CN115475160A

CN115475160A - Application of diosmetin in esophageal squamous cell carcinoma

Info

Publication number: CN115475160A
Application number: CN202211164208.8A
Authority: CN
Inventors: 路静; 陈怡欢; 陈薇; 代晓烁; 张锴; 段晓萱; 董子明
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2022-09-23
Filing date: 2022-09-23
Publication date: 2022-12-16

Abstract

The invention belongs to the technical field of tumor prevention and treatment, and particularly relates to application of diosmetin in esophageal squamous cell carcinoma. Diosmetin is used for inhibiting proliferation, migration and invasion of esophageal squamous carcinoma cells, inducing cycle arrest of esophageal squamous carcinoma cells and promoting apoptosis of esophageal squamous carcinoma cells. Preliminary experiment results show that the single DIOS shows a good prevention and treatment effect on esophageal squamous carcinoma related cell lines, the prevention and treatment effect is dose-dependent to the dosage of the DIOS, and the related prevention and treatment effect is equivalent to the CDDP effect. After the DIOS and the CDDP are jointly applied, the DIOS and the CDDP show a better synergistic effect, the application effect on the prevention and treatment of the esophageal squamous carcinoma is detailed better than that of the DIOS or CDDP experimental group, particularly the nephrotoxicity caused in the application process of the CDDP is obviously reduced, and the better application effect is shown. Based on the research, a new treatment strategy can be provided for the clinical treatment of the esophageal squamous carcinoma.

Description

Application of diosmetin in esophageal squamous cell carcinoma

Technical Field

The invention belongs to the technical field of tumor prevention and treatment, and particularly relates to application of diosmetin in esophageal squamous cell carcinoma.

Background

Esophageal cancer is one of the common malignant tumors of the digestive tract, with the seventh incidence and the sixth incidence worldwide mortality. Currently, the 5-year survival rate of patients with esophageal cancer is only 20%, while the 5-year survival rate of patients who have developed metastasis is only 5%. Esophageal cancer can be divided into esophageal squamous cell carcinoma and esophageal adenocarcinoma by histological type, with esophageal squamous carcinoma being one of the most aggressive squamous cell carcinomas. Statistics show that the incidence rate of esophageal squamous cell carcinoma is high in China, and the number of diseased esophageal squamous cell carcinoma accounts for about half of the global esophageal squamous cell carcinoma cases every year in China. Therefore, the improvement of esophageal squamous carcinoma has important technical significance for further improving the average life of the population in China.

In recent years, the rapid development of endoscope technology makes it possible to discover esophageal squamous cell carcinoma at an early stage, and further lays a foundation for prevention and treatment of esophageal squamous cell carcinoma. In the prior art, many different methods have been used for the treatment of patients with esophageal squamous carcinoma, including endoscopic therapy, surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, etc. Among them, surgical treatment and chemoradiotherapy are the most common treatment means, and improve the prognosis of esophageal squamous cell carcinoma patients to a certain extent, but adverse drug reactions and drug resistance phenomena occur in the treatment process, so that the overall treatment effect is difficult to satisfy. Targeted therapy and immunotherapy have better application potential in cancer treatment, but individual responses of patients are greatly different, so that the application of the targeted therapy and the immunotherapy lacks better universality. Therefore, based on the current status of esophageal squamous cell carcinoma treatment, a need exists for new safe and effective therapeutic drugs.

Diosmetin (DIOS) is a natural flavonoid compound extracted from various plants such as acacia, chrysanthemum, lemon, and citrus, and related chemical synthesis technical routes have been developed in the prior art. Research has shown that DIOS has anti-inflammatory, antibacterial, and antioxidant effects, and in recent years, its anti-tumor effect has been receiving attention. It has been found that DIOS has anticancer effects in liver cancer, colorectal cancer, lung cancer and other tumors, such as inhibiting cell proliferation, migration and invasion, inducing apoptosis and cell cycle arrest. However, the mechanism of action of DIOS in tumors is still not clear enough, and further research and investigation is needed to find out whether DIOS has similar action in the prevention and treatment of other types of tumors.

Disclosure of Invention

Based on the application of diosmetin in the prevention and treatment of other types of tumors, the application aims to try to apply and develop DIOS in the prevention and treatment of esophageal squamous cell carcinoma, so that a certain technical basis is laid for the prevention and treatment of esophageal squamous cell carcinoma.

The technical scheme provided by the application is detailed as follows.

Diosmetin is used for inhibiting proliferation, migration and invasion of esophageal squamous carcinoma cells, inducing cycle arrest of the esophageal squamous carcinoma cells and promoting apoptosis of the esophageal squamous carcinoma cells;

the cycle block is to block the cycle of the esophageal squamous carcinoma cells in a G2/M stage;

when the diosmetin is applied, the dosage and the application time of diosmetin are in positive correlation with the application effect, namely, the diosmetin exerts the corresponding application effect on esophageal squamous cell carcinoma cells in a dosage-dependent and time-dependent manner, and the application effect is improved along with the increase of the dosage and the extension of the application time of diosmetin.

The esophageal squamous carcinoma cell specifically comprises the following components: cell lines of esophageal squamous carcinoma KYSE150, KYSE450 and ECa 109.

The diosmetin is applied together with cisplatin to prevent and treat esophageal squamous carcinoma.

In the prior art, cisplatin (cissplatin, CDDP) is a first-line drug for clinically treating esophageal squamous cell carcinoma, but has serious nephrotoxicity, so that the application of Cisplatin is more limited. Partial research aiming at DIOS shows that DIOS can activate Nrf2/HO-1 antioxidant pathway and increase the expression of antioxidant, thereby eliminating antioxidant products such as free radicals and the like, further maintaining the redox steady state of cells and fully playing the cell protection function. Based on the wide-ranging antioxidant function and the known anti-tumor effect aiming at part of cancer types, the inventor tries to apply DIOS to the prevention and treatment of esophageal squamous carcinoma so as to expand the anti-tumor types of the esophageal squamous carcinoma, and simultaneously tries to apply DIOS and CDDP in a combined way so as to investigate whether the drug combination has a synergistic effect and reduce renal toxicity caused by CDDP, thereby providing a new treatment strategy for the prevention and treatment of esophageal squamous carcinoma.

Preliminary experiment results show that the single DIOS shows a better prevention and treatment effect on the esophageal squamous cell carcinoma related cell line, the prevention and treatment effect is dose-dependent to the dosage of the DIOS, and the related prevention and treatment effect is equivalent to the CDDP effect. After the DIOS and the CDDP are jointly applied, the DIOS and the CDDP show a better synergistic effect, the application effect on the prevention and treatment of the esophageal squamous carcinoma is obviously better than that of the DIOS or CDDP experimental group, particularly the nephrotoxicity caused in the application process of the CDDP is obviously reduced, and the better application effect is shown. Based on the researches, the novel treatment strategy can be provided for the clinical treatment of the esophageal squamous cell carcinoma, and meanwhile, better reference and reference are provided for the prevention and treatment of other tumors.

Drawings

FIG. 1 shows the toxicity test results of DIOS; the results of the cell viability of DIOS with different concentrations on normal esophageal epithelial cells Het-1A and esophageal squamous carcinoma cells KYSE150, KYSE450 and ECa109 5348 h are shown in the figure;

FIG. 2 shows the effect of DIOS on the proliferative capacity of esophageal squamous carcinoma cells; there are shown absorbance values for different concentrations of DIOS on KYSE150, KYSE450,

ECa109 cells

0h, 24h and 48h, respectively;

FIG. 3 shows the cell cycle induction block results of DIOS on esophageal squamous carcinoma cells; the figure shows the results of detecting the distribution ratio of cells in each period by adopting flow cytometry after DIOS with different concentrations respectively acts on KYSE150, KYSE450 and ECa109 cells 24 h;

FIG. 4 shows the result of experiments on apoptosis of esophageal squamous cell carcinoma by DIOS concentration, which indicates that apoptosis is concentration-dependent on DIOS dosage; the flow result graph and the apoptosis cell ratio statistical result of DIOS with different concentrations after respectively acting on KYSE150, KYSE450 and ECa109 cells 24h are shown in the graph;

FIG. 5 shows the result of experiments on apoptosis of esophageal squamous cell carcinoma by DIOS application time, and the results show that apoptosis and DIOS application time are time-dependent; the flow chart and the apoptotic cell ratio statistics result of 40 μ M DIOS on KYSE150, KYSE450 and

ECa109 cells

24h, 48h and 72 h respectively;

FIG. 6 shows the effect of DIOS on the migration and invasion ability of esophageal squamous carcinoma cells; wherein:

(A) The migration capability result of the cells when DIOS with different concentrations acts on 0h, 24h and 48h of different types of esophageal squamous carcinoma cells is detected through a scratch experiment;

(B) The cell migration and invasion capacity after different types of esophageal squamous carcinoma cells 24h are pretreated by DIOS with different concentrations is detected through a Transwell cell migration and invasion experiment;

the scale bar in the figure is 100 μm;

FIG. 7 shows the results of DIOS growth inhibition of transplanted esophageal squamous carcinoma cell tumors in mice; wherein:

(A) Schematic representation of tumor sizes of mice at different stages;

(B) The change curve of the tumor volume of the mice is shown;

(C) A mouse tumor weight statistical chart is obtained;

(D) A tumor inhibition rate statistical chart is shown;

(E) Detecting the expression level result of the tumor tissue Ki67 for immunohistochemistry;

the scale bar in the figure is 25 μm;

FIG. 8 shows the evaluation results of DIOS and CDDP for the toxic and side effects of CDX model mice; wherein:

(A) A graph of the change in body weight of the mice;

(B) HE staining patterns of mouse heart, liver, spleen and lung;

(C) Is a mouse kidney HE staining pattern; wherein: (1) kidney of control mouse; (2) the kidneys of mice in DIOS group have interstitial lymphocyte infiltration; (3) renal tubular epithelial cell apoptosis of CDDP group mice; (4) renal interstitial fibrosis of mice in the CDDP group; (5) infiltrating lymphocytes in the interstitial kidney of mice in the CDDP group; (6) infiltrating interstitial plasma cells in the kidney of CDDP group mice;

(D) Detecting mRNA level changes of kidney injury indexes KIM-1 and NGAL of the mice for qRT-PCR;

the scale bar in the figure is 25 μm;

FIG. 9 shows the calculation results of the combination dose index of DIOS and CDDP; wherein:

(A) The cell survival rate results of CDDP with different concentrations after being acted on KYSE150 and ECa109 cells 48 h;

(B) The cell survival rate of DIOS and CDDP after combined action of different concentrations of 48h is shown;

(C) A combined index analysis chart after DIOS and CDDP are combined to act on cells at different concentrations;

(D) (ii) absorbance values for DIOS and CDDP single and combined drugs on KYSE150 and

ECa109 cells

0h, 24h, 48h and 72 h;

FIG. 10 shows the effect of DIOS and CDDP single and combined drug on the cell cycle and apoptosis of esophageal squamous carcinoma; wherein:

(A) The distribution proportion of DIOS and CDDP single drugs and combined drugs in each period after the DIOS and CDDP single drugs and combined drugs act on KYSE150 and ECa109 cells 24h is increased;

(B) Is a flow result graph and an apoptotic cell proportion statistical graph after DIOS and CDDP single drugs and combined drugs act on KYSE150 and ECa109 cells 24 h;

FIG. 11 shows the results of the growth inhibition of human esophageal squamous carcinoma xenografts (LEG 398 and LEG 404) in mice by DIOS in combination with CDDP; wherein:

(A) Schematic representation of mouse tumor size at different growth stages;

(B) A curve chart of the change of the tumor volume of the mouse is shown;

(C) A mouse tumor weight statistical chart is obtained;

(D) A tumor inhibition rate statistical chart is shown;

FIG. 12 is a graph showing the effect of immunohistochemical detection of the level of Ki67 expression in DIOS in combination with CDDP in human esophageal squamous cell carcinoma xenografts (LEG 398 and LEG 404); the scale bar in the figure is 25 μm.

FIG. 13 shows the evaluation results of the side effects of DIOS and CDDP combinations on PDX model mice; wherein:

(A) LEG398 cases PDX model mice weight change graph and each group of mice spleen index and kidney index statistical graph;

(B) LEG404 cases PDX model mice body weight change graph and spleen index and kidney index statistical graph of each group of mice;

(C) Spleen and kidney morphograms from LEG398 PDX model mice;

(D) LEG404 PDX model mice spleen and kidney morphograms;

FIG. 14 is a graph of the effect of DIOS and CDDP combinations on nephrotoxicity in LEG398 mice in the PDX model CDDP group, showing that combinations can reduce nephrotoxicity of CDDP alone; wherein:

(A) Detecting mRNA level changes of KIM-1 and NGAL of the kidney of the mouse for qRT-PCR;

(B) Detecting results of mouse serum BUN and SCR;

(C) Is a mouse kidney HE staining pattern; wherein: (1) kidney of control mouse; (2) the kidneys of mice in DIOS group have interstitial lymphocyte infiltration; (3) renal tubular epithelial cell apoptosis of CDDP group mice; (4) renal interstitial fibrosis of mice in the CDDP group; (5) infiltrating lymphocytes in the interstitial kidney of mice in the CDDP group; (6) infiltrating interstitial plasma cells in the kidney of CDDP group mice; (7) infiltrating lymphocytes in the interstitium of the kidney of the mice in the combined group;

(D) HE staining patterns of mouse heart, liver, spleen and lung;

FIG. 15 shows the effect of DIOS and CDDP combinations on renal toxicity in mice from the group of LEG404 PDX model CDDP, indicating that combinations can reduce renal toxicity of CDDP alone; wherein:

(A) Detecting mRNA level changes of KIM-1 and NGAL in the kidney of the mouse for qRT-PCR;

(B) Detecting results of mouse serum BUN and SCR;

(C) Is a mouse kidney HE staining pattern; wherein: (1) kidney of control mouse; (2) the kidneys of mice in DIOS group have interstitial lymphocyte infiltration; (3) apoptosis of renal tubular epithelial cells of CDDP group mice; (4) renal interstitial fibrosis of mice in the CDDP group; (5) infiltrating lymphocytes in the interstitial kidney of mice in the CDDP group; (6) infiltrating interstitial plasma cells in the kidney of CDDP group mice; (7) infiltrating lymphocytes in the interstitium of the kidney of the mice in the combined group;

(D) HE staining patterns of mouse heart, liver, spleen and lung;

in the above figures, reference is made to data statisticsp＜0.05，**p＜0.01，***p＜0.001。

Detailed Description

The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.

Biological material:

cell: the esophageal squamous cancer cell lines KYSE150, KYSE450 and ECa109 (on the surface of preliminary experiments, the three esophageal squamous cancer cell lines are sensitive to DIOS reaction, and the research backgrounds of the three cell lines are clear, so that the three cell lines are selected as representatives for research), and the normal esophageal epithelial cells Het-1A are all common cell lines (strains) in the existing medical research, can be obtained from public channels, and are used as professional education research institutions by the applicant, so that related cell lines (strains) are stored for a long time;

experimental animals:

CB17/SCID mice, purchased from Beijing Wintolite laboratory animal technology, inc.;

NOD-SCID mouse, purchased from Gtaso Nanjing Collection drug health Biotech GmbH;

the main experimental reagents are as follows:

diosmetin (greater than or equal to 98 percent (HPLC)), cisplatin, DMSO, trizol and the like, and American SIGMA-ALDRICH products;

corn oil, product of MCE corporation, usa;

RPMI-1640 medium, RPMI-DMEM medium, israel Biological Industries, inc.;

fetal bovine serum, product of Gibco, usa;

ultraView Universal DAB inhibitor, ultraView Universal HRP Multimer, products of Ventana, USA;

ki67 antibody, product of Santa Cruz, USA;

DAB staining kit, product of gold bridge biotechnology limited of China fir in Beijing;

the main apparatus comprises:

flow cytometer BD facscan, usa.

Example 1

In the previous data analysis process, because no report of applications of DIOS to esophageal cancer is seen, from the viewpoint of safety and practicability, the inventor takes a specific esophageal squamous carcinoma cell line (line) as an example and researches the influence of DIOS on the toxic and side effects, cell proliferation, migration and the like of the esophageal cancer cell line (line), and the specific experimental process and results are summarized as follows.

(I) cytotoxicity

The specific process and operation of the toxicity test of DIOS on esophageal squamous carcinoma cells refer to the following steps:

firstly, culturing esophageal squamous carcinoma cells KYSE150, KYSE450, ECa109 and normal esophageal epithelial cells Het-1A conventionally, digesting and centrifuging when the confluence degree of the cells reaches about 80%, discarding supernatant, then using a complete culture medium to resuspend cell precipitates, and respectively adjusting the cell density of KYSE150, KYSE450, ECa109 and Het-1A to 8 multiplied by 10 ⁴ 1X 10 units/mL ⁵ 1X 10 units/mL ⁵ one/mL and 8X 10 ⁴ Per mL;

then, two 96-well plates (one is cultured to 24h, and the other is cultured to 48 h) are taken, cells are paved in the well plates, each hole is 100 mu L, the cells are divided into 7 groups, and each group is provided with 5 multiple holes; DIOS toxicity test drug concentrations were set at 0. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M and 80. Mu.M;

( In the preparation of DIOS, taking the diosmetin mother liquor of 40 mM as an example, the reference operation in the specific preparation is as follows: adding 0.012 g powder into 1 mL DMSO, and dissolving completely to obtain diosmetin mother liquor with concentration of 40 mM, and diluting the mother liquor with corresponding culture medium to corresponding concentration )

The next day, after the cells adhere to the wall, the cells are replaced by fresh culture media containing different drug concentrations;

culturing 48h, taking out a 96-well plate, fixing cells for 5 min by using 4% paraformaldehyde solution at normal temperature, then dyeing for 5 min by using 0.1% crystal violet in an incubator at 37 ℃, cleaning and drying by using tap water after dyeing is finished, adding 1% SDS for dissolving, and measuring the absorbance of 595 nm by using an enzyme labeling instrument;

finally, the absorbance values were statistically aggregated and plotted.

The results of the experiment are shown in FIG. 1. As can be seen from fig. 1: DIOS shows obvious toxic effects on esophageal squamous carcinoma cells KYSE150, ECa109 and KYSE450, the toxic effects are stronger along with the increase of the drug concentration, certain concentration dependence is shown, and the DIOS has a certain influence on the growth of normal esophageal epithelial cells Het-1A, but the overall toxicity is lower. The result shows that DIOS has application potential for pertinence in prevention and treatment of esophageal squamous carcinoma.

(II) Effect on cell proliferation

On the basis of the toxicity test, the inventor further researches the condition of the DIOS on the proliferation of the esophageal squamous cell carcinoma, and the specific process and operation refer to the following steps:

firstly, culturing esophageal squamous carcinoma cells KYSE150, KYSE450 and ECa109 conventionally, digesting and centrifuging when the confluence degree of the cells reaches about 80%, discarding supernatant, then using a complete culture medium to resuspend cell precipitates, and respectively adjusting the cell density of KYSE150, KYSE450 and ECa109 to 5 multiplied by 10 ⁴ 6X 10 pieces/mL ⁴ 6X 10 pieces/mL ⁴ Per mL;

then, three 96-well plates (cultured for 0h, 24h and 48h respectively) were taken, and cells were plated in the well plates at 100. Mu.L per well; DIOS concentration is set to 0 μ M, 20 μ M, 40 μ M, each group is provided with 5 multiple wells;

taking out the 0h plate, fixing the cells with 4% paraformaldehyde solution for 5 min at normal temperature, then dyeing with 0.1% crystal violet for 5 min in an incubator at 37 ℃, after dyeing, washing and drying with tap water, adding 1% SDS for dissolving, and measuring the absorbance of 595 nm by an enzyme labeling instrument;

after culturing for 24h, taking out the 24h plate, and treating by adopting the same treatment mode as the 0h plate; meanwhile, replacing a fresh culture medium for the 96-well plate needing to be continuously cultured, and continuously culturing for 48 hours, and treating in the same treatment mode; finally, the absorbance values were statistically collected and plotted.

The results of the experiment are shown in FIG. 2. As can be seen from fig. 2: DIOS has obvious time-dependent and concentration-dependent inhibition effect on the proliferation capacity of esophageal squamous carcinoma cells KYSE150, ECa109 and KYSE 450. When the time is the same, the inhibition is significantly enhanced with increasing drug concentration; the inhibitory effect is significantly enhanced with increasing duration of action when the drug concentration is constant. On the other hand, DIOS shows a certain difference in the proliferation inhibitory effect on different esophageal squamous carcinoma cell lines (lines).

(III) cell cycle and apoptosis Effect

On the basis of the above experiment, in order to further clarify the reason of the influence of the DIOS on the cell proliferation of the related esophageal squamous carcinoma cell line (line), the inventors studied the cancer cell cycle and apoptosis conditions in the process of inhibiting the cell proliferation of the esophageal squamous carcinoma cell line (line) by the DIOS, and the specific experimental process and operation are referred to as follows.

(1) Cell cycle experiments:

first, human esophageal squamous carcinoma cells KYSE150, KYSE450 and ECa109 were routinely cultured, and the cells were subcultured to 3 6 cm culture dishes (5X 10) ⁵ Per dish) in three groups (DIOS 0. Mu.M, 20. Mu.M and 40. Mu.M);

on the next day, after the cells adhere to the wall, the cells are washed twice by PBS and replaced by serum-free culture medium, and after 24h is cultured, the cells are replaced by complete culture medium containing drugs with corresponding concentrations;

then, continuously culturing for 24h (namely after the drug acts on 24 h), digesting and centrifuging each group of cells, and collecting cell precipitates in a 1.5 mL centrifuge tube;

the collected cell pellet was resuspended in 1 mL PBS and counted 1X 10 ⁶ Centrifuging at 1000 rpm for 5 min for each cell, and repeating twice; in the centrifugation process, 50 mu L of supernatant is left when the supernatant is discarded each time so as to prevent the cells from being discarded;

then adding 250 mu L PBS into the collected cell sediment for resuspension, adding 750 mu L precooled absolute ethyl alcohol after resuspension, and placing the mixture in a refrigerator at the temperature of minus 20 ℃ for fixation overnight;

the next day, taking out the fixed cells from a refrigerator at-20 ℃, centrifuging for 5 min at 1000 g, discarding the supernatant to retain the cell precipitate, resuspending the cell precipitate with PBS, centrifuging for 5 min at 1000 g, discarding the supernatant to retain the precipitate, and repeating twice to wash away residual ethanol;

and finally, dyeing: adding 500 μ L staining buffer to each tube, adding 10 μ L RNase A (50X), placing in water bath at 37 deg.C, and incubating for 30 min; then 25. Mu.L of PI staining solution (20X) was added and incubated at 4 ℃ for 30 min in the absence of light;

after the staining is finished, the cells are filtered, the machine detection is carried out by flow cytometry, and relevant detection results are counted.

The results of the experiment are shown in FIG. 3. Analysis can see that: DIOS has obvious induction blocking effect on the cell cycle of esophageal squamous carcinoma cells KYSE150, KYSE450 and ECa109, and the proportion of cells in the G0/G1 phase and the S phase is reduced along with the increase of the concentration of DIOS, while the proportion of cells in the G2/M phase is obviously increased, so that the cancer cells can be blocked in the G2/M phase, and the proliferation of the cancer cells can be inhibited.

(2) Annexin V-FITC/PI apoptosis assay:

first, human esophageal squamous carcinoma cells KYSE150, KYSE450 and ECa109 were routinely cultured, and the cells were subcultured to 6 cm culture dishes (5X 10) ⁵ Per dish), the experimental groups were designed in three groups (DIOS 0. Mu.M, 20. Mu.M and 40. Mu.M groups, while different time experimental designs of

DIOS

0h, 24h, 48h and 72 h were designed for 40. Mu.M DIOS treated cells). Simultaneously, 3 control groups were designed, which were: 0.μ M DIOS treatment negative group, FITC group and PI group;

on the next day, when the confluence degree of the cells reaches about 70%, washing twice by PBS, and replacing with a complete culture medium containing drugs with corresponding concentrations;

subsequently, after culturing 24h (i.e., after drug action 24 h), each group of cells was digested with 0.25% trypsin digestion solution without EDTA, centrifuged at 300 g for 5 min at 4 ℃ to collect cell pellet;

resuspend the cell pellet with 1 mL precooled PBS and count 1X 10 ⁶ Centrifuging the cells at 4 ℃ for 5 min at 300 g, and washing twice; in the centrifugation process, 50. Mu.L of supernatant was left each time the supernatant was discarded,to prevent the cells from being discarded;

finally, adding 200 mu L of 1 Xannexin V binding buffer solution into each tube of cells, adding 5 mu L of Annexin V-FITC and 5 mu L of PI working solution after heavy suspension, and incubating for 10-15 min on ice in a dark place;

after the incubation is finished, 300. Mu.L of 1 × Annexin V binding buffer solution is added into each tube of cells, the cells are subjected to machine detection by flow cytometry, and related detection results are counted.

The results are shown in FIGS. 4 and 5. As can be seen from FIGS. 4 and 5, DIOS can significantly promote apoptosis of KYSE150, ECa109 and KYSE450 cells of esophageal squamous carcinoma, and the proportion of apoptotic cells is also obviously increased along with the increase of the concentration of DIOS. On the other hand, the proportion of apoptotic cells is also obviously increased along with the prolonging of the DIOS action time, wherein after the cells 72 h are treated by 40 mu M DIOS, the apoptotic proportion almost reaches more than 30 percent, and the application potential is better.

(IV) Effect of cell migration and invasiveness

One of the difficulties in tumor prevention and treatment is that cancer cells have strong migration and invasion abilities, and therefore, the influence of related prevention and treatment measures on the migration and invasion abilities of cancer cells needs to be considered. The relevant experimental procedures and operations are specifically referred to as follows.

(1) Cell scratch test:

first, with reference to the above procedure, after the cancer cells after culture were digested, centrifuged, and resuspended, the cell concentration was adjusted to 2.5X 10 ⁵ Per mL;

then, taking a 12-hole plate, drawing 3 transverse lines on the back side of the hole plate, adding 1 mL cell suspension into each hole, shaking the hole plate in a shape like a Chinese character 'mi', and then placing the hole plate in a 37 ℃ incubator;

observing the growth condition of the cells the next day, when the confluence degree of the cells reaches about 90%, drawing 3 vertical lines by using a 10-microliter gun head to be vertical to the drawn transverse line, and placing the pore plate in an incubator for standing for 3 min;

then, the plate was washed twice with PBS to wash off suspended cells; the experiment was divided into three groups: DIOS 0 μ M, 20 μ M and 40 μ M, adding blank culture medium containing corresponding concentration of medicine, and taking a picture of the scratch under 4 times of objective lens, and recording as 0 h;

finally, when the culture is carried out for 24 hours or 48 hours, photographing again at the same visual field position as 0h, calculating the scratch area by using Image J, calculating the wound healing rate, and carrying out statistical analysis;

wound healing rate = (control-experimental)/control × 100%.

The cell scratch test is mainly used for indicating the inhibition capability of DIOS on the cell migration capability of KYSE150, KYSE450 and ECa 109. The results of the experiment are shown in FIG. 6.

Analysis can see that: 48 After h, the wound healing rate of the DIOS 0 μ M group KYSE150 cells almost reaches 90%, but the wound healing rate of the DIOS 40 μ M treatment group KYSE150 cells is less than 20%, the wound healing rate of the KYSE450 cells is also less than 20%, and even the wound healing rate of the ECa109 cells is less than 10%. This numerical result indicates that DIOS has significant inhibitory ability on the migration of KYSE150, KYSE450 and ECa109 cells.

(2) Transwell cell migration and invasion assay

Firstly, referring to the previous description, human esophageal squamous carcinoma cells KYSE150, KYSE450 and ECa109 are cultured conventionally, and complete culture media containing drugs with corresponding concentrations are respectively added when the cell confluency reaches about 70% (three groups of experimental groups: DIOS 0. Mu.M, 20. Mu.M and 40. Mu.M);

meanwhile, matrigel (product of CORNING corporation, usa) after dilution (with blank medium) was vertically added from above the Transwell chamber, 70 μ L per well, and placed in a cell culture chamber (gel solidified after 2-4 h);

subsequently, after drug treatment 24h, the cancer cell digests were centrifuged, the cell pellet was resuspended in blank medium, and 200 μ L of cell suspension (2X 10) was added to the upper layer of the Transwell chamber ⁴ Individual cells), take care not to have air bubbles; and 750 mul of complete medium was added to the lower chamber layer; placing the mixture in a cell culture box to culture 16 h (culture for 24h in an invasion experiment);

finally, abandoning the culture medium on the upper layer and the lower layer of the chamber, adding 1 mL of 10% TCA into each hole, fixing 1 h in a dark place at 4 ℃, replacing 0.1% crystal violet of 1 mL into each hole, and placing the holes in an incubator at 37 ℃ for dyeing for 30 min;

and after dyeing is finished, cleaning the small chamber with clear water, airing the small chamber, taking a picture, and performing statistical analysis on the number of cells of the picture taken under a 20-time microscope.

The results of the experiment are shown in FIG. 6. Statistics of the relevant data show that the number of migration and invasion cells in the DIOS treatment group is obviously reduced compared with that in the control group (p<0.001). This result indicates that DIOS can significantly inhibit the migration and invasion abilities of KYSE150, KYSE450 and ECa109 cells, and the inhibitory ability thereof increases with increasing drug concentration.

Example 2

Based on the preliminary Cell experiment in example 1, the inventors further constructed a tumor Cell xenograft (CDX) model, and performed practical animal experiments on the inhibition effect of DIOS on the growth of esophageal squamous Cell carcinoma, and the specific experimental procedures are summarized as follows.

24 SCID mice (4-6 weeks old) were divided into three groups: control, DIOS and CDDP groups; each mouse was inoculated with 1X 10 of the vaccine near the left axilla ⁷ KYSE150 cells (after previously digesting and centrifuging the cultured KYSE150 cells, resuspending the cells in PBS and adjusting the concentration);

after inoculation of tumor cells, tumor size was observed, measured and calculated, and the longest (a) and shortest (b) diameters of the tumors in mice were measured every other day and tumor volume = a × b was calculated ² /2；

When the tumor volume of the mouse reaches 100 mm ³ Around time (around one month in need), indicating that the CDX model was successfully constructed, at which time the grouped dosing was started:

the dose of the CDDP group is 5 mg/kg (dissolved in physiological saline), and the administration is carried out once in five days;

referring to the prior art, DIOS combination dosage was designed as 50 mg/kg (10% DMSO +40% PEG300+5% Tween-80 +45% normal saline) administered three times a week;

one month after administration of 24 mice, anesthetizing the mice with a 0.4% sodium pentobarbital solution, bleeding the eyeballs, then killing all mice, removing subcutaneous tumors and organs (heart, liver, spleen, lung, kidney) of the mice, and weighing; immunohistochemical detection is carried out on tumor tissues, HE staining is carried out on visceral organs, and drug toxicity is evaluated.

The results of the experiments are summarized in FIG. 7. Analysis can see that:

in terms of tumor volume and weight, the volume and weight of tumors in the DIOS-treated group were significantly less than those in the control group;

in terms of tumor inhibition effect, the tumor weight inhibition rate of the DIOS group can reach about 50%, and the tumor inhibition effect of the DIOS group is not statistically different from that of the CDDP group;

in terms of the expression of the tumor tissue Ki67, the expression of the tumor tissue Ki67 in the DIOS group and the CDDP group is obviously reduced compared with that in the control group;

overall, DIOS was able to significantly inhibit the growth of esophageal squamous carcinoma in vivo.

In the experimental process, in order to evaluate the safety of DIOS, the inventor comprehensively evaluates the toxic and side effects of the medicament by monitoring the body weight of a mouse, calculating the organ index of the mouse and carrying out HE staining on the organ section of the mouse in the experimental process.

The statistical results of the body weight and HE staining of the mice are shown in fig. 8, and the calculation results of the organ index after the dissection of the mice are shown in table 1 below.

TABLE 1 CDX model index of organs in each group of mice

。

From the results of table 1 above and fig. 8, it can be seen that:

for the visceral index, there was no statistical difference in visceral index between mice in each group;

in terms of the body weight of the mice, the body weight of the mice in the DIOS group is not obviously changed compared with that of the control group, but the body weight of the mice in the CDDP group is obviously reduced;

for the result of HE staining of the internal organs of the mice, the heart, the liver, the spleen and the lung of each group of mice have no obvious abnormality, but in the result of HE staining of the kidney of the mice, DIOS group only has a few lymphocyte infiltrates, and the kidney tissue of CDDP group mice has obvious renal tubular cell apoptosis, interstitial fibrosis, interstitial lymphocyte and plasma cell infiltrates and the like;

further using qRT-PCR to detect related genes shows that the expression levels of kidney injury markers KIM-1 and NGAL of CDDP group mice are obviously increased, wherein: KIM-1 was expressed in almost three times the control, while NGAL was expressed in almost forty times the control.

In conclusion, DIOS had no significant toxic side effects on mice in this study, while CDDP caused some damage to mice kidneys.

Example 3

Based on the above example 2, it can be seen that DIOS has lower cytotoxicity compared with CDDP, especially for renal toxicity due to CDDP, but considering the factors of drug dosage, therapeutic effect, drug resistance, etc., the inventors tried to combine DIOS and CDDP to examine the effect of combined application on the prevention and treatment effect of esophageal squamous cell carcinoma, and the specific experimental conditions are summarized as follows.

(I) cell experiments

(1) Cytotoxicity test and proliferation test

Specific procedures reference is made to the foregoing description for a description of specific experimental designs and results only.

It should be noted that, in the preparation of CDDP, for example, 1 mM cisplatin mother liquor is prepared, 0.003 g powder is added into 10 mL physiological saline, after the drug is completely dissolved, the cisplatin mother liquor of 1 mM is obtained, and after being subpackaged, the cisplatin mother liquor is preserved at-20 ℃ in dark for later use; when in use, the extract is diluted to the corresponding concentration by using normal saline.

When the specific experimental group is designed: firstly, randomly selecting two esophageal squamous carcinoma cell lines KYSE150 and ECa109 to carry out a combined drug experiment, and setting the drug concentration gradient of CDDP toxicity experiment to be 0 mu M, 0.625 mu M, 1.25 mu M, 2.5 mu M, 5 mu M, 10 mu M and 20 mu M; subsequently, IC50 values of the two cell lines for DIOS and CDDP are calculated respectively by using a cytotoxicity experiment, and the combined drug ratio of the two drugs is set according to the ratio of the IC50 values; and then, carrying out a drug combination cytotoxicity experiment according to a set proportion, carrying out combination index calculation by utilizing CompuSyn software, and selecting a group of drug proportions with the minimum combination index value as the proportion for finally carrying out the cell experiment.

In cell proliferation experiments, the experimental groups were: KYSE150 cells (Control, DIOS 21. Mu.M, CDDP 7. Mu.M and Combination group), ECa109 cells (Control, DIOS 18. Mu.M, CDDP 1.5. Mu.M and Combination group).

The specific experimental results are shown in fig. 9. The results show that:

DIOS has an IC50 of 38.58. Mu.M and CDDP of 12.03. Mu.M in KYSE150 cells; in ECa109 cells, DIOS has an IC50 of 43.46. Mu.M and CDDP of 3.615. Mu.M. The ratio of the IC50 of the two drugs was further calculated and the results were: KYSE150 cells were DIOS: CDDP =3, eca109 cells were DIOS: CDDP = 12. Six groups of drug concentrations are set according to the above to detect cytotoxicity and obtain the result of cell survival rate. For KYSE150 cells, the group with the smallest combination index values had concentrations of DIOS 21. Mu.M and CDDP 7. Mu.M, while for ECa109 cells, the group with the smallest combination index values had concentrations of DIOS 18. Mu.M and CDDP 1.5. Mu.M.

And cell proliferation experiments show that: DIOS and CDDP can obviously inhibit the proliferation capacity of esophageal squamous carcinoma cells KYSE150 and ECa109, and the inhibition effect after combined administration is obviously superior to that of a single medicament.

(2) Cell cycle assay and Annexin V-FITC/PI apoptosis assay

Reference is made to the preceding description for specific experimental procedures. Only the specific experimental design and results are described below.

With reference to the foregoing results, a specific experimental group was designed to: KYSE150 cells (Control, DIOS 21. Mu.M, CDDP 7. Mu.M and Combination group), ECa109 cells (Control, DIOS 18. Mu.M, CDDP 1.5. Mu.M and Combination group).

The specific experimental results are shown in fig. 10. The results show that: DIOS blocks cell cycle at G2/M phase, CDDP blocks cell at S phase, and after combined administration, cell blocks at both S phase and G2/M phase. In addition, DIOS and CDDP can obviously promote apoptosis of esophageal squamous carcinoma cells KYSE150 and ECa109, after combined administration, the apoptosis proportion of KYSE150 cells can reach 20%, and ECa109 cells can also reach 14%, which are obviously higher than that of single-medicine-group cells.

(II) animal experiments

In order to more accurately evaluate the prevention and treatment effect of the combined application of DIOS and CDDP on esophageal squamous cell carcinoma, the inventor constructs a human-derived esophageal squamous cell carcinoma PDX animal model and carries out related experiments, and the specific experimental process is briefly described as follows.

Two random samples of esophageal squamous carcinoma tissue from clinical patients (both from volunteers, who had signed informed consent and the relevant experiments approved by the Zhengzhou university ethical Committee) were selected and numbered: LEG398, LEG404, part of the information is shown in table 2 below.

TABLE 2 relevant clinical data for patients with esophageal squamous carcinoma tissue origin

。

Referring to the prior art, by conventional operation, firstly, transplanting human-derived esophageal squamous carcinoma tissues to the back of a mouse close to an armpit, continuously carrying tumors for 3 times (ensuring the stable reproductive capacity of tumor cells), and then starting to construct a human-derived PDX mouse model and carry out related tests. The specific operation is as follows:

cutting the tumor tissue into small blocks with the volume of about 1cm multiplied by 1cm (the mass is about 0.1 g), inoculating the small blocks into the subcutaneous tissues of the mice, and allowing the subcutaneous tumors to grow to 100 mm ³ Around (approximately three weeks in use), the divided dosing (8 per group) was started:

DIOS group was given 50 mg/kg DIOS (10% DMSO +40% PEG300+5% Tween-80 +45% normal saline);

the CDDP group was given 5 mg/kg CDDP (in saline);

the combination group was given 50 mg/kg DIOS and 5 mg/kg CDDP;

the control group was given the corresponding solvent control;

DIOS is administered three times a week and CDDP is administered once in five days; the combination was given three DIOS times a week and one CDDP for five days.

Measuring the long diameter and the short diameter of the tumor of the mouse every other day, and weighing the mouse; one month after administration of the mice, the experiment was ended. The treatment during and after the experiment can be carried out by referring to the operation.

The tumor growth, tumor inhibition rate and tumor tissue Ki67 expression level of the mice of different treatment groups are shown in FIGS. 11 and 12. Specifically, the method comprises the following steps:

in terms of tumor volume, tumor weight and tumor inhibition rate, the tumor volume and weight of DIOS group, CDDP group and combined group mice in the two PDX models are obviously smaller than those of a control group, and the average tumor weight inhibition rate of the combined group mice can reach 80 percent and is obviously higher than Shan Yaozu;

with respect to the expression level of Ki67 in tumor tissues, the expression level of Ki67 in tumor tissues of the DIOS group, the CDDP group and the combination group was significantly reduced as compared with that of the control group.

The results show that the combined use of DIOS and CDDP inhibits the in vivo growth of human esophageal squamous carcinoma xenograft tumor, and the inhibition effect is better than that of single drug.

Further, in order to evaluate the medication safety in the combined medication process, the toxic and side effects of the medicament are evaluated by monitoring the change conditions of the body weight and the organ index of a mouse and combining the technologies of HE staining, qRT-PCR, serum urea nitrogen detection, creatinine detection and the like in the experimental process. The specific results are summarized below.

The statistical results of the organ index of the PDX models of LEG398 and LEG404 are shown in tables 3 and 4 below. Specific body weight statistics, HE staining and detection results of related indicators are shown in fig. 13, 14 and 15.

TABLE 3 LEG398 PDX model mouse organ indexes

（*p＜0.05，***p＜0.001）。

TABLE 4 LEG404 PDX model mouse organ index

（*p＜0.05，**p＜0.01）。

From the results of table 3 and table 4 above, and the statistical results in conjunction with fig. 13, fig. 14, and fig. 15, it can be seen that:

for the body weight statistics of the mice, the body weight of the mice in the CDDP group is obviously reduced compared with that in the control group;

for the index of the visceral organ index, the kidney index and the spleen index of the CDDP group mouse are obviously reduced, the DIOS group has no obvious difference compared with the control group, and the kidney index and the spleen index of the combined group mouse are higher than those of the CDDP group and basically reach the normal level;

further, the mRNA expression level detection results of kidney injury indexes KIM-1 and NGAL in kidney tissues show that the expression level of CDDP mice is obviously increased (p< 0.001), while the expression level of the mice in the combination group is obviously reduced;

serum test results of Blood Urea Nitrogen (BUN) and Serum creatinine (SCr) showed that BUN and SCr levels in the Serum of mice in the CDDP group were significantly elevated compared to the control group: (p< 0.001), and the combined use of DIOS leads to obvious reduction;

the HE staining result of the mouse visceral organ tissues shows that renal tubular epithelial cell apoptosis, interstitial fibrosis, interstitial lymphocyte and plasma cell infiltration are observed in CDDP group kidneys; only local lymphocyte infiltration was observed by renal HE staining in DIOS and combination mice compared to control.

In combination with the above results, it was confirmed that DIOS in combination with CDDP reduced kidney damage caused by CDDP.

Claims

1. The application of diosmetin in preparing the esophageal squamous cell carcinoma medicament is characterized in that diosmetin is used for inhibiting proliferation, migration and invasion of esophageal squamous cell carcinoma cells, inducing esophageal squamous cell carcinoma cell cycle arrest and promoting esophageal squamous cell carcinoma cell apoptosis.

2. The use of diosmetin according to claim 1 for the preparation of an esophageal squamous carcinoma medicament, wherein said cycle arrest is an arrest of the esophageal squamous carcinoma cell cycle in the G2/M phase.

3. The use of diosmetin in the preparation of esophageal squamous carcinoma medicaments according to claim 1, wherein dosage and application time of diosmetin are in positive correlation with application effect.

4. The use of diosmetin according to claim 1 for the preparation of an esophageal squamous carcinoma medicament, wherein said esophageal squamous carcinoma cells are specifically: cell lines of esophageal squamous carcinoma KYSE150, KYSE450 and ECa 109.

5. The use of diosmetin according to claim 1 for the preparation of medicaments for the prevention and treatment of esophageal squamous carcinoma, wherein diosmetin is applied simultaneously with cisplatin for the prevention and treatment of esophageal squamous carcinoma.