CN103877103A - PLCG1 (phospholipase C-gamma 1) gene and new application of specific inhibitor U73122 thereof to radiation injury resistance - Google Patents

Abstract

The invention relates to a PLCG1 (phospholipase C-gamma 1) gene and a new application of a specific inhibitor U73122 (1-[6((17B)-3-oestrone-1,3,5[10]-triene-17-yl)amino] ethyl)]-1H-pyrrole-2,5-diketone) thereof to radiation injury resistance. Specifically, a known anti-radiation drug tissue gene expression profile technology is adopted to discover that the PLCG1 gene is a latent radiation injury drug target, and a specific inhibitor of the PLCG1 gene is used for experimental verification. The U73122 has a dosage-dependent inhibition effect on V79 cell injury caused by <60>Co gamma ray at 1.25-5 micromoles and can be used for increasing the survival rate of C57BL/6J mice subjected to 7.5Gy radiation of the <60>Co gamma ray in a dose-dependent manner at a dosage of 10mg/kg. The abovementioned experiments prove that the U73122 can be used for preventing and treating radiation injury, and the PLCG1 gene is a latent drug target for radiation injury.

Description

The new purposes of PLCG1 gene and specific inhibitor U73122 Antiradiation injury thereof

Technical field

The present invention relates to Phospholipase Cgamma1 (PLCG1) gene and specific inhibitor 1-[6-[((17B thereof)-3-estrone-1,3,5[10]-triolefin-17-yl) amino] ethyl]-1H-pyrroles-2, the new purposes of 5-diketone (U73122) in Antiradiation injury.

Background technology

Ionizing radiation is a kind of harmful factor of serious harm health, body is subject to heavy dose of ionizing radiation meeting and causes that hemopoietic function of bone marrow obstacle, injury of gastrointestinal tract or brain injury are the acute radiation syndrome of basic lesion, are mainly estrogens, ammonia sulfydryl class and cytokine class for clinical radioprotector at present.The comprehensive therapeutic effect of these medicines also do not approved, though or approved because side effect is too large, can not be used for clinically, therefore efficient, the low toxicity of exploitation or nontoxic radioprotector have great society and economic benefit.

Large quantity research shows both at home and abroad, system of defense and the many polygenic expression of radiation-induced DNA Damage in can active cell, the DNA of identification of damage also repairs, in the time that the degree of DNA damage exceedes the repair ability of cell, some signal paths relevant to cell death are activated, cell death inducing.In these processes, the failure of any one link all can cause the accumulation of gene mutation, finally causes the vicious transformation of cell.Therefore radiant energy causes the change of numerous gene expressions, these radiation related genes of screening and identification all sidedly, not only can understand more all sidedly the signal path that participates in radioreaction, further improve our understanding to mechanism of radiation damage, and for the research of radioprotector and clinically the radiation sensitivity of tumor treat new Research Thinking and drug targets be provided.

Phospholipase C (Phospholipase C, PLC) be an important transmembrane signal transduction molecule, the phospholipid molecule that phospholipase C activates on degradation of cell film produces IP3 and two second messenger molecules of DAG, activate respectively Ca2+ approach and the Protein kinase C in downstream, and then regulating cell proliferation and apoptosis process.

Summary of the invention

The object of the invention is the important function in apoptosis according to PLCG1 gene, use its specific inhibitor U73122 (1-[6-[((17B)-3-estrone-1,3,5[10]-triolefin-17-yl) amino] ethyl]-1H-pyrroles-2,5-diketone), radioprotective activity after checking PLCG1 gene inhibition, using and specific inhibitor U73122 as new radiation damage protection medicine.

This laboratory is by known antiradiation drug Gene Expression Profiles technology, find radiation treatment after PLCG1 raise, and after known antiradiation drug (as WR2721, CBLB502, TPO) is processed, PLCG1 is corresponding downward.Consider the critical role of PLCG1 gene in regulating cell proliferation and apoptosis process, choose PLCG1 gene and carry out experimental verification as potential radiation damage drug targets.Experimental results show that the inhibitor U73122 of PLCG1 gene has the effect of Antiradiation injury.At cellular level, after U73122 effect, in the time of 1.25 μ M administration concentration, higher than 70%, can protect 70% mice to avoid the death causing after 7.5Gy radiation to the suppression ratio of radiation damage in animal level (10mg/kg).

The present invention has the following advantages:

1. found the new purposes of PLCG1 gene in Antiradiation injury

2. found that U73122 all has the new purposes of radioprotective activity in vitro and in vivo.

3. the U73122 in invention can be used as new radiation damage protection medicine, and PLCG1 gene is that the effect of a new radiation damage drug targets is clear and definite.

Accompanying drawing explanation

Below by reference to the accompanying drawings, specific embodiment of the invention is elaborated.

Fig. 1 U73122 is the suppression ratio to radiation damage on cellular level

Fig. 2 U73122 prevents administration pair ⁰c ₀the impact of 30 days survival rates of gamma-rays 7.5Gy mono-subtotal body irradiation C57BL/6J mice

Fig. 3 U73122 is the inhibitory action to target gene PLCG1 on cellular level

The specific embodiment

Set forth radiation resistance in the external and body of medicine amentoflavone of the present invention by following examples and evaluate, but be not limited to following approach.These experiments mainly comprise: 1, U73122 suppression ratio to radiation damage on cellular level; 2, U73122 prevention administration pair ⁰c ₀the impact of 30 days survival rates of gamma-rays 7.5Gy mono-subtotal body irradiation C57BL/6J mice; 3, U73122 inhibitory action to target gene PLCG1 on cellular level

The evaluation of [embodiment mono-] U73122 In Vitro Anti radiation activity

Materials and methods

1.V79 cell culture

V79 cell, containing in the DMEM culture medium of 10% hyclone (Gibco) and 100IU/ml penicillin, 100g/ml streptomycin, in 37 ℃, is cultivated in 5%CO2 incubator.Observation of cell growth conditions is good, is cultured to logarithmic growth after date, and V79 cell is inoculated to 96 orifice plates, 5 × 103 cells/well, and 37 ℃, 5%CO2 is hatched 12 hours, after cell converges, carries out administration experiment, jointly hatches after 24h, treats that cell grows to 70～80% ⁶⁰c ₀radiation gamma processing.

2.U73122 couple ⁶⁰c ₀the V79 cell viability that gamma-rays causes is lost the detection of impact

V79 cell, containing in the DMEM culture medium of 10% hyclone (Gibco) and 100IU/ml penicillin, 100g/ml, in 37 ℃, is cultivated in 5%CO2 incubator.Observation of cell growth conditions is good, is cultured to logarithmic growth after date, and V79 cell is inoculated to 96 orifice plates, 5 × 103 cells/well, 37 ℃, 5%CO2 is hatched 12 hours, after cell converges, by compound amentoflavone with final concentration 1.25 μ M, 2.5 μ M, 5 μ M administration respectively, hatches after 24 hours jointly ⁶⁰c ₀gamma-rays 10Gy irradiates, and close rate is 2.3～2.5Gy/min, establishes without drug cell contrast and blank simultaneously, and 37 ℃, 5%CO2 cultivates, observed result after 72 hours.Every hole adds CCK-8 working solution 10 μ l, lucifuge, and 37 ℃, 5%CO2 cultivates 2 hours, and in microplate reader, (BIO-RADModel680) measures the light absorption value A of 450nm place.Simultaneously under inverted microscope every day observation of cell form.

3.U73122 the processing of exercising result and statistics

U73122 couple ⁶⁰c ₀the impact that gamma-rays causes V79 cell viability to be lost represents without the percentage ratio of drug cell matched group relatively with different group absorbances:

Suppression ratio=(A450 administration-A450 blank)/(A450 contrast-A450 blank) * 100

Result is carried out statistical analysis with Origin Pro7.5, with compared with medicine matched group: #, P < 0.05; ##, P < 0.01; Compared with 4Gy group: *, P < 0.05; *, P < 0.01.

Result

Experiment is independent in triplicate, and CCK-8 testing result is shown in Fig. 1.U73122 is at 1.25 μ M-5 μ M couple ⁶⁰c ₀gamma-rays causes V79 cell viability to be lost and is dose-dependent inhibitory action.

[embodiment bis-] U73122 couple ⁶⁰c ₀the impact of 30 days survival rates of gamma-rays 7.5Gy mono-subtotal body irradiation C57BL/6J mice

Materials and methods

1.C57BL/6J mice is raised

Select 40 of 18～22g male C 57 BL/6 J mouses, provided by Military Medical Science Institute's zoopery center.21 ℃ ± 2 ℃ of mice feeding environment temperature, relative humidity 50% ± 10%, 12 hours dark of 12 hours light.

2.U73122 couple ⁶⁰c ₀the impact of 30 days survival rates of gamma-rays 6Gy mono-subtotal body irradiation C57BL/6J mice

Medicine preparation: precision takes positive drug WR2721 powder 30mg, adds normal saline 2ml, is configured to 15mg/ml solution; U73122 powder 2mg, adds normal saline 2ml, is configured to the solution of 1mg/m, for subsequent use.

Animal grouping and processing: 40 of C57BL/6J mices are divided into four groups at random, every group 10, normal group and radiation matched group lumbar injection equal-volume normal saline, positive drug WR2721 group is pressed 150mg/kg pre-irradiation 30min lumbar injection, and before U73122 group is pressed 10mg/kg dose irradiation, 30min lumbar injection once.

Work in 30 days is deposited situation and is observed: ⁶⁰c ₀after gamma-rays 7.5Gy irradiates, BALB/c mouse loses weight, dry skin and hair, and lethargy, is slow in action, group's contracting hogback, visual disorder.Each administration group, compared with model group, the above-mentioned sign of mice all makes moderate progress.

3.U73122 the processing of radiation resistance result and statistics

U73122 prevents administration pair ⁶⁰c ₀the impact of gamma-rays 6Gy mono-subtotal body irradiation C57BL/6J mice current deposit rate.Result is carried out statistical analysis with Origin Pro 7.5.

Result

U73122,30 days current deposit rates of C57BL/6J mice that the 7.5Gy of 10mg/kg prevention administration processing irradiates the results are shown in Figure 2.The administration of the anti-property of U73122 can dose-dependently improve ⁶⁰c ₀the current deposit rate of gamma-rays 7.5Gy mono-subtotal body irradiation C57BL/6J mice.

[embodiment tri-] U73122 impact on target gene PLCG1 protein expression in vitro

Materials and methods

1.V79 cell culture

With embodiment mono-.

2.U73122 is in vitro to target gene PLCG1 protein expression western blotting

By V79 cell suspension inoculation, in 6 well culture plates, every hole inoculated and cultured liquid 2mL, containing 1.5 × 10 ⁵cell.U73122 is mixed with the storage liquid of 20uM.Dosing final concentration of cells is 2.5 μ M, 5 μ M.Cultivate after 72h, RIPA-PICT (Pharmacia) carries total protein of cell, after protein quantification, each extract is adjusted to same concentrations.Every hole 50 μ g total proteins, after 12% polyacrylamide gel electrophoresis, and carry out Western bloting and observe target gene protein expression.

Result

As seen from Figure 3, U73122 is at 2.5 μ M, and 5 μ M have obvious dose-dependent inhibitory action to target gene PLCG1.

Claims (7)

1. The new purposes of the Antiradiation injury effect that 1.Phospholipase C gamma 1 (PLCG1) gene has.
2. 2. according to claim 1, suppress to have radioprotective effect after PLCG1 gene expression by specific inhibitor, this gene has the new function of Antiradiation injury, is a radiation damage potential drug target.
3. 3. specific inhibitor name according to claim 2 is called 1-[6-[((17B)-3-estrone-1,3,5[10]-triolefin-17-yl) amino] ethyl]-1H-pyrroles-2,5-diketone, English referred to as U73122, its structural formula is

   
4. 4. the pharmaceutical composition of compound according to claim 3, containing pharmaceutical carrier, be characterized as: its active component is 1-[6-[((17B) 3-estrone-1,3,5[10]-triolefin-17-yl) amino] ethyl]-1H-pyrroles-2,5-diketone and derivant thereof.
5. 5. according to the pharmaceutical composition of claim 4, it is characterized by: 1-[6-[((17B)-3-estrone-1,3,5[10]-triolefin-17-yl) amino] ethyl]-1H-pyrroles-2, in 5-derovatives, to 1-[6-[((17B)-3-estrone-1,3,5[10]-triolefin-17-yl) amino] ethyl]-1H-pyrroles-2, the chemical constitution that 5-diketone parent nucleus does is modified.
6. 6. pharmaceutical composition claimed in claim 5, wherein said pharmaceutical composition can be formulated into the solid preparations such as tablet, granule, capsule; The liquid preparation such as injection, oral liquid or patch.
7. 7. claim is 3,4, and the pharmaceutical composition described in 5,6 is in the new purposes of preparing in radiation damage protection medicine.

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