Rotundic Acid Induces DNA Damage and Cell Death in Hepatocellular Carcinoma Through AKT/mTOR and MAPK Pathways.

Hepatocellular carcinoma (HCC) is the fourth largest cause of cancer-related deaths worldwide with limited therapeutic interventions. Renewed interest in natural products as drug leads has resulted in a paradigm shift toward the rapid screening of medicinal plants for the discovery of new chemical entities. Rotundic acid (RA), a plant-derived triterpenoid, has been anecdotally reported to possess anti-inflammatory and cardio-protective abilities. The present study highlights the anti-cancer efficacy of RA on HCC in vitro and in vivo. The inhibitory effects of RA on HCC cell viability was determined by MTT. Soft agar colony formation and clonogenic assays also showed that RA inhibited HCC cell proliferation. Flow cytometry, confocal, and western blot results further indicated that RA induced cell cycle arrest, DNA damage, and apoptosis by modulating the AKT/mTOR and MAPK pathways. Besides the suppression of migration and invasion, tube formation and VEGF-ELISA revealed the anti-angiogenic abilities of RA on HCC. Moreover, RA also inhibited tumor growth in a HepG2 xenograft mouse model. To our best knowledge, this is the first extensive study of the anticancer activity of RA on HCC. The results demonstrate that RA could be a potential drug candidate for HCC treatment.

Effects of Lactobacillus delbrueckii on immune response, disease resistance against Aeromonas hydrophila, antioxidant capability and growth performance of Cyprinus carpio Huanghe var.

The aim of the present study was to investigate effects of dietary Lactobacillus delbrueckii (L. delbrueckii) on immune response, disease resistance against Aeromonas hydrophila (A. hydrophila), antioxidant capability and growth performance of Cyprinus carpio Huanghe var. 450 fish (mean weight of 1.05 ± 0.03 g) were randomly distributed into five groups that fed diets containing different levels of L. delbrueckii (0, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 CFU g-1) for 8 weeks. The results showed that intestinal immune parameters such as lysozyme, acid phosphatase, and myeloperoxidase activities, immunoglobulin M content, and the survival rate were improved in fish fed with 1 × 106 and 1 × 107 CFU g-1L. delbrueckii. In addition, 1 × 107 CFU g-1L. delbrueckii supplementation down-regulated mRNA levels of TNF-α, IL-8, IL-1ß and NF-κBp65, and up-regulated IL-10 and TGF-ß mRNA levels in the intestine. The survival rate was significantly (P < 0.05) higher (68.33%) in fish fed 1 × 106 CFU g-1L. delbrueckii than the control diet-fed group (40%) after challenge by A. hydrophila. Fish fed with diet containing 1 × 106 CFU g-1L. delbrueckii showed higher antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and total antioxidant capacity (T-AOC) and lower MDA concentrations than those of the control group (P < 0.05). The relative gene expression (SOD, CAT, GPX) showed the same trend with their activities. In addition, the growth performance was significantly improved in fish fed with the diet containing 1 × 106 and 1 × 107 CFU g-1L. delbrueckii (P < 0.05). These results demonstrated that dietary optimal levels of L. delbrueckii enhanced immunity, disease resistance against A. hydrophila antioxidant capability and growth performance in Cyprinus carpio Huanghe var.

Intervertebral Foramen Injection of Ozone Relieves Mechanical Allodynia and Enhances Analgesic Effect of Gabapentin in Animal Model of Neuropathic Pain.

BACKGROUND: In a 5-year follow-up study in a hospital in southern China, it was shown that intervertebral foramen (IVF) injection of ozone at the involved segmental levels could significantly alleviate paroxysmal spontaneous pain and mechanical allodynia in patients with chronic, intractable postherpetic neuralgia (PHN) and improve the quality of life. However, so far no proof-of-concept studies in animals have been available. OBJECTIVE: This study was designed to investigate whether IVF ozone has an analgesic effect on animal models of neuropathic and inflammatory pain. STUDY DESIGN: Experimental trial in rats. SETTING: Institute for Biomedical Sciences of Pain. METHODS: By IVF injection, a volume of 50 µl containing 30 µg/mL ozone-oxygen mixture or 50 µl air was carried out on male Sprague-Dawley rats of naïve, inflammatory pain states produced by injections of either bee venom or complete Freud's adjuvant, and neuropathic pain state produced by spared nerve injury, respectively. The effects of IVF ozone on pain-related behaviors were evaluated for 2 weeks or one month. Then combined use of gabapentin (100 mg/1 kg body weight) with IVF ozone was evaluated in rats with neuropathic pain by intraperitoneal administration 5 days after the ozone treatment. Finally, the analgesic effects of another 4 drugs, AMD3100 (a CXCR4 antagonist), A-803467 (a selective Nav1.8 blocker), rapamycin (the mTOR inhibitor), and MGCD0103 (a selective histone deacetylase inhibitor) were evaluated for long term through IVF injection, respectively. RESULTS: (1) IVF injection of ozone at L4-5 was only effective in suppression of mechanical allodynia in rats with neuropathic pain but not with inflammatory pain; (2) the analgesic effects of IVF ozone lasted much longer (> 14 days) than other selective molecular target drugs (< 48 hours) inhibiting or antagonizing at Nav1.8 (A-803467), CXCR4 (AMD3100), mTOR (rapamycin), and histone deacetylase (MGCD0103); (3) combined use of systemic gabapentin and IVF ozone produced a synergistic analgesic effect in rats with neuropathic pain. LIMITATIONS: Evaluation of the possible analgesic effects of the intraplantar injection of ozone was not performed. CONCLUSIONS: In the present study, we provided a line of evidence for the first time that IVF injection of ozone selectively relieved neuropathic pain but not inflammatory pain, and enhanced the analgesic effect of gabapentin. KEY WORDS: Chronic pain, neuropathic pain, inflammatory pain, ozone therapy, interventional therapy, gabapentin, spared nerve injury, bee venom, complete Freud's adjuvant.

Effects of Tongmai oral liquid in femoral ateriovenous fistula.

BACKGROUND: This study was conducted to investigate the protective effect of Tongmai oral liquid on arteriovenous fistula function and to provide an effective method to promote fistula maturation. METHODS: Fifteen female and fifteen male SPF New Zealand rabbits were randomly allocated into 3 groups including control, Aspirin and Tongmai oral liquid groups. A side-to-side femoral arteriovenous fistula was established in each rabbit and then animals were treated with Aspirin or Tongmai oral liquid for 2 weeks. The concentrations of circulating ET-1 and NO were determined before and after operation (on preoperative day, operative day, post-D1, post-D3, post-D7 and post-D15), respectively. Blood flow of the fistula stoma and contralateral artery and vein was determined on the 15th postoperative day. Last, the fistula stoma was dissected to observe patency, thrombosis and adhesion with surrounding tissues. RESULTS: 28 rabbits survived during the surgical process and the following 15-day observational period. Tissue adhesion of arteriovenous fistula with surrounding tissues was improved and fistula thrombosis was reduced by treatment with Tongmai oral liquid. NO concentration decreased to a different extent after vascular surgery. Tongmai oral liquid failed to regulate the equilibrium between NO and ET-1, but it improved blood flow of fistula stoma, as compared to control and Aspirin groups. Blood flow of fistula stoma in the three groups was lower than that of the contralateral femoral artery. CONCLUSIONS: Tongmai oral liquid improved the function of femoral ateriovenous fistula in the rabbit model by increasing blood flow and reducing thrombosis, probably not by regulating the dynamic equilibrium between NO and ET-1.

Triptolide synergistically enhances temozolomide-induced apoptosis and potentiates inhibition of NF-κB signaling in glioma initiating cells.

Glioblastoma multiforme (GBM) is a lethal solid cancer in adults. Temozolomide (TMZ) is a first-line chemotherapeutic agent but the efficacy is limited by intrinsic and acquired resistance in GBM. Triptolide (TPL), a derivative from traditional Chinese medicine, demonstrated anti-tumor activity. In this study, we explored the interaction of TPL and TMZ in glioma-initiating cells (GICs) and the potential mechanism. A GIC line (GIC-1) was successfully established. Cell viability of GIC-1 after treatment was measured using a CCK-8 assay. The interaction between TPL and TMZ was calculated from Chou-Talalay equations and isobologram. Self-renewal was evaluated with tumor sphere formation assay. Apoptosis was assessed with flow cytometry and western blot. Luciferase assay was employed to measure NF-κB transcriptional activity. The expression of NF-κB downstream genes, NF-κB nuclear translocalization and phoshorylation of IκBα and p65 were evaluated using western blot. We found that GIC-1 cells were resistant to TMZ, with the expected IC50 of 705.7 µmol/L. Co-treatment with TPL yielded a more than three-fold dose reduction of TMZ. TPL significantly increased the percentage of apoptotic cells and suppressed the tumor sphere formation when combined with TMZ. Phosphorylation of IκBα and p65 coupled with NF-κB nuclear translocalization were notably inhibited after a combined treatment. Co-incubation synergistically repressed NF-κB transcriptional activity and downstream gene expression. TPL sensitizes GICs to TMZ by synergistically enhancing apoptosis, which is likely resulting from the augmented repression of NF-κB signaling. TPL is therefore a potential chemosensitizer in the treatment of GBM.

Effect of Shengmai injection () on diaphragmatic contractility in doxorubicin-treated rats.

OBJECTIVE: To explore the diaphragmatic toxicity in doxorubicin (DOX)-treated rats and the related mechanisms, as well as the effects of Shengmai Injection (SMI, ) on the diaphragmatic dysfunction. METHODS: Thirty Sprague-Dawley male rats were randomly divided into three groups: control, DOX-treated and DOX+SMI treated groups. DOX was given to rats in DOX and DOX+SMI groups in 6 equal doses [2.5 mg/kg, intraperitoneal injection (i.p.)], on alternate days, over a period of 2 weeks for a cumulative dose of 15 mg/kg. SMI was given to DOX+SMI rats in 12 doses (3 mL/kg, i.p.) for a period of 2 weeks before the administration of DOX and 2 weeks during the administration of DOX. The rats in the control group received equal volume of normal saline. Subsequently, the twitch and tetanic characteristics and force-frequency relationships, and the malondialdehyde (MDA) levels and the superoxide dismutase (SOD) activities, as well as the mRNA content and proteins of inducible nitric oxide synthase (iNOS) were determined. RESULTS: The DOX-treated rats had decreased the peak twitch tension (Pt), maximal tetanic tension (P0) and force-frequency relationship as compared with the control rats (P<0.01), while the diaphragm contractility in rats treated with SMI were significantly higher than that in DOX-treated rats (P<0.01). The DOX-treated rats had increased MAD levels and decreased SOD activities (P<0.05), and SMI decreased the MDA levels and increased the SOD activities in DOX-treated rats (P<0.05). Ultrastructure of diaphragm in the DOX-treated rats revealed typical alterations including fracture of diaphragm fibers, and edema and degeneration of mitochondria; these changes were relieved by SMI treatment. The mRNA content and protein of iNOS in DOX-treated rats were remarkably higher than those in control rats (P<0.01), while SMI decreased the mRNA expression level of iNOS in DOX-treated rats (P<0.05). CONCLUSIONS: Lipid peroxidation is responsible for DOX-induced diaphragm toxicity. SMI protects diaphragm muscles and their function from DOX impairment, and these beneficial effects may be somehow correlated with the decrease in expression of iNOS and lipid peroxidation.

Impact of the haplotypes of the human pregnane X receptor gene on the basal and St John's wort-induced activity of cytochrome P450 3A4 enzyme.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Human pregnane X receptor (PXR/NR1I2) is a key regulator of cytochrome P450 3A4. To date, there are 198 reported SNPs for the human PXR/NR1I2 gene. Some of these SNPs are found to affect the inducing ability of PXR to CYP3A4. WHAT THIS STUDY ADDS: This study, for the first time, has investigated the effect of PXR haplotype on basal and St John's wort-induced CYP3A4 activity in humans. H1/H1 of the PXR gene had weaker basal transcriptional activity but greater inducible transcriptional activity to CYP3A4 than H1/H2 and H2/H2. AIMS: Human pregnane X receptor (PXR/NR1I2) is the master regulator of CYP3A4, which metabolizes >50% of drugs on the market. This study investigated the relationship between the two most frequent haplotypes [H1 (TCAGGGGCCACC) and H2 (CCGAAAACTAAT)] of PXR and basal and St John's wort (SJW)-induced CYP3A4 activity. METHODS: Ten healthy subjects carrying H1 and H2 haplotypes (three subjects with H1/H1, four with H1/H2 and three with H2/H2) entered this study. The 10 subjects did not carry CYP3A4*4, *5 and *6. All subjects were administrated a 300-mg SJW tablet three times daily for 14 days, and CYP3A4 activity was measured using nifedipine (NIF) as a probe. The plasma concentrations of NIF and dehydronifedipine (DNIF) were determined by a validated liquid chromatography/mass spectrometry/mass spectrometry method. RESULTS: After administration of SJW, the AUC(0-infinity) of NIF decreased significantly, and the AUC(0-infinity) of DNIF increased significantly (P < 0.05). For H1/H2, the AUC(0-infinity) of NIF decreased by 42.4%, and the AUC(0-infinity) of DNIF increased by 20.2%; for H2/H2, the AUC(0-infinity) of NIF decreased by 47.9%, and the AUC(0-infinity) of DNIF increased by 33.0%; for H1/H1, the AUC(0-infinity) of NIF decreased by 29.0%, yet the AUC(0-infinity) of DNIF increased by 106.7%. The increase of the AUC(0-infinity) of DNIF in H1/H1 was significantly different from the other two haplotype pairs (P < 0.05). Meanwhile, before administration of SJW, the ratio of AUC(0-infinity(DNIF))/AUC(0-infinity(NIF)) was the lowest for H1/H1 (22.1%), compared with H1/H2 (58.7%) and H2/H2 (30.0%). CONCLUSIONS: H1/H1 of the human PXR gene had weaker basal transcriptional activity but greater inducible transcriptional activity to CYP3A4 than H1/H2 and H2/H2.

Shinella kummerowiae sp. nov., a symbiotic bacterium isolated from root nodules of the herbal legume Kummerowia stipulacea.

Bacterial strain CCBAU 25048(T) was isolated from root nodules of Kummerowia stipulacea grown in Shandong province of China. Cells of the strain were Gram-negative, strictly aerobic, non-spore-forming, motile short rods. Phylogeny of 16S rRNA gene sequences revealed that the strain belonged to the genus Shinella, a member of family Rhizobiaceae. Its closest phylogenetic relatives were Shinella granuli Ch06(T) and Shinella zoogloeoides IAM 12669(T), respectively showing 98.3 and 98.9 % 16S rRNA gene sequence similarity. Strain CCBAU 25048(T) had DNA-DNA relatedness of 43.5 and 34.8 %, respectively, with S. zoogloeoides JCM 20728(T) and S. granuli JCM 13254(T). In addition, in TP-RAPD analysis, different patterns were obtained for these three strains and some rhizobial strains. The nifH, nodC and nodD sequences of CCBAU 25048(T) were identical or very similar to those of bean-nodulating Rhizobium tropici strains. Several phenotypic characteristics, including the use of citrate and d-ribose as carbon sources and growth at pH 11.0, as well as the fatty acid composition, could differentiate CCBAU 25048(T) from the two defined Shinella species. Therefore, a novel species Shinella kummerowiae sp. nov. is proposed, with strain CCBAU 25048(T) (=JCM 14778(T) =LMG 24136(T)) as the type strain.

Biotransformation and pharmacokinetics of the novel anticancer drug, SYUIQ-5, in the rat.

SYUIQ-5, a novel telomerase inhibitor, has demonstrated antitumor activity in nude mouse studies. The objective of the present study was to examine the metabolism and pharmacokinetics of SYUIQ-5 in rats. The plasma pharmacokinetics of SYUIQ-5 was nonlinear following i.p. administration at 15, 30 and 60 mg/kg. SYUIQ-5 metabolism in rat liver microsomes followed Michaelis-Menten kinetics, with Km and Vmax values of 12.3 microM and 2.01 nmol/min/mg protein, respectively. Ketoconazole significantly inhibited the metabolism of SYUIQ-5 in liver microsomes from rats pretreated with control vehicle or various inducers, whereas sulphaphenazole, ticlopidine, quinidine, and methylpyrazole had no inhibitory effects on SYUIQ-5 metabolism. Dexamethasone and beta-naphthoflavone (BNF), but not phenobarbital and ethanol, significantly induced SYUIQ-5 metabolism in rats. Alpha-naphthoflavone significantly inhibited SYUIQ-5 metabolism in liver microsomes from BNF-pretreated rats. Similar to other secondary amines, SYUIQ-5 underwent N-demethylation and O-oxygenation to at least two metabolites by rat liver microsomes. Pretreatment of rats with SYUIQ-5 at 0.1, 5 or 10 mg/kg for 5 days significantly induced the expression and activity of rat Cyp1A1/2, and induced Cyp3A1/2 expression at 10 mg/kg, but not Cyp2E1 and 2B1/2. These results indicate that that SYUIQ-5 exhibits dose-dependent pharmacokinetics in rats and it is mainly metabolized by Cyp3A1/2.

Induction of cytochrome P450 3A by the Ginkgo biloba extract and bilobalides in human and rat primary hepatocytes.

Ginkgo biloba is one of the most popular herbal medicines in the world, due to its purported pharmacological effects, including memory-enhancing, cognition-improving, and antiplatelet effects. The study aimed to investigate the activity and expression of cytochrome P450 (CYP) 3A in human and rat primary hepatocytes treated with standardized G. biloba extract (100, 500, and 2500 ng/ml) for 72 hr, and to measure the protein expression of CYP3A in human and rat primary hepatocytes treated with bilobalide (2, 10, and 50 ng/ml) and ginkgolides B (2, 10, and 50 ng/ml). The activity of CYP3A was measured by the quantification of dehydronifedipine formation using a validated tandem liquid chromatography mass spectrometry (LC/MS/MS) method. The levels of mRNA and protein of CYP3A were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western-blotting analysis, respectively. The G. biloba extract at 100-2,500 ng/ml significantly induced the activity, protein and mRNA expression of CYP3A in a dose-dependent manner in human and rat primary hepatocytes. Bilobalide at 2-50 ng/ml significantly increased CYP3A protein expression in a dose-dependent manner in human and rat primary hepatocytes. However, ginkgolide B did not affect CYP3A protein expression in vitro. The results indicate that G. biloba extract pretreatment significantly induced the expression of CYP3A protein and mRNA and increased CYP3A activity, and there was no significant species difference between human and rat. G. biloba may cause potential interactions with substrate drugs of CYP3A. Bilobalide might play a key role in the enzyme-inducing effects of G. biloba extract. Further study is needed to identify the substances in GBE that induce CYPs in vivo, and elucidate the molecular mechanism of CYP3A induction by GBE and bilobalides.

A mechanistic study of the intestinal absorption of cryptotanshinone, the major active constituent of Salvia miltiorrhiza.

The nature of intestinal absorption of most herbal medicine is unknown. Cryptotanshinone (CTS) is the principal active constituent of the widely used cardiovascular herb Salvia miltiorrhiza (Danshen). We investigated the oral bioavailability of CTS in rats and the mechanism for its intestinal absorption using several in vitro and in vivo models: 1) Caco-2 cell monolayers; 2) monolayers of MDCKII cells overexpressing P-glycoprotein (PgP); and 3) single-pass rat intestinal perfusion with mesenteric vein cannulation. The systemic bioavailabilities of CTS after oral and intraperitoneal administration at 100 mg/kg were 2.05 and 10.60%, respectively. In the perfused rat intestinal model, permeability coefficients based on CTS disappearance from the luminal perfusate (Plumen) were 6.7- to 10.3-fold higher than permeability coefficients based on drug appearance in venous blood (Pblood). Pblood significantly increased in the presence of the P-gP inhibitor, verapamil. CTS transport across Caco-2 monolayers was pH-, temperature- and ATP-dependent. The transport from the apical (AP) to the basolateral (BL) side was 3- to 9-fold lower than that from the BL to the AP side. Inclusion of verapamil (50 microM) in both AP and BL sides abolished the polarized CTS transport across Caco-2 cells. Moreover, CTS was significantly more permeable in the BL to AP than in the AP to BL direction in MDCKII and MDR1-MDCKII cells. The permeability coefficients in the BL to AP direction were significantly higher in MDCKII cells overexpressing PgP. These findings indicate that CTS is a substrate for PgP that can pump CTS into the luminal side.

[Study on new extraction technology of astragaloside IV].

OBJECTIVE: To explore the possibility and the optimal extraction technology of astragaloside IV by SFE-CO2. METHODS: According the content of astragaloside IV, the optimum extraction technology parameters such as extraction temperature, pressure, extraction time, velocity of fluid and co-solvent were investigated and the result was compared with that of water extraction. RESULTS: The optimum technical parameters were as follows: Extracting pressure 40 Mpa, temperature 45 degrees C, extracting time 2h, co-solvent was 95% ethanol and its dosage was 4ml/g, the ratio of CO2 fluid was 10 kg/kg x h. CONCLUSION: Extraction technology of astragaloside IV by SFE-CO2 is reliable, stable.

[Effect of soybean isoflavones on heart function of rats with adriamycin-induced heart failure].

OBJECTIVE: To observe the protective effect of soybean isoflavones (SI) on the heart function of the rats with adriamycin induced heart failure. METHODS: Thirty adult male SD rats were divided into 5 groups:normal control (NC) group, adriamycin (ADR) group, L-SI group, M-SI group and H-SI group. SI of 30, 60, 120 mg.kg(-1).d(-1) was orally administered through a stomach tube once a day for 6 days in L-SI group, M-SI group and H-SI group, respectively. The other two groups were given the same amount of normal saline the same way. Then ADR of 10 mg/kg was given intraperitoneally once to copy the model of heart-failure. The MedLab-U/4c biological signal collecting system was used to record and analyze the LVSP of the rats. The pathological changes of the cardiomyocytes were observed. RESULTS: As compared with NC group, the LVSP,+/-dp/dt max, Vpm of the ADR group were significantly lower (P<0.05 or P<0.01), but those of the H-SI group were markedly higher than those of the ADR group (P<0.01). Electron microscopic morphometry of the heart samples of the rats in ADR group revealed typical alterations, consisting an increase of collagen content, vacuolation, diminishing of the cardiomyocyte diameter, alteration of myofilaments and Z-lines of myofibers, and myofibrillar degeneration. SI of 120 mg.kg(-1).d(-1) treatment could prevent the loss of myofibrillae and the reduction of myocyte diameter, and the degeneration of myofilaments and Z-lines were reversed by SI. CONCLUSION: SI of 120 mg.kg(-1).d(-1) treatment can relieve the toxic effect of ADR on myocardium, and also obviously improve the cardiac contractility of heart-failure rats.