CN108607092B - Application of angelica sinensis protein in preparation of medicine for assisting tumor treatment - Google Patents

Application of angelica sinensis protein in preparation of medicine for assisting tumor treatment Download PDF

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CN108607092B
CN108607092B CN201810420895.2A CN201810420895A CN108607092B CN 108607092 B CN108607092 B CN 108607092B CN 201810420895 A CN201810420895 A CN 201810420895A CN 108607092 B CN108607092 B CN 108607092B
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潘剑茹
王香玲
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Fuzhou University
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Abstract

The invention relates to the field of natural traditional Chinese medicine protein, in particular to application of angelica sinensis protein in preparing an auxiliary tumor treatment medicine; the N-terminal sequence of the angelica sinensis protein is GIQKTEVEAPSTVSA. The angelica protein is used for preparing an auxiliary tumor treatment medicament, and the damage of chemotherapy medicament cisplatin to the spleen of a mouse can be relieved by administering the angelica protein before chemotherapy; the angelica sinensis protein is given before radiotherapy, so that the peripheral blood leukocyte number, thymus index, spleen nodule number (CFU-S) and CAT activity of the spleen of a irradiated mouse are respectively and remarkably increased by 135%, 103%, 23%, 219% and 180%, GST and GSH-Px activity of the spleen of the irradiated mouse can be improved, spleen white marrow injury is improved, the overall protection effect is stronger than that of amifostine, and the weight reduction rate of the irradiated mouse can be remarkably reduced by 94%, so that the survival quality of the irradiated mouse is improved.

Description

Application of angelica sinensis protein in preparation of medicine for assisting tumor treatment
Technical Field
The invention relates to the field of natural traditional Chinese medicine protein, and more particularly relates to application of angelica sinensis protein in preparation of an auxiliary tumor treatment medicine.
Background
The chemotherapy drug cisplatin (DDP) is one of platinum-based broad-spectrum anticancer specific drugs with clinical activity, but has strong killing effect on tumor cells due to low selectivity, and can also inhibit the immune system represented by spleen.
Radiation therapy is an effective means for killing tumors, and is used for treating 2/3 tumor patients. However, radiation damage caused by free radicals generated by radiotherapy has large side effects, the local and overall resistance of the body is seriously weakened, and patients are often forced to interrupt the radiotherapy due to the occurrence of the side effects. Therefore, the prevention and treatment of the side effects of tumor radiotherapy directly influences whether the radiotherapy can be continued or not, is one of the main factors for determining whether the tumor can be completely cured or not, and is increasingly concerned and valued by people.
Amifostine (AMFT), also known as WR-2721, is the only radioprotectant approved for clinical use. Amifostine is administered before radiotherapy, and can significantly reduce the toxicity of chemotherapy drugs on kidney, bone marrow, heart, ear and nervous system. However, in the clinical use process, amifostine is gradually found to have higher toxicity, so that most patients can have adverse reactions such as dizziness, nausea, vomiting and the like. Due to the high price of amifostine and the few side effects after use, the clinical use of amifostine is gradually reduced in recent years, even the amifostine is quitted from clinical use. Therefore, the market is urgently in need of developing new chemotherapeutics and radioprotectors.
Angelica sinensis [ Angelica sinensis (Oliv.) Diels]Is a perennial herb belonging to the Angelica of Umbelliferae, and has the good effect of promoting blood circulation and replenishing blood, and the root of the herb can be used as a medicinal material, is sweet and warm and moist. Chinese angelica, one of the famous Chinese herbs, is honored as "Yaowang" and has certain efficacy on the circulatory system, respiratory system, hematopoietic system, immune system, nervous system and the like. The angelica sinensis total protein has the capacity of eliminating DPPH free radicals and can remarkably promote the proliferation of normal liver cell L-02 (P<0.01) and can also remarkably inhibit the proliferation of leukemia cells K562P<0.01）。
The invention obtains angelica PR-10 family protein ASPR from Chinese medicine angelica. The disease course associated protein-10 family (PR-10) proteins are widely distributed in various species and organs of plants, belong to plant stress resistance proteins, and are related to the ability of plants to resist biotic stress (pathogens) and abiotic stress (drought, heat, cold and salinity).
The invention provides angelica protein and application thereof in preparing a medicament for assisting tumor treatment.
Disclosure of Invention
The invention aims to provide application of angelica sinensis protein in preparing an auxiliary tumor treatment medicament so as to develop a novel chemo-treatment and radiotherapy protective agent.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the preparation method of the angelica protein comprises the following steps:
soaking radix Angelicae sinensis decoction pieces in 10 times volume of 0.05 mol/L Tris-HCl buffer solution (pH 8.0), extracting at 4 deg.C overnight, filtering the residue with 4 layers of gauze the next day, centrifuging the filtrate at 12000 rpm and 4 deg.C for 10min, and collecting the supernatant. And carrying out 80% ammonium sulfate precipitation on the obtained supernatant, collecting the precipitate, dissolving the precipitate in 2-fold volume of 0.05 mol/L Tris-HCl buffer solution (pH8.0), separating by using a Sephadex G-50 chromatographic column, collecting a second elution peak, namely the target protein ASPR, wherein the molecular weight is 18.33 kDa, and sequencing to obtain an N-terminal sequence of GIQKTEVEAPSTVSA. The angelica protein can be used for preparing a medicament for preventing and treating radiation injury.
The use concentration of the angelica protein is 0.5-2 mg/mL; sterile filtered angelica ASPR protein was diluted to working concentration with sterile PBS or normal saline. The application method comprises continuously injecting 0.5 mL of Angelica sinensis ASPR protein 3d into muscle or vein before cisplatin or radiotherapy.
The invention has the advantages that:
(1) the invention uses the angelica ASPR protein in cisplatin damage and acute radiation damage models for the first time.
(2) The invention proves that the angelica ASPR protein has a protective effect on spleen injury caused by cisplatin, and can reduce the injury of the cisplatin on the mouse spleen.
(3) The invention proves that the angelica ASPR protein has a protective effect on acute radiation injury, the angelica ASPR protein is given before radiotherapy, the peripheral blood leukocyte number, the thymus index, the spleen nodule number (CFU-S) and the CAT activity of the spleen of a irradiated mouse can be respectively and obviously increased by 135%, 103%, 23%, 219% and 180%, the GST and GSH-Px activity of the spleen of the irradiated mouse can be improved, the spleen white marrow injury can be improved, the overall protection effect is stronger than that of amifostine, and the weight reduction rate of the irradiated mouse can be obviously reduced by 94%, so that the life quality of the irradiated mouse can be improved.
The specific application of the medicine of the invention is as follows:
(1) can be used for preventing side effects of cancer patients caused by chemotherapy or radiotherapy, such as radioactive lung injury, radioactive hematopoietic system injury, and radioactive liver injury.
(2) Can be used for daily prevention of radiation injury for medical and industrial radioactive source operators and the like, such as maintenance of nuclear industrial equipment and rescue of radiation accident patients.
(3) Can be used for astronauts to prevent space cosmic ray damage.
(4) Can be used for preventing other oxidative damages.
The pharmacological action of the medicine of the invention is as follows:
the angelica ASPR protein can obviously increase the CAT activity of spleen tissues of a mouse to be irradiated, can also improve the GST and GSH-Px activity of the spleen, improve the white marrow damage of the spleen, increase the number of spleen nodules (CFU-S) and peripheral blood leukocytes, improve the index of the spleen and thymus, and maintain the weight of the spleen, thereby improving the quality of life of the mouse.
Drawings
FIG. 1 SDS-PAGE of Angelica sinensis ASPR protein. M: a protein Marker; 1: angelica Sinensis Protein (ASPR).
FIG. 2 Effect of Angelica sinensis ASPR protein on spleen damage by cisplatin in mice. A. Mouse spleen appearance map; B. mouse spleen index n =5, compared to normal group,** P<0.01, compared with the cisplatin damage control group,# P<0.05。
FIG. 3 Effect of Angelica sinensis ASPR protein on the body weight gain rate of the mice. n =10, the ratio of the total number of cells in the normal group,** P <0.01, compared with the radiation control group,## P <0.01。
FIG. 4 Effect of Angelica sinensis ASPR protein on peripheral blood leukocytes in mice. n =10, the ratio of the total number of cells in the normal group,** P <0.01, compared with the radiation control group,# P <0.05，## P <0.01。
FIG. 5 Effect of Angelica sinensis ASPR protein on thymus index of irradiated mice. n =10, the ratio of the total number of cells in the normal group,** P <0.01, compared with the radiation control group,## P <0.01，# P <0.05。
FIG. 6 Effect of Angelica sinensis ASPR protein on spleen index of control mice. n =10, the ratio of the total number of cells in the normal group,** P <0.01, compared with the radiation control group,# P <0.05。
figure 7 effect of angelica ASPR protein on spleen appearance and histology characteristics of the control mice (× 200-fold) n = 10.
FIG. 8 Effect of Angelica ASPR protein on spleen nodule counts in control mice. n =10, the ratio of the total number of cells in the normal group,* P <0.05, compared with the radiation control group,## P <0.01。
FIG. 9 Effect of Angelica sinensis ASPR protein on CAT Activity levels in control mice. n =10, the number of the cells in the normal group,* P <0.05, compared with the radiation control group,# P <0.05。
FIG. 10 Effect of Angelica sinensis ASPR protein on GST viability levels in irradiated mice. n =10, the ratio of the total number of cells in the normal group,** P <0.01。
FIG. 11 Effect of Angelica sinensis ASPR protein on GSH-Px viability levels in control mice. n =10, compared to the radiation control group,# P <0.05。
Detailed Description
Example 1 purification of Angelica sinensis ASPR protein
Soaking radix Angelicae sinensis cut into small pieces with 10 times volume of 0.05 mol/L Tris-HCl buffer solution (pH 8.0), standing overnight at 4 deg.C for about 15 hr, filtering residue with 4 layers of gauze the next day, centrifuging filtrate at 12000 rpm at 4 deg.C for 10min, and collecting supernatant as radix Angelicae sinensis protein crude extract; performing ammonium sulfate one-step precipitation (0-80%) on the crude protein solution, precipitating overnight and collecting the precipitated protein, dissolving in 2 times of volume of 0.05 mol/L Tris-HCl buffer solution (pH 8.0), loading the dissolved solution of the ammonium sulfate precipitation on a Sephadex G-50 chromatographic column fully balanced by an equilibrium solution (0.05 mol/L Tris-HCl buffer solution with pH 8.0), eluting by the equilibrium solution and collecting a second elution peak, and detecting the purity by SDS-PAGE electrophoresis.
The experimental results are as follows:
the angelica protein is subjected to crude extraction, ammonium sulfate one-step precipitation and SephadexG-50 gel filtration chromatography to finally obtain the angelica protein which shows a single band on SDS-PAGE and has the molecular weight of 18.33 KDa, as shown in figure 1. The N-terminal sequence of the protein is GIQKTEVEAPSTVSA, the molecular weight of the protein is 18.33 kDa, and the protein is a PR-10 family protein and is named as ASPR.
Example 2: protective effect of angelica sinensis ASPR protein on cisplatin-damaged mice
1. Experimental animals and groups
Male Kunming mice, purchased from Wu's animal, weighing 20 + -2 g. The animals were normally kept for three days before the experiment. Mice were randomly divided into 5 groups of 5 mice each, and each group and treatment were as follows: the normal group (CON) and the cisplatin damage group (DDP) are injected with 0.3 mL of normal saline for 3d continuously, 0.3 mL of normal saline is injected into the CON group 2h after the last administration, and 0.3 mL (7.5 mg/kg) of cisplatin solution is injected into the DDP group 0.5 mg/mL; the protein adding medicine groups are respectively ASPR1 (0.5 mg/mL) + DDP group, ASPR2 (1.25 mg/mL) + DDP group and ASPR3 (2.0 mg/mL) + DDP group, three groups of mice are respectively injected with 0.5 mL of protein solution for 3d, and after 2 hours of last administration, 0.3 mL of cisplatin solution is injected.
2. Detection index-spleen index
All mice were sacrificed after 2 d of molding, spleens of the mice were removed, residual blood was removed, mass weighed and recorded, and indices thereof were calculated.
Spleen index = (spleen mass/pre-sacrifice body weight) × 100%.
The experimental results are as follows:
as shown in fig. 2, spleen was smaller in the cisplatin-damaged control group compared to the normal group, and spleen was larger in each protein-added group compared to the cisplatin-damaged control group. The spleen index measurement results of the mice in each group also show that the spleen index of the cisplatin-damaged control group is very significantly reduced compared with that of the normal group (P<0.01) of the normal group, which is only 57.19% of the normal group. Compared with the cisplatin-damaged control group, spleen indexes of all protein drug-added groups are increased, wherein 40.35% of the angelica ASPR protein middle dose group is remarkably recovered (the ratio of the angelica ASPR protein middle dose group to the cisplatin-damaged control group is high), (the ratio of the angelica ASPR protein middle dose group to the cisplatin-damaged control group is high, and the ratio ofP<0.05）。
Therefore we chose the optimal protein dose point of 1.25 mg/mL as the next example 3.
Example 3: preventive effect on disposable whole body radiation mouse radiation protection
1. Laboratory animal
Male Kunming mice, purchased from Wu's animal, weighing 20 + -2 g. The water and food can be freely drunk, the feed is a special irradiation material, and the air is exhausted at regular time. Three days before the experiment, the breeding had no abnormality.
2. Illumination conditions and Experimental groups
Test kunming mice were randomly divided into 5 groups: normal group CON (no radiation administration), radiation control group XRT (no radiation administration), amifostine group (AMFT + XRT) (200 mg/kg, 30 min injection before radiation), angelica sinensis ASPR protein group (ASPR + XRT) (1.25 mg/mL), pre-radiation administration group was administered with 3d continuous intraperitoneal injection of angelica sinensis ASPR protein, and radiation was performed 2h after the last injection. The radioactive source is X-ray, the irradiation dose is 6 Gy, the dose rate is 2 Gy/min, and disposable whole body irradiation is carried out. And feeding for 7 days after irradiation.
3. Detecting the index
3.1 weight gain in mice
Body weight measurements were taken of all mice before irradiation, after which the mice were weighed daily for a fixed period and recorded, and the weight gain of the mice over 7 days was calculated.
Body weight gain rate = (mouse body weight on day 7-mouse body weight before radiation)/mouse body weight before radiation × 100%.
3.2 peripheral blood leukocyte count variation
All mice were sacrificed by cervical dislocation 7 days after irradiation. The dead mice are subjected to heart blood sampling immediately, 20 mu l of blood is taken from each mouse, 380 ul of leukocyte diluent (2.0 mL of glacial acetic acid and 1% gentian violet 1.0 mL, distilled water is added to a constant volume of 100 mL and mixed evenly) is used for diluting, the diluted liquid is dripped into a counting plate, standing is carried out for 2-3 min, and the leukocyte counting is carried out under a common optical microscope after the leukocytes sink.
3.3 thymus index and spleen index
After the thymus and spleen of the mouse are picked and residual blood is removed, the mass of the mouse is weighed and recorded, and the organ index of the mouse is calculated.
Thymus index = (thymus mass/pre-sacrifice body weight) × 100%.
Spleen index = (spleen mass/pre-sacrifice body weight) × 100%.
3.4 determination of mouse hematopoietic Stem cells by the exogenous spleen nodule method
Reference is made to the classical extrinsic splenic nodule method. A part of spleen is taken out, and is placed in Bouin's solution (30 mL of picric acid saturated aqueous solution, 10 mL of 10% formaldehyde and 2 mL of glacial acetic acid) for fixation for 24 h, and then the spleen is rinsed with 95% alcohol for macroscopic spleen nodule counting, namely, the spleen convex nodule is counted. The ratio of Bouin's fluid to the sum of spleen volumes is greater than 5: 1.
3.5 spleen epigenetics and histology evaluation
Fixing a part of spleen tissues by using a 10% formalin solution, and then embedding the spleen tissues in paraffin according to a tissue embedding program; after fixation, the tissue sections were cut into 4 μm sections and stained with H & E.
3.6 detection of spleen antioxidant capacity
Preparing a part of spleen into 10% tissue homogenate, centrifuging at 4000 rpm for 20 min, subpackaging the supernatant into a to-be-detected liquid, and respectively determining the activities of CAT, GST and GSH-Px in the spleen tissue homogenate by using Nanjing built-up kit.
4. Statistical analysis: student's t-test in Excel 2013 was used toP<0.05 is a significant difference in the number of,P<0.01 is a very significant difference.
5. Results
As shown in FIG. 3, the body weight of the mouse was in a negative growth state on the seventh day after the whole body irradiation with 6 Gy compared with the normal group ((ii)P<0.01). Compared with the radiation control group, the amifostine or the angelica sinensis protein pre-radiated can remarkably inhibit the weight loss of the irradiated mice (the weight loss of the irradiated mice is reduced by the pre-radiated amifostine or the angelica sinensis protein)P<0.01), the amifostine group can reduce the weight loss rate of the mice by 59%, (P<0.01), the weight loss rate of the pre-dosed angelica sinensis ASPR is reduced by 94% (P<0.01）。
As shown in FIG. 4, the peripheral blood leukocyte count of the mice in the radiation control group was extremely significantly reduced compared with that in the normal group (P<0.01). Compared with the radiation control group, the pre-radiation administration of amifostine can significantly increase the number of the leucocytes of the mice by 106 percent (P<0.05), the number of leukocytes in the blood of the irradiated mice pre-dosed with the angelica protein ASPR was significantly increased by 135% (P<0.05). As shown in FIG. 5, the thymus index of the radiation control group was very significantly reduced compared to that of the normal group (P<0.01). Compared with the radiation control group, the thymus index of the mice can be obviously improved by pre-administration of amifostine or angelica sinensis protein (P<0.05， P<0.01), but the effect of angelica sinensis protein ASPR is stronger than that of amifostine.
As shown in FIG. 6, spleen index of mice in the radiation control group was very significantly reduced compared to that in the normal group (P<0.01). Compared with the radiation control group, the pre-administration of amifostine can lead the patients to be suffered from the diseasesSplenic index of control mice is obviously improved by 33%, (P<0.05), the spleen index of the irradiated mice can be obviously increased by 23 percent respectively by pre-administration of angelica sinensis protein before radiation (P<0.05）。
The spleen histological pathological section results are shown in fig. 7, and spleen red and white marrow of mice in the single radiation group are disordered and have no obvious limit. The pre-administration of amifostine or angelica sinensis protein improved this to varying degrees, with well-defined intact white marrow being visible.
As shown in FIG. 7, the spleen surface of normal mice was smooth and flat, and the spleen surface of mice in the single-radiation group was uneven, and prominent splenic nodule vesicles, i.e., spleen colony forming units (CFU-S), were observed with the naked eye. Given amifostine or angelica sinensis protein in advance, the spleen surface of the control mice showed unequal numbers of spleen nodules. The number of spleen colony forming units in each group of mice after treatment with Bouin solution is shown in FIG. 8. As can be seen from FIG. 8, the normal group mice had no CFU-S, and the radiation control group had a small amount of CFU-S. Compared with the radiation control group, the amount of CFU-S of the mice can be improved without significance by pre-administration of amifostine, and the CFU-S of the mice is increased by more than two times by pre-administration of angelica sinensis protein.
As can be seen from FIGS. 9, 10 and 11, the spleen of mice in the radiation-only group had significantly reduced CAT, GSH-Px and GST activities, as compared with those in the normal group (P<0.05；P<0.01). Compared with the simple radiation group, the premonitine or the angelica ASPR protein can improve the activity of the antioxidant enzyme of the spleen of the irradiated mouse to different degrees. The preadministration of amifostine has obvious effect of improving CAT and GSH-Px activity of spleen of a subject mouse, and has certain effect of improving GST activity of spleen of the subject mouse. The angelica ASPR protein has obvious effect of improving CAT activity of the spleen of the irradiated mouse, the effect is slightly stronger than that of amifostine, and the angelica ASPR protein also has certain effect of improving GSH-Px and GST activity of the spleen of the irradiated mouse.
In summary, the pre-irradiation of Angelica sinensis ASPR protein significantly increased the number of peripheral blood leukocytes in the irradiated mice by 135% compared to the irradiation control group (see below)P<0.05), a significant increase in thymic index of 103%, (P<0.05), a significant increase in spleen index of 23%, (P<0.05), a significant increase in CFU-S of 219%, (P<0.05) has obvious improvement effect on spleen white marrow damage, and can obviously increase the CAT activity of the spleen by 180 percent (P<0.05), and finally, the weight loss rate of the irradiated mice is obviously reduced by 94%, (P<0.01）。
CFU-S is an important index for reflecting spleen hematopoiesis function, and although the protection effect of the angelica sinensis protein on a spleen antioxidant system and a spleen index is slightly weaker than that of amifostine, the improvement effect of the angelica sinensis protein on the spleen CFU-S is much stronger than that of the amifostine. In addition, the angelica sinensis protein has better effects of improving WBC, thymus index and CFU-S of the irradiated mice than amifostine, and finally has stronger inhibiting effect on the weight loss caused by the whole body radiation than the amifostine. The weight growth rate of the mouse can intuitively reflect the survival quality of the mouse and is a comprehensive index of the radiation injury degree of the mouse. Therefore, the comprehensive protection effect of the angelica ASPR protein on radiation injury is stronger than that of amifostine.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fuzhou university
Application of angelica sinensis protein in preparation of drugs for assisting tumor treatment
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> PRT
<213> Angelica sinensis protein
<400> 1
Gly Ile Gln Lys Thr Glu Val Glu Ala Pro Ser Thr Val Ser Ala
1 5 10 15

Claims (1)

1. An application of angelica protein in preparing an auxiliary tumor treatment medicament, wherein the N-terminal sequence of the angelica protein is GIQKTEVEAPSTVSA, the molecular weight is 18.33 KDa, and the auxiliary tumor treatment medicament is a medicament for preventing chemotherapy and radiation injury;
the preparation method of the angelica protein comprises the following steps: soaking radix Angelicae sinensis cut into small pieces in 10 times volume of 0.05 mol/L Tris-HCl buffer solution with pH8.0, standing at 4 deg.C overnight, filtering residue with 4 layers of gauze the next day, centrifuging the filtrate at 12000 rpm and 4 deg.C for 10min, and collecting supernatant as radix Angelicae sinensis protein crude extract; performing one-step precipitation by using 0-80% ammonium sulfate, precipitating overnight and collecting precipitated protein, dissolving the precipitated protein in 2-fold volume of 0.05 mol/L Tris-HCl buffer solution with the pH value of 8.0, loading the dissolved solution of the ammonium sulfate precipitation on a Sephadex G-50 chromatographic column which is fully balanced by 0.05 mol/L Tris-HCl buffer solution with the pH value of 8.0 of a balancing solution, eluting by using the balancing solution and collecting a second elution peak as target protein.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101239094A (en) * 2007-02-05 2008-08-13 天津药物研究院 Active component composition used as adjuvant drug for antitumor and preparation and application thereof

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DE3907822A1 (en) * 1989-03-10 1990-09-13 Adolf Dr Loefflmann USE OF A PEROXIDASE ACTIVITY PROTEIN INFRACTION FROM THE PHOTOSYNTHESIS APPARATUS OF PLANTS AS A MEDICINE, IN PARTICULAR TO COMBAT CANCER, AND A METHOD FOR OBTAINING A PROBLEM ADMINISTRATION
CN101020719B (en) * 2006-02-14 2011-01-12 北京美迪克斯生物技术有限公司 Composite angelica polysaccharide and its preparation process and use
CN102526712B (en) * 2012-01-09 2014-03-26 福州大学 Application of super oxygen dehydrogenises-trans-activator transcription (SOD-TAT) fusion protein to radiation injury prevention and cure medicine preparation

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CN101239094A (en) * 2007-02-05 2008-08-13 天津药物研究院 Active component composition used as adjuvant drug for antitumor and preparation and application thereof

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