CN111803477A

CN111803477A - Application of abstinence sulfur in preparation of anti-head and neck cancer and anti-fibrosis drugs

Info

Publication number: CN111803477A
Application number: CN202010802404.8A
Authority: CN
Inventors: 钱峰; 孙磊; 姜洪超; 王炜伟
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-08-11
Filing date: 2020-08-11
Publication date: 2020-10-23

Abstract

The invention discloses application of abstinence sulfur in preparation of a drug for resisting head and neck cancer and a drug for resisting pulmonary fibrosis. The invention discovers the proliferation inhibiting effect and apoptosis promoting effect of the alcohol withdrawal sulfur on head and neck cancer cells for the first time, and proves that the alcohol withdrawal sulfur is a potential clinical treatment medicine for head and neck cancer diseases. The head and neck cancer refers to head and neck cancer including oral and maxillofacial tumors (oral cancer, gingival cancer, buccal cancer) and neck tumors. The invention also discovers that the abstinence sulfur has a relieving effect on the pulmonary fibrosis for the first time, shows that the abstinence sulfur has important significance in the clinical treatment of the pulmonary fibrosis, and provides a new direction for the treatment of the pulmonary fibrosis. The pulmonary fibrosis refers to idiopathic pulmonary fibrosis and clinical secondary pulmonary fibrosis.

Description

Application of abstinence sulfur in preparation of anti-head and neck cancer and anti-fibrosis drugs

Technical Field

The invention relates to the technical field of drug development and application, in particular to application of sulfur disulfide (tetraethylthiuram disulfide) in preparation of a drug for resisting head and neck cancer and a drug for resisting pulmonary fibrosis.

Background

Head and neck cancer is a neoplastic disease characterized by malignant epithelial cell encirclement in the oral, pharyngeal, laryngeal and nasal cavities. Head and neck cancer is the 6 th highest incidence cancer, the 5-year survival rate of head and neck cancer patients is only 40% -50%, the new cases of head and neck cancer are about 650000 every year, and meanwhile, about 350000 of the patients who die due to head and neck cancer every year. Related studies have shown that smoking, drinking and papillomavirus infection are major inducers of head and neck cancer pathogenesis. The current treatment procedures for head and neck cancer are complicated and cause great economic and mental stress to patients, and at the same time, effective head and neck cancer treatment methods and medicines are still lacking.

Idiopathic pulmonary fibrosis is a pulmonary disease that is caused by progressive chronic lung inflammation, with a shift in loss of pulmonary ventilation function. The idiopathic pulmonary fibrosis mainly takes adults as main causes, and the average survival cycle of patients is 3-6 years. Of the drugs discovered in recent years, pirfenidone and nintedanib are the only drugs available for the treatment of pulmonary fibrosis, however, the only effective therapy for pulmonary fibrosis to date is lung transplantation. Although the research on idiopathic pulmonary fibrosis has been ongoing in recent years, effective therapeutic drugs for pulmonary fibrosis are still very deficient. The cells mainly involved in the pathogenesis of idiopathic pulmonary fibrosis include: lymphocytes, macrophages, dendritic cells, neutrophils, epithelial cells, endothelial cells, fibroblasts, and the like. The activated fibroblast is a myofibroblast, and the activated fibroblast secretes collagen, fibronectin and other substances to cause the deposition of extracellular matrix, and finally pulmonary fibrosis is caused.

Abstinence from alcohol and sulfur was first applied to the treatment of intestinal parasitic diseases, and subsequently, the treatment of abnormality of alcohol tolerance response of patients was found to be further studied and applied to abstinence from symptoms such as alcoholism. The drug dosage for abstinence of alcohol and sulfur is higher, the half lethal drug dosage in animal experiments is 8.6g/kg, and the drug dosage of patients is generally 500mg per day. Therefore, the application of the abstinence wine and sulfur to various diseases has high safety. As a small molecule drug, the application of the disulfiram in head and neck cancer and pulmonary fibrosis is not reported.

Disclosure of Invention

The invention aims to provide the application of the sulfur for abstinence (tetraethylthiuram disulfide) in the preparation of medicaments for resisting head and neck cancer and medicaments for resisting pulmonary fibrosis; in particular to application in preparing medicaments for resisting head and neck cancer (comprising oral cavity and maxillofacial tumors (oral cavity cancer, gingival cancer and cheek cancer) and neck tumors) and medicaments for resisting pulmonary fibrosis. The invention discovers the therapeutic effect of the alcohol withdrawal sulfur on the pulmonary fibrosis for the first time. The invention selects bleomycin which generates pulmonary fibrosis side effect after clinical application as an inducer of experimental pulmonary fibrosis, thereby leading a pulmonary fibrosis model to be closer to the disease process of a human body and leading the clinical application of the medicament to be stronger.

The purpose of the invention is realized by the following technical scheme:

the invention relates to an application of abstinence sulfur in preparing a medicine for treating and inhibiting head and neck cancer.

Preferably, the head and neck cancer is a head and neck cancer including oral, maxillofacial and cervical tumors. The oral and maxillofacial tumor comprises oral cancer, gingival cancer and buccal cancer.

Preferably, the anti-head and neck cancer effect is achieved by promoting apoptosis of head and neck cancer cells and inhibiting proliferation of head and neck cancer cells.

Preferably, the abstinence sulfur is used as an active ingredient, and pharmaceutically acceptable auxiliary materials and auxiliary ingredients are added to prepare a pharmaceutical preparation for use.

Preferably, the pharmaceutical preparation is selected from one of tablets, capsules, powders, granules, sprays and sustained-release agents.

The invention also relates to application of the alcohol withdrawal sulfur in preparation of anti-pulmonary fibrosis drugs.

Preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis and clinical secondary pulmonary fibrosis.

Compared with the prior art, the medicine has the following beneficial effects:

1) the existing anti-head and neck cancer drugs are very limited and have obvious side effects; the drug for abstinence of sulfur from alcohol has strong safety and no serious adverse reaction; at present, only two kinds of pulmonary fibrosis drugs (nitazone and pirfenidone) are used, the drug withdrawal is already applied for decades, the drug safety is strong, and no serious adverse reaction exists;

2) the medicine has low production cost and is favorable for clinical application.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a graph of the results of a colony formation assay and a statistical plot of the results; wherein, A is an experimental result chart formed by cloning HN4, HN12, HN30, Cal27, SCC9 and SCC25 cell lines after the stimulation of the alcohol and sulfur withdrawal, B is an experimental result statistical chart formed by cloning HN4 cell lines, C is an experimental result statistical chart formed by cloning HN12 cell lines, D is an experimental result statistical chart formed by cloning HN30 cell lines, E is an experimental result statistical chart formed by cloning Cal27 cell lines, F is an experimental result statistical chart formed by cloning SCC9 cell lines, and G is an experimental result statistical chart formed by cloning SCC25 cell lines;

FIG. 2 is a statistical chart of the results of MTT tests; wherein, A is an experimental result statistical chart formed by cloning an HN4 cell line, B is an experimental result statistical chart formed by cloning an HN30 cell line, C is an experimental result statistical chart formed by cloning a Cal27 cell line, and D is an experimental result statistical chart formed by cloning an HN12 cell line;

FIG. 3 is a cell cycle flow chart and a statistical chart of results of head and neck cancer cells; wherein, A is a chart of cell cycle flow analysis results of HN30 cell line, and B is a statistical chart of cell cycle flow analysis results of HN30 cell line;

FIG. 4 is a graph of apoptosis flow results and a statistical chart; wherein, A is a cell apoptosis flow analysis result chart of HN30 cell line, B is a cell apoptosis flow analysis result statistical chart of HN30 cell line;

FIG. 5 is a schematic of H & E staining of fixed mouse lung tissue;

FIG. 6 is a schematic representation of Masson staining of fixed mouse lung tissue;

FIG. 7 is a diagram showing the quantitative RT-PCR method for detecting the level of fibrinectin mRNA after total RNA extraction from mouse lung tissue.

FIG. 8 is a schematic diagram showing the quantitative RT-PCR method for detecting collagen mRNA level after total RNA extraction from mouse lung tissue;

FIG. 9 is a schematic diagram of the result of measuring the hydroxyproline content in the lung tissue of the mouse by using the hydroxyproline detection kit.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.

The structural formula of the alcohol withdrawal sulfur used in the invention is as follows:

example 1 study of the Effect of alcohol withdrawal and Sulfur withdrawal on the Regulation of head and neck cancer cell function

The abstinence of sulfur can obviously inhibit the proliferation function of human head and neck cancer cells and promote the apoptosis of the human head and neck cancer cells. Provides important basis for the clinical treatment of head and neck cancer. This example further demonstrates the specific mechanism of action of alcohol withdrawal sulfur on head and neck cancer cell lines and also provides the possibility of alcohol withdrawal sulfur on related diseases with similar molecular mechanisms.

1 materials of the experiment

1.1 cell line: HN30 cell line, Cal27 cell line, HN4 cell line, HN12 cell line, SCC9 cell line, SCC25 cell line.

1.2 drugs and reagents: MTT detection kit (Biyun day) and apoptosis detection kit (Biyun day).

1.3 main equipment: BD LSRFortessa flow cytometer; a multifunctional microplate reader;

2 methods and results

2.1 Experimental methods

2.1.1 clone formation experiment:

the cell control to logarithmic growth phase was applied to the head and neck cancer cell line at the time of experimental study was HN 4. The HN4 cell line is adherent cells, the cell culture dish is taken out of the incubator, the sterile operation is carried out in a super clean bench, the supernatant is discarded, PBS is washed twice to remove the residual culture medium, 1ml of pancreatin is added into the cell culture dish which is cultured with the tumor cells HN4, after the cells are observed to be in a round shape under a mirror, 3ml of DMEM culture medium containing 10% FBS is added into the cells for neutralization treatment, after digestion is finished, the digested cells are added into a corresponding centrifuge tube, the supernatant is sucked off after 1000rpm low-speed centrifugation is carried out for 3min, 10ml of DMEM culture medium containing 10% FBS is added into the centrifuge tube, the cells are lightly blown by using a 1ml pipette tip, and the cells are uniformly dispersed in the culture medium. Counting 10. mu.l of the well dispersed cell suspension according to the following: cell density (number/4 × 10)⁴Cell density was calculated from the equation cell density per ml. The volume of cell suspension inoculated into each well was 0.1ml, and it was ensured that the cells were maintained in a uniform state after inoculation, 200 cells were secured in each well, and 100. mu.l of DMEM medium containing 10% FBS was added thereto, and it was ensured that the cells were dispersed after appropriate shaking uniformly. Adding 0.05, 0.10, 0.20 μ M of sulfur for abstinence. The 96-well plate was gently tapped to ensure dispersion, followed by culture at 37 ℃ for 7 days. In this procedure, it was closely observed that the well plate was taken out in the case where formation of cell masses was observed, the supernatant was discarded, and after fixation treatment by adding 200. mu.l of methanol to each well of the well plate, the well plate was left standing at room temperature for 30min, and after treatment by rinsing with pure water gently, 100. mu.l of each Gimsa staining solution (one) was added thereto, and after appropriate mixing, the well plate was left at room temperature for 20 min. Adding 200 μ l Gimsa staining solution, standing at room temperature for 30min, reacting sufficiently, ensuring cell staining, and removing stainingAfter sufficient washing, the number of clones in each well was determined. The results obtained were recorded and compounds with inhibitory effect on tumor proliferation were recorded for rescreening.

2.1.2 MTT assay

(1) Preparation work: during this operation, a stock solution of MTT was prepared as required, and fifty mg of MTT powder was accurately weighed out and protected from light. In the operation, the powder is completely added into a 10ml sterile centrifuge tube, and the powder is completely dissolved in sterile deionized water after ultrasonic treatment. In the sterilization process, a filtration sterilization mode is generally selected, a 0.22 mu M membrane is used for filtration, the corresponding filtrate is transferred into a sterile centrifuge tube, then 1ml of the filtrate is removed, the filtrate is subpackaged into the centrifuge tube, and proper packaging is carried out, and then proper storage is carried out at the temperature of 80 ℃ below zero.

(2) The experimental steps are as follows: HN12, HN4, Cal27 and HN30 cells were plated, digested and then resuspended in DMEM medium containing 10% FBS, and resuspended after appropriate pipetting. The density of the blood counting plate meter is controlled to be 1 multiplied by 10⁵One/ml, appropriate pipetting followed by removal of the 96-well plate and addition of 0.1ml of cell suspension to the target well. Adding 1X 10 to each group during the experiment⁴The number of the cells (the number of the spread holes is determined according to the proliferation capacity of different cells) is determined, the cells are not added into the surrounding holes, and the sterilized phosphate buffer is added to effectively avoid the liquid evaporation. After spraying alcohol on the surface of the pore plate, the culture medium is transferred into an incubator for adherence and growth. Observing about 12h, taking out when the cells can cover the bottom of the pore plate to reach 80%, discarding the supernatant, adding 0.2ml of fresh culture medium to each pore, adding 0, 16, 32 and 64 mu M of sulfur for alcohol withdrawal, wherein each concentration has six multiple pores, slightly beating the side edge of the 96 pore plate to uniformly disperse the drugs in the culture medium as much as possible, taking out the pore plate after sufficient incubation for 24h, adding 0.01ml of MTT solution to each pore under aseptic condition, then returning the pore plate to the cell incubator to continue incubation for 4h, slightly sucking the supernatant by using a 1ml syringe (the inclined plane of the syringe needle is clamped by a pliers to enable the needle to be flat), dissolving by using corresponding dimethyl sulfoxide, adding 0.1ml of methanol to each pore, and then taking out the cell in a shaking table after 0.1ml of methanol is added to each poreAnd (3) shaking for 10min, and gently beating to ensure that crystals can be completely dissolved in DMSO, then carrying out absorbance detection, detecting and determining the absorbance of each group under the condition of 490nm, and carrying out data processing analysis.

(3) The growth rate of tumor cells or the tumor cell growth inhibition rate was calculated according to the following formula:

cell growth rate (OD administration group/OD solvent group). times.100%

The cell growth inhibition rate was (1-OD administration group/OD solvent group). times.100%

2.1.3 cell cycle assays

Experimental procedure for detection of cell cycle distribution by PI dye:

recovering cells and adjusting the state of the cells to be in the logarithmic growth phase at which the state of the cells is optimal, adjusting the cell density after collecting the corresponding cells, and seeding 1X 10 cells in each of six-well plates⁶Inoculating 1ml of cell suspension into each well, supplementing 1ml of DMEM culture medium containing 10% FBS, culturing overnight, removing supernatant, adding compound medicine with corresponding concentration, treating for 24h, sucking the supernatant with a gun head, digesting the cells with 0.25% pancreatin, centrifuging at 1000rpm in a low-speed centrifuge to separate the supernatant, adding 1ml of precooled PBS into each tube to wash the cells twice to remove residual pancreatin and the like, removing supernatant liquid after the centrifugation, continuously adding 0.5ml of PBS into each tube, slightly blowing and suspending cell precipitation with a 1ml gun head to disperse the cells in a single-cell state, uniformly mixing precooled 70% ethanol (taking 50ml as an example, 35ml of absolute ethanol and 15ml of deionized water, and precooling at-20 ℃), adding 4ml of precooled 70% ethanol into each flow tube to ensure the dispersion effect of the cells, uniformly dispersing the cell suspension into 70% ethanol by using a gun head for fixation, wherein the important step is that the cell suspension is added while a flow tube is rotated, liquid is continuously blown to ensure that the cells are completely dispersed into single cells in the 75% ethanol, then the single cells are placed at 4 ℃, or the single cells are placed at-20 ℃ for at least 24 hours, the cell suspension is centrifuged at 1500rpm for 5 minutes before being detected by a flow cytometer, then the supernatant is discarded to remove the ethanol, and in order to further remove the residual ethanol, PBS (phosphate buffer solution) is required for cleaningWashing cell sediment, similarly centrifuging, removing supernatant, gently resuspending the rest cell sediment by 400 mu l PBS, ensuring that the cells are completely single cell, then adding RNase (RNase) into each tube, placing the tube at room temperature for 30min to completely remove RNA in the cells (the RNase use concentration is 10 mu g/ml), directly adding prepared PI dye (the final concentration is 50 mu g/ml) into each tube after 30min, placing the tube at room temperature in a dark condition for 15min, detecting the cell cycle distribution condition and change on a flow cytometer, and analyzing data by Flowjo 7.6 because the PI dye is sticky.

2.1.4 detection of apoptosis

Annexin V-FITC/PI double staining method:

cultured HN30 cells are collected in logarithmic growth phase, digested with 0.25% trypsin, centrifuged at 1000rpm for 3min, the supernatant is removed, resuspended in a suitable amount of DMEM medium containing 10% FBS, counted on a hemocytometer, plated, and generally plated in six well plates 6X 10⁵3 x 10 of twelve hole plate beds⁵And then, culturing overnight. When the cell proliferates to 70% -80%, the culture solution in the pore plate is replaced by new culture solution, and corresponding medicine is added for treatment for 24 h. After labeling the flow tubes, collecting the culture solution of the supernatant, washing the culture solution once with PBS (in order to remove the serum in the residual culture medium), collecting the PBS into a corresponding centrifuge tube, collecting the adherent cells of the lower layer by trypsinization, combining the collected adherent cells with the supernatant, then carrying out low-speed centrifugation for 3min, then washing the centrifuge tube appropriately through phosphate Buffer solution, discarding the supernatant, continuing to carry out low-speed centrifugation, adding 500 ul of 1 × Binding Buffer to the resuspended cells (5 × Binding Buffer is 1 × working solution is diluted with double distilled water), instantly centrifuging the Annexin-FITC staining solution and PI staining solution in the apoptosis detection kit, placing the tubes on ice for temporary storage, carrying out appropriate blowing through a gun head, then separating partial cell suspension from each tube for comparison study, wherein the comparison study comprises a blank group, a group only staining Annexin which the Annexin V-FITC group (only adding Annexin V-FITC 2.5 μ l) and a group in which only staining PI (only adding 6 μ l), the purpose of the three control groups is to adjust the compensation generated between the fluorescence when the detection is carried out on the computer convenientlyMu.l Annexin V-FITC and 10. mu.l PI were added to each tube. After appropriate mixing, incubation was followed for 5min at room temperature. And detecting the two by using an FITC detection channel, and analyzing the obtained result to determine the apoptosis condition of the cell.

2.2 results of the experiment

2.2.1 Effect of drugs on the proliferative Capacity of head and neck cancer cells

HN4, HN12, Cal27, HN3, SCC9 and SCC25 head and neck cancer cells were plated in 6-well plates,

FIG. 1: the abstinence sulfur has anti-tumor activity, different concentrations of the abstinence sulfur (0, 0.05, 0.10 and 0.20 mu M) are set to carry out clone formation experiments (HN4, HN12, Cal27, HN3, SCC9 and SCC25 cells) on multiple head and neck cancer cell lines (figure 1A), meanwhile, the influence of the abstinence sulfur on the clone formation of the head and neck cancer cells is counted, corresponding statistical graphs (figures 1B-G) are drawn, and the experimental results and the statistical results show that the abstinence sulfur has certain inhibition effect on the proliferation function of the cells and the inhibition effect is dose-dependent. Of these, HN30 was most clearly inhibited.

2.2.2 Effect of disulfiram on cell Activity of head and neck cancer cell lines

FIG. 2: the cells were treated with different concentrations (0, 16, 32 and 64 μ M) of disulfur for 24h, MTT assay was used to detect the effect of drug on cell activity and calculate the cell proliferation rate, fig. 2A is HN4 cell line, fig. 2B is HN30 cell line, fig. 2C is Cal27 cell line, fig. 2D is HN12 cell line, experimental statistics showed that disulfur had toxic effect on the above cells and the killing effect was concentration dependent.

2.2.3 Effect of abstinence from alcohol Sulfur on the cycle of head and neck cancer cell lines

FIG. 3: the cell division process is divided into a cell division period (M phase) and a cell division period, and the cells in the division period are divided into a DNA synthesis early stage (G0/G1 phase), a DNA synthesis phase (S phase) and a DNA synthesis late stage (G2). Propidium Iodide (PI) staining binds intracellular DNA, and since the DNA content of intracellular DNA at different stages of cell division is different, we detected the cell division stage by PI staining, treated the cells with different concentrations (0, 10, 30 and 100. mu.M) of disulfur for 24h, detected HN30 cell cycle distribution by flow cytometry, and performed statistical analysis. Experimental statistics results show that the alcohol withdrawal sulfur can arrest HN30 head and neck cancer cells in the S phase and the G2/M phase of cell proliferation, so that the proliferation process of the head and neck cancer cells is effectively blocked, and the dose dependence of the alcohol withdrawal sulfur on the proliferation blocking effect of the head and neck cancer cells is realized.

2.2.4 alcohol withdrawal Sulfur Induction of apoptosis

FIG. 4: a: annexin V-FITC/PI detection is carried out on the apoptosis condition of HN30 cells treated for 24 hours by using different concentrations of abstinence sulfur (0, 10, 30 and 100 mu M) and the proportion of apoptotic cells is counted; the statistical chart of the experimental results shows that the abstinence of sulfur can promote the apoptosis process of head and neck cancer cells, and the apoptosis is obviously promoted when the concentration is 30 mu M.

The research result of the embodiment shows that the alcohol withdrawal sulfur has a remarkable inhibiting effect on the proliferation of head and neck cancer cells, has a promoting effect on the apoptosis of the head and neck cancer cells, and shows that the alcohol withdrawal sulfur has a potential effect and value on the clinical application of the alcohol withdrawal sulfur to head and neck cancer. Provides a new direction for searching the anti-head and neck cancer drugs.

Example 2 study of regulating Effect of alcohol withdrawal and Sulfur withdrawal on pulmonary fibrosis

In this experiment: the preparation method of the abstinence sulfur for animal experiments comprises dissolving abstinence sulfur in DMSO with concentration of 2g/ml, mixing 25 μ l with 400 μ l ethanol and 400 μ l oleum ricini, mixing with 7.2ml PBS, and ultrasonically mixing the obtained mixture to obtain suspension.

The preparation method of the abstinence sulfur used for the cell experiment comprises the following steps: the abstinence sulfur is directly dissolved in DMSO to prepare 100mM stock solution and 10mM stock solution, and the stock solutions are stored in a refrigerator at the temperature of-80 ℃.

In this example, animal experiments show that abstinence of alcohol and sulfur has certain therapeutic effect on pulmonary fibrosis, and the test method and results are as follows:

1 experimental materials:

1.1 Experimental animals: c57BL/6 mouse, male, with weight of about 25g, was bred in Shanghai university of transportation laboratory animal center, air-conditioned at room temperature of 20-25 deg.C, sterilized water, and sterilized rat food.

1.2 reagent: abstinence sulfur (Disulfiam, Sigma), castor oil (biologies), absolute ethanol (biologies).

1.3 main equipment: real-time PCR System (ABI), upright microscope (Olympus).

2 methods and results

2.1 Experimental methods

2.1.1 mouse pulmonary fibrosis model construction

23 mice, randomized into 4 groups, were given bleomycin or PBS and abstinence sulfur or solvent control (6 bleomycin-and solvent-deprived sulfur groups, 5 PBS-and 6 PBS-deprived sulfur groups). The mice are given bleomycin (trachea administration) on the 0 th day of model building, each mouse is given bleomycin according to the weight, the bleomycin is dissolved in PBS to prepare working concentration (1.4U/kg), and the trachea administration is carried out, wherein the day of model building is 0 day. The mice were given 50mg/kg of sulfur for abstinence by intraperitoneal injection every day, and lung tissues and peripheral blood of the mice were collected 21 days later for analysis.

2.1.2 HE staining of lung tissue: fixing lung tissue by paraformaldehyde, dehydrating, embedding, slicing, dyeing by an H & E staining kit for Yunnan after dewaxing treatment according to instructions, and finally sealing and observing.

2.1.3 Masson staining of lung tissue: fixing lung tissue by paraformaldehyde, dehydrating, embedding, slicing, deparaffinizing, dyeing by Masson dyeing kit, and sealing for observation.

2.1.4 Lung tissue hydroxyproline assay: and (3) after the lung tissue is taken out from the ultra-low temperature refrigerator, the lung tissue is processed according to the specification of the hydroxyproline detection kit of Nanjing Makeup biological company, and then the change of the hydroxyproline level is detected.

2.1.5 lung tissue mRNA extraction, reverse transcription and qPCR experiments: after lung tissues are ground at low temperature, the lung tissues are treated by trichloromethane, isopropanol and 75% ethanol and centrifuged to obtain total RNA, then the total RNA is subjected to reverse transcription by a toyobo reverse transcription kit according to the instruction, and the obtained cDNA is subjected to real-time quantitative PCR detection by a toyobo SYBR Green kit.

2.2 results of the experiment

2.2.1 Lung tissue pathological analysis

The pulmonary tissues of the bleomycin-induced pulmonary fibrosis mice can generate focus areas caused by over-repair, and the focus areas can deposit extracellular matrixes such as collagen. We performed analysis of pathological sections of mouse lung tissue (H & E staining and Masson staining).

The mouse lung tissues are fixed, dehydrated, embedded, sliced and stained, H & E staining results show (figure 5) that the lung tissues have focus areas after the bleomycin molding, and the focus areas of the lung tissues of the drug withdrawal group are obviously reduced, which shows that the effect of the drug withdrawal on the pulmonary fibrosis is reduced. Masson staining (fig. 6) reflects collagen deposition in lung tissue, and the results show that collagen deposition occurs in lung tissue after bleomycin molding, and withdrawal of sulfur can reduce collagen deposition caused by pulmonary fibrosis.

2.2.2 RT-PCR detection of pulmonary fibrosis marker molecules mRNA and protein level changes in pulmonary tissue

During pulmonary fibrosis, the expression of Fibronectin and Collagen in mouse lung tissue increased, and we examined the lung tissue for mRNA levels of Fn1, Col1a 1. The results are shown in FIGS. 7 and 8. Carrying out reverse transcription after extracting total RNA of a mouse lung tissue after modeling, and subsequently carrying out real-time PCR to detect the change of Fn1 and Col1a1 mRNA level; meanwhile, as hydroxyproline is a specific amino acid of a collagen component in lung tissues, the content of hydroxyproline in mouse lung tissues is detected (figure 9), and the experimental result shows that the drug withdrawal sulfur can obviously inhibit the expression of pulmonary fibrosis related molecules.

Example 3

Preparation of tablets:

taking 500g of the abstinence sulfur, adding a proper amount of starch to prepare granules, adding a proper amount of magnesium stearate, uniformly mixing, and pressing into 1000 tablets, one tablet at a time every day.

Example 4

Preparation of capsules

Taking 500g of abstinence sulfur, adding an appropriate amount of diluent, mixing uniformly, subpackaging and filling into empty capsules. Making into capsule 1000 granules 1 time per day, 1 granule each time.

Example 5

Preparation of granules

Taking 40g of abstinence sulfur, adding 670g of sucrose and 290g of auxiliary dextrin, mixing uniformly, and granulating by using a roller flat pressing granulator to obtain 1000g of abstinence sulfur granules, wherein 1 bag (10g) is used each time, and 3 times a day.

The experimental research result shows that the alcohol withdrawal sulfur has a remarkable inhibiting effect on pulmonary fibrosis induced by bleomycin, so that the alcohol withdrawal sulfur has an important value on the pulmonary fibrosis clinically, and a new direction is provided for the research and development of anti-fibrosis treatment medicines.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims

1. An application of disulfur in preparing the medicines for treating and suppressing the cancer of head and neck is disclosed.

2. The use according to claim 1, wherein the head and neck cancer is a head and neck cancer comprising a tumor of the oral maxillofacial region, a tumor of the neck.

3. Use according to claim 2, wherein the oromaxillofacial tumor comprises oral cancer, gingival cancer, buccal cancer.

4. The use of any one of claims 1-3, wherein the abstinence sulfur is used as an active ingredient, and is added with pharmaceutically acceptable auxiliary materials and auxiliary ingredients to prepare a pharmaceutical preparation.

5. The use according to claim 4, wherein the pharmaceutical preparation is selected from one of a tablet, a capsule, a powder, a granule, a spray, and a sustained release formulation.

6. An application of sulfur for abstinence from alcohol in preparing medicine for treating pulmonary fibrosis is disclosed.

7. The use according to claim 6, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis and clinical secondary pulmonary fibrosis.

8. The use of claim 6 or 7, wherein the abstinence sulfur is used as an active ingredient, and is added with pharmaceutically acceptable auxiliary materials and auxiliary ingredients to prepare a pharmaceutical preparation.

9. The use according to claim 8, wherein the pharmaceutical preparation is selected from one of a tablet, a capsule, a powder, a granule, a spray, and a sustained release formulation.