Method for screening key active components of various components in traditional Chinese medicine compound through traditional Chinese medicine systematic biology research mode and application of key active components

Abstract

The invention discloses a method for screening key active ingredients of all components in a traditional Chinese medicine compound through a traditional Chinese medicine system biological research mode and application of the method. A traditional Chinese medicine system biological research mode of'compound traditional Chinese medicine-single traditional Chinese medicine-key active ingredients-target identification-disease treatment 'is established, the whole and local parts of the traditional Chinese medicine are considered, basic research and clinical treatment are combined, an experimental basis is provided for explaining the scientific connotation of the coptis chinensis detoxification decoction and expanding indications, and the coptis chinensis detoxification decoction has a wide application prospect. And a new choice is provided for the treatment of APL. The traditional Chinese medicine compound, single traditional Chinese medicine and key active ingredients of the coptis chinensis detoxification decoction are comprehensively researched. The treatment effects of CR, PCC and SR are equivalent to those of HLJDT, and GF does not have a remarkable effect. The HLJDT and the key active ingredient baicalin thereof prolong the survival time of the APL mouse model. Both berberine and baicalin can inhibit the expression of PML-RAR alpha oncoprotein, and geniposide has no influence on the expression of oncoprotein.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and in particular to a method for screening key active ingredients of various components in a traditional Chinese medicine compound through a traditional Chinese medicine system biology research model and its application. Background Art

[0002] Interpreting the scientific connotation of traditional Chinese medicine with modern science and technology is a huge challenge facing the current work of traditional Chinese medicine, and it is also a major strategic need of the country. Traditional Chinese medicine has three major characteristics: holistic view, syndrome differentiation and treatment, and prevention of disease. Compound prescriptions are the essence of clinical use of traditional Chinese medicine. In traditional Chinese medicine theory, the performance and efficacy of drugs are coordinated and promoted with each other, so as to achieve the therapeutic effect of reducing toxicity and increasing efficacy. However, there are two extreme phenomena in the current field of traditional Chinese medicine: first, a small number of traditional Chinese medicine workers are relatively conservative in their thinking, stick to the tradition of traditional Chinese medicine, and find it difficult to accept the combination of traditional Chinese medicine and modern science; second, in the process of modern research on traditional Chinese medicine, some researchers excessively pursue the westernization of traditional Chinese medicine, resulting in the disconnection between traditional Chinese medicine research and the basic theory of traditional Chinese medicine. Both phenomena hinder the healthy development of traditional Chinese medicine. In order to correct the deviation of these two extreme phenomena, it is a very urgent task to propose a model for traditional Chinese medicine research.

[0003] In addition, there are still four other key issues in the research process of traditional Chinese medicine, including unclear medicinal properties and complex mechanisms of action of traditional Chinese medicine; lack of systematic research on key active ingredients of traditional Chinese medicine; lack of pharmacological models of efficacy and disease syndrome; single research model and lack of innovation. In order to better promote the modernization of traditional Chinese medicine, we must return to the roots of traditional Chinese medicine when considering new research models of traditional Chinese medicine, and use traditional Chinese medicine theory as a guide to deeply explore and inherit this treasure. On the basis of the holistic view of traditional Chinese medicine, with the help of contemporary science and technology, we can study the mechanism of action of traditional Chinese medicine and fully reveal the scientific connotation of traditional Chinese medicine. As an interdisciplinary subject of genomics, transcriptomics, proteomics, metabolomics and bioinformatics, systems biology methods have the advantages of high efficiency, fast speed and high success rate compared with traditional drug discovery research. Systems biology has derived the P4 medical model, namely predictive, preventive, personalized and participatory treatment, which has many similarities with traditional Chinese medicine theory. For example, the system theory of systems biology is similar to the holistic view of traditional Chinese medicine, prediction is equivalent to traditional Chinese medicine diagnosis, prevention is equivalent to treatment before disease, and personalized treatment is similar to the syndrome differentiation and treatment of traditional Chinese medicine. Therefore, the application of systems biology in TCM research has received increasing attention in recent years and may become an innovative platform for the integration of Chinese and Western medicine. Therefore, the present invention proposes a TCM systems biology research model of "TCM compound-single TCM-key active ingredients-target identification-disease treatment" (see Figure 1 ), which takes into account both the overall and local aspects of traditional Chinese medicine and combines basic research with clinical treatment.

[0004] APL is the M3 subtype of AML. Approximately 98% of APL is driven by t(15;17)(q22;q21), which causes the promyelocytic leukemia (PML) gene to form a PML-RARα fusion protein with all-trans retinoic acid receptor α (RARα). The combination of arsenic trioxide and all-trans retinoic acid (ATRA) for molecular targeted therapy of PML-RARα has completely changed the prognosis of patients and improved their survival rate, which also fully reflects the successful clinical application of systems biology therapy. However, retinoic acid syndrome is a fatal side effect of retinoic acid induction therapy, accounting for about 25% of APL patients. The use of other hormonal drugs such as dexamethasone can significantly reduce the mortality associated with retinoic acid syndrome. Due to the unclear mechanism of death from retinoic acid syndrome and the lack of preventive measures, there is still a need to find highly effective and low-toxic drugs for the treatment of APL. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for screening the key active ingredients of each component in a traditional Chinese medicine compound through a traditional Chinese medicine systems biology research model.

[0006] Another object of the present invention is to provide an application of the above method.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for screening key active ingredients of each component in a traditional Chinese medicine compound through a traditional Chinese medicine systems biology research model comprises the following steps:

[0009] (1) According to the characteristics of the disease, select a Chinese herbal compound with potential therapeutic effects, then design a disease model and use the compound ingredients to verify the efficacy of the compound;

[0010] (2) Use disease models to verify the effects of single Chinese medicines in the selected Chinese medicine compound and select single Chinese medicines with therapeutic effects;

[0011] (3) Use disease models to verify the effects of key active ingredients contained in single Chinese medicines with therapeutic effects and screen out key active ingredients with therapeutic effects;

[0012] (4) Further verifying the therapeutic effect of the key active ingredient screened in step (3) through animal experiments and target protein capture experiments, and obtaining the target protein of the key active ingredient.

[0013] The disease described in step (1) is human acute promyelocytic leukemia (APL).

[0014] The Chinese medicinal compound described in step (1) is Huanglian Jiedu Decoction.

[0015] The disease model described in step (1) is a NB4 cell model.

[0016] The single Chinese medicine described in step (2) is at least one of coptis chinensis, scutellaria baicalensis and phellodendron chinense.

[0017] The key active ingredient in step (3) is at least one of berberine, baicalin and gardenia jasminoides.

[0018] The animal experiment described in step (4) is a mouse experiment.

[0019] The above method of screening the key active ingredients of each component in a traditional Chinese medicine compound through the research model of traditional Chinese medicine system biology is used in screening the key active ingredients for treating human acute promyelocytic leukemia.

[0020] A method for screening components in a traditional Chinese medicine compound for key active ingredients for treating human acute promyelocytic leukemia comprises the following steps:

[0021] (1) Adding Chinese herbal medicine compound to NB4 cells to verify its effect on cell proliferation;

[0022] (2) Adding individual Chinese herbs in the Chinese herbal medicine compound to NB4 cells to verify their effects on cell proliferation, and screen out individual Chinese herbs with better cell proliferation inhibition effects;

[0023] (3) adding the key active ingredients contained in the single Chinese medicine screened in step (2) to NB4 cells to screen out the key active ingredients with better cell proliferation inhibition effect;

[0024] (4) The key active ingredients with good inhibitory effects screened out in step (3) are further applied to animal models to verify the effects, thereby obtaining key active ingredients with the ability to treat human acute promyelocytic leukemia.

[0025] The Chinese medicinal compound described in step (1) is Huanglian Jiedu Decoction.

[0026] The single Chinese medicine described in step (2) is at least one of coptis chinensis, scutellaria baicalensis and phellodendron chinense.

[0027] The key active ingredient in step (3) is at least one of berberine, baicalin and gardenia jasminoides.

[0028] Compared with the prior art, the present invention has the following advantages and effects:

[0029] (1) The research model of systems biology of traditional Chinese medicine established in this invention takes a three-pronged approach from the perspectives of traditional Chinese medicine compound, single traditional Chinese medicine and key active ingredients, taking into account both the overall and local aspects of traditional Chinese medicine, and combining basic research with clinical treatment. This provides an experimental basis for the interpretation of the scientific connotation and expansion of the indications of Huanglian Jiedu Decoction, and also provides a new option for the treatment of APL.

[0030] (2) Based on the established TCM system biology research model of "TCM compound-single TCM-key active ingredients-target identification-disease treatment", the present invention conducted a comprehensive study on the entire formula components of Huanglian Jiedu Decoction, including the three levels of TCM compound, single TCM and key active ingredients. The therapeutic effects of CR, PCC and SR were comparable to those of HLJDT, while GF had no significant effect. Subsequent studies showed that HLJDT and its key active ingredient baicalin successfully inhibited the infection of NB4 Luc The fluorescence of cancer cells in mice was enhanced and the spleen was enlarged, which prolonged the survival of the APL mouse model. In addition, baicalin and retinoic acid combined to inhibit the expression of PML-RARa oncoprotein and thus prolonged the survival of the APL mouse model. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the research model of traditional Chinese medicine systems biology used in the present invention.

[0032] Figure 2 The effect of HLJDT on the proliferation of NB4 cell line.

[0033] Figure 3 This is the effect of a single Chinese medicine on the proliferation of NB4 cell line.

[0034] Figure 4 It is the effect of the key active ingredients of HLJDT on the proliferation of NB4 cells.

[0035] Figure 5 It is the combined inhibitory effect of HLJDT compound or single Chinese medicine and ATRA on NB4.

[0036] Figure 6 It is the combined inhibitory effect of BBR or Baicalein and ATRA on NB4 cell line.

[0037] Figure 7 It is the synergy index diagram of HLJDT and effective components and ATRA.

[0038] Figure 8 It is NB4 Luc Fluorescence intensity of stable cell lines.

[0039] Fig. 9 Schematic diagram of the establishment of APL mouse xenograft model and disease progression assessment method

[0040] Fig.10 Intravital imaging was performed on days 0, 8, 12, and 16 after dosing in APL mice to monitor disease progression.

[0041] Fig.11 This is a diagram showing the appearance and weight statistics of mice in the APL mouse experiment.

[0042] Fig.12 This is the Kaplan-Meier survival curve of mice in the control group, single drug group and combined drug group.

[0043] Fig.13 Schematic diagram of the capture and identification principle of baicalin target protein.

[0044] Fig.14 It is the capture and analysis of target proteins in K562-IMR cell line.

[0045] Fig.15 It is a schematic diagram of the protein and compound docking simulation process.

[0046] Fig.16 It is a Ramachandran Plot diagram of the amino acid residue status before and after protein structure optimization.

[0047] Fig.17 This is the result diagram of the molecular docking in Example 3.

[0048] Fig.18 These are the three sequences of amino acid polypeptides containing covalent bond sites screened out in Example 3.

[0049] Fig.19 SPRi verifies the affinity of Baicalein to RARα protein and three polypeptide sites.

[0050] Fig. 20 This is a graph showing the kinetic curve of the interaction detected by the SPR device in Example 3.

[0051] Fig.21 This is a graph showing the effects of berberine, baicalin and gardenia jasminoides on the expression of PML-RARα and RARα proteins, respectively.

[0052] Fig. 22 This is a graph showing the effect of baicalin on the expression levels of PML-RARα and RARα proteins.

[0053] Fig.23 This is the result of baicalin combined with ATRA inhibiting the expression of PML-RARα and RARα proteins.

[0054] Fig.24 This is the KEGG enrichment analysis result of baicalin potential target proteins.

[0055] Fig.25 This is a graph showing the results of baicalin increasing the expression level of LC3 protein and decreasing the expression level of P62 protein in a time- and dose-dependent manner.

[0056] Fig.26 This is the experimental result of using autophagy inhibitor to block the autophagic degradation of PML-RARα and RARα by Baicalein. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0058] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0059] Example 1 Detection of the effect of the compound Huanglian Jiedu decoction and its components on NB4 cell proliferation

[0060] 1.1 Basis for topic selection

[0061] Retinoic acid-induced therapy for APL may cause differentiation syndrome in patients. We hope to develop a highly effective, low-toxic and synergistic Chinese medicine for the treatment of APL. In the early stage, we used biosensor technology to find that Huanglian Jiedu Decoction has a targeting effect on RARα protein, and PML-RARα fusion protein is the characteristic oncogene in APL. Therefore, we hypothesized that Huanglian Jiedu Decoction has anti-APL pharmacological effects. In order to verify this hypothesis, we used NB4 cell line to detect the pharmacological effects of Huanglian Jiedu Decoction.

[0062] 1.2 Cell culture

[0063] Take out the cryopreserved tube of human acute promyelocytic leukemia NB4 cell line from liquid nitrogen or -80℃ freezer, quickly place it in a water bath with a preset temperature of 37℃, shake and dissolve, wipe it clean with alcohol, add 2mL complete culture medium in the clean bench, centrifuge at 800rpm for 3min. Then transfer the cells to a culture bottle with an appropriate amount of RPMIMedium 1640 culture medium containing 10% FBS and 1% penicillin / streptomycin, and culture it in a 37℃ incubator with 5% CO2.

[0064] 1.3 Detection of the effect of compound Huanglian Jiedu decoction on NB4 cell proliferation

[0065] To detect the inhibitory effect of Huanglian Jiedu Decoction (HLJDT) on NB4 cells, we treated NB4 cell lines with 7 different concentrations of compound Huanglian Jiedu Decoction. The specific steps are as follows:

[0066] (1) NB4 cells in the logarithmic growth phase were centrifuged at 800 rpm for 3 min and collected. After cell counting, 5×10 4 The cells were plated at a density of 1:1 / mL into a 96-well plate, with 100 μL of cell suspension in each well.

[0067] (2) Grind Huanglian Jiedu Decoction (purchased from Tsumura, granules) into powder and dissolve it in water to prepare a solution.

[0068] (3) According to the preset concentration gradient, calculate the total volume of the corresponding drug, add 1640 culture medium to 100 μL, and shake well.

[0069] (4) Add the drug to the prepared 96-well plate so that the final drug concentration is the preset concentration (0-350 μg / mL), and set up 6 replicate wells for each concentration.

[0070] (5) Place the 96-well plate in a 37°C incubator with 5% CO2 for incubation.

[0071] (6) After 48 h of incubation, 20 μL of thiazolyl blue solution was added to each well and the cells were placed in a 37°C incubator with 5% CO2 for further incubation.

[0072] (7) After 3 h of incubation, the 96-well plate was placed in a centrifuge and centrifuged at 1200 rpm for 5 min.

[0073] (8) Aspirate the culture medium and add 150 μL of DMSO.

[0074] (9) The mixture was shaken and mixed on a 96-well plate mixer, and the absorbance at a wavelength of 570 nm was measured using a multifunctional microplate reader.

[0075] (10) Statistical data were collected and Graphpad Prism 8.0.1 was used to calculate the drug IC50, and the cell survival curve was drawn.

[0076] (11) Repeat the above steps three times and draw a drug IC50 statistical graph.

[0077] The experimental results are as follows Figure 2 As shown, the IC50 of HLJDT was approximately 64.17 μg / mL, and as the drug concentration gradually increased, HLJDT significantly inhibited the proliferation of NB4 cells.

[0078] 1.4 Effects of single Chinese medicines Rhizoma Coptidis, Radix Scutellariae, Cortex Phellodendri, and Fructus Gardeniae on NB4 cell proliferation

[0079] In order to verify the effects of the four single Chinese medicines in Huanglian Jiedu Decoction on the proliferation of NB4 cell lines, we treated the NB4 cell lines with seven different concentrations of coptis chinensis, phellodendron chinense, scutellaria baicalensis and gardenia jasminoides respectively. The treatment method was based on the method in 1.3. The experimental results are shown in Figure 1. Figure 3 shown.

[0080] The cell survival curve showed that as the drug treatment concentration gradually increased, the proliferation of NB4 cells was gradually inhibited. When the treatment concentration of Coptis chinensis and Phellodendron chinense was 4 μg / mL, the cell proliferation inhibition rate was higher than 50%, but when the treatment concentration of Gardenia jasminoides reached 200 μg / mL, the inhibition rate of NB4 cell line did not reach 10% ( Figure 3 A, B, and D in the figure). Among them, the IC50 of coptis chinensis, phellodendron amurense, scutellaria baicalensis, and gardenia jasminoides were 3.562μg / mL, 3.444μg / mL, 33.16μg / mL, and 878.6μg / mL, respectively. Therefore, coptis chinensis and scutellaria baicalensis had significant inhibitory effects on the NB4 cell line, while gardenia jasminoides had no significant inhibitory effect. At the same time, the inhibitory effects of coptis chinensis and phellodendron amurense were stronger than those of scutellaria baicalensis.

[0081] 1.5 Inhibitory effects of key active ingredients berberine, baicalin and gardenia jasminoides on NB4 cell lines

[0082] In order to determine the key active ingredients for drug efficacy, berberine (BBR), baicalein and geniposide (1μM, 5μM, 10μM, 20μM, 40μM, 80μM) were used to treat NB4 cells pre-plated in 96-well plates for 48 hours, and then their effects on the survival rate of NB4 were detected by MTT method. GraphPad Prism was used to calculate the IC50 of the three active ingredients and draw the survival curve. The specific steps refer to the method in 1.3. The experimental results are shown in Figure 1. Figure 4 shown.

[0083] From the cell survival rate curve, it can be seen that with the gradual increase of BBR and Baicalein treatment concentrations, the survival of NB4 cells was significantly inhibited ( Figure 4 (A). Under the same drug treatment concentration, the cell activity inhibition rate of Gardenia glycoside was not as significant as that of berberine and baicalin. After three IC50 statistical analyses, the IC50 of BBR and Baicalein were both lower than 7μM, while the IC50 of Gardenia glycoside was greater than 70μM ( Figure 4 Therefore, Gardenia glycoside had no significant effect on the activity of the cell line.

[0084] 1.6 Detection of the synergistic inhibitory effect of Huanglian Jiedu Decoction and Coptis chinensis, Scutellaria baicalensis, Phellodendron chinense with ATRA

[0085] In order to verify the synergistic effect of Huanglian Jiedu Decoction and the effective single Chinese medicines Coptis chinensis, Scutellaria baicalensis, and Phellodendron chinense with ATRA, we first selected different concentrations of ATRA (2×10 -3 μM, 1μM, 3μM, 6μM, 10μM, 15μM, 20μM, 30μM, and 50μM) were used to treat NB4 cell lines. After 48 hours of drug addition, the cell survival rate was detected by MTT and the drug IC50 was calculated. The specific steps refer to the method in 1.3. The results are shown in Figure 5 shown.

[0086] The results showed that the IC50 of ATRA was 24.17 μM ( Figure 5 At the same time, the concentration of Huanglian Jiedu Decoction 10μg / mL (Huanglian Jiedu Decoction IC50 is about 1 / 6) was selected to treat NB4 cell lines with each ATRA concentration gradient. After 48h, the synergistic inhibition rate of drugs on NB4 cells was detected by MTT method. As shown in the results, after ATRA was combined with Huanglian Jiedu Decoction, the IC50 was reduced to 10.67μM, which was more than doubled compared with ATRA alone ( Figure 5 Then we used Rhizoma Coptidis (0.5μg / mL), Phellodendron Amurense (1μg / mL), Scutellariae Radix (10μg / mL) in combination with ATRA, and the results showed that the IC50 of ATRA decreased from 24.171μM to 14.29μM, 17.21μM, and 4.865μM ( Figure 5 According to the Chou-Tatalay drug synergy algorithm (CI value less than 1 indicates synergistic effect, equal to 1 indicates additive effect, and greater than 1 indicates antagonistic effect), HLJDT, single Chinese medicine and ATRA all showed synergistic effect ( Figure 7 AD).

[0087] 1.7 Detection of the synergistic inhibitory effect of berberine and baicalin with ATRA

[0088] In the screening experiment of key active ingredients, we found that berberine and baicalin can significantly inhibit the proliferation of NB4 cell line. Therefore, in the combined experiment with ATRA, we selected berberine (0.5μM) and baicalin (2μM) to treat NB4 cell line with ATRA. After 48h, MTT was used to detect cell survival rate. The specific steps refer to the method in 1.3. The results are shown in Figure 6 shown.

[0089] The results show that after drug combination treatment, the IC50 was significantly reduced. After combining with BBR and Baicalein, the IC50 of ATRA was reduced from 24.17μM to 6.027μM and 1.660μM ( Figure 6). According to the Chou-Tatalay drug synergy algorithm, Baicalein, BBR and ATRA all showed synergistic effects. The combined effect of Baicalein and ATRA was the most significant, and the CI index was less than 1 ( Figure 7 E, 7F). Therefore, it is believed that the key active ingredient in the synergistic effect with ATRA is Baicalein.

[0090] Example 2 Construction of APL mouse disease model and evaluation of therapeutic effects by drug administration

[0091] 2.1 Build NB4 Luc Stably transfected cell lines

[0092] In order to effectively track the disease progression of APL in vivo, luciferase lentivirus was used to infect the NB4 cell line that had been pre-plated in a 6-well plate at MOI=30. The specific steps are as follows:

[0093] (1) After cell counting, 5×10 5 The density of 100 μg / mL was plated in a 6-well plate.

[0094] (2) Calculate the volume of virus solution corresponding to the optimal MOI value according to the amount of virus added per well (μL) = MOI × number of cells / virus titer (TU / mL) × 1000. Add the virus solution to a 6-well plate.

[0095] (3) Use 1640 medium without dual antibody to make up the volume to 1000 μL.

[0096] (4) Seal the 6-well plate with a membrane and place it in a centrifuge at 1000 rpm for 1 h.

[0097] (5) After centrifugation, place the 6-well plate in a 37°C incubator for continued culture.

[0098] (6) After 4 to 6 hours, add 1000 μL of dual antibody-free culture medium to each well and return the well to the incubator for continued culture.

[0099] (7) After 24 h, the cells were collected by centrifugation at 800 rpm for 3 min, the medium containing the virus solution was discarded, and fresh 1640 complete medium was replaced.

[0100] (8) When the cells were stable, 1 μg / mL of puromycin was added to the culture medium to screen positive cells.

[0101] (9) After 14 days of continuous screening, the cells were counted and plated in a 96-well plate at a number of 500, 1000, 2000, 6000, 10000, and 20000, and 30 mg / mL VivoGlo was added. TMLuciferin stock solution was prepared to a final concentration of 0.15 mg / mL.

[0102] (10) The fluorescence value and fluorescence intensity were measured using an ELISA reader and a small animal live imaging system, respectively.

[0103] (11)NB4 Luc After the cell line was successfully established, 1 μg / mL of puromycin was used to maintain the culture.

[0104] The experimental results show that the cell luminescence value and fluorescence intensity can be gradually enhanced with the increase of density ( Figure 8 ), successfully built NB4 Luc Stable cell lines.

[0105] 2.2 Establishment of APL mouse xenograft model

[0106] (1) 56 severely immunodeficient R-NSG mice (4-5 weeks old, female) purchased from Ruisiyuan Biotechnology Co., Ltd. were housed in the Experimental Animal Management Center of Jinan University. The mice were allowed to freely access water and food.

[0107] (2) After passing the quarantine, 7-week-old mice were injected with 1×10 7 NB4 Luc cell.

[0108] (3) 10 days after cell injection, prepare anesthetic and VivoGlo TM Luciferin working solution was used to detect the disease status of mice using a small animal in vivo imaging system.

[0109] (4) If weak fluorescence is detected in the mouse, the modeling is successful and the mice are divided into groups for subsequent drug administration experiments.

[0110] 2.3 APL mouse model grouping, drug treatment, and efficacy evaluation

[0111] (1) The diseased mice were randomly divided into 7 groups, with 8 mice in each group. The 7 groups were: PBS, ATRA (10 mg / kg), HLJDT (0.6 g / kg), Baicalein (25 mg / kg), Baicalein (50 mg / kg), ATRA (10 mg / kg) combined with Baicalein (25 mg / kg), HLJDT (0.6 g / kg), and ATRA (10 mg / kg) combined with HLJDT (0.6 g / kg).

[0112] (2) The seven groups of mice were administrated once every two days. HLJDT and Baicalein were administered by oral gavage, and ATRA was injected intraperitoneally.

[0113] (3) The drug was administered continuously for two weeks, during which in vivo imaging was performed every 4 days to detect disease progression and the images were recorded. Body weight was also measured using an electronic balance and fluctuations were recorded.

[0114] (4) After the treatment cycle, the spleens of mice were removed, with 3 mice in each group. The size and weight were counted and photographed for record.

[0115] (5) The time of mouse death was recorded in time, and the mouse survival curve was drawn using Graphpad Prism 8.0.1.

[0116] 2.4 Results and Discussion

[0117] Injection of NB4 Luc After the cells were grown, the details of the mouse disease were detected using a small animal in vivo imaging system, such as Fig.10 The results showed that weak fluorescence was produced in all mice on the 10th day after cell injection. The mice were divided into 7 groups, 8 in each group, for drug treatment as planned. The drug was administered every two days as planned, and the treatment cycle was 14 days. After administration, live imaging was performed on the 8th, 12th and 16th days with the help of a small animal imaging system to detect the fluorescence intensity to reflect the progression of the disease. The results showed that from the overall fluorescence intensity of the mice in the Control group, the fluorescence intensity area of ​​the mice was in a strong expansion state from Day0 to Day16. The overall fluorescence enhancement trend of the mice in the drug-treated group was well controlled. Compared with the Control group, the low-dose and high-dose groups of baicalin and the HLJDT group had an inhibitory effect on the fluorescence enhancement of mice, and the effect of the high-dose group of baicalin was more obvious. In addition, compared with the ATRA group and the low-dose group of baicalin, the combined use of baicalin and ATRA had a more significant inhibitory effect on fluorescence enhancement than the ATRA control group.

[0118] As the mice became ill for a longer time, they gradually developed arched backs and erect hair, which indirectly indicated that the APL mouse model had been successfully established ( Fig.11 In A). In order to further analyze the efficacy of Huanglian Jiedu Decoction, baicalein, or combined with ATRA. During the administration process, the mice were weighed every 4 days, and the weight fluctuation curve was drawn. After the two-week treatment period, 3 mice in each group were randomly selected, the spleens were removed, and the size and weight were measured. From the spleen weight statistics, it can be seen that the Huanglian Jiedu Decoction group and the baicalein group both effectively inhibited spleen enlargement, and the significant difference between the ATRA and Baicalein combination group was very significant (P<0.0001). And from the appearance size, the spleen size and weight of the combined group mice were smaller than those of the ATRA control group ( Fig.11 In addition, the low-dose / high-dose baicalin group and the HLJDT group effectively inhibited weight loss ( Fig.11 (middle D).

[0119] After the treatment period, the survival status of the mice was observed every day, and the death time of each mouse was recorded in time. Finally, the survival analysis of the mice was performed. Compared with the Control group, the HLJDT group and the low / high dose baicalin group prolonged the survival time of the mice, among which the high dose baicalin group and the HLJDT group had more obvious effects than the low dose baicalin group ( Fig.12 ), which is consistent with the inhibitory effect on spleen enlargement in mice, indicating that baicalin plays a key therapeutic role. It is worth noting that the combined administration of baicalin and ATRA showed a good therapeutic effect ( Fig.12 The above results indicate that baicalin and ATRA have a synergistic effect, which is consistent with the result that the combination of the two significantly inhibits the fluorescence intensity and spleen enlargement.

[0120] In summary, Huanglian Jiedu Decoction and baicalin significantly inhibited NB4 Luc The fluorescence intensity of cells was increased, and the proliferation of mouse cancer cells was inhibited. The ATRA and baicalin combined administration group had a more significant inhibitory effect. Huanglian Jiedu Decoction and baicalin could slow down the weight loss of mice and effectively inhibit spleen enlargement. The ATRA and baicalin synergistic group had a very high significant difference. Huanglian Jiedu Decoction and baicalin could both prolong the survival of mice. The ATRA and baicalin combined administration group showed a synergistic effect.

[0121] Example 3 Capture and identification of baicalin target protein

[0122] 3.1 Photocrosslinking sensor chip, HPLC-MS / MS to capture potential target proteins

[0123] In order to study the target protein of baicalin, the target protein was captured and identified by SPR-HPLC-MS coupling technology. The specific steps are as follows:

[0124] (1) Using BioDot TM -1520 array printer prints 10mM Baicalein solution array onto the chip surface. Set the dot spacing to 280μm, the dot diameter to 180μm, use a double-needle printing system, 50×50 dot array on the chip surface, repeat the spotting 5 times, and the chip surface spotting volume is 31.25μL (312.5nM).

[0125] (2) After K562-IMR cells were lysed, the protein concentration was measured and adjusted to a final concentration of 200 μg / mL using lysis buffer.

[0126] (3) NanoSensor biochip substrates were purchased from Lumera, USA. Bio-Care Biotech Co., Ltd. performed photo-crosslinking polymer layer modification treatment to make the chip binding amount batch-to-batch variation <0.5%. The resonance angle of the chip was automatically calibrated to the optimal resonance angle using Berthold bScreenLB 991 biochip analyzer and set aside.

[0127] (4) During the test, the K562-IMR protein lysate sample flowed through the chip surface, and the SPR biochip analyzer was used to monitor in real time the binding of Baicalein fixed on the chip surface and the target protein in the lysate.

[0128] (5) After the test, the chip is enzymatically hydrolyzed in situ in the detection device, and then the type of protein captured on the chip surface is identified using HPLC-MS.

[0129] (6) Functional annotation of the captured target proteins was performed using the Uniprot and GO databases, and enrichment analysis was performed using the KEGG signaling pathway database.

[0130] Schematic diagram of the target protein capture and identification process Fig.13 As shown in the figure, after the test, the potential target proteins were enriched, and we used SPR-HPLC-MS technology to identify the target proteins of baicalin. We screened proteins with MS scores between 1600 and 1100 and drew a heat map. According to the screening results, it was determined that baicalin successfully captured the K562-IMR cell line protein RARα ( Fig.14 ).

[0131] 3.2 Molecular docking simulation of the interaction between baicalin and RARα protein

[0132] First, prepare the primary structure sequence and reference crystal structure of RARα protein. The target protein sequence is obtained from the UniprotKB database, and the crystal diffraction structure of the homologous protein required for modeling is obtained from the PDB database. Input the molecular structure of the Baicalein compound. Scan the active center site of the protein by computer and make a prediction, and then perform docking analysis. The process is shown in the figure below: Fig.15 shown.

[0133] To construct native protein structures, use the Preparation Wizard module ( Inc) to calculate the native structure of the protein. First, the protein is assumed to be in a pH 7.0 environment, and hydrogen atoms are added to the protein structure according to the different isoelectric points of different amino acid residues. Then, the structure is optimized by adjusting hydrogen bonds, eliminating atomic collisions, and adding formal charges to hetero groups. Finally, excess water molecules are removed and the generated structure is minimized (folded to the lowest energy steady state) using the OPLS4 force field optimization. The modeled and optimized protein structures are analyzed by Ramachandran Plot, and the Ramachandran Plot ( Fig.16 ), and then the binding sites of the prepared protein structure are calculated.

[0134] The ligand structures of chemical molecules were synthesized using the LigPrep module of Schrodinger, Inc. The molecular docking was tested using the ultra-precision simulation docking module (XP module, Schrodinger, Inc.). The various spatial forms of baicalin tightly bind to the three pockets of RARα ( Fig.17 ). The MMGBSA score of the binding state was calculated using the Schrodinger MMGBSA module. The bond energy and stability of the compound binding to the protein were then tested. The results showed that the compound binding at Site 2 was the most stable.

[0135] 3.3 SPRi verification of the affinity of baicalin to RARα protein and three epitope peptides

[0136] 3.3.1 Peptide extraction scheme and sequence

[0137] According to the docking model, we analyzed the amino acid residues that provided non-covalent bonds for compound binding. We intercepted three amino acid peptides, RARα-Pep1, Pep2, and Pep3, which contained covalent bond sites for subsequent experiments. Fig.17 The colored amino acids provide non-covalent bonds for the binding of the compounds.

[0138] Site 1 epitope sequence RARα-Pep1: VGELIEKVRKAHQETFPALCQLGKYTTNNS;

[0139] Site 2 epitope sequence RARα-Pep2: LRICTRYTPEQDTMTFSDGLTLNRTQMHNA;

[0140] Site 3 epitope sequence RARα-Pep3: FTTLTIADQITLLKAACLDILILRICTRYT.

[0141] 3.3.2 Verification of binding affinity between baicalin and three peptides

[0142] RARα was obtained by biosynthesis and the proteins corresponding to the sequences of RARα-Pep1~3 were obtained by chemical synthesis. Next, the affinity of Baicalein to RARα and three polypeptide sites was determined by the SPRi method, and Mouse IgG and Rabbit IgG were used as controls in this process. The 6 protein sample stock solutions were diluted into 5 concentration gradients, and all samples were circulated on the chip surface in turn for testing. The specific steps are as follows:

[0143] (1) Take out Baicalein and protein samples from the -80°C freezer and dissolve them at room temperature for later use.

[0144] (2) Using Biodot TM The AD1520 chip array printer prints the working solution on the 3D photo-crosslinking chip, prints 4 replicates for each sample, and prints 4 groups of positive control spots (rapamycin) at the four corners.

[0145] (3) Use DMSO to dilute Baicalein to the concentration of the printing working solution. The printed chip is vacuum dried and then placed in a photocrosslinking instrument for photocrosslinking reaction; then DMF, C2H5OH, and H2O are used to shake and wash for 15 minutes, blow dry with nitrogen, and assemble the Flowcell Cover for use.

[0146] (4) The protein sample solution was transferred to a dialysis bag and dialyzed with PBS to remove detergents, glycerol and other additives in the original storage solution. The solution was then concentrated using a microprotein concentrator tube and the concentration was measured using a BCA protein quantification kit and recorded for later use.

[0147] (5) PBST (pH=7.4, 0.1% Tween 20) was added to the protein sample stock solution to dilute it into 5 concentration gradients: 10 nM, 40 nM, 160 nM, 640 nM, 2560 nM. All samples were sequentially circulated on the chip surface for testing.

[0148] (6) In the interaction test phase, the protein sample was subjected to a flow rate of 0.5 μL·s -1 Flowing over the chip surface; in the surface regeneration phase, Glycine-HCl (pH = 2.0) solution was used as the regeneration solution, and the flow rate was 2 μL·s -1 .

[0149] (7) The regeneration phase of the test showed that the six proteins bound to the chip were regenerated and dissociated using the regeneration solution to determine whether the binding of the tested Baicalein with the six proteins conformed to the Langmuir binding model.

[0150] (8) Based on the real-time detection results of the SPR device, the kinetic curve of the interaction is fitted and the affinity parameters are output.

[0151] According to the real-time detection results of the SPR device, the kinetic curve of the interaction was fitted ( Fig.19 A) and output affinity parameters ( Fig. 20 ). It was found that Baicalein had two strong interaction pairs, two weak interaction pairs and two very weak (or no) interaction pairs with the six proteins. The results showed that the compound Baicalein specifically binds to the Human RARα protein ( Fig.19 A), its affinity constant KD is 1.57E-08M (15.70nM) ( Fig. 20 ). In the affinity analysis of the three epitope peptides, Baicalein has a stronger affinity with RARα_Pep2, with an affinity constant KD of 1.02E-06M (1020.00nM); it has a weaker affinity with RARα_Pep1 and RARα_Pep3, which are 1.64E-03M (1.64mM) and 7.73E-03M (7.73mM), respectively. Combined with the simulation docking analysis results, it is speculated that the epitope corresponding to RARα_Pep2 is the binding epitope of Baicalein on the Human RARα protein.

[0152] 3.4 Results and Discussion

[0153] In summary, in this example, baicalin successfully captured about 90 target proteins in K562-IMR lysate, including RARα, through SPR-HPLC-MS technology; through molecular docking, the computer simulated the binding and bonding relationship between baicalin and the target protein RARα and three polypeptide sites respectively; with the help of SPRi technology, the binding affinity of baicalin to RARα protein was verified, and it had a very high affinity with RARα polypeptide site 2.

[0154] Example 4 Study on the mechanism of action of baicalin in targeted degradation of PML-RARα oncoprotein

[0155] 4.1 Detection of the effects of key active ingredients on PML-RARα protein expression

[0156] 4.1.1 Effects of berberine, baicalin and gardenia on PML-RARα protein expression

[0157] In order to verify the effects of berberine, baicalin and gardenia on RARα protein, 1μM, 6μM and 70μM berberine, baicalin and gardenia were used to treat NB4 cell lines. After 48 hours, total protein of cell lines was extracted and Western Blot analysis was used to confirm the effects of BBR, baicalin and gardenia on RARα protein. The specific steps are as follows:

[0158] (1) NB4 cells in the logarithmic growth phase were cultured at 5×10 5 / mL density was plated in a 6-well plate. The 48h drug treatment was set as blank group, Baicalein (3.5μM) and Baicalein (7μM). And Baicalein (7μM) was set at 0h, 24h and 48h.

[0159] (2) Add drugs of corresponding concentrations and place the 6-well plate in an incubator for a further 48 h. After a fixed concentration of 7 μM Baicalein is added, the plates are treated at 0 h, 24 h and 48 h.

[0160] (3) After the culture is completed, the cells in each group are collected by centrifugation and lysed to obtain the total cell protein.

[0161] (4) Using β-actin as an internal reference, the relative expression level of RARα protein in total protein was detected by Western Blot.

[0162] The results are as follows Fig.21 As shown in Figure 2, berberine and baicalin significantly reduced the expression of RARa protein, whereas gardenia jasminoides had no effect on the expression of RARα protein ( Fig.21 A). The three-time statistical analysis showed that compared with the relative expression of PML-RARα, RARα and β-actin protein in the control group, the berberine treatment group and the baicalin treatment group had significant differences, while the gardenia glycoside treatment group had no significant difference ( Fig.21 (middle B).

[0163] 4.1.2 Verification of the effect of baicalin on PML-RARα protein expression

[0164] Next, in order to verify the effect of baicalin on PML-RARα protein expression, NB4 cell lines were treated with 3.5 μM and 7 μM baicalin, and the expression changes of PML-RARα and RARα proteins were detected by Western Blot after 48 hours. The specific steps refer to the method in 4.1.1.

[0165] The results showed that with the increase of baicalin concentration, the expression levels of PML-RARα and RARα proteins in NB4 cells were significantly decreased ( Fig. 22NB4 cells were treated with 7 μM baicalin for 24 and 48 h, and the expression of PML-RARα and RARα proteins was detected by Western Blot. The results showed that baicalin inhibited the expression of RARα in a time-dependent manner ( Fig. 22 (C / D).

[0166] 4.1.3 Detection of the synergistic inhibitory effect of baicalin and ATRA on the expression of PML-RARα and RARα proteins

[0167] Since ATRA can target the RARα part of PML-RARα oncoprotein and thus lead to the degradation of oncoprotein, and MTT detected that baicalin and ATRA can synergistically inhibit the proliferation of NB4 cells. In order to verify whether the combination of baicalin and ATRA can synergistically reduce the degradation of PML-RARα and RARα proteins, we used low concentrations of baicalin and ATRA to treat NB4 cells, extracted total protein after 48 hours, and detected the levels of PML-RARα and RARα proteins by Western Blot after protein quantification. The specific steps refer to the method in 4.1.1.

[0168] The results showed that compared with single drug, the expression levels of PML-RARα and RARα were significantly reduced after ATRA combined with baicalin treatment ( Fig.23 ). Therefore, baicalin and ATRA can jointly inhibit the expression of PML-RARα and RARα proteins.

[0169] 4.2 Detection of the autophagy pathway of baicalin targeting degradation of RARα

[0170] 4.2.1 KEGG enrichment analysis of potential target proteins of baicalin

[0171] Through the above studies, we found that baicalein can directly target RARα and induce the degradation of PML-RARα and RARα, but the specific molecular mechanism remains to be explored. Next, we performed KEGG enrichment analysis on the potential target proteins of Baicalein captured by SPR-HPLC-MS. After pathway screening, we found that Baicalein is involved in AML and autophagy pathways. Therefore, we speculate that the degradation of PML-RARα and RARα proteins by baicalein is related to the activation of the autophagy pathway ( Fig.24 ).

[0172] 4.2.2 Effects of baicalin on autophagy pathway markers

[0173] Through KEGG enrichment analysis of Baicalein's potential target proteins, we found that Baicalein may induce PML-RARα oncoprotein degradation through the autophagy pathway. To verify this hypothesis, Western blot was used to detect changes in the expression of LC3B and P62 proteins, markers of the autophagy pathway.

[0174] First, NB4 cell lines were treated with 3.5 μM Baicalein and 7 μM Baicalein, and the expression levels of LC3I / II and P62 proteins were detected by Western Blot. The specific steps were as described in 4.1.1.

[0175] The experimental results showed that baicalin caused a progressive increase in LC3 protein levels ( Fig.25 In addition, 7μM Baicalein treatment also caused an increase in LC3 protein levels after 24h and 48h treatment ( Fig.25 In contrast, the expression of P62 protein decreased with the increase of baicalin concentration and treatment time ( Fig.25 (middle B / D).

[0176] Next, in order to verify whether autophagy inhibitors can block the autophagic degradation of RARα by Baicalein, NB4 cells were pretreated with 10 μM autophagy inhibitor CQ for 4 hours and then treated with Baicalein. After 48 hours, total cell protein was extracted and the expression levels of LC3I / II and P62 proteins were detected by Western Blot. The specific steps refer to the method in 4.1.1.

[0177] The experimental results showed that CQ significantly inhibited the degradation of PML-RARα oncoprotein and RARα protein ( Fig.26 ). Therefore, baicalin triggers the degradation of PML-RARα oncoprotein and RARα protein mainly through the autophagy pathway.

[0178] 4.3 Results and Discussion

[0179] The experiments in this example prove that the key active ingredients berberine and baicalin can inhibit the expression of PML-RARα oncoprotein and RARα protein, while gardenia jasminoides has no effect on the expression of oncoprotein; baicalin inhibits the expression of PML-RARα oncoprotein and RARα protein in a time- and concentration-dependent manner, and can be combined with ATRA to reduce the expression levels of PML-RARα oncoprotein and RARα protein; KEGG enrichment analysis found that baicalin is associated with AML and autophagy signaling pathways, and baicalin can increase the expression of autophagy marker LC3 and reduce the expression level of P62 protein.

[0180] In summary, this study conducted a comprehensive study of the entire formula components of HLJDT based on the established TCM systems biology research model, including three levels: TCM compound, single TCM, and key active ingredients. The therapeutic effects of CR, PCC, and SR were comparable to those of HLJDT, while GF had no significant effect. Subsequent studies showed that HLJDT and its key active ingredient, baicalin, successfully inhibited the infection of NB4 Luc The enhanced fluorescence and enlarged spleen of cancer cell mice prolonged the survival of the APL mouse model. From the perspective of traditional Chinese medicine, leukemia is classified as a deficiency disease. Due to the internal accumulation of evil toxins, blood heat symptoms can be seen in the course of the disease. HLJDT has the effect of clearing away heat and detoxifying, which is suitable for the symptoms of leukemia, so it is beneficial for the treatment of acute leukemia.

[0181] In summary, the TCM system biology research model established in this study studied the efficacy and molecular mechanism of HLJDT in overcoming APL from three levels: TCM compound, single TCM and key active ingredients. This study provides a new option for the treatment of APL, and more importantly, it provides a new model for the study of traditional TCM compound. This TCM system biology model takes into account TCM compound, single TCM and key active ingredients, combines basic research with clinical treatment, and provides experimental basis for the interpretation of the scientific connotation and expansion of indications of Huanglian Jiedu Decoction.

[0182] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for screening the key active ingredients of each component in a traditional Chinese medicine compound through a traditional Chinese medicine system biology research model, characterized in that The steps include: (1) Establish a research model of TCM systems biology of "TCM compound-single TCM-key active ingredients-target identification-disease treatment", taking into account both the overall and local aspects of TCM, and combining basic research with clinical treatment. (2) Based on the characteristics of the disease, select a Chinese herbal compound with potential therapeutic effects, then design a disease model and use the compound ingredients to verify the efficacy of the compound; (3) Use disease models to verify the effects of single Chinese medicines in the selected Chinese medicine compound and screen out single Chinese medicines with therapeutic effects; (4) Use disease models to verify the effects of key active ingredients contained in single-drug ingredients with therapeutic effects and screen out key active ingredients with therapeutic effects; (5) Further verifying the therapeutic effect of the key active ingredient screened in step (3) through animal experiments and target protein capture experiments, and obtaining the target protein of the key active ingredient.

2. The method according to claim 1, characterized in that: The disease described in step (1) is human acute promyelocytic leukemia (APL); The Chinese medicinal compound described in step (1) is Huanglian Jiedu Decoction.

3. The method according to claim 1, characterized in that: The disease model described in step (1) is a NB4 cell model.

4. The method according to claim 1, characterized in that: The single Chinese medicine described in step (2) is at least one of coptis chinensis, scutellaria baicalensis and phellodendron chinense.

5. The method according to claim 1, characterized in that: The key active ingredient in step (3) is at least one of berberine, baicalin and gardenia jasminoides.

6. The method according to claim 1, characterized in that: The animal experiment described in step (4) is a mouse experiment.

7. Use of the method for screening key active ingredients of each component in a traditional Chinese medicine compound by using a traditional Chinese medicine systems biology research model as described in any one of claims 1 to 6 in screening key active ingredients for treating human acute promyelocytic leukemia.

8. A method for screening the key active ingredients in the components of a traditional Chinese medicine compound for treating human acute promyelocytic leukemia, characterized in that The steps include: (1) Adding Chinese herbal medicine compound to NB4 cells to verify its effect on cell proliferation; (2) Adding individual Chinese herbs in the Chinese herbal medicine compound to NB4 cells to verify their effects on cell proliferation, and screen out individual Chinese herbs with better cell proliferation inhibition effects; (3) adding the key active ingredients contained in the single Chinese medicine screened in step (2) to NB4 cells to screen out the key active ingredients with better cell proliferation inhibition effect; (4) The key active ingredients with good inhibitory effects screened out in step (3) are further applied to animal models to verify the effects, thereby obtaining key active ingredients with the ability to treat human acute promyelocytic leukemia.

9. The method according to claim 8, characterized in that: The Chinese medicinal compound described in step (1) is Huanglian Jiedu Decoction.

10. The method according to claim 8, characterized in that: The single drug in step (2) is at least one of Rhizoma Coptidis, Radix Scutellariae, and Rhizoma Phellodendri; The key active ingredient in step (3) is at least one of berberine, baicalin and gardenia jasminoides.

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