Osthole inhibits the migration and invasion of highly metastatic breast cancer cells by suppressing ITGα3/ITGß5 signaling.

Metastasis is the leading cause of death in breast cancer patients. Osthole, as an active compound detected in the traditional Chinese medicine Wenshen Zhuanggu Formula, has shown a promising anti-metastatic activity in human breast cancer cells, but the underlying mechanisms remain ambiguous. In this study we elucidated the anti-metastatic mechanisms of osthole in highly metastatic breast cancer cells and a zebrafish xenograft model. We showed that the expression of integrin α3 (ITGα3) and integrin ß5 (ITGß5) was upregulated in highly metastatic MDA-MB-231, MDA-MB-231BO breast cancer cell lines but was downregulated in poorly metastatic MCF-7 breast cancer cell line, which might be the key targets of osthole's anti-metastatic action. Furthermore, we showed that knockdown of ITGα3 and ITGß5 attenuated breast cancer cell migration and invasion possibly via suppression of FAK/Src/Rac1 pathway, whereas overexpression of ITGα3 and ITGß5 caused the opposite effects. Consistently, osthole significantly inhibited breast cancer metastasis by downregulating ITGα3/ITGß5 signaling in vitro and in vivo. These results provide new evidence that osthole may be developed as a candidate therapeutic drug for metastatic breast cancer.

Traditional Chinese medicine targeting cancer stem cells as an alternative treatment for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prevalent and highly malignant cancer throughout the world. Effective treatment of this disease is impeded by the high rate of metastasis, recurrence, and chemoresistance. Recent studies have revealed the close relationship between the malignant phenotype of HCC and cancer stem cells (CSCs). Therefore, CSC-targeted therapy is considered a promising strategy to eradicate HCC. Traditional Chinese medicine (TCM) can be effective in preventing recurrence and metastasis of some advanced HCC. A growing amount of literature has discovered that extracts or compounds derived from TCM exert an anti-CSC effect. This review introduces some formulas and chemical compounds derived from TCMs that have been reported to inhibit CSCs of HCC; these TCM-related drugs may help to provide an alternative approach to help manage cancers, especially for HCC which has a great potential of metastasis, recurrence, and chemoresistance.

Highly sensitive aptasensor based on synergetic catalysis activity of MoS2-Au-HE composite using cDNA-Au-GOD for signal amplification.

Single or few-layer nanosheets of MoS2 (MoS2 nanosheets) and a composite composed of MoS2 nanosheets, Au nanoparticles (AuNPs) and hemin (HE) (denoted as MoS2-Au-HE) were prepared. The composites possessed high synergetic catalysis activity towards the electroreduction of hydrogen peroxide. Furthermore, glucose oxidase (GOD) and AuNPs were used as marker of the complementary DNA (cDNA) strand of kanamycin aptamer to prepare a conjugate (reffered as cDNA-Au-GOD) that was designed as the signal probe. Both cDNA-Au-GOD and MoS2-Au-HE were applied to fabricate aptasensor for kanamycin. MoS2-Au-HE acted as solid platform for kanamycin aptamer and signal transmitters. AuNPs were employed as the supporter of cDNA and GOD which catalyze dissolved oxygen to produce hydrogen peroxide in the presence of glucose. Then cathodic peak current of H2O2 was recorded by differential pulse voltammetry (DPV). The electrochemical reduction of H2O2 was catalyzed by MoS2-Au-HE that was modified onto the surface of a glassy carbon electrode (GCE). The cathodic peak current of H2O2 was highly linearly decreased with an increase of kanamycin concentrations from 1.0ng/L to 1.0×105ng/L, with a detection limit of 0.8ng/L. This aptasensor can be used to detect kanamycin in milk with high specificity, sensitivity and selectivity.

Effects of Erlong Zuoci decoction on the age-related hearing loss in C57BL/6J mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Erlong Zuoci decoction (ELZCD), a typical traditional Chinese medicine (TCM) prescription, has long been clinically used in treatment of deafness and tinnitus with the syndrome of "kidney yin deficiency". However, there are few studies to investigate its pharmacological mechanisms. Until now, there is not report about its effects on the age-related hearing loss (ARHL). AIM OF STUDY: The present study was conducted to observe the effects of ELZCD on the ARHL in C57BL/6J mice and explore the mechanisms. MATERIALS AND METHODS: ELZCD was fed to C57BL/6J mice from 3 months to 6 months in ELZCD group as a dose of 6g/kg/d. And the same volume of saline was fed to mice in ARHL group. 3-months-old C57BL/6J mice were used as control group. High performance liquid chromatography (HPLC) was used for the quality control of ELZCD. Auditory brainstem response (ABR) was used to assess the hearing function of mice. The morphologic changes were observed by hematoxylin eosin (HE) staining. Apoptosis was tested by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. Mitochondrial damage was detected by transmission electron microscopy (TEM). Quantitative RT-PCR (qRT-PCR) was used to observe the mRNA expression of p53 and Bak. Fluorescence immunohistochemical technique was used to test the protein expression of p53 and Bak. RESULTS: The hearing threshold of ARHL group was higher than that of control group (P<0.001) and ELZCD decreased the rise of hearing threshold levels of ARHL mice (P<0.001), which suggested ELZCD inhibited the hearing loss of ARHL mice. HE staining showed that ELZCD decreased the spiral ganglion (SG) cell damage and loss in ARHL. TUNEL test showed that the apoptotic SG cells increased in ARHL group compared to control group and decreased in ELZCD group compared to ARHL group. TEM observation showed that mitochondrial damage was obvious in SG cells of ARHL group and ELZCD inhibited the mitochondrial damage. The qRT-PCR results showed that the mRNA expression of p53 and Bak in ARHL group increased compared to that of control group (P<0.05), and ELZCD reduced the elevated mRNA expression levels of p53 and Bak (P<0.01, P<0.05). In addition, ELZCD inhibited the increased proteins expression (green fluorescence) of p53 and Bak. CONCLUSION: The results demonstrated that ELZCD prevented ARHL in C57BL/6J mice and p53/Bak-mediated mitochondrial apoptosis of SG cells might be involved in the mechanisms.

Investigation of the Material Basis Underlying the Correlation between Presbycusis and Kidney Deficiency in Traditional Chinese Medicine via GC/MS Metabolomics.

Objective. To investigate the correlation between presbycusis and kidney deficiency as defined by traditional Chinese medicine (TCM) and its material basis from the perspective of metabolism. Methods. Pure-tone audiometry was used to test auditory function. A kidney deficiency symptom scoring table was used to measure the kidney deficiency accumulated scores of the research subjects. Gas chromatography/mass spectrometry (GC/MS) was used to measure the metabolites in the urine samples from 11 presbycusis patients and 9 elderly people with normal hearing. Results. Hearing loss in the elderly was positively correlated with kidney deficiency score in TCM. There were significant differences in urine metabolite profile between the presbycusis patients and the controls. A total of 23 differentially expressed metabolites were found. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these metabolites were related to glutathione metabolism, amino acid metabolism, glucose metabolism, the N-methyl-D-aspartic acid (NMDA) receptor pathway, and the γ -aminobutyric acid (GABA) receptor pathway. Conclusion. Glutathione metabolism, amino acid metabolism, glucose metabolism, NMDA receptors, and GABA receptors may be related to the pathogenesis of presbycusis and may be the material basis underlying the correlation between presbycusis and kidney deficiency in TCM.

Bioactivity of five components of Chinese herbal formula Jiangzhi granules against hepatocellular steatosis.

OBJECTIVE: This study aims to evaluate the bioactivity of five components of the traditional Chinese medicine complex prescription Jiangzhi granules against hepatocellular steatosis. METHODS: The five major components, including protopanaxadiol, tanshinone IIA, emodin, chlorogenic acid, and nuciferine, were extracted from Jiangzhi granules. Their cytotoxicity was assessed to determine the safe dose of each component for HepG2 cells. HepG2 cellular steatosis was induced using 1 mmol/L of free fatty acids (FFAs) for 24 h, and then treated with each component at high, intermediate, and low doses (500, 50, and 5 µmol/L), respectively for another 24 h. The effects on HepG2 steatosis were observed directly under optical phase microscopy, or through oil red O staining and Nile red assays. In addition, the levels of reactive oxygen species (ROS) in the steatotic HepG2 cells with and without high-dose protopanaxadiol treatment were measured using fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate staining. RESULTS: No obvious cytotoxicity was observed in the HepG2 cells incubated with each of the five components at up to 500 µmol/L. At 24 h after incubation with FFAs, the HepG2 cells swelled and many lipid droplets accumulated. The lipid content was attenuated after 24 h of incubation with protopanaxadiol, tanshinone IIA, and emodin at 500 or 50 µmol/L (P < 0.05), especially with 500 µmol/L protopanaxadiol (P < 0.01). In addition, the ROS level was elevated in steatotic cells, but decreased after intervention with 500 µmol/L protopanaxadiol (P < 0.05). CONCLUSION: Protopanaxadiol, tanshinone IIA, and emodin alleviate hepatocellular steatosis in a dose-dependent manner, and oxidative stress regulation may partially contribute to the effects of protopanaxadiol.

Chinese medicine formula lingguizhugan decoction improves Beta-oxidation and metabolism of Fatty Acid in high-fat-diet-induced rat model of Fatty liver disease.

Lingguizhugan decoction (LGZG), a classic traditional Chinese medicine (TCM) formula, has been used to treat obesity and hyperlipidemia in recent years, but the related mechanisms underlying the regulation of lipid metabolism by LGZG are not clear yet. Here, we reported the effectiveness and possible mechanisms of LGZG on rats with fatty liver disease induced by high-fat diet (HFD). Our results demonstrated that LGZG significantly attenuated HFD-induced fatty liver disease, as measured by body weight, liver index, epididymal fat pad-body weight ratio (EFP/BW), liver injury, and hepatic triglycerides (TG) probably through increasing serum thyroid hormone levels, improving beta-oxidation (via modulation of TR ß 1 and CPT1A expression), metabolism and transport (through modulation of SREBP-1c, ACSL and ApoB100 expression) of fatty acid. In addition, we discovered the herbal combination with the properties of warming yang to relieve water retention in the formula and proposed the biological basis of LGZG conventional effect via further study on disassembled formula. This study, for the first time, revealed the mechanisms through which LGZG regulates lipid metabolism. Furthermore, our study suggested that it might be feasible to understand the scientific implications of TCM from the perspective of classic formulas' conventional efficacy.

[Comparative study on the effects of different therapeutic methods in preventing and treating nonalcoholic fatty liver in rats].

OBJECTIVE: To explore the pathogenesis of nonalcoholic fatty liver (NAFL) in traditional Chinese medicine (TCM) by comparing the therapeutic efficacy of methods for fortifying the spleen and replenishing qi, warming yang and fortifying the spleen and warming yang to move water. METHODS: Male Wistar rats were randomly divided into normal, model, Sijunzi Tang (SJZ), Lizhong Tang (LZ), Linggui Zhugan Tang (LGZG) and Shenzhuo Tang (SZ) groups. Rats in the normal group were fed with ordinary diet, while the rats in the other groups were fed with high fat diet including 88% ordinary food, 10% lard oil and 2% cholesterol. After four weeks of treatment, the weight of liver and epididymal fat was recorded respectively for calculating the indexes of liver (liver weight/body weight) and epididymal fat (weight of epididymal fat pads/body weight); the pathological changes in liver tissues were observed by hematoxylin and eosin (HE) staining; the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triacylglycerol (TAG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and very-low-density lipoprotein cholesterol (VLDL-C) and liver TAG concentration were analyzed by biochemical test. RESULTS: Body weight, liver and epididymal fat indexes and liver TAG level of rats all significantly increased in the model group as compared with the normal group (P<0.05). Hepatic fatty infiltration, TAG concentration and the levels of serum TAG and ALT were significantly decreased in the LGZG and SZ groups when compared to those in the model group (P<0.05). In addition, epididymal fat index and serum TC level also significantly decreased in the LGZG group as compared with the model group (P<0.05). However, there was no significant change in liver TAG concentration in LZ and model groups. CONCLUSION: Method of warming yang or moving water can promote the lipid metabolism. It may be an effective strategy in preventing and treating NAFL by treating with warming yang and moving water together.

Danshensu inhibits acetaldehyde-induced proliferation and activation of hepatic stellate cell-T6.

OBJECTIVE: To evaluate the effects of danshensu, the main component of the extract of Chinese medicine Salvia miltiorrhiza, on the proliferation and activation of hepatic stellate cells (HSCs). METHODS: The activation of HSC-T6 was induced by exposure to acetaldehyde. In the meantime, different doses of danshensu were added to the culture medium. After 24 h of treatment with danshensu in acetaldehyde, the viability of HSC-T6 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cell cycle was determined through flow cytometry, and the gene transcription levels of plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-ß1 (TGF-ß1), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-2 (MMP-2) were analyzed by real-time quantitative polymerase chain reaction. RESULTS: The proliferation of HSCs induced by 200 µmol/L acetaldehyde could be significantly inhibited by danshensu, and the percentage of HSCs in S phase was significantly increased as compared with the control cells (P<0.05), which were respectively evidenced by MTT assay and flow cytometry. Danshensu down-regulated the mRNA expression of TGF-ß1 and PAI-1 and up-regulated the uPA transcription level (P<0.01), while the transcription level of MMP-2 was not significantly affected in HSC-T6. CONCLUSION: Danshensu can inhibit the proliferation and activation of HSC-T6, as well as regulate some cytokines involved in extracellular matrix accumulation, which offers a potential therapeutic alternative for liver fibrosis.

Polygonum cuspidatum Extract Induces Anoikis in Hepatocarcinoma Cells Associated with Generation of Reactive Oxygen Species and Downregulation of Focal Adhesion Kinase.

Anoikis has been recognized as a potential target for anticancer therapy. Polygonum cuspidatum (Huzhang) is a frequently used Chinese herb in hepatocarcinoma. In present study, we evaluated the effects of Polygonum cuspidatum extract (PCE) in hepatocarcinoma cells in suspension. The results showed that PCE inhibited the proliferation of hepatocarcinoma cells in suspension in a dose- and time-dependent manner. PCE also inhibited anchorage-independent growth of hepatocarcinoma cells in soft agar. PCE induced anoikis in human hepatocarcinoma Bel-7402 cells accompanied by caspase-3 and caspase-9 activation and poly(ADP-ribose) polymerase cleavage, which was completely abrogated by a pan caspase inhibitor, Z-VAD-FMK. In addition, PCE treatment induced intracellular reactive oxygen species (ROS) production in Bel-7402 cells. NAC, an ROS scavenger, partially attenuated PCE-induced anoikis and activation of caspase-3 and caspase-9. Furthermore, PCE inhibited expression of focal adhesion kinase (FAK) in Bel-7402 cells. Overexpression of FAK partially abrogated PCE-induced anoikis. These data suggest that PCE may inhibit suspension growth and induce caspase-mediated anoikis in hepatocarcinoma cells and may relate to ROS generation and FAK downregulation. The present study provides new insight into the application of Chinese herb for hepatocarcinoma treatment.

[Effects of the mixture of Swertia pseudochinensis Hara and Silybum marianum Gaertn extracts on CCl(4)-induced liver injury in rats with non-alcoholic fatty liver disease].

OBJECTIVE: To study the mechanism of liver injury induced by carbon tetrachloride (CCl(4)) in rats with non-alcoholic fatty liver disease (NAFLD), and the therapeutic effects of the extract mixture of Dangyao (Swertia pseudochinensis Hara) and Shuifeiji (Silybum marianum Gaertn) on NAFLD rats with liver injury. METHODS: Male Wistar rats were randomized into normal control group, CCl(4) group, high-fat diet group, high-fat diet plus CCl(4) injection group (model group), diammonium glycyrrhizinate group and extract mixture group. Except the normal control and CCl(4) groups, rats were fed with high-fat diet (88% normal chow, 10% lard and 2% cholesterol) to induce NAFLD. Diammonium glycyrrhizinate and extracts were given by gavage. After eight weeks, a nonlethal dose of CCl(4) was injected intraperitoneally to all rats except the normal and high-fat diet groups. And 48 h later, all rats were sacrificed, and serum and liver tissues were collected for further study. Paraffin-processed liver tissue was stained with hematoxylin-eosin (HE) to observe the pathological changes. Serum alamine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. The levels of triacylglycerol (TAG), malondialdehyde (MDA) and glutathione (GSH) in liver tissues were also examined. Expression of uncoupling protein 2 (UCP2) was determined by reverse transcription-polymerase chain reaction and Western blotting. RESULTS: Liver sections stained with HE showed that the histopathological changes in the normal control group and the CCl(4) group were mild; massive hepatosteatosis diffusing in lobules was shown in the high-fat diet groups; steatosis, hepatocellular ballooning degeneration and inflammatory infiltration were severe around the central vein in sections of the model group. Compared with the model group, hepatosteatosis and ballooning were significantly attenuated in the treatment groups. Levels of serum ALT and AST, contents of TAG and MDA and the UCP2 expression in liver tissues of the model group increased obviously, while the level of liver GSH decreased. Compared with rats in the model group, the above biomarkers in the treatment groups were improved significantly. CONCLUSION: The mixture of Dangyao and Shuifeiji extracts can decrease the susceptibility and degree of liver injury induced by hepatotoxin in rats with NAFLD. Regulation of the balance of pro- and anti-oxidative stress factors is involved in the mechanism.

[Effects of Chinese herbal medicine Jiangzhi Granule on expressions of liver X receptor α and sterol regulatory element-binding protein-1c in a rat model of non-alcoholic fatty liver disease].

OBJECTIVE: To study the effects of Jiangzhi Granule (JZG), a compound traditional Chinese herbal medicine, in regulating liver X receptor α (LXRα) and sterol regulatory element-binding protein-1c (SREBP-1c) expressions in a rat model of non-alcoholic fatty liver disease (NAFLD). METHODS: Forty specific pathogen-free Wistar male rats were randomly divided into normal group, untreated group, pioglitazone (PIO) group and JZG group. All rats were fed with high-fat diet (88% normal chow plus 10% lard plus 2% cholesterol) for 4 weeks except for the normal group. After the NAFLD model was established, PIO and JZG were fed to rats in the corresponding groups respectively for another 4 weeks. At the end of the 8th week, liver steatosis level was observed under a light microscope with hematoxylin and eosin (HE) staining; serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and triacylglycerol (TAG) and free fatty acid (FFA) contents in liver tissues were measured. LXRα and SREBP-1c expressions in liver tissues were determined by real-time polymerase chain reaction and Western blot methods. RESULTS: Compared with the normal group, there were physiological changes for hepatic steatosis in liver tissues in the untreated group as observed by HE staining. JZG improved serum ALT and AST levels which were significantly increased in the untreated group. Both JZG and PIO improved FFA and TAG levels in liver tissues which were significantly increased in the untreated group. mRNA and protein levels of LXRα and SREBP-1c in the untreated group were higher than those in the normal group, while the treatment of JZG and PIO lowered their expressions. CONCLUSION: JZG may regulate fatty acid metabolic disorder by decreasing the levels of LXRα and SREBP-1c.

Dangfei liganning capsules attenuate the susceptibility of rat nonalcoholic fatty liver to carbon tetrachloride toxicity.

OBJECTIVE: To test whether nonalcoholic hepatic steatosis sensitizes carbon tetrachloride (CCl4)-induced liver injury, and to assess the therapeutic effect of Chinese medicine extracts of Dangfei Liganning capsules and their potential underlying mechanisms. METHODS: Male Wistar rats were fed a high-fat diet to induce nonalcoholic fatty liver disease (NAFLD) or a normal diet (N). Eight weeks later, a nonlethal dose of CCl4 was applied intraperitoneally. From the start, HF-CCl4 rats were administered daily Dangyao extracts (D), Dangfei Liganning capsules (DF), or Diammonium Glycyrrhizinate (G) intragastrically. Rats were sacrificed 48 h after CCl4 administration. In addition to serum biochemistry, liver histopathology was observed using hematoxylin-eosin (HE) and oil red O staining, and hepatic levels of triglyceride (TG), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), caspase-3 activation and cytochrome P450 (CYP2E1) expression were assessed. RESULTS: There was almost no response to the nonlethal dose of CCl4 in the N control group. However, the HF group demonstrated massive steatosis, and elevated levels of serum ALT and AST, liver MDA, CYP2E1, and caspase-3 activation, whereas the levels of GSH and SOD were significantly decreased. All indexes assessed were dramatically worse in the HF-CCl4 group compared to the HF group, in addition to the more severe steatosis, hepatocyte ballooning, and inflammatory infiltration apparent in the centrilobular area. The medicines we tested affected the pathological changes in HF-CCl4 rats to differing degrees: DF and G led to improvements in all of the above examined indexes, including an obvious improvement in histopathology, and DF improved serum ALT and MDA levels more markedly than G, whereas D extracts produced only mild liver injury attenuation. CONCLUSION: Liver with NAFLD is more sensitive to hepatotoxicity; furthermore, the disrupted balance of oxidative stress and anti-oxidant defense contributes to the underlying mechanisms. Dangfei Liganning capsules potentially decrease this toxic susceptibility and alleviate liver injury in non-alcoholic fatty liver.

[Effects of er-long-zuo-ci-wan on the spontaneous activities of auditory central nucleus in rat model of tinnitus induced by salicylate acid].

AIM: Observe the effects of er-long-zuo-ci-wan (EIZCW, a compound of Chinese Traditional Medicine) on the spontaneous discharge of external cortex of inferior colliculus (ICx) and secondary auditory cortex (AII) of chronic tinnitus model rats induced by salicylate acid, to explore the neural mechanisms underlying ELZCW preventing tinnitus. METHODS: 30 adult SD rats were involved and divided into three groups, normal control group, chronic tinnitus model group and ELZCW prevention group. Extracellular recording techniques and stereotaxic method were used. The spontaneous spikes were recorded and analyzed from ICx and all in different group rats. The average rate of spontaneous discharge and the interspike interval histogram of spontaneous activities were used as indexes. RESULTS: (1) Compared with normal control group, the average rate of spontaneous discharge recorded from the ICx in the chronic tinnitus model group increased significantly (4.57 +/- 0.54 Hz vs. 3.14 +/- 0.40 Hz, P < 0.05). Furthermore analysis showed that the discharge rate of short spike interval from the ICx in the chronic tinnitus model group increased than that of the normal group (0-40 ms: 58% vs. 40%; 0-4 ms: 9% vs. 5%). And there was an increasing tendency of the average rate of spontaneous discharge recorded from the AII in the chronic tinnitus model group compared with that in the normal group. (2) Compared with the chronic tinnitus model group, the average rate of spontaneous discharge recorded from the ICx and AII in the ELZCW prevention group significantly decreased than that in the chronic tinnitus model group (ICx: 2.41 +/- 0.21 Hz vs. 4.57 +/- 0.54 Hz, P < 0.01. AII: 2.24 +/- 0.24 Hz vs. 4.57 +/- 0.54 Hz , P < 0.01). And the discharge rate of short spike interval from the ICx and AII in the chronic tinnitus model group decreased than that in the normal control group (ICx: 0-40 ms 50% vs. 58%, 0-4 ms 4% vs. 9%. All: 0-22 ms: 24% vs. 31%, 0-8 ms 19% vs. 16%). CONCLUSION: If the increasing of the spontaneous activities of ICx and AII in chronic tinnitus rats means tinnitus, the use of ELZCW could decrease this kind of changes.