CN108578403A - Jamaicin is used to prepare the purposes of the peripheral neuropathy drug of prevention chemotherapy induction - Google Patents
Jamaicin is used to prepare the purposes of the peripheral neuropathy drug of prevention chemotherapy induction Download PDFInfo
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- CN108578403A CN108578403A CN201810095430.4A CN201810095430A CN108578403A CN 108578403 A CN108578403 A CN 108578403A CN 201810095430 A CN201810095430 A CN 201810095430A CN 108578403 A CN108578403 A CN 108578403A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/28—Compounds containing heavy metals
- A61K31/282—Platinum compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
Abstract
The present invention provides the new applications of jamaicin peripheral neuropathy caused by preventing chemotherapy.Jamaicin is widely used in treating gastrointestinal tract inflammation as a kind of broad-spectrum antiseptic clinical medicine of high-efficiency low-toxicity, and takes non-evident effect for a long time.This research is by establishing the preventive and therapeutic effect of the peripheral neuropathy that external DRG (dorsal root ganglion, dorsal root ganglion) cell models and internal mouse model overall merit jamaicin induce chemotherapy.The ability of growth of cancer cells is inhibited not influence chemotherapy effect, that is, chemotherapeutic in addition, this result of study also discloses jamaicin, the clinical expansion that peripheral neuropathy caused by chemotherapy is prevented for jamaicin provides strong evidence.The present invention provides the new application of peripheral neuropathy caused by jamaicin treatment chemotherapy for the first time, this result will provide an important evidence for the clinical application of peripheral neuropathy caused by Chinese herb prevention chemotherapy, of far-reaching significance.
Description
Technical field
The present invention relates to the new applications of jamaicin, and god around prevention chemotherapy induction is used to prepare more particularly to jamaicin
Purposes through lesion drug.
Background technology
Peripheral neuropathy (the Chemotherapy-induced peripheral of caused by chemotherapeutic medicines
Neuropathy, CIPN) it is common chemotherapy relevant dose dependence toxicity, clinically very common, especially receiving
Treat the first month incidence highest for the treatment of.The symptom of CIPN is mostly symmetry, is happened at feeling, kinesitherapy nerve and autonomic nerve
On, but especially based on sensory nerve toxic side effect, typical such as paresthesia of skin, numbness, shouting pain, burning, hyperalgia,
Tendon reflex disappears, seismesthesia and proprioceptive sense loss etc..CIPN not only seriously affects the Health and Living of patient, can also limit
The implementation of chemotherapy regimen aggravates disease in turn.But in 2014, ASCO issued the new guideline of prevention and treatment of CIPN, high due to lacking
Quality, consistency evidence, ASCO do not recommend any drug for CIPN prevention (Gansu Medical Journal,
2016, Vol.35, No.5).
It is common that the drug of CIPN is caused to have platinum class (such as oxaliplatin), taxanes (such as taxol) and vinca,
Wherein first two clinical application is the most extensive.Oxaliplatin (oxaliplatin, OXA) is the 3rd generation after cis-platinum, carboplatin
Platinum series antineoplastic medicament, single medicine or combination therapy multiple types tumour all show that good application prospect, joint 5- fluorine ureas are phonetic
Pyridine/Calciumlevofolinate has become the First-line chemotherapy scheme of Advanced colorectal cancer.Taxol (paclitaxel, PTX) is from red bean
A kind of anticarcinogen extracted in the bark of China fir, clinic are widely used in the treatment of breast cancer, oophoroma and non-small cell lung cancer.
It is now recognized that the development of CIPN is primarily due to influence primary sensory's neuron of dorsal root ganglion (DRG), DRG
Sensory neuron it is abundant, and protected without blood-brain barrier, subject to damage.But the pathogenesis of CIPN is indefinite so far.OXA's
Induced neurotoxicity may make protein dyssynthesis to which inhibits rRNA synthesis in sensory neuron cell space kernel is related,
Sensory neuron organelle is caused abnormal morphology variation and corresponding function damage occur.Cell ultrastructure Senile Mouse is sent out
It is existing, neuronal degeneration most significantly (the Journal of China-Japan Friendship in 24~48h after medication, DRG
Hospital,2007Apr,Vol.21.No.4).PTX may be by destroying microstructure on DRG, lead to the information in aixs cylinder
Transmit and energy supply obstacle, cause DRG neuronal cell necrosis, to play toxic effect (Chinese biochemical drug magazine, 2015,
47(35)：185-188).
Jamaicin (berberine) is also known as berberine, is the main active of the medicinal materials such as the Chinese medicine Ranunculaceae coptis.It is one
The common different beautiful jade alkaloid of kind, molecular formula C20H18NO4, can be clinically used for treatment diabetes, hypertension blood fat, arrhythmia cordis,
Bacillary dysentery etc..The study found that diabetes rat pain threshold after jamaicin is treated is significantly raised, nerve conduction velocity adds
Soon, show jamaicin can alleviate diabetes-induced peripheral neuropathy symptom (modern combination of Chinese tradiational and Western medicine magazine, 2009,18
(2):127-129), and it has been reported that jamaicin treats the peripheral neuropathy of diabetes-induced
The mechanism of action of (Diabeticperipheral Neuropathy, DPN) may be to inhibit releasing for cellular inflammation factor TNF-α
Put (Shaanxi medical journal, 2009,38 (4)：392-395), NGS is activated, NO releases are promoted, improving neural blood supply, (the tenth complete
State's cardiovascular pharmacological science meeting and 2010 international cardiovascular diseases and drug summit forum), make the PPAR of DRG caused by high sugar
The change of β, PAR-2, TRPV4, P2X3, the albumen of n NOS and mRNA level in-site tend to restore to it is normal (clinical medical officer's magazine,
2016,44 (8)：It is 847-852) related.
Currently, there has been no the reports that jamaicin can fight peripheral neuropathy caused by chemotherapy.
Invention content
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of jamaicins to be used to prepare prevention chemotherapy induction
Peripheral neuropathy drug purposes.
The present invention has found jamaicin for mitigating chemotherapy induction by two aspects of zoopery and In vitro cell experiment
The purposes of peripheral neuropathy.
The present invention is achieved through the following technical solutions:
For jamaicin in the purposes of the peripheral neuropathy for the treatment of chemotherapy induction, the jamaicin is Berberine hydrochloride.
Further, wherein the induced drug of the peripheral neuropathy is platinum class or taxanes chemotherapy agent.
Further, platinum class is oxaliplatin, and taxanes are taxol.
Further, jamaicin is administered simultaneously with oxaliplatin or taxol.
Further, jamaicin is for mitigating the use during the dorsal root ganglion of oxaliplatin or taxol induced is damaged
On the way.Still further, exploring the mechanism of action of the peripheral neuropathy of the anti-chemotherapy induction of jamaicin.
The present invention has the following advantages and beneficial effects：
The present invention screens and confirms the new application of jamaicin by carrying out experimental study to Chinese Herbal Active Ingredients.On the one hand,
Jamaicin can effectively mitigate the damage to primary Dorsal ganglion cell of oxaliplatin or taxol induced in vitro, and to changing
Treating the anticancer effect of drug oxaliplatin and taxol does not influence.On the other hand, in vivo in pharmacological evaluation, jamaicin can delay
Solve the allodynia reaction of chemotherapy inducing mouse.Therefore jamaicin can be used for treating the peripheral neuropathy of chemotherapy induction.In addition
The also mechanism of action of the peripheral neuropathy of desk study jamaicin prevention induced by chemotherapeutic agents.The present invention provides not only small
The new therapeutical uses of bark of a cork tree alkali also provide new medicine and new thinking for the peripheral neuropathy treatment of chemotherapy induction.
Description of the drawings
Figure 1A is the peripheral neuropathy that mtt assay detects that the jamaicin of various concentration is induced in oxaliplatin (OXA, 3 μM)
On the active influence of DRG cell mitochondrials on cell model；
Figure 1B is the peripheral nerve disease that CCK-8 methods detect that the jamaicin of various concentration is induced in oxaliplatin (OXA, 3 μM)
Become the influence to DRG cell activity on cell model；
Fig. 1 C are to detect the jamaicin of various concentration around oxaliplatin (OXA, 3 μM) induction using ATP kits
To the influence of DRG cell activity on neuropathy cell model；
Fig. 2 is the DRG neuronal cells that immunofluorescence assays jamaicin (3 μM) induces oxaliplatin (OXA, 3 μM)
Whether axon length shortens prevention improvement result；
Fig. 3 A are the peripheral neuropathy that mtt assay detects that the jamaicin of various concentration is induced in taxol (PTX, 300nM)
On the active influence of DRG cell mitochondrials on cell model；
Fig. 3 B are the peripheral nerve disease that CCK-8 methods detect that the jamaicin of various concentration is induced in taxol (PTX, 300nM)
Become the influence to DRG cell activity on cell model；
Fig. 3 C are to detect the jamaicin of various concentration around taxol (PTX, 300nM) induction using ATP kits
To the influence of DRG cell activity on neuropathy cell model；
Fig. 4 is the DRG neuronal cells that immunofluorescence assays jamaicin (3 μM) induces taxol (PTX, 300nM)
Whether axon length shortens prevention improvement result；
Fig. 5 is whether the jamaicin for detecting various concentration has shadow on HT-29 cancer cells to the anti-cancer ability of oxaliplatin
It rings；
Fig. 6 is whether the jamaicin for detecting various concentration has shadow on SUM-159 cancer cells to the anti-cancer ability of taxol
It rings；
Fig. 7 be evaluate jamaicin to the mouse vola epidermis LI nerve fibers caused by chemotherapy (OXA and PTX) reduce whether
There is prevention effect；
Fig. 8 and Fig. 9 is that chemotherapeutics (OXA and PTX) induces on peripheral neurophaty varying model in DRG jamaicin in vivo
The influence of NF- κ B, p38 and ERK1/2 protein expressions.
Specific implementation mode
In order to make the objectives, technical solutions and advantages of the present invention clearer, below by specific embodiment, to this hair
It is bright to be further elaborated.It should be understood that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
It limits the scope of the invention.
Embodiment 1：Jamaicin is on the active influence of chemotherapeutics (OXA) peripheral neuropathy cell model
Instrument：TECAN SPARK10M microplate reader, Olympus IX71 fluorescence microscopes.
Reagent：Poly-D-lysine, taxol, oxaliplatin and pancreatin come from Sigma companies；Clostridiopetidase A by
Worthington companies provide；Culture medium, serum, dual anti-penicillin and streptomysin derive from Gibco companies；MTT powder is purchased
From Beijing Suo Laibao companies；CCK-8 is purchased from eastern benevolence biology；ATP kits are provided by Lonza companies of the U.S.；Antibody is purchased from
Abcam companies.HT-29 cells and SUM-159 cells are received in other laboratories.
Cell culture, modeling and administration grouping：(1) separation and Extraction newborn SD rat dorsal root ganglion is put into containing 1% pair
In the L15 culture mediums of anti-(being 100units/mL).Supernatant is abandoned in centrifugation, adds the 37 DEG C of digestion of 3mg/mL clostridiopetidase As
50min obtains cell mixture after digestion.(2) complete medium that 1mL contains 10%FBS is added and terminates digestion, is abandoned after centrifugation
Supernatant.It is washed 2 times with L15 culture mediums, the Neurobasal-B27 containing 10ng/mLGDNF and 10%FBS is added and cultivates completely
Base is gently blown and beaten with suction pipe and cell suspension is made.(3) with 70 μm of cellular filter filtration cell suspension, cell is dispelled with pipette tips
It can be inoculated in the processed tissue culture plate of poly-D-lysine, be placed in 37 DEG C, 5%CO2It is cultivated in incubator.Cell is hanged
It is 5 × 10 that liquid, which adjusts cell density,4/ mL is inoculated in 96 orifice plate cultures for 24 hours per 100 μ L of hole.(4) 30mM barberry alkali liquors is molten
In Neurobasal-B27 (serum-concentration is reduced to 2%) complete medium gradient dilution containing 3 μM of oxaliplatins at final concentration
For 30 μM, 10 μM, 3 μM, 1 μM, 300nM.Discard old culture medium, blank group is changed to the fresh culture of 2%FBS, model group with
3 μM of oxaliplatin is added in treatment group, while the jamaicin of the various concentration prepared is added in treatment group.6 multiple holes of every group of setting,
Continue to cultivate 48h.
Mtt assay detects cell activity：(1) under the conditions of being protected from light, cell per well is added 10 μ L 5mg/mL's in step 1 (4)
MTT solution is placed in incubator.After 4h, liquid in hole is abandoned in suction, and 110 μ LDMSO solution are added, shake up.With microplate reader in 490nm waves
Long lower measurement OD values.Testing result is as shown in Figure 1A.(2) interpretation of result：It can cause DRG cells after oxaliplatin processing 48h
Nearly 50% neurotoxicity, jamaicin protection significant to neurotoxicity caused by oxaliplatin at a concentration of 3 μM are made
With protective rate is 20% or so.
CCK-8 methods detect cell activity：(1) under the conditions of being protected from light, 10 μ LCCK- are added in cell per well in step 1.1. (4)
8 solution are placed in incubator culture.After 4h, OD values are directly measured under 450nm wavelength with microplate reader.Testing result such as Figure 1B institutes
Show.(2) interpretation of result：It can cause the neurotoxicity of DRG cells nearly 50%, barberry alkali concentration after oxaliplatin processing 48h
At 3 μM or more to the significant protective effect of neurotoxicity caused by oxaliplatin, and the protective rate at a concentration of 30 μM
It can reach 30%.
ATP methods detect cell activity：(1) according to ATP kit specifications, 96 orifice plates is taken out and are restored to room temperature, often
Hole is added 50 μ L Lysis Buffer and reacts 10min.(2) it is added in transfer 100 μ L liquid to opaque 96 orifice plate per hole
100 μ LATP monitoring reagent plus react 2min.Chemiluminescence intensity is measured with microplate reader.Testing result is such as
Shown in Fig. 1 C.(3) interpretation of result：It can cause the neurotoxicity of DRG cells nearly 50% after oxaliplatin processing 48h, it is each dense
Degree jamaicin can have neurotoxicity caused by oxaliplatin prevention improvement result, and when jamaicin is 1 μM, 3 μM a concentration of
The significant meaning of protective effect.
Embodiment 2：Influence of the jamaicin to chemotherapeutics (OXA) peripheral neuropathy cell model axon growth
Laboratory apparatus and reagent are the same as embodiment 1.
(1) cell suspension adjustment cell density is 5 × 10 by the cell culture processes for using embodiment 14/ mL, per hole
500 μ L are inoculated in 24 orifice plate cultures for 24 hours.
(2) 30mM barberry alkali liquors are dissolved in the Neurobasal-B27 containing 3 μM of oxaliplatins (serum-concentration is reduced to
2%) complete medium gradient dilution is at final concentration of 3 μM.Old culture medium is discarded, blank group is changed to the fresh cultured of 2%FBS
Base, 3 μM of oxaliplatin is added with treatment group for model group, while 3 μM of the jamaicin prepared is added in treatment group.Every group of setting 3
A multiple holes continue to cultivate 48h.
(3) fixed：Culture medium is discarded, PBS is washed three times, each 10min.10min is fixed with 4% paraformaldehyde.
(4) it closes：PBS is washed three times, each 10min.With containing 5% normal sheep serum, 0.2%Triton X-100 and
The confining liquid of 0.5%Tween-20 is incubated 1h at room temperature.
(5) primary antibody：With rabbit-anti β III-Tublin polyclonal antibodies (1:1000) incubation at room temperature is stayed overnight.
(6) secondary antibody：PBS is washed three times, each 10min.Under the conditions of being protected from light, with the goat anti-rabbit antibodies containing fluorescein (FITC)
(1:100) it is incubated at room temperature 1h.
(7) it makes film：PBS is washed 3 times again, each 10min.It observes and takes pictures under inverted fluorescence microscope immediately, cell and axis
It is prominent positive for fluorescent staining in green fluorescence.Every group randomly selects 10 positive cells and measures aixs cylinder.Aixs cylinder data are shown in Fig. 2.
(8) interpretation of result：After oxaliplatin handles 48h, cell axon length reduces 40% or so, jamaicin intervention
There are apparent prevention effect, protective rate 33% or so to axon length reduction caused by oxaliplatin after processing.
Embodiment 3：Jamaicin is on the active influence of chemotherapeutics (PTX) peripheral neuropathy cell model
Laboratory apparatus and reagent are the same as embodiment 1.
Cell culture, modeling and administration grouping：(1) separation and Extraction newborn SD rat dorsal root ganglion is put into containing 1% pair
In the L15 culture mediums of anti-(being 100units/mL).Supernatant is abandoned in centrifugation, adds the 37 DEG C of digestion of 3mg/mL clostridiopetidase As
50min obtains cell mixture after digestion.(2) complete medium that 1mL contains 10%FBS is added and terminates digestion, is abandoned after centrifugation
Supernatant.It is washed 2 times with L15 culture mediums, the Neurobasal-B27 containing 10ng/mLGDNF and 10%FBS is added and cultivates completely
Base is gently blown and beaten with suction pipe and cell suspension is made.(3) with 70 μm of cellular filter filtration cell suspension, cell is dispelled with pipette tips
It can be inoculated in the processed tissue culture plate of poly-D-lysine, be placed in 37 DEG C, 5%CO2It is cultivated in incubator.Cell is hanged
It is 5 × 10 that liquid, which adjusts cell density,4/ mL is inoculated in 96 orifice plate cultures for 24 hours per 100 μ L of hole.(4) 30mM barberry alkali liquors is molten
In Neurobasal-B27 (serum-concentration is reduced to 2%) complete medium gradient dilution containing 300nM taxols at final concentration
For 30 μM, 10 μM, 3 μM, 1 μM, 300nM.Discard old culture medium, blank group is changed to the fresh culture of 2%FBS, model group with
The taxol of 300nM is added in treatment group, while the jamaicin of the various concentration prepared is added in treatment group.6 multiple holes of every group of setting,
Continue culture for 24 hours.
Mtt assay detects cell activity：(1) under the conditions of being protected from light, cell per well is added 10 μ L 5mg/mL's in step 1 (4)
MTT solution is placed in incubator.After 4h, liquid in hole is abandoned in suction, and 110 μ LDMSO solution are added, shake up.With microplate reader in 490nm waves
Long lower measurement OD values.Testing result is as shown in Figure 3A.(2) interpretation of result：Taxol treatment can cause DRG cells about after for 24 hours
55% neurotoxicity, each concentration jamaicin can have neurotoxicity caused by taxol prevention improvement result, and barberry
The significant meaning of protective effect when alkali concentration is 3 μM, protective rate nearly 40%.
CCK-8 methods detect cell activity：(1) under the conditions of being protected from light, 10 μ LCCK- are added in cell per well in step 1.1. (4)
8 solution are placed in incubator culture.After 4h, OD values are directly measured under 450nm wavelength with microplate reader.Testing result such as Fig. 3 B institutes
Show.(2) interpretation of result：Taxol treatment can cause the neurotoxicity of DRG cells about 50%, 3 μM or more of jamaicin after for 24 hours
Can there be significant prevention improvement result to neurotoxicity caused by taxol, protective rate is 25% or more.
ATP methods detect cell activity：(1) according to ATP kit specifications, 96 orifice plates is taken out and are restored to room temperature, often
Hole is added 50 μ L Lysis Buffer and reacts 10min.(2) it is added in transfer 100 μ L liquid to opaque 96 orifice plate per hole
100 μ LATP monitoring reagent plus react 2min.Chemiluminescence intensity is measured with microplate reader.Testing result is such as
Shown in Fig. 3 C.(3) interpretation of result：Taxol treatment can cause the neurotoxicity of DRG cells about 65%, barberry alkali dense after for 24 hours
To the significant protective effect of the protective effect of neurotoxicity caused by taxol when degree is 3 μM, 10 μM, protective rate 30% is left
It is right.
Embodiment 4：Influence of the jamaicin to chemotherapeutics (PTX) peripheral neuropathy cell model axon growth
Laboratory apparatus and reagent are the same as embodiment 1.
(1) cell suspension adjustment cell density is 5 × 10 by the cell culture processes for using embodiment 14/ mL, per hole
500 μ L are inoculated in 24 orifice plate cultures for 24 hours.
(2) 30mM barberry alkali liquors are dissolved in the Neurobasal-B27 containing 300nM taxols (serum-concentration is reduced to
2%) complete medium gradient dilution is at final concentration of 3 μM.Old culture medium is discarded, blank group is changed to the fresh cultured of 2%FBS
Base, the taxol of 300nM is added with treatment group for model group, while 3 μM of the jamaicin prepared is added in treatment group.Every group of setting 3
A multiple holes continue culture for 24 hours.
(3) fixed：Culture medium is discarded, PBS is washed three times, each 10min.10min is fixed with 4% paraformaldehyde.
(4) it closes：PBS is washed three times, each 10min.With containing 5% normal sheep serum, 0.2%Triton X-100 and
The confining liquid of 0.5%Tween-20 is incubated 1h at room temperature.
(5) primary antibody：With rabbit-anti β III-Tublin polyclonal antibodies (1:1000) incubation at room temperature is stayed overnight.
(6) secondary antibody：PBS is washed three times, each 10min.Under the conditions of being protected from light, with the goat anti-rabbit antibodies containing fluorescein (FITC)
(1:100) it is incubated at room temperature 1h.
(7) it makes film：PBS is washed 3 times again, each 10min.It observes and takes pictures under inverted fluorescence microscope immediately, cell and axis
It is prominent positive for fluorescent staining in green fluorescence.Every group randomly selects 10 positive cells and measures aixs cylinder.Aixs cylinder data are shown in Fig. 4.
(8) interpretation of result：Taxol treatment for 24 hours after, cell axon length reduce 60% or so, jamaicin intervention processing after
There are apparent prevention effect, protective rate about 35% to axon length reduction caused by taxol.
Embodiment 5：Influence of the jamaicin to chemotherapeutics (OXA) anti-cancer ability
Laboratory apparatus and reagent are the same as embodiment 1.
Cancer cell culture, modeling and administration grouping：(1) cancerous cell line selects：Clinically oxaliplatin has preferably intestinal cancer
Curative effect, therefore select whether colorectal cancer cell HT-29 analysis jamaicins have an impact oxaliplatin (OXA) anticancer effect.(2)
Cancer cell culture：HT-29 cells are recovered according to conventional method, are inoculated in containing 10% fetal calf serum, 1% dual anti-RPMI-1640
Complete medium, in 37 DEG C, 5%CO2, routine culture in saturated humidity incubator, change within 2-3 days liquid and pass on 1 time.(3) cell point
Group：By the HT-29 cell tryptase enzymic digestions in exponential phase, 96 orifice plates are inoculated in, per 100 μ L of hole, after culture for 24 hours at dosing
Reason.Cell is divided into blank group (being not added with any drug-treated, control) model group (only plus OXA processing), barberry alkali process group
(various concentration, OXA+B).(4) cell interference method：30mM barberry alkali liquors are dissolved in containing 30 μM of oxaliplatins (OXA)
RPMI-1640 complete mediums, gradient dilution is at final concentration of 30 μM, 10 μM, 3 μM, 1 μM, 300nM.Old culture medium is discarded, it is empty
White group is changed to fresh RPMI-1640 complete mediums, and 30 μM of oxaliplatin is added in model group, and barberry alkali process group, which is added, to be prepared
Various concentration jamaicin.6 multiple holes of every group of setting continue to cultivate 48h.(5) mtt assay detects cell activity：It is protected from light condition
Under, the MTT solution merging incubator of 10 μ L 5mg/mL is added in cell per well in step 1 (4).After 4h, liquid in hole is abandoned in suction,
110 μ LDMSO solution are added, shake up.OD values are measured under 490nm wavelength with microplate reader.Testing result is as shown in Figure 5.(6) it ties
Fruit is analyzed：It can cause the death of 50% or more HT-29 cancer cells after oxaliplatin processing 48h, the jamaicin of each concentration is to Austria
Husky profit platinum kills the ability of cancer cell without any influence.
Embodiment 6：Influence of the jamaicin to chemotherapeutics (PTX) anti-cancer ability
Laboratory apparatus and reagent are the same as embodiment 1.
Cancer cell culture, modeling and administration grouping：(1) cancerous cell line selects：Clinically taxol is usually used in treating mammary gland
Cancer and there is good therapeutic effect, therefore selects whether breast cancer cell SUM-159 analysis jamaicins have taxol (PTX) anticancer effect
It influences.(2) cancer cell culture：SUM-159 cells are recovered according to conventional method, are inoculated in containing 10% fetal calf serum, 1% dual anti-
DMEM/HIGH GLUCOSE complete mediums, in 37 DEG C, 5%CO2, routine culture in saturated humidity incubator, change liquid within 2-3 days
Passage 1 time.(3) cell is grouped：By the SUM-159 cell tryptase enzymic digestions in exponential phase, 96 orifice plates are inoculated in, per hole
100 μ L, agent-feeding treatment after culture for 24 hours.Cell is divided into blank group (being not added with any drug-treated, control) model group and (only adds
PTX processing), barberry alkali process group (various concentration, PTX+B).(4) cell interference method：30mM barberry alkali liquors are dissolved in and are contained
Have a DMEM/HIGH GLUCOSE complete mediums of 300nM taxols (PTX), gradient dilution at final concentration of 30 μM, 10 μM, 3
μM、1μM、300nM.Old culture medium is discarded, blank group is changed to fresh DMEM/HIGH GLUCOSE complete mediums, and model group adds
Enter the taxol of 300nM, the jamaicin of the various concentration prepared is added in barberry alkali process group.6 multiple holes of every group of setting, continue to train
It supports for 24 hours.(5) mtt assay detects cell activity：Under the conditions of being protected from light, cell per well is added 10 μ L 5mg/mL's in step 1 (4)
MTT solution is placed in incubator.After 4h, liquid in hole is abandoned in suction, and 110 μ LDMSO solution are added, shake up.With microplate reader in 490nm waves
Long lower measurement OD values.Testing result is as shown in Figure 6.(6) interpretation of result：Taxol treatment can cause SUM-159 cancers thin after for 24 hours
The jamaicin intervention of the toxic effect of 50% or more born of the same parents, each concentration is handled to taxol antitumaous effect without any influence.
Embodiment 7：Pain behavior is tested.
Instrument：Plantar test 37370(Ugo Basile,Italy),Dynamic Plantar
Aesthesiometer (DPA, Ugo Basile, Italy), Olympus IX71 fluorescence microscopes, the more work(of day energy 5200Multi
Energy imaging system, Bio-Rad electrophoresis apparatuses.
Reagent：Taxol, oxaliplatin, jamaicin are purchased from Shanghai Yuan Ye companies；Cell factor kit is purchased from south
The bio tech ltd Jing Maibo；PGP 9.5 is purchased from Beijing biotech firm of Zhong Shan Golden Bridge；biotinylated goat
Anti-rabbit IgG are purchased from BIOSS companies；SG Substrate Kit (Vector, PK-4700) and ABC Kit
(Vector, PK-6100) kit is purchased from Vector Laboratories companies；RIPA lysates and BCA kits are purchased from
The green skies Bioisystech Co., Ltd in Shanghai；Protease inhibitors is purchased from triumphant base biology；PAGE gel rapid preparing reagent
Box comes from Bio-Rad companies；Primary antibody NF- κ B, ERK1/2 are purchased from Abcam companies, primary antibody p38, p-p38, p-ERK1/2 and secondary antibody
CST companies are purchased from, GAPDH antibody comes from Proteintech companies.
Experimental animal and grouping：SPF grades of male ICR mouses, weight 18g-22g are purchased from Jiangning, Nanjing area Qinglongshan animal
(the animal quality certification is numbered for breeding farm：NO.201711485).Mouse is randomly divided into 6 groups, respectively blank group (control),
Taxol model group (PTX), oxaliplatin model group (OXA), paclitaxel treatment group (PTX+B), oxaliplatin treatment group (OXA+
B), jamaicin control group (B), every group 8.Before carrying out zoopery, all Group Animals adaptability are raised one week, are given
Give sufficient food and water.Illustrate hereby, this experiment processing all to animal and Pain behaviour test meet international pain
Research association (IASP) about in experimental study use experimental animal the code of ethics (IASP, 1980；Zimmermann,
1983), and passed through the animal committee of Nanjing University of Traditional Chinese Medicine approval (approval number：ACU171001).
Animal model and animal administration：(1) in this experiment neuropathy is built using chemotherapeutics oxaliplatin or taxol
Varying model.(2) taxol is dissolved in polystyrene castor oil and alcohol mixeding liquid (volume ratio 1 with 6mg/mL：1) in, then 1 is pressed：2
Volume ratio 0.9% normal saline dilution to final concentration 2mg/mL.(3) at the 1st, 3,5 day, to taxol model group and Japanese yew
The mouse of alcohol treatment group carries out tail vein injection with the taxol of 20mg/kg concentration.(4) oxaliplatin is dissolved in 5% glucose,
Ultimate density is 1mg/mL.(5) in a few days ago being given to oxaliplatin model group and oxaliplatin treatment group mouse weekly
4mg/kg oxaliplatins are injected intraperitoneally, and biweekly, amount to surrounding (D1,2,8,9,15,16,22,23).(6) by jamaicin with
Concentration 7.5mg/mL is dissolved in 0.5% sodium carboxymethylcellulose (CMC-Na) (7) that daily to paclitaxel treatment group, oxaliplatin is controlled
Treatment group and jamaicin control group mice, which fill, carries out the administration of jamaicin stomach (chemotherapeutics administration previous hour, jamaicin given low
For 150mg/kg).(8) 0.9% physiological saline of blank control group (control), the injection volume with medicine group and period
Unanimously.
Experimentation：One day before administration (being set as D0 days), to experimental animal first time Behavior test.Weight record is every
Zhou Yici (before each behaviouristics starts).Changes of weight is shown in Table 1.(1) the measurement pyrocondensation of the hot threshold of pain sufficient incubation period is heat pain
Threshold with foot sole stimulation pain threshold detector (Plantar test 37370) measure, before administration (D0) and be administered after the 7th, 14,21,
28 days (D7,14,21,28) is carried out.Mouse is set in the organic glass case on the glass plate of 3mm thickness, adapt to environment 30min, used
Thermostimulation instrument stimulates mouse hind leg mid-plantar skin, and record is from starting to be irradiated to the time for paw withdrawal occur.Adjust irradiation
The intensity of light source makes normal mouse incubation period maintain 20s or so；To prevent burned mice skin, setting automatically cuts off the time and is
30s.Mouse is continuously measured 3 times, per minor tick 5min, calculates average value, the as hot pain threshold of mouse.As a result such as 2 institute of table
Show.(2) tail-flick test (crymodynia threshold value determination) is fixed above the waist by mouse, and tail is in free movement state, 2cm sections of leachings of tail point
Enter in 4 DEG C of ice-water baths, starts timing until tail swing, is 15s to protect mousetail frost bitten, setting time to chopping.Weight
Repetition measurement amount 3 times, per minor tick 5min or more.The results are shown in Table 3.(3) the measurement machinery of mechanicalness paw withdrawal reflex threshold is touched a tender spot
It is abnormal to be reflected using mechanicalness paw withdrawal reflex threshold.This experiment uses dynamic plantar esthesiometer (Dynamic Plantar
Aesthesiometer) measure mechanical paw withdrawal reflex threshold, before administration the 7th after (D0) and administration, 14,
21,28 days (D7,14,21,28) is carried out.First mouse is respectively placed in the organic glass case on metallic sieve, it is 30 points quiet
Zhong Hou, gradually giving mouse mid-plantar to increase the intensity stimulated by a 0.5mm filament, (setting frequency is 1g/s, maximum, force
For 10g), it is slowly increased stimulation pressure, when there are the paw withdrawals reflex responses such as lift is sufficient, licks foot, hides, system can automatically record latent
Pressure when time and animal react.Every mouse repeats continuously to measure 3 times respectively, and average value is the machine of every mouse
Tool paw withdrawal reflex threshold.The results are shown in Table 4.(4) interpretation of result：Table 1 be jamaicin in vivo chemotherapeutics (OXA and
PTX the influence on peripheral neurophaty varying model to mouse weight) is induced.
The weight of 1. each group mouse of table in different time points
Whether jamaicin has prevention improvement result to the mice pain allergic phenomena caused by chemotherapy (OXA and PTX).Wherein table
2 be heat pain, and table 3 is crymodynia, and table 4 is machinery pain.As seen in Table 2, taxol can induce mouse Thermal allodynia, gastric infusion jamaicin
It can prevent mouse to hot hyperalgesia phenomenon；But oxaliplatin group mouse is compared with naive mice, does not have Thermal allodynia phenomenon
Occur.Be shown in Table 3 and hot pain experimental result on the contrary, the cryalgesia allergy of mouse, and jamaicin can be caused after oxaliplatin processing
It can prevent this phenomenon, but taxol does not have a significant impact to the crymodynia of mouse.Table 4 shows that oxaliplatin and taxol are equal
Mouse mechanical hyperalgesia can be caused super quick, jamaicin intervention processing can improve this influence.In addition shown in table 2- tables 4, and it is empty
White group is compared, and the simple pain sensation that berberine in mice is administered does not have a significant impact.
The hot threshold of pain of 2. each group mouse of table in different time points
*With#It is the comparison among groups at same time point；**vs Control,P<0.01；#vs PTX,P<0.05, α=
0.05
The crymodynia threshold of 3. each group mouse of table in different time points
*With#It is the comparison among groups at same time point；*vs Control,P<0.05；#vs OXA,P<0.05, α=0.05
The mechanical threshold of pain of 4. each group mouse of table in different time points
*,#With^It is the comparison among groups at same time point；**vs Control,P<0.01；***vs Control,P<
0.001；
##vs OXA,P<0.01；^vs PTX,P<0.05, α=0.05
Embodiment 8：ELISA method measures the content of Mice Body based intracellular cvtokine.
Laboratory apparatus, reagent, experimental animal and grouping, animal model and animal administration are the same as embodiment 7.
Experimentation：(1) jamaicin is determined to internal cell factor using enzyme-linked immunosorbent assay (ELISA)
The adjustment effect of (NGF, IL-6, TNF-α and IL-1 β).(2) it after last time behaviouristics, is acquired using eye socket blood taking method
Blood sample, every mouse amount for taking blood are about 250 μ L.Whole blood centrifuged after condensing at room temperature about 1 hour (1000 × g, 20
Minute), supernatant is taken, and serum is preserved in -80 DEG C of refrigerators.(3) use Mouse NGF ELISA kits (MBE10062),
IL-6ELISA kits (MBE10288), TNF-α ELISA kits (MBE10037) and mouse IL-1 β ELISA kits
(MBE10289), operation detection is carried out according to the specification of kit.(4) the OD values in each hole are measured at 450nm wavelength.With institute
The OD values for surveying standard items are abscissa, and the concentration values of standard items is ordinate, with Excel Software on Drawing standard curves, and are obtained
The OD values of sample are substituted into equation, calculate the concentration of sample by linear regression equation.It the results are shown in Table 5.(5) interpretation of result：And sky
White group is compared, and oxaliplatin and taxol can influence Mice Body based intracellular cvtokine NGF, IL-6, TNF-α and IL-1 with conspicuousness
The level of β, but these influences can not be improved or be adjusted by jamaicin completely.Chemotherapeutics oxaliplatin and taxol draw
Playing mouse NGF levels reduces, and jamaicin can obviously adjust mouse NGF levels, and the horizontal conspicuousnesses of mouse NGF is made to increase；Barberry
Alkali can improve the mouse IL-6 levels of the raising of mouse IL-1 β levels and taxol induced caused by oxaliplatin and increase, and shadow
Ring significant difference.In addition, the cytokine levels of jamaicin control group mice are compared with blank group, there was no significant difference.
The influence of the 5. berberine in mice tumor growth factor of table
*vs Control,P<0.05；#vs OXA,P<0.05；^vs PTX,P<0.05, α=0.05
Embodiment 9：The immunohistochemical analysis of epidermis nerve fibre (IENF) density and quantitative.
Laboratory apparatus, reagent, experimental animal and grouping, animal model and animal administration are the same as embodiment 7.
Experimentation：(1) after animal blood taking, mouse is put to death.One piece is taken out among mouse vola with operating scissors
Skin；(2) the vola skin histology taken out stays overnight (18-22h) in PLP (periodate-lysine-paraformaldehyde) solution,
It is then stored in anti-icing fluid；(3) in experiment the previous day, vola skin is placed in the PB solution containing 30% sucrose, 4% refrigeration
It preserves overnight；(4) cryogenic thermostat slice (30 μm) after embedding (OCT) freezing；(5) OCT is washed away in frozen section TBS；(6) it floats
In vain：0.025M potassium permanganate handles 10min；(7) TBS washes 10min, and slice, which is placed in 5% ethanedioic acid (oxalic acid), handles 5min；
(8) after TBS washes (each 10min) twice, room temperature is incubated in the TBS containing 5% normal sheep serum and 10%triton x-100
Educate 4h；(9) the rabbit anti-humann PGP9.5 primary antibodies (1: 200, za-0263) 4 containing 5% normal sheep serum are used
DEG C be incubated overnight；(10) after TBS cleanings, in the goat anti-rabbit IgG (1 of biotin labeling:1000, bs-0295g-
Bio 1h is incubated in)；(11) it is infiltrated 30 minutes in methanol/hydrogen peroxide/PBS；(12) after washing (each 5min) twice in PBS,
It is placed again 1 hour in the reagent that ABC kits (vector, PK-6100) are prepared；(13) it is washed in PBS (each 5min) twice
Afterwards, it is dyed with SG Substrate Kit (Vector, PK-4700) (vector, PK-4700), until brown occurs；
(14) PBS is washed twice, then naturally dry, and carrying out endochylema with Yihong dyes 1min；(15) gradient alcohol dehydration drying (95%~
100%)；(16) transparent 3 times of dimethylbenzene；(17) neutral gum sealing.(18) fluorescence microscope (Olympus) 40x object lens are used,
The form of the table underlying nerve nerve fibre (intraepidermal nerve fiber, IENF) of observation and the skin-deep company of computational chart
And number.Every group of at least 4 slices are counted.As a result such as Fig. 7.(19) interpretation of result：Compared with blank group, oxaliplatin and
The processing of taxol can cause mouse vola epidermis LI nerve fibers (IENF) conspicuousness to reduce.The mouse of jamaicin treatment group
Vola epidermis LI nerve fibers conspicuousness is higher than oxaliplatin model group and taxol model group, and jamaicin control group mice
Vola epidermis LI nerve fibers and blank group no significant difference.It follows that jamaicin can cause mouse with Prophylactic chemotherapy
Vola epidermis LI nerve fibers reduce.
Embodiment 10：Western Blot experiments.
Laboratory apparatus, reagent, experimental animal and grouping, animal model and animal administration are the same as embodiment 7.
Experimentation：(1) tissue total protein is extracted：The DRG tissues that -80 DEG C of mouse freezes are taken out, is put into homogenizer, presses
100:500 μ l RIPA lysates and 5 μ l protease inhibitors are added in 1 ratio, and grinding homogenate operates in mixture of ice and water
It carries out.It after the completion of homogenate, is centrifuged (4 DEG C, 12000rpm, 15min), supernatant is sample protein liquid, -80 DEG C of preservations.(2)
BCA methods (kit is purchased from the green skies company in Shanghai) measure sample albumen concentration.(3) sds gel electrophoresis：1) it is denaturalized：Albumen sample
This is mixed with 5 × sample-loading buffer, and 95 degrees Celsius are boiled 10min.2) glue：Quick glue kit prepare 10% separation gel and
5% concentration glue.3) it is loaded：It after gelling is solid, is put into electrophoresis tank, electrophoresis liquid is added, set sequence in advance and added with micropipettor
Sample, is 30 μ g sample albumen per hole applied sample amount, and Marker amounts are 5 μ L.4) electrophoresis：200V constant pressure electrophoresis waits for that sample reaches separation
When glue bottom end, terminate electrophoresis.(4) transferring film：Pvdf membrane and filter paper are cut by gel size, it is solidifying according to cathode-sponge-filter paper-
The sequence of glue-pvdf membrane-filter paper-sponge-anode closely stacks, and is put into transferring film slot, and 250m A constant currents 60min is gone to
Pvdf membrane.(5) it closes：TBST prepares 5% skimmed milk power and 5%BSA, and the pvdf membrane that transferring film is completed is submerged confining liquid completely
In, shaking table closes 1h.(6) primary antibody (1：4000) 4 DEG C of overnight incubations.(7) secondary daily TBST is cleaned 4 times, is placed on shaking table every time
Secondary antibody (1: 8000 dilution, CST companies) is added in 8min, and room temperature is incubated 1h, and TBST washes film 4 times, is placed in 8min on shaking table every time.
(8) it is imaged：ECL chemical luminescence for liquid develops the color, and is taken pictures using imaging system analyzer (Shanghai Tian Neng companies) exposure.(9) it uses
Image J softwares carry out gray value analysis to band.As a result such as Fig. 8 and Fig. 9.(10) interpretation of result：It is compared with blank group, it is difficult to understand husky
Sharp platinum and taxol can cause NF- κ B, pp38 protein expression levels to rise, and significant difference, and barberry alkali process is added
Afterwards, above-mentioned protein expression level has different degrees of decline (P ＜ 0.05).With blank group comparative analysis, oxaliplatin can draw
It plays pERK1/2 protein expressions to increase, but there is no significant difference, taxol can improve to conspicuousness the table of pERK1/2 albumen
Up to level, and jamaicin can reduce this raising effect, there is significant difference (P ＜ 0.05).
The present invention is enterprising by jamaicin peripheral neuropathy In vitro cell model caused by chemotherapy and internal animal model
Row verification test finds that the effect of the peripheral neuropathy of the anti-chemotherapy induction of jamaicin may be by inhibiting oxaliplatin or purple
P38, ERK1/2 phosphorylation activation caused by China fir alcohol reduce inflammation to inhibit the Nuclear Factor kappa B Mobilizations to enter core
Reaction, while inhibiting neurotrosis by regulating and controlling nerve growth factor.
Claims (5)
1. jamaicin is in the application for the peripheral neuropathy drug for preparing anti-chemotherapy induction, it is characterised in that：The jamaicin is
Berberine hydrochloride.
2. application according to claim 1, it is characterised in that：The wherein described chemotherapy induced drug is platinum class or taxanes
Chemotherapeutics.
3. application according to claim 2, it is characterised in that：Platinum class is oxaliplatin, and taxanes are taxol.
4. application according to claim 3, it is characterised in that：The medication of jamaicin and oxaliplatin or taxol is
It is administered simultaneously.
5. application according to claim 1, it is characterised in that：Peripheral nerve disease becomes dorsal root ganglion damage.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115607533A (en) * | 2022-09-29 | 2023-01-17 | 华中科技大学协和深圳医院 | Application of tranilast in preventing and treating chemotherapy-induced peripheral neuralgia |
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