Synergic effects and possible mechanism of emodin and stilbene glycosides on colorectal cancer.

BACKGROUND: Polygonum multiflorum (PM) is a core herb that enhances immunity. It can also detoxify, reduce swelling, and intercept malaria. Its main components, emodin (EMD) and 2,3,5,4'-Tetrahydroxy stilbene-2-O-ß-D-glucoside (stilbene glycoside, TSG), have good anti-cancer potential. PURPOSE: The study aims to investigate synergic effects of EMD and TSG on CRC and its possible mechanism. METHODS: Network pharmacology and bioinformatics were used to identify targets. HPLC was used to analyze the effective ingredients in PM and to determine the content of the main ingredients. HT-29 cells were used for in vitro experiments. Cell Counting Kit-8 (CCK8) and scratch test were used to detect the effects of various chemical components of PM on the proliferation and migration of HT-29 cells, and Western Bolt (WB) test was used to evaluate the effects of EMD and TSG on P53 pathway. In vivo experiments, the effects of EMD and TSG were evaluated by measuring tumor weight and tumor volume in CRC mice model and histological analysis were carried out with HE staining. The expressions of HSP90, P53, COX2, and ROS were detected by quantitative reverse transcription polymerase chain reaction (PCR), and IL-1ß, IL-4, IL-6, IL-10, TGF-ß and IFN-γ were detected by enzyme linked immunosorbent assay (ELISA). WB and Immunohistochemistry (IHC) were used to detect the expression of P53 related proteins. RESULTS: Network pharmacology showed PM closely related to colorectal cancer pathway and the core targets included STAT3 and P53; bioinformatics indicated P53 played an important role in the development and prognosis of CRC; chemical analysis showed identified and quantified gallic acid (GA), cis-TSG, trans-TSG, Emodin glucoside(EMDG), physcion glucoside (PHYG), EMD in PM; EMD induced apoptosis and TSG inhibited migration of HT-29 cells; EMD and TSG could coordinately shrink tumor size of CRC mice, elevate expressions of F4/80, decrease the content of IL-6 and TGF-ß, promote tumor oxidized and reduce expression of P53 and STAT3 in the tumor. CONCLUSIONS: In vitro experiments showed that TSG inhibited cancer cell migration and EMD induced apoptosis. EMD and TSG had synergic effects on CRC, whose possible mechanism might be to regulate the expression of cytokines and inhibit P53 pathway.

Gallic acid ameliorates dextran sulfate sodium-induced ulcerative colitis in mice via inhibiting NLRP3 inflammasome.

Background: Ulcerative colitis (UC) is a chronic recurrent inflammatory bowel disease (IBD). The conventional drugs for UC may induce severe side effects. Herbal medicine is considered as a complementary and alternative choice for UC. Purpose: This study aims to estimate the effect of natural polyphenol gallic acid (GA) on the NLRP3 inflammasome with dextran sulfate sodium (DSS)-induced colitis in mice. Study design: The body weights and symptoms of BALB/c mice were recorded. Histological evaluation, ELISA, q-PCR, immunohistochemistry, and western blotting were carried out to observe the morphology, cytokine contents, mRNA expressions, and protein expressions, respectively. Lipopolysaccharide (LPS)-induced RAW264.7 macrophage was used to probe GA's effect on relative protein expression. Results: GA attenuated weight loss (p < 0.05), relieved symptoms, and ameliorated colonic morphological injury (p < 0.05) in mice with colitis induced by DSS. GA also lowered the contents of TNF-α, IL-1ß, IL-18, IL-33, and IFN-γ in the serum and colon of mice, which were elevated by DSS, downregulated protein, and mRNA expressions of the NLRP3 pathway in the colon tissue. Furthermore, GA downregulated the expressions of NLRP3 (p < 0.05), iNOS (p < 0.01), COX2 (p < 0.01), and P-p65 (p < 0.05), and suppressed NO release (p < 0.001) in LPS-induced RAW264.7 cells. Conclusion: GA ameliorated DSS-induced UC in mice via inhibiting the NLRP3 inflammasome. These findings furnish evidence for the anti-inflammatory effect of herbal medicines containing GA on UC.

Bufothionine induces autophagy in H22 hepatoma-bearing mice by inhibiting JAK2/STAT3 pathway, a possible anti-cancer mechanism of cinobufacini.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinobufacini is extracted from the skins and parotid venom glands of the toad for treating symptoms like swelling and pain in ancient times. Nowadays, cinobifucini injection has also achieved satisfactory therapeutic effects on hepatocellular carcinoma (HCC) in China. AIM OF THE STUDY: Our previous work found that bufothionine, an alkaloid abundant in cinobufacini injection, induced mitochondria-mediated apoptosis. In this work, the underlying effects of bufothionine on autophagy in HCC and its possible dependent pathway were investigated. METHODS: CCK-8 and Hoechst staining assays were performed to verify effects of drugs on proliferation and apoptosis of SMMC7721 cell. H22-tumor-bearing mice model was established by inoculating ascites fluid. HE staining was used to observe pathological changes in liver and tumor tissues. ELISA and Western blot experiments were conducted to investigate IL-6/JAK2/STAT3 signaling pathway. The effects of drugs on expressions of autophagic relative proteins were investigated by Western blot in vitro and in vivo. RESULTS: In vitro, CCK-8 and Hoechst staining assays showed that bufothionine inhibited SMMC7721 cell proliferation and promoted apoptosis at 100 µM. In vivo, bufothionine relieved symptoms of H22-tumor-bearing mice and exerted anti-inflammation activity. ELISA and Western blot demonstrated that bufothionine significantly reduced serum IL-6 concentration, suppressed p-Stat3tyr705, p-Stat3ser727 and Jak2 expressions in tumor tissues and upregulated Atg5, Atg7 and LC3Ⅱ expressions in SMMC7721 cell and H22 tumor. CONCLUSION: This is the first report showing that bufothionine might induce autophagy in HCC by inhibiting JAK2/STAT3 pathway, presenting a possible anti-cancer mechanism of bufothionine in cinobufacini injection.

Ingredients, Anti-Liver Cancer Effects and the Possible Mechanism of DWYG Formula Based on Network Prediction.

BACKGROUND: Hepatitis virus infection plays a critical role in liver cancer initiation and development; so the purpose of this study was to investigate the anti-liver cancer effects of DiWuYangGan (DWYG) which was effective for hepatitis. METHODS: Network predictions were performed. Next, several tests, including HPLC, Caco-2 absorption models, MMT, protein chip, Western blotting and H22-tumor-bearing mouse, were carried out to investigate the effects and possible mechanism of DWYG. RESULTS: Network results showed DWYG might be involved in some processes such as STAT cascade. Some target genes may correspondingly participate in these procedures, such as IL-6, CASP3, AKT1, PPAR, and TP53. Diseases associated with DWYG formula may be liver cancer and hepatitis. Potential active compounds might be CUR and ISO. Chemical analysis results showed that ingredients in the formula, including DEO, SCHB, SOLA, SOLB, SCHA, LIQ, ISO, POT, and CHL, could be determined, indicating that DWYG samples for the following experiments were controllable and consistent. Caco-2 absorption of ingredients in DWYG, including DEO, SCHB, SOLA, SOLB, and LIQ, worked very well. In vitro experiment results showed that DWYG could inhibit the growth of cell lines and its effective ingredients might be SCHB, SOLB, SINA, SINB, SOLB, CUR, DEM, BIS, and GER. Further protein results showed that DWYG could upregulate the expressions of some proteins, including ERK1/2, AKT Ser473, BAD Ser112, PRAS40, Thr246, P38, Gsk-3ß, and Ser9. In vivo experiment results showed that DWYG could shrink tumor size, recover ALT and AST, and decrease IL-6 levels. Their possible mechanism might be through the JAK/STAT3 pathway. CONCLUSION: Besides the known pharmacological function of anti-hepatitis, DWYG extract expressed anti-liver cancer effects and the results were consistent partly with network predictions.

trans-δ-Viniferin inhibits Ca2+-activated Cl- channels and improves diarrhea symptoms.

Ca2+-activated Cl- channels (CaCCs) wildly exist in many tissues which play an important role in ion transport and excitation conduction, especially fluid secretion and smooth muscle contraction in epithelial tissues. TMEM16A as a classic CaCC expresses in the intestine, and has become a potential target of intestinal physiological and pathological researches and therapeutic drug screening. In this study, we identified trans-δ-viniferin (TVN), a resveratrol dimmer, could inhibit TMEM16A activity in TMEM16A expressed FRT cells with IC50 of 19.7 µM, it also prevented Ca2+-activated Cl- current in HT-29 cells with IC50 of 4.65 µM and in colonic mucosa. In the mechanism studies, TVN showed no significant inhibition on CFTR and basal Na+/K+-ATPase in both intestinal epithelial cells and colonic tissues, except for inhibition of calcium concentration and Ca2+-activated K+ channel to some degree. In anti-diarrheal studies, TVN could effectively prevent diarrhea caused by rotavirus infection and reduce the pellet number in IBS-D mice. These physiological effects are at least partially attributed to the inhibitory effect of TVN on CaCC-mediated intestinal fluid secretion and the reduction of smooth muscle contraction force by inhibiting TMEM16A. Collectively, the present study identified a new pharmacological target of TVN which provided the theoretical basis for the application of TVN in the treatment of rotavirus-infected diarrhea and IBS-D.

Effects and possible mechanism of Ruyiping formula application to breast cancer based on network prediction.

Ruyiping (RYP), a Chinese herbal formula, can remove toxin and clear nodular, showing ability of preventing postoperative recurrence of breast cancer. In this study, network was performed to predict possible targets, genes and pathways associated with RYP and breast cancer. Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) were used to quantitatively study RYP formula and its single herbs. MTT methods, Luciferase reporter systems, zebrafish model and western blotting were respectively adopted to verify network prediction. Results showed that the quality of RYP could be controlled and icariin could be selected as mark ingredient; RYP expressed anti-breast tumor effects, which could be associated with inhibiting expression of Transforming Growth Factor ß (TGFß), promoting cells apoptosis and anti-angiogenesis. Parts of these results were consistent with network predictions in some degree, but not all. Network can help us narrow areas, focus on crucial factors, save money as well as time, but the results predicted by network should be confirmed by further experiments.

Amelioration of acute myocardial infarction by saponins from flower buds of Panax notoginseng via pro-angiogenesis and anti-apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Panax notoginseng is traditionally used as an anti-hemorrhagic agent to promote blood circulation without causing "congealed" blood. Furthermore, the flower of P. notoginseng is a popular, traditional medicine taken daily for the preventing of hypertension and for reducing blood cholesterol profiles. Besides, the flower of P. notoginseng contains a higher level of saponins, particularly protopanaxadiol-type ginsenosides, as compared to the root. However, detailed pharmacological studies on this flower have rarely been conducted. MATERIAL AND METHODS: In this study, the saponins extracted from the flower of P. notoginseng (FS) were examined on the endothelial cell migration assay, chemically induced vascular insufficiency model in zebrafish larvae and myocardial infraction (MI) model in rats, for determination of their pro-angiogenic and therapeutic effects on MI treatment. RESULTS: Our results demonstrate that FS significantly promoted VEGF-induced migration of human umbilical vein endothelial cells (HUVECs) and partially restored defective intersegmental vessels (ISV) in a chemically induced vascular insufficiency model of zebrafish larvae. When compared to MI group, two weeks post-treatment of FS (25-50mg/kg/day) induced approximately 3-fold upregulation of VEGF mRNA expression and a concomitant increase in blood vessel density in the peri-infarct area of the heart. Moreover, TUNEL analysis indicates a reduction in the mean apoptotic nuclei per field in peri-infarct myocardium upon FS treatment. CONCLUSIONS: The pro-angiogenic effects of FS demonstrated in in vitro and in vivo experimental models suggest that the purified saponin preparation from flowers of P. notoginseng may potentially provide preventive and therapeutic agent for cardiovascular diseases.

Bufothionine induced the mitochondria-mediated apoptosis in H22 liver tumor and acute liver injury.

BACKGROUND: Bufothionine is an alkaloid in Cinobufacini (Huachansu). This study aims to investigate the effects of bufothionine on liver tumors and acute liver injury. METHODS: In the hepatoprotective experiment, fifty rats were randomly divided into five groups (n = 10): normal saline group, model group, compound glycyrrhizin injection (9.14 mL/kg); cinobufacini injection (3.42 mL/kg) (InjA) and bufothionine (9.77 mL/kg) (BufoA) group. Liver weight indices were recorded to judge the degree of liver swelling, hematoxylin and eosin (H&E) staining of liver tissues was carried out to observe liver histological morphology injury and biochemical indicators including aspartate aminotransferase (AST); alanine aminotransferase (ALT); alkaline phosphatase (ALP); and total bilirubin (TBIL) were determined by modular auto-analyzer. In anti-tumor experiment, H22-tumor-bearing mice were randomly divided into five groups (n = 10): normal saline group, model group, cinobufacini injection (InjB) (5.14 mL/kg), bufothionine (8.02 mL/kg) (BufoB) and 5-fluorouracil (5-Fu) (3.42 mL/kg). Tumors were picked out and determined with vernier calipers. Histological morphology of tumors was observed by H&E staining. In SMMC-7721 cells, expressions of proteins related to mitochondria-mediated apoptosis pathway including Bcl-2, Bax, caspase-3, caspase-9, cyto-c, Bid, and p53 were analyzed by western blotting at low, medium, high concentrations of bufothione (3.62 µg/mL, 18.12 µg/mL,90.62 µg/mL). RESULTS: Butothionine relieved CCl4-induced liver morphology, decreased the level of ALT (P =2.46 × 10(-2)) and expressed tendency to decrease other biochemical markers including AST, ALP and TBIL. Butothionine could also promote necrosis of tumor tissue in H22-tumor-bearing mice and restrained tumor growth with 65.16% inhibition rate. Its mechanism might relate to up-regulation of p53 (at low, mediate and high concentration, corresponding P values were 0.142, 0.0257, 0.0162), caspase-3 (P = 0.246, 0.0267 and 0.0236), cyto-c (P = 0.276, 0.0343 and 0.0429), Bid (P = 0.0125, 0.0395 and 0.0132) and Bax (P = 0.563, 0.0492 and 0.0357) in a dose-dependent manner, down-regulation of Bcl-2 expression (P = 0.0232, 0.0178 and 0.0464), but had no significant effects on caspase-9 (P = 0.253, 0.147 and 0.287). CONCLUSION: Bufothionine induced the proteins for the mitochondria-mediated apoptosis that inhibits liver tumors and protects the liver against acute injury.

Study on spectrum-effect relationship of rhizoma Rhei, cortex Magnoliae Officinalis, fructus Aurantii Immaturus and their formula.

Rhizoma Rhei, cortex Magnoliae Officinalis and fructus Aurantii Immaturus compose dachengqi tang (DCQT), a classical formula of traditional Chinese medicine (TCM) that is used for acute intestinal obstruction and has been proven to be effective and economic. However, the ingredients of TCM are complicated, and it is unclear which ingredients are the most important for its effects. In this paper, the relationship between the spectra and effects is discussed to provide a powerful method and some insights into the quality control of the herbs and their formula. High-performance liquid chromatographic (HPLC) fingerprint analysis was performed to investigate the chemical structures in different batches of rhizoma Rhei, cortex Magnoliae Officinalis, fructus Aurantii Immaturus and DCQT. Hierarchical clustering analysis was employed to evaluate the similarities between fingerprints. Animal model of small intestinal propulsion was established to study the purgative functions of the herbs and DCQT. The relationship between the chemical ingredients and the effects was explored by regression analysis. HPLC fingerprint analysis results demonstrated variations between ingredients in different batches of rhizoma Rhei, cortex Magnoliae Officinalis, fructus Aurantii Immaturus and DCQT. The origin, collection time and preparation process may have contributed to these differences. Small intestinal propulsion results showed that, compared with the control group, the positive and therapeutic groups including single herbs and formula were significantly effective (P < 0.05). Spectrum-effect relationship results indicated that seven peak ingredients, hesperidin, aloe-emodin, honokiol, rhein, magnolol, emodin and sennoside A, were inducted in the regression equation, among which, the influence of sennoside A was the largest and most positively associated with the effects. The data analysis results indicated that many ingredients contributed to the purgative effects, among which, sennoside A might be the most important effective component; therefore, sennoside A should be determined for quality control. Furthermore, the spectrum-effect relationship is simple, operative and suitable for the quality evaluation of TCM.

Bufothionine, a possible effective component in cinobufocini injection for hepatocellular carcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinobufacini has been traditionally used in China for the treatment of tumor since hundreds years ago. For recent years, its modern preparation,cinobifucini injection has also obtained satisfactory therapeutic functions for cancer. MATERIALS AND METHODS: High performance liquid chromatography (HPLC) analysis was applied to determine the content of cinobufagin, resibufogenin and bufothionine in cinobufacin extract liquid and injection; MTT assay and flow cytometric analysis were also respectively used to study the effect of cinobufacini extract liquid, injection and three chemical structures on cells and cell cycles. RESULTS: HPLC results demonstrated that in cinobufacini extract liquid three ingredients (cinobufagin, resibufogenin and bufothionine) were all monitored while in cinofacini injection only bufothinone was detected; MTT assays showed bufothionine could obviously inhibit the proliferation of human hepatocellular carcinoma cell lines such as SMMC-7721 and BEL-7402 in a dose- and time-dependent manner as well as cinobufagin and resibufogenin; further flow cytometric analysis indicated obvious increases in G2/M phase and decrease in G0/G1 phase when SMMC-7721 cell line exposure to bufothionine (480 µg/ml). CONCLUSIONS: These results suggested bufothionine could be involved in treatment of human cancer for cinobufacini injection and the mechanism might be relative to induce G2/M phase cell cycle arrest.