Metabolic regularity of bioactive compounds in Bufei Jianpi granule in rats using ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry analysis technology.

Bufei Jianpi granule (BJG) is clinically effective for treating chronic obstructive pulmonary disease (COPD). At present, there is no report regarding the drug metabolism of BJG in vivo. This work developed an ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry method with high accuracy and sensitivity to determine drug metabolism of this compound in vivo. After continuous administration of BJG, the concentrations of 10 components in rat plasma, namely betaine, peimine, peiminine, astragaloside A, sinensetin, nobiletin, naringin, calycosin, formononetin, and magnolol, were determined at different time points. Meanwhile, the pharmacokinetic parameters and metabolic rules of these 10 components were evaluated: Cmax , 8.624-574.645 ng/mL; Tmax , 0.250-8.667 h; AUC0-t , 17.640-8947.393 ng h/mL; T1/2 , 3.405-66.014 h; mean residence time (MRT), 6.893-11.223 h. All these components possessed anti-inflammatory, antioxidant, and other biological activities to varying degrees, contributing to improving lung function, mitigating pneumonia and pulmonary fibrosis, and preventing and treating chronic obstructive pulmonary disease. Exploring the pharmacokinetic parameters and the laws of chemical components in BJG forms the scientific basis for applying the compound clinically and identifying quality markers for the control of the compound.

[Mechanism of famous classical formula Huaihua Powder in treatment of ulcerative colitis based on metabonomics].

Ultra-high performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry(UHPLC-Q-TOF-MS) was employed in this study to observe the effect of Huaihua Powder on the serum metabolites of mice with ulcerative colitis and reveal the mechanism of Huaihua Powder in the treatment of ulcerative colitis. The mouse model of ulcerative colitis was established by dextran sodium sulfate salt(DSS). The therapeutic effect of Huaihua Powder on ulcerative colitis was preliminarily evaluated based on the disease activity index(DAI), colon appearance, colon tissue morphology, and the content of inflammatory cytokines such as tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1ß(IL-1ß). UHPLC-Q-TOF-MS was employed to profile the endogenous metabolites of serum samples in blank control group, model group, and low-, medium-, and high-dose Huaihua Powder groups. Multivariate analyses such as principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and orthogonal partial least squares discriminant analysis(OPLS-DA) were performed for pattern recognition. Potential biomarkers were screened by Mass Profiler Professional(MPP) B.14.00 with the thresholds of fold change≥2 and P<0.05. The metabolic pathways were enriched by MetaboAnalyst 5.0. The results showed that Huaihua Powder significantly improved the general state and colon tissue morphology of mice with ulcerative colitis, reduced DAI, and lowered the levels of TNF-α, IL-6, and IL-1ß in serum. A total of 38 potential biomarkers were predicted to be related to the regulatory effect of Huaihua Powder, which were mainly involved in glycerophospholipid metabolism, glycine, serine, and threonine metabolism, mutual transformation of glucuronic acid, and glutathione metabolism. This study employed metabolomics to analyze the mechanism of Huaihua Powder in the treatment of ulcerative colitis, laying a foundation for the further research.

Qualitative, quantitative, and pharmacokinetic study on the absorbed components of Ardisia japonica (Thunb.) Blume in rat plasma based on molecular networking combined with quadrupole time-of-flight LC/MS and triple quadrupole LC/MS.

Isolation and screening of different compounds from plant extracts are always the key for natural drug research, and the absorbed prototype components have been considered as potential active ingredients. UHPLC combined with quadrupole time-of-flight mass spectrometry (Q-TOF-LC/MS) has been widely used in the research of natural drugs; however, we still need a more effective tool to compare and treat from a raw data. In this study, we provided a fast analytical method to measure the absorbed prototype components and their metabolites both qualitatively and quantitatively based on molecular networking (MN). For example, in Ardisia japonica (Thunb.) Blume, a total of eight absorbed prototype components in rat plasma were identified. Furthermore, pharmacokinetic study was also successfully performed on the eight absorbed prototype components in rat plasma. Our findings have provided important information on the investigation of A. japonica in vivo. More importantly, the MS network analysis pattern serves as an integral solution for qualitative and quantitative determination of phytochemical compounds in natural drugs.

Analysis of plasma migration components in Patrinia villosa (Thunb.) Juss. effective parts by UPLC-Q-TOF-MS.

Patrinia villosa (Thunb.) Juss. (PVJ) is described as pungent, bitter and slightly cold in Chinese medicine, and is associated with the large intestine, stomach and liver meridians. The preliminary experiments of our research team proved that PVJ total flavonoids have excellent inhibitory effects on liver cancer cells. The present experiment uses the UPLC-Q-TOF-MS technology and serum pharmacochemistry methods to analyze the chemical components in vitro and in vivo of PVJ antiliver tumors. A total of 14 chemical components were identified in the total flavonoids extract of PVJ, and it is mainly composed of flavonoids, flavonones, flavonols and phenolic acids. At the same time, seven prototypical components and seven metabolic components were detected in the drug-containing plasma. Hydrocaffeate and scutellarein are the phase I metabolites of caffeic acid and scutellarin, respectively. Sulfated apigenin, sulfated luteolin, sulfated kaempferol and methylated kaempferol are the II phase metabolites of apigenin, luteolin, kaempferol, respectively. The experiment provides a reference for the research and development of antitumor drug candidates, and provides a basis for revealing the bioactive components of PVJ and the antitumor mechanism.

Simultaneous determination of eight bioactive components of Cirsium setosum flavonoids in rat plasma using triple quadrupole LC/MS and its application to a pharmacokinetic study.

Cirsium setosum (Willd.) MB. has been reported to exert significant anti-hemorrhagic, anti-inflammation, antimicrobial, sedative and detoxicating efficacy. It has been widely used to treat gastrointestinal bleeding, uterine bleeding, infectious hepatitis and cardiovascular disease in China. Recent studies have shown that flavonoids are the main active components in C. setosum. Nevertheless, to the best of our knowledge, there is no report concerning the simultaneous determinations and pharmacokinetics of constituents in C. setosum flavonoids in rat plasma. In this study, a rapid, sensitive and selective triple quadrupole liquid chromatography-mass spectrometry method was developed to determine eight analytes from the flavonoids of C. setosum in rat plasma. In addition, the pharmacokinetic study of the eight analytes in rats after oral administration of C. setosum flavonoids was successfully completed through this method. According to the pharmacokinetic parameters of the eight analytes, rutin, naringin, quercetin, acacetin, wogonin were the long-acting components of the C. setosum flavonoids, with long elimination time and high bioavailability. Of note, the method developed in this study fills a blank in pharmacokinetic studies of C. setosum flavonoids. Our findings provide valuable views on the understanding of the absorption mechanism of C. setosum flavonoids and their clinical efficacy.

[Study of cancer cell apoptosis induced by Schizonepeta tenuifolia with microfluidic chip technology].

This study was designed to elucidate the chemical composition and anti-cancer effects of Schizonepeta tenuifolia's ethanol extracts. Microfluidic technology was used in the study of Schizonepeta tenuifolia from 9 different geographic regions. The ethanol extracts were examined with HPLC to establish their Fingerprints in order to analyze the relationship between the spectrum and efficacy index through Grey Correlation software, and a rapid HPLC-Q-TOF/MS method was established. The result shows that chromatographic peaks of the 19, 6, 11, 16, 18th are the representative diosmetin, luteoloside, hesperidin, luteolin, and apigenin. The 10, 12, 20th peaks may be naringenin-7-O-glucuronide or quercitrin, rosmarinate or acetylcorynoline, and 5,7-dihydroxy-6,4-dimethoxy flavone. The major chemical composition of Schizonepeta tenuifolia was found to have the anti-lung-tumor effects. A new method was established for the quality control of traditional Chinese medicine.

[Study on relationship between efficacy against lung cancer and different parts of Schizonepeta tenuifolia based on microfluidic chip technology].

In order to further clarify the rational use of different medicinal parts of Schizonepeta, microfluidic technology was used in this study to investigate the differences in drug efficacy against lung cancer in vitro. The ethanol extracts were examined with HPLC to establish their fingerprints in order to analyze the relationship between the spectrum and efficacy index through Grey Correlation software, and a rapid HPLC-Q-TOF/MS method was established. The result in vitro shows that the effect and components of different medicinal parts had a certain differences, and apoptosis and necrosis rate from big to small in turn is leaf, flower, root, stem. The chromatographic peaks of the 26, 12, 2, 6 and 15th are the luteolin, icynaroside, rosmarinic, caffeic acid, and hesperidin, while the 20 and 10th may be dan phenolic acid L and benzoic acid. On the one hand, preliminary study reflects that the root of Schizonepeta tenuifolia may be developed into the medicinal parts in future. On the other hand, the major chemical composition of S. tenuifolia was found to have the anti-lung-tumor effects. This new method was established for the quality control and the rational use of different parts of traditional Chinese medicine.

Two new steroidal glycosides from Cynanchum otophyllum Schneid.

Two new C21 steroidal glycosides were isolated from Cynanchum otophyllum Schneid. Their structures were elucidated as qinyangshengenin-3-O-ß-d-digitoxopyranoside (1) and rostratamine-3-O-ß-d-cymaropyranosyl-(1 â†’ 4)-ß-d-digitoxopyranoside (2) on the basis of chemical and spectroscopic methods, including 1D and 2D NMR experiments.

[Optimization of Extraction Process of Total Flavonoids from Schizonepeta tenuifolia Based on Analytical Hierarchy Process with Dose-Effect Comparison Method].

OBJECTIVE: To optimize conditions for extracting total flavonoids from Schizonepeta tenuifolia by the method of analytical hierarchy process with the correlation method of dose-effect. METHODS: Take the amount and concentration of ethanol,the extraction time and times as examine factors, with the inhibition rate of Caco-2 human cloning colonic cancer cell as index to optimize the optimum conditions of total flavonoids from Schizonepeta tenuifolia by orthogonal experiment design, and analyze the Pearson correlation coefficient between the total flavonoids content, hesperidin content, extraction rate and pharmacodynamics indexes through SPSS 19.0 software to determine the dose-effect comparative indicators, and then analytic hierarchy process is adopted to define the weight coefficients range of the content of total flavonoids and hesperidin. RESULTS: The best extraction process conditions were as follows:15 times the amount of 75% ethanol, water circumfluence extracting for 3 times,each time for 2 hours; The weight coefficient ranges of the content of total flavonoids and hesperidin were 0.7500-0.9000 and 0.1000-0.2500 respectively. CONCLUSION: Using the comprehensive scores of the content of total flavonoids and hesperidin, which is calculated by weight coefficient ranges that based on the analytical hierarchy process,to replace the inhibition rate as the indicator to optimize the optimum extraction process is scientific and reasonable. This optimization process is simple and practical,which provides a new method for selecting the best extraction indicators to get effective ingredients from traditional Chinese medicine.

Two new steroidal glycosides from Cynanchum wallichii.

Two new C21 steroidal glycosides were isolated from Cynanchum wallichii Wight. Their structures were elucidated as caudatin-3-O-ß-d-glucopyranosyl-(1 → 4)-ß-d-oleandropyranosyl-(1 → 4)-ß-d-cymaropyranosyl-(1 → 4)-ß-d-digitoxopyranoside (1) and caudatin-3-O-ß-d-glucopyranosyl-(1 → 4)-ß-d-cymaropyranosyl-(1 → 4)-ß-d-oleandropyranosyl-(1 → 4)-ß-d-cymaropyranosyl-(1 → 4)-ß-d-digitoxopyranoside (2) by spectroscopic methods including 1D and 2D NMR experiments.

[Establishment of spectrum-effect relationship network model of qizhiweitong granules promoting gastrointestinal motility].

OBJECTIVE: To establish the spectrum-effect relationship network model of Qizhiweitong granules promoting gastrointestinal motility for providing scientific basis for its quality control and efficacy evaluation. METHODS: The Latin hypercube sampling was used to establish full-time multi-wavelength fusion fingerprints of different compatibility groups of Qizhiweitong granules. At the same time, the appreciation rate, the contents of cGMP and NO in small intestine smooth muscle cells after administrated drugs were determined. Then the spectrum-effect relationship of different compatibility groups were correlated, and the network model was established with gray correlation method and BP neural network. RESULTS: 20 compounds with correlation index more than 0.9 were found from 36 constituents in Qizhiweitong granules. The spectrum-effect relationship network model of Qizhiweitong granules promoting gastrointestinal motility was successfully established. And through this model to forecast the result of the test, the absolute value of the relative error was less than 6.83%. CONCLUSION: In this study, a spectrum-effect relationship network model of Qizhiweitong granules promoting gastrointestinal motility is established successfully, which provides a new method for its reasonable quality control and efficacy evaluation, lays the experiment basis for component-effect study, and provides reference for other TCM's study.

[HPLC fingerprint analysis of flavonoids of phyllanthi fructus from different habitats].

OBJECTIVE: To establish the HPLC fingerprint of flavonoids of Phyllanthi Fructus from different habitats. METHODS: HPLC method was adopted. The flavonoids composition of Phyllanthi Fructus from 10 different habitats was determined on an Agilent C, chromatographic column with 0. 5% formic acid water (A)-acetonitrile (B) as the mobile phase in gradient elution under the wavelength of 254 nm. The HPLC fingerprints of flavonoids composition of Phyllanthi Fructus were established to evaluate the qualitiy of them. RESULTS: The HPLC fingerprints of flavonoids composition of Phyllanthi Fructus from 10 different habitats were established. 18 common peaks were found and the similarities of them were more than 0. 90 except the ones from Guangxi and Guangdong. CONCLUSION: The method is simple, accurate and repeatable. It can be used for research and quality control of the effective components in Phyllanthi Fructus.

Classical prescription Floris Sophorae Powder treat colorectal cancer by regulating KRAS/MEK-ERK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Colorectal cancer (CRC) belongs to the category of intestinal wind, anal ulcer, abdominal mass and other diseases in traditional Chinese medicine (TCM). Floris Sophorae Powder (F.S), is a classical prescription is recorded in Puji Benshi Fang for the treatment of intestinal carbuncle. It has been incorporated into the prescriptions for the treatment of intestinal diseases and achieved remarkable results in modern medicine. However, the mechanism of F.S in the treatment of colorectal cancer remains unclear and requires further study. AIM OF THE STUDY: To investigate F.S in treating CRC and clarify the underlying mechanism. MATERIALS AND METHODS: This study was based on Dextran Sulfate Sodium Salt (DSS) combined with Azoxymethane (AOM) induced CRC mouse model to clarify the pharmacological effects of F.S. The serum metabolomics was used to study the mechanism of action, and the chemical composition of F.S was found by UPLC-Q-TOF-MS. The rationality of serm metabolomics results was verified through the clinical target database of network pharmacology, and the upstream and downstream targets of related pathways were found. The mechanism pathway was verified by Western blot to clarify its mechanism of action. RESULTS: In vivo pharmacological experiments showed that F.S inhibited tumor growth and improved hematochezia. The vital signs of mice in the high-dose F.S group approached to those in the control group. A total of 43 differential metabolites were found to be significantly changed by serum metabolomics. F.S could modulate and recover most of the differential metabolites, which proved to be closely related to the KRAS/MEK-ERK signaling pathway. A total of 46 compounds in F.S were identified, and the rationality of serm metabolic pathway was verified by network pharmacology. Western blot results also verified that the expression of KRAS, E2F1, p-MEK and p-ERK were significantly decreased after F.S treatment. CONCLUSION: Classical prescription Floris Sophorae Powder treat colorectal cancer by regulating KRAS/MEK-ERK signaling pathway.

[Study on chromatography-efficacy relationship of anti-inflammatory activity of qizhi weitong particle compound herbs with neural network and gray correlation method].

OBJECTIVE: To establish the chromatography-efficacy relation method for analyzing the anti-inflammatory activity of Qizhi Weitong particles, in order to lay a foundation for quality control and pharmacodynamic evaluation of traditional Chinese medicine compounds. METHOD: On the basis of a full-time multi-wavelength fusion fingerprint of Qizhi Weitong particles, the latin hypercube sampling was used to divide six herbs in Qizhi Weitong particles into groups of different proportions to determine their inhibition ratios of TNF-alpha, IL-6 and NO released by LPS-induced RAW264. 7 cells. Pharmaeodynamic data and chemical information of HPLC fingerprints of each group were analyzed with the gray correlation method to get the anti-inflammatory effect of each chromatographic peak, and then fitted with BP neural network to establish the chromatography-efficacy relation. RESULT: There were 25 peaks closely related to the anti-inflammatory activity. With the 25 peaks as input items, the 3-BP network was adopted to establish the neural network model for anti-inflammatory effect of Qizhi Weitong particles. CONCLUSION: With an error of less than 7%, the model could better fit with the complicated non-linear relation of the compound, and applied in studying the chromatography-efficacy relation. In this study on the chromatography-efficacy relation, a new method is established to evaluate the anti-inflammatory activity of Qizhi Weitong particles. It is of practical significance as an effective approach for controlling quality and exploring the material basis for efficacy of traditional Chinese medicine compounds.

[Optimized extraction technology of flavonoid compounds with anti-SMMC-7721 tumor activities in bark of Juglans mandshurica by orthogonal experimental design based on dose-effect fusion evaluation method].

OBJECTIVE: To optimize the extraction technology of total flavonoids with antineoplastic activities in Juglans mandshurica, and explore the correlation between total flavonoids and pharmacodynamics indicators. METHODS: The quantity of antineoplastic components, ratio of extraction and cell inhibition rate were taken as the comprehensive indexes to optimize the main factors that influence the extraction of effective components by orthogonal experiment design. SPSS 17.0 software was used to analyze the Pearson correlation between effective components and pharmacodynamics indexes. RESULTS: The best extracting condition of total flavonoids were as follows: the ratio of 60% ethanol to Juglans mandshurica was 20: 1, extracting for 3 times, each time for 2 hour at 70 degrees C. Flavonoids extraction yield and cell inhibition rate was positively related in the straight line. CONCLUSION: This study provides a new insight into the optimization of extraction technology for traditional Chinese medicine, and lays a safe and reliable experimental basis for the clinical application of Juglans mandshurica.

[Study on all-time multi-wavelength fusion fingerprint of Qizhiweitong granules and multi-component quantitative analysis].

OBJECTIVE: To control the quality of Qizhiweitong granules with the all-time multi-wavelength fusion fingerprint quantification as the major technique. METHOD: Agilent TC-C18 (4.6 mm x 250 mm, 5 microm) chromatographic column was adopted, with 0.02% formic acid water-acetonitrile as the mobile phase for linear gradient elution. The flow rate was 1 mL x min(-1), column temperature was 30 degrees C, and detector wavelength was 230, 254, 283 nm. Matlab was adopted for all-time multiple-wavelength fusion for data in dif format. RESULT: A good relationship was shown for albiflorin in 56.5-452 mg x L(-1) (r = 0.999 8), paeoniflorin in 107-856 mg x L(-1) (r = 0.999 8), licorice glycoside in 73.4-687 mg x L(-1) (r = 0.999 8), naringin in 109-872 mg x L(-1) (r = 0.999 8), neohesperidin in 48.0-384 mg L(-1) (r = 0.999 8), and glycyrrhizic acid in 38.6-308 mg x L(-1) (r = 0.999 8), with recoveries of 0.999 8. CONCLUSION: The method is simple, accurate and highly reproducible, and can provide basis for quality control of Qizhiweitong granules.

[Effect of flavonoids of polygoni orientails fructus on human hepatoma cell line SMMC-7721].

OBJECTIVE: To investigate the inhibitory effects of flavonoids of Polygoni Orientails Fructus on cell cycle and apoptosis of human hepatoma cell line SMMC-7721. METHODS: MTT method was used to study the inhibitory rate and time-dose relationship of flavonoids on SMMC-7721. Flow cytometry PI staining and Annexin V-EGFP/PI double staining were used to measure the DNA concentration and apoptosis rate in SMMC-7721. RESULTS: Flavonoids of Polygoni Orientails Fructus had obvious inhibitory effect and with good linear relationship between time and dose. Flavonoids of Polygoni Orientails Fructus could regulate the G1/S transition in hepatoma cell, which could make the cell in S phase arrest, result in the accumulation of cells in S phase, blocked DNA synthesis and replication of cells, blocked tumor cells into the G2/M phase, so as to restrain the proliferation of tumor cells and induce the apoptosis of SMMC-7721 cells. And this inductive effect showed obvious time-dose-effect relationship. CONCLUSION: The flavonoids of Polygoni Orientails Fructus could inhibit the hepatoma cell line SMMC-7721 in vitro by inhibiting cell proliferation and inducing apoptosis of the cell in a time-dose dependence maner.

Patrinia villosa treat colorectal cancer by activating PI3K/Akt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: At present, the colorectal cancer (CRC) is a malignant tumor of the colon and rectum that is often found at the junction of the two, and it will invade many visceral organs and organizations, causing very serious damage to the body of the patient. Patrinia villosa Juss. (P.V), is a well-known traditional chinese medicine (TCM), and is recorded in the Compendium of Materia Medica as a necessary article for the treatment of intestinal carbuncle. It has been incorporated into traditional cancer treatment prescriptions in modern medicine. While the mechanism of action of P.V in the treatment of CRC remains unclear. AIM OF THE STUDY: To investigate P.V in treating CRC and clarify the underlying mechanism. MATERIALS AND METHODS: This study was based on Azoxymethane (AOM) combined with the Dextran Sulfate Sodium Salt (DSS)-induced CRC mouse model to clarify the pharmacological effects of P.V. The mechanism of action was found by metabolites and metabolomics. The rationality of metabolomics results was verified through the clinical target database of network pharmacology, and find the upstream and downstream target information of relevant action pathways. Apart from that, the targets of associated pathways were confirmed, and the mechanism of action was made clear, using quantitative PCR (q-PCR) and Western blot. RESULTS: The number and the diameter of tumors were decreased when mice were treated with P.V. P.V group section results showed newly generated cells which improved the degree of colon cell injury. Pathological indicators presented a trend of recovery to normal cells. Compared to the model group, P.V groups had significantly lower levels of the CRC biomarkers CEA, CA19-9, and CA72-4. Through the evaluation of metabolites and metabolomics, it was found that a total of 50 endogenous metabolites had significant changes. Most of these are modulated and recovered after P.V treatment. It alters glycerol phospholipid metabolites, which are closely related to PI3K target, suggesting that P.V can treat CRC though the PI3K target and PI3K/Akt signaling pathway. q-PCR and Western blot results also verified that the expression of VEGF, PI3K, Akt, P38, JNK, ERK1/2, TP53, IL-6, TNF-α and Caspase-3 were significantly decreased, whereas that of Caspase-9 was increased after treatment. CONCLUSION: P.V is dependent on PI3K target and PI3K/Akt signaling pathway for CRC treatment.

Effect of Oroxylum indicum on hepatocellular carcinoma via the P53 and VEGF pathways based on microfluidic chips.

BACKGROUND: Hepatocellular carcinoma (HCC), abbreviated as liver cancer, is one of the most common cancers in clinics. HCC has a wider spread and higher incidence due to its high malignancy and metastasis. In HCC, effective strategies to block cancer cell migration, invasion, and neovascularization need to be further studied. Consumption of flavonoid-rich Oroxylum indicum (OI) has been associated with multiple beneficial effects, including anti-inflammatory and anticancer properties, but the potential effects on HCC have not been thoroughly investigated. OBJECTIVE: In this study, we aimed to reveal the effect of OI on HCC and its potential mechanism through microfluidic technology. METHODS: We designed microfluidic chips for cell migration, invasion, and neovascularization to evaluate the effect of OI on HepG2 cells. To further explore the mechanism of its anti-liver cancer action, the relevant signaling pathways were studied by microfluidic chips, RT‒qPCR and immunofluorescence techniques. Compared to the control group, cell migration, invasion, and angiogenesis were significantly reduced in each administration group. According to the P53 and VEGF pathways predicted by network pharmacology, RT‒qPCR and immunofluorescence staining experiments were conducted. RESULTS: The results showed that OI upregulated the expression of Bax, P53 and Caspase-3 and downregulated the expression of Bcl-2 and MDM2. It has been speculated that OI may directly or indirectly induce apoptosis of HepG2 cells by regulating apoptosis-related genes. OI blocks the VEGF signaling pathway by downregulating the expression levels of VEGF, HIF-1α and EGFR and inhibits the migration and invasion of HepG2 cells and the formation of new blood vessels. CONCLUSION: Our findings suggest that OI may inhibit the migration, invasion, and neovascularization of HepG2 cells, and its regulatory mechanism may be related to the regulation of the P53 and VEGF pathways.

Possible mechanisms associated with immune escape and apoptosis on anti-hepatocellular carcinoma effect of Mu Ji Fang granules.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common digestive system cancers with high mortality rates worldwide. The main ingredients in Mu Ji Fang Granules (MJF) are alkaloids, flavonoids, and polysaccharides. MJF has been used in the clinical treatment of hepatitis, cirrhosis and HCC for more than 30 years. Few previous studies have focused on the mechanism of MJF on tumor immu-nology in the treatment of HCC. AIM: To explore the mechanism of action of MJF on tumor immunology in the treatment of HCC. METHODS: The absorbable ingredients of MJF were identified using Molecule Network related to High Performance Liquid Chromatography-Electron Spray Ionization-Time of Flight- Mass Spectrometry, and hub potential anti-HCC targets were screened using network pharmacology and pathway enrichment analysis. Forty male mice were randomly divided into the Blank, Model, and MJF groups (1.8, 5.4, and 10.8 g/kg/d) following 7 d of oral administration. Average body weight gain, spleen and thymus indices were calculated, tumor tissues were stained with hematoxylin and eosin, and Interferon gamma (IFN-γ), Tumor necrosis factor α (TNF-α), Interleukin-2, aspartate aminotransferase, alanine aminotransferase, alpha-fetoprotein (AFP), Fas, and FasL were measured by Enzyme-linked Immunosorbent Assay. Relevant mRNA expression of Bax and Bcl2 was evaluated by Real Time Quantitative PCR (RT-qPCR) and protein expression of Transforming growth factor ß1 (TGF-ß1) and Mothers against decapentaplegic homolog (SMAD) 4 was assessed by Western blotting. The HepG2 cell line was treated with 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL of MJF, and another 3 groups were treated with TGF-ß1 inhibitor (LY364947) and different doses of MJF. Relevant mRNA expression of TNF-α, IFN-γ, Bax and Bcl2 was evaluated by RT-qPCR and protein expression of TGF-ß1, SMAD2, p-SMAD2, SMAD4, and SMAD7 was assessed by Western blotting. RESULTS: It was shown that MJF improved body weight gain and tumor inhibition rate in H22 tumor-bearing mice, protected immune organs and liver function, reduced the HCC indicator AFP, affected immunity and apoptosis, and up-regulated the TGF-ß1/SMAD signaling pathway, by increasing the relative expression of TGF-ß1, SMAD2, p-SMAD2 and SMAD4 and decreasing SMAD7, reducing immune factors TNF-α and IFN-γ, decreasing apoptosis cytokines Fas, FasL and Bcl2/Bax, and inhibiting the effect of LY364947 in HepG2 cells. CONCLUSION: MJF inhibits HCC by activating the TGF-ß1/SMAD signaling pathway, and affecting immune and apoptotic cytokines, which may be due to MJF adjusting immune escape and apoptosis.