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

Abstract

The present invention relates to PLCG1 gene and its specific inhibitor U73122 (1-[6-[((17B)-3-estrone-1,3,5[10]-triene-17-yl)amino]ethyl] -1H-pyrrole-2,5-dione) in the new application of radiation damage. Specifically, it was discovered that the PLCG1 gene is a potential drug target for radiation damage by using the tissue gene expression profiling technology of known radiation-resistant substances, and its specific inhibitor was used for experimental verification. U73122 at 1.25μM-5μM for ^60C _. γ-rays can inhibit the injury of V79 cells in a dose-dependent manner; ⁶⁰ C can be increased in a dose-dependent manner at 10 mg/kg _. The survival rate of C57BL/6J mice irradiated with 7.5Gy gamma rays. The above shows that U73122 can be used to prevent and treat radiation damage, and the PLCG1 gene is a potential drug target for radiation damage.

Description

New application of PLCG1 gene and its specific inhibitor U73122 in resisting radiation damage

technical field

The present invention relates to Phospholipase Cgamma1 (PLCG1) gene and its specific inhibitor 1-[6-[((17B)-3-estrone-1,3,5[10]-triene-17-yl)amino]ethyl Base]-1H-pyrrole-2,5-dione (U73122) in the new application of radiation damage.

Background technique

Ionizing radiation is a harmful factor that seriously endangers human health. The body is exposed to large doses of ionizing radiation, which can cause bone marrow hematopoietic dysfunction, gastrointestinal tract damage or brain damage as the basic pathological changes. Acute radiation syndrome is currently used in clinical radiation protection drugs Mainly estrogens, aminothiols and cytokines. The comprehensive curative effect of these drugs has not yet been recognized, or even though recognized, they cannot be used clinically because of too many side effects. Therefore, the development of high-efficiency, low-toxicity or non-toxic radiation protection drugs has great social and economic benefits.

A large number of studies at home and abroad have shown that radiation-induced cellular DNA damage can activate the defense system and the expression of many genes in the cell, recognize the damaged DNA and repair it. When the degree of DNA damage exceeds the repair ability of the cell, some cells are related to cell death Related signaling pathways are activated, inducing apoptosis. The failure of any link in these processes will lead to the accumulation of gene mutations, and finally lead to malignant transformation of cells. Therefore, radiation can cause changes in the expression of many genes. Comprehensive screening and identification of these radiation-related genes can not only provide a more comprehensive understanding of the signaling pathways involved in the radiation response, further improve our understanding of the mechanism of radiation damage, but also pave the way for the development of radiation protection drugs. Research and clinical radiosensitization therapy of tumors provide new research ideas and drug targets.

Phospholipase C (Phospholipase C, PLC) is an important transmembrane signal transduction molecule. Phospholipase C activates and degrades phospholipid molecules on the cell membrane to produce two second messenger molecules, IP3 and DAG, which activate the downstream Ca2+ pathway and protein kinase C respectively. And then regulate the process of cell proliferation and apoptosis.

Contents of the invention

The object of the present invention is to use its specific inhibitor U73122 (1-[6-[((17B)-3-estrone-1,3,5[10]) according to the important role of PLCG1 gene in apoptosis -trien-17-yl)amino]ethyl]-1H-pyrrole-2,5-dione), to verify the anti-radiation activity of PLCG1 gene inhibition, and its specific inhibitor U73122 as a new radiation damage protection drug .

Through the tissue gene expression profiling technology of known anti-radiation drugs, our laboratory found that PLCG1 was up-regulated after radiation treatment, and PLCG1 was correspondingly down-regulated after treatment with known anti-radiation drugs (such as WR2721, CBLB502, TPO). Considering the important position of the PLCG1 gene in the regulation of cell proliferation and apoptosis, the PLCG1 gene was selected as a potential radiation-damaged drug target for experimental verification. Experiments have proved that the inhibitor of PLCG1 gene U73122 has the effect of resisting radiation damage. At the cellular level, after U73122 acts, at 1.25 μ M administration concentration, the inhibition rate of radiation damage is higher than 70%, and at the animal level (10mg/kg) can protect 70% of mice from 7.5Gy radiation. die.

The present invention has the following advantages:

1. Discovered the new use of PLCG1 gene in radiation damage resistance

2. Discovered a new application of U73122 having anti-radiation activity both in vitro and in vivo.

3. U73122 in the invention can be used as a new radiation damage protection drug, and PLCG1 gene is a new radiation damage drug target with a clear role.

Description of drawings

The specific implementation of the present invention will be described in detail below in conjunction with the accompanying drawings.

Fig. 1 Inhibition rate of radiation damage by U73122 at the cellular level

Fig. 2 The effect of U73122 prophylactic administration on the 30-day survival rate of C57BL/6J mice irradiated with ⁰ C ₀ γ-ray 7.5Gy once

Fig. 3 Inhibitory effect of U73122 on the target gene PLCG1 at the cellular level

Detailed ways

The following examples are used to illustrate the evaluation of anti-radiation effects of the drug Amantiobiflavone in vitro and in vivo, but are not limited to the following methods. These experiments mainly include: 1. The inhibitory rate of U73122 on radiation damage at the cellular level; 2. The effect of U73122 prophylactic administration on the 30-day survival rate of C57BL/6J mice irradiated with ⁰ C ₀ γ-ray 7.5Gy once; 3. Inhibitory effect of U73122 on the target gene PLCG1 at the cellular level

[Example 1] Evaluation of U73122 anti-radiation activity in vitro

Materials and Methods

1. V79 cell culture

V79 cells were cultured in a DMEM medium containing 10% fetal bovine serum (Gibco), 100 IU/ml penicillin, and 100 g/ml streptomycin at 37°C in a 5% CO2 incubator. Observe that the cells are in good growth state. After culturing to the logarithmic growth phase, inoculate V79 cells in a 96-well plate, 5×103 cells/well, and incubate at 37°C with 5% CO2 for 12 hours. After the cells are confluent, perform the drug administration experiment and incubate together for 24 hours Afterwards, the cells were irradiated with ⁶⁰ C ₀ gamma rays until they grew to 70-80%.

2. Detection of the effect of U73122 on the loss of viability of V79 cells induced by ⁶⁰ C ₀ γ-rays

V79 cells were cultured in a DMEM medium containing 10% fetal bovine serum (Gibco), 100 IU/ml penicillin, and 100 g/ml at 37°C in a 5% CO2 incubator. Observe that the cells are in good growth state. After culturing to the logarithmic growth phase, inoculate V79 cells in a 96-well plate, 5×103 cells/well, and incubate at 37°C with 5% CO2 for 12 hours. The final concentrations of 1.25 μM, 2.5 μM, and 5 μM were administered separately, and after 24 hours of co-incubation, ⁶⁰ C ₀ γ-rays 10Gy were irradiated at a dose rate of 2.3-2.5Gy/min. Incubate in % CO2 and observe the results after 72 hours. Add 10 μl of CCK-8 working solution to each well, protect from light, incubate at 37° C., 5% CO 2 for 2 hours, and measure the absorbance A at 450 nm on a microplate reader (BIO-RADModel680). At the same time, the cell morphology was observed daily under an inverted microscope.

3. Processing and statistics of U73122 action results

The effect of U73122 on the loss of viability of V79 cells caused by ⁶⁰ C ₀ γ-rays is expressed as the percentage of absorbance values of different groups relative to the control group without drugs:

Inhibition rate=(A450 administration-A450 blank)/(A450 control-A450 blank)*100

The results were statistically analyzed using Origin Pro7.5. Compared with the control group without drugs: #, P<0.05; ##, P<0.01; compared with the 4Gy group: *, P<0.05; **, P<0.01.

result

The experiment was repeated three times independently, and the results of CCK-8 detection are shown in Figure 1. U73122 has a dose-dependent inhibitory effect on the loss of viability of V79 cells induced by ⁶⁰ C ₀ γ-rays at 1.25μM-5μM.

[Example 2] Effect of U73122 on the 30-day survival rate of C57BL/6J mice irradiated once with ⁶⁰ C ₀ gamma rays 7.5Gy

Materials and Methods

1. Breeding of C57BL/6J mice

Forty male C57BL/6J mice weighing 18-22 g were selected and provided by the Animal Experiment Center of the Academy of Military Medical Sciences. The mice were kept at an ambient temperature of 21°C±2°C, a relative humidity of 50%±10%, and 12 hours of light and 12 hours of darkness.

2. Effect of U73122 on the 30-day survival rate of C57BL/6J mice irradiated with ⁶⁰ C ₀ gamma rays 6Gy once

Drug preparation: Precisely weigh 30 mg of positive drug WR2721 powder, add 2 ml of normal saline to make a 15 mg/ml solution; add 2 mg of U73122 powder, add 2 ml of normal saline, make a 1 mg/m solution for later use.

Animal grouping and treatment: 40 C57BL/6J mice were randomly divided into four groups, 10 in each group. The normal group and the radiation control group were intraperitoneally injected with an equal volume of normal saline. In the U73122 group, a dose of 10 mg/kg was injected intraperitoneally 30 minutes before irradiation.

30-day survival observation: After being irradiated with 7.5Gy of γ-rays at ⁶⁰ C ₀ , BALB/c mice lost weight, with dry fur, listlessness, sluggish movement, shrunken and arched back, and visual impairment. In each administration group, compared with the model group, the above signs of the mice were all improved.

3. Processing and statistics of U73122 anti-radiation results

The effect of U73122 preventive administration on the survival rate of C57BL/6J mice irradiated with ⁶⁰ C ₀ gamma rays 6Gy once. The results were statistically analyzed with Origin Pro 7.5.

result

The results of the 30-day survival rate of 7.5Gy irradiated C57BL/6J mice treated with U73122, 10mg/kg prophylactic administration are shown in Fig. 2 . Prophylactic administration of U73122 can dose-dependently increase the survival rate of C57BL/6J mice irradiated with ⁶⁰ C ₀ γ-ray 7.5Gy once.

[Example 3] Influence of U73122 on the expression of target gene PLCG1 protein in vitro

Materials and Methods

1. V79 cell culture

Same as embodiment one.

2. U73122 expressed western blotting of target gene PLCG1 protein in vitro

The V79 cell suspension was inoculated in a 6-well culture plate, and each well was inoculated with 2 mL of culture solution, containing 1.5×10 ⁵ cells. U73122 was formulated as a 20uM stock solution. The final concentration of drug-added cells is 2.5 μM, 5 μM. After culturing for 72 hours, total cell protein was extracted by RIPA-PICT (Pharmacia), and each extract was adjusted to the same concentration after protein quantification. 50 μg of total protein per well was electrophoresed on a 12% polyacrylamide gel, and Western blotting was performed to observe the protein expression of the target gene.

result

It can be seen from Fig. 3 that U73122 has an obvious dose-dependent inhibitory effect on the target gene PLCG1 at 2.5 μM and 5 μM.

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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