Traditional Chinese medicine compound preparation for treating lung cancer and application

Abstract

The invention discloses a traditional Chinese medicine compound preparation for treating lung cancer. The traditional Chinese medicine compound preparation is prepared from the following raw material medicines in parts by weight: 6-9 parts of ginkgo leaves, 6-9 parts of schisandra chinensis, 15-45 parts of astragalus membranaceus, 9-15 parts of bighead atractylodes rhizome, 15-30 parts of coix seeds, 15-30 parts of Chinese yam, 12-24 parts of akebia fruits, 15-30 parts of salvia chinensis, 15-30 parts of selaginella doederleinii hieron and 15-30 parts of oldenlandia diffusa. The invention finds that the traditional Chinese medicine compound preparation inhibits invasion and metastasis of lung cancer cells through a TGF-beta / STAT3 / PD-L1 signal channel, reveals that the traditional Chinese medicine compound preparation plays a role in killing lung cancer tumor cells, also has a remarkable inhibiting role in metastasis and relapse of tumors, and is clear and reliable in anti-tumor effect.

Description

Technical Field

[0001] The invention belongs to the field of research, development and utilization of traditional Chinese medicine, and specifically relates to a traditional Chinese medicine compound preparation for treating lung cancer and its use. Background Art

[0002] Lung cancer is one of the most deadly and dangerous cancers in the world, killing many men and women every year. Despite advances in chemotherapy, immunotherapy, and targeted therapy, lung cancer remains the leading cause of cancer-related deaths. Distant metastasis of tumors is the cause of most cancer deaths, and preventing and controlling tumor recurrence and distant metastasis is the hallmark of effective cancer treatment.

[0003] Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule that inhibits immune response. The immune escape process of tumor cells is closely related to PD-L1. Targeting PD-L1 may be a promising treatment for cancer patients. We can also inhibit the expression of PD-L1 by inhibiting the upstream STAT3 signal. Compared with the TNM staging system, p-STAT3 and PD-L1 can be used as an excellent indicator to predict the prognosis of patients after tumor surgery. Inhibiting the activation of STAT3 to reduce the expression of PD-L1 can inhibit the invasion and metastasis of tumor cells. Chemotherapeutic drugs and targeted drugs are accompanied by great adverse reactions, and immune checkpoint inhibitors have low efficacy. Although there have been great advances in the diagnosis and treatment of lung cancer in recent years, it is still far from achieving the goal of basically controlling the disease. Therefore, exploring new treatment approaches remains an urgent task. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, according to an embodiment of the present invention, it is hoped to provide a compound preparation for treating lung cancer that has the effect of significantly improving the general condition of lung cancer patients and inhibiting the metastasis and recurrence of lung cancer.

[0005] The present invention is based on the clinical practice and working basis of traditional Chinese medicine in treating lung cancer, takes improving the general condition of lung cancer patients or animal models and inhibiting lung cancer metastasis and recurrence as efficacy indicators, and combines the compound prescription of the present invention through dialectical analysis of the pathogenesis of lung cancer and sorting out and screening the efficacy of Chinese herbal medicines.

[0006] According to the embodiments, the invention provides a compound preparation for treating lung cancer, the innovation of which is that it is made of the following raw materials in parts by weight: 6-9 parts of Ginkgo biloba leaves, 6-9 parts of Schisandra chinensis, 15-45 parts of Astragalus membranaceus, 9-15 parts of Atractylodes macrocephala, 15-30 parts of Coix seeds, 15-30 parts of Dioscorea opposita, 12-24 parts of Semen Preparata, 15-30 parts of Ilex erythrorhizon, 15-30 parts of Platycodon grandiflorum, and 15-30 parts of Hedyotis diffusa.

[0007] According to one embodiment, in the aforementioned compound preparation for treating lung cancer of the present invention, the raw materials and their weight proportions are preferably: 9 parts of Ginkgo biloba leaves, 9 parts of Schisandra chinensis, 30 parts of Astragalus membranaceus, 9 parts of Atractylodes macrocephala, 30 parts of Coix seeds, 15 parts of Dioscorea opposita, 15 parts of Semen Preparata, 30 parts of Lithospermum officinale, 30 parts of Cephalotaxus chinensis, and 30 parts of Hedyotis diffusa.

[0008] According to one embodiment, in the aforementioned compound preparation for treating lung cancer of the present invention, the compound preparation is a decoction, tablet, granule, capsule or oral liquid.

[0009] According to one embodiment, in the aforementioned compound preparation for treating lung cancer of the present invention, the lung cancer is non-small cell lung cancer.

[0010] There is no special requirement for the preparation method of the compound preparation for treating lung cancer of the present invention. It can be prepared into any dosage form in pharmacy, such as decoction, granules, tablets, capsules or oral liquid, by conventional methods of Chinese medicine preparation.

[0011] The properties of the raw materials included in the present invention are as follows:

[0012] Ginkgo biloba: sweet, bitter, astringent, neutral. Enters the heart and lung meridians. It has the effects of astringing the lungs and relieving asthma, promoting blood circulation and removing blood stasis, dredging collaterals and relieving pain, and removing turbidity and reducing lipids.

[0013] Schisandrae Chinensis: sour, sweet, warm in nature. Enters the lung, heart, and kidney meridians. It has the effects of astringing, strengthening qi, promoting the production of body fluids, and nourishing the kidneys and calming the mind.

[0014] Astragalus: sweet in taste, slightly warm in nature. Enters the spleen and lung meridians. It has the effects of strengthening the spleen and replenishing the middle, raising yang and lifting the sunken, strengthening the body's defense system, promoting urination, and removing toxins and promoting tissue regeneration.

[0015] Atractylodes macrocephala: bitter, sweet, warm in nature. Enters the spleen and stomach meridians. Has the effects of replenishing qi and strengthening the spleen, drying dampness and promoting diuresis, stopping sweating, and calming the fetus.

[0016] Coix seeds: sweet, light, cool in nature. Enter the spleen, stomach, and lung meridians. It has the effects of promoting diuresis and eliminating dampness, strengthening the spleen and stopping diarrhea, removing numbness, discharging pus, and detoxifying and dispersing nodules.

[0017] Chinese Yam: sweet in taste, neutral in nature; enters the spleen, lung and kidney meridians. It has the effects of strengthening the spleen, nourishing the lungs, consolidating the kidneys and improving the essence.

[0018] Precipitous Seed: sweet, cold. Enter the liver, spleen, and kidney meridians. Soothe the liver and regulate qi, promote blood circulation, disperse blood stasis and relieve pain, and relieve restlessness and promote urination.

[0019] Lithospermum officinale: pungent, bitter, slightly cold. Liver meridian, spleen meridian. Clears away heat and detoxifies, reduces swelling and resolves nodules.

[0020] Lithospermum officinale: sweet, slightly bitter, astringent, cool in nature. Enters the lung and liver meridians. Has the effects of clearing away heat and detoxification, anti-cancer, and hemostasis.

[0021] Hedyotis diffusa: slightly bitter, cold in nature. Stomach, large intestine, small intestine meridians. Clears away heat, detoxifies, removes dampness and relieves stranguria.

[0022] The compound medicine of the present invention is based on the theory that the etiology and pathogenesis of lung cancer are generally deficiency of the root and excess of the superficial. The deficiency and dispersion of the ancestral qi is the root, and qi stagnation, blood stasis and toxic evil are the superficial. Ginkgo leaf is the main drug, which belongs to the heart and lung meridians, and has the effects of astringing the lungs and calming asthma, promoting blood circulation and removing blood stasis. Schisandra chinensis and Astragalus membranaceus are the assistant drugs, which help Ginkgo leaf to astringe the lungs and replenish qi, and are assisted by Atractylodes macrocephala, Coix seeds, and Chinese yam to promote dampness and invigorate the spleen, help qi and blood to be transformed, and Premonition Seed can soothe the liver, regulate qi and activate blood circulation, so as to regulate the ancestral qi, help Longhu Huihuan, Lithospermum officinale, Cypress on the stone, and Hedyotis diffusa to detoxify and disperse stagnation, so as to remove stagnation and lumps.

[0023] Studies on Chinese medicine components show that the ginkgo biloba flavonoids, ursolic acid, luteolin and other monomers contained in the raw materials of the present invention have the effect of inhibiting tumor metastasis. The subsequent examples and test examples will prove that the compound preparation of the present invention can improve the general condition of lung cancer patients, regulate the occurrence and development of tumors, inhibit tumor metastasis and recurrence, and can also enhance anti-tumor immune response and prevent tumor immune escape by regulating the expression of PD-L1. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Figure 3 shows that the Ginkgo Gujin Decoction granules provided in the embodiments of the present invention inhibit the cell proliferation of PC-9 and A549 cells. Figure 1 middle: Figure 1 (A) CCK-8 assay was used to detect the effects of Ginkgo Gujin Decoction granules at concentrations of 0, 0.75, 1.5, 3, 6, 12, 24, 48, 96, and 192 mg / ml on the growth and proliferation of PC-9 and A549 cells. The results showed that after 24 h of treatment with the above different concentrations of Ginkgo Gujin Decoction granules, Ginkgo Gujin Decoction granules at 12 mg / ml had an inhibitory effect on the growth of lung cancer cells, and the inhibitory effect became more obvious as the drug concentration increased. Figure 1 (B) Compared with the control group, when the concentration of Ginkgo Gujin Tang granules was 6 mg / ml, cell proliferation did not change significantly (P>0.05); when the concentration of Ginkgo Gujin Tang granules was 12 mg / ml, cell proliferation was inhibited (P<0.05). In order to exclude the influence of the cytotoxic effect of Ginkgo Gujin Tang granules on PC-9 and A549 cells, the subsequent in vitro studies on the anti-lung cancer cell metastasis related experiments of Ginkgo Gujin Tang granules were carried out at drug concentrations of 1.5, 3, and 6 mg / ml.

[0025] Figure 2 The scratch test results of the present invention show that the Yinxing Gujintang granules inhibit the migration of lung cancer cells. Figure 2(A) shows that in PC-9 cells, compared with the Ginkgo Gujin Decoction Granule group, the scratch repair rate of the control group was significantly increased, and the migration ability was significantly enhanced (P < 0.01). The Ginkgo Gujin Decoction Granule group inhibited the metastasis ability of lung cancer cells in a drug concentration gradient-dependent manner. Figure 2 (B) is a statistical bar graph of PC-9 cell scratch area. Figure 2 (C) showed that in A549 cells, compared with the Ginkgo Gujin Decoction Granule group, the scratch repair rate of the control group was significantly increased, and the migration ability was significantly enhanced (P < 0.01). The Ginkgo Gujin Decoction Granule group inhibited the metastasis ability of lung cancer cells in a drug concentration gradient-dependent manner. Figure 2 (D) is a statistical bar graph of the scratch area of ​​A549 cells.

[0026] Figure 3 : is a Western Blot result diagram of the effect of Ginkgo Gujin Decoction granules on lung cancer cells provided by an embodiment of the present invention, wherein: Figure 3 (A) is a graph showing the changes in protein expression in PC-9 and A549 cells regulated by Ginkgo Gujin Tang granules; Figure 3 (B) is a statistical graph of Western Blot results for PC-9 and A549 cells.

[0027] Figure 4 (A) Effect of Ginkgo Gujin Decoction Granules on TGF-β-induced migration of lung cancer PC-9 cells in scratch test. Figure 4 (B) is a statistical bar graph of PC-9 cell scratch area. Figure 4 (C) Effect of Ginkgo Gujin Decoction Granules on TGF-β-induced migration of lung cancer cells A549 using scratch test. Figure 4 (D) is a statistical bar graph of the scratch area of ​​A549 cells.

[0028] Figure 5 (A) Western Blot results of Ginkgo Gujin Decoction granules regulating protein changes in PC-9 and A549 cells induced by TGF-β. Figure 5 (B) is a statistical graph of Western Blot results for PC-9 and A549 cells.

[0029] Figure 6 (A) is a graph showing the Western Blot results of Si PD-L1 in PC-9 cells. Figure 6 (B) is a statistical graph of Western Blot results of PC-9 cells. Figure 6 (C) is a graph showing the Western Blot results of Si PD-L1 in A549 cells. Figure 6 (D) is a statistical graph of Western Blot results of PC-9 cells. Figure 6(E) is a graph showing the results of a scratch assay after Si PD-L1 in PC-9 cells. Figure 6 (F) Statistical graph of the results of Si PD-L1 scratch assay on PC-9 cells. Figure 6 (G) is a graph showing the results of a scratch assay after Si PD-L1 in A549 cells. Figure 6 (H) Statistical graph of the results of Si PD-L1 scratch assay in A549 cells. Figure 6 (I) is a graph showing the results of a scratch assay in which PC-9 cells were induced by TGF-β after Si PD-L1. Figure 6 (J) is a statistical graph of the scratch assay results induced by TGF-β after Si PD-L1 in PC-9 cells. Figure 6 (K) is a graph showing the results of a scratch assay in A549 cells induced by TGF-β after Si PD-L1. Figure 6 (L) is a statistical graph of the scratch assay results induced by TGF-β after Si PD-L1 in A549 cells.

[0030] Figure 7 (A) is an in vivo experiment of the Ginkgo Gujin Decoction granules provided in an embodiment of the present invention. Figure 7 (A) The results show the tumor burden of lung tissue in mice in the YXGJD1 (Ginkgo Biloba Granules 3.3 g / kg), YXGJD2 (Ginkgo Biloba Granules 6.6 g / kg) and Cisplatin groups compared with the Control group. Figure 7 (B) is the HE results of lung tissues of mice in each group. Figure 7 (C) is a statistical graph of lung tissue weight of mice in each group. Figure 7 (DF) are statistical graphs of liver and kidney function of mice in each group. Figure 7 (G) is a statistical graph of the body weight of mice in each group. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. These embodiments should be understood to be only used to illustrate the present invention and not to limit the scope of protection of the present invention. After reading the contents of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0032] Example 1

[0033] Weigh 9 g of Ginkgo biloba leaves, 9 g of Schisandra chinensis, 30 g of Astragalus membranaceus, 9 g of Atractylodes macrocephala, 30 g of Coix seeds, 15 g of Dioscorea opposita, 15 g of Semen prunifolia, 30 g of Ilex erythrorhizon, 30 g of Cypress and 30 g of Hedyotis diffusa, and prepare them into granules (Ginkgo Gujintang granules) by conventional methods of Chinese medicine preparation, with 11 g granules in each package, and each gram of granules is equivalent to 9.4 g of crude drug, which are diluted with pure water to 0.75, 1.5, 3, 6, 12, 24, 48, 96, and 192 mg / ml for acting on cells; or diluted with pure water to 3.3 g / kg and 6.6 g / kg for oral administration to mice.

[0034] In the following Test Examples 1-4 of the present invention, Ginkgo Solid Gold Granules were purchased from Wanshicheng Chinese Medicine Products Co., Ltd., and the raw materials, their proportions, and preparation methods were the same as those in Example 1.

[0035] Ginkgo Gujin Granules are the empirical prescription of Deng Haibin, chief physician of Longhua Hospital. Does it have the effect of inhibiting the invasion and metastasis of lung cancer cells? In-depth clarification of the anti-invasion and metastasis effect of Ginkgo Gujin Granules on lung cancer cells and its safety is the focus of expanding the medicinal value of Ginkgo Gujin Granules, and it is also the difficulty in developing anticancer drugs. The technical scheme of the present invention is further described below in combination with Test Examples 1-5.

[0036] S101, cell culture: cell recovery, cell culture and cell passage. The CCK-8 (cell counting kit 8, CCK-8) experiment was used to detect the effects of different concentrations of Ginkgo Gujin Decoction granules on the growth and proliferation of PC-9 and A549 cells.

[0037] S102, scratch test was used to detect the inhibitory effect of Ginkgo Gujin Decoction granules on the migration of PC-9 and A549 cells.

[0038] S103, Western blot experiments were used to detect the effect of Ginkgo Gujin Tang granules on the expression of MMP2 and MMP9 proteins related to invasion and metastasis of PC-9 and A549 lung cancer cells, and to detect the expression of STAT3, p-STAT3, and PD-L1 proteins related to the TGF-β / STAT3 / PD-L1 signaling pathway.

[0039] S104, Transforming growth factor-β1 (TGF-β1) was used to induce invasion and metastasis of PC-9 and A549 cells; the effect of Ginkgo Gujin Tang granules on the migration of PC-9 and A549 cells induced by TGF-β1 was observed by scratch assay; the effect of Ginkgo Gujin Tang granules on the expression of invasion and metastasis-related proteins and TGF-β / STAT3 / PD-L1 signaling pathway of PC-9 and A549 cells induced by TGF-β1 was detected by Western blot assay. After knocking down PD-L1 in PC-9 and A549 cells, the effect of PD-L1 on the invasion and metastasis of lung cancer cells was observed by scratch assay. TGF-β induced invasion and metastasis of PC-9 and A549 cells with PD-L1 knockdown, and the effect of PD-L1 on invasion and metastasis in lung cancer cells was further observed.

[0040] S105, an in vivo lung cancer metastasis model was established by injecting A549 cells into the tail vein of BALB / c nude mice; different concentrations of Ginkgo Gujin Decoction granules were used for intervention, and the lung weight of mice was observed; the liver and kidney function and body weight of mice were tested, and the toxic and side effects of the drug were observed.

[0041] Experimental Example 1 - Effects of Ginkgo Gujin Decoction Granules on the Growth and Proliferation of PC-9 and A549 Lung Cancer Cells.

[0042] 1. Experimental Materials

[0043] 1.1 Experimental cells

[0044] Human non-small cell lung cancer PC-9 cells and A549 cells were purchased from Wuhan Pronocell Life Science Co., Ltd. PC-9 and A549 cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin. Both cells were cultured at 37°C and 5% CO 2 Culture in a constant temperature incubator with saturated humidity.

[0045] 1.2 Experimental drugs

[0046] Ginkgo Gujin Decoction granules were purchased from Wanshicheng Chinese Medicine Products Co., Ltd., containing 9g of ginkgo leaves, 9g of Schisandra chinensis, 30g of Astragalus membranaceus, 9g of Atractylodes macrocephala, 30g of Coix lachryma-jobi, 15g of Dioscorea opposita, 15g of Precocious Seed, 30g of Ilex erythrorhizon, 30g of Platycodon grandiflorum, and 30g of Hedyotis diffusa. Each package contains 11g of granules, and each gram of granules is equivalent to 9.4g of the crude drug.

[0047] 1.3 Main reagents for the experiment

[0048]

[0049] 1.4 Main instruments of the experiment

[0050]

[0051] 2. Cell Culture

[0052] 2.1 Cell recovery

[0053] Take out the frozen cells from the -80℃ refrigerator, quickly put the cryopreserved tube into a preheated 37℃ water bath, and shake it until the liquid in the cryopreserved tube melts, spray alcohol for disinfection, and put it into the clean bench; carefully aspirate the cell suspension and put it into a centrifuge tube containing 10mL of culture medium, blow the cells slightly, put it into a centrifuge at 1000rpm for 5min, discard the supernatant, blow the cells again with 8-10mL of culture medium, and transfer them into a culture dish and put them into a 37℃, 5% CO 2 Culture in an incubator.

[0054] 2.2 Cell culture

[0055] The cells were cultured with complete culture medium containing 10% fetal bovine serum, 1% double antibody (penicillin-streptomycin mixed solution), and 89% DMEM medium at 37°C with 5% CO. 2 Cultured in a constant temperature incubator.

[0056] 2.3 Cell passaging

[0057] When the cells are cultured to cover about 80-90% of the bottom area of ​​the 10cm culture dish, they can be subcultured. Take 2ml PBS buffer and gently rinse the adherent cells in the culture dish twice. Use a 1ml pipette to add 1ml 0.25% phenol red-free trypsin to the 10cm culture dish. Shake the culture dish to make 1ml phenol red-free trypsin fully cover the bottom of the entire culture dish. Use a 1ml pipette to gently blow the residual cells at the bottom of the 10cm culture dish. After gently blowing into a cell suspension, collect all of them into a 5ml EP tube. After balancing, put 5ml in a centrifuge. EP tube, centrifuge at 1300rpm, 3min, discard the supernatant, be careful not to suck the cells in, add 1ml complete culture medium, blow gently, stir evenly, centrifuge the cell suspension, add 7ml complete culture medium to the 10cm culture dish in advance, and distribute the cell suspension to each 10cm culture dish according to the 1:3 subculture ratio. Mark the cell name, subculture date, name and other information, place in the incubator, and continue to culture until the cells are completely attached. The cells are subcultured 3-4 times a week.

[0058] 3. Cell Proliferation Assay

[0059] Take PC-9 and A549 cells that are in good growth and in the logarithmic growth phase, treat the cells according to the subculture steps, digest and centrifuge, blow and mix, take 20μl of the cell suspension and vertically enter the cell counting plate, and count the cells with a cell counter. Take a disposable sample tank to hold complete culture medium, dilute the cells to the required concentration, mix and inoculate each well of the 96-well plate. Use a spray gun to blow and mix the cells several times during each plating process to ensure the consistency of the number of cells in each well. Add 100μl of PBS solution to each well around the 96-well plate. After the plating is completed, mark the cell type, name, time, and experimental type, and place it in an incubator to continue culturing until the cells are completely attached. Set the drug concentration in advance before adding the drug: the concentration of Ginkgo Gujintang granules is set to 0, 0.75, 1.5, 3, 6, 12, 24, 48, 96, and 192 mg / ml respectively. Set 3 replicates for each concentration. 24 hours after plating, observe whether the cells adhere to the wall. When the cells grow well, perform drug intervention, use a pipette to discard the old culture medium, and then use a 100μl pipette to aspirate the remaining culture medium. According to the above drug concentration, the solution is configured in the multi-channel sample tank and stirred evenly with a 1ml pipette. After 24 hours of drug addition and culture, the CCK-8 proliferation kit is used to detect the growth and proliferation of cells after intervention. The operation should be kept away from light. After the culture medium was fully aspirated with a pistol, the residual culture medium was aspirated again with a new pistol tip. 100 μl of DMEM culture medium without FBS containing 10% CCK-8 solution was added to each well and placed in an incubator to allow the CCK-8 reagent to fully react. After 1 hour, the 96-well plate was taken out and the absorbance value (OD value) of the solution in each well was measured with a multifunctional microplate reader at a wavelength of 450 nm. The cell survival rate was calculated using the equation [(As (experimental well) - Ab (blank well) / Ac (control well) - Ab (blank well)] × 100%. After saving the data, Graph Pad Prism 9.0 was used for data analysis and graphing.

[0060] 4. Experimental results: Ginkgo Gujin Decoction granules inhibited the cell proliferation of PC-9 and A549 cells. Figure 1 middle: Figure 1 (A) CCK-8 assay was used to detect the effects of Ginkgo Gujin Decoction granules at concentrations of 0, 0.75, 1.5, 3, 6, 12, 24, 48, 96, and 192 mg / ml on the growth and proliferation of PC-9 and A549 cells. The results showed that after 24 h of treatment with the above different concentrations of Ginkgo Gujin Decoction granules, Ginkgo Gujin Decoction granules at 12 mg / ml had an inhibitory effect on the growth of lung cancer cells, and the inhibitory effect became more obvious as the drug concentration increased. Figure 1(B) Compared with the control group, when the concentration of Ginkgo Gujin Tang granules was 6 mg / ml, cell proliferation did not change significantly (P>0.05); when the concentration of Ginkgo Gujin Tang granules was 12 mg / ml, cell proliferation was inhibited (P<0.05). In order to exclude the influence of the cytotoxic effect of Ginkgo Gujin Tang granules on PC-9 and A549 cells, the subsequent in vitro studies on the anti-lung cancer cell metastasis related experiments of Ginkgo Gujin Tang granules were carried out at drug concentrations of 1.5, 3, and 6 mg / ml.

[0061] Experimental Example 2-Effect of Ginkgo Gujin Decoction Granules on the Migration Ability of PC-9 and A549 Cells.

[0062] 1. Experimental Materials

[0063] 1.1 Experimental cells

[0064] The experimental cells were the same as those in 1.1 of Experimental Example 1.

[0065] 1.2 Experimental drugs

[0066] Ginkgo Gujin Decoction Granules were purchased from Wanshicheng Chinese Medicine Products Co., Ltd., containing 9g of ginkgo leaves, 9g of schisandra chinensis, 30g of astragalus, 9g of atractylodes macrocephala, 30g of coix seeds, 15g of yam, 15g of cyperus rotundus, 30g of lithospermum officinale, 30g of cyperus rotundus, and 30g of oldenlandia diffusa. Each package contained 11g of granules, and each gram of granules was equivalent to 9.4g of crude drug, which was diluted to concentrations of 1.5, 3, and 6mg / ml to act on lung cancer cells, respectively.

[0067] 1.3 Main reagents and instruments for the experiment

[0068] The main reagents and instruments are the same as those in 1.3 and 1.4 of Experiment 1.

[0069] 2. Cell Culture

[0070] Same as 2. Cell culture method in Experimental Example 1.

[0071] 3. Scratch test

[0072] This part was divided into different concentrations of Ginkgo Gujin Decoction granules. PC-9 and A549 cells in the logarithmic growth phase were obtained, digested with trypsin and centrifuged, and then mixed with serum-free DMEM medium to adjust the cell concentration to 2.5×10 5 / ml were inoculated in a six-well plate, and 3ml of culture medium was added to each well. 24h after plating, check whether the cells have reached 80%-90%. When the cells are in good growth condition, scratch the plate with a 100μL pipette tip perpendicular to the scratch on the plane of the six-well plate, and use PBS solution to gently wash the bottom of the six-well plate twice. After cleaning the scratch area as much as possible, 3ml of serum-free DMEM culture medium was added to the six-well plate, and photos were taken under a microscope, and then placed in the culture medium for continued culture. At 24h, the repair of each group of scratches was observed and photographed under a microscope, and the experimental data were analyzed using ImageJ software and calculated using Graphpad.

[0073] Increased migration ability is a decisive feature of tumor cell metastasis. The scratch test can detect the migration ability of tumor cells within a certain period of time. The results of the 24h scratch test show Figure 2 (A) shows that in PC-9 cells, compared with the Ginkgo Gujin Decoction Granule group, the scratch repair rate of the control group was significantly increased, and the migration ability was significantly enhanced (P < 0.01). The Ginkgo Gujin Decoction Granule group inhibited the metastasis ability of lung cancer cells in a drug concentration gradient-dependent manner. Figure 2 (B) is a statistical bar graph of PC-9 cell scratch area. Figure 2 (C) showed that in A549 cells, compared with the Ginkgo Gujin Decoction Granule group, the scratch repair rate of the control group was significantly increased, and the migration ability was significantly enhanced (P < 0.01). The Ginkgo Gujin Decoction Granule group inhibited the metastasis ability of lung cancer cells in a drug concentration gradient-dependent manner. Figure 2 (D) is a statistical bar graph of the scratch area of ​​A549 cells.

[0074] Experimental Example 3 - Protein level detection of the effect of Ginkgo Gujin Decoction Granules in inhibiting the invasion and metastasis of lung cancer.

[0075] 1. Western blot experiments were used to detect the effects of Ginkgo Gujin Tang granules on the expression of invasion and metastasis-related proteins MMP2 and MMP9 in PC-9 and A549 lung cancer cells and the inhibition of TGF-β / STAT3 / PD-L1.

[0076] Digest the cells and plate them, place them in an incubator for culture, discard the cell culture medium in the culture dish, wash twice with pre-cooled PBS solution, add an appropriate amount of lysis solution (RIPA lysis solution, PMSF, protease inhibitors, phosphatase inhibitors), shake gently to make the bottom of the dish completely in contact with the lysis solution, then place it on ice for lysis for 25 minutes, scrape the cells at one end of the bottom of the dish, and then collect them into a marked EP tube, centrifuge at 4℃, 12000rpm for 15 minutes, move the supernatant to the newly marked EP tube, and be careful not to aspirate the cell fragments at the bottom of the dish. When the BSA standard is diluted to 2mg / ml, dilute it in half according to the number of sample wells required, and take 20μl and add it to the 96-well plate. Take 4μl protein sample, add 16μl ddH2O to the 96-well plate, mix thoroughly, add 200μl BCA working solution to each well and culture in a 37℃ incubator for 30min, use a microplate reader to read the absorbance at a wavelength of 562nm, make a standard curve and count the protein concentration in the sample. Mix 5× loading buffer and protein sample in a ratio of 1:4, shake and stir evenly, cook the protein at 100℃ for 10min, open the lid once before cooking to avoid the EP tube lid bursting during the cooking process, cool to room temperature, store in a -20℃ refrigerator, and can also be used for subsequent experiments.

[0077] Place clean thick and thin glass plates neatly, pay attention to the front and back, and then vertically clamp them on the rack to prepare for glue filling. Prepare the glue according to the proportions in the instructions and mix them evenly. After preparing the separation gel, add an appropriate amount of anhydrous ethanol to seal. When a clear dividing line appears (about 15 minutes), pour out the ethanol and absorb the excess ethanol. Add the concentrated gel and insert a ten-hole comb, and slowly insert it vertically to prevent bubbles. After the protein sample is dissolved, shake and mix it evenly. Heat it at 100℃ for 5 minutes to return to room temperature, centrifuge it slightly, and then load it. Put it in the electrophoresis tank, fill the inner tank with 1× running buffer, completely cover the loading hole, and load the sample without liquid leakage. Put the marker first, then the protein, add the remaining 1× running buffer to the outer tank of the electrophoresis, cover the lid, pay attention to the positive and negative poles, and turn on the power supply. The upper layer of gel is 80V. After the marker runs out of the upper layer of gel (about 25 minutes), change the voltage to 120V and then run the lower layer of gel. The marker in the protein sample runs to the bottom of the lower layer of gel. The electrophoresis is over, and then the membrane is transferred.

[0078] The transfer solution prepared in advance was precooled in a 4°C refrigerator. The PVDF membrane (5.5 cm × 8.5 cm) was soaked in methanol for 10 seconds and then transferred to the transfer solution together with the sponge. After the electrophoresis, the glass plate was pried open to remove all the concentrated gel, and a sandwich structure was made. In the order of "sponge-filter paper-PVDF membrane-gel-filter paper-sponge", following the principle of "black gel and white membrane", the clamps were fastened, and the transfer tank was placed. The precooled transfer solution was poured to the Blotting line, and the lid was covered. Pay attention to the electrode correspondence. The transfer tank was placed in a basin and wrapped with ice for transfer to avoid overheating of the PVDF membrane. Select different transfer currents and transfer times according to the molecular weight of the antibody. After the transfer, place the PVDF membrane in the pre-prepared skim milk and block it on a shaker for 2 hours. The blocking time can be appropriately extended in winter. After blocking, wash it 3 times with 1×TBST, 5 minutes each time, cut the membrane according to the molecular weight of the target protein, prepare the primary antibody at a ratio of 1:1000 between the primary antibody diluent and the antibody. After washing, place the strips in an incubation box and add the primary antibody for overnight incubation.

[0079] Wash the membrane with TBST 3 times × 10min on a shaker, prepare the secondary antibody at a ratio of 1:10000 of TBST to the secondary antibody, and incubate the secondary antibody on a shaker at room temperature for 1h after washing. After incubation, wash the membrane with TBST 3 times × 10min on a shaker, prepare the ECL colorimetric solution (about 300μl / membrane) in advance, and wrap it with tin foil to avoid light. First, remove the membrane from TBST, gently press the liquid with a paper towel, place it in the center of the imaging system, add the pre-prepared developer, so that the membrane is completely covered with the developer without overflowing, wait for 30s of the developer to react, and then develop it with the machine. After the membrane shows obvious immunoblot bands in the machine, click save to save the results in your own folder. Use ImageJ software to analyze the experimental data, and use GraphPad Prism9.0 for data analysis and mapping.

[0080] Western blot experiments were used to detect the expression changes of invasion and metastasis-related marker proteins in each group of lung cancer cells (PC-9 and A549 cells) after intervention with Ginkgo Gujin Decoction Granules. The results showed that Figure 3 (A) Different concentrations of Ginkgo Gujintang granules regulate the changes of invasion and metastasis-related proteins (MMP2, MMP9) in PC-9 and A549 cells and inhibit the TGF-β / STAT3 / PD-L1 signaling pathway; Figure 3 (B) is a statistical graph of Western Blot results for PC-9 and A549 cells.

[0081] Experimental Example 4-Effect of Ginkgo Gujin Decoction Granules on TGF-β-induced lung cancer cell migration.

[0082] 1. Use transforming growth factor-β1 (TGF-β1) to induce invasion and metastasis of PC-9 and A549 cells.

[0083] Preparation of TGF-β1 working solution: Take an appropriate amount of sterilized sodium citrate solution, dissolve TGF-β1 lyophilized powder, prepare a 2.5ug / ml working stock solution, and store in a -20℃ refrigerator.

[0084] 1.1 The scratch test was used to observe the effect of Ginkgo Gujin Decoction Granules on TGF-β1-induced PC-9 and A549 cell migration.

[0085] The scratch test was divided into 4 groups, namely, blank control group, Ginkgo Gujintang granule group, TGF-β group, and TGF-β+Ginkgo Gujintang granule group (Ginkgo Gujintang granule group concentration was 6 mg / ml). PC-9 and A549 cells in the logarithmic growth phase were obtained, trypsin digested and centrifuged, and then mixed and plated. After 24 hours of plating, observe whether the cells have reached 80%-90%. Scratch operation was performed under the condition of good cell growth. Take 100μl sterile pipette tip vertically to the plane of the six-well plate to scratch, wash twice with PBS solution to remove the scratched cells, add Ginkgo Gujintang granules, TGF-β, Ginkgo Gujintang granules + TGF-β to serum-free culture medium, and then add six-well plates. After taking pictures under a microscope, put them into culture and continue to culture. After 24 hours, observe the changes in migration ability of each group under a microscope and take pictures. Image J software was used to analyze the experimental data, and Graph PadPrism 9.0 was used to draw graphs.

[0086] The scratch test was used to detect the lung cancer cells (PC-9 and A549 cells) in the NC group, Ginkgo Gujin Tang granules, TGF-β group and TGF-β+ Ginkgo Gujin Tang granules group, and the changes in the migration ability of the four groups of cells were observed. Figure 4 (A) Effect of Ginkgo Gujin Decoction Granules on TGF-β-induced migration of lung cancer PC-9 cells in scratch test. Figure 4 (B) is a statistical bar graph of PC-9 cell scratch area. Figure 4 (C) Effect of Ginkgo Gujin Decoction Granules on TGF-β-induced migration of lung cancer cells A549 using scratch test. Figure 4 (D) is a bar graph showing the scratch area of ​​A549 cells. Ginkgo Gujin Decoction Granules significantly inhibited the metastasis of lung cancer cells induced by TGF-β.

[0087] 1.2 Western blot experiments were used to detect the effects of Ginkgo Gujin Decoction Granules on the expression of invasion and metastasis-related proteins of PC-9 and A549 induced by TGF-β1 and the TGF-β / STAT3 / PD-L1 signaling pathway.

[0088] Western blot experiments were performed to detect the mechanism of Ginkgo Gujin Decoction Granules on the invasion and metastasis of lung cancer cells (PC-9 and A549 cells). The results showed: Figure 5 (A) Ginkgo Gujin Decoction granules inhibited the changes of TGF-β-induced invasion and metastasis-related proteins (MMP2, MMP9) in PC-9 and A549 cells, inhibited the expression of proteins related to the TGF-β / STAT3 / PD-L1 signaling pathway, and inhibited the expression of p-STAT3 and PD-L1.

[0089] Figure 5 (B) is a statistical graph of Western Blot results for PC-9 and A549 cells. Figure 6 (A) is a graph showing the Western Blot results of SiPD-L1 in PC-9 cells. Figure 6 (B) is a statistical graph of Western Blot results of PC-9 cells. Figure 6 (C) is a graph showing the Western Blot results of Si PD-L1 in A549 cells. Figure 6 (D) is a statistical graph of Western Blot results of PC-9 cells. Figure 6 (E) is a graph showing the results of a scratch assay after Si PD-L1 in PC-9 cells. Figure 6 (F) Statistical graph of the results of Si PD-L1 scratch assay on PC-9 cells. Figure 6 (G) is a graph showing the results of a scratch assay after Si PD-L1 in A549 cells. Figure 6 (H) Statistical graph of the results of Si PD-L1 scratch assay in A549 cells. Figure 6 (I) is a graph showing the results of a scratch assay in which PC-9 cells were induced by TGF-β after Si PD-L1. Figure 6 (J) is a statistical graph of the scratch assay results induced by TGF-β after Si PD-L1 in PC-9 cells. Figure 6 (K) is a graph showing the results of a scratch assay in A549 cells induced by TGF-β after Si PD-L1. Figure 6 (L) is a statistical graph of the scratch test results induced by TGF-β after Si PD-L1 in A549 cells. The experimental results show that inhibiting the expression of PD-L1 can inhibit the invasion and metastasis of lung cancer cells.

[0090] Experimental Example 5—In vivo animal experiments verified that Ginkgo Gujin Decoction Granules inhibited the metastasis of lung cancer cells.

[0091] 1. An in vivo lung cancer metastasis model was established by injecting A549 cells into the tail vein of BALB / c nude mice.

[0092] When the A549 cell density in the culture dish reaches more than 80%, the cells are digested with trypsin, counted, and resuspended in PBS to a cell concentration of 4 × 10 8 / ml; fix the mouse, mix the cell suspension thoroughly with a pipette to prevent cell aggregation and embolism, draw 100μl of cell suspension with an insulin needle, wipe the blood vessels on both sides of the mouse's tail with alcohol to fill the blood vessels of the mouse's tail, and insert the needle 3 / 4 of the way from the root of the tail. When inserting the needle, hold the upper end with the index and middle fingers of the left hand, and hold the lower end with the thumb and ring finger. The needle and the blood vessel are about 30°, with the needle tip bevel facing up. After gently piercing the skin, the needle is immediately parallel to the blood vessel. If the liquid is pushed smoothly, it means that the needle is successfully inserted. After injecting 100μl of suspension, press the needle hole with a dry cotton ball to stop bleeding and lift the tail for 30s.

[0093] 2. Different concentrations of Ginkgo Gujin Decoction granules were used to intervene and observe the changes in lung tissue and toxic and side effects in mice.

[0094] Male BALB / c nude mice were randomly divided into 4 groups with 6 mice in each group, including model control group (Control), low-dose Ginkgo Gujin Tang granule group (YXGJD1), high-dose Ginkgo Gujin Tang granule group (YXGJD2) and cisplatin group (Cisplatin). Each mouse was injected with 4×10 7 A549 cells were used to construct a mouse lung metastasis model. After 14 days of modeling, drug treatment was started. The control group was gavaged with normal saline solution every day, and the YXGJD1 and YXGJD2 groups were gavaged with Ginkgo Gujin Tang granules (3.3g / kg and 6.6g / kg) every day, respectively (the dose selection was based on our preliminary experimental results, dose conversion standards and related studies. The low-dose Ginkgo Gujin Tang granule group 3.3g / kg was equivalent to 9 times the human dose, and each gram of granules was equivalent to 9.4g of crude drug). The Cisplatin group was injected with 2mg / kg of cisplatin every other day. Each BALB / c nude mouse was weighed once every 2 days. Finally, euthanasia was performed according to animal welfare ethics. Lung tissues were removed from the mice, photographed and the number of lung nodules was counted, and divided into PBS, 10% neutral formalin, and stored in a -80℃ refrigerator for subsequent experiments.

[0095] After 24 hours of treatment with bouin fixative, white deposits can be seen on the surface of lung tissue. Observe and take photos of mouse lung tissue, and record the number of lung nodules. Then, fix the lung tissue by immersion in 10% neutral formalin. After immersion and fixation of lung tissue in 10% formalin for 24 hours, dehydrate, embed in conventional paraffin, and slice. Place the paraffin sections in an oven and bake at 60°C for 1-2 hours, dewax with conventional xylene and ethanol, stain with hematoxylin for 10 minutes, rinse with sterile water, and wash away the remaining color. After differentiation with 0.7% hydrochloric acid ethanol, rinse with sterile water, the sections turn blue for about 15 minutes, soak in 95% ethanol for 30 seconds, and then stain with alcohol eosin for 30 seconds, dehydrate with different concentrations of ethanol in turn, fully soak in xylene, seal with neutral gum, and randomly shoot 3 fields of view under an optical microscope.

[0096] The observation showed that the morphology of lung tissues in the Control group, YXGJD1 group, YXGJD2 group and Cisplatin group changed, and several irregular growths visible to the naked eye were generated on the surface, either large or small. After 24 hours of treatment with bouin fixative, white deposits were visible on the surface of lung tissues. Compared with the Control group, the white deposits on the surface of the YXGJD1 group, YXGJD2 group and Cisplatin group were reduced. The HE-stained pathological sections of the lung tissues of mice in each group were observed under the microscope. The alveolar and interstitial cancer cells in the lung tissues of the mice in the Control group occupied the normal tissues, lost the normal structure, and most of the alveolar cells were atrophied and arranged in disorder; compared with the Control group, the alveolar morphology of the YXGJD1 group, YXGJD2 group and Cisplatin group was relatively normal, and the area of ​​cell canceration was reduced. The tumor burden of each group of lung tissues was calculated by Image J software. The results showed that compared with the Control group, the average tumor burden of the lung tissues of mice in the YXGJD1, YXGJD2 and Cisplatin groups was reduced, and the difference was statistically significant (P < 0.01). Experimental results: In vivo experiments showed that the effect of Ginkgo Gujin Decoction Granules on lung cancer cell metastasis was concentration gradient dependent, and the difference was statistically significant compared with the control group (P < 0.01). Figure 7 (A) is an in vivo experiment of the Ginkgo Gujin Decoction granules provided in an embodiment of the present invention. Figure 7 (A) The results show the tumor burden of lung tissue in mice in the YXGJD1 (Ginkgo Biloba Granules 3.3 g / kg), YXGJD2 (Ginkgo Biloba Granules 6.6 g / kg) and Cisplatin groups compared with the Control group. Figure 7 (B) is the HE results of lung tissues of mice in each group. Figure 7 (C) is a statistical graph of lung tissue weight of mice in each group. Figure 7 (DF) are statistical graphs of liver and kidney function of mice in each group. Figure 7(G) is a statistical chart of the weight of mice in each group. Blood tests of the liver and kidney functions of mice in each group found that there was no obvious liver and kidney damage in the Ginkgo Gujin Decoction Granule group compared with the control group.

[0097] The metastasis of lung cancer indicates a decrease in survival time and quality of life, and the existing treatment methods have limited efficacy. Traditional Chinese medicine has a multi-target effect. The innovation of the present invention is that it has been found that Ginkgo Gujin Decoction Granules inhibit the invasion and metastasis of lung cancer cells through the TGF-β / STAT3 / PD-L1 signaling pathway. Therefore, the present invention provides a potential new anti-tumor drug for the treatment of lung cancer, which has not been reported in the public literature. The specific mechanism of its anti-cancer effect is clear, which is of great significance for the prevention and treatment of malignant tumors and has broad prospects.

[0098] In summary, the Chinese medicine compound preparation of the present invention has an inhibitory effect on the proliferation of lung cancer cells and increases in a concentration-dependent manner; the inhibitory effect on migration ability increases in a concentration-dependent manner. The compound has good efficacy, is easy to use, safe and has no toxic side effects, and is suitable for all stages of lung cancer. The present invention reveals that while Ginkgo Gujin Decoction Granules have a killing effect on lung cancer tumor cells, they also have a significant inhibitory effect on tumor metastasis and recurrence, and its anti-tumor effect is clear and reliable.

Claims

1. A Chinese medicinal compound preparation for treating lung cancer, characterized in that: The invention is prepared from the following raw materials in parts by weight: 6-9 parts of ginkgo leaves, 6-9 parts of schisandra chinensis, 15-45 parts of astragalus, 9-15 parts of atractylodes macrocephala, 15-30 parts of coix seeds, 15-30 parts of yam, 12-24 parts of cyperus rotundus, 15-30 parts of lithospermum officinale, 15-30 parts of cyperus rotundus and 15-30 parts of oldenlandia diffusa.

2. The Chinese medicinal compound preparation for treating lung cancer according to claim 1, characterized in that: The raw materials and their weight portions are: 9 parts of ginkgo leaves, 9 parts of schisandra chinensis, 30 parts of astragalus, 9 parts of atractylodes macrocephala, 30 parts of coix seeds, 15 parts of yam, 15 parts of cyperus rotundus, 30 parts of lithospermum officinale, 30 parts of cyperus rotundus, and 30 parts of oldenlandia diffusa.

3. The Chinese medicinal compound preparation for treating lung cancer according to claim 1 or 2, characterized in that: The compound preparation is in the form of decoction, granules, tablets, capsules or oral liquid.

4. The Chinese medicinal compound preparation for treating lung cancer according to claim 1 or 2, characterized in that: The lung cancer is non-small cell lung cancer.

5. Use of the Chinese medicinal compound preparation according to claim 1, 2 or 3 in the preparation of a medicine for treating lung cancer.

6. The use according to claim 5, characterized in that The lung cancer was non-small cell lung cancer.

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