Application of drug serum prepared from common clubmoss herb in liver cancer cells

Abstract

The invention discloses an application of medicated serum prepared from common clubmoss herb in liver cancer cells, and belongs to the technical field of medicines. The invention provides the application of the drug serum prepared from the common clubmoss herb in preparation of drugs for inhibiting proliferation and invasion of liver cancer cells and promoting apoptosis of the liver cancer cells. The invention verifies that the herb of common clubmoss herb drug-containing serum can effectively inhibit HepG2 cell proliferation and invasion, promote cell apoptosis, inhibit HepG2 cell mTOR signal channel mTOR protein phosphorylation, up-regulate expression of pro-apoptotic proteins caspase-9, caspase-3 and bax, and down-regulate expression of anti-apoptotic protein bcl-2. According to the application disclosed by the invention, the influence of the medicinal serum of the thunbergia grandiflora on related apoptosis genes in an mTOR molecular signal channel of HepG2 liver cancer cells and proliferation, invasion and apoptosis of the HepG2 liver cancer cells is researched from the aspect of in-vitro experiments, so that an experimental basis is expected to be provided for clinical application.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to application of drug-containing serum prepared from Herba Lycopodii in liver cancer cells. Background Art

[0002] Liver cancer has become one of the common malignant tumors, with high incidence and mortality, which seriously endangers human health. Clinical treatment of liver cancer is mainly based on surgery, chemotherapy, interventional therapy and liver transplantation, but it causes great damage to surrounding tissues and has great side effects. Developing effective drugs to treat the disorder of proliferation and apoptosis is an effective goal of clinical treatment of liver cancer. Studies have found that traditional Chinese medicine has the effects of inhibiting the proliferation of liver cancer cells, inducing apoptosis of liver cancer cells, inhibiting the invasion and metastasis of liver cancer cells, reversing the drug resistance of liver cancer cells and enhancing the body's immunity.

[0003] Sarcopyramis nepalensis Wall is the dried whole herb of Sarcopyramis nepalensis Wall, a plant of the family Melastomataceae. It is a traditional Chinese medicine commonly used by the folk and traditional Chinese medicine in southern Fujian. It has the effects of clearing the liver and purging fire, clearing heat and detoxifying. It is widely used in the folk to treat acute hepatitis and protect the liver. Clinically, it can improve the clinical efficacy in the treatment of acute and chronic hepatitis, cirrhosis and adjuvant treatment of refractory ascites. The mammalian target of rapamycin (mTOR) signaling pathway is widely involved in regulating various biological processes such as cell growth, proliferation, metabolism, autophagy, survival and cell cycle. Its disorder is related to many diseases and is an important target for cancer treatment and metabolic disease intervention. The activation of mTOR signaling is related to the process of promoting cancer cells, making mTOR a promising target for new combination therapy. At present, there is no research on the effect of Sarcopyramis nepalensis Wall-containing serum on the related apoptosis genes in the mTOR molecular signaling pathway of HepG2 liver cancer cells and the proliferation, invasion and apoptosis of HepG2 liver cancer cells. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a medicated serum prepared from Herba Lycopodii in liver cancer cells to solve the problems existing in the above-mentioned prior art. The present invention proves that the medicated serum of Herba Lycopodii can effectively inhibit the proliferation and invasion of HepG2 cells, promote cell apoptosis, and inhibit the phosphorylation of mTOR protein in the mTOR signaling pathway of HepG2 cells, upregulate the expression of pro-apoptotic proteins caspase-9, caspase-3, and bax, and downregulate the expression of anti-apoptotic protein bcl-2.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical solution 1: Application of a drug-containing serum prepared from Herba Lycopodii in the preparation of drugs for inhibiting the proliferation and invasion of liver cancer cells.

[0007] Furthermore, the drug-containing serum is obtained by gavage of the alcohol extract of Herba Lycopodii into mice and then collecting blood.

[0008] The preparation method of the alcohol extract of the herb of Psoralea corylifolia is to dry the whole herb of the herb at low temperature, crush it, reflux extract it with 75% ethanol by volume for 3 times, each time for 1.5 hours, combine the filtrate, recover the ethanol under reduced pressure, and evaporate it in a water bath to obtain the alcohol extract.

[0009] Furthermore, the inhibition of liver cancer cell proliferation and invasion is achieved by upregulating the expression of caspase-9, caspase-3 and bax proteins.

[0010] Technical solution 2: Application of a drug-containing serum prepared from Herba Lycopodii in the preparation of drugs that promote apoptosis of liver cancer cells.

[0011] Furthermore, the drug-containing serum is obtained by gavage of the alcohol extract of Herba Lycopodii into mice and then collecting blood.

[0012] Furthermore, the promotion of liver cancer cell apoptosis is achieved by down-regulating the expression of the anti-apoptotic protein bcl-2.

[0013] Technical solution three: Application of a drug-containing serum prepared from Herba Lycopodii in the preparation of drugs that inhibit the phosphorylation of mTOR.

[0014] Furthermore, the drug-containing serum is obtained by gavage of the alcohol extract of Herba Lycopodii into mice and then collecting blood.

[0015] Furthermore, the liver cancer cells are human liver cancer HepG2 cells.

[0016] The present invention discloses the following technical effects:

[0017] The present invention studies the effect of medicated serum of Herba Eupatoriae on related genes in the mTOR signaling pathway and cell proliferation and apoptosis of HepG2 liver cancer cells. The results show that each medicated serum group of Herba Eupatoriae can reduce the proliferation ability and cell viability of HepG2 cells (P<0.01), inhibit the clone formation rate of HepG2 cells (P<0.01 or P<0.05) and invasion ability, and induce apoptosis of HepG2 cells. The medicated serum group of Herba Eupatoriae can upregulate the expression of caspase-9, caspase-3, bax, p-mTOR / mTOR proteins (P<0.01), downregulate the expression of bcl-2 protein (P<0.01), and has a certain concentration dependence; the medium-dose medicated serum of Herba Eupatoriae combined with rapamycin inhibitor (Rapamycin) can synergistically enhance the expression of caspase-9, caspase-3, bax, p-mTOR / mTOR proteins (P<0.01), and downregulate the expression of bcl-2 protein (P<0.01). In summary, the present invention verifies that the medicated serum containing Herba Eupatoriae can effectively inhibit the proliferation and invasion of HepG2 cells, promote cell apoptosis, and inhibit the phosphorylation of mTOR protein in the mTOR signaling pathway of HepG2 cells, upregulate the expression of pro-apoptotic proteins caspase-9, caspase-3, and bax, and downregulate the expression of anti-apoptotic protein bcl-2. The present invention studies the effect of the medicated serum containing Herba Eupatoriae on the related apoptotic genes in the mTOR molecular signaling pathway of HepG2 liver cancer cells and the proliferation, invasion, and apoptosis of HepG2 liver cancer cells from the aspect of in vitro experiments, in order to provide an experimental basis for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Photos of the plate cloning experiment of each dose group (A) and their statistics (B); n = 6; **P < 0.01 compared with the control group;

[0020] Figure 2 The green fluorescence expression of cells in each dose group (A) and the statistical effect of the drug-containing serum in the high-dose group on the viability of HepG2 cells at different time points (B); n = 6, compared with the control group, **P < 0.01;

[0021] Figure 3 Results of the effects of drug-containing serum in each dose group on HepG2 cell viability; n = 6, compared with the control group, **P < 0.01;

[0022] Figure 4 The results of the effects of drug-containing serum in the high-dose group on HepG2 cell viability at different times; n = 6, compared with the control group, **P < 0.01;

[0023] Figure 5 Results of cell invasion experiments in each dose group; n = 6 (200 times magnification);

[0024] Figure 6 The statistics of cell invasion experiment in each dose group; n = 6, compared with the control group, **P < 0.01;

[0025] Figure 7 The statistical results of the drug-containing serum in each dose group on the apoptosis of HepG2 cells; n = 6, compared with the control group, **P < 0.01;

[0026] Figure 8 The results of the AnnexinV-FITC / PI double staining experiment on HepG2 cell apoptosis by drug-containing serum in the control group; n=6;

[0027] Fig. 9 The results of the AnnexinV-FITC / PI double staining experiment on HepG2 cell apoptosis by drug-containing serum in the blank serum group; n=6;

[0028] Fig.10 The results of the AnnexinV-FITC / PI double staining experiment on HepG2 cell apoptosis by the low-dose group drug-containing serum; n=6;

[0029] Fig.11 The results of the AnnexinV-FITC / PI double staining experiment on HepG2 cell apoptosis by the drug-containing serum in the medium-dose group; n=6;

[0030] Fig.12 The results of the AnnexinV-FITC / PI double staining experiment on HepG2 cell apoptosis by drug-containing serum in the high-dose group; n=6;

[0031] Fig.13 Statistical results of the effects of drug-containing serum on the protein expression of Caspase-9 (A), Caspase-3 (B), Bax (C) and Bcl-2 (D) in HepG2 cells and Western blot results (E); n = 6, **P < 0.01 compared with the control group;

[0032] Fig.14 Statistical results of the effect of drug-containing serum on the expression of mTOR (A) and p-mTOR (B) proteins in HepG2 cells and Western blot results (C); n = 6, **P < 0.01 compared with the control group;

[0033] Fig.15 Statistical data and Western blot results (E) of the effects of drug-containing serum combined with Rapamycin on the expression of Caspase-9 (A), Caspase-3 (B), Bax (C) and Bcl-2 (D) proteins in HepG2 cells; n=6, **P<0.01 compared with the control group;

[0034] Fig.16 Statistical results of the effects of drug-containing serum combined with Rapamycin on the expression of mTOR (A) and p-mTOR (B) proteins in HepG2 cells and Western blot detection results (C); n=6, **P<0.01 compared with the control group. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] The invention divides in vitro cultured liver cancer HepG2 cells into a control group, a high-dose, a medium-dose and a low-dose group of medicated serum of Herba Lycopodii, adopts CCK-8 and cell clone formation to detect the proliferation of HepG2 cells, adopts FDA staining to observe the viability of HepG2 cells, adopts Transewell to detect the invasion ability of HepG2 cells; adopts Annexin V-FITC / PI double staining to detect the apoptosis of HepG2 cells; and adopts Western blot to detect the expression levels of Caspase-9, Caspase-3, Bax, Bcl-2, mTOR and p-mTOR proteins in each group of cells, so as to study the influence of medicated serum of Herba Lycopodii on related genes in the mTOR signaling pathway of liver cancer HepG2 cells and cell proliferation and apoptosis.

[0041] Example 1

[0042] 1. Instruments and reagents

[0043] 1.1 Main drugs and reagents

[0044] Fengguidoucao was purchased from the medicinal material market in Zhangzhou, Fujian Province, and was identified as Sarcopyramis nepalensis Wall, a plant of the Melastomataceae family, by Professor Teng Jianbei of Guangxi University of Chinese Medicine. After low-temperature drying, it was crushed and refluxed with 75% ethanol for 3 times, each time for 1.5 hours. The filtrates were combined, the ethanol was recovered under reduced pressure, and the extract was evaporated in a water bath to obtain a standby extract.

[0045] DMEM medium (Gibco, batch number: 8118189); PBS buffer (Wuhan Google Biotechnology Co., Ltd., batch number: 190123); BSA (Biyuntian Biotechnology Co., Ltd.); methanol (Nanjing Chemical Reagent Co., Ltd., batch number: 190423445K); 5× protein loading buffer (Tiangen Biochemical Technology (Beijing) Co., Ltd., batch number: 190218); pre-stained marker (Thermo, batch number: 18K14); skim milk powder (BioFroxx, batch number: EZ2811C382); developer (New Saimei Biotechnology Co., Ltd., batch number: 190120115); polyclonal antibodies Caspase 3, Caspase 9, BAX, BCL2, mTOR, GAPDH, Goat Anti-Rabbit IgG (H+L) HRP conjugate (Proteintech, catalog numbers: 19677-1-AP, 10380-1-AP, 50599-2-Ig, 12789-1-AP, 20657-1-AP, 10494-1-AP, SA00001-2); Anti-mTOR (phospho S2481) (abcam, catalog number: ab137133).

[0046] 1.2 Experimental animals and cells

[0047] SPF grade KM mice, half male and half female, 18-22g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., license number: SCXK Xiang 2016-0002, animal qualification certificate number: 430727231102020062. The experimental animals were kept in an SPF barrier facility equipped with a high-efficiency air filter (HEPA), the temperature was maintained at 20-24°C, the relative humidity was 40-70%, and the light cycle was 12 hours of light / 12 hours of darkness. The animals were kept in independent ventilated cages (IVC), and sterilized feed and drinking water were provided unlimitedly, and the bedding was changed twice a week. This experiment was approved by the Experimental Animal Center of Guangxi University of Chinese Medicine, and the ethical approval number is: DW20240919-326. HepG2 cells were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences. The cell line was cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 Culture in an incubator.

[0048] 1.3 Main instruments

[0049] High-speed low-temperature centrifuge (Thermo Fisher Scientific, model: MicroCL 17R); analytical balance (Serdorisco Instrument (Beijing) Co., Ltd., model: BSA124S); ultra-low temperature refrigerator (Siemens, Japan, model: KK24T18TI); continuous wavelength microplate reader (TECAN, Switzerland, model: infinite M200pro); fluorescence microscope (Nikon, Japan, model: TE2000-U); multifunctional molecular imager (Jena, Germany, model: UVP ChemStudio815); universal electrophoresis instrument (Bio-Rad, USA, model: PowerPac).

[0050] 2 Experimental methods

[0051] 2.1 Preparation of serum containing ethanol extract of Herba Lysimachiae

[0052] 40 SPF Kunming mice were randomly divided into a blank group, a high-dose, a medium-dose, and a low-dose group of ethanol extract of Psoralea corylifolia (dose was 8.8, 4.4, and 2.2 g of crude drug / kg, respectively), with 10 mice in each group. Each group of mice was given the drug by gavage (20 mL / kg), twice a day, for 5 days, and the blank group was replaced with normal saline. Before the last administration, the mice were fasted and did not take water for 12 hours, anesthetized 1 hour later, blood was collected from the eyeball, left to stand at 4°C for 1 hour, centrifuged at 3000 r / min for 10 minutes, and the upper serum was taken and placed in a 56°C water bath for inactivation for 30 minutes, filtered and sterilized with a 0.22 μm microporous filter, mixed according to the grouping, and stored at -80°C for use.

[0053] 2.2 Detection of the inhibitory rate of HepG2 cell proliferation by serum containing herba syringae pumila by CCK-8 method

[0054] HepG2 cells in the logarithmic growth phase were obtained and digested with trypsin. Single-cell suspension was prepared in DMEM medium containing 10% fetal bovine serum at a concentration of 5×10 4 Each well was inoculated with 100 μL / well of 100 μL / mL and inoculated in a 96-well plate. After the cells adhered to the wall, they were divided into a control group, a blank serum group, and a high, medium, and low dose group of drug-containing serum of Fenggui Doucao. Each group had 6 duplicate wells and was cultured in a cell culture incubator for 24 hours. Except for the control group, each well was added with 100 μL of 10% FBS complete medium. The remaining groups were added with 100 μL of drug-containing serum or blank mouse serum and complete medium mixture (final serum concentration was 20%), and continued to be cultured for 24 hours, 48 ​​hours, and 72 hours. The original culture solution was gently sucked off, and 100 μL of culture medium containing 10% CCK-8 solution was added to each well, and the cells were placed in an incubator and incubated again for 2 hours. The absorbance (OD value) of each group of cells was detected by an enzyme marker, and the cell proliferation inhibition rate was calculated. Inhibition rate (%) = (1-average OD value of the drug group / average OD value of the control group) × 100%.

[0055] 2.3 FDA method to determine the effect of serum containing Herba Lycopodii on HepG2 cell viability

[0056] After the cells were treated with drugs for 24 hours as described in 2.2, 100 μL of 5 μg / mL FDA / PBS solution was added to each well and incubated for 10 minutes. The liquid in the well was discarded, and PBS buffer was added to rinse twice. Then, photos were taken under a fluorescence microscope to observe the cell viability of HepG2 cells after 0, 6, 12, 24, and 48 hours of action of the drug-containing serum of Fengguidoucao. Six fields of view were randomly selected for photography, and the fluorescence values ​​were finally analyzed using Image J software to calculate the average fluorescence value of each group.

[0057] 2.4 Effect of serum containing Herba Lycopodii on HepG2 cell clone formation

[0058] Take HepG2 cells in the logarithmic growth phase, digest them with trypsin, prepare a single cell suspension, take 5 mL of each inoculated culture dish, and the number of cells in each dish is 300. Put it in the cell culture incubator and culture it for 24 hours. After the cells adhere to the wall, add 5 mL of the mixture of drug-containing serum and culture medium (the final concentration of drug-containing serum is 20%), add culture medium to the control group, and continue to culture for 24 hours, 48 ​​hours, and 72 hours. When visible clones appear in the culture dish, rinse with PBS twice and fix with 4% paraformaldehyde for 10 minutes. Discard the fixative, add an appropriate amount of 10% Giemsa staining solution for 30 minutes, wash off the staining solution, and after drying, place the culture dish under a microscope, randomly select six fields of view, and count the number of cell clones.

[0059] 2.5 Transwell chamber invasion assay to detect the effect of serum containing Herba Lycopodii on the invasion ability of HepG2 cells

[0060] HepG2 cells in the logarithmic growth phase were collected and 2×10 5 The cells were seeded into the transwell chamber of a 24-well plate. The experimental groups were the same as above. Different treatment drugs were added to the upper chamber, and complete culture medium was added to the lower chamber. The chamber was returned to the incubator. After 24 hours of intervention, the upper chamber was fixed with 4% paraformaldehyde for 15 minutes, stained with 1% crystal violet for 1 hour, rinsed with tap water, and dried. Three randomly selected areas of each chamber were photographed under a microscope, and the number of cells that passed through the matrix gel to reach the upper chamber membrane was calculated using ImageJ 1.8.0 software.

[0061] 2.6 Annexin V-FITC / PI double staining method to detect the effect of serum containing Herba Lycopodii on the apoptosis of HepG2 cells

[0062] HepG2 cells were treated with the drug-containing serum of Herba Lysimachiae for 72 h according to the grouping method in 2.2, and then digested with trypsin, resuspended in 1×PBS precooled at 4°C, centrifuged at 1000-2000 rpm for 5 min, the supernatant was discarded, and 195 μL of binding solution was added to resuspend the cells. 1×10 cells were taken from each well. 5 Then, 5 μL Annexin V-FITC and 10 μL PI were added and evenly mixed. The mixture was reacted in a dark room for 20 min and the cell apoptosis was detected by flow cytometry.

[0063] 2.7 Western blot method to detect the effect of serum containing herba syringae on the expression of proteins related to mTOR signaling pathway

[0064] HepG2 cells were cultured at a concentration of 1×10 5 / mL, inoculated in 6-well plates, experimental grouping as above, cells adhered to the wall and acted on the drug for 24h, total protein was extracted by protein lysis buffer, total protein concentration was determined by BCA method, and protein was denatured in a 95℃ metal bath. Protein samples were loaded with 10μL / well, electrophoresed, transferred to the membrane, blocked with 5% skim milk powder in TBST solution for 2h, primary antibodies (GAPDH, Caspase-3, Caspase-9, Bax, Bcl-2, mTOR, p-mTOR) were added, incubated overnight at 4℃, primary antibodies were collected, rinsed 3 times with TBST, incubated with secondary antibodies at room temperature for 1h, rinsed 3 times with TBST, PVDF membrane was developed with ECL chemiluminescent substrate, and grayscale values ​​of Caspase-3, Caspase-9, Bax, Bcl-2, mTOR, and p-mTOR protein bands were analyzed using ImageJ2 (version 2.1.4.7) software.

[0065] 2.8 Western blot analysis of the effect of serum containing Herba Lysimachiae combined with mTOR inhibitors on the expression of proteins related to the mTOR signaling pathway

[0066] HepG2 cells were inoculated in 6-well plates and cultured for 24 hours. After the cells attached to the wall, they were divided into a control group (normal saline), a medium-dose group containing serum of Herba Eupatorii (4.4 g crude drug / kg), an mTOR inhibitor Rapamycin (10 nmol / well), and a medium-dose group containing serum of Herba Eupatorii (4.4 g crude drug / kg) + mTOR inhibitor Rapamycin (10 nmol / well). After 24 hours of intervention, the grayscale values ​​of Caspase-3, Caspase-9, Bax, Bcl-2, mTOR, and p-mTOR protein bands in each group were detected.

[0067] 2.9 Statistical methods

[0068] SPSS 22.0 software was used to analyze the data. P<0.05 was considered statistically significant. Two independent sample t-tests were used for pairwise comparisons between groups, and SNK tests were used for pairwise comparisons between multiple groups.

[0069] 3 Experimental results

[0070] 3.1 Effect of serum containing Herba Lycopodii on proliferation of HepG2 cells

[0071] The results of the cell proliferation experiment showed (Table 1) that, compared with the control group, the serum groups containing Herba Lycopodii inhibited the proliferation of HepG2 (P<0.01), and were positively correlated with the drug concentration.

[0072] Table 1 Results of the inhibition of HepG2 cell proliferation by drug-containing serum (n=6)

[0073] Group Dosage (g / kg) OD value Inhibition rate (%) Control group -- 0.88±0.05 -- Blank group -- 0.85±0.08 3.81 High dose group 8.8 0.39±0.06** 57.76 Medium dose group 4.4 0.57±0.07** 35.34 Low dose group 2.2 0.71±0.06** 30.47

[0074] Note: **P<0.01 compared with the control group.

[0075] Results of clone formation experiments ( Figure 1 ) showed that the average clone numbers of the control group and the drug-containing serum groups of Fengguidoucao were (224.33), (147.67), (89.67), and (49.00), respectively. The variance analysis showed significant differences in the clone numbers among the four groups (P<0.01), and was positively correlated with the drug concentration.

[0076] 3.2 Effect of serum containing Herba Lycopodii on the viability of HepG2 cells

[0077] The results of cell viability experiments showed that ( Figure 2-Figure 4 ), compared with the blank group, the green fluorescence expression of cells in each Herba Lycopodii drug-containing serum group was weakened, which could inhibit cell viability and was positively correlated with the drug concentration; the Herba Lycopodii drug-containing serum high-dose group showed that the green fluorescence intensity of HepG2 cells gradually weakened from 0 to 48 h, indicating that the inhibition of drug on cell viability was positively correlated with the action time.

[0078] 3.4 Effect of Herba Lycopodii containing serum on the invasion ability of HepG2 cells

[0079] The results of cell invasion assay showed that ( Figure 5 and Figure 6 ), the number of HepG2 cells that passed through the bottom of the chamber membrane in the control group and the blank group was large, indicating that the cells had strong invasion ability. After the treatment of each drug-containing serum group of Fengguidoucao, the number of HepG2 cells that passed through the bottom of the chamber membrane decreased, indicating that the drug-containing serum had a certain inhibitory effect on the invasion ability of HepG2.

[0080] 3.5 Effect of serum containing Herba Lycopodii on apoptosis of HepG2 cells

[0081] The results of the Annexin V-FITC / PI double staining experiment showed that the apoptosis rates of HepG2 cells in the control group and the high, medium and low levels of Psoralea corylifolia were 0.185%, 33.04%, 36.90% and 40.59%, respectively, and there were significant differences among the groups (P<0.01)( Figure 7-Figure 12 ).

[0082] 3.6 Effect of Herba Lycopodii Medicinal Serum on the Expression of Proteins Related to the mTOR Signaling Pathway

[0083] Western blot results showed that ( Fig.13 and Fig.14 ), compared with the control group, the serum groups containing Herba Lycopodii could up-regulate the expression of Caspase-9, Caspase-3, and Bax proteins in HepG2 cells (P<0.01), down-regulate the expression of Bcl-2 protein (P<0.01), and inhibit the expression of p-mTOR protein in HepG2 cells (P<0.01), and the expression was positively correlated with the dose.

[0084] 3.7 Effects of Herba Lycopodii-containing serum combined with mTOR inhibitors on the expression of proteins related to the mTOR signaling pathway

[0085] Western blot results showed that ( Fig.15 and Fig.16 ), compared with the control group, the herbaceous medicated serum group, the rapamycin group, and the herbaceous medicated serum + rapamycin combined group could up-regulate the expression of Caspase-9, Caspase-3, and Bax proteins in HepG2 cells, down-regulate the expression of Bcl-2 protein, and inhibit the expression of p-mTOR protein in the mTOR signaling pathway (P<0.01). The combined drug group had a stronger effect than the herbaceous medicated serum and rapamycin alone.

[0086] The present invention adopted CCK-8 method, FDA fluorescence method and cell clone formation method to preliminarily detect the proliferation activity of HepG2 cells by serum containing Herba Lycopodii, and transewell method to detect the cell invasion ability. The experimental results showed that the serum containing Herba Lycopodii could inhibit the proliferation and invasion of liver cancer cells in a dose-dependent manner (P<0.01 or P<0.05).

[0087] Inducing cell apoptosis is considered to be a potential strategy for treating liver cancer. The experimental results of the Annexin V-FITC / PI double staining method of the present invention show that the serum containing Herba Lycopodii induced apoptosis of HepG2 cells in a concentration-dependent manner.

[0088] The PI3K / AKT signaling pathway is a classic cell proliferation and anti-apoptosis pathway, which is significantly activated in liver cancer. A variety of anti-tumor drugs inhibit the proliferation and apoptosis of liver cancer cells by inhibiting the PI3K / AKT pathway, and the mTOR protein is a driving molecule in the downstream of the PI3K / AKT signal transduction pathway. It is an important eukaryotic cell signal and plays a key role in regulating the growth, apoptosis, autophagy, and protein translation of tumor cells. After mTOR phosphorylation is activated, it will inhibit the phosphorylation of Beclin-1 by downregulating ubiquitin expression proteins, thereby inhibiting cell apoptosis. In the present invention, it is found that after the Fenggui Doucao drug-containing serum acts on liver cancer cells, the phosphorylation of mTOR is inhibited, resulting in a decrease in the expression of p-mTOR, which in turn leads to an increase in the expression of downstream ubiquitin proteins, thereby promoting apoptosis of liver cancer cells. The group verification experiment of the present invention (control group, medium-dose group containing serum of Herba Lycopodii, mTOR inhibitor Rapamycin (10nmol / well), medium-dose group containing serum of Herba Lycopodii + mTOR inhibitor Rapamycin (10nmol / well)) proved that the serum containing Herba Lycopodii has the effect of inhibiting the phosphorylation of mTOR.

[0089] The Bcl-2 protein family is divided into pro-apoptotic proteins and anti-apoptotic proteins according to their functions. The interaction between the two restricts cell apoptosis. Bcl-2 companion X protein (Bax) is a pro-apoptotic protein that regulates cell mitochondrial apoptosis. When Bax is activated, it can promote mitochondrial outer membrane permeability (MOMP), diffuse mitochondrial membrane gap protein cytochrome C, activate apoptotic protease activating factor (APAF-1), form a Caspase activation platform, and promote the activation of cell apoptosis initiator caspase-9, and then activate the downstream execution factors of the cascade reaction caspase-3 and caspase-7, cut the cell substrate and cause cell apoptosis. The Caspase family is a class of cysteine ​​proteases that can highly specifically break the peptide bond after the aspartic acid residue, thereby affecting the activity function of the protein. Among them, Caspase-3 functional typing is a cell apoptosis execution factor, which is located in the downstream of the cascade reaction and is the main effector in the process of cell apoptosis. Under normal circumstances, it exists in the form of zymogen and can be activated by the upstream cell apoptosis initiator Caspase-9 of the cascade reaction. When Caspase-3 is activated, cleaved Caspase-3 is formed, which inactivates the DNA repair enzyme-poly [ADP-ribose] polymerase 1 (PARP-1) by shearing, thereby leading to cell apoptosis. The anti-apoptotic protein Bcl-2 inhibits the activity of the pro-apoptotic protein Bax by forming a heterodimer with Bax, and can also promote cell survival by anti-oxidation and regulating calcium ion concentration. The Western blot results of the present invention show that the drug-containing serum of Fenggui Doucao activates the downstream Caspase cascade protein family by upregulating Bax protein and downregulating Bcl-2 protein expression, thereby promoting the apoptosis of liver cancer cells.

[0090] In summary, the serum containing Herba Lycopodii can inhibit the proliferation activity of HepG2 cells and induce apoptosis. Its mechanism of action is related to upregulating the expression of pro-apoptotic proteins caspase-9, caspase-3, and bax in HepG2 cells, downregulating the expression of anti-apoptotic protein bcl-2, and inhibiting the phosphorylation expression of mTOR protein in the mTOR signaling pathway. Moreover, the combined use of the mTOR inhibitor Rapamycin has a better effect than using either drug alone.

[0091] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a medicated serum prepared from Herba Lycopodii in the preparation of a drug for inhibiting the proliferation and invasion of liver cancer cells.

2. The use according to claim 1, characterized in that: The drug-containing serum is obtained by gavage of the alcohol extract of Herba Lycopodii into mice and then collecting blood.

3. The use according to claim 1, characterized in that: The inhibition of liver cancer cell proliferation and invasion is achieved by up-regulating the expression of caspase-9, caspase-3 and bax proteins.

4. Application of a medicated serum prepared from Herba Lycopodii in the preparation of a drug for promoting apoptosis of liver cancer cells.

5. The use according to claim 4, characterized in that: The drug-containing serum is obtained by gavage of the alcohol extract of Herba Lycopodii into mice and then collecting blood.

6. The use according to claim 4, characterized in that: The promotion of liver cancer cell apoptosis is achieved by down-regulating the expression of anti-apoptotic protein bcl-2.

7. Use of a medicated serum prepared from Herba Lycopodii in the preparation of a drug for inhibiting the phosphorylation of mTOR.

8. The use according to claim 7, characterized in that: The drug-containing serum is obtained by gavage of the alcohol extract of Herba Lycopodii into mice and then collecting blood.

9. The use according to any one of claims 1 to 8, characterized in that: The liver cancer cells are human liver cancer HepG2 cells.

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