Application of drug-containing serum prepared from Herba Lycopodii in liver cancer cells

Abstract

The present invention discloses an application of a medicated serum prepared from Herba Lycopodii in liver cancer cells, and belongs to the field of medical technology. The present invention provides a medicated serum prepared from Herba Lycopodii in the preparation of a drug for inhibiting the proliferation and invasion of liver cancer cells, and promoting apoptosis of liver cancer cells. The present invention verifies that the Herba Lycopodii medicated serum 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 effects of the Herba Lycopodii medicated serum on 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 from the perspective of in vitro experiments, in order to provide an experimental basis for clinical application.

Description

Technical Field

[0001] The present 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 a common malignant tumor with high incidence and mortality, posing a serious threat to human health. Clinical treatments for liver cancer primarily rely on surgery, chemotherapy, interventional therapy, and liver transplantation, but these methods can cause significant damage to surrounding tissues and severe side effects. Developing effective drugs to treat dysregulation of proliferation and apoptosis is a promising goal in the clinical treatment of liver cancer. Studies have shown that traditional Chinese medicine (TCM) can inhibit liver cancer cell proliferation, induce apoptosis, inhibit invasion and metastasis, reverse drug resistance, and enhance immunity.

[0003] Sarcopyramis nepalensis Wall, the dried whole herb of the Melastomataceae family, is a traditional Chinese herbal medicine used in folk and traditional Chinese medicine in southern Fujian. It has the effects of clearing the liver and purging fire, clearing heat and detoxifying, and is widely used in folk medicine to treat acute hepatitis and protect the liver. Clinically, it has been shown to improve clinical efficacy as an adjunctive treatment for acute and chronic hepatitis, cirrhosis, and refractory ascites. The mammalian target of rapamycin (mTOR) signaling pathway is widely involved in regulating multiple biological processes, including cell growth, proliferation, metabolism, autophagy, survival, and the cell cycle. Its dysregulation is associated with numerous diseases and is an important target for cancer therapy and metabolic disease intervention. Activation of mTOR signaling is associated with cancer-promoting processes, making mTOR a promising target for new combination therapies. Currently, the effects of serum containing Sarcopyramis nepalensis on apoptosis-related genes in the mTOR molecular signaling pathway and on the proliferation, invasion, and apoptosis of HepG2 liver cancer cells remain unknown. Summary of the Invention

[0004] The present invention aims to provide a medicated serum prepared from Herba Eupatorii for use in liver cancer cells to address the problems of the prior art. The present invention demonstrates that Herba Eupatorii medicated serum 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 the 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 Chinese yew herb is to dry the whole Chinese yew 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 filtrates, recover the ethanol under reduced pressure, and evaporate it to dryness 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 downregulating the expression of the anti-apoptotic protein bcl-2.

[0013] Technical solution three: Use 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 studied the effects of medicated serum containing Herba Eupatorii on related genes in the mTOR signaling pathway and cell proliferation and apoptosis in HepG2 liver cancer cells. The results showed that all medicated serum groups containing Herba Eupatorii could 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 containing Herba Eupatorii could upregulate the expression of caspase-9, caspase-3, bax, and p-mTOR / mTOR proteins (P<0.01), and downregulate the expression of bcl-2 protein (P<0.01), and there was a certain concentration dependence; the medium-dose medicated serum containing Herba Eupatorii combined with rapamycin inhibitor (Rapamycin) could synergistically enhance the expression of caspase-9, caspase-3, bax, and 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 Fengguidoucao 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 Fengguidoucao on 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 from 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

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

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

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

[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, **P < 0.01 compared with the control group;

[0023] Figure 5 The cell invasion assay results of each dose group; n = 6 (200x microscope);

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

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

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

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

[0029] Figure 11 Results of the Annexin V-FITC / PI double staining assay of HepG2 cell apoptosis by the drug-containing serum in the medium-dose group; n=6;

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

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

[0032] Figure 14 Statistical analysis of the effects 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] Figure 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] Figure 16 Statistical data and Western blot results (C) of the effects of drug-containing serum combined with Rapamycin on the expression of mTOR (A) and p-mTOR (B) proteins in HepG2 cells; 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be 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 present invention divides in vitro cultured liver cancer HepG2 cells into a control group and high-, medium- and low-dose groups of Herba Lycopodii medicated serum, uses CCK-8 and cell clone formation to detect HepG2 cell proliferation, uses FDA staining to observe HepG2 cell viability, and uses Transewell to detect HepG2 cell invasion ability; uses Annexin V-FITC / PI double staining to detect HepG2 cell apoptosis; and uses Western blot to detect the expression levels of Caspase-9, Caspase-3, Bax, Bcl-2, mTOR and p-mTOR proteins in cells of each group, so as to study the effect of Herba Lycopodii medicated serum on related genes in the mTOR signaling pathway and cell proliferation and apoptosis of liver cancer HepG2 cells.

[0041] Example 1

[0042] 1. Instruments and reagents

[0043] 1.1 Main drugs and reagents

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

[0045] Duplex medium (Gibco, lot number: 8118189); PBS buffer (Wuhan Google Biotechnology Co., Ltd., lot number: 190123); BSA (Biyuntian Biotechnology Co., Ltd.); methanol (Nanjing Chemical Reagent Co., Ltd., lot number: 190423445K); 5× protein loading buffer (Tiangen Biochemical Technology (Beijing) Co., Ltd., lot number: 190218); prestained marker (Thermo, lot number: 18K14); skim milk powder (BioFroxx, lot number: EZ2811C382); developer (New Saimei Biotechnology Co., Ltd., lot number: 190120115); polyclonal antibodies to Caspase 3, Caspase 9, BAX, BCL2, mTOR, GAPDH, and 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, weighing 18-22 g, were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd., license number: SCXK-Xiang 2016-0002, animal qualification certificate number: 430727231102020062. Experimental animals were housed in an SPF-grade barrier facility equipped with a high-efficiency air filter (HEPA), maintained at a temperature of 20-24°C, a relative humidity of 40-70%, and a photoperiod of 12 h light / 12 h dark. Animals were housed in individually ventilated cages (IVCs) and provided free access to sterilized feed and purified drinking water. Bedding was changed twice weekly. This experiment was approved by the Experimental Animal Center of Guangxi University of Chinese Medicine, ethics approval number: DW20240919-326. HepG2 cells were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences and cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 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 freezer (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 Eupatorii

[0052] Forty SPF Kunming mice were randomly divided into a blank group and high-, medium-, and low-dose groups (8.8, 4.4, and 2.2 g of the raw herb / kg, respectively) of ethanol extract from Psoralea corylifolia (10 mice per group). Each group received the drug by gavage (20 mL / kg) twice daily for 5 days, while the blank group received saline instead. Mice were fasted for 12 hours before the final dose. One hour after the final dose, blood was collected from the eyeballs, incubated at 4°C for 1 hour, and centrifuged at 3000 rpm for 10 minutes. The upper serum layer was collected and inactivated in a 56°C water bath for 30 minutes. The serum was sterilized by filtration through a 0.22 μm microporous filter, mixed according to grouping, and stored at -80°C until use.

[0053] 2.2 Detection of the inhibitory rate of HepG2 cell proliferation by serum containing Herba Lycopodii var.

[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. 4 Cells were inoculated into 96-well plates at a concentration of 100 μL / well. After cell attachment, the cells were divided into a control group, a blank serum group, and high-, medium-, and low-dose groups of medicated serum containing 100 μL of 10% FBS complete medium. Each group was cultured in a cell culture incubator for 24 hours. Except for the control group, which received 100 μL of 10% FBS complete medium per well, all other groups received 100 μL of a mixture of medicated serum or blank mouse serum and complete medium (final serum concentration was 20%). Cultures were continued for 24, 48, and 72 hours. The original culture medium was gently aspirated, and 100 μL of culture medium containing 10% CCK-8 solution was added to each well. The cells were then incubated again in the incubator for 2 hours. The absorbance (OD value) of each group of cells was measured by a microplate reader, and the cell proliferation inhibition rate was calculated. Inhibition rate (%) = (1-mean OD value of the drug group / mean OD value of the control group) × 100%.

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

[0056] After 24 hours of drug treatment as described in 2.2, 100 μL of a 5 μg / mL FDA / PBS solution was added to each well and incubated for 10 minutes. The liquid in the wells was discarded and the cells were rinsed twice with PBS buffer. Cell viability of HepG2 cells was observed under a fluorescence microscope after 0, 6, 12, 24, and 48 hours of treatment with the drug-containing serum. Six randomly selected fields of view were photographed, and fluorescence values ​​were analyzed using Image J software. The mean fluorescence value for each group was calculated.

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

[0058] Take HepG2 cells in the logarithmic growth phase, digest them with trypsin, and prepare a single cell suspension. Take 5 mL of each culture dish and inoculate 300 cells per dish. Place in a cell culture incubator and culture for 24 hours. After the cells adhere to the wall, add 5 mL of a 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 twice with PBS and fix with 4% paraformaldehyde for 10 minutes. Discard the fixative, add an appropriate amount of 10% Giemsa staining solution and stain for 30 minutes. Wash off the staining solution. 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 syringa syringae-containing serum on the invasion ability of HepG2 cells

[0060] HepG2 cells in the logarithmic growth phase were collected and 2×10 5 Cells were seeded into 24-well transwell chambers. 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 chambers were returned to the incubator. After 24 hours of intervention, the upper chambers were 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 ImageJ 1.8.0 software was used to count the number of cells that passed through the matrix gel and reached the upper chamber membrane.

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

[0062] HepG2 cells were treated with Herba Hedyotis diffusae serum according to grouping in 2.2 for 72 h, digested with trypsin, resuspended in 4°C pre-cooled 1× PBS, centrifuged at 1000-2000 rpm for 5 min, the supernatant discarded, and resuspended in 195 μL binding buffer. 1×10 cells were collected per well. 5 Then, 5 μL Annexin V-FITC and 10 μL PI were added to the cells and evenly mixed. The cells were reacted in a dark room for 20 min and the cell apoptosis was detected by flow cytometry.

[0063] 2.7 Western blot analysis of the effect of serum containing Herba Lycopodii on the expression of proteins related to the mTOR signaling pathway

[0064] HepG2 cells were cultured at a concentration of 1 × 10 5 Cells were plated at 400 μg / mL and seeded in 6-well plates. Experimental grouping was the same as above. After cell attachment, the cells were treated with drugs for 24 h. Total protein was extracted using protein lysis buffer and determined by the BCA assay. Proteins were denatured in a 95°C metal bath. Protein samples were loaded at 10 μL / well for electrophoresis and transferred to the membrane. The membrane was blocked with 5% skim milk powder in TBST for 2 h and then incubated overnight at 4°C. Primary antibodies were collected, the membranes were rinsed three times with TBST, and then incubated with secondary antibodies at room temperature for 1 h. The membranes were then rinsed three times with TBST. ECL chemiluminescent substrate was added to the PVDF membrane for development, and the 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 effects of Herba Lycopodii medicated serum combined with mTOR inhibitors on the expression of proteins related to the mTOR signaling pathway

[0066] HepG2 cells were inoculated into 6-well plates and cultured for 24 hours. After the cells adhered to the wall, they were divided into a control group (normal saline), a medium-dose herbaceous medicated serum group (4.4 g crude drug / kg), an mTOR inhibitor Rapamycin (10 nmol / well), and a medium-dose herbaceous medicated serum group (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 perform statistical analysis on 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 Herba Lycopodii-containing serum on HepG2 cell proliferation

[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 Inhibition results 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 cell clone numbers in the control group and the Fengguidoucao drug-containing serum groups were (224.33), (147.67), (89.67), and (49.00), respectively. Variance analysis showed significant differences in the clone numbers among the four groups (P<0.01) and were positively correlated with drug concentrations.

[0076] 3.2 Effect of Herba Lycopodii-containing serum on HepG2 cell viability

[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 hours, indicating that the drug's inhibition 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 cell membrane bottom was higher in the control group and blank group, indicating that the cells had strong invasive ability. After treatment with the various sera containing the herb, the number of HepG2 cells that passed through the cell membrane bottom decreased, indicating that the sera contained a certain inhibitory effect on the invasive ability of HepG2.

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

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

[0082] 3.6 Effect of Herba Lycopodii-containing serum on the expression of proteins related to the mTOR signaling pathway

[0083] Western blot test results showed that ( Figure 13 and Figure 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 medicated serum combined with mTOR inhibitors on the expression of proteins related to the mTOR signaling pathway

[0085] Western blot test results showed that ( Figure 15 and Figure 16 Compared with the control group, the herbaceous medicated serum group, the rapamycin group, and the herbaceous medicated serum + rapamycin combination group all upregulated the expression of Caspase-9, Caspase-3, and Bax proteins in HepG2 cells, downregulated the expression of Bcl-2 protein, and inhibited the expression of p-mTOR protein in the mTOR signaling pathway (P<0.01). The combined drug group had a stronger effect than either herbaceous medicated serum or rapamycin alone.

[0086] The present invention used CCK-8 method, FDA fluorescence method and cell clone formation method to preliminarily detect the proliferation activity of HepG2 cells by the medicated serum containing Herba Lycopodii, and transewell method to detect the cell invasion ability. The experimental results showed that the medicated 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 a potential strategy for treating liver cancer. The results of the Annexin V-FITC / PI double staining assay of the present invention showed that 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. 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 inhibits the phosphorylation of Beclin-1 by downregulating ubiquitin expression proteins, thereby inhibiting cell apoptosis. In the present invention, it was found that after the Fenggui Doucao drug-containing serum acted on liver cancer cells, the phosphorylation of mTOR was inhibited, resulting in a decrease in the expression of p-mTOR, which in turn led to an increase in the expression of downstream ubiquitin proteins, thereby promoting the apoptosis of liver cancer cells. The group verification experiment of the present invention (control group, medium-dose group of medicated serum of Herba Eupatorii, mTOR inhibitor Rapamycin (10nmol / well), medium-dose group of medicated serum of Herba Eupatorii + mTOR inhibitor Rapamycin (10nmol / well)) proved that the medicated serum of Herba Eupatorii has the effect of inhibiting the phosphorylation of mTOR.

[0089] The Bcl-2 protein family is functionally divided into pro-apoptotic and anti-apoptotic proteins, and the interaction between the two regulates apoptosis. Bcl-2 associate X protein (Bax) is a pro-apoptotic protein that regulates apoptosis via the mitochondrial pathway. Upon activation, Bax promotes mitochondrial outer membrane permeabilization (MOMP), leading to the diffusion of cytochrome C, a protein in the mitochondrial intermembrane space. This activates apoptosis protease activating factor (APAF-1), forming a caspase activation platform. This promotes the activation of the apoptosis initiator caspase-9, which subsequently activates the downstream executioners of the cascade, caspase-3 and caspase-7, cleaving cellular substrates and inducing apoptosis. The caspase family is a class of cysteine ​​proteases that are highly specific for cleaving peptide bonds following aspartic acid residues, thereby affecting protein activity and function. Among them, Caspase-3 is functionally classified as an apoptosis execution factor, located downstream of the cascade reaction and the main effector in the apoptosis process. Under normal circumstances, it exists in the form of a zymogen and can be activated by the upstream apoptosis initiator Caspase-9 in the cascade reaction. When Caspase-3 is activated, it forms cleaved Caspase-3, which cleaves and inactivates the DNA repair enzyme poly (ADP-ribose) polymerase 1 (PARP-1), 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. It also promotes cell survival through antioxidant and calcium ion concentration regulation. Western blot results of the present invention show that the Fengguidoucao-containing serum promotes apoptosis of liver cancer cells by upregulating Bax protein and downregulating Bcl-2 protein expression, thereby activating the downstream Caspase cascade protein family.

[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 Herba Lycopodii with the mTOR inhibitor Rapamycin has a better effect than either drug alone.

[0091] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An application of a drug-containing serum prepared from Herba Lycopodii in the preparation of a drug for treating liver cancer, characterized in that: The drug-containing serum is obtained by gavage of the alcohol extract of the Herba Lycopodii on mice and then collecting blood; the preparation method of the alcohol extract is as follows: the whole Herba Lycopodii is reflux-extracted with 75% volume concentration ethanol for 3 times, each time for 1.5 hours, the filtrates are combined, and the ethanol is recovered under reduced pressure to obtain the drug-containing serum.

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