Method for evaluating tobacco harm by detecting cytokines

Abstract

The invention relates to a method for evaluating tobacco harm by detecting cytokines, is suitable for evaluating macrophage inflammation and oxidative stress induction level of a tobacco smoke aqueous extract, and belongs to the technical field of tobacco harm evaluation. According to the cytokine detection method, the tobacco smoke aqueous extract is adopted to directly treat the tested cells, and the cytokine induction effect of the tobacco smoke aqueous extract can be quantitatively judged. The method makes up the blank of in-vitro detection of risk of cardiovascular diseases possibly caused by tobacco-induced cytokines, and can be suitable for different types of tobaccos in the market, such as cigarettes, electronic cigarettes and simulated cigarettes.

Description

Technical Field

[0001] The invention relates to a detection method for cytokines induced by water extract of tobacco smoke, and belongs to the technical field of tobacco hazard evaluation. Background Art

[0002] The smoke produced by tobacco combustion is a complex mixture of more than 7,000 compounds, of which 95% are gases, such as carbon monoxide, hydrogen cyanide, volatile nitrosamines, etc., and 5% are particulate matter, including semi-volatile and non-volatile substances, such as tar, nicotine, etc. Most of these compounds are harmful to the human body, and at least 69 of them are known carcinogens, such as polycyclic aromatic hydrocarbons and nitrosamines. New tobacco converts e-cigarette liquid into a mixture similar to cigarette smoke through electric heating or electronic atomization. The smoke liquid is generally composed of nicotine, propylene glycol, glycerol and flavors and fragrances, and the ingredients can be as many as hundreds. After these ingredients are converted into smoke by high-temperature atomization, they form many substances that are harmful or potentially harmful to human health, such as formaldehyde, acetaldehyde, acrolein, crotonaldehyde, lead, chromium, nickel, diacetyl, propylene glycol, etc. These smoke products may have cardiovascular, respiratory and growth toxicity hazards to the human body.

[0003] Atherosclerotic heart disease (ASCVD) is a common disease that can affect all blood vessels in the body. It is a heart disease caused by atherosclerotic lesions in the coronary arteries, which causes stenosis or obstruction of the vascular lumen, myocardial ischemia, hypoxia or necrosis, and is often called "coronary heart disease". Smoking has always been one of the main risk factors for atherosclerosis. Some studies in the past few decades have identified the adverse effects of smoking on traditional cardiovascular risk factors, including blood pressure, blood lipids and diabetes. Smoking has an impact on inflammation, endothelial function and platelet aggregation. Smoking causes endothelial cell damage or excessive apoptosis, which increases vascular permeability. Low-density lipoprotein (LDL) then infiltrates and accumulates under the endothelium, and is oxidized by lipoxygenase and reactive oxygen groups into oxidized low-density lipoprotein (OX-LDL), which causes macrophages to phagocytose LDL and then develop into foam cells. Foam cells undergo programmed cell death or necrosis, and necrotic foam cells release a large amount of interleukins, such as IL-1β, TNF-α and other inflammatory factors. Therefore, by detecting the inflammatory factors interleukins and oxidative stress factors in cells, we can reversely understand the harmful effects of smoking on the body. Summary of the invention

[0004] The purpose of the present invention is to provide a detection method suitable for evaluating the induction effect of tobacco smoke water extract on cytokines, wherein the cytokines include proinflammatory factors interleukin IL-6 or / and IL-8 or / and TNF-α, or / and oxidative stress factors including SOD and MDA. The method can detect the harmful effects of tobacco smoke on inflammation and oxidative stress of the body.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] Peritoneal macrophage extraction:

[0007] Macrophages naturally exist in the peritoneal cavity of mice. Macrophages can be obtained by flushing the peritoneal cavity with normal saline and then extracting the peritoneal lavage fluid.

[0008] Preparation before the experiment: 3 days before extracting macrophages, grab a healthy adult SPF mouse and inject 1 mL of 3% thioglycolate broth intraperitoneally. The mouse is fasted for 8 hours. Before the experiment, metal instruments such as ophthalmic scissors and ophthalmic forceps are sterilized by high pressure, and centrifuge tubes are sterilized by radiation. Before the experiment, the clean bench is ultraviolet disinfected for 30 minutes.

[0009] Cell extraction: Kill the mouse by dislocating the neck, immerse the whole mouse in 70% alcohol for 3-5 seconds, and then lie on the stainless steel square plate. Use a syringe to draw 5mL of saline, extract the skin and peritoneum in the middle of the mouse's abdomen, let the needle pierce into the mouse's abdominal cavity from the extraction site, slowly push the saline, gently rub the mouse's abdomen several times, and let it stand for 3-5 minutes to allow the liquid to flow fully in the abdominal cavity. Use tweezers to extract the skin of the lower abdomen and separate it from the peritoneum. Cut a small hole in the skin at the extraction site with the tweezers, then cut the entire abdominal skin, pinch the skin on both sides and slowly tear it apart to expose the peritoneum. Clamp the skin on one side of the mouse with your hand to squeeze the internal organs to form a cavity in the abdominal cavity, insert the syringe needle into the gap to extract the peritoneal fluid, and then inject the peritoneal fluid into the centrifuge tube.

[0010] Cell culture: The collected peritoneal perfusion fluid was centrifuged at 1000r / min for 10min, the supernatant was discarded, the cell culture medium was added to resuspend and the cells were counted, the cell concentration was adjusted and inoculated into the cell culture bottle, and the culture was incubated at 37℃ for 4-6h. After the macrophages fully adhered to the wall, the supernatant was discarded, the new culture medium was replaced, and the culture medium was returned to continue the culture. After the cells adhered to 80-90%, the cells were collected, which were the primary peritoneal macrophages.

[0011] Sterile smoke solution collection

[0012] The present invention provides a method for evaluating tobacco harm by detecting cytokines, which is characterized by comprising the following steps: firstly, using DMEM cell culture medium to collect tobacco smoke water extract, capturing or diluting to prepare tobacco smoke water extract solutions with a series of concentrations; then adding test cells to the cell culture medium of the tobacco smoke water extract solution for poisoning treatment, and finally detecting the content of cytokines.

[0013] The preparation of the flue gas aqueous extract solution comprises: using a linear smoking machine, continuously inhaling 100 puffs of an electronic cigarette under the conditions of a puffing capacity of 55.0 ml, a puffing duration of 3 s, and a puffing frequency of 30 s, capturing the smoke with a capture bottle filled with DMEM cell culture medium, the capture concentration being 20 puffs / mL, filtering and sterilizing the captured liquid with a 0.22 μm sterile filter, obtaining a smoke aqueous extract of tobacco smoke as a test liquid, and storing it at -80°C for later use.

[0014] Test steps

[0015] (1) Cell culture: Primary macrophages with good growth status were obtained, digested and subcultured, and the cell density was adjusted to 1×10 5 100 μL (1×10 cells / mL) of cell suspension was added to each well of a 96-well culture plate. 4 / well), at 37°C, 5% CO 2 Culture in an incubator overnight (about 16 to 24 hours). Discard the original culture medium the next day.

[0016] (2) Group treatment: Test group: 100 μL / well of different concentrations of test substances were added to each well, including 20 μg / mL, 10 μg / mL, 5 μg / mL, and 2.5 μg / mL; positive control group: 100 μL / well of 20 μg / mL LPS was added to each well; vehicle control group: 100 μL / well of F12K complete medium was added to each well. Three replicate wells were set for each concentration group, and the cells were incubated at 37°C and 5% CO2 for about 24 hours.

[0017] (3) Collect the cell supernatant, which can be frozen at -80°C.

[0018] ELISA kit for detecting cytokines

[0019] Pro-inflammatory factor kit and oxidative stress kit were used to detect cytokine content.

[0020] Wherein, a positive control group and a solvent control group are set up during the poisoning treatment, lipopolysaccharide LPS is added to the positive control group, and DMEM cell culture medium is added to the solvent control group. The pro-inflammatory factors IL-6, IL-8, and TNF-α content are significantly increased, representing positive harm; or / and the oxidative stress SOD content is significantly reduced, representing positive harm; or / and the oxidative factor MDA content is significantly increased, representing positive harm. DETAILED DESCRIPTION

[0021] Case 1: Purchase a brand of cigarettes called “Zhonghua” from the market and test its cytokine induction level.

[0022] The steps include:

[0023] 1. Collection of peritoneal cells

[0024] Take a healthy adult SPF mouse and inject 1 mL of 3% thioglycolate broth intraperitoneally. The mouse should be fasted for 8 hours before the experiment.

[0025] The mice were killed by dislocating the neck, and the whole mouse was immersed in 70% alcohol for 3 to 5 seconds, and then lying on the stainless steel square plate. 5 mL of saline was drawn with a syringe, and the skin and peritoneum of the middle abdomen of the mouse were extracted. The needle was inserted into the mouse's abdominal cavity from the extraction site, and the saline was slowly injected. The mouse's abdomen was gently rubbed several times, and it was left to stand for 3-5 minutes to allow the liquid to flow fully in the abdominal cavity. The skin of the lower abdomen was extracted with tweezers to separate it from the peritoneum. A small incision was made in the skin at the extraction site with the tweezers, and then the entire abdominal skin was cut open. The skin on both sides was pinched and slowly torn to both sides to expose the peritoneum. The skin of one side of the mouse was clamped by hand to squeeze the internal organs to form a cavity in the abdominal cavity. The needle of the syringe was inserted into the gap to extract the peritoneal fluid, and then the peritoneal fluid was injected into the centrifuge tube.

[0026] The collected peritoneal perfusion fluid was centrifuged at 1000r / min for 10min, the supernatant was discarded, the cell culture medium was added to resuspend and the cells were counted, the cell concentration was adjusted and inoculated into the cell culture bottle, and incubated in a 37℃ incubator for 4-6h. After the macrophages fully adhered to the wall, the supernatant was discarded, the new culture medium was replaced, and the culture medium was returned to continue culturing. After the cells adhered to 80-90%, the cells were collected and used as test cells.

[0027] 2. Sample Collection

[0028] A linear smoking machine was used to connect cigarettes purchased on the market, and the cigarettes were lit. The smoking machine parameters were set as follows: the puffing capacity was 55.0 ml, the puffing duration was 3 s, and the puffing frequency was 30 s. When the cigarette was finished, a new cigarette was manually replaced. 100 puffs were taken continuously in this way. The smoke was captured in a capture bottle filled with DMEM cell culture medium. The capture concentrations were 20 puffs / mL, 10 puffs / mL, 5 puffs / mL, and 2.5 puffs / mL. The capture liquid was filtered and sterilized with a 0.22 μm sterile filter to obtain the smoke water extract of the electronic cigarette smoke as the test liquid, which was stored at -80°C for use.

[0029] 3. Toxic treatment

[0030] (1) Cell culture: Primary macrophages with good growth status were obtained, digested and subcultured, and the cell density was adjusted to 1×10 5 100 μL (1×10 cells / mL) of cell suspension was added to each well of a 96-well culture plate. 4 / well), at 37°C, 5% CO 2 Culture in an incubator overnight (about 16 to 24 hours). Discard the original culture medium the next day.

[0031] (2) Group treatment: Test group: 100 μL / well of different concentrations of test substances were added to each well, including 20 μg / mL, 10 μg / mL, 5 μg / mL, and 2.5 μg / mL; positive control group: 100 μL / well of 20 μg / mL LPS was added to each well; vehicle control group: 100 μL / well of DMEM complete medium was added to each well. Three replicate wells were set for each concentration group, and the cells were incubated at 37°C and 5% CO2 for about 24 hours.

[0032] (3) Collect the cell supernatant.

[0033] 4. Detection

[0034] The cytokine levels were detected using ELISA kits.

[0035] 5. The test results are as follows

[0036] 1) Cellular inflammatory factors

[0037] Table 1 Effects of Zhonghua cigarettes on inflammatory factors in peritoneal macrophages of mice

[0038]

[0039]

[0040] 2) Oxidative stress factors

[0041] Table 2 Effects of Zhonghua cigarettes on oxidative stress factors in mouse peritoneal macrophages

[0042]

[0043]

[0044] Case 2: Purchase an electronic cigarette "ZIP ME" from the market and test its cytokine induction level. The steps include:

[0045] Please refer to Case 1 for specific steps.

[0046] 1) Cellular inflammatory factors

[0047] Table 3 Effects of electronic cigarette “ZIP ME” on inflammatory factors in peritoneal macrophages of mice

[0048]

[0049]

[0050] 2) Oxidative stress factors

[0051] Table 4 Effects of electronic cigarette “ZIP ME” on oxidative stress factors in mouse peritoneal macrophages

[0052]

[0053] Notes: *: p<0.05, **: p<0.01.

[0054] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0055] The above-described embodiments are merely explanations of the present application and are not limitations of the present application. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for evaluating tobacco harm by detecting cytokines, characterized in that: The method comprises the following steps: firstly, using DMEM cell culture medium to collect tobacco smoke water extract, capturing or diluting to prepare tobacco smoke water extract solutions of a series of concentrations; then adding the test cells to the cell culture medium of the tobacco smoke water extract solution for poisoning treatment, and finally detecting the content of cytokines.

2. The cytokine detection method according to claim 1, characterized in that: The cytokines include pro-inflammatory factors IL-6 and / or IL-8 and / or TNF-α and / or oxidative stress factors SOD and MDA.

3. The cytokine detection method according to claim 1, characterized in that: The preparation of the tobacco smoke water extract solution comprises: using a linear smoking machine, continuously inhaling 100 puffs of an electronic cigarette under the conditions of a puffing capacity of 55.0 ml, a puffing duration of 3 s, and a puffing frequency of 30 s / puff, and using a capture bottle filled with a DMEM cell culture medium to capture smoke, capturing tobacco smoke water extract solutions of a series of concentrations, filtering and sterilizing the captured solution with a 0.22 μm sterile filter, and obtaining a tobacco smoke water extract as a test solution.

4. The cytokine detection method according to claim 1 or 3, characterized in that: The capture series concentrations of tobacco smoke water extract solutions are 20 puffs / mL, 10 puffs / mL, 5 puffs / mL, and 2.5 puffs / mL respectively.

5. The cytokine detection method according to claim 4, characterized in that: The test cells are mouse peritoneal macrophages.

6. The cytokine detection method according to claim 5, characterized in that: During the poisoning treatment, a positive control group and a solvent control group were additionally set up. Lipopolysaccharide LPS was added to the positive control group, and DMEM cell culture medium was added to the solvent control group.

7. According to the cytokine detection method of claim 5 or 6, the content of the pro-inflammatory factors IL-6, IL-8, and TNF-α is significantly increased, representing a positive hazard; or / and the content of the oxidative stress SOD is significantly decreased, representing a positive hazard; or / and the content of the oxidative factor MDA is significantly increased, representing a positive hazard.

8. A method for determining the cellular damage of aqueous extracts of tobacco smoke, characterized in that: The method comprises the following steps: firstly, using DMEM cell culture medium to collect tobacco smoke water extract, capturing or diluting to prepare water extract solutions with a series of concentrations; then adding the test cells to the cell culture medium of the tobacco smoke water extract solution for poisoning treatment, and finally calculating the cytokine induction level of the tobacco smoke water extract.

9. The method for determining cell damage according to claim 8, characterized in that: During the poisoning treatment, a positive control group and a solvent control group were additionally set up. Lipopolysaccharide LPS was added to the positive control group, and DMEM cell culture medium was added to the solvent control group.

10. The method for determining cell damage according to claim 9, characterized in that: The content of the pro-inflammatory factors IL-6, IL-8, and TNF-α is significantly increased, indicating a positive hazard; or / and the content of the oxidative stress factor SOD is significantly decreased, indicating a positive hazard; or / and the content of the oxidative stress factor MDA is significantly increased, indicating a positive hazard.

11. The method for determining cell damage according to claim 10, characterized in that: ELISA kit was used for detection.

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