce cisplatin-induced nephrotoxicity, but the clinical OCT2 inhibitor attempted is (such as
Cimetidine) often due to there is stronger suppression to being arranged outside the cis-platinum of MATE1 mediations, cis-platinum is accumulated more in kidney on the contrary
More, toxicity is stronger.The study find that suppression of the tetrahydropalmatine to OCT2 is noticeably greater than the suppression to MATE1, can significantly reduce
Renal toxicity, ototoxicity and the neurotoxicity of cis-platinum；(3) OCT2 is expressed because kidney, cochlear hair cell and DRGs are high,
And often lack OCT2 expression on tumour cell, because cis-platinum has certain passive permeability, therefore, tetrahydropalmatine with
Cis-platinum shares, and while decrease cis-platinum institute is causing toxicity, can't reduce the antitumous effect of cis-platinum.
Brief description of the drawings
Fig. 1 cis-platinums concentration dependent toxicity on MDCK-hOCT2, MDCK-hOCT2/hMATE1 and mock cells.
Using solvent control group cytoactive average as 100%, data are represented with mean ± SD in figure, n=3；Compared with mock cells, *P<
0.05, * *P<0.01 and * * *P<0.001。
Fig. 2 (-)-THP, (+)-THP and (±)-THP concentration suppress MPP according to lazyness+On MDCK-hOCT2 cells
Accumulation.With solvent control group MPP+ Uptake values are 100%, and data are represented with mean ± SD in figure, n=3.
Fig. 3 (-)-THP, (+)-THP and (±)-THP concentration suppress MPP according to lazyness+On MDCK- hMATE1 cells
Accumulation.With solvent control group MPP+ Uptake values are 100%, and data are represented with mean ± SD in figure, n=3.
Fig. 4 (-)-THP, (+)-THP and (±)-THP is to the concentration of cis-platinum toxicity on MDCK-hOCT2 cells according to lazyness
Property inhibitory action.Using solvent control group numerical value as 100%, data are represented with mean ± SD in figure, n=3；Compared with control group,### P
<0.001；Compared with cis-platinum group, * * *P<0.001。
Fig. 5 (-)-THP, (+)-THP and (±)-THP toxicity on MDCK-hOCT2/hMATE1 cells to cis-platinum
Concentration is according to lazyness inhibitory action.Using solvent control group numerical value as 100%, data are represented with mean ± SD in figure, n=3；With control group
Compare,### P<0.001；Compared with cis-platinum group, *P<0.05, * *P<0.01 and * * *P<0.001。
Fig. 6 (-)-THP, (+)-THP and (±)-THP concentration reduce MDCK-hOCT2 cells breast caused by cis-platinum according to lazyness
The seepage of acidohydrogenase (LDH).Using solvent control group LDH vigor as 100%, data are represented with mean ± SD in figure, n=3；With
Control group compares,### P<0.001；Compared with cis-platinum group, * *P<0.01 and * * *P<0.001。
Fig. 7 (-)-THP, (+)-THP and (±)-THP concentration reduce cis-platinum according to lazyness and cause MDCK-hOCT2/hMATE1
The seepage of cell lactic dehydrogenase (LDH).Using solvent control group LDH vigor as 100%, data are represented with mean ± SD in figure,
n=3；Compared with control group,### P<0.001；Compared with cis-platinum group, *P<0.05, * *P<0.01 and * * *P<0.001。
Fig. 8 (-)-THP, (+)-THP and (±)-THP toxicity on mouse primary renal cells to cis-platinum
Concentration is according to lazyness inhibitory action.Using solvent control group numerical value as 100%, data are represented with mean ± SD in figure, n=3；With control group
Compare,### P<0.001；Compared with cis-platinum group, * * *P<0.001。
Distributions of Fig. 9 (-)-THP in mice plasma and renal tissue.Data are represented with mean ± SD in figure, n=5.
Figure 10 (-)-THP (20 mg/kg, tail vein injection,iv) to cis-platinum (10 mg/kg, iv) in mouse kidney
The influence of concentration.Data are represented with mean ± SD in figure, n=6-10；Compared with cis-platinum group, * * *P<0.001。
Figure 11 (-)-THP (20mg/kg, tail vein injection,iv) mouse weight is administered to cis-platinum (10 mg/kg, iv)
Influence.Data are represented with mean ± SD in figure, n=6-10；Compared with control group,### P<0.001；Compared with cis-platinum group, *P<
0.05 and * *P<0.01。
Figure 12 (-)-THP (20 mg/kg, tail vein injection,iv) to cis-platinum (10 mg/kg, iv) mouse renal function
Influence.Using solvent control group biochemical indicator average as 100%, data are represented with mean ± SD in figure, n=6-10；With control group
Compare,### P<0.001；Compared with cis-platinum group, * * *P<0.001.
The representative collection of illustrative plates of Figure 13 kidney of mouse pathological examinations.(A) solvent control group (control) mouse
Kidney sections；(B) cis-platinum group (10mg/kg,iv) mouse kidney section；(C) merge give cis-platinum (10mg/kg,iv)
With (-)-THP (20mg/kg,iv) group mouse kidney section.Renal tissues pathology changes：In black arrow meaning renal tubule
Cast, the necrosis of green arrow meaning renal cells.Leica DM2500 microscopes, 10*20 times is taken pictures.
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
The cis-platinum of embodiment 1 concentration dependent poison on MDCK-hOCT2, MDCK-hOCT2/hMATE1 and mock cells
Property
1.1 on 96 orifice plates inoculating cell
It is (big by Zhejiang to choose MDCK-hOCT2, MDCK-hOCT2/hMATE1 and the mock cell in exponential phase
Pharmaceutical college's structure is learned, specific construction method is shown in a:Lei Hongmei etc., it is stable express hMATE1 and coexpression hMATE1 with
The structure of hOCT1 or hOCT2 cell models.Acta Pharmaceutica Sinica, 2015,50 (7)：842-847.b:Wang K., et al.,
Involvement of organic cation transporter 2 inhibition in potential
mechanisms of antidepressant action.Prog Neuropsychopharmacol Biol Psychiatry, 2014,53:90-98), digest, collect cell.It is close with fresh culture again suspension cell, adjustment cell
Spend for 4.0 × 104Individual/mL, 96 porocyte plates cultures are inoculated in 0.2 mL/ holes.
1.2 MTT experiment
After the h of cell culture 24, reject culture medium, the culture medium of addition medicine containing various concentrations (cis-platinum), with containing corresponding
The culture medium of concentration solvent (DMSO) is blank control group.48 h are cultivated after administration and carry out MTT experiments, add 5 mg/ per hole
The μ L of mL MTT (thiazole bromide blue tetrazolium) reagent 20, gently shake up, and after being incubated 4h, discard Incubating Solution, are added in every hole
150 μ L DMSO, micro shaker vibrate 10 min, after formazan crystallization is completely dissolved, enzyme-linked immunosorbent assay instrument measure 570
The absorbance of each sample in nm wavelength (using 630 nm as reference) place, with the average of absorbance/blank control group absorbance
Represent the survival rate of the sample cell.Data are represented with mean ± SD (n=3), to be not added with the control group of cis-platinum
(control) cell survival rate is 100%；Compared with control group, * * *P <0.001, * *P<0.01 and *P< 0.05。
1.3 result
MTT experiment result shows, MDCK-hOCT2, MDCK-hOCT2/hMATE1 and mock cells and cis-platinum (0.01-
1000 μm of ol/L) be incubated 48 h altogether after, MDCK-hOCT2 and MDCK-hOCT2/hMATE1 cells are sensitive to the toxicity of cis-platinum
Property is significantly larger than mock cells, its LD50Value is respectively 8.77 ± 0.21 μm of ol/L, 5.49 ± 0.18 μm of ol/L and
14.8 ± 3.5 μm of ol/L, referring to Fig. 1（Using solvent control group cytoactive average as 100%, data are with mean ± SD in figure
Represent, n=3；Compared with mock cells, *P<0.05, * *P<0.01 and * * *P<0.001）.The above results prompting hOCT2 is situated between
Leading cis-platinum cellular uptake increases cells accumulation, causes its cytotoxicity to strengthen.
The tetrahydropalmatine of embodiment 2, including (-)-THP, (+)-THP and (±)-THP are to MPP+In MDCK-hOCT2
With the inhibitory action gathered on MDCK- hMATE1 cells
Choose in exponential phase MDCK-hOCT2, MDCK-hOCT2/hMATE1 and MDCK-hMATE1 cell (by
Pharmaceutical college of Zhejiang University builds, and specific construction method is shown in a:Lei Hongmei etc. is stable to express hMATE1 and coexpression hMATE1
With the structure of hOCT1 or hOCT2 cell models.Acta Pharmaceutica Sinica, 2015,50 (7)：842-847.b:Wang K., et
al., Involvement of organic cation transporter 2 inhibition in potential
mechanisms of antidepressant action.Prog Neuropsychopharmacol Biol Psychiatry, 2014,53:90-98), digest, collect cell.It is close with fresh culture again suspension cell, adjustment cell
Spend for 2.0 × 105Individual/mL, 24 porocyte plates cultures are inoculated in 0.5 mL/ holes, when cell growth is i.e. available up to 90% degrees of fusion
Tested in accumulation.
MDCK-hOCT2 and MDCK-hMATE1 cells are high expression people OCT2 and the mdck cell of MATE1 transporters.Research
0.001 ~ 100 μm of ol/L (-)-THP, (+)-THP and (±)-THP have been investigated to MPP+(OCT2 and MATE1 transporters warp
Allusion quotation substrate, 1 μm of ol/L, 3min) influence gathered in above-mentioned cell.As a result show：(-)-THP, (+)-THP and
(±)-THP can significantly inhibit MPP+Accumulation on MDCK-hOCT2 and MDCK-hMATE1 cells, to OCT2 inhibition constants
IC50Respectively 2.6,9.5,0.24 μm of ol/L, to MATE1 inhibition constants IC50Respectively 7.4,8.6,2.6 μm of ol/L, ginseng
Fig. 2, Fig. 3 are seen, with solvent control group MPP+Uptake values are 100%, and data are represented with mean ± SD (n=3) in figure, with not
The cell survival rate for adding THP is 100%.The above results are prompted：(-)-THP, (+)-THP and (±)-THP to OCT2 and
MATE1 is respectively provided with stronger inhibitory action, and (-)-THP and (±)-THP are weaker than the suppression to OCT2 to MATE1 rejection abilities
System.
Embodiment 3 (-)-THP, (+)-THP and (±)-THP is to cis-platinum in MDCK-hOCT2 and MDCK-hOCT2/
The influence of toxicity on hMATE1 cells
3.1 MTT experiment
MDCK-hOCT2 and MDCK-hOCT2/hMATE1 in exponential phase are chosen (by pharmaceutical college of Zhejiang University structure
Build, specific construction method is shown in a:Lei Hongmei etc. is stable to express hMATE1 and coexpression hMATE1 and hOCT1 or hOCT2
The structure of cell model.Acta Pharmaceutica Sinica, 2015,50 (7)：842-847.b:Wang K., et al., Involvement of
organic cation transporter 2 inhibition in potential mechanisms of
antidepressant action.Prog Neuropsychopharmacol Biol Psychiatry, 2014,53: 90-
98), digest, collect cell.With fresh culture again suspension cell, adjustment cell density is 4.0 × 104mL-1, with 0.2mL/
Hole is inoculated in 96 porocyte plates cultures.After the h of cell culture 24, reject culture medium, addition contains 1 ~ 100 μm of ol/L (-)-THP,
(+)-THP and (±)-THP cis-platinum culture medium solution, using the culture medium of solvent containing respective concentration (DMSO) as blank control
Group.48 h are cultivated after administration and carry out MTT experiments, add 5 mg/mL MTT (thiazole bromide blue tetrazolium) reagent 20 per hole
μ L, are gently shaken up, and after being incubated 4h, discard Incubating Solution, and 150 μ L DMSO, micro shaker vibration 10 are added in every hole
Min, after formazan crystallization is completely dissolved, enzyme-linked immunosorbent assay instrument measure 570 nm wavelength (using 630 nm as reference) place
The absorbance of each sample, the survival rate of the sample cell is represented with the average of absorbance/blank control group absorbance.
The measure of LDH vigor in 3.2 cell culture fluids
The cell culture based specimen collected after 48 h of administration culture carries out LDH vitality tests, according to lactic dehydrogenase
(LDH) determine kit specification and require LDH vigor in measure measure cell culture fluid.The solvent control being administered with each concentration
Group enzyme activity is 100% progress relative quantification.
3.3 result
(-)-THP is investigated with the LDH vigor in MTT experiments and cell culture fluid, (+)-THP and-THP pairs of (±)
The influence of cis-platinum toxicity on MDCK-hOCT2 and MDCK-hOCT2/hMATE1 cells.As a result show, cis-platinum significantly reduces
Cell survival rate, and (-)-THP, (+)-THP and (±)-THP concentration resist cis-platinum according to lazyness (1 ~ 100 μm of ol/L)
The cell survival rate of (cisplatin, 15 μm of ol/L) induction reduces, referring to Fig. 4, Fig. 5（In figure using solvent control group numerical value as
100%, data are represented with mean ± SD in figure, n=3；Compared with control group,### P<0.001；Compared with cis-platinum group, *P<
0.05, * *P<0.01 and * * *P<0.001）.Meanwhile (-)-THP, (+)-THP and (±)-THP (1 ~ 100 μm of ol/L) can be bright
The aobvious rise for reducing cell LDH vigor caused by cis-platinum, referring to Fig. 6, Fig. 7, using solvent control group LDH vigor as 100% in figure,
Data are represented with mean ± SD in figure, n=3；Compared with control group,### P<0.001；Compared with cis-platinum group, *P<0.05, * *P<
0.01 and * * *P<0.001。
Embodiment 4 (-)-THP, (+)-THP and (±)-THP toxicity on mouse primary renal cells to cis-platinum
Influence
The separation and culture of 4.1 mouse primary renal cellses
Healthy ICR mouse (male, ~ 25g), 75% alcohol-pickled 1 minute, is fixed on operating table after etherization.Split
Abdominal cavity, with without Ca2+HBSS solution from vena portae hepatica perfusion to kidney existing canescence, remove rapidly double kidneys be placed in it is sterile added with double
In the PBS solution of anti-(streptomysin of 1% penicillin+1%).Cleaned several times with added with dual anti-PBS solution in sterile super-clean bench,
Kidney coating is removed, is shredded；After cleaning 3 ~ 5 times, shredded with aseptic operation blade, be placed in 0.1% type Ⅳ collagenase and 0.1% pancreatin
Mixed solution in concussion digestion about 40min.Supernatant liquid (contains DMEM/ in 80 mesh sieve net filtrations with DMEM/F12 culture mediums
F12 culture mediums, 10% hyclone, 1% is dual anti-, the agent of 1% Insulin-Transferrin-selenium three) eccentric cleaning 2 times, it is resuspended in training
Support in base, planted after crossing 200 eye mesh screens in 96 orifice plates, and change liquid every other day, growth can be used to cytotoxicity experiment after about 5 ~ 6 days.
4.2 MTT experiment
After Primary kidney cells culture about 5 days, reject culture medium, add containing 1 ~ 100 μm of ol/L (-)-THP, (+)-THP and
(±)-THP cis-platinum culture medium solution, using the culture medium of solvent containing respective concentration (DMSO) as blank control group.Trained after administration
Support 48 h and carry out MTT experiments, add the μ L of 5 mg/mL MTT (thiazole bromide blue tetrazolium) reagent 20 per hole, gently shake
It is even, after being incubated 4h, Incubating Solution is discarded, adds 150 μ L DMSO in every hole, micro shaker vibrates 10 min, Dai formazan knots
After crystalline substance is completely dissolved, enzyme-linked immunosorbent assay instrument determines the extinction of each sample in 570 nm wavelength (using 630 nm as reference) place
Degree, the survival rate of the sample cell is represented with the average of absorbance/blank control group absorbance.
4.3 result
MTT experimental results show, (-)-THP, (+)-THP and (±)-THP can concentration according to lazyness (1 ~ 100
μm ol/L) toxicity of the cis-platinum (cisplatin, 15 μm of ol/L) on mouse primary nephrocyte is reduced, increase cell survival
Rate, referring to Fig. 8, using solvent control group numerical value as 100% in figure, data are represented with mean ± SD in figure, n=3；With control group ratio
Compared with,### P<0.001；Compared with cis-platinum group, * * *P<0.001。
Influence to induced by Cisplatin renal toxicity
5.1 experimental animal
ICR mouse (25 ~ 30g), male, (cleaning two level, animal license are provided by Zhejiang Academy of Medical Sciences animal center
Card number：SCXK (Zhejiang) 20140001).All animals are raised according to the requirement of Zhejiang University's experimental animal Operating Guideline, raise
Support environmental Kuznets Curves suitable temperature (20 ~ 23 DEG C) and humidity (50 ~ 60%), 12 h fasting, free water before experiment.
5.2 (-)-THP renal tissues are distributed
5.2.1 animal administration and sample collection
ICR mouse 25, it is randomly divided into 5 groups, every group 5.Tail vein injection gives 20 mg/kg (-)-THP solution,
10 min after administration, 20 min, 30 min, 60 min and 120 min time points take blood plasma and kidney respectively.By tissue with life
Reason salt solution washes away blood, and filter paper is blotted and is precisely weighed, and dispersal mechanism is homogenized into 1g/15mL tissue homogenate using tissue
(acetonitrile：Water=1:1), -80 DEG C of preservations, it is to be measured.
5.2.2 biological sample pre-processes
The blood plasma or tissue sample of -80 DEG C of preservations, thaw at RT, take blood plasma and each 50 μ L of tissue homogenate to put 1.5 mL
In centrifuge tube, add 200 μ L and 450 μ L acetonitriles (ng/mL of containing the internal standard Loratadine 50) respectively, blood plasma or tissue sample revolve and shaken
2 min protein precipitations are swung, 15 min is centrifuged in 13000rpm, takes supernatant, carry out LC-MS/MS analyses.With determinand and internal standard
Peak area ratio, retinue standard curve is substituted into, carries out quantitative analysis.
Influences of 5.3 (-)-THP to cis-platinum concentration in kidney
5.3.1 animal administration and sample collection
ICR mouse 16 (male, 25 ~ 30g of body weight), are randomly divided into 10 mg/kg cis-platinums groups and 10 mg/kg cis-platinums
+ 20 mg/kg (-)-THP groups.After tail vein single injection gives (-)-THP solution, then the mg/kg cisplatin solutions of intravenous 10.Give
After the h of medicine 72, cut open to kill and take renal tissue, blood is washed away with physiological saline, filter paper blots, -80 DEG C of preservations, to be measured.
5.3.2 kidney samples A pre-processes
The kidney samples A of -80 DEG C of preservations, thaw at RT, the accurately weighed mg of renal tissue 50 put in 2.0 mL centrifuge tubes, added
200 μ L nitric acid and 400 μ L H2O2, in 80 DEG C of water-bath vibration 1.5 h of resolution, taking-up, which is placed in ice bath, is cooled to room temperature.Add
200 μ L ammoniacal liquor are neutralized, 0.22 μM of water-based membrane filtration, and are settled to 3.0 mL with 0.1% nitric acid, and sample is laggard through ultrasound
Row ICP-MS analyzes the concentration of platinum.
Influences of 5.4 (-)-THP to cis-platinum induced mice renal toxicity
5.4.1 animal administration and sample collection
22 ICR mouse (male, 25 ~ 30g of body weight), are randomly divided into control groups, 10 mg/kg cis-platinums groups and 10
Mg/kg cis-platinums+20 mg/kg (-)-THP groups (n=6-10).After tail vein single injection first gives (-)-THP solution, then it is quiet
The mg/kg cisplatin solutions of note 10, control groups give the physiological saline of same volume.The body weight of record mouse daily, close observation
Record mouse skin, the change of hair and general activity situation.72 h before administration and after administration, the blood sampling of eye socket angular vein clump, take
Virus monitory renal function biochemical indicator (including urea nitrogen BUN, creatinine CRE, uric acid URA), and cut open and kill mouse, take out kidney, essence
It is close to weigh, after portion of tissue is fixed with 40% formalin buffer, FFPE, pathological section is done, carry out HE dyeing, it is micro-
The pathologic change of Microscopic observation nephridial tissue structure.
5.5 experimental result 5.5.1 (-)-THP renal tissues are distributed
After mouse mainline 20 mg/kg (-)-THP solution, (-)-THP is distributed rapidly in renal tissue, 10 min
When up to 22.6 μM, about 5 times of Plasma (4.5 μM), referring to Fig. 9.Prompt (-)-THP concentration in nephridial tissue
It is higher, interaction may occur with transporter OCT2/MATE1 in kidney and influence the kidney concentration of cis-platinum, thus may
Influence its Toxicity of Kidney.
5.5.2 (-)-THP reduces the kidney concentration of cis-platinum
In order to illustrate whether (-)-THP can exist by suppressing the cis-platinum renal transport of OCT2/MATE1 mediations to influence cis-platinum
The concentration of kidney, we further compare single and give 10 mg/kg cis-platinums, and merge and give cis-platinum and 20 mg/kg
After the h of (-)-THP 72, the difference of cis-platinum concentration in kidney.As a result show, individually give cis-platinum in cis-platinum group mouse kidney
At concentrations up to 141.9 ppb, and it is 81.7 ppb that (-)-THP group mouse kidney concentration is given in merging, referring to Figure 10, explanation
(-)-THP can significantly inhibit the kidney intake of cis-platinum and reduce its kidney concentration.
5.5.3 animal general status in toxicity of cisplatin experiment
With the cis-platinum of 10 mg/kg dosage to 24 h after mouse tail vein single injection after, mouse mouse fur starts to hold up,
And occur it is dispirited, blunt to extraneous stimulate the reaction, activity reduce, anorexia, body weight starts to be decreased obviously, and control rats
Behavior and activity reaction it is without exception, and body weight increases steadily, and both have significant difference, illustrate cis-platinum to mouse mouse entirety
Level has certain toxicity.And merging the mouse for giving 20 mg/kg (-)-THP, body weight caused by cis-platinum reduces obvious slow
Solution, (-)-THP is prompted to suppress the mouse kidney toxicity caused by cis-platinum, referring to Figure 11.
5.5.4 Biochemical Indices In Serum
Urea nitrogen (BUN), creatinine (CRE) and the uric acid (URA) of 72 h mice serums after preceding and administration is administered in detection
Value.As seen from Figure 12, cis-platinum group mice serum renal function index BUN values are individually given and are significantly higher than solvent control group
（control）, after cis-platinum shares 20 mg/kg (-)-THP, BUN values substantially reduce and close to control groups.Above-mentioned knot
Fruit is prompted, and 10 mg/kg cis-platinums of intravenous injection can cause serious renal toxicity in Mice Body；(-) of 20 mg/kg dosage-
THP has stronger mitigation to renal toxicity caused by cis-platinum.
5.5.5 renal pathology inspection
HE is dyed, and pathological analysis of cutting into slices, cisplatin administration group kidney of mouse pathologic finding finds there are damage situations：It is main
The pathological change wanted is cast in renal tubule, and part renal cells is downright bad, and shares 20 mg/kg (-)-THP groups
Renal tissues damage significantly takes a turn for the better, and has no that obvious pathology sexually revises.Solvent control group (control) group renal pathology checks not
Any exception is seen, referring to Figure 13.
Claims (2)
1. application of a kind of tetrahydropalmatine in anti-toxicity of cisplatin medicine is prepared, it is characterised in that the toxicity is kidney poison
Property, the tetrahydropalmatine is rotundine, dextrorotation tetrahydropalmatine or dltetrahydropalmatine.
2. application according to claim 1, it is characterised in that the auxiliary material that the medicine is allowed by tetrahydropalmatine and preparation
It is made, the method for administration of the medicine is intravenous or oral.
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