CN112472792B

CN112472792B - Application of cyclosporine A and tripterine in preparation of medicine for treating lung cancer

Info

Publication number: CN112472792B
Application number: CN202011580954.6A
Authority: CN
Inventors: 谭克; 樊玉梅; 孟艳秀; 曹鹏秀
Original assignee: Hebei Normal University
Current assignee: Hebei Normal University
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2022-12-13
Anticipated expiration: 2040-12-28
Also published as: CN112472792A

Abstract

The invention discloses an application of cyclosporine A (CysA) and tripterine in preparing a medicament for treating lung cancer. Non-small cell lung cancer cells are used as experimental materials, the death of tumors is focused, the effect and the molecular mechanism of CysA with different concentrations combined with tripterine induced cell death are deeply researched, and the cancer cell death is promoted by reducing the toxic and side effect of the tripterine, so that a theoretical basis and a new way are provided for the treatment of the cancers.

Description

Application of cyclosporine A and tripterine in preparation of medicine for treating lung cancer

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to cyclosporine A _， CysA) and tripterine in preparation of medicine for treating lung cancer.

Background

Lung cancer is one of the malignant tumors which has the fastest increase of morbidity and mortality and the greatest threat to human life, and has become the first cause of death of malignant tumors in urban population in China. Non-small cell lung cancers include squamous cell carcinomas, adenocarcinomas, large cell carcinomas, which have slower growth and division of cancer cells and relatively late metastatic spread as compared to small cell carcinomas. Non-small cell lung cancer accounts for approximately 80% of all lung cancers, with a low 5-year survival rate as approximately 75% of patients develop in the middle and advanced stages. The method has important significance for deeply understanding the pathogenic factors of the lung cancer and developing appropriate clinical treatment medicines.

Apoptosis is divided into endogenous apoptosis and exogenous apoptosis. The endogenous apoptotic pathway, also known as the mitochondrial pathway, is often activated under conditions of intense oxidative stress and DNA damage, among others. Bcl-2 family proteins primarily regulate endogenous apoptotic pathways, with family members including both pro-apoptotic factors (Bad, bax, bid, bak, etc.) and anti-apoptotic factors (Bcl-2, bcl-Xl, bcl-w, etc.). These proteins localize to the mitochondria and collectively control permeability of the mitochondrial membrane. This pathway, when activated, can lead to the release of cytochrome C and other pro-apoptotic factors from the mitochondria. In the cytoplasm, cytochrome C forms a complex with APAF1, which then recruits and activates caspase-9 and caspase-3 to initiate the apoptotic pathway. The extrinsic pathway is also called a death receptor pathway, and signals are transmitted from outside the cell to inside the cell, and the formation of a Death Inducing Signaling Complex (DISC) is promoted by the binding of a pro-apoptotic ligand and a pro-apoptotic receptor, thereby inducing the apoptosis of the cell.

Tripterine (celastrol), also known as celastrol, is a quinone methyl triterpene compound extracted from common plants such as tripterygium wilfordii, celastrus orbiculatus and striga asiatica. The molecular formula is C ₂₉ H ₃₈ O ₄ And has a molecular weight of about 450.61. According to various reports in the literature, tripterine has multiple significant and effective pharmacological activities, such as inhibiting inflammatory response, resisting tumor growth, resisting oxidative stress, treating obesity, and inhibiting pathological processes of different types of neurodegenerative diseases (such as Alzheimer's disease, parkinson's disease, huntington's chorea, etc.). Meanwhile, tripterine has potential clinical application prospect in the process of treating allergic asthma, leprosy, rheumatic arthritis and other autoimmune diseases.

Although tripterygium wilfordii has good pharmaceutical activity, the clinical application of tripterygium wilfordii in tumor treatment is limited due to the damage of reproductive system, endocrine system and digestive system, especially the damage of blood system and skin mucosa. However, at low concentrations, tripterine is not likely to produce toxic side effects, and its significant activity in inducing tumor cell apoptosis is being continuously confirmed by experiments. Therefore, in the use of tripterine and preparations thereof, attention should be paid to the dosage, the medication safety is improved, and the toxic and side effects are reduced, and the combined treatment with other medicines becomes one of the important means for reducing the toxic and side effects of the medicines and improving the drug effect.

Therefore, how to combine other drugs with tripterine to significantly induce cancer cell apoptosis is a technical problem that needs to be solved by those skilled in the art.

Disclosure of Invention

In view of the above, the invention provides the application of CysA and tripterine in the medicine for treating lung cancer, non-small cell lung cancer cells are used as experimental materials, the death of tumors is focused, the effect and the molecular mechanism of CysA and tripterine induced cell death at different concentrations are deeply researched, and the death of cancer cells is promoted by reducing the toxic and side effects of tripterine, so that a theoretical basis and a new way are provided for the treatment of cancers.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of CysA and tripterine in preparing medicine for treating lung cancer is provided.

The technical effect achieved by the technical scheme is as follows: cysA is generally used as an immunosuppressant to inhibit the proliferation and differentiation of T cells and B cells in vivo, and has the function of inhibiting mitochondrial permeability transition pore, and is combined with cyclophilin D in mitochondria to block the combination of the cyclophilin D and mitochondrial inner membrane, so that the opening degree of the mitochondrial permeability transition pore is inhibited.

As a preferable technical scheme of the invention, in the medicament, the effective concentration of CysA is 1-5 mu M, and the effective concentration of tripterine is 1-5 mu M.

As a preferred technical scheme, cysA and tripterine can promote the expression of ROS in non-small cell lung cancer cells.

Reactive Oxygen Species (ROS) are partial reduction products of oxygen during the metabolism of the body. ROS play an important role in vital activities, and low levels of ROS help to suppress inflammatory responses and promote cell proliferation. However, high levels of ROS can promote anti-tumor signaling and induce tumor cell senescence and death. Because tumor cells need to maintain the steady state of ROS at a higher level, the ROS-regulating anticancer therapy has potential application value, and CysA and tripterine can promote the expression of ROS, so that the combined use of the CysA and the tripterine can effectively activate the transduction of antitumor signals, thereby achieving the antitumor effect.

As a preferred technical scheme, cysA and tripterine can promote expression of apoptosis-promoting protein BAX in non-small cell lung cancer cells.

As a preferred technical scheme, cysA and tripterine can promote the expression of clear Caspase-3 in non-small cell lung cancer cells.

As a preferred technical scheme, cysA and tripterine can inhibit the expression of apoptosis inhibitory protein Bcl-2 in non-small cell lung cancer cells.

Through the technical scheme, compared with the prior art, the technical effects achieved by the invention comprise that: (1) The two medicines are combined to act on the non-small cell lung cancer, the medicine composition has simple use mode and better medicine effect, and the proliferation inhibition effect of the non-small cell lung cancer cells is enhanced along with the prolonging of the acting time;

(2) The safety is high, the side effect is low, the cell death of cancer cells can be obviously induced, the survival rate of lung cancer cells is reduced, and no damage is caused to other functions of an organism;

(3) Low cost and suitability for large-scale production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the effect of the CCK-8 method of the present invention on the activity of human NSCLC cells at different concentrations;

FIG. 2 is a diagram illustrating the effect of the CCK-8 method of detecting tripterine of different concentrations on the activity of human NSCLC cells in example 2 of the present invention;

FIG. 3 is a graph showing the effect of the CCK-8 method of the present invention on the viability of human NSCLC cells when detecting CysA and tripterine simultaneously;

FIG. 4 is a graph showing the effect of flow cytometry for detecting the simultaneous treatment of CysA and tripterine on ROS levels in human NSCLC cells in example 4 of the present invention;

FIG. 5 is a graph showing the effect of simultaneous treatment of CysA and tripterine on the morphology of human NSCLC cells in example 5 of the present invention;

FIG. 6 is a graph showing the effect of flow cytometry in example 6 on the apoptosis rate of NSCLC cells by simultaneous treatment with CysA and tripterine;

FIG. 7 is a graph showing the effect of Western Blot to detect the effect of simultaneous treatment of CysA and tripterine on the expression of apoptosis-controlling protein of non-small cell lung cancer cells in example 7;

FIG. 8-1 is a schematic diagram of the in vivo index assay of CysA and tripterine for simultaneous treatment of no toxic or side effects on the heart, kidney, spleen, liver and lung of nude mice in example 8; FIG. 8-2 is a graph comparing the conventional effects of control and treatment groups on mouse blood; fig. 8-3 are graphs illustrating the effect of different treatments on tumor volume size.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

CCK-8 method for detecting influence of CysA on human HCC827 cell viability

1) Laboratory instruments and materials

Cell culture incubator (Thermo Fisher), ELX-800 microplate reader (BioTek), inverted microscope (LEICA DMIRB), fetal bovine serum (Biological Industries), DMEM (high sugar) medium (GIBCO), CCK-8 (MCE), DMSO (Sigma), cysA (BBI), 96-well cell culture plate (NEST), non-small cell lung cancer cell (HCC 827 cell, shanghai cell institute of the Central academy of sciences).

2) The process is as follows:

HCC827 cells were seeded at a density of about 7000 cells/well in a 96-well plate, and 5% CO was assayed at 37 ℃% ₂ And culturing in a cell culture box containing DMEM high-sugar culture solution containing 10% fetal calf serum. When the cell density reaches 70-80%, 100 μ L of the culture medium is addedCysA was added at final concentrations of 0, 1, 2.5, 5, 10, 20, 40. Mu.M to the gradient, and DMSO-treated cells were used as negative controls, with three duplicate wells per cell. After respectively continuing culturing for 24 hours, observing the shape under an inverted microscope; then, a medium containing 10% of CCK-8 was prepared, added to each well in the form of a stock solution, incubated in an incubator for 30min, and then OD was measured _450nm The effect of different concentrations of CysA on the viability of HCC827 cells was examined and the results are shown in fig. 1.

3) Principle of experiment

The CCK-8 reagent contains WST-8 (chemical name: 2- (2-Methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfonic acid benzene) -2H-tetrazole monosodium salt), which is a compound similar to MTT, and can be reduced to a highly water-soluble orange Formazan product, formazan, by mitochondrial dehydrogenase in the presence of an electron coupling reagent, 1-Methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS), and the amount of Formazan generated is proportional to the number of living cells. Therefore, the cell proliferation and toxicity analysis can be directly carried out by utilizing the characteristic, and the more the cell proliferation is faster, the darker the color is; the more cytotoxic, the lighter the color. The absorbance was measured with a microplate reader at a wavelength of 450nm and the amount of Formazan product formed was directly proportional to the number of cells. The number of viable cells was determined based on the measured OD value, and the larger the OD value, the stronger the cell activity.

Cell viability was calculated as follows: cell viability = experimental OD value/blank control OD value × 100%; data statistics are expressed as mean ± standard deviation (x ± s) using the t-test.

As can be seen from fig. 1, the survival rate of HCC827 cells gradually decreased with the increase in CysA concentration. The results show that CysA can reduce the survival rate of human HCC827 cells, and the survival rate is reduced in a concentration-dependent manner.

Example 2

CCK-8 method for detecting influence of tripterine on activity of human HCC827 cells

1) Laboratory instruments and materials

Cell culture incubator (Thermo Fisher), ELX-800 microplate reader (BioTek), inverted microscope (LEICA DMIRB), fetal bovine serum (Biological Industries), DMEM (high-sugar) medium (GIBCO), CCK-8 (MCE), DMSO (Sigma), tripterine (Sigma), 96-well cell culture plate (NEST), HCC827 cells (Shanghai cell institute of the department of China).

2) Experimental procedure

HCC827 cells were seeded at a density of about 7000 cells/well in a 96-well plate, and 5% CO was assayed at 37 ℃% ₂ And culturing in a cell culture box containing DMEM high-sugar culture solution containing 10% fetal calf serum. When the cell density reaches 70-80%, adding tripterine with final gradient concentration of 0, 0.5, 1, 2.5, 5, 10, 20, 40 μ M, and using DMSO-treated cells as negative control group, wherein each cell has three multiple wells. After further culturing for 24 hours, the morphology was observed under an inverted microscope. Then preparing a medium containing 10% of CCK-8, adding the medium to each well in the form of a changing solution, incubating the medium in an incubator for a certain period of time, and measuring OD _450nm . The effect of tripterine at different concentrations on the activity of human HCC827 cells was examined, and the results are shown in fig. 2.

3) Principle of experiment same as example 1

As can be seen from fig. 2, the survival rate of HCC827 cells gradually decreased with the increase of tripterine concentration. The results show that tripterine can reduce the survival rate of HCC827 cells, and the survival rate is reduced in a concentration-dependent manner.

Example 3

CCK-8 method for detecting influence of CysA and tripterine on activity of human HCC827 cells

1) Laboratory instruments and materials

Cell culture chambers (Thermo Fisher), ELX-800 microplate reader (BioTek), inverted microscope (LEICA DMIRB), fetal bovine serum (Biological Industries), DMEM (high-sugar) medium (GIBCO), CCK-8 (MCE), DMSO (Sigma), cysA (BBI), tripterine (Sigma), 96-well cell culture plates (NEST), HCC827 cells (Shanghai institute of China academy of sciences).

2) Experimental procedure

HCC827 cells were seeded at a density of about 7000 cells/well in a 96-well plate, and 5% CO was assayed at 37 ℃% ₂ And culturing in a cell culture box containing DMEM high-sugar culture solution containing 10% fetal calf serum. When the cell density reaches 70-80%, 5 μ M CysA and tripterine with final gradient concentrations of 0, 1, 1.5, 2, 2.5 μ M are added, and DMSO-treated cells are used as negative control group, and each cell is provided with three multiple wells. After further culturing for 24 hours, the morphology was observed under an inverted microscope. Then preparing a medium containing 10% of CCK-8, adding the medium to each well in the form of a changing solution, incubating the medium in an incubator for a certain period of time, and measuring OD _450nm . The results of detecting the effect of simultaneous treatment of CysA and tripterine on the activity of human HCC827 cells are shown in FIG. 3.

3) Principle of experiment same as example 1

Cell viability was calculated as follows: cell viability = experimental OD value/blank control OD value × 100%; statistics are expressed as mean ± standard deviation (x ± s) using the t-test.

As shown in FIG. 3, when HCC827 cells were treated with 5. Mu.M CysA and 2.5. Mu.M tripterine simultaneously, the cell death rate was increased in a tripterine concentration-dependent manner.

As can be seen from FIGS. 1, 2 and 3, the most prominent concentration of CysA and tripterine in inhibiting cell viability is 40 μ M, but the CysA concentration is only 5 μ M and the tripterine concentration is only 2.5 μ M when the CysA and the tripterine are used in combination, which indicates that the combined use of the CysA and the tripterine has unexpected technical effect and greatly reduces the concentration of the drug.

Example 4

Flow cytometry for detecting influence of CysA and tripterine on ROS level in HCC827 cells

1) Laboratory instruments and materials

Cell culture incubator (Thermo Fisher), DCFH-DA (Sigma), flow cytometer FACSCalibur (BD), fetal bovine serum (Biological Industries), DMEM (high sugar) medium (GIBCO), DMSO (Sigma), cysA (BBI), tripterine (Sigma), 10cm cell culture plate (NEST), HCC827 cells (shanghai institute of china).

2) Experimental procedure

HCC827 cells at about 2X 10 ⁶ Cell/plate density was seeded in 10cm cell culture dishes at 37 ℃ and 5% CO ₂ And culturing in a cell culture box containing DMEM high-sugar culture solution containing 10% fetal calf serum. When the cell density reached 70-80%, 5. Mu.M CysA and 2.5. Mu.M tripterine were added for 24h, and DMSO-treated cells were used as negative control groups, with three replicates for each cell. After the treatment, the cells were washed gently with 1 XPBS for 2-3 times. PBS was discarded and 8. Mu.L of DCFH-DA ROS probe dilution at a final concentration of 10uM was added. The cells were returned to 37 ℃ and 5% CO ₂ And incubating the incubator in dark. After 30min the supernatant was discarded, followed by gentle washing 2 times with 1 × PBS to wash off excess ROS probe. The cells were then digested with 1mL of trypsin and centrifuged at 800rpm at 4 ℃ for 3min. After collecting cells, the cells were gently washed 1 time with 1 × PBS, then 500ul of 1 × PBS was added to each centrifuge tube, the cells were resuspended with a pipette, transferred to a BD Calibur flow-through loading tube, and DCFH-DA staining positive cells were detected using a flow cytometer FL1 channel. At least 10000 cells are detected in each sample, the change of intracellular ROS level is calculated and analyzed by combining with flow cytometry analysis software FlowJo after data are obtained, and a ROS distribution graph is drawn, and the result is shown in figure 4.

3) Principle of experiment

DCFH-DA (2,7-dichlorofluorescein protein acetate) is a cell permeable, non-labeled, oxidation sensitive fluorescent probe. DCFH-DA (2,7-dichlorofluorescein protein acetate) was hydrolyzed by cellular esterase to 2,7-dichlorotetrafluorodioxane (2,7-dichlorofluorescein, DCFH) whereas DCFH was unable to permeabilize the cell membrane, thus allowing the probes to be loaded into the cells. Reactive oxygen species in the cell can then oxidize non-fluorescent DCFH to produce fluorescent DCF, thereby determining the level of intracellular ROS as a function of fluorescence intensity. The probe is widely used for detecting cell redox reaction.

As can be seen from FIG. 4, although a single treatment of 5. Mu.M CysA and 2.5. Mu.M Tripterine slightly increased the intracellular ROS level, the combined treatment of CysA and Tripterine significantly increased the intracellular ROS level.

Example 5

Effect of CysA and tripterine on human HCC827 cell morphology by simultaneous treatment

1) Laboratory instruments and materials

Cell culture incubator (Thermo Fisher), inverted microscope (Olympus), fetal bovine serum (Biological Industries), DMEM (high-sugar) medium (GIBCO), CCK-8 (MCE), DMSO (Sigma), cysA (BBI), tripterine (Sigma), 10cm cell culture dish (NEST), HCC827 cells (Shanghai cell institute of Central academy of sciences).

2) Experimental procedure

HCC827 cells were cultured at about 2X 10 ⁶ Cell/dish density was inoculated into 10cm cell culture dishes at 37 ℃ and 5% CO ₂ And culturing in a cell culture box containing DMEM high-sugar culture solution containing 10% fetal calf serum. When the cell density reached 70-80%, 5. Mu.M CysA and 2.5. Mu.M tripterine were added, and DMSO-treated cells were used as negative control. After further culturing for 24 hours, the morphology was observed under an inverted microscope.

The effect of simultaneous treatment of CysA and tripterine on HCC827 cell morphology was observed under an inverted microscope, and the results are shown in FIG. 5.

As shown in FIG. 5, when 5. Mu.M CysA and 2.5. Mu.M tripterine were treated simultaneously, the cell morphology was significantly changed, inducing cell death.

The result shows that after the HCC827 cells are treated by the low-concentration CysA and the low-concentration tripterine in a combined mode, cell death of cancer cells can be remarkably induced, the survival rate of lung cancer cells is reduced, and therefore the effect of treating lung cancer is achieved.

Example 6

Flow cytometry for detecting influence of combined treatment of CysA and tripterine on apoptosis rate of HCC827 cells

Laboratory instruments and materials

Cell culture box (Thermo Fisher), flow cytometer FACSCalibur (BD), annexin V-FITC Apoptosis Detection Kit (BD), RNase A (Solarbio), fetal bovine serum (Biological Industries), DMEM (high sugar) culture medium (GIBCO), DMSO (Amresco), cysA (CysA), 6cm cell culture dish (NEST), HCC827 cells (Shanghai institute of China academy), electric heating constant temperature water tank (DK-8D type).

Experimental procedure

HCC827 cells were cultured at a temperature of about 1.0X 10 ⁶ Cell/plate density was seeded in 6cm cell culture dishes at 37 ℃ and 5% CO ₂ And DMEM high-glucose culture solution containing 10% fetal calf serum. When the cell density reaches 70-80%, 5 μ M CysA and 2.5 μ M tripterine are added for treatment for 24h, and DMSO-treated cells are used as negative control groups, and each cell is provided with three multiple wells. Detecting cell distribution by using a flow cytometer: collecting all HCC827 cells including adherent living cells and floating dead cells in culture dish, centrifuging at 4 deg.C for 5min at 1500rpm, washing HCC827 cells with PBS for 2-3 times, counting cells, centrifuging at 4 deg.C 1500rpm for 5min, discarding supernatant, and collecting 1-5 × 10 ⁵ And (4) cells. 500 μ l of Binding buffer was added to resuspend the cells. Adding 5 μ l Annexin V-FITC, mixing, adding 10 μ l Propidium Iodid, mixing, and reacting at room temperature in dark for 5-15min. Within 1h, the Annexin V-FITC fluorescence signal was detected by the FL1 channel and the Propidium Iodide fluorescence signal was detected by the FL2 or FL3 channel. An Annexin V-FITC single positive tube and a Propidium Iodide (PI) single positive tube are simultaneously arranged during each detection, and are used for determining a fluorescence compensation value and the position of a cross quadrant gate. Filtering the stained Cell suspension by using a filter screen, collecting the Cell suspension into a flow cytometer detection tube, starting the flow cytometer to detect, detecting at least 10000 cells in each sample, calculating and analyzing the apoptosis distribution condition by Cell Quest software carried by the system after obtaining data and combining with other flow cytometer analysis software FlowJo, and drawing an apoptosis distribution graph, wherein the result is shown in figure 6.

3) Principle of experiment

Apoptosis refers to an autonomous and ordered death pattern of cells under the control of genes in order to maintain the stability of the internal environment. Apoptosis is a basic biological phenomenon of cells, and plays an important role in the processes of evolution of organisms, stabilization of internal environment, inhibition of tumor proliferation, deterioration and the like. Apoptosis is an active process, and relates to the activation, expression, regulation and the like of a series of genes, such as Bcl-2 family, caspase family, cancer suppressor gene p53 and the like.

The Annexin V-FITC apoptosis detection kit is an apoptosis kit for detecting phosphatidylserine on the surface of a cell membrane during apoptosis by using FITC labeled recombinant human Annexin V. Annexin V is a kind of phospholipid binding protein which is widely distributed in eukaryotic cell cytoplasm and depends on calcium ions, and participates in signal transduction in cells. Annexin V selectively binds Phosphatidylserine (PS). PS is distributed mainly inside the cell membrane, i.e. on the side adjacent to the cytoplasm. In the early stages of apoptosis, different types of cells evert the PS to the cell surface, i.e., outside the cell membrane. PS promotes coagulation and inflammatory responses upon exposure to the cell surface. And after Annexin V is combined with PS which is everted to the cell surface, procoagulant and proinflammatory activities of the PS can be blocked. Annexin V labeled by a fluorescent probe FITC with green fluorescence, namely Annexin V-FITC, can be used for conveniently and directly detecting the eversion of PS, which is an important characteristic of apoptosis by using a flow cytometer or a fluorescence microscope. Propidium Iodide (PI) is a nucleic acid dye. It cannot penetrate intact cell membranes, but can stain either bad cells or cells with late apoptosis that lose cell membrane integrity, exhibiting red fluorescence. In the case of necrotic cells, annexin V-FITC can enter the cytoplasm due to the loss of the integrity of the cell membrane and bind to the PS detected in the cell membrane, thus causing necrotic cells to also exhibit green fluorescence. Therefore, by matching annexin V with PI, cells at different apoptosis stages can be distinguished.

Example 7

WesternBlot detection of influence of CysA and tripterine combined treatment on HCC827 apoptosis regulatory protein expression

1) Laboratory instruments and materials

Cell culture incubator (Thermo Fisher), basic electrophoresis apparatus power supply (Bio-rad), electrophoresis apparatus (six one), transfer tank (Bio-rad), fetal bovine serum (Biological Industries), DMEM (high sugar) medium (GIBCO), DMSO (Amresco), cysA (BBI), 10cm cell culture dish (NEST), HCC827 cells (Hospital Shanghai cell division), PVDF membrane (Roapplied Science), BAX (50599-2-GT, proteinTech), bcl-2 (8978 zx8978-1-AP, proteinTech), clear Caspase-3 (9661, che) and β -actin (20536-1-AP, proteinTenti-RB/HRP (Kirkegard & Perry).

2) Experimental procedure

HCC827 cells were cultured at a temperature of about 2.0X 10 ⁶ Cell/dish density was inoculated into 10cm cell culture dishes at 37 ℃ and 5% CO ₂ And DMEM high-glucose culture solution containing 10% fetal calf serum. When the cell density reaches 70-80%, 5 μ M CysA and 2.5 μ M tripterine are added for treatment for 24h, and DMSO-treated cells are used as negative control groups, and each cell is provided with three multiple wells. Collecting HCC827 cells, extracting total protein, performing sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) electrophoretic separation, transferring the protein onto a polyvinylidene fluoride (PVDF) membrane, sealing with 5% skimmed milk powder for 1h, incubating at 4 ℃ for one time overnight, detecting the change conditions of BAX, bcl-2 and cleared Caspase-3 related proteins of cell cycle regulation, adopting beta-actin as an internal reference, rinsing with TBST, adding a secondary antibody, incubating at normal temperature for 1h, washing with TBST for 3 times, developing with ECL luminescence solution serving as a chemiluminescence agent, and developing in a chemiluminescence imaging system, wherein the result is shown in FIG. 7.

3) Principle of experiment

WesternBlot uses polyacrylamide gel electrophoresis, and the test substance is a protein, "probe" is an antibody, "and" color development "is with the labeled secondary antibody. SDS-PAGE allows protein samples to be separated and transferred to a solid support, polyvinylidene difluoride membrane (PVDF). The solid phase carrier can adsorb protein and keep the type of polypeptide separated by electrophoresis and the biological activity of the polypeptide unchanged. The transferred PVDF membrane is called a blot and treated with a protein solution (e.g., 5% skim milk powder) to block the hydrophobic binding sites on the PVDF membrane. The PVDF membrane is treated with an antibody (primary antibody) to the target protein-only the protein to be investigated binds specifically to the primary antibody to form an antigen-antibody complex, and thus after washing away the unbound primary antibody, only the primary antibody is bound at the site of the target protein. The PVDF membrane treated with the primary antibody is treated with a labeled secondary antibody, which is an antibody against the primary antibody, and if the primary antibody is obtained from a mouse, the secondary antibody is an antibody against mouse IgG. After treatment, the labeled secondary antibody binds to the primary antibody to form an antibody complex, which indicates the location of the primary antibody, i.e., the location of the protein to be studied.

As shown in FIG. 7, cysA and tripterine combined treatment can significantly increase the expression of the cell apoptosis-promoting proteins BAX and clear Caspase-3, and reduce the expression of the apoptosis-inhibiting protein Bcl-2. The above results demonstrate that CysA and tripterine combined treatment can inhibit the proliferation of HCC827 cells by inducing the expression of apoptosis-related proteins.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Example 8

Male nude mice were purchased from huafukang (beijing, china) company (5-6 weeks) and fed with standard animal diet and water. HCC827 cells were mixed with Matrigel at a ratio of 1:1, mixing uniformly. The cells were inoculated subcutaneously in the right hind leg of nude mice. Mice were treated once daily by intraperitoneal injection of cyclosporin A or/and tripterine. Body weight and tumor volume were measured. Mice were euthanized 21 days after treatment, and tumors and major organs were harvested and fixed with formalin or stored by snap freezing. The organs of heart, liver, spleen, lung and kidney were HE stained, as can be seen from fig. 8, cyclosporin a and tripterine treatment had no significant toxic side effects on the major organs of mice. And no abnormality is found by detecting indexes such as blood conventional Red Blood Cells (RBC), white Blood Cells (WBC), hemoglobin (HGB), lymphocytes (LYM) and the like of the nude mice. In addition, cyclosporin a and tripterine combined treatment significantly inhibited tumor growth compared to the single treatment group.

Claims

1. The application of the combination of the cyclosporin A and the tripterine in the preparation of the medicine for treating the lung cancer is characterized in that in the medicine, the effective concentration of the cyclosporin A is 5 mu M, and the effective concentration of the tripterine is 2.5 mu M.